CHARACTERISTIC ANALYSIS METHOD AND CLASSIFICATION OF PHARMACEUTICAL COMPONENTS BY USING TRANSCRIPTOMES

Abstract
The present invention provides a novel method for the classification of adjuvants. In one embodiment, the present invention provides a method for generating organ transcriptome profiles for adjuvants, said method comprising: (A) a step for obtaining expression data by performing transcriptome analysis for at least one organ of a target organism by using at least two adjuvants; (B) a step for clustering the adjuvants with respect to the expression data; and (C) a step for generating the organ transcriptome profile for the adjuvants on the basis of the clustering.
Description
STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 690188_409C1_SEQUENCE_LISTING.txt. The text file is 2 KB, was created on Apr. 18, 2022, and is being submitted electronically via EFS-Web.


TECHNICAL FIELD

The present invention relates to a feature analysis method and classification of components used in drugs (hereinafter, referred to as “drug component” unless specifically noted otherwise, and refers to a component such as active ingredients, additives, or adjuvants). More specifically, the present invention relates to classification and feature analysis methodologies based on transcriptome analysis of a drug component such as an adjuvant.


BACKGROUND ART

Evaluation of efficacy and safety (toxicity) of a drug component (e.g., active ingredient, additive, adjuvant, or the like) or the drug itself serves a critical role in determining whether the drug is approved and allowed to be distributed to the market.


Nonclinical trials are proactively conducted for active ingredients with regard to efficacy and safety from the active pharmaceutical ingredient stages. However, additional components (additive) and adjuvants are not proactively tested. Currently, safety and efficacy are empirically tested without a systematic approach.


Adjuvants in particular have been recognized as supplemental components, rather than drawing attention for their own efficacy. The term adjuvant is derived from “adjuvare” which means “help” in Latin. Adjuvant is a collective term for substances (agents) administered with the primary agent such as a vaccine for use in enhancing the effect thereof (e.g., immunogenicity). Research and development of classical adjuvants (i.e., immunoadjuvants) have a long history of about 90 years, but research on the mechanisms of adjuvants themselves was not very active until recently. Recently, research and development thereon is active, triggered by immunological and microbiological research as well as research on natural immunity and dendritic cells. For empirically conducted adjuvant development, scientific approach is about to become possible lately from molecular to organism levels.


SUMMARY OF INVENTION
Solution to Problem

The present invention has been completed after finding that by clustering results of transcriptome analysis for a plurality of drug components (e.g., active ingredients, additives, or adjuvants), each cluster can be clustered by each feature of the components (e.g., active ingredients, additives, or adjuvants) to systematically classify the drug components. It was also found that known drug components (e.g., active ingredients, additives, or adjuvants) have a typical reference drug component (reference active ingredient for active ingredients, reference additive for additives, or reference adjuvant for adjuvants), such that the present invention also provides a technology that can identify whether novel substances or substances with an unknown specific effect or function (e.g., efficacy of an active ingredient, assistive function of an additive, or adjuvant function) are substances belonging to separate (e.g., 6 types) categories or others.


Therefore, the present invention provides the following.


(Item a1)

A method of generating an organ transcriptome profile of an adjuvant, the method comprising: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more adjuvants; (B) clustering the adjuvants with respect to the expression data; and (C) generating a transcriptome profile of the organ of the adjuvants based on the clustering.


(Item a2)

The method of item a1, wherein the transcriptome analysis comprises administering the adjuvants to the target organism and comparing a transcriptome in the organ at a certain time after administration with a transcriptome in the organ before administration of the adjuvants, and identifying a set of differentially expressed genes (DEGs) as a result of the comparison.


(Item a3)

The method of item a2, comprising integrating the set of DEGs in two or more adjuvants to generate a set of differentially expressed genes (DEGs) in a common manner.


(Item a4)

The method of item a3, comprising identifying a gene whose expression has changed beyond a predetermined threshold value as a result of the comparison, and selecting a differentially expressed gene in a common manner among identified genes to generate a set of significant DEGs.


(Item a5)

The method of item a4, wherein the predetermined threshold value is identified by a difference in a predetermined multiple and predetermined statistical significance (p value).


(Item a6)

The method of any one of items a2 to a5, comprising performing the transcriptome analysis for at least two or more organs to identify a set of differentially expressed genes only in a specific organ and using the set as the organ specific gene set.


(Item a7)

The method of any one of items a1 to a6, wherein the transcriptome analysis is performed on a transcriptome in at least one organ selected from the group consisting of a liver, a spleen, and a lymph node.


(Item a8)

The method of any one of items a1 to a7, wherein a number of the adjuvants is a number that enables statistically significant clustering analysis.


(Item a9)

The method of any one of items a1 to a8, comprising providing one or more gene markers unique to a specific adjuvant or an adjuvant cluster and a specific organ in the profile as an adjuvant evaluation marker.


(Item a10)


The method of any one of items a1 to a9, further comprising analyzing a biological indicator to correlate the adjuvants with a cluster.


(Item a11)


The method of item a10, wherein the biological indicator comprises at least one indicator selected from the group consisting of a wounding, cell death, apoptosis, NFκB signaling pathway, inflammatory response, TNF signaling pathway, cytokines, migration, chemokine, chemotaxis, stress, defense response, immune response, innate immune response, adaptive immune response, interferons, and interleukins.


(Item a12)


The method of item a10, wherein the biological indicator comprises a hematological indicator.


(Item a13)


The method of item a12, wherein the hematological indicator comprises at least one selected from the group consisting of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), relative (%) content of lymphocytes (LY %), relative (%) content of monocytes (MO %), relative (%) content of granulocytes (GR %), red blood cells (RBC), hemoglobins (Hb, HGB), hematocrits (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), platelet concentration (PCT), mean platelet volume (MPV), and platelet distribution width (PDW).


(Item a14)


The method of item a10, wherein the biological indicator comprises a cytokine profile.


(Item a15)


A program for implementing a method of generating an organ transcriptome profile of an adjuvant on a computer, the method comprising: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more adjuvants; (B) clustering the adjuvants with respect to the expression data; and (C) generating a transcriptome profile of the organ of the adjuvants based on the clustering.


(Item a15A)


The program of item a15, further comprising a feature of any one of items a1 to a14.


(Item a16)


A recording medium storing a program for implementing a method of generating an organ transcriptome profile of an adjuvant on a computer, the method comprising: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more adjuvants; (B) clustering the adjuvants with respect to the expression data; and (C) generating a transcriptome profile of the organ of the adjuvants based on the clustering.


(Item a16A)


The recording medium of item a16, further comprising a feature of any one of items a1 to a14.


(Item a17)


A system for generating an organ transcriptome profile of an adjuvant, the system comprising: (A) an expression data acquiring unit for obtaining or inputting expression data by performing transcriptome analysis on at least one organ of a target organism using two or more adjuvants; (B) a clustering computing unit for clustering the adjuvants with respect to the expression data; and (C) a profiling unit for generating a transcriptome profile of the organ of the adjuvants based on the clustering.


(Item a17A)


The system of item a17, further comprising a feature of any one of items a1 to a14.


(Item a18)


A method of providing feature information of an adjuvant, the method comprising: (a) providing a candidate adjuvant in at least one organ of a target organism; (b) providing a reference adjuvant set with a known function; (c) obtaining gene expression data by conducting transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) providing a feature of a member of the reference adjuvant set belonging to the same cluster as that of the candidate adjuvant as a feature of the candidate adjuvant.


(Item a19)


The method of item a18, further comprising a feature of any one of items a1 to a14.


(Item a20)


A program for implementing a method of providing feature information of an adjuvant on a computer, the method comprising: (a) providing a candidate adjuvant in at least one organ of a target organism; (b) providing a reference adjuvant set with a known function; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) providing a feature of a member of the reference adjuvant set belonging to the same cluster as that of the candidate adjuvant as a feature of the candidate adjuvant.


(Item a20A)


The program of item a19, further comprising a feature of any one of items a1 to a14.


(Item a21)


A recording medium for storing a program for implementing a method of providing feature information of an adjuvant on a computer, the method comprising: (a) providing a candidate adjuvant in at least one organ of a target organism; (b) providing a reference adjuvant set with a known function; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) providing a feature of a member of the reference adjuvant set belonging to the same cluster as that of the candidate adjuvant as a feature of the candidate adjuvant.


(Item a21A)


The recording medium of item a20, further comprising a feature of any one of items a1 to a14.


(Item a22)


A system for providing feature information of an adjuvant, the system comprising: (a) a candidate adjuvant providing unit for providing a candidate adjuvant; (b) a reference adjuvant providing unit for providing a reference adjuvant set with a known function; (c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) a feature analysis unit for providing a feature of a member of the reference adjuvant set belonging to the same cluster as that of the candidate adjuvant as a feature of the candidate adjuvant.


(Item a22A)


The system of item a22, further comprising a feature of any one of items a1 to a14.


(Item a23)


A method of controlling quality of an adjuvant by using the method of item a1 to item a14 or item a18 or item a19, the program of item a15, 15A, item a20, or item a20A, the recording medium of item a16, item a16A, item a21, or item a21A, or the system of item a, item a17, item a17A, item a22, or item a22A.


(Item a24)


A method of testing safety of an adjuvant by using the method of item a1 to item a14 or item a18 or item a19, the program of item a15, 15A, item a20, or item a20A, the recording medium of item a16, item a16A, item a21, or item a21A, or the system of item a, item a17, item a17A, item a22, or item a22A.


(Item a25)


A method of determining an effect of an adjuvant by using the method of item a1 to item a14 or item a18 or item a19, the program of item a15, 15A, item a20, or item a20A, the recording medium of item a16, item a16A, item a21, or item a21A, or the system of item a, item a17, item a17A, item a22, or item a22A.


<Classification Method>
(Item b1)

A method of classifying an adjuvant comprising classifying an adjuvant based on transcriptome clustering.


(Item b2)

The method of item b1, wherein the classification further comprises classification by at least one feature selected from the group consisting of classification based on a host response, classification based on a mechanism, classification by application based on a mechanism or cells (liver, lymph node, or spleen), and module classification.


(Item b3)

The method of item b1 or b2, wherein the classification comprises at least one classification selected from the group consisting of G1 to G6:


(1) G1 (interferon signaling);


(2) G2 (metabolism of lipids and lipoproteins);


(3) G3 (response to stress);


(4) G4 (response to wounding);


(5) G5 (phosphate-containing compound metabolic process); and


(6) G6 (phagosome).


(Item b4)

The method of item b3,


wherein the classification of G1 to G6 is performed by comparison with transcriptome clustering of a reference adjuvant,


wherein

    • a reference adjuvant of G1 is a STING ligand,
    • a reference adjuvant of G2 is a cyclodextrin,
    • a reference adjuvant of G3 is an immune reactive peptide,
    • a reference adjuvant of G4 is a TLR2 ligand,
    • a reference adjuvant of G5 is a CpG oligonucleotide,


      and/or
    • a reference adjuvant of G6 is a squalene oil-in-water emulsion adjuvant.


(Item b5)

The method of item b3 or b4,


wherein the classification of G1 to G6 is performed by comparison with transcriptome clustering of a reference adjuvant,


wherein reference adjuvant of G1 is selected from the group consisting of cdiGMP, cGAMP, DMXAA, PolyIC, and R848,


wherein a reference adjuvant of G2 is bCD (β cyclodextrin),


wherein a reference adjuvant of G3 is FK565,


wherein a reference adjuvant of G4 is MALP2s,


wherein a reference adjuvant of G5 is selected from the group consisting of D35, K3, and K3SPG, and/or


wherein a reference adjuvant of G6 is AddaVax.


(Item b6)

The method of any one of items b3 to b5,


wherein the classification of G1 to G6 is performed based on an expression profile of a gene (identification marker gene; DEG) with a significant difference in expression in transcriptome analysis,


wherein a DEG of the G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216,


wherein a DEG of the G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5,


wherein a DEG of the G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1,


wherein a DEG of the G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b,


wherein a DEG of the G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and


wherein a DEG of the G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


(Item b7)

A method of classifying an adjuvant, the method comprising:


(a) providing a candidate adjuvant in at least one organ of a target organism;


(b) providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6 of any one of items b3 to b6;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and


(d) determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item b8)

A method of manufacturing an adjuvant composition having desirable function, comprising:


(A) providing an adjuvant candidate,


(B) selecting an adjuvant candidate having a transcriptome expression pattern corresponding to a desirable function, and


(C) manufacturing an adjuvant composition using a selected adjuvant candidate.


(Item b9)

The method of item b8, wherein the desirable function comprises any one or more of G1 to G6 of any one of items b3 to b6.


(Item b10)


An adjuvant composition for exerting a desirable function, comprising an adjuvant exerting the desirable function, wherein the desirable function comprises any one or more of G1 to G6 of any one of items b3 to b6.


(Item b11)


A method of controlling quality of an adjuvant by using the method of any one of items b1 to b7.


(Item b12)


A method of testing safety of an adjuvant by using the method of any one of items b1 to b7.


(Item b13)


A method of determining an effect of an adjuvant by using the method of any one of items b1 to b7.


(Item b14)


A program for implementing an adjuvant classification method comprising classifying an adjuvant based on transcriptome clustering on a computer.


(Item b14A)


The program of item b14, wherein the transcriptome clustering further comprises one or more features of any one of items b2 to b7.


(Item b15)


A recording medium storing a program for implementing an adjuvant classification method comprising classifying an adjuvant based on transcriptome clustering on a computer.


(Item b15A)


The recording medium of item b15, wherein the transcriptome clustering further comprises one or more features of any one of items b2 to b7.


(Item b16)


A system for classifying an adjuvant based on transcriptome clustering, comprising a classification unit for classifying an adjuvant.


(Item b16A)


The system of item b16, wherein the transcriptome clustering further comprises one or more features of any one of items b2 to b7.


(Item b17)


A program for implementing an adjuvant classification method comprising classifying an adjuvant on a computer, the method comprising:


(a) providing a candidate adjuvant in at least one organ of a target organism;


(b) providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6 of any one of items b3 to b6;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and


(d) determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item b17A)


The program of item b17, further comprising one or more features of any one of items b2 to b7.


(Item b18)


A recording medium storing a program for implementing an adjuvant classification method on a computer, the method comprising:


(a) providing a candidate adjuvant in at least one organ of a target organism;


(b) providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6 of any one of items b3 to b6;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and


(d) determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item b18A)


The recording medium of item b18, further comprising one or more features of any one of items b2 to b7.


(Item b19)


A system for classifying an adjuvant, the system comprising:


(a) a candidate adjuvant providing unit for providing a candidate adjuvant in at least one organ of a target organism;


(b) a reference adjuvant storing unit for providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6 of any one of items b3 to b6;


(c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and


(d) a determination unit for determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item b19A)


The system of item b19, further comprising one or more features of any one of items b2 to b7.


(Item b20)


A gene analysis panel for use in classification of an adjuvant to G1 to G6 of any one of items b3 to b6 and/or to others, the gene analysis panel comprising means for detecting at least one DEG selected from the group consisting of a DEG of G1, a DEG of G2, a DEG of G3, a DEG of G4, a DEG of G5, and a DEG of G6,


wherein the DEG of G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216,


wherein the DEG of G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5,


wherein the DEG of G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1,


wherein the DEG of G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b,


wherein the DEG of G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and


wherein the DEG of G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


(Item b21)


The gene analysis panel of item b20, wherein the gene analysis panel comprises means for detecting at least a DEG of G1, means for detecting at least a DEG of G2, means for detecting at least a DEG of G3, means for detecting at least a DEG of G4, means for detecting at least a DEG of G5, and means for detecting at least a DEG of G6.


<Adjuvant of “Adjuvant”>

(Item bX1)


A composition for eliciting or enhancing adjuvanticity of an antigen, comprising δ inulin (β-D-[2→1]poly(fructo-furanosyl)α-D-glucose) or a functional equivalent thereof.


(Item bX2)


The composition of item bX1, wherein the equivalent has a transcriptome expression profile equivalent to δ inulin.


<Dendritic Cell Activation>

(Item bA1)


A composition for activating a dendritic cell, comprising δ inulin or a functional equivalent thereof.


(Item bA2)


The composition of item bA1, wherein the activation is performed in the presence of a macrophage.


(Item bA3)


The composition of item bA1 or bA2 comprising S inulin or a functional equivalent thereof, wherein the composition is administered with an enhancer of a macrophage.


<Th Orientation>

(Item bB1)


A composition for enhancing a Th1 response of a Th1 type antigen and a Th2 response of a Th2 type antigen, comprising S inulin or a functional equivalent thereof.


<TNFα>

(Item bC1)


An adjuvant composition comprising S inulin or a functional equivalent thereof, wherein the composition is administered while TNFα is normal or enhanced.


<Same Adjuvant/Adjuvant Determination Method+Manufacturing Method>

(Item bD1)


A method of determining whether a candidate adjuvant elicits or enhances adjuvanticity of an antigen, the method comprising: (a) providing a candidate adjuvant; (b) providing δ inulin or a functional equivalent thereof as an evaluation reference adjuvant; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the evaluation reference adjuvant to cluster the gene expression data; and (d) determining the candidate adjuvant as eliciting or enhancing adjuvanticity of an antigen if the candidate adjuvant is determined to belong to the same cluster as the evaluation reference adjuvant.


(Item bE1)


A method of manufacturing a composition comprising an adjuvant that elicits or enhances adjuvanticity of an antigen, the method comprising: (a) providing one or more candidate adjuvants; (b) providing δ inulin or a functional equivalent thereof as an evaluation reference adjuvant; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the evaluation reference adjuvant to cluster the gene expression data; (d) if there is an adjuvant belonging to the same cluster as the evaluation reference adjuvant among the candidate adjuvants, selecting the adjuvant as an adjuvant that elicits or enhances adjuvanticity of an antigen, and if not, repeating (a) to (c); and (e) manufacturing a composition comprising the adjuvant that elicits or enhances adjuvanticity of an antigen obtained in (d).


(Item c1)

A method for classifying a drug component comprising classifying a drug component based on transcriptome clustering.


(Item c2)

The method of item c1, wherein the step of classifying comprises a) generating a reference component based on the transcriptome clustering; and b) classifying a candidate drug component based on the reference component.


(Item c3)

The method of item c1 or c2, wherein the drug component is selected from the group consisting of an active ingredient, an additive, and an adjuvant.


(Item c4)

The method of any one of items c1 to c3, wherein the drug component is an adjuvant.


(Item c5)

The method of any one of items c1 to c4, wherein the classification further comprises classification by at least one feature selected from the group consisting of classification based on a host response, classification based on a mechanism, classification by application based on a mechanism or cells (liver, lymph node, or spleen), and module classification.


(Item c6)

The method of any one of items c1 to c5, wherein the classification comprises at least one classification selected from the group consisting of G1 to G6:


(1) G1 (interferon signaling);


(2) G2 (metabolism of lipids and lipoproteins);


(3) G3 (response to stress);


(4) G4 (response to wounding);


(5) G5 (phosphate-containing compound metabolic process); and


(6) G6 (phagosome).


(Item c7)

The method of item c6,


wherein the drug component is an adjuvant, and the classification of G1 to G6 is performed by comparison with transcriptome clustering of a reference drug component,


wherein a reference drug component of G1 is a STING ligand,


wherein a reference drug component of G2 is a cyclodextrin,


wherein a reference drug component of G3 is an immune reactive peptide,


wherein a reference adjuvant of G4 is a TLR2 ligand,


wherein a reference drug component of G5 is a CpG oligonucleotide, and/or


wherein a reference drug component of G6 is a squalene oil-in-water emulsion adjuvant.


(Item c8)

The method of item c6 or c7,


wherein the classification of G1 to G6 is performed by comparison with transcriptome clustering of a reference drug component,


wherein a reference component of G1 is selected from the group consisting of cdiGMP, cGAMP, DMXAA, PolyIC, and R848,


wherein a reference drug component of G2 is bCD (P cyclodextrin),


wherein a reference drug component of G3 is FK565,


wherein a reference drug component of G4 is MALP2s,


wherein a reference drug component of G5 is selected from the group consisting of D35, K3, and K3SPG, and/or


wherein a reference drug component of G6 is AddaVax.


(Item c9)

The method of any one of items c6 to c8,


wherein the classification of G1 to G6 is performed based on an expression profile of a gene (identification marker gene; DEG) with a significant difference in expression in transcriptome analysis,


wherein a DEG of the G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216,


wherein a DEG of the G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5,


wherein a DEG of the G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1,


wherein a DEG of the G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b,


wherein a DEG of the G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and


wherein a DEG of the G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


(Item c10)


A method of classifying a drug component, the method comprising:


(a) providing a candidate drug component;


(b) providing a reference drug component set;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in the reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c11)


The method of classifying a drug component of item c10, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set classified to at least one selected from the group consisting of G1 to G6 of any one of items c6 to c8;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c12)


A method of manufacturing a composition having a desirable function, comprising:


(A) providing a candidate drug component,


(B) selecting a candidate drug component having a transcriptome expression pattern corresponding to a desirable function, and


(C) manufacturing a composition using a selected candidate drug component.


(Item c13)


A method of screening for a composition having a desirable function, comprising;


(A) providing a candidate drug component; and


(B) selecting a candidate drug component having a transcriptome expression pattern corresponding to a desirable function.


(Item c14)


The method of item c12 or c13, wherein the desirable function comprises any one or more of G1 to G6 of any one of items c6 to c8.


(Item c15)


A composition for exerting a desirable function, comprising a drug component exerting the desirable function, wherein the desirable function comprises one or more classifications specified by the method of any one of items c1 to c11.


(Item c16)


The composition of item c15 for exerting a desirable function, comprising a drug component exerting the desirable function, wherein the desirable function comprises any one or more of G1 to G6 of any one of items c6 to c8.


(Item c17)


A method of controlling quality of a drug component by using the method of any one of item c1 to c11.


(Item c18)


A method of testing safety of a drug component by using the method of any one of items c1 to c11.


(Item c19)


A method of providing a toxicity bottleneck gene, comprising:


identifying a candidate of a toxicity bottleneck gene by using the method of any one of items c1 to c11;


making a knockout animal by knocking out the toxicity gene in another animal species; and


determining whether toxicity is reduced or eliminated in the knockout animal to select a gene with a reduction or elimination as a toxicity bottleneck gene.


(Item c20)


A method of determining toxicity of an agent, comprising:


determining whether activation of gene expression is observed for at least one of toxicity bottleneck genes for a candidate drug component such as an adjuvant; and


determining a candidate drug component observed to have the activation as having toxicity.


(Item c21)


A method of determining an effect of a drug component by using the method of any one of items c1 to c11.


(Item c22)


A method of providing an efficacy bottleneck gene, comprising:


identifying an efficacy determination gene by using the method of any one of items c1 to c11;


making a knockout animal by knocking out the toxicity gene in another animal species; and


determining whether efficacy is reduced or eliminated in the knockout animal to select a gene with a reduction or elimination as an efficacy bottleneck gene.


(Item c23)


A method of determining efficacy of an agent, comprising;


determining whether activation of gene expression is observed for at least one of efficacy bottleneck genes for a candidate drug component such as an adjuvant; and


determining a candidate drug component observed to have the activation as having efficacy.


(Item c24)


A program for implementing a drug component classification method comprising classifying a drug component based on transcriptome clustering on a computer.


(Item c25)


A recording medium storing a program for implementing a drug component classification method comprising classifying a drug component based on transcriptome clustering on a computer.


(Item c26)


A system for classifying a drug component based on transcriptome clustering, comprising a classification unit for classifying a drug component.


(Item c27)


A program for implementing a method of classifying a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) calculating a reference drug component set;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in a reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c28)


The program of item c27 for implementing a method of classifying a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set classified to at least one selected from the group consisting of G1 to G6 of any one of items c6 to c8;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c29)


A recording medium storing a program for implementing a method of classifying a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) calculating a reference drug component set;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in a reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c30)


The recording medium of item c29 storing a program for implementing a method of classifying a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set classified to at least one selected from the group consisting of G1 to G6 of any one of items c6 to c8;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c31)


A system for classifying a drug component, the system comprising:


(a) a candidate drug component providing unit for providing a candidate drug component in at least one organ of a target organism;


(b) a reference drug component calculating unit for calculating a reference drug component set;


(c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) a determination unit for determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in a reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster.


(Item c32)


The system of item c31 for classifying a drug component, the system comprising:


(a) a candidate drug component providing unit for providing a candidate drug component in at least one organ of a target organism;


(b) a reference drug component storing unit for providing a reference drug component set classified to at least one selected from the group consisting of G1 to G6 of any one of items c6 to c8;


(c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determination unit for determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the candidate drug cluster does not belong to any group.


(Item c33)


A gene analysis panel for using a drug component in classification specified by the method of any one of items c1 to c11.


(Item c34)


A gene analysis panel for use in classification of an adjuvant to G1 to G6 of any one of items c6 to c8 and/or to others, the gene analysis panel comprising means for detecting at least one DEG selected from the group consisting of a DEG of G1, a DEG of G2, a DEG of G3, a DEG of G4, a DEG of G5, and a DEG of G6,


wherein the DEG of G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216,


wherein the DEG of G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5,


wherein the DEG of G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1,


wherein the DEG of G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b,


wherein the DEG of G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and


wherein the DEG of G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


(Item c35)


The gene analysis panel of item c34, wherein the gene analysis panel comprises means for detecting at least a DEG of G1, means for detecting at least a DEG of G2, means for detecting at least a DEG of G3, means for detecting at least a DEG of G4, means for detecting at least a DEG of G5, and means for detecting at least a DEG of G6.


(Item c36)


A method of generating an organ transcriptome profile of a drug component, the method comprising:


obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components;


clustering the drug components with respect to the expression data; and generating a transcriptome profile of the organ of the drug components based on the clustering.


(Item c37)


The method of item c36, wherein the transcriptome analysis comprises administering the drug component to the target organism and comparing a transcriptome in the organ at a certain time after administration with a transcriptome in the organ before administration of the drug component, and identifying a set of differentially expressed genes (DEG) as a result of the comparison.


(Item c38)


The method of item c37, comprising integrating the set of DEGs in two or more drug components to generate a set of DEGs with a same change.


(Item c39)


The method of item c37 or c38, comprising selecting a DEG whose expression has changed beyond a predetermined threshold value among the DEGs with the same change as a result of the comparison to generate a set of significant DEGs.


(Item c40)


The method of item c39, wherein the predetermined threshold value is identified by a difference in a predetermined multiple and predetermined statistical significance (p value).


(Item c41)


The method of any one of items c38 to c41, comprising performing the transcriptome analysis for at least two or more organs to identify a set of differentially expressed genes (DEG) only in a specific organ and using the set as the organ specific DEG set.


(Item c42)


The method of any one of items c36 to c41, wherein the transcriptome analysis is performed on a transcriptome in at least one organ selected from the group consisting of a liver, a spleen, and a lymph node.


(Item c43)


The method of any one of items c36 to c42, wherein a number of types of the drug components is a number that enables statistically significant clustering analysis.


(Item c44)


The method of any one of items c36 to c43, comprising providing one or more gene markers unique to a specific drug component or a drug component cluster and a specific organ in the profile as a drug component evaluation marker.


(Item c45)


The method of any one of items c36 to c44, further comprising analyzing a biological indicator to correlate the drug components with a cluster.


(Item c46)


The method of item c45, wherein the biological indicator comprises at least one indicator selected from the group consisting of a wounding, cell death, apoptosis, NFκB signaling pathway, inflammatory response, TNF signaling pathway, cytokines, migration, chemokine, chemotaxis, stress, defense response, immune response, innate immune response, adaptive immune response, interferons, and interleukins.


(Item c47)


The method of item c46, wherein the biological indicator comprises a hematological indicator.


(Item c48)


The method of item c47, wherein the hematological indicator comprises at least one selected from the group consisting of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), relative (%) content of lymphocytes (LY %), relative (%) content of monocytes (MO %), relative (%) content of granulocytes (GR %), red blood cells (RBC), hemoglobins (Hb, HGB), hematocrits (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), platelet concentration (PCT), mean platelet volume (MPV), and platelet distribution width (PDW).


(Item c49)


The method of item c45, wherein the biological indicator comprises a cytokine profile.


(Item c50)


A program for implementing a method of generating an organ transcriptome profile of a drug component on a computer, the method comprising:


(A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components;


(B) clustering the drug components with respect to the expression data; and


(C) generating a transcriptome profile of the organ of the drug components, based on the clustering.


(Item c51)


A recording medium storing a program for implementing a method of generating an organ transcriptome profile of a drug component on a computer, the method comprising:


(A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components;


(B) clustering the drug components with respect to the expression data; and


(C) generating a transcriptome profile of the organ of the drug components based on the clustering.


(Item c52)


A system for generating an organ transcriptome profile of a drug component, the system comprising:


(A) an expression data acquiring unit for obtaining or inputting expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components;


(B) a clustering computing unit for clustering the drug components with respect to the expression data; and


(C) a profiling unit for generating a transcriptome profile of the organ of the drug components based on the clustering.


(Item c53)


A method of providing feature information of a drug component, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set with a known function;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) providing a feature of a member of the reference drug component set belonging to the same cluster as that of the candidate drug component as a feature of the candidate drug component.


(Item c54)


The method of item c53, further comprising a feature of any one of items c36 to c49.


(Item c55)


A program for implementing a method of providing feature information of a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set with a known function;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) providing a feature of a member of the reference drug component set belonging to the same cluster as that of the candidate drug component as a feature of the candidate drug component.


(Item c56)


A recording medium for storing a program for implementing a method of providing feature information of a drug component on a computer, the method comprising:


(a) providing a candidate drug component in at least one organ of a target organism;


(b) providing a reference drug component set with a known function;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) providing a feature of a member of the reference drug component set belonging to the same cluster as that of the candidate drug component as a feature of the candidate drug component.


(Item c57)


A system for providing feature information of a drug component, the system comprising:


(a) a candidate drug component providing unit for providing a candidate drug component;


(b) a reference drug component providing unit for providing a reference drug component set with a known function;


(c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) a feature analysis unit for providing a feature of a member of the reference drug component set belonging to the same cluster as that of the candidate drug component as a feature of the candidate drug component.


(Item c58)


A method of controlling quality of a drug component by using the method of items c36 to c49 or item c53.


(Item c59)


A method of testing safety of a drug component by using the method of items c36 to c49 or item c53.


(Item c60)


A method of determining an effect of a drug component by using the method of items c36 to c49 or item c53.


The present invention is intended so that one or more of the aforementioned features can be provided not only as the explicitly disclosed combinations, but also as other combinations thereof. Additional embodiments and advantages of the invention are recognized by those skilled in the art by reading and understanding the following detailed description as needed.


Advantageous Effects of Invention

The present invention provides a technology that can systematically classify a drug component (active ingredient, additive, adjuvant, or the like), and analyze and accurately predict a function thereof (e.g., detailed properties, safety, efficacy, or the like of an active ingredient, additive, or adjuvant) without detailed experimentation even for a drug component (e.g., active ingredient, additive, or adjuvant) with an unknown function. The present invention also provides a technology that can systematically classify a drug component (active ingredient, additive, adjuvant, or the like), and analyze whether a function is the same as one of the known reference drug components (e.g., reference adjuvants of G1 to G6 of the adjuvants exemplified herein) or others even for a drug component (e.g., active ingredient, additive, or adjuvant) with an unknown function.





BRIEF DESCRIPTION OF DRAWINGS


FIG. 1A shows an adjuvant gene space constituting a significantly differentially expressed gene (sDEG) from each organ. The sDEG for each gene is shown in a Venn diagram. A set unique to the lympho node (LN), liver (LV), and spleen (SP) was analyzed using a TargetMine pathway annotation with a p value. Likewise, a gene set shared by all three organs (LV, SP, and LN) was annotated by pathway analysis with a p value.



FIG. 1B shows an adjuvant gene space constituting a significantly differentially expressed gene (sDEG) from each organ. The sDEG for each gene is shown in a Venn diagram. A set unique to the lymph node (LN), liver (LV), and spleen (SP) was analyzed using a TargetMine pathway annotation with a p value. Likewise, a gene set shared by all three organs (LV, SP, and LN) was annotated by pathway analysis with a p value.



FIG. 2A shows relative comparison of adjuvants targeting the same receptor in the lymph node. A preferentially induced gene was selected by representing the value of fold changes among adjuvants targeting the same receptor such as 35/K3/K3SPG (Table 20) or cdiGMP/cGAMP/DMXAA (Table 21) as a z-score. The figure shows Venn diagrams (a, d), and mapping of selected genes to 40 modules (c, f).



FIG. 2B shows relative comparison of adjuvants targeting the same receptor in the lymph node. A preferentially induced gene was selected by representing the value of fold changes among adjuvants targeting the same receptor such as 35/K3/K3SPG (Table 20) or cdiGMP/cGAMP/DMXAA (Table 21) as a z-score. The figure shows Venn diagrams (a, d), and mapping of selected genes to 40 modules (c, f).



FIG. 3A shows adjuvant induced hematological changes. Hematological change of peripheral blood after adjuvant injection (a). Black solid lines, two gray dotted lines on the outside thereof, and two red dotted lines on the outside thereof indicate the average of mice treated with a buffer control, standard deviation (SD) level for 1 SD, and SD level for 2 SD, respectively. A change in parameter over 1 SD is indicated by a red bar (1 SD, light red; 2 SD, dark red), and other changes in parameter are indicated by a black bar. The number of correlated genes and representative list thereof are shown (b). Correlation plot for the white blood cell (WBC) count in the blood and CXCL9 expression level in the liver (LV) (c). The red sloped line indicates the linearly fitted line. Adjuvants that changed the WBC count more than 1 SD are indicated by a (light) red font. It should be noted that the hematological data for samples from Exp5 (bCD_ID, D35_ID, K3SPG_ID) and Exp10 (DMXAA_ID, MALP2s_ID, MPLA_ID, R848_ID) were obtained by independent experiments. For this reason, these are not physically associated with gene expression data of an organ (indicated by black×in the plot), but still exhibited an excellent correlation.



FIG. 3B shows adjuvant induced hematological changes. Hematological change of peripheral blood after adjuvant injection (a). Black solid lines, two gray dotted lines on the outside thereof, and two red dotted lines on the outside thereof indicate the average of mice treated with a buffer control, standard deviation (SD) level for 1 SD, and SD level for 2 SD, respectively. A change in parameter over 1 SD is indicated by a red bar (1 SD, light red; 2 SD, dark red), and other changes in parameter are indicated by a black bar. The number of correlated genes and representative list thereof are shown (b). Correlation plot for the white blood cell (WBC) count in the blood and CXCL9 expression level in the liver (LV) (c). The red sloped line indicates the linearly fitted line. Adjuvants that changed the WBC count more than 1 SD are indicated by a (light) red font. It should be noted that the hematological data for samples from Exp5 (bCD_ID, D35_ID, K3SPG_ID) and Exp10 (DMXAA_ID, MALP2s_ID, MPLA_ID, R848_ID) were obtained by independent experiments. For this reason, these are not physically associated with gene expression data of an organ (indicated by black×in the plot), but still exhibited an excellent correlation.



FIG. 4 is a schematic diagram showing the configuration for practicing the system of the invention.



FIG. 5 shows the correlation between the number of upregulated gene probes and consistency among adjuvant treated mice (overlapping portion in the Venn diagram). The horizontal axis indicates the percentage of probes at an overlapping portion among the total number of gene probes excluding overlaps (overlap between two samples for some of the adjuvants). The vertical axis indicates the number of upregulated (mean FC>2) gene probes. The red line (sloped line) indicates linear fitting. The gray region indicates the 99% confidence region. The names of adjuvants appearing outside the 99% confidence region are shown. The analysis shows that a potent gene response induced by an adjuvant in each organ is positively correlated with consistency of gene responses among individual mice.



FIG. 6A shows 40 modules from each organ, and the difference and commonality in the relationship among adjuvant group associated genes. Adjuvant group associated upregulated genes were selected based on the z-score of the gene expression value (Table 17). The selected probes were analyzed with respect to the distribution within 40 modules for each organ. G1 associated genes were distributed to a single interferon associated module (Tables 17 and 18). Genes associated with other groups were distributed broadly and differently to several modules for each organ. G1 in LN (5 types of adjuvants) indicates results of distribution of genes associated with 5 types of adjuvants (cdiGMP, cGAMP, DMXAA, PolyIC, and R848). The bars and numbers indicate the percentage of a gene probe within each group. G4 in LV and G3 and G6 in LN each comprise only one type of adjuvant (MalP2s, FK565, and AddaVax, respectively). These results have limited available data, so that interpretation requires care.



FIG. 6B shows 40 modules from each organ, and the difference and commonality in the relationship among adjuvant group associated genes. Adjuvant group associated upregulated genes were selected based on the z-score of the gene expression value (Table 17). The selected probes were analyzed with respect to the distribution within 40 modules for each organ. G1 associated genes were distributed to a single interferon associated module (Tables 17 and 18). Genes associated with other groups were distributed broadly and differently to several modules for each organ. G1 in LN (5 types of adjuvants) indicates results of distribution of genes associated with 5 types of adjuvants (cdiGMP, cGAMP, DMXAA, PolyIC, and R848). The bars and numbers indicate the percentage of a gene probe within each group. G4 in LV and G3 and G6 in LN each comprise only one type of adjuvant (MalP2s, FK565, and AddaVax, respectively). These results have limited available data, so that interpretation requires care.



FIG. 6C shows 40 modules from each organ, and the difference and commonality in the relationship among adjuvant group associated genes. Adjuvant group associated upregulated genes were selected based on the z-score of the gene expression value (Table 17). The selected probes were analyzed with respect to the distribution within 40 modules for each organ. G1 associated genes were distributed to a single interferon associated module (Tables 17 and 18). Genes associated with other groups were distributed broadly and differently to several modules for each organ. G1 in LN (5 types of adjuvants) indicates results of distribution of genes associated with 5 types of adjuvants (cdiGMP, cGAMP, DMXAA, PolyIC, and R848). The bars and numbers indicate the percentage of a gene probe within each group. G4 in LV and G3 and G6 in LN each comprise only one type of adjuvant (MalP2s, FK565, and AddaVax, respectively). These results have limited available data, so that interpretation requires care.



FIG. 7 shows a Venn diagram of genes significantly upregulated with a CpG adjuvant and annotation analysis thereof. Significantly differentially expressed genes were analyzed with a Venn diagram, and the shown gene sets were further analyzed for biological annotation by TargetMine. Shared genes of D35_K3_K3SPG (including 75 gene probes) were strongly associated with the interferon associated biological process.



FIG. 8 shows a Venn diagram of genes significantly upregulated with a sting ligand adjuvant and annotation analysis thereof. Significantly differentially expressed genes were analyzed with a Venn diagram, and the shown gene sets were further analyzed for biological annotation by TargetMine. Shared genes of cdiGMP_cGAMP_DMXAA (including 1491 gene probes) were strongly associated with the interferon associated biological process.



FIG. 9 shows grouping of adjuvants when determined by cluster analysis of a plurality of organs. AS04 was G1 in LN and G2 in LV. G2 was a unique group in SP. The adjuvant cluster structures did not change overall after adding data for AS04.



FIG. 10A shows that Advax™ induces a Th2 response when combined with a Th2 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering SV and an adjuvant. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of SV and adjuvant and stimulated with an MHC class I or II nucleoprotein epitope peptide. After the stimulation, IFN-7, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, †P<0.05, †††P<0.001 in Dunnett's multiple comparison test.



FIG. 10B shows that Advax™ induces a Th2 response when combined with a Th2 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering SV and an adjuvant. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of SV and adjuvant and stimulated with an MHC class I or II nucleoprotein epitope peptide. After the stimulation, IFN-γ, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, †P<0.05, †††P<0.001 in Dunnett's multiple comparison test.



FIG. 10C shows that Advax™ induces a Th2 response when combined with a Th2 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering SV and an adjuvant. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of SV and adjuvant and stimulated with an MHC class I or II nucleoprotein epitope peptide. After the stimulation, IFN-γ, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, †P<0.05, †††P<0.001 in Dunnett's multiple comparison test.



FIG. 11A shows that Advax™ exhibits a Th1 response when combined with a Th1 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering WV and an adjuvant. The mice were immunized on day 0 and day 14 using 15 μg of SV with alum. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE titer were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of WV and adjuvant and stimulated with an MHC class I or II specific nucleoprotein epitope peptide. After the stimulation, IFN-γ, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, and P<0.05 in Dunnett's multiple comparison test.



FIG. 11B shows that Advax™ exhibits a Th1 response when combined with a Th1 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering WV and an adjuvant. The mice were immunized on day 0 and day 14 using 15 μg of SV with alum. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE titer were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of WV and adjuvant and stimulated with an MHC class I or II specific nucleoprotein epitope peptide. After the stimulation, IFN-γ, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, and P<0.05 in Dunnett's multiple comparison test.



FIG. 11C shows that Advax™ exhibits a Th1 response when combined with a Th1 type antigen. (A to D) On day 0 and day 14, C57BL/6J mice (n=3) were immunized by i.m. administering WV and an adjuvant. The mice were immunized on day 0 and day 14 using 15 μg of SV with alum. Serum antigen specific total IgG, IgG1, and IgG2c and total IgE titer were measured by ELISA on days 14 and 28. (E to G) On day 28, spleen cells were prepared from mice immunized with 15 μg of WV and adjuvant and stimulated with an MHC class I or II specific nucleoprotein epitope peptide. After the stimulation, IFN-γ, IL-13, and IL-17 in the supernatant were measured by ELISA. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, and P<0.05 in Dunnett's multiple comparison test.



FIG. 12 shows that Advax™ does not induce an immune response in Tlr7−/− mice or when combined with a Th0 antigen. On day 0 and day 14, (A) C57BL/6J mice (n=4 or 5) or (B) Tlr7−/− mice (n=5) were each immunized by i.m. administering 100 μg of OVA and an adjuvant, or 1.5 μg of WV alone, or 1.5 μg of WV and an adjuvant. Serum antigen specific total IgG titer was measured by ELISA on days 14 and 28. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05 and **P<0.01 in Dunnett's multiple comparison test.



FIG. 13 shows that Advax™ activates DC in vivo, but not in vitro. (A to C) Bone marrow derived DC was stimulated in vitro for 24 hours with 1 mg/ml alum, 1 mg/ml Advax™, or 50 ng/ml LPS, and then CD40 expression on pDC was evaluated. (D to F) 0.67 mg of alum, 1 mg of Advax™, or 50 ng of LPS was injected into the base of the tail of C57BL/6J mice. After 24 hours from the injection, the groin lymph nodes were collected and treated with DNasel and collagenase. The cells were then stained and analyzed by FACS. The results represent three separate experiments. The results for saline are indicated by the gray region and a line, and results for adjuvant are indicated only by a line without further filling in with color.



FIG. 14A shows that a macrophage is required for the adjuvant effect of Advax™. Two-photon excitation microscopy on the lymph nodes (A to C) after 1 hour and (D to F) after 24 hours from i.d. administration of Brilliant Violet 421 labeled Advax delta inulin particles. CD169+ and MARCO+ macrophages were stained by i.d. injecting anti-CD169-FITC and anti-MARCO-phycoerythrin antibodies 30 minutes prior to the Advax™ administration. (A, D) Blue (left) indicates Advax™, (B, E) green (middle) indicates CD169+ macrophage, and (C, F) red (right) indicates MARCO+ macrophage. (G, H) Phagocytes in the lymph node were depleted by chlodronate liposome injection on the indicated date (days −2 and −7) and then WV+Advax™ were i.d. administered on day 0 for immunization. Serum antigen specific total IgG or IgG2 titer was measured by ELISA on days 14 and 28. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, and ***P<0.001 in Student's t-test.



FIG. 14B shows that a macrophage is required for the adjuvant effect of Advax™. Two-photon excitation microscopy on the lymph nodes (A to C) after 1 hour and (D to F) after 24 hours from i.d. administration of Brilliant Violet 421 labeled Advax delta inulin particles. CD169+ and MARCO+ macrophages were stained by i.d. injecting anti-CD169-FITC and anti-MARCO-phycoerythrin antibodies 30 minutes prior to the Advax™ administration. (A, D) Blue (left) indicates Advax™, (B, E) green (middle) indicates CD169+ macrophage, and (C, F) red (right) indicates MARCO+ macrophage. (G, H) Phagocytes in the lymph node were depleted by chlodronate liposome injection on the indicated date (days −2 and −7) and then WV+Advax™ were i.d. administered on day 0 for immunization. Serum antigen specific total IgG or IgG2 titer was measured by ELISA on days 14 and 28. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, and ***P<0.001 in Student's t-test.



FIG. 15A shows that Advax™ changes the gene expression of IL-1β, CLR, and TNF-α signaling pathway. The transcriptome of all organs (lung; LG, liver; LV, spleen; SP, kidney; KD, lymph node; LN) after 6 hours of administration of Advax™ alone (i.d., or i.p.) was obtained by Affimetrix Gene Chip (n=3). (C) IPA upstream regulator of Advax™ induced genes was analyzed in SP.



FIG. 15B is an expanded view of the explanation of symbols in IPA upstream regulator analysis of Advax™ induced genes in SP of FIG. 15A.



FIG. 15C is an expanded view of the correlation diagram according to IPA upstream regulator analysis of Advax™ induced genes in SP of FIG. 15A.



FIG. 16A shows that TNF-α is required for the adjuvant effect of Advax™ (A) The abdominal cavity macrophage was stimulated for 8 hours with Advax™ or alum, and TNF-α in the supernatant was measured by ELISA. (B) 1 hour after 1 mg of Advax™ or 0.67 mg of alum was i.p. injected, serum and peritoneal lavage fluid were collected, and the TNF-α level therein was measured by ELISA. (C to E) On day 0 and day 14, heterozygous or Tnfa−/− mice (n=5) were immunized by i.m. administering 1.5 μg of WV and an adjuvant. Serum antigen specific total IgG, IgG1, and IgG2c titers were measured by ELISA on days 14 and 28. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, and ***P<0.001 in Dunnett's multiple comparison test and Student's t-test.



FIG. 16B shows that TNF-α is required for the adjuvant effect of Advax™ (A) The abdominal cavity macrophage was stimulated for 8 hours with Advax™ or alum, and TNF-α in the supernatant was measured by ELISA. (B) 1 hour after 1 mg of Advax™ or 0.67 mg of alum was i.p. injected, serum and peritoneal lavage fluid were collected, and the TNF-α level therein was measured by ELISA. (C to E) On day 0 and day 14, heterozygous or Tnfa−/− mice (n=5) were immunized by i.m. administering 1.5 μg of WV and an adjuvant. Serum antigen specific total IgG, IgG1, and IgG2c titers were measured by ELISA on days 14 and 28. The results represent three separate experiments. The median value and SEM are shown for each group. Statistical significance is indicated by *P<0.05, **P<0.01, and ***P<0.001 in Dunnett's multiple comparison test and Student's t-test.



FIG. 17 shows a schematic diagram for creating a “toxic” group and “non-toxic” group based on a toxiogenomic database.



FIG. 18A shows that probes (genes) selected for a necrosis prediction model were concentrated into 5 pathways associated with immune response (1) and metabolism (4).



FIG. 18B calculated the “toxicity” score of each adjuvant by comparing gene variation patterns at 6 hours and 24 hours after administration of each adjuvant with the “toxic” gene pattern and “nontoxic” gene pattern after 6 and 24 hours in the toxiogenomic database. The top part of the figure shows adjuvants exhibiting a high toxicity score in the comparative result after 6 hours (left) and after 24 hours (right). The bottom part shows the toxicity score of each adjuvant in the comparative result after 24 hours.



FIG. 19 shows the ALT activity at 6 hours (top) and 24 hours (bottom) after actually administrating each adjuvant.



FIG. 20 shows results of staining liver collected on day 1, day 2, day 3, and day 5 of intraperitoneal administration of FK565 to mice with hematoxylin and eosin and performing histological analysis. The arrow indicates liver damage. The scale bar indicates 100 km.



FIG. 21 shows results of staining a liver collected after intraperitoneally administering FK565 into mice with TUNEL and checking for apoptosis. The left side shows results of a control PBS administration, and the right side shows results of FK565 administration.



FIG. 22 Blood was collected on day 1 and day 2 after 3 hours from intraperitoneally administering PBS, FK565 (1 μg/kg, 10 μg/kg, or 100 μg/kg), or LPS (1 mg/kg) to mice.



FIG. 22 shows results of biochemical analysis on the serum level of aspartate transaminase (AST) and alanine transaminase (ALT) by using the blood.



FIG. 23 shows gene clusters including Osmr obtained as a result of analysis the database.



FIG. 24 The top row shows a change in expression (fold change) of gene Y in the liver after 6 hours from administering each adjuvant to rats. The bottom row shows results of staining liver collected by administering FK565 to wild-type (left) or Osmr knockout (right) mouse with TUNEL.



FIG. 25 Blood was collected one day after intraperitoneally administering PBS, FK565 (1 μg/kg, 10 μg/kg, or 100 μg/kg), or LPS (1 mg/kg) to wild-type or Osmr knockout mice.



FIG. 25 shows results of biochemical analysis on the serum level of aspartate transaminase (AST) and alanine transaminase (ALT) by using the blood.



FIG. 26 shows a schematic diagram for creating an adjuvanticity prediction model.



FIG. 27 shows that probes (genes) selected for an adjuvanticity prediction model are concentrated into 42 pathways associated with cell death (4), immune response (2), and metabolism (36). The figure shows a Venn diagram for genes constituting pathways associated with cell death (4) and immune response (2).



FIG. 28 shows scores for drug, immunostimulant, LPS, and TNF calculated by an adjuvanticity prediction model (top). PO indicates oral administration, IV indicates intravenous administration, and IP indicates intraperitoneal administration. The ROC curve of the adjuvanticity prediction model is shown (bottom).



FIG. 29 Ovalbumin as well as alum, CpGk3 or 5 types of drugs (ACAP, BOR, CHX, COL, PHA) were intradermally administered to mice on day 0 and day 14 with 2 to 3 doses, and blood and spleens were collected on day 21. The results of measuring anti-ovalbumin (ova) antibody titer (IgG1, IgG2, and total IgG) on day 21 are shown. The Y axis indicates the increase in antibody titer on the scale of log 10. The number within the parenthesis indicates the dosage (μg/dose/mouse). Ovalbumin was administered at 10 μg/dose/mouse.



FIG. 30 Spleen cells were stimulated by adding ovalbumin (ova) 257-264 peptide (OVA-MHC1), ova 323-339 peptide (OVA-MHC2), or ova protein (OVA-whole) in addition to each drug in vitro, or treated without additional stimulation. The supernatant was collected. The results of measuring Th1 (IL-2 and IFN-γ) cytokines in the supernatant by ELISA are shown. The number within the parenthesis indicates the dosage (μg/dose/mouse). Ovalbumin was administered at 10 μg/dose/mouse.



FIG. 31 Spleen cells were stimulated by adding ovalbumin (ova) 257-264 peptide (OVA-MHC1), ova 323-339 peptide (OVA-MHC2), or ova protein (OVA-whole) in addition to each drug in vitro, or treated without additional stimulation. The supernatant was collected. The results of measuring Th2 (IL-4 and IL-5) cytokines in the supernatant by ELISA are shown. The number within the parenthesis indicates the dosage (μg/dose/mouse). Ovalbumin was administered at 10 μg/dose/mouse.



FIG. 32 shows result of administering ovalbumin as well as alum, CpGk3 or 5 types of drugs (ACAP, BOR, CHX, COL, PHA) to mice, and collecting blood after 3 hours (day 0), and performing biochemical analysis on the serum level of aspartate transaminase (AST) and alanine transaminase (ALT). The number within the parenthesis indicates the dosage (μg/dose/mouse). Ovalbumin was administered at 10 μg/dose/mouse.



FIG. 33 shows results of collecting blood after 6 hours and 24 hours from intraperitoneally administering each drug to mice and analyzing miRNA in the circulating blood. The vertical axis indicates −log 10 (p value), and the horizontal axis indicates −log 2 (miRNA volume).



FIG. 34 shows failed QC examples with unknown reason.





DESCRIPTION OF EMBODIMENTS

The present invention is explained hereinafter with the best modes thereof. Throughout the entire specification, a singular expression should be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Thus, singular articles (e.g., “a”, “an”, “the”, and the like in the case of English) should also be understood as encompassing the concept thereof in the plural form, unless specifically noted otherwise. Further, the terms used herein should be understood as being used in the meaning that is commonly used in the art, unless specifically noted otherwise. Therefore, unless defined otherwise, all terminologies and scientific technical terms that are used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of a contradiction, the present specification (including the definitions) takes precedence.


(Classification of Drug Component)


There are many drug components with known effects that are not systematically classified. For example, immune adjuvants have an important role in improving the potency of a vaccine. A wide range of substances have been reported as functioning as an immune adjuvant, but the mode of action and safety profiles are not fully understood. The present invention has found that the mechanism of action and potential safety profile of drug components can be predicted and classified by performing transcriptome analysis on all organs in animals (e.g., mice) using a large number (21 in the Examples) of different drug components (e.g., adjuvant), creating integrated data for drug component induced responses thereof (adjuvant induced response for adjuvants), and elucidating (extracting) detailed features of the drug components. The present invention has also found that drug components can be divided into specific groups, and a standard drug component (e.g., adjuvant) can be determined for each group. The present invention provides a flexible standardizing method for comprehensively and systematically evaluating any drug component (e.g., adjuvant) and a framework thereof (classification method). The present invention demonstrates, for example, that each drug component can be classified to at least a characteristic adjuvant group named G1 to G6 or another group. Such classification can identify or specifically predict the attribute of a target substance by performing transcriptome analysis on the target substance such as a substance with an unknown drug component function (e.g., adjuvant function) or a novel substance, performing clustering analysis by including transcriptome analysis data on a reference drug component (e.g., reference adjuvant) with confirmed function, and determining reference drug component which is classified to the same cluster as the target substance. The present invention provides a flexible standardizing method for comprehensively and systematically evaluating any drug component (active ingredient, additive, adjuvant, or the like), and a framework thereof.


Adjuvants, which are exemplary classification targets of the invention, have been traditionally used as an additive in a vaccine. Various substances such as oil emulsion, small molecules that are often formulated as nanoparticles or aluminum salt, lipids, and nucleic acids are known to function as an adjuvant with many vaccine antigens (Coffman, R. L., Sher, A. & Seder, R. A. Immunity 33, 492-503 (2010); Reed, S. G., Orr, M. T. & Fox, C. B. Nat Med 19, 1597-1608 (2013) and Desmet, C. J. & Ishii, K. J. Nature reviews. Immunology 12, 479-491 (2012)). The modes of action of adjuvants are categorized empirically into two classes (immunostimulant and antigen delivery system), but are not systematically classified. It is proposed that immunostimulants be further classified into pathogen associated molecular patterns (PAMPs) (Janeway, C. A., Jr. Cold Spring Harbor symposia on quantitative biology 54 Pt 1, 1-13 (1989)) or damage associated molecular patterns (DAMPs) (Matzinger, P. Annu Rev Immunol 12, 991-1045 (1994)) in accordance with the exogenous origin or endogenous origin, which is recognized by a germ line coding pattern recognition receptor, resulting in induction of interferon or pro-inflammatory cytokine secretion (Kawai, T. & Akira, S. Nature immunology 11, 373-384 (2010); Matzinger, P. Annu RevImmunol 12, 991-1045 (1994)). Since many adjuvants are understood as affecting multiple signaling pathways in a recipient, the underlining mechanism of each adjuvant enhancing or directing an immune response in vivo was not classifiable with conventional technologies, but the present invention enables the classification thereof. The present invention can categorize all adjuvants in terms of host responses.


To use a new drug component, establishment of appropriate formulation and GMP production method must be cleared. For example, to use a new adjuvant in a clinical vaccine, multiple steps need to be cleared including establishment of appropriate formulation and GMP production method, and more importantly, the risk and benefit (safety and efficacy) of the adjuvant utilization (Shoenfeld, Y. & Agmon-Levin, N. Journal of autoimmunity 36, 4-8 (2011); Batista-Duharte, A., Lindblad, E. B. & Oviedo-Orta, E. Toxicology letters 203, 97-105 (2011); Zaitseva, M. et al. Vaccine 30, 4859-4865 (2012); Stassijns, J., Bollaerts, K., Baay, M. & Verstraeten, T. Vaccine 34, 714-722 (2016)). Since the results in animal experiments can be extrapolated to humans to some degree, preclinical trial animal research is needed for vaccine development. (Batista-Duharte, A., Lindblad, E. B. & Oviedo-Orta, Toxicology letters 203, 97-105 (2011); Sun, Y., Gruber, M. & Matsumoto, M. Journal of pharmacological and toxicological methods 65, 49-57 (2012)). In fact, careful assessment of biological and immunopathological effects in animals is essential for development of new adjuvanted vaccines (Mastelic, B. et al. Biologicals 41, 115-124 (2013)). Currently approved adjuvanted vaccines have cleared these preclinical toxicology evaluations before human use, and after commercialization their safeties are tracked by side effect investigative monitoring such as pharmacovigilance monitoring. However, a variety of substances with very different physicochemical properties function as adjuvants, which has not been provided with a logical explanation. While there was no simple and straightforward method to evaluate vaccine adjuvant's mode of action and potential safety concerns in animal models, the approaches used in this invention, enable this evaluation.


A systems vaccinology approach (Pulendran, B. Proc Natl Acad Sci USA 111, 12300-12306 (2014)) represents a relatively new research approach to vaccine science, in particular for humans, by identifying correlates of protection (Ravindran, R. et al. Science 343, 313-317 (2014); Tsang, J. S. et al. Cell 157, 499-513 (2014); Nakaya, H. I. et al. Immunity 43, 1186-1198 (2015); Sobolev, O. et al. Nature immunology 17, 204-213 (2016)). By investigating gene expression profiles induced early after vaccination, these approaches can predict adaptive immune responses that follow.


The present invention provides a systematic and comprehensive research approach for drug components (e.g., active ingredient, additive, and adjuvant). For example, various analysis methods are studied for vaccine adjuvants and molecular signatures are reported in the adjuvant field, but this has not lead to a systematic solution. Rather, it is reported that various known classification methods are not capable of classification (Olafsdottir, T., Lindqvist, M. & Harandi, A. M. Vaccine 33, 5302-5307 (2015)). Therefore, the present invention systematically and comprehensively classifies drug components (e.g., active ingredient, additive, and adjuvant) to provide reliable prediction means for efficacy and safety of novel drug components (e.g., active ingredient, additive, and adjuvant). Furthermore, the present invention provides reliable prediction means for identifying the function of a substance with unknown or indefinite drug component function (e.g., efficacy of active ingredient, assistive function of additive, or adjuvant function). Furthermore, substances with known function can also be utilized for quality control thereof and as part of safety management, and as part of efficacy and safety verification for a new drug component (e.g., adjuvant).


The inventors obtained gene expression data of approximately 330 microarrays from mouse liver (LV), spleen (SP), and draining inguinal lymph nodes (LN) after administration of a wide range of different adjuvants. An adjuvant induced gene (transcriptome) panel was able to be defined by integrating the data. This is also referred to as “adjuvant gene space” herein. Analyzing the adjuvant induced gene expression data (transcriptome) in this space revealed properties of each adjuvant in vivo. This approach was used to predict the unknown mechanism of action of two relatively new adjuvants. One such prediction matched the results that were independently studied, confirming that the approach of the invention provides correct prediction results (Hayashi, M. et al., Scientific Reports 6: 29165.doi:10.1038/srep29165.). This approach is not limited to adjuvants and is envisioned to be applicable to all drug components such as active ingredients and additives. In other words, the inventors can define a drug component induced gene (transcriptome) panel by obtaining gene expression data after administration of a wide range of different drug components and integrating the data. This is also referred to as “drug component gene space” herein. Analyzing the drug component induced gene expression data (transcriptome) in this space can reveal properties of each drug component in vivo. Such a method can be implemented by using artificial intelligence (AI) that utilizes machine learning or the like.


Definitions

The definitions of the terms and/or basic technologies particularly used herein are explained hereinafter as appropriate.


As used herein, “drug component” refers to any component or ingredient that can constitute a drug or pharmaceutical. Examples thereof include active ingredients (those exhibiting efficacy on their own), additives (components that are not expected to have efficacy on their own, but are expected to serve a certain role (e.g., excipient, lubricant, surfactant, or the like) when contained in a drug), adjuvants (those enhancing the efficacy (e.g., ability to elicit immune responses) of the primary drug (e.g., antigen for vaccines or the like)), and the like. Examples of drug components include pharmaceutically acceptable carrier, diluent, excipient, buffer, binding agent, blasting agent, diluent, flavoring agent, lubricant, and the like. A drug component can be an individual substance or a combination of a plurality of substances or agents. A combination of an active ingredient and an additive can include any combination, such as a combination of an adjuvant and an active ingredient.


As used herein, “active ingredient” refers to a component exerting the intended efficacy. One or more components can fall under an active ingredient.


As used herein, “additive” refers to any component not expected to have efficacy, but serves a certain role when contained in a drug such as a pharmaceutically acceptable carrier, diluent, excipient, buffer, binding agent, blasting agent, diluent, flavoring agent, lubricant, and the like. As used herein, R cyclodextrin and the like are encompassed as an additive, but such components can also be found to be effective as an adjuvant. In such a case, those skilled in the art determine whether such a component is an adjuvant or an additive depending on the objective.


As used herein, “adjuvant” refers to a compound that enhances an immune response of a subject to an antigen (e.g., vaccines or the like) when co-administered with the antigen. Adjuvant mediated enhancement of an immune response can be typically evaluated by any method known in the art, including, but not limited to, one or more of (i) increase in the number of antibodies generated in response to immunization with the above adjuvant/antigen combination to the number of antibodies generated in response to immunization with the above antigen alone; (ii) increase in the number of T cells recognizing the antigen or the adjuvant; (iii) increase in the level of one or more type I cytokines; and (iv) in vivo defense after raw challenge.


As used herein, “transcriptome” is a term referring to the entirety of all transcriptional products (e.g., mRNA, primary transitional product (set of all RNA molecules including mRNA, rRNA, tRNA and other noncoding RNA), and transcripts) in cells (one cell or a population of cells) under a specific condition. A transcriptome, in relation to the present invention, refers to a cell, a population of cells, preferably a population of cancer cells, or a set of all RNA molecules produced in all cells of a given individual at a specific time.


As used herein, “exome” refers to an aggregate of all exons in the human genome, referring to the entire part of the genome of an organism formed by an exon which is the coded portion of the expressed gene. An exome provides a genetic blueprint used in the synthesis of a protein and other functional gene products. This is functionally the most important part of the genome, so that it was considered to be most likely to contribute to the phenotype of an organism.


Any suitable sequencing method can be used in accordance with the present invention for “transcriptome analysis” herein. Next-generation sequencing (NGS) technology is preferred. As used herein, the term “next generation sequencing” or “NGS” refers to all new high-throughput sequencing technologies, which divide the entire genome into small pieces to randomly read nucleic acid templates in parallel along the entire genome, in comparison to “conventional” sequencing known as Sanger chemistry. The NGS technologies (also known as massive parallel sequencing technologies) can deliver nucleic acid information of the full genome, exome, transcriptome (all transcribed sequences of the genome) or methylome (all methylated sequences of the genome) in a very short period, such as 1 to 2 weeks or less, preferably 1 to 7 days or less, or most preferably less than 24 hours, which enables a single cell sequencing approach in principle. Any NGS platform that is commercially available or mentioned in a reference can be used for practicing the present invention. As used herein, “transcriptome expression profile” refers to a profile of the expression status of each gene when performing transcriptome analysis on a certain agent.


As used herein, “transcriptome expression profile equivalent to . . . ” means that the “transcriptome expression profile” is substantially identical or identical, or substantially similar for a certain objective. Identity of expression profiles can be determined by whether expression profiles are similar for a molecule of a drug component (e.g., adjuvant) or the like or a part thereof. In this regard, whether profiles are similar can be determined by the degree of gene expression of sDEG or the like as defined herein and determined based on the degree of expression, amount, active amount, or the like. Although not wishing to be bound by any theory, in some embodiments of the invention, it is understood that drug components (e.g., adjuvants) belonging to the same cluster by classifying drug components (e.g., adjuvants) based on the similarity have the same feature as the drug components (e.g., adjuvants) in the same category. Therefore, features of novel drug components (e.g., adjuvants) or drug components (e.g., adjuvants) with an unknown function can be analyzed by investigating whether drug components belong to the same drug component cluster (e.g., adjuvant cluster) by using the approach of the invention. To study the similarity herein, a “similarity score” can be used. “Similarity score” refers to a specific numerical value indicating similarity. For example, a suitable score can be used appropriately in accordance with the technique used for calculating the transcriptome expression pattern or the like. A similarity score can be calculated using a regressive approach, neural networking method, machine learning algorithm such as support vector machine or random forest, or the like.


As used herein, “clustering” or “cluster analysis” or “clustering analysis” are interchangeably used, referring to a method of classifying a subject by aggregating subjects that are similar to one another among a population (subjects) of subjects having different properties to create (dividing) a cluster. Each subset after the division is referred to as a cluster. There are several types of division methods. In some cases, each of all subjects of classification is an element of one cluster (referred to as a hard or crisp cluster), and in some cases, a cluster partially belongs to a plurality of clusters simultaneously (referred to as soft or fuzzy cluster). Hard cluster analysis is generally used herein. Examples of typical cluster analysis include hierarchical cluster analysis, non-hierarchical cluster analysis, and the like. Hierarchical cluster is generally used, but analysis is not limited thereto. As used herein, “transcriptome clustering” refers to clustering based on results of transcriptome analysis.


As used herein, “cluster” generally refers to a collection of similar elements of a certain population (e.g., drug component (e.g., adjuvant)) from a distribution of elements in a multidimensional space without external criteria or designation of the number of groups. As used herein, a “cluster” of drug components (e.g., adjuvants) refers to a collection of similar drug components among a large number of drug components (e.g., adjuvants). Drug components (e.g., adjuvants) belonging to the same cluster have the same (e.g., identical or similar) effect (e.g., adjuvant function (e.g., cytokine stimulation or the like)). This can be classified by multivariate analysis. A cluster can be constituted by using various cluster analysis approaches. The cluster of drug components (e.g., adjuvants) provided by the present invention is capable of classification by the function of the drug components (e.g., adjuvants) by showing that a drug component belongs to the cluster. Further, drug components (e.g., adjuvants) belonging to the same cluster after classification can be predicted as having a property that is characteristic to the cluster with high precision and reasonable probability. A reasonable probability can be appropriately set at, for example, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or the like depending on the parameter used in cluster analysis.


As used herein, “identical” or “similar” function is used for results after cluster analysis. Functions, when having substantially the same degree of activity, are referred to as identical, and when having qualitatively the same activity but different amount for a property, are referred to as “similar”. Such a degree of similarity can be appropriately determined such as 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, or the like. As used herein, “identical cluster” refers to being in the same cluster in cluster analysis. Whether it is possible to be in the same cluster can be determined by similarity.


As used herein, “similarity” refers to the degree of similarity of expression profiles for molecules of drug components such as an active ingredient, additive, or adjuvant or a part thereof. Similarity can be defined by the degree of gene expression of the sDEG defined herein or the like and determined based on the degree of expression, amount, active amount, or the like. Although not wishing to be bound by any theory, in some embodiments of the invention, it is understood that drug components (e.g., active ingredients, additives, or adjuvants) belonging to the same cluster by classifying drug components (e.g., active ingredients, additives, or adjuvants) based on similarity have the same feature as the drug components (e.g., active ingredients, additives, or adjuvants) in the same category. Therefore, features of novel drug components (e.g., active ingredients, additives, or adjuvants) or drug components (e.g., active ingredients, additives, or adjuvants) with an unknown function can be analyzed by investigating whether drug components belong to the same drug component cluster (e.g., adjuvant cluster) by using the approach of the invention. To study the similarity herein, a “similarity score” can be used. “Similarity score” refers to a specific numerical value indicating the similarity. For example, a suitable score can be used appropriately in accordance with the technique used for calculating the transcriptome expression pattern or the like. A similarity score can be calculated using a technology used in artificial intelligence (AI) such as a regressive approach, neural networking method, machine learning algorithm such as support vector machine or random forest, or the like.


As an important indicator, it is suitable to determine a threshold value so that expression patterns of drug components (e.g., active ingredient, additive, or adjuvant) known to have identical or similar function match well. If statistical significance is prioritized, other threshold values can also be used. Those skilled in the art can appropriately determine a threshold value by referring to the descriptions herein depending on the situation. For example, values with a maximum distance found from cluster analysis using a hierarchical clustering approach (e.g., group average method (average linkage clustering)), nearest neighbor method (NN method), K-NN method, Ward's method, furthest neighbor method, or centroid method) less than a specific value can be considered as the same cluster. Examples of such a value include, but are not limited to, less than 1, less than 0.95, less than 0.9, less than 0.85, less than 0.8, less than 0.75, less than 0.7, less than 0.65, less than 0.6, less than 0.55, less than 0.5, less than 0.45, less than 0.4, less than 0.35, less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1, less than 0.05, and the like. Clustering approaches are not limited to hierarchical approaches (e.g., nearest neighbor method). A non-hierarchical approach (e.g., k-means method) can be used. A hierarchical clustering can be preferably used.


Those skilled in the art can appropriately determine the distance (similarity) between elements to be classified in clustering. Examples of distances between elements that are commonly used include Euclidian distance, Mahalanobis distance, or cosine similarity (distance), and the like.


Software for performing hierarchical clustering is not limited. Examples thereof include Java®-based free software, Clustering Calculator (Brzustowski, J.) (http://www2.biology.ualberta.ca/jbrzusto/cluster.php/).


The following output can be obtained by inputting vector data into such software:


Height of connection position of tree (arbitrary unit);


Topology of tree;


Distance between each node of tree (arbitrary unit)


Bootstrap value of each connection (e.g., 1000 runs). Other outputs can also be optionally obtained.


A tree diagram can be drawn by using a suitable software from such output data (including, but not limited to, Phylip/DRAWTREE format, and (hierarchical trees) using Tree Explorer software, Tamura, K., available at http://www.evolgen.biol.)


In addition to bootstrap values (also called bp), values such as p-value of multiscale bootstrap (Au) can also be outputted. Such values indicate the mathematical stability of clustering. AU is parameter that is often used in sequence analysis or the like, which can be suitable for indicating the stability of a phylogenetic tree.


Hierarchical clustering is classified into divisive and agglomerative clustering. For agglomerative clustering that is typically used, an initial state with N clusters comprising only one subject is first created when data consisting of N subjects are given. From this state, distance d between clusters (C1, C2) is calculated from distance d between subjects x1 and x2 (x1, x2) (dissimilarity), and two clusters with the shortest distance are successively merged. Such merging is repeated until all subjects are merged into a single cluster to obtain a hierarchical structure. The hierarchical structure is displayed as a dendrogram. A dendrogram is a binary tree in which each terminal node represents each subject, and a cluster formed by merging is represented by a non-terminal node. The horizontal axis of a non-terminal node represents the distance between clusters when merged. There are several approaches depending on the difference in the distance function d (C1, C2) of clusters C1 and C2, including nearest neighbor method or single linkage method; furthest neighbor method or complete linkage method; group average method; Ward's method (Ward's method minimizes the sum of square of distance from each subject to the centroid of the cluster including the subject), and the like. The nearest neighbor method, furthest neighbor method, and group average method can be applied when the distance d (xi, xj) between any subjects are given. The distance after merging clusters can be updated with constant time by the Lance-Williams update formula (G. N. Lance and W. T. Williams, The Computer Journal, vol. 9, pp. 373-380 (1967)). This can be applied by finding the Euclidean distance between vectors if subjects are described as a numerical vector. For Ward's method, a predetermined formula can be directly applied if subjects are given as numerical vectors. If only the distance between subjects is given, this can be applied by updating the distance using the Lance-Williams update formula. The amount of calculation in a normal case where the distance can be updated with a constant time by the Lance-Williams update formula can be found by 0(N2 log N). Meanwhile, the distance updating approach described above has a property of reducibility. An algorithm (F. Murtagh, The Computer Journal, vol. 26, pp. 354-359 (1983)) is known which can calculate in the time 0(N2) by utilizing the property of the nearest neighbor graph. The algorithm can be implemented by using known information in the Olson document (C. F. Olson: Parallel Computing, Vol. 21, pp. 1313-1325 (1995)) or the like for the amount of calculation on a coloring parallel computer. An example of the hierarchical clustering analysis of the invention is provided in the Examples.


Hierarchical clustering can be performed in each organ for each drug component (e.g., active ingredient, additive, or adjuvant) and for each gene probe. In such analysis, the ratio of cell population responding to a drug component (e.g., adjuvant) can also be analyzed. Cells can also be analyzed for each type of immune cells.


In one embodiment of the invention herein, transcriptome analysis can be performed by administering a target drug component (e.g., active ingredient, additive, or adjuvant) to a target organism and comparing a transcriptome in a target organ at a certain time after administration with a transcriptome in the same or corresponding organ prior to administration of the drug component (e.g., adjuvant), and identifying a set of differentially expressed genes (DEGs) as a result of the comparison.


As used herein, “differentiently expressed gene” or “differentially expressed gene” (DEG) refers to a gene whose expression has changed (e.g., increased, decreased, manifested, or eliminated) as a result of administering a drug component (e.g., active ingredient, additive, or adjuvant) to the target organism and comparing a transcriptome in the organ at a certain time after administration with a transcriptome in the organ prior to administration of the drug component (e.g., active ingredient, additive, or adjuvant). If the change is “significant”, the gene is referred to as “significant DEG” or “sDEG”. In this regard, significant change generally refers to, but is not limited to, a statistically significant change. If suitable for the objective of the invention, significance can be determined using appropriate criteria. DEG determination methods are exemplified in the Examples. Representative examples include, but are not limited to the following: the change can be defined as a statistically significant change (upregulation or downregulation) satisfying all of the following conditions: a predetermined threshold value when determining a significant DEG is identified by a predetermined difference in multiple and predetermined statistical significance (p value); typically the mean fold change (FC) is >1.5 of <0.667, the p value in associated t-test is <0.01 without multiple testing correction, and customized PA call is 1. Other identification methods can also be used. In a preferred embodiment, a gene whose expression has changed beyond a predetermined threshold value is identified as a result of comparison and differentially expressed genes in the common manner among identified genes are selected to generate a set of significant DEG. Analysis of DEGs can use any approach that can analyze differential expression. The volcano plot used in the Examples is a scatter diagram arranging the statistical effect on the y axis and the biological effect on the x axis in all individuals/property matrices. The only limitation is that only examination of the difference between the levels of qualitative explanatory variables of two levels can be executed. In a volcano plot, the coordinate of the y axis is generally scaled by −log 10 (p value) to facilitate reading of the diagram. High values reflect the most significant effect, while low values correspond to a barely significant effect. Since a statistically significant effect is not necessarily interesting in the biological scale, use of a volcano plot can reduce the risk of experiments involving very accurate measurements due to a large number of repetitions possibly providing a lower p value associated with a very small biological difference. Therefore, analysis focusing not only on the p value but also the biological effect can be performed.


When analyzing DEGs, a plurality of samples are generally processed, while response can vary in such a plurality of samples. In such a case, the reaction is represented as a Venn diagram. A large overlap thereof can be determined as consistent expression with high universality. The present invention has found that a ratio of overlap in a Venn diagram is correlated with the number of upregulated gene probes. By plotting in this manner, potent gene responses of a drug component induced property (e.g., efficacy of active ingredient, assistive function of additive, and adjuvant function) can be determined by Venn diagram analysis. FIG. 7 provides an example of annotation analysis and Venn diagram of genes significantly upregulated with a CpG adjuvant. Likewise, FIG. 8 provides an example thereof for cdiGMP.


As used herein, a set of all sDEGs for each organ and drug component is referred to as a “drug component gene space”. As used herein, a set of all sDEGs for each organ and adjuvant is referred to as an “adjuvant gene space”. Likewise, this can be referred to as “active ingredient gene space” for active ingredients and “additive gene space” for additives.


As used herein, integration of a set of DEGs for two or more drug components (e.g., active ingredient, additive, or adjuvant) to generate a set of differentially expressed genes (DEG) in the common manner can be referred to as shared DEG set generation. An embodiment herein can perform the transcriptome analysis for at least two or more organs to identify a set of differentially expressed genes only in a specific organ and using the set as the organ specific gene set. Therefore, “organ specific gene set” as used herein refers to a set of differentially expressed genes specifically to a certain organ.


As used herein, “organ” refers to a unit constituting the body of a multicellular organism such as an animal or plant among organisms, which is morphologically distinct from the surroundings and serves a set of functions as a whole. Representative examples include, but are not limited to, liver, spleen, and lymph nodes, as well as other organs such as kidney, lung, adrenal glands, pancreas, and heart.


As used herein, “number enabling statistically significant clustering analysis” is the number related to adjuvants, referring to the number of samples from which a statistically significant difference can be detected upon clustering analysis. Those skilled in the art can appropriately determine the detection power or the like and determine the number based on conventional techniques in the field of statistics.


As used herein, “gene marker” refers to a substance used as an indicator for evaluating the condition or action of a target, referring to a gene related substance when correlating with the expression level of a gene herein. As used herein, “gene marker” can also be referred to as a “marker”, unless specifically noted otherwise.


A gene (marker) group associated with a drug component (e.g., active ingredient, additive, or adjuvant) can be represented using, but not limited to, a z-score heat map approach.


As used herein, “drug component evaluation marker” refers to a gene marker that is unique to or specific to a specific drug component or a drug component cluster and a specific organ. A drug component evaluation marker can be unique to or specific to a plurality of organs or drug components or drug component clusters, but in such a case the specific organs or drug components or drug component clusters can be identified by concurrently using another marker. In regard to such a relationship, a gene with a significant relationship shared among drug component related genes is selected. A drug component group of upregulated genes can be selected based on the z-score of the expression (see FIG. 6).


If a drug component is an adjuvant, a drug component evaluation marker is referred to as an “adjuvant evaluation marker”. In other words, “adjuvant evaluation marker” as used herein refers to a gene marker that is unique to or specific to a specific adjuvant or adjuvant cluster and a specific organ. An adjuvant evaluation marker can be unique to or specific to a plurality of organs or adjuvants or adjuvant clusters, but in such a case the specific organs or adjuvants or adjuvant clusters can be identified by concurrently using another marker. In regard to such a relationship, a gene with a significant relationship shared among adjuvant related genes is selected. An adjuvant group of upregulated genes can be selected based on the z-score of the expression (see FIG. 6). This can be referred to as an “active ingredient evaluation marker” if a drug component is an active ingredient, and as an “additive evaluation marker” for additives.


A biological process can be annotated by analyzing transcriptome profile data. Such annotation can be performed using software such as TargetMine. Annotation can be represented by a keyword. Wounding, cell death, apoptosis, NFκB signaling pathway, inflammatory response, TNF signaling pathway, cytokines, migration, chemokine, chemotaxis, stress, defense response, immune response, innate immune response, adaptive immune response, interferons, interleukins, or the like can be used. This can be separated for each organ, route of administration, or the like. Examples of annotations for wounding include regulation of response to wounding; response to wounding; and positive regulation of response to wounding. Examples of annotations for cell death include cell death; death; programmed cell death; regulation of cell death; regulation of programmed cell death; positive regulation of programmed cell death; positive regulation of cell death; negative regulation of cell death; and negative regulation of programmed cell death. Examples of annotations for apoptosis include apoptotic process; regulation of apoptotic process; apoptotic signaling pathway; intrinsic apoptotic signaling pathway; positive regulation of apoptotic process; regulation of apoptotic signaling pathway; negative regulation of apoptotic process; and regulation of intrinsic apoptotic signaling pathway. Examples of annotations for NFκB signaling pathway include NF-kappa B signaling pathway; I-kappa B kinase/NF-kappa B signaling; positive regulation of I-kappa B kinase/NF-kappa B signaling; and regulation of I-kappa B kinase/NF-kappa B signaling. Examples of annotations for inflammatory response include inflammatory response; regulation of inflammatory response; positive regulation of inflammatory response; acute inflammatory response; and leukocyte migration involved in inflammatory response. Examples of annotations for TNF signaling pathway include TNF signaling pathway. Examples of annotations for cytokines include response to cytokine; Cytokine-cytokine receptor interaction|Endocytosis; cellular response to cytokine stimulus; Cytokine-cytokine receptor interaction; cytokine production; regulation of cytokine production; cytokine-mediated signaling pathway; cytokine biosynthetic process; cytokine metabolic process; positive regulation of cytokine production; negative regulation of cytokine production; regulation of cytokine biosynthetic process; regulation of tumor necrosis factor superfamily cytokine production; tumor necrosis factor superfamily cytokine production; positive regulation of cytokine biosynthetic process; cytokine secretion; and positive regulation of tumor necrosis factor superfamily cytokine production. Examples of annotations for migration include positive regulation of leukocyte migration; cell migration; leukocyte migration; regulation of leukocyte migration; neutrophil migration; positive regulation of cell migration; granulocyte migration; myeloid leukocyte migration; regulation of cell migration; and lymphocyte migration. Examples of annotations for chemokine include chemokine-mediated signaling pathway; chemokine production; regulation of chemokine production; and positive regulation of chemokine production. Examples of annotations for chemotaxis include cell chemotaxis; chemotaxis; leukocyte chemotaxis; positive regulation of leukocyte chemotaxis; taxis; granulocyte chemotaxis; neutrophil chemotaxis; positive regulation of chemotaxis; regulation of leukocyte chemotaxis; regulation of chemotaxis; and lymphocyte chemotaxis. Examples of annotations for stress include response to stress; regulation of response to stress; and cellular response to stress. Examples of annotations for defense response include defense response; regulation of defense response; positive regulation of defense response; defense response to other organism; defense response to bacterium; defense response to Gram-positive bacterium; defense response to protozoan; defense response to virus; regulation of defense response to virus; regulation of defense response to virus by host; and negative regulation of defense response. Examples of annotations for immune response include immune response; positive regulation of immune response; regulation of immune response; activation of immune response; immune response-activating signal transduction; immune response-regulating signaling pathway; negative regulation of immune response; and production of molecular mediator of immune response. Examples of annotations for innate immune response include innate immune response; regulation of innate immune response; positive regulation of innate immune response; activation of innate immune response; innate immune response-activating signal transduction; and negative regulation of innate immune response. Examples of annotations for adaptive immune response include adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains; adaptive immune response; positive regulation of adaptive immune response; regulation of adaptive immune response; and regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains. Examples of annotations for interferons include response to interferon-alpha; interferon-alpha production; cellular response to interferon-alpha; positive regulation of interferon-alpha production; regulation of interferon-alpha production; cellular response to interferon-beta; response to interferon-beta; positive regulation of interferon-beta production; regulation of interferon-beta production; interferon-beta production; response to interferon-gamma; cellular response to interferon-gamma; interferon-gamma production; and regulation of interferon-gamma production. Examples of annotations for interleukins include interleukin-6 production; regulation of interleukin-6 production; positive regulation of interleukin-6 production; interleukin-12 production; regulation of interleukin-12 production; and positive regulation of interleukin-12 production. Examples of biological indicators include cytokine profiles. Cytokine profiles include, but are not limited to IFNA2; IFNB1; IFNG; IFNL1; IFNA1/IFNA13; IL15; IL4; IL1RN; IFNK; IFNA4; IL1B; IL12B; TNFSF10; TNF; IFNA10; IFNA21; IFNA5; IFNA7; IFNA14; IFNA6; IFNE; IFNA8; IFNA16; CD40LG; IL6; IL2; IL12A; IL27; OSM; IFNA17; EBI3; IL10; IFNW1; TNFSF11; IL7; and the like.


As analyzed items, a plot of a hematological indicator and gene expression can also be applied. In this regard, examples of hematological indicators include, but are not limited to, white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), relative (%) content of lymphocytes (LY %), relative (%) content of monocytes (MO %), relative (%) content of granulocytes (GR %), red blood cells (RBC), hemoglobins (Hb, HGB), hematocrits (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), platelet concentration (PCT), mean platelet volume (MPV), platelet distribution width (PDW), and the like.


By using the present invention, drug components (e.g., active ingredients, additives, or adjuvants) can be grouped by cluster analysis (FIG. 9).


Biological activity can also be represented, for example, by an absolute value or relative value of absorbance or the like for data using a binding constant or dissociation constant of an antigen-antibody reaction or binding constant or dissociation constant to an antigen of each antibody when using two or more antibodies in a binding assay or the like, or data from ELISA or the like.


The detecting agent or detecting means used in the analysis of the invention can be any means, as long as a gene or the expression thereof can be detected.


The detecting agent or detecting means of the present invention may be a complex or composite molecule in which another substance (e.g., label or the like) is bound to a portion enabling detection (e.g., antibody or the like). As used herein, “complex” or “composite molecule” refers to any construct comprising two or more portions. For instance, when one portion is a polypeptide, the other portion may be a polypeptide or other substances (e.g., sugar, lipid, nucleic acid, other carbohydrate or the like). As used herein, two or more constituent portions of a complex may be bound by a covalent bond or any other bond (e.g., hydrogen bond, ionic bond, hydrophobic interaction, Van der Waals force, the like). When two or more portions are polypeptides, the complex may be called a chimeric polypeptide. Thus, “complex” as used herein includes molecules formed by linking a plurality of types of molecules such as a polypeptide, polynucleotide, lipid, sugar, or small molecule.


As used herein, “detection” or “quantification” of polynucleotide or polypeptide or polypeptide expression can be accomplished by using a suitable method including, for example, an immunological measuring method and measurement of mRNAs, including a bond or interaction to a marker detecting agent. However, measurement can be performed with the amount of PCR product in the present invention. Examples of a molecular biological measuring method include northern blot, dot blot, PCR and the like. Examples of an immunological measurement method include ELISA using a microtiter plate, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), single radial immunodiffusion (SRID), turbidimetric immunoassay (TIA), western blot, immunohistochemical staining and the like. Further, examples of a quantification method include ELISA, RIA and the like. Quantification may also be performed by a gene analysis method using an array (e.g., DNA array, protein array). DNA arrays are outlined extensively in (Ed. by Shujunsha, Saibo Kogaku Bessatsu “DNA Maikuroarei to Saishin PCR ho” [Cellular engineering, Extra issue, “DNA Microarrays and Latest PCR Methods” ]. Protein arrays are discussed in detail in Nat Genet. 2002 December; 32 Suppl: 526-32. Examples of a method of analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid system, in vitro translation and the like, in addition to the methods discussed above. Such additional analysis methods are described in, for example, Genomu Kaiseki Jikkenho Nakamura Yusuke Labo Manyuaru [Genome analysis experimental method Yusuke Nakamura Lab Manual], Ed. by Yusuke Nakamura, Yodosha (2002) and the like. The entirety of the descriptions therein is incorporated herein by reference.


As used herein, “means” refers to anything that can be a tool for accomplishing an objective (e.g., detection, diagnosis, therapy). In particular, “means for selective recognition (detection)” as used herein refers to means capable of recognizing (detecting) a certain subject differently from others.


As used herein, “(nucleic acid) primer” refers to a substance required for initiating a reaction of a polymeric compound to be synthesized in a polymer synthesizing enzyme reaction. A synthetic reaction of a nucleic acid molecule can use a nucleic acid molecule (e.g., DNA, RNA or the like) complementary to a portion of a sequence of a polymeric compound to be synthesized. A primer can be used herein as a marker detecting means.


Examples of a nucleic acid molecule generally used as a primer include those having a nucleic acid sequence with a length of at least 8 contiguous nucleotides, which is complementary to a nucleic acid sequence of a gene of interest (e.g., marker of the invention). Such a nucleic acid sequence may be a nucleic acid sequence with a length of preferably at least 9 contiguous nucleotides, more preferably at least 10 contiguous nucleotides, still more preferably at least 11 contiguous nucleotides, at least 12 contiguous nucleotides, at least 13 contiguous nucleotides, at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, at least 25 contiguous nucleotides, at least 30 contiguous nucleotides, at least 40 contiguous nucleotides, or at least 50 contiguous nucleotides. A nucleic acid sequence used as a probe comprises a nucleic acid sequence that is at least 70% homologous, more preferably at least 80% homologous, still more preferably at least 90% homologous, or at least 95% homologous to the aforementioned sequence. A sequence that is suitable as a primer may vary depending on the property of a sequence intended for synthesis (amplification). However, those skilled in the art are capable of designing an appropriate primer in accordance with an intended sequence. Design of such a primer is well known in the art, which may be performed manually or by using a computer program (e.g., LASERGENE, PrimerSelect, or DNAStar).


As used herein, “probe” refers to a substance that can be means for search, which is used in a biological experiment such as in vitro and/or in vivo screening. Examples thereof include, but are not limited to, a nucleic acid molecule comprising a specific base sequence, a peptide comprising a specific amino acid sequence, a specific antibody, a fragment thereof, and the like. As used herein, a probe can be used as marker detecting means.


If a drug component is an adjuvant herein, examples of classification of adjuvant include, but are not limited to G1 (interferon signaling); G2 (metabolism of lipids and lipoproteins); G3 (response to stress); G4 (response to wounding); G5 (phosphate-containing compound metabolic process); G6 (phagosome), and the like. Such classification was only found by executing the method of generating an organ transcriptome profile of a drug component (e.g., adjuvant) of the invention.


A reference drug product (also referred to as a standard drug component) for classifying drug components can be determined by using the present invention. This is exemplified herein. If a drug component is an adjuvant, a reference adjuvant (standard adjuvant) for adjuvant classification can be identified. For example for the G1 to G6, the reference adjuvant (standard adjuvant) of G1 is selected from the group consisting of cdiGMP, cGAMP, DMXAA, PolyIC, and R848, the reference adjuvant (standard adjuvant) of G2 is bCD, the reference adjuvant (standard adjuvant) of G3 is FK565, the reference adjuvant (standard adjuvant) of G4 is MALP2s, the reference adjuvant (standard adjuvant) of G5 is selected from the group consisting of D35, K3, and K3SPG, and/or the reference adjuvant (standard adjuvant) of G6 is AddaVax. The cdiGMP, cGAMP, DMXAA, PolyIC, and R848 are RNA related adjuvants (STING ligands), and cdiGMP elicits a Th1 response and DMXAA elicits a Th2 response. G1 can be deemed as biological function: interferon response (type I, type II), and stress related drug component (e.g., active ingredient, additive, or adjuvant). G2 is a metabolism of lipids and lipoproteins, and bCD is a typical drug component (e.g., active ingredient, additive, or adjuvant) while ALM also has a similar action, and biological function includes inflammatory cytokine, lipid metabolism, and DAMP (action with host derived dsDNA) action. G3 is a response to stress cluster, and representative drug components (e.g., adjuvant) include FK565, and examples of biological function include T-cell cytokine, NK cell cytokine, stress response, wounding response, PAMP, and the like. G4 is response to wounding, and representative drug components (e.g., adjuvants) include MALP2s, and examples of biological function include TNF response, stress response, wounding response, and PAMP. G5 is a phosphate-containing compound metabolic process, and CpG (D35, K3, K3SPG) as well as TLR9 ligands are representative drug components (e.g., active ingredients, additives, or adjuvants), and examples of biological functions include nucleic acid metabolism and phosphoric acid containing compound metabolism. G6 is phagosome, and AddaVax (MF59) is a representative drug component (e.g., adjuvant), and examples of biological functions include phagosome (phagocytosis), ATP, and the like. If a drug component is an active ingredient, the above term can be referred to as a reference (standard) active ingredient or the like. For an additive, the term can be referred to as a representative additive, reference (standard) additive, or the like.


For G1 (interferon signaling), typically a STING ligand is a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant). Typical examples thereof include cdiGMP, cGAMP, DMXAA, PolyIC, and R848, including an RNA related adjuvant (STING ligand). CdiGMP elicits a Th1 response, and DMXAA elicits a Th2 response. G1 can be deemed as biological function: interferon response (type I, type II), and stress related drug component (e.g., active ingredient, additive, or adjuvant). “STING” (stimulator of interferon genes) is an adaptor protein, identified as a membrane protein localized in endoplasmic reticulum, activating TBK1 and IRF3 by stimulation of dsDNA to induce the expression of type I interferon. A STING ligand is a ligand to STING. Interferon is secreted by stimulation of STING. Examples of STING include cdiGMP, cGAMP, DMXAA, PolyIC, R848, 2′3′-cGAMP, and the like. cdiGMP is a cyclic diGMP. cGAMP is cyclic AMP-AMP. DMXAA is 5,6-dimethyxanthenone-4-acetic acid. PolyIC is also referred to as Poly I:C. R848 is resiquimod.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216.


G2 (metabolism of lipids and lipoproteins) is a lipid and lipoprotein metabolizing property. β cyclodextrin (bCD) is a representative drug component (e.g., representative active ingredient, representative additive, or representative adjuvant) and a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant). Meanwhile, ALM (D35, K3 (LV)) also has a similar action. Examples of biological functions include inflammatory cytokine, lipid metabolism and DAMP (action with host derived dsDNA) action. bCD is an abbreviation of R cyclodextrin and a representative example used as an adjuvant.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5.


G3 (response to stress) is a stress responsive cluster. Representative adjuvants include FK565 (heptanoyl-γ-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine), which is an immune reactive peptide. Examples of biological functions include T cell cytokine, NK cell cytokine, stress response, wounding response, PAMP, and the like.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1.


G4 (response to wounding) is a wounding responsive drug component (e.g., active ingredient, additive, or adjuvant), which can be expressed as a toll-like receptor (TLR) 2 ligand. Representative drug components (e.g., adjuvant) include MALP2s (macrophage activating lipopeptide 2). Examples of biological functions include TNF response, stress response, wounding response, and PAMP.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b.


G5 (phosphate-containing compound metabolic process) is a drug component (e.g., active ingredient, additive, or adjuvant) with a phosphate-containing compound metabolic process. CpG (D35, K3, K3SPG, and the like) is a representative drug component (e.g., representative adjuvants) and a reference drug component (e.g., reference adjuvant). TLR9 ligands and the like are also representative examples. Examples of biological functions include nucleic acid metabolism and phosphoric acid containing compound metabolism.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn.


G6 (phagosome) is an adjuvant with phagosome. Squalene oil-in-water emulsion adjuvant such as AddaVax and MF59 are representative drug components (e.g., representative active ingredients, representative additives, or representative adjuvants) and reference drug components (e.g., reference active ingredients, reference additives, or reference adjuvants). Examples of biological functions include phagosome (phagocytosis), ATP, and the like.


A gene with a significant difference in the expression (significant DEG) in transcriptome analysis of G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


While the drug components described above (e.g., active ingredients, additives, and adjuvants) can be denoted by an abbreviation, the full name thereof (if any), typical source, physical property, receptor (if any), and reference articles were summarized in the following table.

















Abbrev-
Typical
Full name/


Reference


iation
source
explanation
Physical property
Receptor
article















TABLE 1-1












AddaVax
InvivoGen
Emulsion
Squalene oil-in-
Unknown
Calabro S et al.,



(VacciGrade)
similar to
water emulsion

Vaccine, 2013




AddaVax ®
adjuvant

Jul 18; 31(33):




MF59


3363-9.;







Caproni E et







al., J Immunol.







2012 April 1;







188 (7): 3088-







98.





ADX
Vaxine
Advax ®
Delta inulin semi-
Unknown
Honda-Okubo





crystalline

Y et al.,





particle

Vaccine. 2012







August3;







30(36): 5373-







81.: Saade F et







al., Vaccine.







2013 April 8;







31 (15): 1999-







2007





ALM
Brenntag
Aluminum
Gel suspension
Unknown





hydroxide gel







(ALUM)








bCD
ISP
Hydroxy-
Cyclodextrin
Unknown




Cavitron
propyl-β-






W7 HP7
cyclodextrin






Pharma









cdiGMP
Yamasa
Cyclic
Cyclic
STING




Corporation
diguanylate
dinucleotide






mono-







phosphate








cGAMP
InvivoGen
Cyclic
Cyclic
STING





[G(2′5′)pA
dinucleotide






(3′,5′)p]








D35
GeneDesign
A class CpG
Synthetic
TLR9
Verthelyi D,




ODN (5′-
oligodeoxyribonu

Ishii KJ, Gursel




GGT GCA
cleotide

M, Takeshita F,




TCG ATG


Klinman DM, J




CAG GGG


Immunol. 2001




GG-3′)


Feb 15; 166(4):







2372-7.; Aoshi







T et al., J







Immunol Res.







2015;2015:







316364.





DMXAA
Sigma-
5,6-dimethyl-
Synthetic
STING




Aldrich
9-oxo-9H-
chemical






xanthene-4-
substance






acetate








ENDCN
Eurocine
Endocine ®
Monoolein and
Unknown
Falkeborn T et



Vaccines

oleic acid

al., PLoS One.







2013 August 8;







8(8); e70527.;







Mal tai s AK et







al., Vaccine.







2014 May 30;







32(26): 3307-







15.





FCA
Sigma-
Complete
Mycobacterium/
CLR and




Aldrich
Freund's
water-in-oil
others (but





adjuvant
emulsion
unknown)











Table 1-2












FK565
Astellas
Heptanoyl-γ-
Synthetic peptide
NODI




Pharma
D-glutamyl-
glycan






(L)-meso-







diamino-







pimelyl-(D)-







alanine








ISA51VG
SEPPIC
Incomplete
Water-in-oil
Unknown





Freund's
emulsion






adjuvant








K3
GeneDesign
B class CpG
Synthetic
TLR9
Verthelyi D,




ODN
oligodeoxy-

Ishii KJ, Gursel




(ATCGACT
ribonucleotide

M, Takeshita F,




CTCGAGCG


Klinman DM, J




TTCTC)


Immunol. 2001







Feb 15; 166(4):







2372-7.





K3SPG
K3-sAs40
Complex of
Deoxyribonucleotide/
TLR9
Kobiyama K et



from
K3CpG
glucan complex

al., Proc Natl



GeneDesi
ODNs and β


Acad Sci USA.



gn was
glucan


2014 Feb 25;



combined
(schizo-


111(8): 3086-



with SPG
phyllan, SPG)


91.



in-house









MALP2s
Campridge
Macrophage
Lipoprotein
TLR2/6
Sawahata R et



Peptide
activating


al., Microbes



Provided
lipopeptide


Infect.2011



by
2 short


Apr; 13(4):



Tsukasa
lipopeptide


350-8.



Seya
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DE)








MBT
InvivoGen
N-acetyl-
Synthetic peptide
NOD2





muranyl-L-
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PolylC
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polycytidylic







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R848
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4-amino-2-
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Sciences
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methyl)-a,a-







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sHZ
Nippon
Synthetic
Hematin crystal
Unknown
Coban C et al.,



Zenyaku
hemozoin β


CellHost



Kogyo



Microbe. 2010







Jan 21; 7(1):







50-61; Onishi







M et al.,







Vaccine. 2014







May23;







32(25); 3004-9.









bCD is an abbreviation of β cyclotextrin and is a representative example used as an adjuvant. bCD can also be an additive.


FK565 is a type of immunoreactive peptide. The chemical name is heptanoyl-γ-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine, which is explained in detail in J Antibiot (Tokyo). 1983 August; 36(8): 1045-50.


MALP2s is an abbreviation of macrophage-activating lipopeptide-2, a Toll-like receptor (TLR) 2 ligand similar to BCG-CWS. This is more specifically represented as [S-(2,3)-bispalmitoyloxy propyl Cys (P2C)-GNNDESNISFKEK, which is described in detail in Akazawa T. et al., CancerSci 2010; 101: 1596-1603 and the like.


As used herein, “CpG motif” refers to a non-methylated dinucleotide moiety of an oligonucleotide, comprising a cytosine nucleotide and the subsequent guanosine nucleotide. This is used as a representative example of an adjuvant. As a result of the analysis of the invention, CpG motifs were found to belong to G5, i.e., nucleic acid metabolism or phosphoric acid containing compound metabolism. 5-methylcytosine may also be used instead of cytosine. CpG oligonucleotides (CpG ODN) are short (about 20 base pairs) single-stranded synthetic DNA fragments comprising an immunostimulatory CpG motif. A CpG oligonucleotide is a potent agonist of Toll-like receptor 9 (TLR9), which activates dendritic cells (DCs) and B cells to induce type I interferons (IFNs) and inflammatory cytokine production (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006)), and acts as an adjuvant of Th1 humoral and cellular immune responses including cytotoxic T lymphocyte (CTL) responses (Brazolot Millan, C. L., Weeratna, R., Krieg, A. M., Siegrist, C. A. & Davis, H. L. Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998); Chu, R. S., Targoni, O. S., Krieg, A. M., Lehmann, P. V. & Harding, C. V. The Journal of experimental medicine 186, 1623-1631 (1997)). In this regard, CpG ODNs were considered to be a potential immunotherapeutic agent against infections, cancer, asthma, and hay fever (Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006); Klinman, D. M. Nature reviews. Immunology 4, 249-258 (2004)). A CpG oligodeoxynucleotide (CpG ODN) is a single stranded DNA comprising an immunostimulatory non-methylated CpG motif, and is an agonist of TLR9. There are four types of CpG ODNs, i.e., K type (also called B type), D type (also called A type), C type, and P type, each with a different backbone sequence and immunostimulatory properties (Advanced drug delivery reviews 61, 195-204 (2009)).


Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P types) have been reported. A/D type ODNs are oligonucleotides characterized by a poly-G motif with a centrally-located palindromic (palindromic structure) CpG-containing sequence of phosphodiester (PO) and a phosphorothioate (PS) bond at the 5′ and 3′ ends, and by high interferon (IFN)-α production from plasmacytoid dendritic cells (pDC). Types other than the A/D type consist of a PS backbone. B/K type ODN contains multiple nonmethylated CpG motifs, typically with a non-palindromic structure. B/K type CpG primarily induces inflammatory cytokines such as interleukin (IL)-6 or IL-12, but has low IFN-α production. B/K type ODN is readily formulated using saline, some of which is still under clinical trial. Two modified ODNs including D35-dAs40 and D35core-dAs40 were discovered by the inventors, which are similarly immunostimulatory as the original D35 in human PBMC and induces high IFN-α secretion in a dose dependent manner. C type and P type CpG ODNs comprise one and two palindromic structure CpG sequences, respectively. Both are capable of activating B cells like K types and activating pDCs like D types, but C-type CpG ODNs more weakly induce IFN-α production than P type CpG ODNs (Hartmann, G., et al. European journal of immunology 33, 1633-1641 (2003); Marshall, J. D., et al. Journal of leukocyte biology 73, 781-792(2003); and Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010)).


AddaVax refers to a squalene based oil-in-water adjuvant. MF59 also have a similar structure. As used herein, “squalene based oil-in-water adjuvant” refers to an adjuvant, which is an emulsion with an oil-in-water structure comprising squalene.


The drug component used (e.g., active ingredient, additive, or adjuvant) herein can be isolated or purified. As used herein, a “purified” substance or biological agent (e.g., protein or nucleic acid such as a gene marker or the like) refers to a biological agent having at least a part of a naturally accompanying agent removed. Therefore, the purity of a purified biological agent is generally higher than the normal state of the biological agent (i.e., concentrated). The term “purified” as used herein refers to the presence of preferably at least 75 wt %, more preferably at least 85 wt %, still more preferably 95 wt %, and most preferably at least 98 wt % of the same type of biological agent. The substance used in the present invention is preferably a “purified” substance. As used herein, “isolated” refers to removal of at least one of any accompanying agent in a naturally occurring state. For example, removal of a specific genetic sequence from a genomic sequence is also referred to as isolation. Therefore, the gene used herein can be isolated.


As used herein, “subject” refers to a target (e.g., organisms such as humans, or cells, blood, serum, or the like extracted from an organism) subjected to the diagnosis, detection, therapy, or the like of the present invention.


As used herein, “agent” broadly may be any substance or another element (e.g., light, radiation, heat, electricity, and other forms of energy) as long as the intended objective can be achieved. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including for example DNAs such as cDNAs and genomic DNAs, and RNAs such as mRNAs), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, information transmitting substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as a medicament (e.g., small molecule ligands and the like) and composite molecules thereof).


As used herein, “therapy” refers to the prevention of exacerbation, preferably maintaining the current condition, more preferably alleviation, and still more preferably elimination of a disease or disorder (e.g., cancer or allergy) in case of such a condition, including being capable of exerting an effect of improving or preventing a patient's disease or one or more symptoms accompanying the disease. Preliminary diagnosis conducted for suitable therapy may be referred to as a “companion therapy”, and a diagnostic agent therefor may be referred to as “companion diagnostic agent”.


As used herein, “therapeutic agent” broadly refers to all agents that are capable of treating the condition of interest (e.g., diseases such as cancer or allergies). In one embodiment of the invention, “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. A pharmaceutical composition can be manufactured, for example, by mixing an active ingredient with the aforementioned carriers by any method that is known in the technical field of pharmaceuticals. Further, usage form of a therapeutic agent is not limited, as long as it is used for therapy. A therapeutic agent may consist solely of an active ingredient or may be a mixture of an active ingredient and any ingredient. Further, the shape of the carriers is not particularly limited. For example, the carrier may be a solid or liquid (e.g., buffer). Therapeutic agents for cancer, allergies, or the like include drugs (prophylactic agents) used for the prevention of cancer, allergies, or the like, and suppressants of cancer, allergies, or the like.


As used herein, “prevention” refers to the act of taking a measure against a disease or disorder (e.g., diseases such as cancer or allergy) from being in such a condition, prior to the onset of such a condition. For example, it is possible to use the agent of the invention to perform diagnosis, and use the agent of the invention, as needed, to prevent or take measures to prevent allergies or the like.


As used herein, “prophylactic agent” broadly refers to all agents that are capable of preventing the condition of interest (e.g., disease such as cancer or allergies).


As used herein, “kit” refers to a unit providing portions to be provided (e.g., testing agent, diagnostic agent, therapeutic agent, antibody, label, manual, and the like), generally in two or more separate sections. This form of a kit is preferred when intending to provide a composition that should not be provided in a mixed state and is preferably mixed immediately before use for safety reasons or the like. Such a kit advantageously comprises instructions or a manual preferably describing how the provided portions (e.g., testing agent, diagnostic agent, or therapeutic agent) should be used or how a reagent should be processed. When the kit is used herein as a reagent kit herein, the kit generally comprises an instruction describing how to use a testing agent, diagnostic agent, therapeutic agent, antibody, and the like.


As used herein, “instruction” is a document with an explanation of the method of use of the present invention for a physician or for other users. The instruction describes a detection method of the invention, how to use a diagnostic agent, or a description instructing administration of a medicament or the like. An instruction may also have a description instructing oral administration, or administration to the esophagus (e.g., by injection or the like) as the site of administration. The instruction is prepared in accordance with a format specified by a regulatory authority of the country in which the present invention is practiced (e.g., Ministry of Health, Labour and Welfare in Japan, Food and Drug Administration (FDA) in the U.S., or the like), with an explicit description showing approval by the regulatory authority. The instruction is a so-called “package insert”, and is generally provided in, but not limited to, paper media. The instructions may also be provided in a form such as electronic media (e.g., web sites provided on the Internet or emails).


As used herein, “diagnosis” refers to identifying various parameters associated with a disease, disorder, condition or the like in a subject to determine the current or future state of such a disease, disorder, or condition. The condition in the body can be examined by using the method, apparatus, or system of the invention. Such information can be used to select and determine various parameters of a formulation, method, or the like for treatment or prevention to be administered, disease, disorder, or condition in a subject or the like. As used herein, “diagnosis” when narrowly defined refers to diagnosis of the current state, but when broadly defined includes “early diagnosis”, “predictive diagnosis”, “prediagnosis”, and the like. Since the diagnostic method of the invention in principle can utilize what comes out from a body and can be conducted away from a medical practitioner such as a physician, the present invention is industrially useful. In order to clarify that the method can be conducted away from a medical practitioner such as a physician, the term as used herein may be particularly called “assisting” “predictive diagnosis, prediagnosis or diagnosis”.


The procedure for formulation as the drug of the invention or the like is known in the art and is described in the Japanese Pharmacopoeia, US Pharmacopoeia, pharmacopoeia of other countries, and the like. Therefore, those skilled in the art can determine the amount to be used without undue experimentation with the descriptions herein.


As used herein, “program” is used in the general meaning used in the art. A program describes the processing to be performed by a computer in order, and is legally considered a “product”. All computers are operated in accordance with a program. Programs are expressed as data in modern computers and stored in a recording medium or a storage device.


As used herein, “recording medium” is a recording medium storing a program for executing the present invention. A recording medium can be anything, as long as a program can be recorded. For example, a recording medium can be, but is not limited to, a ROM or HDD or a magnetic disk that can be stored internally, or an external storage device such as flash memory such as a USB memory.


As used herein, “system” refers to a configuration that executes the method of program of the invention. System fundamentally means a system or organization for executing an objective, wherein a plurality of elements are systematically configured to affect one another. In the field of computers, system refers to the entire configuration such as the hardware, software, OS, and network.


DESCRIPTION OF PREFERRED EMBODIMENTS

The preferred embodiments of the invention are described hereinafter. It is understood that the embodiments provided hereinafter are provided to better facilitate the understanding of the present invention, and thus the scope of the invention should not be limited by the following descriptions. Thus, it is apparent that those skilled in the art can refer to the descriptions herein to make appropriate modifications within the scope of the present invention. Those skilled in the art can also appropriately combine any embodiments.


<Transcriptome Analysis of Drug Component>


The present invention provides a method of generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant). The method comprises: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components (e.g., active ingredients, additives, or adjuvants); (B) clustering the drug components (e.g., active ingredients, additives, or adjuvants) with respect to the expression data; and (C) generating a transcriptome profile of the organ of the drug components (e.g., active ingredients, additives, or adjuvants) based on the clustering.


The transcriptome analysis used in the method of the invention can be embodied by using any approach. Transcriptome analysis can be transcriptome analysis of an organ of a drug component (e.g., active ingredient, additive, or adjuvant) performing transcriptome analysis of an organ of a target organism without administering the drug component (e.g., active ingredient, additive, or adjuvant), obtaining a control transcriptome, performing transcriptome analysis of the same organ of the target organism after administering a candidate drug component (e.g., active ingredient, additive, or adjuvant), and normalizing as needed using the control transcriptome. The same procedure can also be performed on a second or another subsequent drug component (e.g., active ingredient, additive, or adjuvant). Clustering analysis of each drug component (e.g., active ingredient, additive, or adjuvant) can be performed by using expression data obtained from transcriptome analysis for two or more drug components (e.g., active ingredients, additives, or adjuvants). Each drug component (e.g., active ingredient, additive, or adjuvant) can be analyzed based on cluster information of the drug component (e.g., active ingredient, additive, or adjuvant) obtained based on gene expression data, which is the result of clustering analysis. In particular, a drug component (e.g., active ingredient, additive, or adjuvant) belonging to the same cluster as a standard drug component (e.g., active ingredient, additive, or adjuvant) or reference drug component (reference drug component and standard drug component refer to the same component herein) can be estimated to have the same function as the reference drug component (standard drug component). Generation of a transcriptome profile of the organ of the adjuvant based on the clustering can be embodied by using any approach that is known in the art. For example, a profile can use a dendrogram or expressed using a spreadsheet software such as Excel®, but the profile is not limited thereto.


(Functional Classification of Drug Component (e.g., Active Ingredient, Additive, or Adjuvant))


In one aspect, the present invention provides a method of classifying an adjuvant comprising classifying a drug component (e.g., active ingredient, additive, or adjuvant) based on transcriptome clustering. The classification based on transcriptome clustering in the present invention can comprise classifying a target drug component (e.g., active ingredient, additive, or adjuvant) based on a result of transcriptome clustering of a reference drug component (e.g., active ingredient, additive, or adjuvant). There are various examples of classification of a drug component (e.g., active ingredient, additive, or adjuvant) including classification by at least one feature selected from the group consisting of classification based on a host response, classification based on a mechanism, classification by application based on a mechanism or cells (liver, lymph node, or spleen), and module classification.


In a representative example, classification of a drug component (e.g., active ingredient, additive, or adjuvant) provided by the present invention comprises at least one classification selected from the group consisting of (1) G1 (interferon signaling); (2) G2 (metabolism of lipids and lipoproteins); (3) G3 (response to stress); (4) G4 (response to wounding); (5) G5 (phosphate-containing compound metabolic process); and (6) G6 (phagosome). For G1 to G6, the corresponding portions can be classified for adjuvants, but some, albeit a small number, are not classified thereto. They can be deemed as not classified to G1 to G6. Additional transcriptome analysis can be performed for further classification as needed.


In a preferred embodiment of the invention, a target substance that has not been classified can be classified by clustering a result of transcriptome analysis using each reference drug component (e.g., active ingredient, additive, or adjuvant) of G1 to G6 and comparing them. In this regard, the reference drug component of G1 is selected from the group consisting of cdiGMP, cGAMP, DMXAA, PolyIC, and R848, the reference drug component of G2 is bCD, the reference drug component of G3 is FK565, the reference drug component of G4 is MALP2s, the reference drug component of G5 is selected from the group consisting of D35, K3, and K3SPG, and/or the reference drug component of G6 is AddaVax. These reference drug components are representative. Other drug components (e.g., active ingredients, additives, or adjuvants) determined as belonging to G1 to G6 can be used instead.


In one embodiment, classification of G1 to G6 is performed based on an expression profile of a gene (identification marker gene; DEG) with a significant difference in expression in transcriptome analysis. The DEG of G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216, the DEG of G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5, the DEG of G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1, the DEG of G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b, the DEG of G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and the DEG of G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


These DEGs can be used when identifying an alternative to a reference drug component (e.g., active ingredient, additive, or adjuvant). A drug component (e.g., active ingredient, additive, or adjuvant) with substantially the same pattern of the DEG can be used as a reference drug component (e.g., active ingredient, additive, or adjuvant).


Therefore, in one aspect, the present invention provides a gene analysis panel for use in classification of an adjuvant to G1 to G6 or to others. The gene analysis panel comprises a detecting agent or detecting means for detecting at least one DEG selected from the group consisting of a DEG of G1, a DEG of G2, a DEG of G3, a DEG of G4, a DEG of G5, and a DEG of G6, wherein the DEG of G1 comprises at least one selected from the group consisting of Gm14446, Pm1, H2-T22, Ifit1, Irf7, Isg15, Stat1, Fcgr1, Oas1a, Oas2, Trim12a, Trim12c, Uba7, and Ube216, wherein the DEG of G2 comprises at least one selected from the group consisting of Elovl6, Gpam, Hsd3b7, Acer2, Acox1, Tbl1xr1, Alox5ap, and Ggt5, wherein the DEG of G3 comprises at least one selected from the group consisting of Bbc3, Pdk4, Cd55, Cd93, Clec4e, Coro1a, and Traf3, Trem3, C5ar1, Clec4n, Ier3, Il1r1, Plek, Tbx3, and Trem1, wherein the DEG of G4 comprises at least one selected from the group consisting of Ccl3, Myof, Papss2, Slc7a11, and Tnfrsf1b, wherein the DEG of G5 comprises at least one selected from the group consisting of Ak3, Insm1, Nek1, Pik3r2, and Ttn, and wherein the DEG of G6 comprises at least one selected from the group consisting of Atp6v0d2, Atp6v1c1, and Clec7a.


Preferably, the gene analysis panel of the invention comprises a detecting agent or detecting means for detecting at least a DEG of G1, a detecting agent or detecting means for detecting at least a DEG of G2, a detecting agent or detecting means for detecting at least a DEG of G3, a detecting agent or detecting means for detecting at least a DEG of G4, a detecting agent or detecting means for detecting at least a DEG of G5, and a detecting agent or detecting means for detecting at least a DEG of G6.


The detecting agent or detecting means contained in the gene analysis panel of the invention can be any means, as long as a gene can be detected.


In one aspect, the present invention provides a method of classifying a drug component, the method comprising:


(a) providing a candidate drug component;


(b) providing a reference drug component set;


(c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and


(d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in the reference drug component set, and determining as impossible to classify if the candidate drug component does not belong to any cluster. The drug component can be, for example, an active ingredient, additive, adjuvant, or the like.


In one embodiment, the present invention provides a method of classifying an adjuvant. The method comprises: (a) providing a candidate drug component (candidate adjuvant) in at least one organ of a target organism; (b) providing a reference drug component (reference adjuvant) set classified to at least one selected from the group consisting of G1 to G6; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (candidate adjuvant) and the reference drug component (reference adjuvant) set to cluster the gene expression data; and (d) determining that the candidate drug component (candidate adjuvant) belongs to the same group if a cluster to which the candidate drug component (candidate adjuvant) belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


In the present invention, (a) providing a candidate drug component (candidate adjuvant) in at least one organ of a target organism can be performed by any approach. For example, a novel substance can be obtained or synthesized, or an already commercially available substance can be obtained and provided as a candidate drug component (e.g., candidate adjuvant). Examples of candidate drug components (e.g., candidate adjuvant) include, but are not limited to, a protein, polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide, nucleotide, nucleic acid, polysaccharide, oligosaccharide, lipid, liposome, oil-in-water molecule, water-in-oil molecule, organic small molecule (e.g., hormone, ligand, information transmitter, organic small molecule, molecule synthesized by combinatorial chemistry, small molecule that can be used as a pharmaceutical product or additive, or the like) and composite molecule thereof.


In the present invention, (b) providing a reference drug component set (e.g., reference adjuvant set classified to at least one selected from the group consisting of G1 to G6) can be performed by any approach. In this regard, the exemplified features of G1 to G6 are described in other parts herein. While anything can be used as a reference drug component (reference adjuvant), typically a reference drug component (e.g., reference adjuvant) of G1 is selected from the group consisting of cdiGMP, cGAMP, DMXAA, PolyIC, and R848, a reference drug component (e.g., reference adjuvant) of G2 is bCD, a reference drug component (e.g., reference adjuvant) of G3 is FK565, a reference drug component (e.g., reference adjuvant) of G4 is MALP2s, a reference drug component (e.g., reference adjuvant) of G5 is selected from the group consisting of D35, K3, and K3SPG, and/or a reference drug component (e.g., reference adjuvant) of G6 is AddaVax. These drug components (e.g, reference adjuvant) can utilize a commercially available, newly synthesized, or manufactured drug component.


In the present invention, (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate adjuvant) and the reference drug component (e.g., reference adjuvant) set to cluster the gene expression data can be performed by any approach. Transcriptome analysis and clustering can be performed by appropriately combining known approaches in the art.


In one embodiment, the transcriptome analysis performed in the present invention administers the drug components (e.g., adjuvants) to the target organism and compares a transcriptome in the organ after a certain time after administration with a transcriptome in the organ before administration of the drug components (e.g., adjuvants), and identifies a set of differentially expressed genes (DEG) (preferably a gene with a statistically significant change, i.e., significant DEG) as a result of the comparison. The series of operations can be standardized. Such a standardized procedure can be that described for example at http://sysimg.ifrec.osaka-u.ac.jp/adjvdb/. It is understood that technologies known in the art can be appropriately applied in this manner for administration of a drug component (e.g., adjuvant), harvesting an organ, RNA extraction, and GeneChip data acquisition. The procedures can be those complying with the law and guidelines meeting the appropriate standards of the facility, and approved by an appropriate committee of the facility to comply with the regulation and ethical standards of authorities.


Examples of administration of a drug component (e.g., adjuvant) include, but are not limited to, administration to a site with low stimulation such as the base of the tail. The administration method can be intradermal (id) administration to the base of a tail, or intraperitoneal (ip), i.n. (intranasal), or oral administration, or the like. The dosage of a drug component (e.g., adjuvant) is selected to induce an excellent effect (e.g., adjuvant function) without inducing severe reactogenicity in a target animal by referring to information known in the art and information in the Examples. A negative control experiment is conducted using a suitable buffer subject group in addition to the drug component (e.g., adjuvant) administration group.


As needed, a preliminary experiment can be conducted to investigate a differentially expressed gene in an organ after administration of a drug component (e.g., adjuvant) and investigate genes with a dramatic change or genes that return to normal after an appropriate period of time has passed. As shown in the Examples, it was found in an example that it is preferable to see the gene expression at 6 hours after administration. In addition, many return to normal after 24 hours. Thus, a change in gene expression can be preferable at, for example, 1 to 20 hours after administration, preferably after 3 to 12 hours such as any point after 4 to 8 hours or about 6 hours. Gene expression can be investigated at this time or by using a gene chip (e.g., Affimetrix GeneChip microarray system (Affymetrix)) or the like. A sample for testing with a gene chip can be prepared by, for example, preparing Total RNA with an appropriate kit.


Expression can be analyzed by using any approach known in the art. For example, software that is a part of a system such as Affimetrix GeneChip microarray system (Affymetrix) can be used, software can be self-created, or another program available on the Internet or the like can be used.


In one embodiment, the method of the invention comprises integrating the set of DEGs in two or more drug components (e.g., adjuvants) to generate a set of differentially expressed genes (DEG) in the common manner in transcriptome analysis in the present invention. In this regard, the DEG can be preferably a significant DEG. A significant DEG can be extracted by setting any threshold value. Meanwhile in a specific embodiment, a predetermined threshold value used in the present invention can be identified by a difference in a predetermined multiple and a predetermined statistical significance (p value). For example, the value can be defined as a statistically significant change (upregulation or downregulation) satisfying all of the following conditions: Mean fold change (FC) is >1.5 or <0.667; p value of associated t-test is <0.01 without multiple testing correction; and customized PA call is 1. In this regard, other values can be set for FC, which can be greater than 1 fold and 10 fold or less (the other is an inverse thereof), such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold (and the other is an inverse thereof), or the like. The set of values are generally in a relationship of being inverses, but a combination that is not in a relationship of inverses can also be used. For p values, a baseline other than <0.01 can also be used, such as <0.05, <0.04, <0.03, <0.02, or the like, or <0.009, <0.008, <0.007, <0.006, <0.005, <0.004, <0.003, <0.002, <0.001, or the like.


In one embodiment, the method of the invention comprises identifying a gene whose expression has changed beyond a predetermined threshold value as a result of the comparison before and after administration of a drug component (e.g., adjuvant), and selecting differentially expressed genes in the common manner among identified genes to generate a set of significant DEGs. The predetermined threshold value used in this regard can be any threshold value explained in other parts herein. Genes selected as having the same change in the present invention are used as a set of significant DEGs. Such a set of significant DEGs can be used for classification of drug components (e.g., adjuvants).


In one embodiment, the method of the invention comprises performing the transcriptome analysis for at least two or more organs to identify a set of differentially expressed genes only in a specific organ and using the set as the organ specific gene set. If such an organ specific gene set is used, a drug component (e.g., adjuvant) can be classified by only performing transcriptome analysis in a specific organ. A drug component (e.g., adjuvant) can be classified by utilizing comparison with a reference drug component (e.g., reference adjuvant), a standard drug component (e.g., standard adjuvant) or the like.


In another embodiment, the transcriptome analysis performed in the present invention is performed on a large number of organs, preferably on a transcriptome in at least one organ selected from the group consisting of a liver, a spleen, and a lymph node. Although not wishing to be bound by any theory, this is because these organs exhibit results that enable clear identification of a property of an adjuvant as shown in the Examples, but the present invention is not limited thereto. A drug component other than adjuvant (e.g., active ingredient, additive, or the like) and other organs (e.g., kidney, lung, adrenal glands, pancreas, heart, or the like) can also be selected.


In one embodiment, the number of drug components (e.g., adjuvants) to be analyzed by the present invention is a number that enables statistically significant clustering analysis. Such a number can be identified by using common general knowledge associated with statistics. Identification of the number is not the essence of the present invention.


In one embodiment, the method of the invention comprises providing one or more gene markers unique to a specific drug component (e.g., adjuvant) and a specific organ in the determined profile as a drug component (e.g., adjuvant) evaluation marker. By using the drug component (e.g., adjuvant) of the invention, an assay that was not achievable with conventional technology, e.g., unknown drug component (e.g., adjuvant) or a known drug component (e.g., adjuvant) that has not been analyzed, can be evaluated without a large number of experiments. In the present invention, (d) determining that the candidate drug component (e.g., candidate adjuvant) belongs to the same group if a cluster to which the candidate drug component (e.g., candidate adjuvant) belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster, can also be determined by analyzing the cluster explained in (c) in the art. Although not wishing to be bound by any theory, as demonstrated by the Examples, gene unique to a specific organ and a specific drug component (e.g., adjuvant) identified by the present invention can be used in cluster analysis. As a result thereof, a drug component (e.g., adjuvant) can be evaluated by comparison with a reference drug component (e.g., reference adjuvant) or standard drug component (e.g., standard adjuvant).


In one embodiment, the method of the invention further comprises analyzing a biological indicator for a drug component (e.g., adjuvant) and correlating with a cluster. Any indicator can be used as the analyzed biological indicator as long as the indicator enables analysis. A biological indicator is an item that is objectively measured/evaluated as an indicator for a normal process or pathological process, or a pharmacological reaction to therapy. The indicator can be measured with a biomarker or the like. Examples of typical biological indicators include, but are not limited to, at least one indicator selected from the group consisting of a wounding, cell death, apoptosis, NFκB signaling pathway, inflammatory response, TNF signaling pathway, cytokines, migration, chemokine, chemotaxis, stress, defense response, immune response, innate immune response, adaptive immune response, interferons, and interleukins. A biomarker characterizing a condition or change in a disease or the degree of healing is used as a surrogate marker for checking the efficacy of a new drug in a clinical trial. The blood sugar or cholesterol levels or the like are typical biomarkers as indicators for lifestyle diseases. This also includes not only substances derived from an organism contained in the urine or blood, but also electrocardiogram, blood pressure PET image, bone density, lung function, and SNPs. Various biomarkers related to DNA, RNA, biological protein and the like have been found by the advancement in genomic analysis and proteomic analysis.


In one representative embodiment, the biological indicator comprises a hematological indicator.


Examples of such a hematological indicator include, but are not limited to, white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), relative (%) content of lymphocytes (LY %), relative (%) content of monocytes (MO %), relative (%) content of granulocytes (GR %), red blood cells (RBC), hemoglobins (Hb, HGB), hematocrits (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), platelet concentration (PCT), mean platelet volume (MPV), and platelet distribution width (PDW). One or more, or all of these hematological indicators can be measured.


In one embodiment, the biological indicator analyzed in the present invention comprises a cytokine profile. The term “cytokine profile” refers to the amount of various cytokines and the types of cytokines produced in a patient at a certain time. Cytokines are proteins released by white blood cells, having an immunological effect. Examples of cytokines include, but are not limited to, interferon (such as (y-interferon), tumor necrosis factor, interleukin (IL) 1, IL-2, IL-4, IL-6, and IL-10. Examples of cytokines of the cardiocirculatory system include, but are not limited to, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CRP, CSF, CXCL16, Erythropoietin (EPO), FGF, Fractalkine (CXC3L1), G-CSF, GM-CSF, IFNγ, IL-1, IL-2, IL-5, IL-6, IL-8, IL-8 (CXCL8), IL-10, IL-15, IL-18, M-CSF, PDGF, RANTES (CCL5), TNFα, VEGF, and the like. In another aspect, the present invention provides a program for implementing a method of generating an organ transcriptome profile of a drug component (e.g., adjuvant) on a computer. The method of implementing a program comprises: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components (e.g., adjuvants); (B) clustering the drug components (e.g., adjuvants) with respect to the expression data; and (C) generating a transcriptome profile of the organ of the drug components (e.g., adjuvants) based on the clustering. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof.


In another aspect, the present invention provides a method of manufacturing a composition having a desirable function. The method comprises: (A) providing a candidate drug component; (B) selecting a candidate drug component having a transcriptome expression pattern corresponding to a desirable function; and (C) manufacturing a composition using a selected candidate drug component. In this regard, (A) and (B) can use any feature of providing a candidate drug component (e.g., adjuvant), transcriptome analysis, clustering, and the like in the method of classifying a drug component (e.g., adjuvant) of the invention described herein.


In the present invention, (C) manufacturing a composition using a selected candidate drug component (e.g., candidate adjuvant) can be carried out using any approach that is known in the art. Such manufacturing of a composition can be accomplished, preferably, by mixing a pharmaceutically acceptable carrier, a diluent, an excipient, and/or an active ingredient (antigen or the like for an adjuvant or vaccine) with the selected candidate drug components (e.g., adjuvants). Excipients can include buffer, binding agent, blasting agent, diluent, flavoring agent, lubricant, and the like.


In a specific embodiment, the desirable function comprises one or more of G1 to G6 in the step of selecting a candidate drug component (e.g., candidate adjuvant) having a transcriptome expression pattern corresponding to a desirable function of the invention.


In another aspect, the present invention provides a composition for exerting a desirable function, comprising a drug component (e.g., adjuvant) exerting the desirable function, wherein the desirable function preferably comprises one or more of G1 to G6. The drug component (e.g., adjuvant) exerting the desirable function contained in the drug component (e.g., adjuvant) contained in the composition of the invention can be a drug component identified by the method of the invention. Preferably, the drug component (e.g., adjuvant) exerting the desirable function contained in the drug component (e.g., adjuvant) contained in the composition of the invention is not a reference drug component (e.g., reference adjuvant), but can be a drug component whose function (G1 to G6 or others) is newly identified.


The present invention also provides a method of controlling quality of a drug component (e.g., active ingredient, additive, or adjuvant) by using the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. Quantity control reviews a result of analysis of transcriptome clustering used in the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) and determines whether a gene expression pattern that is similar to a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) in a reference animal or the like to make a judgment. If a gene expression pattern that is substantially the same as a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) of a group to which the target drug component (e.g., active ingredient, additive, or adjuvant) belongs or a substitute thereof is found, the target drug component (e.g., target active ingredient, target additive, or target adjuvant) can be determined as having good quality. If a difference in the gene expression pattern is found, the level of quality can be identified in accordance with the degree thereof.


The present invention provides a method of testing safety of a drug component (e.g., active ingredient, additive, or adjuvant) by using the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. A safety test reviews a result of analyzing transcriptome clustering used in the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) described herein, and determines whether a gene expression pattern that is similar to a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) is found in a reference animal or the like to make a judgment. If a gene expression pattern that is substantially the same as a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) of a group to which the target drug component (e.g., active ingredient, additive, or adjuvant) belongs or a substitute thereof is found, the target drug component (e.g., target active ingredient, target additive, or target adjuvant) can be determined to be highly safe. If a difference in a gene expression pattern is found, the level of safety can be identified in accordance with the degree thereof.


The present invention also provides a method of testing an effect (efficacy) of a drug component (e.g., active ingredient, additive, or adjuvant) by using the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. An effect test (efficacy test) reviews a result of analyzing transcriptome clustering used in the method of classifying a drug component (e.g., active ingredient, additive, or adjuvant), and determines whether a gene expression pattern that is similar to a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) is found in a reference animal or the like to make a judgment. If a gene expression pattern that is substantially the same as a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) of a group to which the target drug component (e.g., active ingredient, additive, or adjuvant) belongs or a substitute thereof is found, the target drug component (e.g., target active ingredient, target additive, or target adjuvant) can be determined as having the same degree of effect as the drug component (e.g., active ingredient, additive, or adjuvant). If a difference in a gene expression pattern is found, the level of effect can be identified in accordance with the degree thereof.


In one aspect, the present invention can identify a bottleneck gene for efficacy of toxicity (safety).


As used herein, “bottleneck gene” refers to a gene that has a fundamental effect on arriving at a phenomenon (e.g., efficacy is found, or toxicity is found).


For example, if the phenomenon is safety or toxicity, a toxicity bottleneck gene can be identified. A toxicity bottleneck gene refers to a gene that can determine whether a target (e.g., drug component) has or does not have toxicity if a change in the expression of the gene is observed (e.g., change from absence to presence of expression, presence to absence of expression, or increase or decrease of expression). Typically, a toxicity bottleneck gene is examined after a target is administered, and it can be determined that the target is toxic if expression of the gene is observed or increases.


A toxicity bottleneck gene can be identified by using the approach of the invention. For example, a candidate gene of a toxicity bottleneck gene can be determined by performing transcriptome analysis on a substance known as having toxicity using the method of the invention, identifying the pattern thereof, and identifying a gene having a pattern that is at least partially similar to the target substance. For the candidate gene, it is possible to knock out, in another animal species, a corresponding gene in the another animal species to prepare a knockout animal, determine whether toxicity is reduced or eliminated in the other animal species compared to a no knockout animal, and select the gene with reduction or elimination in toxicity as a toxicity bottleneck gene. Reduction or elimination is preferably statistically significant. The present invention also provides a method of providing or identifying such a toxicity bottleneck gene.


In one aspect, the present invention provides a method of determining toxicity of a drug component (e.g., active ingredient, additive, or adjuvant). The method comprises: determining whether a change (e.g., activation) in gene expression is observed for at least one of toxicity bottleneck genes for a candidate drug component such as a candidate adjuvant; and determining a candidate drug component having the change (e.g., activation) observed as having toxicity. In determining toxicity, a combination of a plurality of components or a combination in the final formulation can also be tested in addition to testing on individual drug components. In some cases, a toxicity test for the final combination can be important.


In another example, an efficacy bottleneck gene can be identified if the target phenomenon is efficacy. An efficacy bottleneck gene refers to a gene that can determine whether a target (e.g., drug component) has or does not have efficacy if a change in the expression of the gene is observed (e.g., change from absence to presence of expression, presence to absence of expression, or increase or decrease of expression). Typically, an efficacy bottleneck gene is examined after a target is administered, and it can be determined that the target has efficacy if expression of the gene is observed or increases. At least one efficacy bottleneck gene can be identified, but is provided as a set in some cases.


An efficacy bottleneck gene can be identified by using the approach of the invention. For example, a candidate gene of an efficacy bottleneck gene can be determined by performing transcriptome analysis on a substance known as having efficacy using the method of the invention, identifying the pattern thereof, and identifying a gene having a pattern that is at least partially similar to the target substance. For the candidate gene, it is possible to knock out, in another animal species, a corresponding gene in the another animal species to prepare a knockout animal, determine whether efficacy is increased or expressed in the knockout animal compared to a no-knockout animal, and select the gene with increase or expression as an efficacy bottleneck gene. Increase or expression is preferably statistically significant. The present invention also provides a method of providing or identifying such an efficacy bottleneck gene.


In one aspect, the present invention provides a method of determining efficacy of a drug component (e.g., active ingredient, additive, or adjuvant). The method comprises: determining whether a change (activation) in gene expression is observed for at least one of efficacy bottleneck genes for a candidate drug component such as a candidate adjuvant; and determining a candidate drug component having the change (activation) observed as having efficacy. If efficacy can also be defined for an additive, efficacy can be similarly determined using an efficacy bottleneck gene. Adjuvants are envisioned to be tested with the primary drug (e.g., antigen for a vaccine).


(Computer Program, System, and Recording Medium)


In yet another aspect, the present invention provides a recording medium storing a program for implementing a method of generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) on a computer. The method of executing a program stored in the recording medium comprises: (A) obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components (e.g., active ingredients, additives, or adjuvants); (B) clustering the drug components (e.g., active ingredients, additives, or adjuvants) with respect to the expression data; and (C) generating a transcriptome profile of the organ of the drug components (e.g., active ingredients, additives, or adjuvants) based on the clustering. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof.


In yet another aspect, the present invention provides a system for generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant). The system comprises: (A) an expression data acquiring unit for obtaining or inputting expression data by performing transcriptome analysis on at least one organ of a target organism using two or more adjuvants (e.g., active ingredients, additives, or adjuvants); (B) a clustering computing unit for clustering the drug components (e.g., active ingredients, additives, or adjuvants) with respect to the expression data; and (C) a profiling unit for generating a transcriptome profile of the organ of the drug components (e.g., active ingredients, additives, or adjuvants) based on the clustering. Each unit of the system of the invention (expression data acquiring unit, clustering computing unit, profiling unit, and the like) can employ any configuration for embodying any embodiment that can be employed in the method of the invention or a combination thereof, and can be implemented in any embodiment.


In this regard, the expression data acquiring unit of the system of the invention is configured to be able to generate data by performing transcriptome analysis, or obtain data as a result thereof, using a drug component (e.g., active ingredient, additive, or adjuvant).


In one aspect, the present invention provides a program for implementing a classification method of a drug component (e.g., active ingredient, additive, or adjuvant) comprising classifying a drug component (e.g., active ingredient, additive, or adjuvant) based on transcriptome clustering on a computer. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof described herein.


In another aspect, the present invention provides a recording medium storing a program for implementing a classification method for a drug component (e.g., active ingredient, additive, or adjuvant) comprising classifying a drug component (e.g., active ingredient, additive, or adjuvant) based on transcriptome clustering on a computer. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof described herein.


In another aspect, the present invention provides a system for classifying a drug component (e.g., active ingredient, additive, or adjuvant) comprising a classification unit for classifying a drug component (e.g., active ingredient, additive, or adjuvant), based on transcriptome clustering. Each unit of the system of the invention (classification unit, and the like) can employ any configuration for embodying any embodiment that can be employed in the method of the invention or a combination thereof, and can be implemented in any embodiment.


In this regard, the classification unit of the system of the invention is configured to be able to generating data by performing transcriptome analysis, or obtain a data as a result thereof, using a drug components (e.g., active ingredients, additives, or adjuvants).


The configuration of the system of the invention is now explained by referring to the functional block diagram in FIG. 4. It is understood that the figure shows the invention embodied as a single system, but an invention embodied with a plurality of systems is also within the scope of the present invention. The method embodied by the system can be written as a program (e.g., program for implementing classification of a drug component (e.g., active ingredient, additive, or adjuvant) on a computer). Such a program can be recorded on a recording medium and embodied as a method.


The system 1000 of the invention is constituted by connecting a RAM 1003, a ROM, SSD, or HDD or a magnetic disk, an external storage device 1005 such as flash memory such as a USB memory, and an input/output interface (I/F) 1025 to a CPU 1001 built into a computer system via a system bus 1020. An input device 1009 such as a keyboard or a mouse, an output device 1007 such as a display, and a communication device 1011 such as a modem are each connected to the input/output I/F 1025. The external storage device 1005 comprises an information database storing unit 1030 and a program storing unit 1040. Both are certain storage areas secured within the external storage apparatus 1005.


In such a hardware configuration, various instructions (commands) are inputted via the input device 1009 or commands are received via the communication I/F, communication device 1011, or the like to call up, deploy, and execute a software program installed on the storage device 1005 on the RAM 1003 by the CPU 1001 to accomplish the function of the invention in cooperation with an OS (operating system). Of course, the present invention can be implemented with a mechanism other than such a cooperating setup.


In the implementation of the present invention, data used as transcriptome clustering, such as expression data obtained as a result of performing transcriptome analysis on at least one organ of a target organism for a drug component (e.g., active ingredient, additive, or adjuvant) or information equivalent thereto (e.g., data obtained by simulation) can be inputted via the input device 1009, inputted via the communication I/F, communication device 1011, or the like, or stored in the database storing unit 1030. The step of obtaining expression data by performing transcriptome analysis on at least one organ of a target organism using two or more drug components (e.g., active ingredients, additives, or adjuvants) and/or implementation of transcriptome clustering for classification can be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. As such software for performing transcriptome analysis, software shown in the Examples can be used, but software is not limited thereto. Any software known in the art can be used. Analyzed data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The step of clustering the drug components (e.g., active ingredients, additives, or adjuvants) with respect to the expression data can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The created clustering analysis data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The step of generating a transcriptome profile of the organ of the drug component (e.g., active ingredient, additive, or adjuvant) based on clustering can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The created transcriptome profile data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The data processing or storage of various features of transcriptome profile data can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The profile feature or information can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030.


The data or calculation result or information obtained via the communication device 1011 or the like is written and updated immediately in the database storing unit 1030. Information attributed to samples subjected to accumulation can be managed with an ID defined in each master table by managing information such as each of the sequences in each input sequence set and each genetic information ID of a reference database in each master table.


The above calculation result can be associated with known information such as various information on drug component (e.g., active ingredient, additive, or adjuvant) or biological information and stored in the database storing unit 1030. Such association can be performed directly to data available through a network (Internet, Intranet, or the like) or as a link to the network.


A computer program stored in the program storing unit 1040 is configured to use a computer as the above processing system, e.g., a system for performing the process of data provision, transcriptome analysis, expression data analysis, clustering, profiling, and other processing. Each of these functions is an independent computer program, a module thereof, or a routine, which is executed by the CPU 1001 to use a computer as each system or device. It is assumed hereinafter that each function in each system cooperates to constitute each system.


<Method of Analyzing Drug Component with Unknown Function>


In another aspect, the present invention provides feature information of a drug component (e.g., active ingredient, additive, or adjuvant). The method comprises: (a) providing a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant); (b) providing a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data; and (d) providing a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant). The feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention is provided using the transcriptome analysis technology of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention, which can comprise one or a combination of any features in <Transcriptome analysis of drug component> described herein.


In this regard, the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) can be a novel substance or a known substance. A property as a conventional drug component (e.g., active ingredient, additive, or adjuvant) can be known or unknown. A candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) is intended to be provided to at least one organ of a target organism. Examples of such an organ include, but are not limited to, liver, spleen, lymph node.


A reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function can be provided using any adjuvant belonging to G1 to G6 specifically mentioned in <Transcriptome analysis of drug component>, or using a drug component (e.g., active ingredient, additive, or adjuvant) separately identified using an approach described in <Transcriptome analysis of drug component> or a set thereof.


Obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set can use any approach known in the art. For the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set, already analyzed data can be used or new data can be reacquired. When using already analyzed data, transcriptome analysis of a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) can be preferably performed under conditions that were used for the already analyzed data (e.g., dosage form, dosage and administration, or the like), but the analysis is not necessarily limited thereto. Gene expression data, when obtained, is clustered. Clustering can use any approach. A method mentioned in <Transcriptome analysis of drug component> described herein can be used.


Once a result of analyzing clustering of gene expression data is obtained after transcriptome analysis, the drug component (e.g., active ingredient, additive, or adjuvant) cluster (group) to which the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) belongs is determined, and a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) can be provided as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant). Information provided by such a method of providing a feature information is highly likely to be a feature that the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) actually has. The method can be considered very useful for predicting a property of a novel substance or a known substance with an unknown function as a drug component (e.g., active ingredient, additive, or adjuvant).


In one aspect, the present invention provides a program for implementing a method of providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) on a computer. The method implemented by the program comprises: (a) providing a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant); (b) providing a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data; and (d) providing a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant). The feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention is provided using the transcriptome analysis technology of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention, which can comprise one or a combination of any features in <Transcriptome analysis of drug component> described herein.


In one aspect, the present invention provides a recording medium for storing a program for implementing a method of providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) on a computer. The method executed by a program stored in the recording medium comprises: a) providing a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant); (b) providing a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data; and (d) providing a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant). The feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention is provided using the transcriptome analysis technology of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention, which can comprise one or a combination of any features in <Transcriptome analysis of drug component> described herein.


In one aspect, the present invention provides a system for providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant). The system comprises: a) a candidate drug component providing unit for providing a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant); (b) a reference drug component providing unit for providing a reference drug component (e.g., active ingredient, additive, or adjuvant) set with a known function; (c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., active ingredient, additive, or adjuvant) and the reference drug component (e.g., active ingredient, additive, or adjuvant) set to cluster the gene expression data; and (d) a feature analysis unit for providing a feature of a member of the reference drug component (e.g., active ingredient, additive, or adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., active ingredient, additive, or adjuvant) as a feature of the candidate drug component (e.g., active ingredient, additive, or adjuvant). The feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention is provided using the transcriptome analysis technology of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention, which can comprise one or a combination of any features in <Transcriptome analysis of drug component> described herein. Each unit of the system of the invention (candidate drug component providing unit, reference drug component providing unit, transcriptome clustering analysis unit, feature analysis unit, and the like) can employ any configuration for embodying any embodiment that can be employed in the method of the invention or a combination thereof, and can be implemented in any embodiment.


In one embodiment, the candidate drug component providing unit can have any configuration, as long as the unit has a function and arrangement for providing a candidate drug component (e.g., active ingredient, additive, or adjuvant). The unit can be provided as the same or different structure as the analysis unit or profiling unit. A candidate drug component (e.g., active ingredient, additive, or adjuvant) is intended to be provided to at least one organ of a target organism.


In one embodiment, a reference drug component (e.g., active ingredient, additive, or adjuvant) providing unit provides a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function. A reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) can be stored in advance in the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) providing unit, or the unit may be configured to receive a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) provided separately from the outside. The candidate drug component providing unit and reference drug component providing unit can be different or the same. If the same, a configuration or a function can be provided so that a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) is not mixed in with a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant).


In one embodiment, a transcriptome clustering analysis unit obtains gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data. For a transcriptome analysis of a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant), a transcriptome clustering analysis unit itself can comprise all of the function, or the transcriptome clustering analysis unit can be configured to have a function of performing transcriptome analysis on a result after externally obtaining gene expression data and inputting the data.


In one embodiment, a feature analysis unit provides a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant).


Next, a system for providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) can also perform the same processing as the system for generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) (see the functional block diagram in FIG. 4).


In another aspect, the present invention provides a program for implementing a method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) on a computer. The method comprises: (a) providing a candidate drug component in at least one organ of a target organism; (b) calculating a reference drug component set; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and (d) determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in a reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster. In this method, a drug component can be an active ingredient, additive, adjuvant, or combination thereof. In another aspect, the present invention provides a recording medium storing a program for implementing the above method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) on a computer.


In one embodiment, the present invention provides a program for implementing a method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) on a computer, the method comprising: (a) providing a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) in at least one organ of a target organism; (b) providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6; (c) obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data; and (d) determining that the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) belongs to the same group if a cluster to which the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the candidate drug component does not belong to any cluster. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof. In another aspect, the present invention provides a recording medium storing a program for implementing the above method of classifying a drug component (e.g., active ingredient, additive, or adjuvant) on a computer.


In another embodiment, the present invention provides a program for implementing a method of classifying an adjuvant on a computer and a recording medium storing the program. In this regard, the method comprises: (a) providing a candidate adjuvant in at least one organ of a target organism; (b) providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster. Each step used therein can be carried out in any embodiment that can be employed in the method of the invention or a combination thereof.


In another aspect, the present invention provides a system for classifying a drug component (e.g., active ingredient, additive, or adjuvant). The system comprises: (a) a candidate drug component providing unit for providing a candidate adjuvant in at least one organ of a target organism; (b) a reference drug component calculating unit for calculating a reference drug component set; (c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and (d) a determination unit for determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in a reference drug component set, and determining as impossible to classify if the cluster does not belong to any cluster. In this system, the drug component can be an active ingredient, additive, adjuvant, or combination thereof.


In one embodiment, the present invention provides a system for classifying a drug component. The system comprises: (a) a candidate drug component providing unit for providing a candidate drug component in at least one organ of a target organism; (b) a reference drug component storing unit for providing a reference drug component set classified to at least one selected from the group consisting of G1 to G6 of the invention; (c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and the reference drug component set to cluster the gene expression data; and (d) a determination unit for determining that the candidate drug component belongs to the same group if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster.


In another embodiment, the present invention provides a system for classifying an adjuvant, the system comprising: (a) a candidate adjuvant providing unit for providing a candidate adjuvant in at least one organ of a target organism; (b) a reference adjuvant storing unit for providing a reference adjuvant set classified to at least one selected from the group consisting of G1 to G6; (c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the reference adjuvant set to cluster the gene expression data; and (d) a determination unit for determining that the candidate adjuvant belongs to the same group if a cluster to which the candidate adjuvant belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster. Each unit of the system of the invention (candidate drug component providing unit, reference adjuvant storing unit, transcriptome clustering analysis unit, determination unit, and the like) can employ any configuration for embodying any embodiment that can be employed in the method of the invention or a combination thereof, and can be implemented in any embodiment.


In this regard, the classification unit of the system of the invention is configured to be able to generating data by performing transcriptome analysis, or obtain data as a result thereof, using a drug component (e.g., active ingredient, additive, or adjuvant).


The configuration of the system of the invention is now explained by referring to the functional block diagram in FIG. 4. It is understood that the figure shows the invention embodied as a single system, but an invention embodied with a plurality of systems is also within the scope of the present invention. The method embodied by the system can be written as a program. Such a program can be recorded on a recording medium and embodied as a method.


The system 1000 of the invention is constituted by connecting a RAM 1003, a ROM, SSD or HDD or a magnetic disk, an external storage device 1005 such as flash memory such as a USB memory, and an input/output interface (I/F) 1025 to a CPU 1001 built into a computer system via a system bus 1020. An input device 1009 such as a keyboard or a mouse, an output device 1007 such as a display, and a communication device 1011 such as a modem are each connected to the input/output I/F 1025. The external storage device 1005 comprises an information database storing unit 1030 and a program storing unit 1040. Both are certain storage areas secured within the external storage apparatus 1005.


In such a hardware configuration, various instructions (commands) are inputted via the input device 1009 or commands are received via the communication I/F, communication device 1011, or the like to call up, deploy, and execute a software program installed on the storage device 1005 on the RAM 1003 by the CPU 1001 to accomplish the function of the invention in cooperation with an OS (operating system). Of course, the present invention can be implemented with a mechanism other than such a cooperating setup.


In the implementation of the present invention, if gene expression data was obtained by transcriptome analysis on a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set with a known function, expression data obtained by transcriptome analysis or information equivalent thereto (e.g., data obtained by simulation) can be inputted via the input device 1009, inputted via the communication I/F, communication device 1011, or the like, or stored in the database storing unit 1030. The step of obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set to cluster the gene expression data can be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. As such software for performing transcriptome analysis or expression analysis, software shown in the Examples can be used, but software is not limited thereto. Any software known in the art can be used. Analyzed data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The step of providing a feature of a member of the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set belonging to the same cluster as that of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) as a feature of the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The created data of a feature of a candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The data processing or storage of various features of transcriptome profile data can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The profile feature or information can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030.


In another implementation of the present invention, data related to a candidate adjuvant provided by the step of providing the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) in at least one organ of a target organism can be inputted via the input device 1009, inputted via the communication I/F, communication device 1011, or the like, or stored in the database storing unit 1030. Data for a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set provided by the step of providing a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set classified to at least one selected from the group consisting of G1 to G6 can also be similarly stored or inputted. Alternatively, data for a reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) set can be called out and used from the database storing unit 1030, or via the communication I/F, communication device 1011, or the like. The step of obtaining gene expression data by performing transcriptome analysis on the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) and the reference drug component (e.g., reference active ingredient, reference additive, or reference adjuvant) to cluster the gene expression data can be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. As such software for performing transcriptome analysis and/or clustering, software shown in the Examples can be used, but software is not limited thereto. Any software known in the art can be used. Analyzed data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030. The step of determining that the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) belongs to the same group if a cluster to which the candidate drug component (e.g., candidate active ingredient, candidate additive, or candidate adjuvant) belongs is classified to the same cluster as at least one in groups G1 to G6, and determining as impossible to classify if the cluster does not belong to any cluster can also be executed with a program stored in the program storing unit 1040, or a software program installed in the external storage device 1005 by inputting various instructions (commands) via the input device 1009 or by receiving commands via the communication I/F, communication device 1011, or the like. The determination data can be outputted through the output device 1007 or stored in the external storage device 1005 such as the information database storing unit 1030.


The data or calculation result or information obtained via the communication device 1011 or the like is written and updated immediately in the database storing unit 1030. Information attributed to samples subjected to accumulation can be managed with an ID defined in each master table by managing information such as each of the sequences in each input sequence set and each genetic information ID of a reference database in each master table.


The above calculation result can be associated with known information such as various information on drug component (e.g., active ingredient, additive, or adjuvant) or biological information and stored in the database storing unit 1030. Such association can be performed directly to data available through a network (Internet, Intranet, or the like) or as a link to the network.


A computer program stored in the program storing unit 1040 is configured to use a computer as the above processing system, e.g., a system for performing the process of data provision, transcriptome analysis, expression data analysis, clustering, profiling, and other processing. Each of these functions is an independent computer program, a module thereof, or a routine, which is executed by the CPU 1001 to use a computer as each system or device. It is assumed hereinafter that each function in each system cooperates to constitute each system.


<Application for Quality Control, Safety Test, and Effect Determination>


In one aspect, the present invention provides a method of controlling quality of a drug component (e.g., active ingredient, additive, or adjuvant) using the method of generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention and/or the method of providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. Quality control of a drug component (e.g., active ingredient, additive, or adjuvant) maintains quality at a certain level or higher by testing in advance whether quality of a drug component (e.g., active ingredient, additive, or adjuvant) in each lot is suitable upon distribution as a pharmaceutical product in particular. An organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) can analyze a property of various drug components (e.g., active ingredient, additive, or adjuvant) by using a significant DEG, so that quality can be maintained at a certain level without actually conducting complex tests.


Examples of specific approaches of quality control include, but are not limited to, performing the above analysis on a drug component (e.g., active ingredient, additive, or adjuvant) subjected to quality control to obtain the organ transcriptome profile thereof, and comparing the standard organ transcriptome profile (also referred to as reference transcriptome profile) estimated for the drug component (e.g., active ingredient, additive, or adjuvant) with an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) subjected to quality control, and if there is no significant difference, determining that estimated quality is retained. Alternatively, if there is a significant difference, it is determined that quality standard is not satisfied. When a significant difference is not found, whether a quality standard is satisfied can be determined by further testing.


In another aspect, the present invention provides a method of testing safety of a drug component (e.g., active ingredient, additive, or adjuvant) by using the method of generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention and/or the method of providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. A novel drug component (e.g., active ingredient, additive, or adjuvant) would require toxicological evaluation. For adjuvants, toxicological evaluation as a formulation comprising a novel adjuvant and an antigen is also required. Even known drug components (e.g., active ingredients, additives, or adjuvants) and antigens require toxicological evaluation depending on the combination thereof. As a part or the entire toxicological evaluation, such an organ transcriptome profile or feature information of a drug component (e.g., active ingredient, additive, or adjuvant) can be utilized. This is useful for extrapolation to safety after human injection.


Examples of specific approaches to determine safety include, but are not limited to, performing the above analysis on a drug component (e.g., active ingredient, additive, or adjuvant) subjected to the determination of safety to obtain an organ transcriptome profile thereof, comparing the standard organ transcriptome profile (also referred to as reference transcriptome profile) estimated for the drug component (e.g., active ingredient, additive, or adjuvant) with an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) subjected to determination of safety, and if there is no significant difference, determining that estimated safety is retained. Alternatively, if there is a significant difference, it is determined that a safety standard is not satisfied. When a significant difference is not found, whether a safety standard is satisfied can be determined by further testing.


In another aspect, the present invention provides a method of determining an effect of a drug component (e.g., active ingredient, additive, or adjuvant) by using the method of generating an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention and/or the method of providing feature information of a drug component (e.g., active ingredient, additive, or adjuvant) of the invention. A novel drug component (e.g., active ingredient, additive, or adjuvant) would require efficacy evaluation. For adjuvants, efficacy evaluation as a formulation comprising a novel adjuvant and an antigen is also required. Even known adjuvants and antigens require efficacy evaluation depending on the combination thereof. As a part or the entire efficacy evaluation, such an organ transcriptome profile or feature information of a drug component (e.g., active ingredient, additive, or adjuvant) can be utilized. This is useful for extrapolation to efficacy evaluation after human intake.


Examples of specific approaches to determine efficacy evaluation include, but are not limited to, performing the above transcriptome analysis on a drug component (e.g., active ingredient, additive, or adjuvant) subjected to the determination of efficacy to obtain an organ transcriptome profile thereof, comparing the standard organ transcriptome profile (also referred to as reference transcriptome profile) estimated for the drug component (e.g., active ingredient, additive, or adjuvant) with an organ transcriptome profile of a drug component (e.g., active ingredient, additive, or adjuvant) subjected to determination of efficacy, and if there is no significant difference, determining that estimated efficacy is attained. Alternatively, if there is a significant difference, it is determined that an efficacy standard is not satisfied. When a significant difference is not found, whether an efficacy standard is satisfied can be determined by further testing.


In the present invention, intra-database analysis can be performed using toxiogenomic data base (GATE) or the like in addition to an adjuvant database. For example, an adjuvant database is demonstrated to be usable in humans, monkeys, mice, rats, and the like. It is also understood that the database can be used similarly in other animals. Further, a toxiogenomic database is open to the public for humans and rats. Intra-database analysis can be performed using other available databases. They are data for 6 hours and 24 hours with a single agent administration. A gene expression profile (liver, kidney, lymph node, spleen, and the like), hematology (white blood cell, red blood cell, platelet, and the like), biochemical testing (aspartate transaminase (AST), alanine transaminase (ALT), creatinine (CRE), and the like, as well as serum miRNA profiles and the like can also be tested. Databases thereof are also available.


While the present invention is capable of transcriptome based toxicity and efficacy prediction, a prediction model can also be generated for prediction by using machine learning (e.g., support vector machine) instead.


For example, for a toxic group (10) and a non-toxic group (10) in a publicly opened toxiogenomic gate (150), a group from which pathological findings were obtained from four administrations and a group from which a toxicity related feature is not observed are identified. A sample exhibiting an expression pattern similar to the toxic group can be predicted as toxic, and a sample exhibiting a pattern similar to the non-toxic group can be determined as non-toxic. In such a case, a prediction model can be generated by machine learning. Such models and prediction methods based on the models are also within the scope of the invention.


Efficacy can be determined by using an adjuvant database associated with efficacy such as an adjuvant database. Databases for particles, emulsions, DNA/RNA, TLR ligands or the like can be used.


The present invention can be practiced using artificial intelligence (AI) that utilizes machine learning. As used herein, “machine learning” refers to a technology that imparts a computer with an ability to learn without explicit programming. It is a process for improving its own performance by a functional unit acquiring new knowledge/skill, or reconstructing existing knowledge/skill. Most of the labor required for programming details can be reduced by programming a computer to learn from experience. In the field of machine learning, a method of constructing a computer program that can automatically improve from experience has been debated. The role of data analysis/machine learning is as an underlying technology that is the foundation of intelligent processing, together with the field of algorithms. It is generally used in conjunction with other technologies. Knowledge in the collaborating field (domain knowledge, e.g., medical field) is required. The scope of application thereof includes roles in prediction (collecting data to predict something would occur), searching (finding a notable feature from collected data), and testing/description (studying relationship among various elements in data). Machine learning is based on an indicator indicating the degree of achievement of a goal in the real world. A user of machine learning needs to understand the goal in the real world. When the goal is achieved, an indicator that would improve needs to be formulated. Machine learning can use linear regression, logistic regression, support vector machine, or the like. In addition, the precision of determination of each model can be calculated by cross validation. After ranking, a feature amount can be increased one by one and perform machine learning (linear regression, logistic regression, support vector machine, or the like) and cross validation to calculate the precision of determination of each model. A model with the highest precision can be selected in this manner. In the present invention, any machine learning can be used. As supervised machine learning, linear, logistic, support vector machine (SVM) or the like can be used.


Machine learning uses logical reasoning. There are roughly three types of logical reasoning, i.e., deduction, induction, and abduction. Deduction, under the hypothesis that Socrates is a human and all humans die, reaches a conclusion that Socrates would die, which is a special conclusion. Induction, under the hypothesis that Socrates would die and Socrates is a human, reaches a conclusion that all humans would die, and determines a general rule. Abduction, under a hypothesis that Socrates would die and all humans die, arrives at Socrates is a human, which falls under a hypothesis/explanation. However, it should be noted that how induction generalizes is dependent on the premise, so that this may not be objective.


Impossible has three basic principles, i.e., impossible, very difficult, and unsolved. Further, impossible includes generalization error, no free lunch theorem, and ugly duckling theorem and true model observation is impossible, so that it is not possible to verify. Such an ill-posed problem should be noted.


Feature/attribute in machine learning represents the state of a subject of prediction when viewed from a certain aspect. A feature vector/attribute vector combines features (attribute) describing a subject of prediction in a vector form.


As used herein, “model” or “hypothesis” are used synonymously, which is expressed using mapping describing the relationship of inputted prediction subjects to prediction results, or a mathematical function or Boolean expression of a candidate set thereof. For learning with machine learning, a model considered the best approximation of the true model is selected from a model set by referring to training data.


Examples of models include generation model, identification model, function model, and the like. Models show a difference in the direction of classification model expression of the mapping relationship between the input (subject of prediction) x and output (result of prediction) y. A generation model expresses a conditional distribution of output y given input x. An identification model expresses a joint distribution of input x and output y. The mapping relationship is probabilistic for an identification model and a generation model. A function model has a definitive mapping relationship, expressing a definitive functional relationship between input x and output y. While identification is sometimes considered slightly more precise in an identification model and a generation model, there is basically no difference in view of the no free lunch theorem.


For learning in machine learning, a model considered the best approximation of the true model is selected from a model set by referring to training data. There are various learning methods depending on the “approximation”. A typical method is the maximum likelihood estimation, which is a standard of learning that selects a model with the highest probability of producing training data from a probabilistic model set. Maximum likelihood estimation can select a model that best approximates the true model. KL divergence to the true distribution becomes small for greater likelihood. There are various types of estimation that vary by the type of form for finding a parameter or estimated prediction value. Point estimation finds only one value with the highest certainty. Maximum likelihood estimation, MAP estimation and the like use the mode of a distribution or function and are most often used. Meanwhile, interval estimation is often used in the field of statistics in a form of finding a range in which an estimated value falls, where the probability of an estimated value falling in the range is 95%. Distribution estimation is used in Bayesian estimation or the like in combination with a generation model introducing a prior distribution for finding a distribution in which an estimated value falls.


(Adjuvant of Adjuvant)


Vaccine adjuvants are very important for initiating, maximizing and extending the immunogenicity of many vaccines and the potency thereof. For over 80 years, aluminum salt (alum) was the only adjuvant conventionally used in humans. In recent years, additional adjuvants such as alum combined with monophosphoryl lipid A and squalene oil emulsion, while limited, have been approved for human use. A suitable adjuvant is ideally selected based on vaccine properties that provide the best pathogen protection, including the immune response type. Unfortunately, the number of adjuvants approved for human use is currently lacking. Thus, precise adjustment in immune response induction is limited. There is an urgent need for the development of different types of adjuvants.


The most known adjuvant targets a pathogen recognition receptor (PRR) such as TLR, NOD, or inflammasome receptor and induces upregulation of a costimulatory molecule on an antigen presenting cell (APC) and production of inflammatory cytokines and type I interferon (IFN) (Olive, 2012, Expert review of vaccines 11, 237-256). Pathogen associated molecule patterns (PAMPs) contained in a vaccine such as microorganism nucleic acids, glycolipids, or proteins function as an endogenous innate adjuvant and elicit an innate immune response, which then potentiates an adaptive immune response induced by a specific antigen (Desmet, C. J., and Ishii, K. J. (2012). Nature reviews Immunology 12, 479-491). For example, viral RNA in an influenza WV vaccine activates TLR7, which induces a response biased to Th1 against a WV antigen (Koyama, S., Aoshi, T., Tanimoto, T., Kumagai, Y., Kobiyama, K., Tougan, T., Sakurai, K., Coban, C., Horii, T., Akira, S., et al. (2010). Science translational medicine 2, 25ra24.)


However, the mechanism of inducing or enhancing adjuvant activity is not known.


Thus, in another aspect, the present invention is based on a result of studying the immunological feature and mechanism of action of Advax™, a delta inulin which is a microparticle derived from inulin developed as a vaccine adjuvant. In this regard, delta inulin becomes a type 2 adjuvant when combined with a Th2 type antigen, an influenza split vaccine, but exhibits a behavior as a type 1 adjuvant when combined with a Th1 type antigen, influenza inactivated whole virion (WV). Its adjuvant effect to WV of delta inulin is lost in TLR7 knockout mice which lack the built-in RNA adjuvant effect of WV, and showed no adjuvant effect for ovalbumin, a neutral Th0-type antigen. Therefore, unlike other adjuvants, delta inulin potentiates the intrinsic property of co-administered antigen, while its adjuvanticity absolutely requires not only dendritic cells but also phagocytic macrophages and TNF-α. These results demonstrated that delta inulin is a unique class of adjuvant which can potentiate the endogenous adjuvant effect of the vaccines through a not yet fully-identified, unique mechanisms of action.


As used herein, the term “inulin” is understood as a simple inactive polysaccharide consisting of β-D-[2→1]-polyfructofuranosyla-D-glucose family wherein fructose is bound straight without a side chain and a glucose is bound to the end, and includes not only inulin and β-D-[2→1]-polyfructofuranosyla-D-glucose, but also inulin derivatives (includes functional equivalents in some cases), including for example β-D-[2→1]-polyfructose which is potentially obtained by enzymatic removal of the terminal glucose from inulin by using an invertase or inulase enzyme that can remove the glucose at the end thereof. Other derivatives included within the scope of the term or functional equivalent are, for example, inulin derivatives with a free hydroxyl group etherified or esterified by a chemical substitution with alkyl, aryl, or acyl group by a known method. While an inulin composition has a known constitution consisting of a simple and neutral polysaccharide, the molecular weight varies, ranging from 16 kilodaltons (kD) or less to greater. Inulin is a reserve carbohydrate of Compositae and is available at low cost from the bulb of dahlia. Inulin has a relatively hydrophobic polyoxyethylene-like backbone. In addition to this rare structure, the non-ionized property enables preparation of very pure inulin readily by recrystallization. In nature, inulin consists of fructose with a degree of polymerization (DP) of about 60 or greater and has various solubilities, properties, and the like.


While the molecular composition of inulin is well known, the reported solubily varies. Currently at least 5 types of inulin are known, i.e., alpha inulin (aIN; see Phelps, C F. The physical properties of inulin solutions. Biochem J95: 41-47(1965)), beta inulin (bIN; see Phelps, C F. The physical properties of inulin solutions. Biochem J95: 41-47(1965)), gamma inulin (gIN), delta inulin (dIN, also known as deltin), and epsilon inulin (eIN). aIN to dIN are characterized by different dissolution rates in an aqueous medium, i.e., from a form that rapidly dissolves at 23° C. (beta inulin; 323° inulin) through a form that is soluble with a half-life of 8 minutes at 37° C. (alpha inulin; α378 inulin), and a form that is substantially insoluble at 37° C. (gamma inulin) and a form that is substantially insoluble at 50° C. (delta inulin) (for gamma inulin, see WO87/02679 and Cooper, P. D. and Carter, M., 1986 and Cooper, P. D. and Steele, E. J., 1988). As discussed below, eIN was identified as having a different property from aIN to dIN. For delta inulin, an adjuvant product sold as Advax™ is known. Delta inulin is insoluble to water at 50° C. Delta inulin is disclosed in WO 2006/024100, the content of which is incorporated herein by reference. Delta inulin is soluble only when heated to 70 to 80° C. in a concentrated solution (e.g., 50 mg/ml). The 50% OD700 thermal transition point in a non-concentrated solution is 53 to 58° C. Delta inulin can be readily prepared by heating a concentrated gamma inulin solution to 55° C. or higher. Alpha inulin (aIN) is obtained by precipitation from water. Beta inulin is obtained by precipitation from ethanol. Epsilon inulin preferably has a 50% OD700 thermal transition point of a diluted suspension (<0.5 mg/ml) in the range of about 58° C. to about 80° C., and has a low solubility to a water solvent at 59° C. or lower, more preferably 75° C. or lower. A single molecule of an eIN particle has a molecular weight in the range from about 5 to about 50 kilodaltons (kD). The degree of polymerization (DP) of a single molecule of an eIN particle is high in many cases (i.e., degree of polymerization of fructose of 25 or greater, preferably 35 or greater). eIN exhibits a lower solubility to dimethyl sulfoxide (solvent known to neutralize a hydrogen bond) compared to each of aIN, bIN, gIN, and dIN.


Gamma inulin is substantially insoluble to water at 37° C., but is soluble to a concentrated solution (e.g., 50 mg/ml) only at a temperature of 45° C. or higher, as in a and β polymorphic forms. Delta inulin is particulate and has a sharp melting point, i.e., 50% OD700 thermal transition point (dissolution phase transition of non-concentrated solution) of 47±1° C. Delta inulin is insoluble to water at 50° C., and soluble to a concentrated solution (e.g., 50 mg/ml) only when heated to 70 to 80° C. as described in WO 2006/024100. dIN is characterized by a 50% OD700 thermal transition point in a non-concentrated solution of 53 to 58° C. dIN can be readily prepared by heating a concentrated gIN solution to 55° C. or higher. Delta inulin (dIN) and gamma inulin (gIN) are insoluble at 37° C. It is already known that even if introduced into an organism such as a human with this temperature, particulate forms thereof can be maintained, and they are immunologically active and effective especially as an adjuvant of a vaccine, alone or with an antigen binding carrier material such as aluminum hydroxide (Cooper, P D and E J Steele, 1991, Cooper, P D et al., 1991a, Cooper, P D et al., 1991b. WO90/01949 and WO2006/024100). Episilon inulin is also immunologically active and can have the same or higher immunological activity relative to gamma inulin and delta inulin. Epsilon inulin is the most thermostable among the five inulin polymorphic forms. A suspension of the particles thereof remains insoluble even at temperatures at which other polymorphic forms dissolve. Epsilon inulin is the most thermostably advantageous even when heated to 85° C. When used as an adjuvant requiring thermostability, epsilon inulin particles can be stable at high temperatures.


As used herein, “δ inulin (β-D-[2→1]poly(fructo-furanosyl)α-D-glucose” is a substance constituting an adjuvant. An adjuvant consisting of microparticles constituted thereby is typically available as an adjuvant product known as Advax™. Advax™, which is a delta inulin adjuvant, is a microparticulate adjuvant. The microparticles thereof are derived from microparticles of polyfructo furanosyl-d-glucose (delta inulin). It is shown that this improves the immunogenicity and potency of various vaccines including vaccines to influenza, hepatitis B, Japanese encephalitis, West Niles virus, HIV, Bacillus anthracis, and Listeria (Dolter et al., 2011; Feinen et al., 2014; Honda-Okubo et al., 2012; Larena et al., 2013; Petrovsky et al., 2013; Rodriguez-Del Rio et al., 2015; Saade et al., 2013). A vaccine comprising the Advax™ adjuvant such as a vaccine for hepatitis B, influenza, and allergy due to an insect bite has been evaluated in a human clinical trial (Gordon et al., 2014; Heddle et al., 2013; Nolan et al., 2008).


While excellent immunogenicity and tolerance have been demonstrated in these clinical trials, the mechanism of action of Advax™ is still unknown. In the present invention, various types of vaccine antigens were used to test the adjuvant effect of delta inulin. Unexpectedly, delta inulin did not bias an immune response to a co-administered antigen, unlike TLR agonists. Interestingly, delta inulin instead enhanced the immune bias of the vaccine antigen itself. This suggests that delta inulin functions in a new form of “adjuvant of adjuvant” and has action to amplify the innate adjuvant activity of the antigen itself.


As used herein, “elicit or enhance adjuvantivity of antigen” means that for an antigen, antigen's own adjuvantivity is elicited (e.g., generated when absent) or enhanced (e.g., increases an already present activity).


As used herein, “activate dendritic cells” refers to dendritic cells reaching a state where they can exert the innate function thereof or increasing the degree of the state. Examples thereof include elevating the expression of co-stimulatory molecules and presenting an antigen to naïve T cells that have never encountered the antigen to give or enhance the function to activate naïve T cells, and the like. Dendritic cells that have never encountered a foreign object are called immature dendritic cells, which are significantly different from activated dendritic cells, including the expression of cell surface molecules and the like. While immature dendritic cells are highly phagocytic, expression levels of NMC class II molecules and co-stimulatory molecules such as CD80, CD86, and CD40 are low. Dendritic cells intake antigens even without an onset of infection or the like, but are not able to active naïve T cells due to the lower expression of NMC class II or co-stimulatory molecules. Upon bacterial or viral infection, dramatic change is induced in dendritic cells. Dendritic cells that are activated and mature due to various stimulations with the infection would express a large amount of NMC class II presenting an antigen peptide from bacteria or virus and have increased expression of co-stimulatory molecules, and migrate to the T cell region of their lymph node through the lymph duct in a chemokine receptor CCR7 dependent manner. An antigen is presented to naïve T cells in the T cell region of the lymph node, and various cytokines are simultaneously released to induce differentiation from naïve T cells to effector T cells. It is understood that the following three types of signals are involved in activation of dendritic cells upon an infection. The first activating signal is from cytokines such as TNFα that release neutrophils, macrophages, or the like which have infiltrated the infected site, second activating signal is from a dead cell derived component from neutrophils, macrophages, or the like that have died upon the infection, and third activating signal is from Toll-like receptors (TLR) (see the section of macrophage in part 7 for details) that recognize a component from bacteria or virus (e.g., lippolysaccharide from gram negative bacteria, or the like). Dendritic cells remain at a site of infection for several hours, take in antigens sufficiently and become activated, then migrate their lymph node through the lymph duct, activate naïve T cells, and end their life in about a week. New dendritic cells are supplied from the bone marrow to the infected site where dendritic cells are no longer moving. As long as the infection persists, the step of activation of dendritic cells at the focus of infection→migration to their lymph node is repeated.


As used herein, “in the presence of macrophage” refers to any environment where an innate or exogenous macrophage is present.


As used herein, “macrophage enhancer” refers to any agent that imparts or enhances the function or activity of a macrophage. Examples of macrophage enhancers include picolinic acid, crystalline silica, conventional adjuvant such as aluminum salt, and the like.


As used herein, “Th1 type antigen” refers to an antigen associated with Th1 cells, preferably any antigen that elicits or enhances a Th1 immune response. As explained below, Th1 type antigen especially refers to antigens that enhance cellular immunity (engulfs pathogen cells).


As used herein, “Th2 type antigen” refers to an antigen associated with Th2 cells, preferably any antigen that elicits or enhances a Th2 immune response. As explained below, Th2 type antigen especially refers to antigens that enhance humoral immunity (neutralizes toxin of pathogens).


Although the present invention will be explained while presuming Th1 cells and Th2 cells as having any property or function known in the art, the properties and functions of Th1 and Th2 cells that are especially noteworthy herein are further explained hereinafter. Lymphocytes are divided into T cells and B cells that produce an antibody (immunoglobulin). T cells are further divided into helper T cells (CD4 antigen positive) that regulate immune reactions with presentation of an antigen from a monocyte/macrophage and killer T cells (CD8 antigen positive) that kills viral infection cells or the like. Helper T cells are divided into Th1 cells (type 1 T helper cells) and Th2 cells (type 2 T helper cells). Whether antigen presenting cells produce interleukin (IL)-12 or prostaglandin (PG) E2 determines which of Th1 cells (responsible for cellular immunity) and Th2 cells (responsible for humoral immunity) are dominant.


IL-12 secreted by an antigen presenting cell macrophage when presenting an antigen to a T cell differentiates Th0 cells (naïve Th cells) to Th1 cells. Th1 cells produce IL-2, interferon (IFN)-γ (suppress production of IgE antibody), tumor necrosis factor (TNF)-α, TNF-β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 to increase the activity of phagocytes such as monocytes and are involved in cellular immunity (tuberculin reaction or the like). IFN-7 produced by Th1 cells promotes differentiation of Th0 cells into Th1 cells. PGE2 secreted by a macrophage when presenting an antigen to T cells induces differentiation of Th0 cells into Th2 cells. Th2 cells produce IL-3, IL-4 (cytokine increasing the production of immunoglobulin (Ig) E antibodies; also produced from mastocytes and natural killer (NK) T cells), IL-5, IL-6, IL-10, and IL-13, and are involved in humoral immunity (antibody production or the like). IL-10 suppresses the production of IL-12 and the production of IFN-7 from Th1 cells. IL-4 or IL-6 produced by Th2 cells promotes differentiation of Th0 cells into Th2 cells. For differentiation of Th0 cells into Th2 cells, PGE2 produced from arachidonic acid is understood to be more important than IL-4. Th2 cells proliferate even with an antigen stimulation from B cells (does not produce IL-12) as an antigen presenting cell.


In an immune response by Th1 cells, cellular immunity results in an inflammatory reaction mainly around mononuclear cells such as lymphocytes and macrophage. For example in an immune response to fungus Cryptococcus, Th1 cells predominantly act to form a strong granuloma to confine the infection locally. Meanwhile, if Th2 cells predominantly act, inflammatory cell infiltration is extremely poor. For example, humoral immunity does not kill intracellular parasites such as Cryptococcus. For this reason, Cryptococcus fills the alveolar space, such that the infection readily spreads hematogenously to result in the onset of meningitis or the like. It is understood that IgE antibody production increases so that a subject is more likely to have an allergic disposition when Th2 cells are more predominant than Th1 cells.


Fungal components (Pathogen-associated molecular pattern: PAMP) act on dendritic cells, promote differentiation of Th0 cells into Th1 cells and create a Th1 cell predominant state to improve allergic disposition. This is the basis for the so-called sanitary hypothesis to consider intake of food such as natto or yogurt improving allergic disposition. Type 1 interferon (IFN-α and IFN-β) is produced in a viral infection. Type 1 interferon acts on T cells to induce production of IFN-γ or IL-10.


In a bacterial infection, type 2 interferon IFN-γ is produced to induce Th1 cells. In an infection by intracellular parasitic bacteria (tubercle bacillus, salmonella, Listeria, or the like), mainly Th1 cells are induced, phagocytes (macrophage) are activated due to IFN-α produced from Th1 cells, and CD8 positive killer T cells are activated due to IL-2 produced from Th1 cells to kill bacteria or the like.


In an infection by extracellularly proliferating bacteria (gram positive coccus such as Staphylococcus or the like), mainly Th2 cells are induced and antibodies are produced by cytokines produced from Th2 cells to kill bacteria or the like. Th1 cells produce IL-2, activate killer T cells, NK cells, or the like, and activate cellular immunity. Th2 cells produce IL-4, activate B cells via a CD40 ligand (CD40L, gp39), and promote production of type I allergy causing IgE antibodies to activate humoral immunity. Th1 cells also produce IFN-γ, but IFN-γ suppresses the CD40 ligand (CD40L) expression of Th2 cells to suppress IgE antibody production. IL-10 and IL-4 produced by Th2 cells suppress the reaction of Th1 cells.


Antigen presenting cells (dendritic cells) identify whether a pathogen (bacteria or virus), toxin, or the like has infiltrated the body by the TLRs on the surface, and produce, in response, inflammatory cytokine, interferon, or the like. As a result, Th0 cells differentiate into Th1 cells or Th2 cells.


In cellular immunity, macrophage is activated by IFN-γ produced by Th1 cells to kill intracellular parasitic bacteria. Further, killer T cells are activated by IL-2 produced by Th1 cells to damage viral infection cells.


In humoral immunity, B cells differentiate and proliferate due to IL-4, IL-5, IL-6, and IL-13 produced by Th2 cells, and antibodies (immunoglobulin) are produced. Antibodies neutralize extrabacterial toxins produced by a pathogen, opsonize extracellular parasitic bacteria, promote engulfment by macrophage, activate the complement system, and dissolve bacteria.


As used herein, “Th1 response” refers to the immune response by Th1 cells described above. In an immune response by Th1 cells, cellular immunity acts to induce an inflammatory reaction mainly around mononuclear cells such as lymphocytes and macrophage as described above in detail. In an immune response to fungal Cryptococcus, Th1 cells predominantly act to form a strong granuloma to confine the infection locally. As used herein, “Th2 response” refers to Th2 cells predominantly acting. As described above in detail, inflammatory cell infiltration is extremely poor. For example, humoral immunity cannot kill intracellular parasites such as Cryptococcus. For this reason, Cryptococcus fills the alveolar space, such that the infection readily spreads hematogenously to result in the onset of meningitis or the like.


As used herein, “normal or enhanced state of TNFα” refers to a state where tumor necrosis factor α (TNFα) is maintained at a normal level in vivo or normal level of TNFα in vivo is replicated. “Enhanced” refers to a state where there is a higher level of TNFα than normal level of TNFα in vivo, or higher TNFα than the normal level in vivo is replicated.


As used herein, “adjuvant of adjuvant” is understood as a concept encompassing imparting adjuvant activity to a substance comprising activity to enhance the adjuvant activity of a compound already known to be an adjuvant and other substances that are lacking or unknown to be adjuvant.


As used herein, “candidate adjuvant” is a type of a candidate drug component, referring to any substance or a combination thereof considered as an adjuvant. A candidate adjuvant can be a TLR independent adjuvant, TLR dependent adjuvant, or the like as described below, but a compound whose function or property is unknown as an adjuvant, other substances, and combinations thereof can also be used. Examples of TLR independent adjuvant include, but are not limited to the following: alum (aluminum phosphate/aluminum hydroxide; inorganic salt exhibiting various adaptations); AS03 (GSK; squalene) (10.68 mg), DL-α-tocopherol (11.86 mg), and polysorbate 80 (4.85 mg), oil-in-water emulsion used in pandemic influenza; MF59 (Novartis; 4 to 5% (w/v) squalene, 0.5% (w/v) Tween 80, 0.5% Span 85, optionally variable amounts of muramul tripeptide phosphatidyl-ethanolamine (MTP-PE)), oil-in-water emulsion used in influenza): Provax (Biogen Idec; squalene+Pluronic L121), an oil in water emulsion); Montanide (Seppic SA; Bioven; Cancervax; mannide oleate and mineral oil), water-in-oil emulsion used in treating malaria and cancer); TiterMax (CytRx; squalene+CRL-8941), water-in-oil emulsion); QS21 (Antigenics; fraction of Quil A), plant-derived composition used in treating melanoma, malaria, HIV, and influenza); Quil A (Statens Serum Institute; purified fraction of Quillaja saponaria), plant-derived composition used in various treatments); ISCOM (CSL; Isconova; saponin+sterol plus+optionally phospholipid), plant-derived composition used in various treatments including influenza); liposomes (Crucell; Nasvax; synthetic phospholipid spheres consisting of lipid), used in treating various disease), and the like.


TLR-dependent adjuvants include, but are not limited to the following: Ampligen (Hemispherx; synthetic specifically configured double-stranded RNA containing regularly occurring regions of mismatching), effected by activation of TLR3 and used as a vaccine against pandemic flu); AS01 (GSK; MPL, liposomes, and QS-21), effected by MPL-activation of TLR4, liposomes provide enhanced antigen delivery to APCs, QS-21 provides enhancement of antigen presentation to APCs and induction of cytotoxic T cells, also used as a vaccine against malaria and tuberculosis.); AS02 (GSK; MPL, o/w emulsion, and QS-21) is effected by MPL-activation of TLR4, the o/w emulsion provides innate inflammatory responses, APC recruitment and activation, enhancement of antigen persistence at injection site, presentation to immune-competent cells, elicitation of different patterns of cytokines, and the QS-21 provides enhancement of antigen presentation to APCs and induction of cytotoxic T cells; this is also used as a vaccine against malaria, tuberculosis, HBV, and HIV; AS04 (GSK; MPL, aluminum hydroxide/aluminum phosphate) is effected by MPL-activation of TLR4, alum provides a depot effect, local inflammation, and increase in antigen uptake by APCs; this is also used as a vaccine for HBV, HPV, HSV, RSV, and EBV.); MPL RC-529 (Dynavax; MPL) is effected by activation of TLR4 and is used as a vaccine against HBV); E6020 (Eisa/Sanofi Pasteur; synthetic phospholipid dimer) is effected by activation of TLR4); TLR-technology (Vaxinnate; antigen and flagellin, effected by activation of TLR5 and used in vaccines against influenza); PF-3512676 (CpG 7909) (Coley/Pfizer/Novartis; immunomodulating synthetic oligonucleotide), effected by activation of TLR9 and used in vaccines against HBV, influenza, malaria, and anthrax.); ISS (Dynavax; short DNA sequences), effected by activation of TLR9 and used in vaccines against HBV and influenza.); IC31 (Intercell; peptide and oligonucleotide), effected by activation of TLR9, formation of an injection site depot, and enhancing of antigen uptake into APCs and used as a vaccine against influenza, tuberculosis, malaria, meningitis, allergy, and cancer indications.); and the like.


As used herein, “evaluation reference adjuvant” is also called “reference adjuvant” or “standard adjuvant”, which is a reference drug component and is referred to as an adjuvant with a known function. Such an adjuvant has a property or function determined by a method known in the art. For example, the function of δ inulin (β-D-[2→1]poly(fructo-furanosyl)α-D-glucose) or a function equivalent thereof as an adjuvant is known, so that the present invention can use this as a reference.


As used herein “gene expression data” refers to any expression data of various genes.


<δ Inulin which is an Adjuvant of “Adjuvant”>


In one aspect, the present invention provides a composition for eliciting or enhancing adjuvanticity of an antigen comprising δ inulin (β-D-[2→1]poly(fructo-furanosyl)α-D-glucose) or a functional equivalent thereof. In this regard, examples thereof include, but are not limited to, an adjuvant product known as Advax™ of δ inulin (β-D-[2→1]poly(fructo-furanosyl)α-D-glucose) or a functional equivalent thereof.


In one embodiment, an equivalent of δ inulin used herein has a transcriptome expression profile that is equivalent to that of δ inulin. Such a transcriptome expression profile can be used in practice by performing transcriptome analysis and analysis of a gene expression profile. Transcriptome analysis can be practiced by any approach described herein.


<Dendritic Cell Activation>


In another aspect, the present invention provides a composition for activating a dendritic cell, comprising δ inulin or a functional equivalent thereof. In this regard, activation can be performed, for example, in the presence of a macrophage. Alternatively, the composition comprising δ inulin or a functional equivalent thereof can be administered with a macrophage enhancer. This is because the present invention has found that 6 inulin or a functional equivalent thereof activates dendritic cells under conditions where a macrophage is normal or enhanced. Therefore, the present invention can be the basis for activation or an indicator when using δ inulin or a functional equivalent thereof as an adjuvant. For δ inulin or a functional equivalent thereof used herein, any substance explained in the section of <Adjuvant of “adjuvant”> or a combination thereof can be used. Further, an adjuvant having the same dendritic cell activation as δ inulin or a functional equivalent thereof can be identified by using transcriptome analysis explained in <Adjuvant of “adjuvant”> or <Same adjuvant/adjuvant determination method and manufacturing method>.


<Th Orientation>


In another aspect, the present invention provides a composition for enhancing a Th1 response of a Th1 type antigen and a Th2 response of a Th2 type antigen, comprising S inulin or a functional equivalent thereof. For δ inulin or a functional equivalent thereof used herein, any substance explained in the section of <Adjuvant of “adjuvant”> or a combination thereof can be used. Further, an adjuvant having the same Th orientation as δ inulin or a functional equivalent thereof can be identified by using transcriptome analysis explained in <Adjuvant of “adjuvant”> or <Same adjuvant/adjuvant determination method and manufacturing method>.


<Technology Based on TNFα KO Mice>


In another aspect, the present invention provides an adjuvant composition comprising δ inulin or a functional equivalent thereof, wherein the composition is administered while TNFα is normal or enhanced. For δ inulin or a functional equivalent thereof used herein, any substance explained in the section of <Adjuvant of “adjuvant”> or a combination thereof can be used. Further, an adjuvant having the same property under conditions where TNFα is normal or enhanced as δ inulin or a functional equivalent thereof can be identified by using transcriptome analysis explained in <Adjuvant of “adjuvant”> or <Same adjuvant/adjuvant determination method and manufacturing method>.


<Same Adjuvant/Adjuvant Determination Method and Manufacturing Method>


In one aspect, the present invention provides a method of determining whether a candidate adjuvant elicits or enhances adjuvanticity of an antigen. The method comprises: (a) providing a candidate adjuvant; (b) providing δ inulin or a functional equivalent thereof as an evaluation reference adjuvant; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the evaluation reference adjuvant to cluster the gene expression data; and (d) determining the candidate adjuvant as eliciting or enhancing adjuvanticity of an antigen if the candidate adjuvant is determined to belong to the same cluster as the evaluation reference adjuvant. For δ inulin or a functional equivalent thereof used herein, any substance explained in the section of <Adjuvant of “adjuvant”> or a combination thereof can be used.


A candidate adjuvant can be provided in any form in the method of the invention. 6 inulin or a functional equivalent thereof can also be provided as an evaluation reference adjuvant in any form. For example, delta inulin such as Advax™ can be used as an evaluation reference adjuvant. Advax™ is a crystalline nanoparticles of inulin, which is a biological polymer used in vaccines against hepatitis B (prophylactic and therapeutic), influenza, Bacillus anthracis, Shigella, Japanese encephalitis, rabies, bee toxin, or allergy, and cancer immunotherapy (sold by Vaxine Pty).


In another aspect, the present invention provides a method of manufacturing a composition comprising an adjuvant that elicits or enhances adjuvanticity of an antigen. The method comprises: (a) providing one or more candidate adjuvants; (b) providing δ inulin or a functional equivalent thereof as an evaluation reference adjuvant; (c) obtaining gene expression data by performing transcriptome analysis on the candidate adjuvant and the evaluation reference adjuvant to cluster the gene expression data; (d) if there is an adjuvant belonging to the same cluster as the evaluation reference adjuvant among the candidate adjuvants, selecting the adjuvant as an adjuvant that elicits or enhances adjuvanticity of an antigen, and if not, repeating (a) to (c); and (e) manufacturing a composition comprising an adjuvant that elicits or enhances adjuvanticity of an antigen obtained in (d). For δ inulin or a functional equivalent thereof used herein, any substance explained in the section of <Adjuvant of “adjuvant”> or a combination thereof can be used.


<Drug Using Adjuvant of Adjuvant>


An adjuvant or “adjuvant of adjuvant” used in the present invention is provided as a pharmaceutical product or pharmaceutical composition.


The composition of the invention can be prepared as an injection, or oral, enteral, transvaginal, transdermal, or transocular agent with a pharmaceutically acceptable carrier, diluent, or excipient. The composition can also be a composition comprising an active ingredient such as a vaccine antigen (including genetically recombinant antigen), antigen peptide, or anti-idiotypic antibody. An additional or alternative active ingredient can be a lymphokine, cytokine, thymocyte simulation factor, macrophage simulating factor, endotoxin, polynucleotide molecule (e.g., encoding a vaccine antigen) or recombinant viral vector, microorganisms (e.g., microorganism extract), or virus (e.g., inactivated or attenuated virus). In fact, the composition of the invention is especially suitable for use when an inactivated or attenuated virus is an active ingredient.


When the present invention is used as an adjuvant composition, preferred target vaccine antigens include some or all of antigens of bacteria, virus, yeast, mold, protozoa, and other microorganisms, human, animal, or plant derived pathogens, pollen, and other allergens, especially toxins (e.g., toxin of honey bees or wasps) and allergens inducing asthma such as house dust mites and dog or cat dandruff.


Particularly preferred vaccine antigens are HA protein of an influenza virus (e.g., inactivated seasonable influenza virus and seasonal H1, H3, or B strain or pandemic H5 strain recombinant HA antigen), influenza nucleoprotein, rotavirus outer capsid protein, human immunodeficiency virus (HIV) antigen such as gp120, RS virus (RSV) surface antigen, human papilloma virus E7 antigen, herpes simplex virus antigen, hepatitis B virus antigen (e.g., HBs antigen), hepatitis C virus (HCV) surface antigen, inactivated Japanese encephalitis, lyssavirus surface antigen (inducing rabies), and other viral antigens, Shigella, Porphyromonas gingivalis (e.g. proteases and adhesin protein), Helicobacter pylori (e.g., urease), Listeria monocytogenes, Mycobacterium tuberculosis (e.g., BCG), Mycobacterium avium (e.g., hsp65), Chlamydia trachomatis, Candida albicans (e.g., the outer membrane proteins), pneumococcus, meningococcus (e.g., class 1 outer membrane protein), Bacillus anthracis (anthrax causing bacteria), Coxiella burnetti (Q fever causing bacteria that can induce a long-term defense response against autoimmune diabetes (i.e. type 1 diabetes)), other microorganism derived antigens, and malaria causing protozoa (especially Plasmodium falciparum and Plasmodium vivax). Other particularly preferred antigens are cancer antigens (i.e., antigen associated with one or more cancers) such as carcinoembryonic antigen (CEA), mucin-1 (MUC-1), epithelial tumor antigen (ETA), abnormal products of p53 and ras, and melanoma antigens (MAGE).


When the composition of the invention is a vaccine antigen, the composition preferably comprises an antigen binding carrier material. An antigen binding carrier material is, for example, one or more of magnesium, calcium, or aluminum phosphate, sulfate, hydroxide (e.g., aluminum hydroxide and/or aluminum sulfate) and other metal salts or precipitates, and/or one or more of protein, lipid, sulfated or phosphorylated polysaccharide (e.g., heparin, dextran, or cellulose derivative) containing organic acid and chitin (poly N-acetylglucosamine), deacetylated derivative thereof, base cellulose derivative, other organic bases, and/or other antigens. An antigen binding carrier material can be particles of any material with poor solubility (aluminum hydroxide (alum) gel or hydrated salt complex thereof). Typically, an antigen binding carrier material does not have a tendency to aggregate, or is treated to avoid aggregation. Most preferably, an antigen binding carrier material is an aluminum hydroxide (alum) gel, aluminum phosphate gel, or calcium phosphate gel.


The present invention can be provided as a kit. As used herein, “kit” refers to a unit providing portions to be provided (e.g., testing agent, diagnostic agent, therapeutic agent, antibody, label, manual, and the like), generally in two or more separate sections. This form of a kit is preferred when intending to provide a composition that should not be provided in a mixed state and is preferably mixed immediately before use for safety reasons or the like. Such a kit advantageously comprises instructions or a manual preferably describing how the provided portions (e.g., testing agent, diagnostic agent, or therapeutic agent) should be used or how a reagent should be processed. When the kit is used herein as a reagent kit herein, the kit generally comprises an instruction describing how to use a testing agent, diagnostic agent, therapeutic agent, antibody, and the like.


In this manner in another aspect of the invention, the present invention is directed to a kit, the kit further comprising: (a) a container comprising the pharmaceutical composition of the invention in a solution form or a lyophilized form, (b) optionally a second container comprising a diluent or a reconstitution solution for the lyophilized formulation, and (c) optionally a manual directed to (i) use of the solution or (ii) reconstitution and/or use of the lyophilized formulation. The kit further has one or more of (iii) buffer, (iv) diluent, (v) filter, (vi) needle, or (v) syringe. The container is preferably a bottle, vial, syringe, or a test tube or a multi-purpose container. The pharmaceutical composition is preferably lyophilized.


The kit of the invention preferably has a manual for the lyophilized formulation of the invention and reconstitution and/or use thereof in a suitable container. Examples of the suitable container include a bottle, vial (e.g., dual chamber vial), syringe (dual chamber syringe or the like), and test tube. The container can be made of various materials such as glass or plastic. Preferably, the kit and/or container comprises a manual showing the method of reconstitution and/or use on the container or accompanying the container. For example, the label thereof can have an explanation showing that the lyophilized formulation is reconstituted to have the aforementioned peptide concentration. The label can further have an explanation showing that the formulation is useful for, or is for subcutaneous injection. The kit of the invention can have a single container including a formulation of the pharmaceutical composition of the invention with or without other constituent elements (e.g., other compounds or pharmaceutical composition of the other compounds) or have separate containers for each constituent element.


The pharmaceutical composition of the invention is suitable for administering the peptide via any acceptable route such as oral (enteral), transnasal, transocular, subcutaneous, intradermal, intramuscular, intravenous, or transdermal route. Preferably, the administration is subcutaneously administration, and most preferably intradermal administration. Administration can use an infusion pump. Therefore, the medicament of the invention can be provided as a therapeutic or prophylactic method. Such a method for treating or preventing a disease comprises administering an effective amount of the composition, adjuvant, or medicament of the invention to a subject in need thereof with an effective amount of vaccine antigen or the like.


As used herein, “or” is used when “at least one or more” of the listed matters in the sentence can be employed. When explicitly described herein as “within the range” of “two values”, the range also includes the two values themselves.


(General Technology)


Any molecular biological approaches, biochemical approaches, microbiological approaches, and bioinformatics that is known in the art, well known, or conventional can be used herein.


Reference literatures such as scientific literatures, patents, and patent applications cited herein are incorporated herein by reference to the same extent that the entirety of each document is specifically described.


As described above, the present invention has been described while showing preferred embodiments to facilitate understanding. The present invention is described hereinafter based on Examples. The above descriptions and the following Examples are not provided to limit the present invention, but for the sole purpose of exemplification. Thus, the scope of the present invention is not limited to the embodiments and Examples specifically described herein and is limited only by the scope of claims.


EXAMPLES

The Examples are described hereinafter. When necessary, all experiments were conducted in compliance with the guidelines approved by the ethics committee of the Osaka University in the following Examples. For reagents, the specific products described in the Examples were used. However, the reagents can be substituted with an equivalent product from another manufacturer (Sigma-Aldrich, Wako Pure Chemical, Nacalai Tesque, R & D Systems, USCN Life Science INC, or the like). PBS used as a control reagent and DMSO were obtained from Nacalai Tesque. Tris-HCl was obtained from Wako Pure Chemical.


(Method)


Standard Operation Protocol


All procedures adhere to each of the following standard operation protocols (standard procedure: adjuvant administration and organ sampling (standard procedure 1), RNA extraction and GeneChip data acquisition (standard procedure 2), and quality control and final data inclusion to the database (standard procedure 3). Detailed information on these protocols is summarized below. All experiments were conducted under the appropriate laws and guidelines and approved by the National Institutes of Biomedical Innovation, Health and Nutrition.


(Standard Procedure 1)


(Adjuvant Administration and Sampling)


C57BL/6 mice (male, 5-week old, C57BL/6JJc1) were purchased from CLEA Japan and acclimated for at least one week (day −7 to day −10). On day 1 at 10 AM: start administration of buffer or adjuvant solution (finish administration within 30 minutes).


i.d.: administer a total of 100 μL to the base of the tail (left side 50 μL+right side 50 μL)


*A total of 200 μL of bCD was i.d. administered.


i.p.: administer a total of 200 μL to the lower quadrant of abdomen.


i.n.: subcutaneously inject 50 μL of ketamine/xylazine mixture (90 mg/kg of ketamine and 10 mg/kg of xylazine) for anesthesia. Slowly drip 10 μL of the solution into the nose (5 μL into each nasal cavity) with a P20 PIPETMAN. 4 PM: Start sampling of organ (finish sampling from all mice within 30 minutes)


(Blood Sampling)


Take about 200 μL of blood from the retro-orbital venous plexus with a non-heparinized capillary tube and place the blood into a 1.5 mL tube comprising 2 μL of 10% EDTA-2K for hematological testing. Thoroughly mix the sample by gentle tapping. Stored at room temperature until hematological testing.


(Organ Sampling)


LN: Expose and remove inguinal lymph nodes (both sides). Remove adipose tissue to reduce adipose tissue contamination as much as possible under a stereoscopic microscope in a 35 mm dish containing about 1 mL of RNAlater. After cleaning, transfer lymph nodes of both sides to a 2.0 mL Eppendorf Protein LoBind tube containing 1 mL of RNAlater. SP: Expose and remove spleen. Remove adipose tissue and pancreas tissue as much as possible. After cleaning, the divide spleen into three parts with a razor blade. Transfer each part individually to 2.0 mL Eppendorf Protein LoBind tubes containing 1 mL of RNAlater (total of three tubes).


LN: Expose and remove the left lobe of a liver. Punch out three parts with a Biopsy Punch (φ 5 mm). Transfer each part to 2.0 mL Eppendorf Protein LoBind tubes containing 1 mL of RNAlater (total of three tubes). Place each harvested organ in a tube containing RNAlater. Maintain the tubes at 4° C. overnight and store at −80° C. until use.


(Hematological Cell Count)


The hematological cell count was found using VetScan HMII (Abaxis). 50 μl of EDTA-2K blood sample was diluted by adding 250 μl of saline. Measurements are taken with VetScan HMII by following the instruction.


(Standard Procedure 2)


(RNA Extraction and GeneChip Data Acquisition)


This protocol was established by Molecular Toxicology, Biological Safety Research Center, National Institute of Health Sciences and Toxicogenomics informatics project (TGP2), National Institutes of Biomedical Innovation, Health and Nutrition and Division of Cellular by reference to the manufacture's manuals.


(1. Homogenization of Animal Tissues)


*1.1. Reagents and equipments


*1.1.1 Reagents
1) RNeasy® Mini Kit (QIAGEN, cat. #74106)
2) Buffer RLT*
3) 2-Mercapto Ethanol (β-ME)
1.1.2. Equipments

1) Zirconium beads (diameter of 5 mm, Tosoh, cat. #YTZ-5)


2) Electronic scale


3) Aspirator
4) Mixer Mill MM300 (QIAGEN)
5) Microcentrifuge
*1.2. Preparation of Reagents
*1.2.1. Buffer RLT*

1) Add 10 μL of 2-Mercapto Ethanol per 1 mL Buffer RLT before use


*stored at room temperature for up to 1 month


*1.3. Homogenization of Animal Tissues Procedures

1) Dissolve the samples at room temperature.


Confirm that no crystals or a precipitate are present in RNAlater.


*Generally, this will not affect subsequent RNA purification. However, in rare cases, this may lead to RNA instability.


2) Measure organ weight.


*The weight of samples should be about 30-100 mg.


*The weight of muscle samples should be about 30-50 mg. Excessive muscle sample weight leads to the coagulation of homogenate.


3) Remove the RNAlater reagent by aspiratoration. Any crystals that may have formed should be removed at this time.


4) Place Zirconium beads into the tubes (1 bead per tube) using flame-sterilized tweezers.


5) Add 400 μL of Buffer RLT.

*In the case of muscle samples, add 600 μL of Buffer RLT in order to prevent coagulation of homogenate.


6) Place the tubes on the Mixer Mill adaptor. At the TGP2 laboratory, the tubes are placed only on each side of the Mixer Mill Adaptor. Placing tubes on the center of the Mixer Mill Adaptor may cause incomplete disruption.


7) Disrupt and homogenize organ slice by the Mixer Mill under optimal conditions.


*At the TGP2 laboratory, disruption is carried out for 3 min at 25 Hz at room temperature. After the initial disruption step, the Mixer Mill Adapter should be rotated to ensure that each tube is homogenized equally. The second disruption step is also carried out for 3 min at 25 Hz at room temperature.


8) Remove bubbles arising during homogenization by centrifugation for 1 min at 300×g at room temperature.


9) Store homogenate at −80° C.


(2. Purification of Total RNA from Animal Tissues (TRI-Easy Method)


This chapter describes the extraction and purification of total RNA from animal tissue. The “TRI-easy method” combines acid guanidinium-phenol-chloroform (AGPC) extraction and RNeasy technology.


*2.1. Reagents and equipments


*2.1.1. Reagents
1) TRIzol® LS Reagent (Invitrogen, cat. #10296-028)
2) RNeasy® Mini Kit (QIAGEN, cat. #74106)
Buffer RLT*
Buffer RW1*
Buffer RPE

RNase-Free water


RNeasy mini spin columns


Collection tubes (1.5 mL)


Collection tubes (2 mL)


3) DNase (QIAGEN, cat. #79254)

DNase I, RNase-Free (lyophilized)


Buffer RDD

DNase-RNase-Free water


4) 2-Mercapto Ethanol (β-ME)
5) Ethanol
6) Chloroform

7) DEPC-Treated water (Ambion, cat. #9920)


*2.1.2. Equipments

1) Electronic scale


2) Aspirator
3) Mixer Mill

4) Microcentrifuge (rotor for 2 ml tubes)


5) Multiskan Spectrum (Themo Labsystems)
6) Gene Quant pro (Amersham Pharmacia Biotech)
*2.2 Preparation of Reagents
*2.2.1. Buffer RLT*

1) Add 10 μL of R-ME per 1 mL Buffer RLT before use.


*stored at room temperature for up to 1 month


*2.2.2. Buffer RPE

1) Before use for the first time, add 4 volumes of ethanol.


*stored at room temperature


*2.2.3. 50% ethanol


1) Add 1 volume of DEPC-Treated water to the ethanol.


*Do not add bleach or acidic solutions directly to Buffer RLT, Buffer RW1, and the sample-preparation waste.


*2.2.4. DNase

*1) Dissolve the lyophilized DNase I (1500 Kunitz units) in 560 μL of the DNase-RNase-Free water.


*Thawed aliquots can be stored at 2-8° C. for up to 6 weeks.


*For long-term storage, aliquots can be stored at −20° C. for up to 9 months.


*Do not vortex the reconstituted DNase I.


*Do not refreeze the aliquots after thawing.


2) Add 10 μl DNase I stock solution (see above) to 70 μL Buffer RDD. Mix by gently inverting the tube, and centrifuge briefly to collect residual liquid from the sides of the tube.














TABLE 2







reagents
one sample
25 samples
50 samples









DNase I
10 μL
 250 μL
 490 μL



Buffer RDD
70 μL
1750 μL
3430 μL











*DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex.


*2.3. [Optional] calculate the volume of Spike RNA


*2.4. Total RNA purification procedures (TRI-easy method)


1) Dissolve the tissue homogenate at room temperature.


2) Adjust the volume to 150 μL with RLT Buffer to prepare the appropriate concentration of the sample homogenate in Collection tubes (2 mL).


3) Add 3 volume of TRIzol LS Reagent (450 μL) and incubate for 5 minutes at room temperature after mixing by vortexing.


4) Add 1 volume of chloroform (150 μL) and incubate for 2 to 15 minutes at room temperature after shaking vigorously by hand for 30 seconds.


5) Centrifuge at no more than 12000×g for 15 minutes at room temperature.


6) Carefully separate the upper aqueous phase and transfer into a new 1.5 mL tube.


7) Add 1 volume of 50% ethanol and mix by pipetting.


*Using 50% ethanol (instead of 70% ethanol) may increase RNA yields from liver samples.


8) Transfer the supernatant to an RNeasy mini spin column placed in a 2 mL collection tube.


9) Close the lid gently, and centrifuge for 15 seconds at no more than 8,000×g at room temperature.


10) Discard the flow-through and set the column again.


11) Add 350 μL of Buffer RW1 to the RNeasy spin column to wash the spin column membrane.


12) Close the lid gently, and centrifuge for 15 seconds at no more than 8,000×g at room temperature.


13) Discard the flow-through and set the column again.


14) [Optional] Add 80 μL of DNase solution to the column and incubate for 15 minutes at room temperature.


15) Add 350 μL of Buffer RW1 to the column.


16) Close the lid gently, and centrifuge for 15 seconds at no more than 8,000×g at room temperature to wash the spin column membrane.


17) Place the column in a new collection tube.


18) Add 500 μL of RPE to the RNeasy spin column.


19) Close the lid gently, and centrifuge for 15 seconds at no more than 8,000×g at room temperature.


20) Discard the flow-through and set the column again.


21) Add 500 μL of RPE to the RNeasy spin column.


22) Close the lid gently, and centrifuge for 2 minutes at no more than 8,000×g at room temperature.


23) Place the column in a new collection tube.


24) Close the lid gently, and centrifuge for 1 minute at no more than 15,000×g at room temperature.


25) Place the column in a new 1.5 mL collection tube.


26) Add 40 μL of DNase-RNase-Free water to eluted RNA.


27) Incubate the tubes for 3 minutes at room temperature.


28) Close the lid gently, and centrifuge for 1 minute at no more than 15,000×g at room temperature.


29) Add the total amount of eluate onto the column to re-elute RNA for high RNA yield.


30) Incubate the tubes for 3 minutes at room temperature.


31) Close the lid gently, and centrifuge for 1 minute at no more than 15,000×g at room temperature.


*When the RNA concentration of eluate is lower than the required concentration, repeat steps 29 to 31.


32) Measure OD260, OD280, OD975, OD900 of the eluate by Multiskan Spectrum. The total RNA is required a higher concentration than 500 ng/μL.


33) [QC] The reading OD values, variability, and OD260/OD280


34) [QC] 28S and 18S ribosomal RNA by electrophoresis


(3. Synthesis of cDNA)


*3.1. Reagents and equipments


*3.1.1. Reagents

1) One-Cycle cDNA Synthesis Kit (Affymetrix, cat. #900431, store at −20° C.)


T7-Oligo(dT) Primer, 50 μM


5×1st Strand Reaction Mix


DTT, 0.1M


dNTP, 10 mM


SuperScript II, 200 U/μL


5×2nd Strand Reaction Mix



E. coli DNA Ligase, 10 U/μL



E. coli DNA Polymerase I, 10 U/μL


RNase H, 2 U/μL


T4 DNA Polymerase, 5 U/μL


EDTA, 0.5M (store at room temperature)


RNase-free Water (store at room temperature)


*SuperScript for One-Cycle cDNA kit for use with Affymetrix One-Cycle Assays and/or equivalent components from Invitrogen can be used.


2) Sample Cleanup Module (Affymetrix, cat. #900371)

cDNA Binding Buffer


cDNA Wash Buffer, 6 mL concentrate


cDNA Elution Buffer


3) DEPC-Treated water (Ambion, cat. #9915G)


4) 0.5M EDTA Disodium Salt (Sigma, cat. #E-7889)
5) Ethanol
*3.1.2. Equipments
1) Sample Cleanup Module (Affymetrix, cat. #900371)

cDNA Cleanup Spin Column


2 mL Collection Tube


1.5 mL microtube, DNase/RNase/pyrogen free


*Do not use 1.5 mL Collection Tube in the kit, because their lids can break in rare cases.


2) Heat block


3) Microcentrifuge

*3.2. Preparation of reagents


*3.2.1. First-Strand Master Mix

1) Prepare sufficient First-Strand Master Mix in a 1.5 mL tube. See the following table.


2) Mix by flicking the tube and spin down briefly after dissolving the solution.


3) Prepare First-Strand Master Mix immediately before use and place on ice.












TABLE 3





reagents
one sample
24 samples
48 samples







5× First Strand
4 μL
100 μL
200 μL


Buffer


0.1M DTT
2 μL
 50 μL
100 μL


10 mM dNTPs mix
1 μL
 25 μL
 50 μL


total
7 μL
175 μL
350 μL









*3.2.2. Second-Strand Master Mix

1) Prepare sufficient Second-Strand Master Mix in a 15 mL tube. See the following table.


2) Mix by flicking the tube and spin down briefly after dissolving the solution.


3) Mix well by gently flicking the tube and spin down briefly.


4) Prepare Second-Strand Master Mix immediately before use and place on ice.












TABLE 4





reagents
one sample
24 samples
48 samples





















DEPC-Treated
91
μL
2,275
μL
4,550
μL


water


5× First Strand
30
μL
750
μL
1,500
μL


Buffer


10 mM dNTP Mix
3
μL
75
μL
150
μL



E. coli DNA Ligase

1
μL
25
μL
50
μL



E. coli DNA

4
μL
100
μL
200
μL


Polymerase I



E. coli DNA RNase H

1
μL
25
μL
50
μL


total
130
μL
3,250
μL
6,500
μL










*3.2.3. cDNA Wash Buffer


1) Add 24 mL of ethanol to obtain a working solution and checkmark the box to avoid confusion.


*Store at room temperature.


*When the entire amount of buffer is not used within a month, prepare the required amount of buffer in DNase/RNase free tube.


*3.3. cDNA synthesis procedures


*3.3.1. First Strand cDNA synthesis


1) Prepare 5 μg/10 μL of RNA samples using RNase-free water.


2) Mix well by flicking the tube and spin down briefly after dissolving RNA samples stored at −80° C.


3) Add 2 μL of 50 μM T7-Oligo(dT) Primer. Mix well by flicking the tube and spin down briefly.


4) Incubate the tubes for 10 minutes at 70° C.


5) Cool the sample at 4° C. for 2 minutes and spin down briefly.


6) Add 7 μL of First-Strand Master Mix to each reaction mixtures for a final volume of 19 μL. Mix thoroughly by flicking the tube and spin down briefly to collect the reaction mixtures at the bottom of the tube.


7) Immediately incubate the tubes at 42° C. for 2 minutes.


8) Add 1 μL of SuperScript II to the reaction mixtures. Mix thoroughly by flicking the tube and spin down briefly.


9) Incubate the tubes at 42° C. for 1 hour.


10) Cool the samples at 4° C. for 2 minutes.


11) Centrifuge the tube briefly (about 5 seconds) to collect the reaction mixtures at the bottom of the tube and immediately proceed to next step.


*3.3.2. Second Strand cDNA synthesis


1) Add 130 μL of Second-Strand Master Mix to each first-strand synthesis sample. Mix well by flicking the tube and spin down briefly.


2) Incubate the tubes at 16° C. for 2 hours.


3) Add 2 μL of T4 DNA Polymerase. Mix well by flicking the tube and spin down briefly.


4) Incubate the tubes at 16° C. for 5 minutes.


5) Add 10 μL of EDTA, 0.5M. Mix well by vortexing and spin down briefly.


*Double-stranded cDNA sample can be stored at −20° C.


*3.3.3. Cleanup of Double-Stranded cDNA


*All steps of this protocol should be performed at room temperature.


2) Add 600 μL of cDNA Binding Buffer to the double-stranded cDNA sample. Mix by vortexing for 3 minutes and spin down briefly.


*Check that the color of the mixture is yellow (same for color of cDNA Binding Buffer without the cDNA synthesis reaction).


*If the color of the mixture is orange or violet, add 10 μL of 3M sodium acetate, pH 5.0, and mix. Check that the color of the mixture is yellow.


3) Set the cDNA Cleanup Spin Column on a 2 mL Collection Tube


4) Mark the lids of spin column with the sample number to avoid the misidentification of samples.


5) Place 500 μL of the sample in the cDNA Cleanup Spin Column and close the lids of the column. Then centrifuge for 1 minute at 8,000×g (10,500 rpm) at room temperature.


6) After centrifuge, discard flow-through and set again the cDNA Cleanup Spin Column on a 2 mL Collection Tube.


7) Load the remaining mixture (262 μL) in the spin column and close the lids of the column. Then centrifuge for 1 minute at 8,000×g (10,500 rpm) at room temperature.


8) After centrifuge, discard flow-through and Collection Tube.


9) Transfer the spin column into a new 2 mL Collection Tube.


10) Pipet 700 μL of the cDNA Wash Buffer onto the spin column and close the lids of the column. Then centrifuge for 1 minute at 8,000×g (10,500 rpm) at room temperature.


11) After centrifuge, discard flow-through and Collection Tube. Then, transfer spin column into a new 2 mL Collection Tube.


12) Open the cap of the spin column and centrifuge for 5 minutes at 10,000×g (15,000 rpm) at room temperature.


13) After centrifuge, discard flow-through and Collection Tube.


14) Transfer spin column into a 1.5 mL Collection Tube.


15) Pipet 14 μL of cDNA Elution Buffer directly onto the spin column membrane.


16) Incubate for 1 minute at room temperature and centrifuge for 1 minute at 10,000×g (15,000 rpm) to elute.


*The average volume of eluate is 12 μL.


(4. Synthesis of cRNA)


*4.1. Reagents and equipments


*4.1.1. Reagents

1) IVT labeling Kit (Affymetrix, cat. #900449, store at −20° C.)


10×IVT Labeling Buffer


IVT Labeling Enzyme Mix


IVT Labeling NTP Mix


3-Labeling Control (0.5 μg/μL)


RNase-free Water


2) GeneChip Sample Cleanup Module (Affymetrix, cat. #900371)

IVT cRNA Binding Buffer


IVT cRNA Wash Buffer, 5 mL concentrate


RNase-free Water


5× Fragmentation Buffer


cDNA Binding Buffer


cDNA Wash Buffer, 6 mL concentrate


cDNA Elution Buffer


3) DEPC-Treated water (Ambion, cat. #9920, store at room temperature)


4) Ethanol
4.1.2. Equipments
1) GeneChip Sample Cleanup Module (Affymetrix, cat. #900371)

IVT cRNA Cleanup Spin Columns


1.5 mL Collection Tubes (for elution)


2 mL Collection Tubes


1.5 mL microtube, DNase/RNase/pyrogen free


*Be careful not to confuse cRNA Cleanup Spin Columns with cDNA Cleanup Spin Columns.


2) Heat block


3) Microcentrifuge
*4.2. Preparation of Reagents
*4.2.1. IVT Reaction Mix

1) Prepare sufficient IVT Reaction Mix in a 1.5 mL tube. See the following table.


2) Mix by flicking the tube and spin down briefly after dissolving each solution.


*Prepare First-Strand Master Mix immediately before use and place on ice.












TABLE 5





reagents
one sample
24 samples
48 samples







RNase-free Water
10 μL
260 μL
500 μL


10X IVT Labeling
 4 μL
104 μL
200 μL


Buffer


IVT Labeling NTP
12 μL
312 μL
600 μL


Mix


IVT Labeling
 4 μL
104 μL
200 μL


Enzyme Mix





total
30 μL
780 μL
1500 μL 










*4.2.2. IVT cRNA Wash Buffer (store at room temperature)


1) Add 20 mL of ethanol to obtain a working solution, and checkmark the box on the bottle label to avoid confusion.


*If necessary, dissolve a precipitate by warming the precipitate in a water bath at 30° C., and then place the buffer at room temperature.


*4.2.3. 80% Ethanol (store at room temperature)


1) Mix ethanol and DEPC-Treated water in a ratio of 4:1, in an RNase/DNase free tube.


*4.3. IVT reaction procedure


*4.3.1. IVT reaction


1) Prepare 30 μL of IVT Reaction Mix in a 1.5 mL tube.


2) Add 10 μL of the double-stranded cDNA sample. Mix by flicking the tube and spin down briefly after dissolving each solution.


3) Incubate the tubes for 16 hours at 37° C. with mixing at 300 rpm.


4) Store labeled cRNA at −80° C. if not purifying immediately


*4.3.2. cRNA clean-up


1) Add 60 μL of RNase-free Water to the samples (40 μL) by vortexing for 3 seconds.


2) Add 350 μL IVT cRNA Binding Buffer to the mixture by vortexing for 3 seconds.


*Exchange the tip if there is a possibility of contamination.


3) Add 250 μL 100% ethanol to the mixture, and mix well by pipetting.


*Exchange the tip if there is a possibility of contamination.


4) Place the mixture (700 μL) on the IVT cRNA Cleanup Spin Column sitting in a 2 mL


Collection Tube.

5) Centrifuge for 15 seconds at 8,000×g (10,500 rpm).


6) Discard the flow-through and Collection Tube.


7) Transfer the spin column in a new 2 mL Collection Tube.


8) Pipet 500 μL IVT cRNA Wash Buffer onto the spin column.


*Exchange the tip if there is a possibility of contamination.


9) Centrifuge for 15 seconds at 8,000×g (10,500 rpm) and discard flow-through.


10) Pipet 500 μL 80% (v/v) ethanol onto the spin column.


*Exchange the tip if there is a possibility of contamination.


11) Centrifuge for 15 seconds at 8,000×g (10,500 rpm) and discard flow-through.


12) With open caps, centrifuge for 5 minutes at 8,000×g (10,500 rpm) and discard flow-through and Collection Tube.


13) Transfer spin column into a new 1.5 mL Collection Tube, and pipet 11 μL of RNase-free Water directly onto the spin column membrane.


*Exchange the tip if there is a possibility of contamination.


14) With closed caps, centrifuge for 1 minute 10,000×g (15,000 rpm) to elute.


15) Pipet 10 μL of RNase-free Water directly onto the spin column membrane.


*Exchange the tip if there is a possibility of contamination.


16) With closed caps, centrifuge for 1 minute 10,000×g (15,000 rpm) to elute.


17) Determine the RNA concentration by measurement of O.D.


*4.3.3. Fragmentation of cRNA


1) Calculate the amount of cRNA and RNase-free water to adjust cRNA concentration to 20 μg/μL.


*In the case of whole blood sample, cRNA concentration is 10 μg/μL


*In TGP, in order to exclude the carry-over of total RNA, calculate the amount of cRNA according to the formula described below.





[amount of cRNA]=RNAm−totalRNAi*Y


RNAm: apparent amount of cRNA measured after IVT reaction


total RNAi1): amount of total RNA of starting sample






Y:[amount of cDNA solution used in IVT reaction]2)/[amount of cDNA solution]3)



1) 5 μg in TGP, 2) 10 μL in TGP, 3) 12 μL in TGP


2) Dispense RNase-free Water and cRNA calculated in step 1) into 1.5 mL microtube.


3) Mark the sample number on the lid of the tube.


*Exchange the tip if there is a possibility of contamination.


*In TGP, barcode labels were placed on the residual cRNA samples and stored at −80° C.


4) Add 8 μL of the 5× fragmentation buffer.


*Exchange the tip if there is a possibility of contamination.


5) Mix by tapping the tube and spin down briefly.


6) For the quantification of the cRNA (before reaction), dispense 1 μL of these reaction


mixtures into the 8-strip PCR reaction tubes for electrophoresis.


7) Incubate the tubes for 35 minutes at 94° C.


8) For the quantification of the cRNA (after reaction), dispense 1 μL of these fragmented


reaction mixtures into the 8-strip PCR reaction tubes for electrophoresis.


9) [QC] Fragmentation pattern of cRNA by electrophoresis on a denaturing agarose


*Generally do not store fragmented cRNA samples. If necessary, store at −80° C.


*4.3.4. Brief quality check of the cRNA and their fragmentation


1) Criterion of the quality check of the cRNA


CRNA electrophoresis pattern does not look smeared entirely, bands around 1k bp are strongly stained (Lane 1).


CRNA electrophoresis pattern does not look smeared entirely, bands around 1k bp are not strongly stained (Lane 3).


The size distribution of cRNA is under 1k bp (Lane 7). These samples are not suitable for the subsequent reactions, because they often result in showing high backgrounds.


2) Examination of the fragmented cRNA


*The size distribution of fragmented cRNA displays 35-200 bp, which is at the tip of gel electrophoresis (Lanes 2 and 4).


(5. Hybridization)


*5.1. Reagents and equipments


*5.1.1. Reagents
1) GeneChip Eukaryotic Hybridization Control kit (Affymetrix, cat. #900454 or #900457) 20× Eukaryotic Hybridization Control

Control Oligonucleotide B2


*Control Oligonucleotide B2 (150 μL) were dispended into three tubes and stored at −20° C.


2) MES-free acid monohydrate (Sigma, cat. #M5287, 250 g, stored at room temperature)


3) MES Sodium Salt (Sigma, cat. #M5057, 100 g, stored at room temperature)


4) 5M NaCl (Ambion, cat. #9760, 100 mL, stored at room temperature)


5) 0.5M EDTA (Sigma, cat. #E7889, 100 mL, stored at room temperature)


6) 10% Tween20 (Surfact-Amps 20, PIERCE, cat. #9005-64-5, 10 mL, stored at room temperature for up to 1 year)


7) 10 mg/mL Herring Sperm DNA (Promega, cat. #D1811, stored at −20° C. for up to 1 year or at 4° C. for up to 1 month)


8) 50 mg/mL Bovine Serum Albumin (Invitrogen, cat. #15561-020, 3 mL, stored at −20° C. for up to 1 year or at 4° C. for up to 1 month)


9) DEPC-Treated water (Ambion, cat. #9920, stored at room temperature))


10) 20×SSPE Buffer (Invitrogen, cat. #15591-043, 1 L, stored at room temperature)


*5.1.2. Equipments

1) 50 mL tube, DNase/RNase/pyrogen free


2) 15 mL tube, DNase/RNase/pyrogen free


3) 1.5 mL microtube, DNase/RNase/pyrogen free


4) Bottle Top Vacuum Filter (Iwaki, cat. #8024-045)
5) Storage Bottle (Iwaki, cat. #8930-001)

6) Tough-Spots (Toho, ½ inch diameter, white, T-SPOTS-50)


7) Gene Chip Hybridization Oven 640 (Affymetrix)
8) Cool Incubator (Mitsubishi Electronic Engineering, CN-25A)
*5.2. Preparation of Reagents

5.2.1. 12×MES stock Buffer (protect from light, stored at 4° C. for up to 3 months)


1) Mix and adjust volume to 1,000 mL. The pH should be between 6.5 and 6.7.


2) Filter through a 0.22 μM filter.


*This manipulation is for degassing and removal of dust rather than for sterilization.


*Do not autoclave MES buffer.


*If MES buffer turns yellow, do not use them.













TABLE 6









reagents
1000
mL



MES-free acid monohydrate
70.4
g



MES Sodium Salt
193.3
g



DEPC-Treated water
800
mL











*5.2.2. 2× Hybridization Buffer (protect from light, stored at 4° C. for up to 1 month) Final 1× concentration buffer is 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween-20.


1) Prepare in 50 mL tube, and mix by inverting.













TABLE 7









reagents
50
mL



12x MES Stock Buffer
8.3
mL



5M NaCl
17.7
mL



0.5M EDTA
4.0
mL



10% Tween20
0.1
mL



DEPC-Treated water
19.9
mL



Total
50
mL











*5.2.3. 1× Hybridization Buffer (protect from light, stored at 4° C. for up to 1 month)


1) Prepare in 50 mL tube, and mix by inverting.












TABLE 8









reagents
50 mL



2x Hybridization Buffer
25 mL



DEPC-Treated water
25 mL



Total
50 mL











*5.2.4. Wash A solution (Non-Stringent Wash Buffer, stored at room temperature for up to 1 month)


1) Prepare in beaker using measuring cylinder, and mix well by a stirrer.


2) Transfer the prepared Wash A solution to a clean dedicated bottle.


*This manipulation is for degassing and removal of dust rather than for sterilization.













TABLE 9









reagents
1000
mL



20x SSPE
300
mL



10% Tween-20
1
mL



DEPC-Treated water
699
mL



total
1000
mL










*4.1. Hybridization

1) Equilibrate GeneChip to room temperature 30 minutes before use to prevent dew condensation and cracking of the rubber septa.


*Check the GeneChip (glass surface scratch etc.)


2) Transfer 30 μL of each fragmented and labeled cRNA samples to the 1.5 mL microtubes.


3) Prepare sufficient Hybridization Mix in a 15 mL tube. See the following.












TABLE 10





reagents
one sample
24 samples
48 samples





















Control
5
μL
125
μL
245
μL


Oligonucleotide B2


20x Eukaryotic
15
μL
375
μL
735
μL


Hybrydization


Control (heated to


65° C. for 5 minutes)


10 mg/mL Herring
3
μL
75
μL
147
μL


Sperm DNA


50 mg/mL Bovine
3
μL
75
μL
147
μL


Serum Albumin


2x Hybridization
150
μL
3.75
mL
7.35
mL


Buffer


DMSO
30
μL
0.75
mL
1.47
mL


DEPC-Treated water
64
μL
1.6
mL
3.136
mL


total
270
μL
6.75
mL
13.23
mL










4) Add 270 μL of the Hybridization Mix to the fragmented and labeled cRNA samples. Mix by flicking the tube and spin down briefly.


*Exchange the tip if there is a possibility of contamination.


5) Incubate the tubes for 5 minutes at 99° C. with heat block.


6) Incubate the tubes for 5 minutes at 45° C. with heat block.


7) Centrifuge for 5 minutes at 10,000×g (15,000 rpm) at room temperature to collect any insoluble material from the hybridization cocktail.


8) While centrifuging the hybridization cocktail, wet the array with 200 μL of 1× Hybridization Mix.


*Vent the GeneChip through the septa in the top right by inserting a clean, unused RAININ pipette tip.


*Fill 1× Hybridization Mix (pre-hybridization buffer) through the septa in the bottom left with a clean, unused RAININ pipette tip.


9) Incubate the GeneChip for 10 minutes at 45° C. with a 60 rpm rotation using Hybridization Oven (pre-hybridization).


10) Check for a buffer leak from the GeneChip.


11) Vent the array with a clean, unused RAININ pipette tip and remove the 1× Hybridization Mix from the GeneChip with a clean, unused RAININ pipette tip.


12) Refill 200 μL of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube with a clean, unused RAININ pipette tip.


13) Place ½ inch Tough Spots on the septa to prevent leakage.


14) Store microtubes after taking out the supernatants (appx. 100 μL of hybridization cocktail remain) at −80° C.


*In TGP, place the sample ID barcode on the tube.


*Do not touch the glass surface of the GeneChip.


15) Place GeneChip into the Hybridization Oven and incubate for 18 hours at 45° C. with a 60 rpm rotation (hybridization).


*To avoid applying stress on the motor, load probe arrays in a balanced configuration around the axis.


16) After hybridization, remove the hybridization cocktail from the GeneChip with a clean, unused RAININ pipette tip.


*In TGP, this cocktail is stored at −80° C. as the hybridization cocktail for rehybridization, in addition to the remaining hybridization cocktail.


17) Fill the GeneChip completely with the appropriate volume of Wash Buffer A (appx. 260 μL)


*In TGP, place the GeneChip in a cool incubator until the next step.


(Standard Protocol 3)


(Quality Control (QC) and Final Data Inclusion to the Database)


All gene expression in Adjuvant Database Project passed quality control (QC). Data acquisition was performed in-house with GeneChip® Scanner 3000 7G (Affymetrix). The acquired data was further analyzed for the background signal, the corner signal, the number of the presence/absence calls, and the expression values of housekeeping genes. Intra- and inter-group reproducibilities were then checked by scatter plot analysis. X-axis and Y-axis indicate the gene expression value of each gene from two different samples of the same (intra-group) or different (inter-group) adjuvant treated mouse groups. Red dots indicate genes exhibiting high coefficient of correlation (CV).


(Representative QC Examples)


The following are 5 types of representative QC examples observed in this project.


1) Failed QC Examples with Unknown Reason (Re-Assayed with Spare Samples)(FIG. 34)


DMXAA_ID_SP_x1 (a-1) exhibited a severely distorted scatter plot for unknown reasons. sHz_ID_LV_x3 (a-2) and MBT_ID_LV_x3 (a-3) exhibited an unusually curved scatter plot for another sample treated with the same adjuvant from the same organ. Other (spare) tissue fragments, which were simultaneously sampled from the same sample but were stored separately as spares, were used for re-assay. If data from the spare fragment also exhibited a distorted scatter plot, the sample was excluded from subsequent analysis. In these three cases, re-assay data for DMXAA_ID_SP_x1, sHz_ID_LV_x3, and MBT_ID_LV_x3 passed QC and were used for subsequent analysis.


2) Failed QC Sample with Severe Contamination of Other Tissues (Excluded from Subsequent Analysis)


The scatter plot between ADX_ID_LN_c1 and ADX_ID_LN_c3 exhibited one sided placement. CV analysis revealed that the ADX_ID_LN_c1 sample was heavily contaminated with adipose tissue surrounding the inguinal lymph nodes. Even with a CV filter, adipose tissue derived genes could not be eliminated (indicated by black dots broadly distributed from the center line). Therefore, these samples were completely excluded from subsequent analysis to avoid the significant effect due to such contamination.


3) Passed QC with Unavoidable Weak Contamination by Other Tissues


ADX_IP_SP_c2 (c-1) and cdiGMP_ID_SP_c1 (c-2) exhibited contamination by pancreas on a normal scatter plot in spleen (e-2). Since the pancreas and the spleen are proximately located, it was technically challenging to completely remove pancreatic tissues from the spleen even with careful cleaning. For this reason, a CV filter was applied to remove the contamination derived genes (generally exhibiting high CV) by a data processing method.


4) Passed QC with Different Magnitude of Host Responses


While samples exhibiting a broad distribution (d-1, 2) was able to be detected due to individual differences among mice, this reflects the difference in the magnitude of host responses after adjuvant administration.


5) Passed QC with No Problem


(Synthetic Virtual Control)


To obtain at least three control samples for statistical analysis on the samples in experiment 3, “03PBS_LN_c1” was replaced with synthetic virtual control “03PBS_LN_c1v”. The synthetic virtual control “03PBS_LN_c1v” was created using the average of individual gene expression values among pooled control samples from a total of 10 experiments. For each probe set, average values were calculated after removing probes with the highest and lowest values from the pooled control samples.


(Sample Exclusion Based on Further Evaluation of Post QC Dataset)


Two other samples K3SPG_IP_LV_x2 and K3SPG_IP_SP_x2 (both samples from the #2 mouse in Experiment 2) were removed for exhibiting gene responses that were too strong compared to other two mice (not shown). For ADX_ID_LN, K3SPG_IP_LV, and K3SPG_IP_SP, only two treated samples were used for subsequent analysis. Other samples were analyzed with three samples.


(CV Filtering)


During QC analysis on GeneChip data, some samples contained weak but a substantial level of contamination from genes derived from other tissues (for example, red in the above QC scatter plots). It was technically difficult to completely remove the microcontamination (QC example 3) by sampling organs after careful cleaning. Therefore, a coefficient of variation (CV) filter was developed to reduce microcontamination derived genes from the target organ gene analysis. Each gene probe's baseline variations were calculated using GeneChip data from a total of 33 buffer injected control mice (not shown). The origin of high CV gene probes in each organ was further analyzed by utilizing the Gene Expression Barcode 3.0 (http://barcode.luhs.org/) database. Analysis with the Barcode 3.0 revealed that these high CV gene probes were mainly from the neuronal tissue in LV (not shown), pancreas tissue in SP (not shown), and adipose tissues in LN (not shown). To remove the effect of these microcontaminations, the data was filtered with CV value <1, and the filtered data (43200 out of 45037 probes) were used for further analysis.


(Administration of Adjuvant and Sampling)


Detailed information on adjuvants used in this Example is further described. C57BL/6 mice (male, 5-week old, C57BL/6JJc1) were purchased from CLEA Japan and acclimated for at least one week. Each adjuvant was prepared as described in standard procedure 1. The dose/volume (Table 11) indicated for each adjuvant was administered (intradermally; i.d.) mainly to the base of the tail of the mice (n=3 for each group). Five types of adjuvants (ADX, ALM, bCD, K3, and K3SPG) were i.p. administered. ENDCN was i.n. (intranasally) administered. Since a genetic profile with very large variability and low reproducibility was obtained in a preliminary experiment from intramuscular administration, this Example did not use intramuscular administration. However, intramuscular injection can also be used if conditions are adjusted. The dose of each adjuvant was selected to induce excellent adjuvant function without severe reactogenicity in the mice based on the inventors' experiment (ALM, K3 and K3SPG, bCD, cdiGMP and cGAMP, D35, DMXAA, FK565, sHZ), previous report (AddaVax, ADX, ENDCN, PolyIC, Pam3CSK4, MPLA, MALP2s, R848), or the following common protocol (1:1 mixture of FCA and ISA51VG) (details described below).


While the selected dose of MBT was the same as the dose of FK565, experiment was not conducted in this regard. This was determined based on preliminary data (data not shown) for FK565 and other NOD ligands. Among the total of 21 different types of adjuvants, 3 to 5 different types of adjuvants were used in one experiment, and a suitable control buffer group was used (for most adjuvants, PBS; for ENDCN, Tris-HCl; and for DMXAA, MALP2s, MPLA, and R848, 5% DM standard procedure BS). In this study, a total of 10 independent experiments were conducted (see standard procedure 1, e.g., Table 11).





















TABLE 11






Test substance

Time
Dose

Dose volume
# of
Group






Exp. No.
(abbreviation)
Route
(hrs)
(/body)
Conc.
(/body)
mice
No.
LV
SP
LN
Kit






























Exp. 1
PBS
i.p.
6
0
mg
0
mg/mL
0.1
mL
3
1



E



ADX
i.p.
6
1
mg
10
mg/mL
0.1
mL
3
2



E


Exp. 2
PBS
i.p.
6
0
mg
0
mg/mL
0.1
mL
3
1



E



ALM
i.p.
6
1
mg
10
mg/mL
0.1
mL
3
2



E



K3
i.p.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
3



E



K3SPG
i.p.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
4
✓*1
✓*1

E


Exp. 3
PBS
i.d.
6
0
mg
0
mg/mL
0.1
mL
3
1


✓*2
E



ADX
i.d.
6
1
mg
10
mg/mL
0.1
mL
3
2


✓*3
E



ALM
i.d.
6
1
mg
10
mg/mL
0.1
mL
3
3



E



K3
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
4



E





















Exp. 4
PBS
i.p.
6


 0%
0.2
mL
3
1



E



bCD
i.p.
6


30%
0.2
mL
3
2



E



Tris-HCl
i.n.
6


 0%
0.005
mL
3
3



E



ENDCN
i.n.
6


 2%
0.005
mL
3
4



E






















Exp. 5*4
PBS
i.d.
6
0
mg
0
mg/mL
0.1
mL
3
1



E



D35
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
2



E



K3SPG
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
3



E






















bCD
i.d.
6


30%
0.2
mL
3
4



E


Exp. 6
PBS
i.d.
6


 0%
0.1
mL
3
1



E



FCA
i.d.
6


50%
0.1
mL
3
4



E






















Exp. 7
PBS
i.d.
6
0
mg
0
mg/mL
0.1
mL
3
1



P



sHZ
i.d.
6
0.2
mg
2
mg/mL
0.1
mL
3
2



P



FK565
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
3



P



MBT
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
4



P


Exp. 8
PBS
i.d.
6
0
mg
0
mg/mL
0.1
mL
3
1



P



PolyIC
i.d.
6
0.1
mg
1
mg/mL
0.1
mL
3
2



P



Pam3CSK4
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
3



P



cdiGMP
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
4



P





















Exp. 9
PBS
i.d.
6


0
0.1
mL
3
1



P



Addavax
i.d.
6


50%
0.1
mL
3
2



P



ISA51VG
i.d.
6


50%
0.1
mL
3
3



P























cGAMP
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
4



P


Exp. 10*4
PBS DMSO5%
i.d.
6
0
mg
0
mg/mL
0.1
mL
3
1



P



MPLA
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
2



P



MALP2s
i.d.
6
0.01
mg
0.1
mg/mL
0.1
mL
3
3



P



R848
i.d.
6
0.1
mg
1
mg/mL
0.1
mL
3
4



P



DMXAA
i.d.
6
0.1
mg
1
mg/mL
0.1
mL
3
5



P









In a preliminary experiment, FCA and ALM were tested at different points up to 72 hours. In the tested dose range, changes in gene expression reached a peak at the 6 hour mark in many cases, and majority of changes subsided at the 24 hours mark. With a preliminary experiment, changes in gene expression in an organ was studied after adjuvant administration, consistently at 6 hours after administration, revealing that the changes generally returned to a normal level in mice and rats after 24 hours. For this reason, the 6 hours mark after administration was chosen for sampling. At 6 hours after adjuvant administration, LV, SP, and LN (both sides) were removed to study the gene expression thereof by using Affimetrix Genechip microarray system (Affymetrix). The sampled organs were each placed in a tube containing RNAlater. The tube was maintained at 4° C. overnight, and stored at −80° C. until use. Blood was also collected after 6 hours from adjuvant administration for general hematological testing. About 200 μL of blood was taken from the retro-orbital venous plexus with a non-heparinized capillary tube and placed into a 1.5 mL tube comprising 2 μL of 10% EDTA-2K for hematological testing. The hematological cell count was found using VetScan HMII (Abaxis). 50 μl of EDTA-2K blood sample was diluted by adding 250 μl of saline. Measurements were taken with VetScan HMII by following the instruction.


(Data Acquisition and Quality Control)


Microarray data was acquired in accordance with Igarashi et al (Igarashi, Y. et al. Open TG-GATEs: a large-scale toxicogenomics database. Nucleic acids research 43, D921-927 (2015)). Briefly, RLT buffer (Qiagen GmbH., Germany) was added to a tissue block stored in RNAlater, and homogenized by shaking with 5 mm diameter zirconium beads (ASONE Corporation, Japan) for 5 minutes at 20 Hz in a Mixer Mill 300 (Qiagen, Germany). Total RNA was isolated from LV, SP, and LN by using Trizol LS (Life Technologies, CA) and RNeasy Mini Kit (Qiagen) in accordance with the instruction of the manufacturer. GeneChip® 3′IVT Express Reagent Kit or GeneChip® 3′IVT PLUS Reagent Kit was used to prepare a sample. The gene expression profile was determined using GeneChip® Mouse Genome 2.0 Array (Affymetrix, CA) in accordance with the instruction of the manufacturer. The washing step and the staining step were carried out using GeneChip® Hybridization, Wash, and Stain kit on a fluidics station 450 (Affymetrix). The GeneChip® array was scanned with GeneChip® Scanner 3000 7G (Affymetrix). The resulting digital image files (DAT file) were converted to CEL files using Affymetrix® GeneChip® Command Console® software (standard procedure 2).


Ultimately, 330 (99 controls and 231 treatment groups) GeneChip data files and hematological parameters were obtained from 10 separately conducted experiments (Table 11). Notably, one PBS control and one ADX_ID-treated LN sample in Experiment 3 had to be removed because of the large amount of adipose tissue-derived gene expression (see standard procedure 3). Consequently, the ADX_ID-treated LN sample data was excluded and the PBS control sample was replaced with the synthetic virtual control data (standard procedure 3) for further analysis on the Experiment 3 dataset. Furthermore, the K3SPG_IP-treated #2 mouse-derived LV and SP samples were excluded from the analysis, because i.p. K3-SPG injection was not performed properly (standard procedure 3). Any gene probes that exhibited high coefficients of variation in the control buffer-treated mice was also excluded to reduce the overall experimental condition-dependent factors including microcontamination of non-target tissue (standard procedure 3).


(Determination of the Presence or Absence of Gene Expression (Customized PA Call))


The presence or absence (PA) call in MAS5.0 was customized as follows. The normalized MAS5.0 expression data from each of the two groups of three mice that were treated with a single adjuvant or its appropriate vehicle control were averaged, and the average expression ratio for each group was calculated. In this process, the MAS5.0 PA calls (customized PA call) were integrated as follows. When the PA calls from the three control samples were [“P”, “P”, and “A” ], “P” was chosen as their dominant PA calls (over half were “P”). The same strategy was applied to the three adjuvant-treated samples. When the expression ratio of each gene was >1.0, the customized PA call was determined by the dominant call of the treated group. When the expression ratio was <1.0, the call was determined by the dominant call of the vehicle-control group. The resultant customized PA call was processed as “P” is “1”, and “A” is “0”, i.e., when the ratio is greater than 1 and the PA calls of the treated samples were “P,” “P,” and “A,” the customized PA call of this gene set was “1”. In the case of a two sample analysis (ADX_ID_LN, K3SPG_IP_LV, and K3SPG_IP_SP), [“P”, “A” ] was processed as “A”.


(Significantly Differentially Expressed Genes (Genes with a Significant Change in Expression=sDEGs or Significant SEGs))


sDEGs were defined as statistically significant changes (up- or down-regulation) satisfying all of the following conditions: an average fold change (FC) of >1.5 or <0.667, an associated t-test p-value of <0.01 with no multiple test corrections, and a customized PA call of 1.


(Biological Themes Enrichment Analysis with TargetMine)


A defined sets of genes were selected according to specified criteria (e.g., FC, PA-call, threshold values). Their functional enrichment values were then obtained from TargetMine (Chen, Y. A., Tripathi, L. P. & Mizuguchi, K. PloS one 6, e17844 (2011)) (http://targetmine.mizuguchilab.org/) using the API interface. The following resources were used throughout the analysis via TargetMine Interface (GO, GOSlim, Integrated Pathway, KEGG, Reactome, and the NCI pathway). The Holm-Bonferroni method was used for multiple testing corrections.


(Biological Annotation Analysis of Individual Adjuvants Using Whole Organ Transcriptomics (Table 12))


















TABLE 12











(10)R848.
(10)R848.
(10)R848.
(8)Poly_IC.
(8)Poly_IC.
(8)Poly_IC.


Group
Probe Index
Gene Symbol
Modules
ID.LV.x3
ID.LV.x1
ID.LV.x2
ID.LV.x3
ID.LV.x1
ID.LV.x2





G1
1449025_at
Ifit3
20
1.49
2.19
1.76
1.95
3.24
3.25


G1
1456890_at
Ddx58
20
0.95
2.42
1.34
1.87
3.02
2.77


G1
1450783_at
Ifit1
20
0.63
1.54
1.14
1.28
2.54
2.35


G1
1450034_at
Stat1
20
1.52
2.37
1.92
1.94
2.89
3.04


G1
1436562_at
Ddx58
20
1.6
2.64
2.33
1.64
2.92
2.82


G1
1437503_a_at
Shisa5
20
2.49
3.09
2.43
1.52
2.49
2.66


G1
1418825_at
Irgm1
20
2.02
2.87
2.12
1.84
2.78
2.84


G1
1426906_at
Mndal
20
1.21
1.65
1.42
2.37
3.14
2.4


G1
1450033_a_at
Stat1
20
1.05
1.4
1.2
1.92
2.73
2.67


G1
1436058_at
Rsad2
20
0.67
1.97
1.77
1.97
3
3.3


G1
1423986_a_at
Shisa5
20
2.23
2.93
2.32
1.61
2.5
2.99


G1
1434268_at
Adar
20
1
2.77
1.8
1.28
2.82
2.17


G1
1425405_a_at
Adar
20
0.96
1.63
1.17
1.58
3.45
2.75


G1
1435526_at
Tor1aip2
20
1.22
3.12
2.18
1.58
2.88
2.62


G1
1431591_s_at
Gm9706///Isg15
20
1.95
2.55
2.1
2.13
3.28
3.62


G1
1455500_at
Rnf213
20
0.72
1.29
1.03
1.47
2.64
3.01


G1
1418115_s_at
Tor1aip2
20
0.54
1.53
1.08
1.13
2.38
2.68


G1
1418116_at
Tor1aip2
20
0.89
1.45
1.22
0.96
2.73
2.94


G1
1426970_a_at
Uba7
20
2.27
2.64
2.42
1.71
3
3.13


G1
1421323_a_at
G3bp2
20
1.04
2.13
1.21
1.31
2.83
2.85


G1
1422005_at
Eif2ak2
20
1.09
2.43
1.56
1.83
2.89
2.87


G1
1448757_at
Pml
20
0.66
1.77
1.53
2
3.58
3.3


G1
1417244_a_at
Irf7
20
2.37
2.27
2.39
2.38
3.53
3.38


G1
1426276_at
Ifih1
20
2.36
3.25
2.46
2.12
3.06
3.34


G1
1449875_s_at
H2-T10///H2-
20
1.77
2.05
2.01
2.21
2.89
3.28




T22///H2-T9


G1
1437432_a_at
Trim12a
20
1.68
2.13
1.94
2.9
3.39
3.59


G1
1456103_at
Pml
20
1.29
3.66
1.62
2.12
3.26
2.5


G1
1426716_at
Tdrd7
20
0.94
1.79
1.21
1.44
3.28
3.24



















(10)DMXAA.
(10)DMXAA.
(10)DMXAA.
(9)cGAMP.
(9)cGAMP.
(9)cGAMP.
(8)cdiGMP.
(8)cdiGMP.


Group
ID.LV.x3
ID.LV.x1
ID.LV.x2
ID.LV.x3
ID.LV.x1
ID.LV.x2
ID.LV.x1
ID.LV.x2





G1
0.74
1.39
1.3
2.06
2.13
1.95
1.7
1.75


G1
1.56
1.67
1.93
2.38
2.26
2.28
1.2
1.45


G1
1.04
2.21
1.54
2.63
2.75
2.63
2.21
2.28


G1
1.16
1.53
1.96
2.26
2.46
1.97
0.88
1.02


G1
1.31
2.07
1.54
2.6
2.04
2.13
0.94
0.88


G1
1.72
2.12
1.62
2.09
1.57
1.63
1.05
1.12


G1
0.79
1.39
1.33
2.14
2.25
2.09
1.33
1.29


G1
0.73
1.42
1.19
2.19
2.08
2.11
1.77
2.45


G1
1.15
1.5
1.73
2.85
3.11
2.52
1.49
1.44


G1
0.44
1.56
1.25
1.75
2.08
1.89
2.39
2.41


G1
1.42
2.03
1.81
2.36
1.7
1.43
0.72
1.07


G1
0.92
2.15
1.52
1.8
2.22
1.71
1.7
1.83


G1
0.93
2.03
1.5
2.37
2.95
2.38
1.58
1.67


G1
1.01
2.23
1.48
2.32
2.32
2.28
0.94
0.83


G1
0.8
1.28
1.39
1.73
2.13
1.62
1.01
0.94


G1
0.97
1.79
1.2
2.18
2.58
2.34
2.07
2.08


G1
0.98
2.45
1.52
2.71
2.9
2.68
1.81
1.67


G1
1.37
2.19
1.92
2.68
2.41
2.58
1.58
1.33


G1
1.21
1.81
1.59
1.91
2.06
1.28
0.53
1.46


G1
0.71
1.32
1.22
2.43
3.19
2.6
1.74
1.5


G1
0.68
1.36
1.3
2.73
3.11
2.21
1.02
1.38


G1
0.83
1.62
1.35
2.49
2.13
2.02
1.41
1.57


G1
1
1.05
1.18
1.62
1.6
1.62
0.85
1.21


G1
0.78
1.25
1.09
1.66
1.73
1.62
0.89
1.09


G1
0.51
1.27
1.64
2.45
2.29
1.8
1.07
1.04


G1
1.11
1.82
1.56
1.61
2.07
1.46
0.73
0.83


G1
0.34
2.18
1.34
1.62
1.62
1.48
1.31
1.3


G1
1.09
1.6
1.74
2.5
2.18
2.34
1.47
1.15



















(8)cdiGMP.
(9)AddaVax.
(9)AddaVax.
(9)AddaVax.
(9)ISA51VG.
(9)ISA51VG.



Group
ID.LV.x3
ID.LVx2
ID.LVx1
ID.LVx3
ID.LV.x1
ID.LV.x2







G1
2.34
−0.5
−0.5
−0.5
−0.5
−0.5



G1
2.07
−0.5
−0.7
−0.6
−0.6
−0.6



G1
2.4
−0.5
−0.5
−0.5
−0.5
−0.5



G1
2.07
−0.5
−0.6
−0.6
−0.6
−0.6



G1
1.49
−0.5
−0.6
−0.4
−0.5
−0.6



G1
1.32
−0.6
−0.7
−0.7
−0.6
−0.8



G1
1.81
−0.5
−0.6
−0.6
−0.6
−0.7



G1
2.76
−0.4
−0.6
−0.6
−0.5
−0.7



G1
2.11
−0.5
−0.6
−0.6
−0.6
−0.6



G1
2.39
−0.5
−0.5
−0.5
−0.5
−0.5



G1
1.57
−0.2
−0.7
−0.6
−0.5
−0.6



G1
2.95
−0.3
−0.7
−0.7
−0.8
−0.9



G1
1.66
−0.3
−0.4
−0.6
−0.5
−0.5



G1
1.54
−0.4
−0.4
−0.3
−0.4
−0.5



G1
2.02
−0.5
−0.5
−0.5
−0.5
−0.5



G1
3.16
−0.5
−0.6
−0.6
−0.6
−0.6



G1
2.42
−0.5
−0.9
−0.6
−0.7
−0.8



G1
2.19
−0.4
−0.7
−0.6
−0.5
−0.8



G1
1.41
−0.5
−0.6
−0.5
−0.5
−0.4



G1
2.34
−0.5
−0.6
−0.6
−0.6
−0.5



G1
1.93
−0.4
−0.6
−0.6
−0.6
−0.6



G1
2.16
−0.4
−0.5
−0.5
−0.3
−0.5



G1
1.92
−0.5
−0.5
−0.6
−0.5
−0.7



G1
1.65
−0.5
−0.5
−0.4
−0.5
−0.6



G1
2.06
−0.5
−0.6
−0.6
−0.5
−0.6



G1
1.46
−0.5
−0.5
−0.5
−0.5
−0.6



G1
2.58
−0.6
−0.6
−0.5
−0.5
−0.6



G1
2.22
−0.3
−0.5
−0.5
−0.4
−0.5

























(9)ISA51VG.
(3)K3.
(3)ADX.
(3)K3.
(3)ALM.
(3)ALM.
(3)ALM.


Group
Probe Index
Gene Symbol
Modules
ID.LVx3
ID.LV.x1
ID.LV.x1
ID.LV.x2
ID.LV.x2
ID.LV.x1
ID.LV.x3





G1
1449025_at
Ifit3
20
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1456890_at
Ddx58
20
−0.6
−0.4
−0.5
−0.4
−0.4
−0.3
−0.5


G1
1450783_at
Ifit1
20
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1450034_at
Stat1
20
−0.6
−0.4
−0.5
−0.5
−0.5
−0.5
−0.6


G1
1436562_at
Ddx58
20
−0.5
−0.3
−0.6
−0.4
−0.3
−0.6
−0.3


G1
1437503_a_at
Shisa5
20
−0.7
−0.5
−0.5
−0.3
−0.5
−0.6
−0.3


G1
1418825_at
Irgm1
20
−0.6
−0.5
−0.6
−0.6
−0.6
−0.5
−0.5


G1
1426906_at
Mndal
20
−0.6
−0.3
−0.3
−0.3
−0.4
−0.4
−0.3


G1
1450033_a_at
Stat1
20
−0.6
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1436058_at
Rsad2
20
−0.5
−0.5
−0.5
−0.5
−0.4
−0.4
−0.5


G1
1423986_a_at
Shisa5
20
−0.6
−0.5
−0.6
−0.5
−0.6
−0.5
−0.5


G1
1434268_at
Adar
20
−0.7
−0.6
−0.6
−0.5
−0.6
−0.5
−0.6


G1
1425405_a_at
Adar
20
−0.6
−0.3
−0.4
−0.7
−0.5
−0.5
−0.4


G1
1435526_at
Tor1aip2
20
−0.4
−0.4
−0.3
−0.5
−0.5
−0.4
−0.5


G1
1431591_s_at
Gm9706///Isg15
20
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1455500_at
Rnf213
20
−0.6
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1418115_s_at
Tor1aip2
20
−0.5
−0.3
−0.4
−0.3
−0.4
−0.3
−0.4


G1
1418116_at
Tor1aip2
20
−0.5
−0.4
−0.3
−0.5
−0.5
−0.5
−0.6


G1
1426970_a_at
Uba7
20
−0.3
−0.2
−0.6
−0.3
−0.6
−0.5
−0.6


G1
1421323_a_at
G3bp2
20
−0.6
−0.4
−0.4
−0.5
−0.3
−0.6
−0.6


G1
1422005_at
Eif2ak2
20
−0.6
−0.5
−0.6
−0.5
−0.6
−0.5
−0.4


G1
1448757_at
Pml
20
−0.4
−0.4
−0.5
−0.6
−0.2
−0.4
−0.4


G1
1417244_a_at
Irf7
20
−0.6
−0.4
−0.5
−0.4
−0.4
−0.4
−0.5


G1
1426276_at
Ifih1
20
−0.5
−0.4
−0.5
−0.4
−0.5
−0.5
−0.5


G1
1449875_s_at
H2-T10///H2-
20
−0.4
−0.3
−0.4
−0.5
−0.6
−0.6
−0.5




T22///H2-T9


G1
1437432_a_at
Trim12a
20
−0.5
−0.6
−0.4
−0.5
−0.4
−0.5
−0.3


G1
1456103_at
Pml
20
−0.5
−0.5
−0.4
−0.6
−0.4
−0.4
−0.5


G1
1426716_at
Tdrd7
20
−0.5
−0.3
−0.2
−0.6
−0.4
−0.6
−0.4


















(10)MPLA.
(10)MPLA.
(10)MPLA.
(2)K3.
(2)K3.
(2)K3SPG.
(2)K3.


Group
ID.LV.x2
ID.LV.x1
ID.LV.x3
IP.LV.x2
IP.LV.x3
IP.LV.x3
IP.LV.x1





G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.1
−0.2
−0.4
−0.3
−0.4
−0.4
−0.4


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.5
−0.4
−0.5
−0.6
−0.6
−0.6
−0.5


G1
−0.4
−0.3
−0.5
−0.2
−0.4
−0.3
−0.5


G1
−0.3
−0.3
−0.4
−0.4
−0.6
−0.5
−0.5


G1
−0.5
−0.4
−0.6
−0.5
−0.6
−0.6
−0.5


G1
−0.4
−0.2
−0.5
−0.4
−0.4
−0.5
−0.5


G1
−0.5
−0.5
−0.6
−0.5
−0.5
−0.6
−0.6


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.4
−0.2
−0.3
−0.5
−0.8
−0.6
−0.6


G1
−0.3
−0
−0.3
−0.8
−0.8
−0.7
−0.6


G1
−0.4
−0.2
−0.6
−0.5
−0.2
−0.5
−0.5


G1
−0.1
−0.2
−0.5
−0.2
−0.5
−0.5
−0.3


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.5
−0.4
−0.5
−0.4
−0.5
−0.6
−0.5


G1
−0.4
−0.4
−0.6
−0.2
−0.2
−0.1
−0.5


G1
−0.3
−0
−0.2
−0
−0.2
−0.1
−0.4


G1
−0.2
−0.1
−0.6
−0.4
−0.3
−0.6
−0.5


G1
−0.6
−0.3
−0.5
−0.4
−0.4
−0.4
−0.6


G1
−0.4
−0.3
−0.4
−0.7
−0.5
−0.6
−0.6


G1
−0.3
−0.4
−0.4
−0.3
−0.6
−0.4
−0.5


G1
−0.5
−0.4
−0.5
−0.6
−0.5
−0.6
−0.5


G1
−0.4
−0.4
−0.4
−0.3
−0.5
−0.4
−0.4


G1
−0.3
−0.3
−0.4
−0.2
−0.4
−0.3
−0.2


G1
−0.6
−0.4
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.4
−0.3
−0.3
−0.3
−0.6
−0.4
−0.5


G1
−0.3
−0.3
−0.4
−0.1
−0.3
−0.2
−0.2



















(2)K35SPG.
(7)sHz.
(7)MBT.
(7)sHz.
(7)sHz.
(7)MBT.



Group
IP.LV.x1
ID.LV.x2
ID.LV.x2
ID.LV.x3
ID.LV.x1
ID.LV.x1







G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.3
−0.5
−0.6
−0.8
−0.3
−0.4



G1
−0.4
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.4
−0.5
−0.5
−0.6
−0.6
−0.6



G1
0.09
−0.5
−0.6
−0.7
−0.7
−0.6



G1
−0.2
−0.5
−0.5
−0.6
−0.6
−0.7



G1
−0.4
−0.5
−0.5
−0.6
−0.6
−0.6



G1
−0.4
−0.4
−0.4
−0.4
−0.4
−0.6



G1
−0.4
−0.5
−0.4
−0.5
−0.6
−0.5



G1
−0.4
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.3
−0.3
−0.4
−0.5
−0.5
−0.6



G1
−0.3
−0.5
−0.5
−0.7
−0.6
−0.9



G1
−0.2
−0.4
−0.2
−0.5
−0.5
−0.6



G1
−0.3
−0.3
−0.8
−0.8
−0.4
−0.8



G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.3
−0.5
−0.4
−0.5
−0.6
−0.6



G1
−0.2
−0.3
−0.5
−0.7
−0.6
−0.8



G1
0.11
−0.3
−0.5
−0.9
−0.7
−0.9



G1
−0.3
−0.7
−0.5
−0.4
−0.6
−0.7



G1
−0.6
−0.3
−0.6
−0.7
−0.7
−0.9



G1
−0.5
−0.3
−0.6
−0.7
−0.5
−0.5



G1
−0.3
−0.4
−0.4
−0.2
−0.5
−0.6



G1
−0.3
−0.5
−0.3
−0.4
−0.4
−0.4



G1
−0.3
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.1
−0.3
−0.4
−0.6
−0.4
−0.6



G1
−0.4
−0.4
−0.4
−0.4
−0.5
−0.6



G1
−0.4
−0.4
−0.5
−0.4
−0.5
−0.5



G1
0.12
−0.2
−0.3
−0.4
−0.3
−0.7
























(7)MBT.
(4)bCD.
(4)bCD.
(4)bCD.
(5)K3SPG.
(5)K3SPG.


Group
Probe Index
Gene Symbol
Modules
ID.LV.x3
IP.LV.x1
IP.LV.x2
IP.LV.x3
ID.LV.x1
ID.LV.x2





G1
1449025_at
Ifit3
20
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1456890_at
Ddx58
20
−0.5
−0.6
−0.4
−0.3
−0.6
−0.4


G1
1450783_at
Ifit1
20
−0.4
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1450034_at
Stat1
20
−0.5
−0.5
−0.5
−0.5
−0.6
−0.6


G1
1436562_at
Ddx58
20
−0.4
−0.4
−0.3
−0.1
−0.4
−0.5


G1
1437503_a_at
Shisa5
20
−0.6
−0.4
−0.3
−0.3
−0.3
−0.4


G1
1418825_at
Irgm1
20
−0.5
−0.5
−0.6
−0.5
−0.6
−0.6


G1
1426906_at
Mndal
20
−0.4
−0.3
−0.3
−0.5
−0.5
−0.5


G1
1450033_a_at
Stat1
20
−0.4
−0.5
−0.6
−0.5
−0.5
−0.5


G1
1436058_at
Rsad2
20
−0.5
−0.5
−0.4
−0.4
−0.5
−0.5


G1
1423986_a_at
Shisa5
20
−0.6
−0.6
−0.3
−0.3
−0.7
−0.7


G1
1434268_at
Adar
20
−0.3
−0.3
−0.4
−0.4
−0.5
−0.6


G1
1425405_a_at
Adar
20
−0.4
−0.5
−0.4
−0.3
−0.3
−0.3


G1
1435526_at
Tor1aip2
20
−0.6
−0.4
−0.3
−0.6
−0.4
−0.6


G1
1431591_s_at
Gm9706///Isg15
20
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1455500_at
Rnf213
20
−0.4
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1418115_s_at
Tor1aip2
20
−0.5
−0.4
−0.5
−0.4
−0.5
−0.5


G1
1418116_at
Tor1aip2
20
−0.6
−0.3
−0.3
−0.5
−0.4
−0.6


G1
1426970_a_at
Uba7
20
−0.6
−0.5
−0.5
−0.4
−0.6
−0.8


G1
1421323_a_at
G3bp2
20
−0.5
−0.4
−0.3
−0.6
−0.4
−0.8


G1
1422005_at
Eif2ak2
20
−0.3
−0.5
−0.4
−0.5
−0.6
−0.6


G1
1448757_at
Pml
20
−0.4
−0.3
−0.3
−0.4
−0.5
−0.6


G1
1417244_a_at
Irf7
20
−0.4
−0.6
−0.7
−0.5
−0.5
−0.4


G1
1426276_at
Ifih1
20
−0.5
−0.4
−0.5
−0.4
−0.5
−0.6


G1
1449875_s_at
H2-T10///H2-
20
−0.4
−0.3
−0.4
−0.3
−0.3
−0.6




T22///H2-T9


G1
1437432_a_at
Trim12a
20
−0.4
−0.5
−0.6
−0.6
−0.5
−0.4


G1
1456103_at
Pml
20
−0.4
−0.5
−0.4
−0.6
−0.3
−0.5


G1
1426716_at
Tdrd7
20
−0.2
−0.3
−0.1
−0.3
−0.4
−0.3


















(5)K3SPG.
(5)D35.
(5)D35.
(5)D35.
(2)ALM.
(2)ALM.
(2)ALM.


Group
ID.LV.x3
ID.LV.x1
ID.LV.x3
ID.LV.x2
IP.LV.x2
IP.LV.x1
IP.LV.x3





G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.4
−0.3
−0.4
−0.3
−0.6
−0.6
−0.8


G1
−0.5
−0.5
−0.4
−0.4
−0.5
−0.5
−0.5


G1
−0.6
−0.5
−0.5
−0.5
−0.6
−0.6
−0.6


G1
−0.5
−0.4
−0.4
−0.1
−0.6
−0.6
−0.7


G1
−0.4
−0.4
−0.4
0.15
−0.8
−0.8
−1.1


G1
−0.6
−0.5
−0.5
−0.3
−0.6
−0.6
−0.6


G1
−0.6
−0.5
−0.4
−0.3
−0.6
−0.7
−0.7


G1
−0.6
−0.5
−0.5
−0.4
−0.5
−0.6
−0.6


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.4
−0.5
−0.6
−0
−0.8
−0.8
−0.9


G1
−0.4
−0.3
−0.4
−0.3
−0.5
−0.6
−0.7


G1
−0.5
−0.1
−0.3
−0.3
−0.6
−0.7
−0.9


G1
−0.6
−0.4
−0.5
−0.4
−0.7
−0.6
−0.7


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5


G1
−0.5
−0.4
−0.4
−0.4
−0.6
−0.6
−0.7


G1
−0.7
−0.4
−0.6
−0.1
−0.5
−0.6
−0.6


G1
−0.6
−0.4
−0.5
−0.2
−0.5
−0.6
−0.7


G1
−0.6
−0.8
−0.6
−0.5
−0.4
−0.7
−0.8


G1
−0.8
−0.5
−0.4
−0.4
−0.3
−0.3
−0.6


G1
−0.6
−0.5
−0.4
−0.4
−0.6
−0.6
−0.6


G1
−0.5
−0.7
−0.4
−0.3
−0.7
−0.6
−0.8


G1
−0.3
−0.3
−0.2
−0.3
−0.7
−0.7
−0.6


G1
−0.5
−0.5
−0.5
−0.3
−0.5
−0.5
−0.6


G1
−0.6
−0.4
−0.3
−0.2
−0.8
−0.8
−0.8


G1
−0.6
−0.4
−0.5
−0.4
−0.5
−0.5
−0.6


G1
−0.3
−0.4
−0.3
−0.3
−0.6
−0.7
−0.7


G1
−0.2
−0.4
−0.4
−0.1
−0.9
−0.8
−0.9




















(5)bCD.
(6)FCA.
(4)ENDCN.
(5)bCD.
(5)bCD.
(4)ENDCN.
(4)ENDCN.



Group
ID.LV.x3
ID.LV.x3
IN.LV.x2
ID.LV.x2
ID.LV.x1
IN.LV.x1
IN.LV.x3







G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.6
−0.8
−0.5
−0.7
−0.7
−0.4
−0.7



G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.5
−0.5
−0.6
−0.6
−0.6
−0.5
−0.6



G1
−0.7
−0.8
−0.6
−0.7
−0.7
−0.6
−0.4



G1
−0.4
−0.7
−0.4
−0.7
−0.5
−0.5
−0.5



G1
−0.5
−0.5
−0.6
−0.7
−0.6
−0.6
−0.6



G1
−0.5
−0.5
−0.6
−0.6
−0.6
−0.4
−0.4



G1
−0.6
−0.5
−0.5
−0.6
−0.6
−0.5
−0.5



G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.6
−0.7
−0.3
−0.7
−0.8
−0.3
−0.4



G1
−0.1
−0.5
−0.7
−0.5
−0.3
−0.7
−0.9



G1
−0.4
−0.7
−0.8
−0.6
−0.7
−0.5
−0.4



G1
−0.4
−0.3
−0.2
−0.7
−0.4
−0.1
−0.4



G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5
−0.5



G1
−0.5
−0.5
−0.6
−0.6
−0.6
−0.5
−0.6



G1
−0.2
−0.5
−0.5
−0.7
−0.6
−0.3
−0.4



G1
−0.4
−0.5
−0.6
−0.8
−0.6
−0
−0.3



G1
−0.6
−0.5
−0.6
−0.7
−0.6
−0.5
−0.5



G1
−0.3
−0.2
−0.3
−0.4
−0.3
−0.4
−0.4



G1
−0.4
−0.5
−0.6
−0.6
−0.5
−0.5
−0.5



G1
−0.6
−0.4
−0.7
−0.7
−0.7
−0.6
−0.4



G1
−0.4
−0.6
−0.6
−0.4
−0.5
−0.6
−0.7



G1
−0.5
−0.5
−0.5
−0.6
−0.6
−0.4
−0.4



G1
−0.6
−0.8
−0.4
−0.8
−0.6
−0.4
−0.5



G1
−0.5
−0.5
−0.6
−0.5
−0.5
−0.6
−0.5



G1
−0.6
−0.4
−0.7
−0.6
−0.5
−0.6
−0.5



G1
−0.7
−0.6
−0.9
−1
−0.9
−0.4
−0.3























(3)K3.
(6)FCA.
(6)FCA.
(10)MALP2s.
(10)MALP2s.


Group
Probe Index
Gene Symbol
Modules
ID.LV.x3
ID.LV.x1
ID.LV.x2
ID.LV.x2
ID.LV.x1





G1
1449025_at
Ifit1
20
−0.5
−0.5
−0.5
−0.5
−0.4


G1
1456890_at
Ddx58
20
−0.6
−0.5
−0.9
−0.5
−0.4


G1
1450783_at
Ifit1
20
−0.5
−0.5
−0.5
−0.5
−0.5


G1
1450034_at
Stat1
20
−0.5
−0.4
−0.5
−0.1
0.19


G1
1436562_at
Ddx58
20
−0.5
−0.6
−0.7
−0.5
−0.4


G1
1437503_a_at
Shisa5
20
−0.5
−0.7
−0.7
−0.3
−0.1


G1
1418825_at
Irgm1
20
−0.5
−0.4
−0.5
−0.3
0.35


G1
1426906_at
Mndal
20
−0.5
−0.5
−0.6
−0.4
0.4


G1
1450033_a_at
Stat1
20
−0.5
−0.5
−0.5
−0.2
−0


G1
1436058_at
Rsad2
20
−0.5
−0.5
−0.5
−0.4
−0.4


G1
1423986_a_at
Shisa5
20
−0.5
−0.7
−0.4
−0.2
−0


G1
1434268_at
Adar
20
−0.1
−0.5
−0.5
0.04
0.15


G1
1425405_a_at
Adar
20
−0.5
−0.7
−0.6
−0.5
−0.7


G1
1435526_at
Tor1aip2
20
−0.1
−0.2
−0.3
−0.4
−0.3


G1
1431591_s_at
Gm9706///Isg15
20
−0.5
−0.5
−0.5
−0.4
−0.3


G1
1455500_at
Rnf213
20
−0.5
−0.5
−0.5
−0.4
−0.2


G1
1418115_s_at
Tor1aip2
20
−0.1
−0.1
−0.3
−0.5
−0.5


G1
1418116_at
Tor1aip2
20
−0.1
−0.1
−0.3
−0.3
−0.5


G1
1426970_a_at
Uba7
20
−0.4
−0.4
−0.6
−0
0.6


G1
1421323_a_at
G3bp2
20
−0.5
−0.2
−0.5
−0.4
0.05


G1
1422005_at
Eif2ak2
20
−0.5
−0.4
−0.6
−0.3
0.07


G1
1448757_at
Pml
20
−0.5
−0.1
−0.4
−0.6
−0.4


G1
1417244_a_at
Irf7
20
−0.4
−0.5
−0.5
−0.4
−0.3


G1
1426276_at
Ifih1
20
−0.5
−0.5
−0.6
−0.5
−0.3


G1
1449875_s_at
H2-T10///H2-
20
−0.7
−0.7
−0.7
−0.6
−0.3




T22///H2-T9


G1
1437432_a_at
Trim12a
20
−0.4
−0.5
−0.4
−0.6
−0.3


G1
1456103_at
Pml
20
−0.6
−0.4
−0.4
−0.3
0.04


G1
1426716_at
Tdrd7
20
−0.6
−0.4
−0.6
−0.9
−0.9



















(10)MALP2s.
(1)ADX.
(1)ADX.
(1)ADX.
(3)ADX.
(3)ADX.
(7)FK565.
(7)FK565.


Group
ID.LV.x3
IP.LV.x1
IP.LV.x2
IP.LV.x3
IP.LV.x2
IP.LV.x3
ID.LV.x3
ID.LV.x1





G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.4
−0.4
−0.4


G1
−0.5
−0.4
−0.4
−0.4
−0.2
−0.4
−0.5
−0.5


G1
−0.5
−0.5
−0.5
−0.5
−0.5
−0.4
−0.4
−0.4


G1
0.05
−0.3
−0.4
−0.4
−0.5
−0.4
−0.1
−0.1


G1
−0.5
−0.4
−0.4
−0.5
−0.6
−0.4
−0.2
−0.3


G1
−0.4
−0.1
−0.3
−0.4
−0.6
−0.3
−0.2
−0.3


G1
−0
−0.4
−0.4
−0.5
−0.5
−0.4
0.07
0.13


G1
0
−0.6
−0.7
−0.7
−0.7
−0.6
−0.4
−0.5


G1
−0
−0.3
−0.4
−0.4
−0.5
−0.4
−0.1
−0


G1
−0.4
−0.4
−0.5
−0.5
−0.5
−0.5
−0.4
−0.4


G1
−0.2
−0.3
−0.4
−0.4
−0.6
−0.3
−0.1
0.01


G1
0.2
−0.5
−0.6
−0.7
−0.7
−0.5
−0.1
−0.1


G1
−0.7
−0.4
−0.2
−0.6
−0.3
−0.3
−0.4
−0.5


G1
−0.5
−0.4
−0.4
−0.6
−0.6
−0.5
−0.8
−0.9


G1
−0.3
−0.5
−0.5
−0.5
−0.5
−0.4
−0.1
−0.1


G1
−0.4
−0.3
−0.4
−0.5
−0.4
−0.3
0.02
0.06


G1
−0.5
−0.4
−0.5
−0.7
−0.6
−0.4
−0.6
−0.5


G1
−0.7
−0.3
−0.6
−0.6
−0.9
−0.6
−0.7
−0.9


G1
0.08
−0.2
−0.6
−0.3
−0.5
−0.2
−0.4
−0.5


G1
−0.2
−0.4
−0.6
−0.4
−0.7
−0.6
−0.3
−0.3


G1
−0.4
−0.3
−0.4
−0.5
−0.6
−0.5
0.05
0.09


G1
−0.4
−0.4
−0.4
−0.4
−0.7
−0.4
−0.5
−0.6


G1
−0.3
−0.4
−0.5
−0.4
−0.3
−0.4
−0.3
−0.3


G1
−0.4
−0.4
−0.5
−0.5
−0.5
−0.4
−0.3
−0.3


G1
−0.5
−0.4
−0.5
−0.4
−0.7
−0.3
0.21
0.04


G1
−0.4
−0.4
−0.4
−0.5
−0.5
−0.2
0.1
−0.1


G1
−0.2
−0.4
−0.5
−0.5
−0.4
−0.3
−0.5
−0.5


G1
−0.9
−0.4
−0.5
−0.7
−0.7
−0.5
−0.5
−0.4



















(7)FK565.
(8)Pam3CSK4.
(8)Pam3CSK4.
(8)Pam3CSK4.

Group



Group
ID.LV.x2
ID.LV.x3
ID.LV.x1
ID.LV.x2
Group
Z Ave







G1
−0.4
−0.4
−0.5
−0.5
G1
1.95



G1
−0.1
−0.4
−0.4
−0.4
G1
1.95



G1
−0.4
−0.4
−0.5
−0.4
G1
1.94



G1
−0
−0.5
−0.5
−0.4
G1
1.93



G1
−0.2
−0.5
−0.6
−0.5
G1
1.93



G1
−0.1
−0.4
−0.6
−0.4
G1
1.93



G1
0.08
−0.3
−0.4
−0.3
G1
1.93



G1
−0.5
−0.6
−0.6
−0.4
G1
1.93



G1
0
−0.4
−0.5
−0.3
G1
1.92



G1
−0.4
−0.4
−0.5
−0.4
G1
1.92



G1
0.18
−0.5
−0.5
−0.3
G1
1.91



G1
−0.1
−0.2
−0.4
−0.4
G1
1.91



G1
−0.4
−0.3
−0.5
−0.2
G1
1.91



G1
−0.9
−0.5
−0.4
−0.4
G1
1.9



G1
−0.2
−0.4
−0.5
−0.5
G1
1.9



G1
0.03
−0.2
−0.3
−0.3
G1
1.9



G1
−0.5
−0.3
−0.4
−0.3
G1
1.9



G1
−0.7
−0.3
−0.4
−0.3
G1
1.9



G1
−0.3
−0.4
−0.6
−0.6
G1
1.9



G1
−0.2
−0.6
−0.4
−0.4
G1
1.89



G1
−0.1
−0.5
−0.5
−0.3
G1
1.89



G1
−0.8
−0.2
−0.2
−0.4
G1
1.89



G1
−0.4
−0.4
−0.6
−0.3
G1
1.89



G1
−0.3
−0.4
−0.5
−0.4
G1
1.89



G1
0.29
−0.5
−0.6
−0.5
G1
1.89



G1
−0.2
−0.3
−0.4
−0.4
G1
1.88



G1
−0.5
−0.2
−0.5
−0.5
G1
1.88



G1
−0.5
−0.1
−0.4
−0.2
G1
1.88










Whole organ transcriptome data for each organ from each mouse were analyzed as follows to obtain biological themes for individual samples from each adjuvant-administered group of three mice. For a particular experimental condition (e.g., DMXAA, LV, i.d.), eij is the expression value of the ith gene in the jth sample (where j=1, 2, 3) and ei(C) is the mean expression value of the corresponding control samples. A set of Pj genes was first defined, where eij/ei(c)>2.0. Biological themes enriched within Pj were then identified using TargetMine (Chen, Y. A. et al., PloS one 6, e17844 (2011)) (http://targetmine.mizuguchilab.org/). Tj denote the resulting list of biological themes, with associated p-values from Fischer's exact test.


Next, the biological themes called by all the individual samples in the group were scored as follows. Having obtained three lists (T1, T2 and T3) for a given experimental condition, a consensus list T of themes was created as T={(t1, p1), (t2, p2), . . . }, where ti is a biological theme that appears in all of T1, T2 and T3, and pi is the smallest of the associated p-values. Only the themes with the p-value of <0.05 were included in T.


To summarize the biological themes in T further, a list of terms considered to be associated with adjuvant activity in general was separately prepared. AT={at1, at2, . . . }, where ati is a pre-selected adjuvant-associated term (e.g., “stress” or “cytokine”). Finally, an enrichment score S for an adjuvant-associated term ati was defined as: S(ati)=Σ−log(pj), where the name of tj in T includes a term ati as a substring and pj is the associated p-value.


This scoring scheme summarizes the relative enrichment of pre-selected adjuvant-associated terms in genes responding to each adjuvant in each organ.


(Hierarchical Clustering)


The union of all sDEGs for adjuvants and each organ was defined as “adjuvant gene space”. Hierarchical clustering of adjuvants and genes in the adjuvant gene space was performed by the hclust function of the R package, with the 1-Pearson correlation coefficient as the distance measure and the Ward D2 algorithm. The resultant gene cluster, defined here as “gene module” (labeled Mix, where i is the module number and x is the organ), assumes that genes within a module behave similarly upon administration of a given adjuvant. The 21 adjuvants used herein were also hierarchically clustered, and grouped as Gjy, where (j) is the group number and (y) is the organ.


(Cell Population Profiling Based on the ImmGen Database)


The ImmGen database (Heng, T. S. et al., Nature immunology 9, 1091-1094 (2008)) (http://www.immgen.org/) provides the gene expression profiles of various immune cell types under steady-state conditions. Their expression profiles were used to estimate the origin of the cell type from which the genes were derived. Initially, from the ImmGen expression profile, each gene (i) was weighted in all ten immune cell types (j).










w
ij

=


e
ij





j
=
1

10


e
ij







[

Numeral


1

]







Here, it is assumed that the weight of a cell type (e.g., neutrophil) for a given gene depends only on the expression level of that gene in that particular cell type, independent of its expression level in other cell types (e.g., macrophage, B cell). Subsequently, for the expression profile of each adjuvant, genes were selected based on their fold changes, p-value cutoffs, and PA call thresholds. The cell type contribution in each sample (k) for cell type (j) is calculated as:










S
jk

=



s
ik



w
ij


G





[

Numeral


2

]







where (s) is the gene's differential expression and G is the total number of genes that satisfy the given cutoff.


(Z-Score-Based Differential Gene Expression Analysis)


Each sample's FC value was converted into a z-score on a gene basis by using the entire data set for each organ. The Z-score of the triplicates of interest were summed for each adjuvant. For example, the three sample z-score in the LV sample following cdiGMP administration was summed on a gene-by-gene basis, after which the summed scores of >3 genes were selected as relatively preferentially expressed genes in the LV after cdiGMP administration. The following threshold was applied for selections. In the LV, G1LV-associated genes were selected with cdiGMP(z>3) & cGAMP(z>3) & DMXAA(z>3) & PolyIC(z>3) & R848(z>3). G2LV: ALM_IP(z>1) & bCD(z>1) & ENDCN(z>1) & FCA(z>1). G3LV: ADX(z>1) & Pam3CSK4(z>1) & FK565(z>1). G4LV: MALP2s(z>3). In the SP, G1SP: cdiGMP(z>3) & cGAMP(z>3) & DMXAA(z>3) & PolyIC(z>3) & R848(z>3). G2SP: ENDCN_x2+ENDCN_x3(z>2) & ALM_IP(z>3) & bCD(z>3). G3SP: FCA(z>3) & FK565(z>3) & Pam3CSK4(z>3). G4SP: ADX_ID_x2+ADX_ID_x3(z>2) & ADX_IP(z>3) & MALP2s(z>3). In the LN, G1LN(5adj): cdiGMP(z>3) & cGAMP(z>3) & DMXAA(z>3) & PolyIC(z>3) & R848(z>3). G1LN(8adj): cdiGMP(z>0) & cGAMP(z>0) & DMXAA(z>0) & PolyIC(z>0) & R848(z>0) & MALP2s(z>575 0) & MPLA_x1+MPLA_x3(z>0) & Pam3CSK4_x2+Pam3CSK4_x3(z>0). G2LN: bCD(z>3) & FCA(z>3). G3LN: FK565(z>3). G5LN: K3(z>1.5) & K3SPG_x1+K3SPG_x2(z>1) & D35_x1+D35_x3(z>1). G6LN: AddaVax(z>3).


Hematological Data and Gene Expression Correlation Analysis


Pearson's correlation analysis was applied to the hematological data and to the gene expression changes in each organ. The data used for the analysis were selected using the following criteria. The genes should be cPA=1 and the hematology data should be >1 standard deviation (1 SD) from the mean of the pooled control. The resultant hematological parameter pairs and genes were further selected by Welch's t-test. The pairs with p-values of <0.05 and with correlation coefficient absolute values of <0.8 and with an absolute value of the slope (incline) of <1.2 were selected. The hematological and gene data derived from Exp. 5 and Exp. 10 were obtained in separate experiments, so these unlinked data were not used in this analysis. Importantly, when these unlinked sample data were plotted as test samples, these data were also a good fit with the correlation lines.









TABLE 13







Adjuvants and control used in this Example












Name
Full Name/Description
Physical property
Receptor















1
AddaVax
similar to MF59
squalene oil-in-water emulsion
Unknown


2
ADX
Advax ™
semi-crystalline particles of delta inulin
Unknown


3
ALM
Aluminium hydroxide gel
gel suspension
Unknown


4
bCD
Hydroxypropyl-beta-cyclodextrin
cyclodexstrin
Unknown


5
cdiGMP
Cyclic diguanylate monophosphate
cyclic di-nucleotide
STING


6
cGAMP
Cyclic [G(2′,5′)pA(3′,5′)p]
cyclic di-nucleotide
STING


7
D35
Type A CpG ODNs
syntheitc oligodeoxyribonucleotide
TLR9


8
DMXAA
5,6-dimethylxanthenone-4-acetic acid
Synthetic chemicals
STING


9
ENDCN
Endocine ™
Mono-olein and oleic acid
Unknown


10
FCA
Complete Freund's Adjuvant
Mycobacterium/water-in-oil emulsion
CLR and unkown


11
FK555
heptanoyl-r-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine
Synthetic peptidoglycans
NOD1


12
ISA51VG
Incomplete Freund's Adjuvant
water-in-oil emulsion
Unknown


13
K3
Type B CpG ODNs
syntheitc oligodeoxyribonucleotide
TLR9


14
K3SPG
Complex of K3 CpG ODNs and beta-glucan(SPG)
deoxyribonucleotide/glucan complex
TLR9


15
MALP2s
macrophage-activating lipopeptide-2 short lipopeptide
lipopeptide
TLR2/6


16
MBT
N-Acetyl-muramyl-L- Alanyl-D-Glutamin-n-butyl-ester
Synthetic peptidoglycans
NOD2


17
MPLA
Monophosphoryl lipid A
low-toxicity derivative of lipopolysaccharide
TLR4


18
Pam3CSK4
Pam-3-Cys-Ser-Lys-Lys-Lys-Lys
synthetic triacylated lipopeptide
TLR1/2


19
PolylC
Polyinosine-polycytidylic acid
double stranded ribonucleotide polymer
TLR3 and MDA5


20
R548
imidazoquinoline compound
guanosine derivative
TLR7


21
sHZ
synthetic hemozoin
beta hematin crystals
Unknown









Next, the adjuvants and the dosages used in this Example are shown.









TABLE 14







Adjuvants and dosages thereof used in each experiment















Test



Dose
Group



Exp. No.
substance
Route
Dose
Conc.
volume
No.
Kit



















Exp. 1
PBS
i.p.
0
mg
0
mg/mL
0.1 mL
1
E



ADX
i.p.
1
mg
10
mg/mL
0.1 mL
2
E


Exp. 2
PBS
i.p.
0
mg
0
mg/mL
0.1 mL
1
E



ALM
i.p.
1
mg
10
mg/mL
0.1 mL
2
E



K3
i.p.


0.1
mg/mL
0.1 mL
3
E



K3SPG
i.p.


0.1
mg/mL
0.1 mL
4
E


Exp. 3
PBS
i.d.
0
mg
0
mg/mL
0.1 mL
1
E



ADX
i.d.
1
mg
10
mg/mL
0.1 mL
2
E



ALM
i.d.
1
mg
10
mg/mL
0.1 mL
3
E



K3
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
4
E















Exp. 4
PBS
i.p.


 0%
0.2 mL
1
E



bCD
i.p.


30%
0.2 mL
2
E



Tris-HCl
i.n.


 0%
0.005 mL 
3
E



ENDCN
i.n.


 2%
0.005 mL 
4
E
















Exp. 5
PBS
i.d.
0
mg
0
mg/mL
0.1 mL
1
E



D35
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
2
E



K3SPG
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
3
E
















bCD
i.d.


30%
0.2 mL
4
E


Exp. 6
PBS
i.d.


 0%
0.1 mL
1
E



FCA
i.d.


50%
0.1 mL
4
E
















Exp. 7
PBS
i.d.
0
mg
0
mg/mL
0.1 mL
1
P



sHZ
i.d.
0.2
mg
2
mg/mL
0.1 mL
2
P



FK565
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
3
P



MBT
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
4
P


Exp. 8
PBS
i.d.
0
mg
0
mg/mL
0.1 mL
1
P



PolyIC
i.d.
0.1
mg
1
mg/mL
0.1 mL
2
P



Pam3CSK4
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
3
P



cdiGMP
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
4
P















Exp. 9
PBS
i.d.


 0%
0.1 mL
1
P



Addavax
i.d.


50%
0.1 mL
2
P



ISA51VG
i.d.


50%
0.1 mL
3
P

















cGAMP
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
4
P


Exp. 10
PBS DMSO5%
i.d.
0
mg
0
mg/mL
0.1 mL
1
P



MPLA
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
2
P



MALP2s
i.d.
0.01
mg
0.1
mg/mL
0.1 mL
3
P



R848
i.d.
0.1
mg
1
mg/mL
0.1 mL
4
P



DMXAA
i.d.
0.1
mg
1
mg/mL
0.1 mL
5
P









Table 15 Number of gene probes with statistically significant changes after adjuvant administration. The significantly changed genes, which are described as “significantly differentially expressed genes” (sDEGs), are defined by the following criteria: fold change >1.5 (up) or <0.667 (down) with p-value of <0.01 and customized PA call=1. Liver is indicated as LV. Spleen is indicated as SP. Lymph node is indicated as LN. Blanks indicate that the samples were not examined.





















TABLE 15










AddaVax
ADX
ALM
bCD
cdlGMP
cGamp
D35
DMXAA
ENDCN*
FCA





i.d.
LV
Up
29
11
17
107
280
841
15
162
100
198


i.n.text missing or illegible when filed

Down
18
14
15
315
218
248
36
101
24
139



SP
Up
16
14
13
111
707
680
23
121
14
136




Down
13
13
8
59
261
93
19
48
13
39



LN
Up
61
25
38
89
1579
1135
69
1342

640




Down
73
25
16
110
908
255
12
261

284


i.p.
LV
Up

160
198
14




Down

88
552
20



SP
Up

346
136
4




Down

129
21
5






















FK565
ISA61VG
K3
K3SPG
MALP2s
MST
MPLA
Pam3CSK4
PolylC
R845
sHz





i.d.
2094
17
10
19
628
14
9
114
516
702
12


i.n.text missing or illegible when filed
1972
27
22
81
1248
28
24
51
1122
1462
8



1672
9
11
12
774
34
9
200
1360
3879
26



624
18
18
59
311
25
22
50
738
1313
12



526
16
53
57
1895
19
8
23
2203
3091
8



306
12
18
99
718
13
18
27
1460
1193
5


i.p.


18
20





50
33





24
20





10
14






text missing or illegible when filed indicates data missing or illegible when filed














TABLE 16







Identification information of genes mentioned in this Example










Gene



Gene name
ID:
NCBI reference number












Gm14446
667373
NM_001101605.1; NM_001110517.1


Pml
18854
NM_001311088.1; NM_008884.5;




NM_178087.4


H2-T22
15039
NM_010397.4


Ifit1
15957
NM_008331.3


Irf7
54123
NM_001252600.1; NM_001252601.1;




NM_016850.3


Isg15
1E+08
NM_015783.3


Stat1
20846
NM_001205313.1; NM_001205314.1;




NM_009283.4


Fcgr1
14129
NM_010186.5


Oas1a
246730
NM_145211.2


Oas2
246728
NM_145227.3


Trim12a
76681
NM_023835.2


Trim12c
319236
NM_001146007.1; NM_175677.4


Uba7
74153
NM_023738.4


Ube2l6
56791
NM_019949.2


Elovl6
170439
NM_130450.2


Gpam
14732
NM_008149.3


Hsd3b7
101502
NM_001040684.1; NM_133943.2


Acer2
230379
NM_001290541.1; NM_001290543.1;




NM_139306.3


Acox1
11430
NM_001271898.1; NM_0157.29.3


Tbl1xr1
81004
NM_030732.3


Alox5ap
11690
NM_001308462.1; NM_009663.2


Ggt5
23887
NM_011820.4


Bbc3
170770
NM_133234.2


Pdk4
27273
NM_013743.2


Cd55
13136
NM_010016.3


Cd93
17064
NM_010740.3


Clec4e
56619
NM_019948.2


Coro1a
12721
NM_001301374.1; NM_009898.3


Traf3
22031
NM_001286122.1; NM_011632.3


Trem3
58218
NM_021407.3


G5ar1
12273
NM_001173550.1; NM_007577.4


Clec4n
56620
NM_001190320.1; NM_001190321.1;




NM_020001.2


Ier3
15937
NM_133662.2


Il1r1
16177
NM_001123382.1; NM_008362.2


Plek
56193
NM_019549.2


Tbx3
21386
NM_011535.3; NM_198052.2


Trem1
58217
NM_021406.5


Ccl3
20302
NM_011337.2


Myof
226101
NM_001099634.1; NM_001302140.1


Papss2
23972
NM_001201470.1; NM_011864.3


Slc7a11
26570
NM_011990.2


Tnfrsf1b
21938
NM_011610.3


Ak3
56248
NM_021299.1


Insm1
53626
NM_016889.3


Nek1
18004
NM_001293637.1; NM_001293638.1;




NM_001293639.1; NM_175089.4


Pik3r2
18709
NM_008841.3


Ttn
22138
NM_011652.3; NM_028004.2


Atp6v0d2
242341
NM_175406.3


Atp6v1c1
66335
NM_025494.3


Clec7a
56644
NM_001309637.1; NM_020008.3









(Results)


Adjuvant Gene Space—the Union of sDEGs Characterizes Organ Responses to Adjuvants


A total of 21 adjuvants (Table 1) were administered to mice at the tail base (intradermally; i.d.), intraperitoneally (i.p.) or intranasally (i.n.) Six hours after adjuvant administration, whole organ transcriptomes of the LV, SP (as systemic organs) and LNs (as local lymphoid tissues) were obtained, and a set of significantly differentially expressed genes (sDEGs) for each organ-adjuvant pair was defined (Table 15). Next, the adjuvant-induced gene responses were integrated by combining all the sDEGs on a per-organ basis. The union of sDEGs from the LV, SP, and LNs resulted in a total of 8049, 8449 and 9451 gene probes, respectively (FIG. 1). The adjuvant gene spaces included genes whose expression was significantly changed by the administration of at least one of the 21 different adjuvants examined in vivo. Overall, 3874 (48% of the LV-induced genes), 2331 (28% of the SP-induced genes), and 2991 (31% of the LNs-induced genes) genes were unique to each organ. Pathway analysis with TargetMine (Chen, Y. A. et al., PloS one 6, e17844 (2011)) showed that these unique genes were related to lipid metabolism, transcription, and the immune system in the LV, SP and LNs, respectively (FIG. 1). The three organs shared 2299 genes with pathway enrichments related to interferons, cytokines, NF-κB, and TNF signaling (FIG. 1).


According to the volcano plot data, half of the adjuvants (AddaVax, ADX, ALM, D35, ISA51VG, K3, K3SPG, MBT, MPLA, and sHZ) produced modest responses (<100 sDEGs) when they were administered locally. This is consistent with the fact that the dose levels were chosen to mimic real vaccination situations. bCD, ENDCN, and Pam3CSK4 induced intermediate (100-200 sDEGs) responses. By comparison, cdiGMP, cGAMP, DMXAA, FCA, FK565, MALP2s, PolyIC, and R848 induced strong (>500 sDEGs) gene responses in at least one organ. These adjuvants tended to elicit larger gene responses in directly draining LNs, and relatively weaker responses in systemic (LV and SP) organs (Table 15). Interestingly, FK565, the synthetic NOD1 ligand, induced greater responses in LV and SP than in LNs after i.d. administration (Table 15). ADX and ALM induced obvious gene expression changes in LV and SP, whereas K3, K3SPG and bCD induced relatively weaker responses (Table 15). MPLA, Pam3CSK4, and other relatively mild adjuvants tended to exhibit varying gene responses among the individual mice and organs (FIG. 5).


As half of the adjuvants induced only limited numbers of sDEGs, it was difficult to characterize them by regular gene annotation analysis. Therefore, another gene selection criterion (fold change >2) was also tested for its ability to extract meaningful biological annotations for each adjuvant. In fact, this analysis obtained enough and reasonable in vivo biological annotations from even a practical dose (relatively small amount) of adjuvant. Almost all the adjuvants induced inflammation, cytokine/chemokine responses, chemotaxis/migration, and stress/defense/immune responses, relatively more strongly in LNs than in LV and SP after i.d. administration. With the tissue damage-associated annotations (wounding, cell death, apoptosis, NFκB signaling pathway), ADX, D35, K3, K3SPG, MBT, and sHZ did not induce cell death and wounding in LNs, a finding consistent with the good local tolerability profiles of ADX and sHZ. Interferon and interleukin responses against most adjuvants (except AddaVax, ADX, ISA51VG, MBT, and sHZ) in LNs were also detected. In spite of relatively strong inflammation and stress responses in LNs, ALM, K3SPG and MPLA exhibited limited responses in LV and SP, indicating that these adjuvant effects were locally restricted. In contrast, with the exception of ISA51VG and sHZ, the other adjuvants appeared to cause detectable levels of systemic organ responses even after i.d. local administration. Intriguingly, MBT, a NOD2 ligand, appeared to induce more gene responses in the LV than in SP and LNs after i.d. administration, a result similar to that for FK565. Ip. administration of ADX, ALM, and K3SPG induced relatively stronger inflammation- and stress-associated responses in LV and SP than i.d. administration. These results indicate that this dataset without p-value selection provides considerable information about adjuvant-induced gene responses, at a level sufficient to retrieve each adjuvant-induced biological response in vivo with substantial reliability, thus supporting the whole organ transcriptome approach.


To obtain more details of the adjuvant gene spaces that clarifies adjuvant induce host responses in three organs, hierarchical clustering of genes and adjuvants was performed (see also http://sysimg.ifrec.osaka-u.ac.jp/adjvdb/methodologies/adjv_space.html). This analysis found that separating approximately 10,000 gene probes for each organ into a total of 40 clusters (a few hundred genes per cluster) was suitable for obtaining biological annotations for each cluster using TargetMine. Hereafter, to discriminate the gene clusters from the adjuvant clusters discussed below, these 40 clusters for each organ are referred to as “modules”. Gene Ontology (GO) enrichment analysis of these modules revealed that adjuvant administration induced a broad range of biological processes in each organ, such as immune-related processes and more basic biological cellular processes. The types of cells that responded to each adjuvant were also identified by comparing the gene expression profiles with those publicly available from the ImmGen database (Heng, T. S. et al., Nature immunology 9, 1091-1094 (2008)) (https://www.immgen.org/), as described in Methods. This analysis revealed that the LV and SP responses were markedly associated with neutrophils and stromal cells. In LNs, in addition to neutrophils and stromal cells, the responses were associated with macrophages. Interestingly, one third of the genes in SP clustered in modules 14-19, all of which were related to RNA processing, and were strongly associated with B cells. In contrast, in LV, almost no association with T cells was observed, a finding consistent with general knowledge of relatively low T cell residency in the LV. In LNs, a greater variety of immune cell types were associated with each module. Genes in module 16 of the LNs (M16LN), which were upregulated by Pam3CSK4, K3, K3SPG and FCA, were phagosome-related and were correspondingly associated with macrophage populations. The same “immune system process” annotation appeared three times and modules (M)15LN, M27LN and M40LN were associated with different cell types. M15LN (weakly induced by ALM and K3) was associated with T cells, M27LN (induced by most of the adjuvants) was strongly associated with neutrophils, and M40LN (strongly induced by cdiGMP, cGAMP, DMXAA, PolyIC, and R848) was associated with a broader range of immune cells including dendritic cells, macrophages, neutrophils, and stromal cells. These data show that the adjuvant gene space approach can provide multilayered information, including each adjuvant's biological properties and the responses of different cell types to the adjuvants.


Classification of Adjuvants into 6 Groups within the Adjuvant Gene Space


Adjuvants have been categorized according to their stimulating properties (e.g., PAMPs or DAMPs) or their physicochemical features (e.g., solute, particles, or emulsions). The qualitative differences of these descriptors make it difficult to categorize adjuvants without bias. Therefore, the hierarchical clustering results were utilized to categorize the adjuvants. The cluster analysis showed an interesting and insightful grouping within the adjuvant gene space for each organ. Although each organ had a characteristic gene profile (FIG. 1), rather consistent adjuvant groupings was observed among the LV, SP, and LNs. In the cluster trees, most of the three replicates of the same adjuvant were closely connected (forming the first and the second dendrogram nodes), indicating that individual mice administered with the same adjuvant exhibited similar gene expression changes. Some interesting exceptions were D35_ID_x2 and K3_ID_x3 in LN. At a higher clustering threshold level (cutoff height=1.0), four major groups were formed in LV, and similarly in SP, excluding batch effects (Leek, J. T. et al., Nat Rev Genet 11, 733-739 (2010)). In LNs, the cutoff height of 1.0 gave 10 groups, indicating that LNs responded to each adjuvant more strongly and diversely than did LV or the SP. The cutoff height of 1.5 for LNs gave a total of five groups excluding the batch effect. This cutoff setting also gave more comparable groupings to those of LV and SP.


The majority of the clusters were consistent across the organs. Five adjuvants (cdiGMP, cGAMP, DMXAA, PolyIC, and R848) were fixed to a single cluster in LV, SP, and LNs, which are denoted as group (G) 1. From a similar perspective, it was found that bCD, FK565, and MALP2s served as references for G2, G3, and G4, respectively, because they constantly appeared in a separate cluster. In LNs, two additional groups, G5 and G6, were observed.


When similar transcriptome analysis was performed on rats, the results were obtained. The results are nearly the same as the results for mice, showing that transcriptome analysis is also useful for grouping of adjuvants in rats.


(Each Adjuvant Group Associated with Characteristic Biological Responses)


To characterize further each adjuvant group at the gene level, genes that were preferentially upregulated in each adjuvant group (G1 to G6) compared with the other groups were first identified by converting their expression fold-change values into z-scores (Table 17 (partially excerpted)).



















TABLE 17












bCD.


D35.




Term

AddaVax.
ADX.
ALM.
LV.
cdiGMP.
cGAMP.
LV.
DMXAA.
ENDCN.


(Keywords)
GO annotation
LV.ID
LV.ID
LV.ID
ID
LV.ID
LV.ID
ID
LV.ID
LV.ID





wounding
regulation of response
0
0
0
6.31
10.7
3.31
0
3.49
0



to wounding


wounding
response to wounding
0
0
0
5.85
11.4
0
0
0
0


wounding
positive regulation of
0
0
0
0
0
0
0
0
0



response to wounding


cell death
cell death
5.38
5.36
0
5.8
17.7
10.2
5.7
6.04
13.7


cell death
death
0
0
0
0
12.1
4.77
0
0
8.85


cell death
programmed cell death
0
0
0
0
14
6.49
0
0
6.75


cell death
regulation of cell death
0
0
0
0
4.48
3.82
0
0
9.15


cell death
regulation of programmed
0
0
0
0
5.36
4.93
0
0
6.22



cell death


cell death
positive regulation of
0
0
0
0
0
0
3.05
0
3.68



programmed cell death


cell death
positive regulation of
0
0
0
0
0
0
0
0
4.51



cell death


cell death
negative regulation of
0
0
0
0
0
0
0
0
0



cell death


cell death
negative regulation of
0
0
0
0
0
0
0
0
0



programmed cell death


apoptosis
apoptotic process
0
0
0
0
12.1
4.46
0
0
7.32


apoptosis
regulation of apoptotic
0
0
0
0
4.93
4.63
0
0
5.49



process


apoptosis
apoptotic signaling
0
0
0
0
0
0
0
0
3.18



pathway


apoptosis
intrinsic apoptotic
0
0
0
0
0
0
0
0
4.34



signaling pathway


apoptosis
positive regulation of
0
0
0
0
0
0
3.18
0
0



apoptotic process


apoptosis
regulation of apoptotic
0
0
0
0
0
0
0
0
0



signaling pathway


apoptosis
negative regulation of
0
0
0
0
0
0
0
0
0



apoptotic process


apoptosis
regulation of intrinsic
0
0
0
0
0
0
0
0
0



apoptotic signaling



pathway


NF-kappa B
NF-kappa B signaling
0
11.9
0
6.86
15.7
4.86
5.27
3.77
0


signaling
pathway


pathway


NF-kappa B
I-kappaB kinase/NF-
0
3.98
0
4.21
13.8
0
0
0
0


signaling
kappaB signaling


pathway


NF-kappa B
positive regulation
0
0
0
0
7.15
0
0
0
0


signaling
of I-kappaB kinase/


pathway
NF-kappaB signaling


NF-kappa B
regulation of I-kappaB
0
0
0
0
8.16
0
0
0
0


signaling
kinase/NF-kappaB


pathway
signaling


inflammatory
inflammatory response
7.12
12.6
0
10.9
26.1
11.5
0
0
0


response


inflammatory
regulation of inflammatory
0
0
0
7.33
12.3
0
0
0
0


response
response


inflammatory
positive regulation of
0
0
0
0
0
0
0
0
0


response
inflammatory response


inflammatory
acute inflammatory response
0
0
0
0
0
0
0
0
0


response


inflammatory
leukocyte migration involved
0
0
0
0
0
0
0
0
0


response
in inflammatory response


TNF signaling
TNF signaling pathway
5.94
4.51
0
10.6
31.3
27.6
12.1
10.7
8.38


pathway


cytokine
response to cytokine
0
0
9.11
7.77
56.3
45.9
8.12
26.5
7.7


cytokine
Cytokine-cytokine receptor
0
9.49
0
7.76
33.9
24.6
5.2
15.4
0



interaction|Endocytosis


cytokine
cellular response to cytokine
0
3.74
9.43
0
38.8
32.9
3.28
16.7
0



stimulus


cytokine
Cytokine-cytokine receptor
0
5.61
0
4.24
13.8
5.57
0
0
0



interaction


cytokine
cytokine production
0
5.88
0
3.82
31.3
17.4
0
6.98
0


cytokine
regulation of cytokine production
0
5.41
0
4.06
31.6
19.1
0
8.37
0


cytokine
cytokine-mediated signaling
0
0
7.05
0
20
16.4
0
0
4.84



pathway


cytokine
cytokine biosynthetic process
0
0
0
0
7.93
6.84
0
0
0


cytokine
cytokine metabolic process
0
0
0
0
7.39
8.21
0
0
0


cytokine
positive regulation of cytokine
0
0
0
0
23.6
11.1
0
7.86
0



production


cytokine
negative regulation of cytokine
0
0
0
0
6.2
3.21
0
0
0



production


cytokine
regulation of cytokine
0
0
0
0
3.81
0
0
0
0



biosynthetic process


cytokine
regulation of tumor necrosis factor
0
0
0
0
4.03
0
0
0
0



superfamily cytokine production


cytokine
tumor necrosis factor superfamily
0
0
0
0
3.79
0
0
0
0



cytokine production


cytokine
positive regulation of cytokine
0
0
0
0
3.62
0
0
0
0



biosynthetic process


cytokine
cytokine secretion
0
0
0
0
0
0
0
0
0


cytokine
positive regulation of tumor
0
0
0
0
0
0
0
0
0



necrosis factor superfamily



cytokine text missing or illegible when filed


migration
positive regulation of
0
12.3
0
0
3.33
0
0
0
0



leukocyte migration


migration
cell migration
3.44
4.19
0
0
0
0
0
0
0


migration
leukocyte migration
0
8.85
0
0
0
0
0
0
0


migration
regulation of leukocyte migration
0
9.34
0
0
0
0
0
0
0


migration
neutrophil migration
0
0
0
0
0
0
0
0
0


migration
positive regulation of cell
0
0
0
0
0
0
0
0
0



migration


migration
granulocyte migration
0
0
0
0
0
0
0
0
0


migration
myeloid leukocyte migration
0
0
0
0
0
0
0
0
0


migration
regulation of cell migration
0
0
0
0
0
0
0
0
0


migration
lymphocyte migration
0
0
0
0
0
0
0
0
0


chemokine
chemokine-mediated signaling
0
3.8
0
0
5.9
0
0
0
0



pathway


chemokine
chemokine production
0
0
0
0
8.52
0
0
0
0


chemokine
regulation of chemokine
0
0
0
0
9.64
0
0
0
0



production


chemokine
positive regulation of
0
0
0
0
8.31
0
0
0
0



chemokine production


chemotaxis
cell chemotaxis
7.11
13.1
0
5.73
0
4.4
4.51
0
5.77


chemotaxis
chemotaxis
6.22
4.82
0
0
0
0
0
0
0


chemotaxis
leukocyte chemotaxis
0
7.8
0
0
0
0
0
0
0


chemotaxis
positive regulation of
0
4.39
0
0
0
0
0
0
0



leukocyte chemotaxis


chemotaxis
taxis
6.2
4.79
0
0
0
0
0
0
0


chemotaxis
granulocyte chemotaxis
0
0
0
0
0
0
0
0
0


chemotaxis
neutrophil chemotaxis
0
0
0
0
0
0
0
0
0


chemotaxis
positive regulation of chemotaxis
0
0
0
0
0
0
0
0
0


chemotaxis
regulation of leukocyte
0
3.26
0
0
0
0
0
0
0



chemotaxis


chemotaxis
regulation of chemotaxis
0
0
0
0
0
0
0
0
0


chemotaxis
lymphocyte chemotaxis
0
0
0
0
0
0
0
0
0


stress
response to stress
9.84
13.2
0
10.4
56.2
44.6
9.13
40.4
17.2


stress
regulation of response to stress
0
0
0
0
42.9
25.3
0
21
0


stress
cellular response to stress
0
0
0
0
0
0
0
0
3.17


defense response
defense response
3.3
20.5
0
8.51
80.9
62.7
5.76
48.4
0


defense response
regulation of defense response
4.39
9.94
0
6.85
56.5
37.5
0
26.8
0


defense response
positive regulation of
0
9.54
0
0
35.8
24.7
0
19
0



defense response


defense response
defense response to
0
6.37
0
0
44.1
37
0
23.6
0



other organism


defense response
defense response to bacterium
0
0
0
0
10.5
8.38
0
0
0


defense response
defense response to Gram-
0
0
0
0
7.18
0
0
0
0



positive bacterium


defense response
defense response to protozoan
0
0
0
0
5.31
4.37
0
3.52
0


defense response
defense response to virus
0
0
0
0
24.6
19.8
0
15
0


defense response
regulation of defense response
0
0
0
0
0
0
0
0
0



to virus


defense response
regulation of defense response
0
0
0
0
0
0
0
0
0



to virus by host


defense response
negative regulation of defense
0
0
0
0
8.05
0
0
0
0



response


immune response
immune response
0
12.2
0
0
52.3
42.2
0
25.5
0


immune response
positive regulation of immune
0
0
0
0
24.4
15.7
0
10.2
0



response


immune response
regulation of immune response
0
0
0
0
29.4
20.7
0
12.4
0


immune response
activation of immune response
0
0
0
0
17.9
12.6
0
5.67
0


immune response
immune response-activating
0
0
0
0
19.3
14.1
0
5.98
0



signal transduction


immune response
immune response-regulating
0
0
0
0
23.5
17.6
0
8.39
0



signaling pathway


immune response
negative regulation of immune
0
0
0
0
6.54
0
0
0
0



response


immune response
production of molecular
0
0
0
0
0
0
0
0
0



mediator of immune response


innate immune
innate immune response
0
4.13
0
0
40.7
31.4
0
34.9
0


response


innate immune
regulation of innate immune
0
3.19
0
0
33.4
24.8
0
19.8
0


response
response


innate immune
positive regulation of innate
0
0
0
0
22.3
17.2
0
12.4
0


response
immune response


innate immune
activation of innate immune
0
0
0
0
21.2
16.9
0
9.24
0


response
response


innate immune
innate immune response-
0
0
0
0
20.3
16.2
0
7.99
0


response
activating signal transduction


innate immune
negative regulation of innate
0
0
0
0
10.4
0
0
0
0


response
immune response


adaptive immune
adaptive immune response based
0
0
0
0
10.6
3.78
0
0
0


response
on somatic recombination of



immune receptors built from



immunoglobulin superfamily



domains


adaptive immune
adaptive immune response
0
0
0
0
10
0
0
0
0


response


adaptive immune
positive regulation of adaptive
0
0
0
0
0
0
0
0
0


response
immune response


adaptive immune
regulation of adaptive immune
0
0
0
0
8.01
0
0
0
0


response
response


adaptive immune
regulation of adaptive immune
0
0
0
0
3.64
0
0
0
0


response
response based on somatic



recombination of immune



receptors built from



immunoglobulin superfamily



domains


IFN alpha
response to interferon-alpha
0
0
0
0
12.6
14.8
0
11.3
0


IFN alpha
interferon-alpha production
0
0
0
0
7.69
3.79
0
6.81
0


IFN alpha
cellular response to interferon-
0
0
0
0
7.42
6.72
0
9.3
0



alpha


IFN alpha
positive regulation of interferon-
0
0
0
0
8.97
4.81
0
7.85
0



alpha production


IFN alpha
regulation of interferon-alpha
0
0
0
0
8.3
4.28
0
7.3
0



production


IFN beta
cellular response to interferon-
0
0
0
0
23.1
17.6
0
16.7
0



beta


IFN beta
response to interferon-beta
0
0
0
0
26.5
23.5
0
17.9
0


IFN beta
positive regulation of interferon-
0
0
0
0
13.2
9.2
0
5.66
0



beta production


IFN beta
regulation of interferon-beta
0
0
0
0
9.22
5.76
0
3.09
0



production


IFN beta
interferon-beta production
0
0
0
0
8.61
5.22
0
0
0


IFN gamma
response to interferon-gamma
0
0
0
0
10.8
10
0
10.3
0


IFN gamma
cellular response to interferon-
0
0
0
0
10.1
7.44
0
7.37
0



gamma


IFN gamma
interferon-gamma production
0
0
0
0
5.35
0
0
0
0


IFN gamma
regulation of interferon-gamma
0
0
0
0
0
0
0
0
0



production


interleukin-6
interleukin-6 production
0
0
0
0
7.8
0
0
0
0


interleukin-6
regulation of interleukin-6
0
0
0
0
8.27
0
0
0
0



production


interleukin-6
positive regulation of interleukin-
0
0
0
0
0
0
0
0
0



6 production


interleukin-12
interleukin-12 production
0
0
0
0
6.51
0
0
0
0


interleukin-12
regulation of interleukin-12
0
0
0
0
6.99
0
0
0
0



production


interleukin-12
positive regulation of interleukin-
0
0
0
0
0
0
0
0
0



12 production
























Term
FCA.
FK565.
ISA51VG.
K3.
K3SPG.
MALP2s.
MBT.
MPLA.
Pam3CSK4.
Poly_IC.
R848.
sHz.



(Keywords)
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID
LV.ID







wounding
3.82
12.1
0
0
0
9.89
0
0
11.1
16.7
11.1
0



wounding
5.68
12.8
0
0
0
10.3
0
0
12.2
14.8
9.74
0



wounding
0
9.45
0
0
0
0
0
0
3.54
9.8
4.53
0



cell death
14.9
21.7
0
18.3
0
17.5
0
0
20.3
22.6
23.1
3.62



cell death
11.7
16.4
0
13.9
0
12.6
0
0
14.9
17.8
18.3
0



cell death
8.34
15.7
0
14.9
0
13.3
0
0
15.2
18.1
19.4
0



cell death
5.76
14
0
10.5
0
9.13
0
0
6.86
15.4
15.5
0



cell death
5.11
12.9
0
10.9
0
8.86
0
0
6.45
15.6
15.5
0



cell death
0
7.1
0
7.25
0
0
0
0
0
4.25
4.66
0



cell death
0
7.29
0
6.67
0
0
0
0
0
3.46
4.64
0



cell death
0
0
0
0
0
0
0
0
0
4.38
0
0



cell death
0
0
0
0
0
0
0
0
0
4.89
0
0



apoptosis
9.08
15.2
0
15.7
0
12.4
0
0
15
15.7
18.6
0



apoptosis
5.44
13
0
10.1
0
8.76
0
0
6.84
14.8
15.5
0



apoptosis
4.48
19.9
0
10.3
0
9.05
0
0
3.68
12.4
10.6
0



apoptosis
0
13.9
0
0
0
5.47
0
0
0
7.32
5.18
0



apoptosis
0
6.58
0
5.99
0
0
0
0
0
3.59
4.1
0



apoptosis
0
13.2
0
3.19
0
0
0
0
0
7.04
5.48
0



apoptosis
0
0
0
0
0
0
0
0
0
4.79
0
0



apoptosis
0
8.33
0
0
0
0
0
0
0
0
0
0



NF-kappa B
10.8
19.8
0
6.19
0
8.36
6.87
0
29.6
18
8.86
0



signaling



pathway



NF-kappa B
8.18
12.9
0
0
0
3.13
6.14
0
16.9
13.5
7.93
0



signaling



pathway



NF-kappa B
3.69
3.05
0
0
0
0
0
0
0
8.57
5.3
0



signaling



pathway



NF-kappa B
0
5.82
0
0
0
0
0
0
5.45
9.04
5.28
0



signaling



pathway



inflammatory
19
29.8
0
0
0
23.4
0
0
38
32.2
22.2
0



response



inflammatory
5.87
11.4
0
0
0
9.25
0
0
13.6
14.4
6.29
0



response



inflammatory
0
3.69
0
0
0
0
0
0
5.39
5.99
0
0



response



inflammatory
0
0
0
0
0
0
0
0
5.25
0
0
0



response



inflammatory
0
0
0
0
0
0
0
0
3.41
0
0
0



response



TNF signaling
14
30.9
3.76
14.5
0
18.7
6.37
3.93
35.8
22.1
16.1
0



pathway



cytokine
12.8
37.8
0
0
0
34.9
3.82
0
46.5
66.1
44.8
4.53



cytokine
5.33
32.6
0
0
0
12.8
6.94
0
25.9
48.5
30.5
0



cytokine
3.22
23.6
0
0
0
24.8
0
0
31.5
42.3
30.4
0



cytokine
5.96
17.1
0
0
0
8.95
3.53
0
13.6
12.1
11.1
0



cytokine
10.7
30.2
0
0
0
20.9
0
0
32.6
45.6
30.4
0



cytokine
5.96
27.5
0
0
0
15.5
0
0
29.8
43.5
26.5
0



cytokine
0
8.69
0
0
0
11.3
0
0
17
20.9
16.5
0



cytokine
7.49
16.2
0
0
0
6.41
0
0
19.7
7.3
5.44
0



cytokine
9.31
17
0
0
0
7.38
0
0
21.6
8.4
4.8
0



cytokine
0
18.6
0
0
0
6.74
0
0
21.5
34.3
20.3
0



cytokine
0
3.46
0
0
0
4.78
0
0
5.39
12.6
5.01
0



cytokine
4.48
14
0
0
0
5.33
0
0
17
4.18
0
0



cytokine
0
7.57
0
0
0
4.82
0
0
11.6
15
5.55
0



cytokine
0
7.13
0
0
0
4.49
0
0
11.3
14.5
6.84
0



cytokine
0
10.1
0
0
0
0
0
0
14.5
3.4
0
0



cytokine
0
0
0
0
0
5.26
0
0
0
3.66
0
0



cytokine
0
0
0
0
0
0
0
0
0
5.29
0
0



migration
0
13.6
0
0
0
10.3
4.41
0
21.1
22.1
8.03
0



migration
0
8.98
0
0
0
0
4.69
0
14.7
6.99
0
0



migration
0
14.2
0
0
0
15.1
0
0
27.6
23.4
7.89
0



migration
0
7.86
0
0
0
7.84
0
0
15.4
18.5
5.8
0



migration
0
7.57
0
0
0
3.05
0
0
14
9
3.14
0



migration
0
6.55
0
0
0
3.39
0
0
11.4
9.34
3.96
0



migration
0
6.19
0
0
0
0
0
0
14.1
8.14
3.31
0



migration
0
4.49
0
0
0
0
0
0
11.1
7.59
0
0



migration
0
0
0
0
0
0
0
0
4.46
6.05
0
0



migration
0
0
0
0
0
0
0
0
0
3.46
0
0



chemokine
0
0
0
0
0
0
0
0
5.38
6.25
3.95
0



chemokine
0
0
0
0
0
0
0
0
4.48
7.84
0
0



chemokine
0
0
0
0
0
0
0
0
5.3
9.13
0
0



chemokine
0
0
0
0
0
0
0
0
0
9
0
0



chemotaxis
5.33
16.1
0
0
0
11.8
10.2
3.38
31.4
20.7
11
0



chemotaxis
0
0
0
0
0
3.46
5.4
0
17.6
6.87
0
0



chemotaxis
0
15.7
0
0
0
10.7
0
0
24.2
22
8.03
0



chemotaxis
0
5.48
0
0
0
7.96
0
0
13.8
13.1
3.91
0



chemotaxis
0
0
0
0
0
3.41
5.38
0
17.5
6.8
0
0



chemotaxis
0
5.83
0
0
0
3.47
0
0
15.3
7.42
4.44
0



chemotaxis
0
6.49
0
0
0
3.65
0
0
14.7
7.6
3.7
0



chemotaxis
0
0
0
0
0
3.06
0
0
10.9
8.31
3.31
0



chemotaxis
0
0
0
0
0
5.59
0
0
11.3
9.92
0
0



chemotaxis
0
0
0
0
0
0
0
0
6.79
6.5
3.62
0



chemotaxis
0
0
0
0
0
0
0
0
0
3.15
0
0



stress
23.7
57.7
3.9
12.9
0
41.9
3.75
0
47.4
58.8
63.8
7.7



stress
0
28.5
0
0
0
17.4
0
0
17.9
40.8
45.6
0



stress
4.33
9.02
0
0
0
7.06
0
0
0
5.39
0
0



defense response
15.3
57.8
0
0
0
39.7
8.88
0
64.1
90.6
71.2
0



defense response
8.02
29.6
0
0
0
17.8
0
0
29.2
57.5
50.3
0



defense response
5.46
24.9
0
0
0
11
0
0
19.9
41.3
33.9
0



defense response
0
18.4
0
0
0
22.2
0
0
19.3
48.9
46.5
0



defense response
0
5.98
0
0
0
7.44
0
0
11.4
15.8
18.6
0



defense response
0
6.22
0
0
0
3.07
0
0
7.28
6.93
8.29
0



defense response
0
0
0
0
0
6.08
0
0
0
4.67
5.54
0



defense response
0
0
0
0
0
5.29
0
0
0
25.8
20
0



defense response
0
0
0
0
0
0
0
0
0
6.75
6.02
0



defense response
0
0
0
0
0
0
0
0
0
4.62
4.72
0



defense response
0
0
0
0
0
0
0
0
0
0
0
0



immune response
8.95
46.1
0
0
0
22.5
12.8
0
45.5
79.8
54.5
0



immune response
0
20.8
0
0
0
7.16
0
0
15.2
35.5
27.8
0



immune response
0
19.5
0
0
0
6.54
0
0
12.2
39.5
34.2
0



immune response
0
11.9
0
0
0
0
0
0
13.2
23.2
14.6
0



immune response
0
9.97
0
0
0
0
0
0
13.2
20.5
12.6
0



immune response
0
12.1
0
0
0
0
0
0
14.3
23.7
15.1
0



immune response
0
0
0
0
0
0
0
0
0
3.8
0
0



immune response
0
0
0
0
0
0
0
0
0
4.58
0
0



innate immune
0
33.7
0
0
0
13.1
6.73
0
24.9
59.5
49.6
0



response



innate immune
0
19.7
0
0
0
7.53
0
0
12.9
40.7
37.9
0



response



innate immune
0
16.7
0
0
0
3.39
0
0
11.3
27.7
22.9
0



response



innate immune
0
16.2
0
0
0
0
0
0
10.3
23.3
17.9
0



response



innate immune
0
14.3
0
0
0
0
0
0
11.2
18.5
13.8
0



response



innate immune
0
0
0
0
0
0
0
0
0
7.74
8.91
0



response



adaptive immune
0
8.99
0
0
0
0
0
0
10.5
11.9
4.42
0



response



adaptive immune
0
8.71
0
0
0
0
0
0
11.7
13
6.23
0



response



adaptive immune
0
3.56
0
0
0
0
0
0
0
7.16
4.06
0



response



adaptive immune
0
0
0
0
0
0
0
0
0
6.84
0
0



response



adaptive immune
0
0
0
0
0
0
0
0
0
0
0
0



response



IFN alpha
0
4.54
0
0
0
0
0
0
0
13.3
10.2
0



IFN alpha
0
0
0
0
0
0
0
0
0
4.7
3.15
0



IFN alpha
0
0
0
0
0
0
0
0
0
4.95
0
0



IFN alpha
0
0
0
0
0
0
0
0
0
5.95
0
0



IFN alpha
0
0
0
0
0
0
0
0
0
5.3
0
0



IFN beta
0
9.65
0
0
0
15.2
0
0
7.13
17.2
17.9
0



IFN beta
0
17.3
0
0
0
23.8
0
0
11.6
23
23.9
0



IFN beta
0
0
0
0
0
0
0
0
0
8.94
6.66
0



IFN beta
0
0
0
0
0
0
0
0
0
7.54
5.68
0



IFN beta
0
0
0
0
0
0
0
0
0
9.35
7.51
0



IFN gamma
0
11
0
0
0
13.3
0
0
3.17
22.3
15.7
0



IFN gamma
0
0
0
0
0
8.03
0
0
0
10.5
11.2
0



IFN gamma
0
0
0
0
0
0
0
0
0
5.43
0
0



IFN gamma
0
0
0
0
0
0
0
0
0
5.32
0
0



interleukin-6
0
3.6
0
0
0
5.33
0
0
7.44
9.62
7.8
0



interleukin-6
0
4.17
0
0
0
5.87
0
0
7.82
10.2
6.67
0



interleukin-6
0
0
0
0
0
0
0
0
6.14
5.06
4.53
0



interleukin-12
0
8.76
0
0
0
0
0
0
9.58
6.82
0
0



interleukin-12
0
9.49
0
0
0
0
0
0
10.1
7.39
0
0



interleukin-12
0
3.24
0
0
0
0
0
0
0
4.28
0
0








text missing or illegible when filed indicates data missing or illegible when filed







In the 40 modules of the adjuvant gene space, high z-scored genes in G1 from LV and SP were almost exclusively found in the modules of M20LV (annotated as “response to biotic stimulus”) and M23SP (annotated as “response to virus”), respectively (FIG. 6). Similarly, in LNs, the genes preferentially upregulated in the 5 reference adjuvants of G1 (cdiGMP, cGAMP, DMXAA, PolyIC, and R848) were found in the M40LN module (annotated as “immune system process”) (FIG. 6). The associated genes from the other groups (G2, G3, G4, G5, and G6) were distributed in several modules and clearly differed from the G1-associated modules (FIG. 6). TargetMine (Table 18) and Ingenuity Pathways Analysis™ (IPA) upstream cytokine (Table 19) analyses provided further details of the adjuvant group-associated biological features.













TABLE 18





Organ
group
Term
Pvalue
Genelist







LV
G1
Interferon Signaling
  1E−08
Uba7, Irf7, Irf9, Stat1, Trim12a, Trim12c, Trim30d, Pml, Oas1c, Ifit1


LV
G1
Interferon gamma signaling
6.4E−07
Irf7, Irf9, Stat1, Trim12a, Trim12c, Trim30d, Pml, Oas1c


LV
G1
response to interferon-beta
1.7E−06
Iigp1, Xaf1, Stat1, Igtp, Ifit3, Ifit1


LV
G1
Influenza A
  2E−06
Irf7, Irf9, Eif2ak2, Stat1, Adar, Ifih1, Ddx58, Pml, Rsad2


LV
G1
response to cytokine
3.1E−06
Gbp7, Iigp1, Xaf1, Irf7, Eif2ak2, Stat1, Adar, Parp9, Igtp, Pml, Ifit3, Ifit1


LV
G1
Measles
8.6E−06
Ccnd2, Irf7, Irf9, Eif2ak2, Stat1, Adar, Ifih1, Ddx58


LV
G1
Herpes simplex infection
1.3E−05
Irf7, Irf9, Eif2ak2, Stat1, Ifih1, H2-T10, Ddx58, Pml, Ifit1


LV
G1
cellular response to interferon-beta
4.6E−05
Iigp1, Stat1, Igtp, Ifit3, Ifit1


LV
G1
response to other organism
6.6E−05
Gbp7, Iigp1, Eif2ak2, Stat1, Adar, Ifih1, Ddx58, Dhx58, Cd47, Pml, Ifit3, Rsad2,






Ifit1


LV
G1
response to stress
9.4E−05
Gbp7, Ascc3, Iigp1, Pgap2, Irf7, Zfyve26, Eif2ak2, Shisa5, Stat1, Adar, Trim12c,






Ifih1, Irgm1, Ddx58, Parp9, Dhx58, Cd47, Pml, Rsad2


LV
G1
response to virus
0.0003
Eif2ak2, Adar, Ifih1, Ddx58, Dhx58, Pml, Ifit3, Rsad2, Ifit1


LV
G1
innate immune response
0.00037
Gbp7, Irf7, Stat1, Adar, Trim12c, Ifih1, Ddx58, Parp9, Dhx58, Pml


LV
G1
cellular response to cytokine stimulus
0.00108
Gbp7, Iigp1, Irf7, Stat1, Adar, Igtp, Pml, Ifit3, Ifit1


LV
G1
defense response
0.0013
Gbp7, Iigp1, Irf7, Stat1, Adar, Trim12c, Ifih1, Irgm1, Ddx58, Parp9, Dhx58, Cd47,






Pml, Rsad2


LV
G1
symbiosis, encompassing mutualism through parasitism
0.00239
Gbp7, Eif2ak2, Stat1, Adar, Pml, Rsad2


LV
G1
Hepatitis C
0.00472
Irf7, Irf9, Eif2ak2, Stat1, Ddx58, Ifit1


LV
G1
type 1 interferon production
0.00553
Irf7, Irf9, Ifih1, Ddx58, Dhx58


LV
G1
regulation of innate immune response
0.00809
Irf7, Adar, Trim12c, Ddx58, Parp9, Dhx58, Rsad2


LV
G1
Immune System
0.00897
Uba7, Irf7, Irf9, Stat1, Trim12a, Trim12c, Ifih1, Trim30d, Ddx58, Mndal, Pml,






Oas1c, Ifit1


LV
G1
Innate Immune System | Adaptive Immune System |
0.0118
Ccnd2, Uba7, Irf7, Irf9, Eif2ak2, Stat1, Trim12a, Adar, Trim12c, Ifih1, H2-T10,




Hemostasis | Pathways in cancer | Developmental

Trim30d, Irgm1, Ddx58, Lama3, Dhx58, Cd47, Mndal, Igtp, Pml, Oas1c, Rsad2, Ifit1




Biology | PI3K-Akt signaling pathway | Signaling




by Rho GTPases


LV
G1
TRAF3-dependent IRF activation pathway
0.01547
Irf7, Ifih1, Ddx58


LV
G1
immune system process
0.01883
Gbp7, Irf7, Eif2ak2, Stat1, Adar, Trim12c, Ifih1, Ddx58, Parp9, Dhx58, Cd47, Pml,






Rsad2


LV
G1
positive regulation of type I interferon production
0.02291
Irf7, Ifih1, Ddx58, Dhx58


LV
G1
multi-organism cellular process
0.02396
Eif2ak2, Stat1, Adar, Ddx58, Dhx58, Pml, Rsad2


LV
G1
negative regulation of viral process
0.03055
Eif2ak2, Stat1, Adar, Pml, Rsad2


LV
G1
negative regulation of multi-organism process
0.03779
Eif2ak2, Stat1, Adar, Dhx58, Pml, Rsad2


LV
G1
regulation of defense response
0.05868
Irf7, Adar, Trim12c, Ddx58, Parp9, Dhx58, Cd47, Pml, Rsad2


LV
G1
immune response
0.06777
Gbp7, Irf7, Stat1, Adar, Trim12c, Ifih1, Ddx58, Parp9, Dhx58, Pml, Rsad2


LV
G1
pattern recognition receptor signaling pathway
0.08153
Irf7, Trim12c, Ddx58, Dhx58, Rsad2


LV
G1
Antiviral mechanism by IFN-stimulated genes
0.10767
Uba7, Stat1, Ifit1


LV
G1
response to interferon-alpha
0.11458
Eif2ak2, Adar, Ifit1


LV
G1
multi-organism process
0.23618
Gbp7, Iigp1, Eif2ak2, Stat1, Adar, Ifih1, Ddx58, Dhx58, Cd47, Tdrd7, Pml, Ifit3,






Rsad2, Ifit1


LV
G1
positive regulation of defense response
0.27807
Irf7, Trim12c, Ddx58, Dhx58, Cd47, Rsad2


LV
G1
response to organic substance
0.57043
Hap1, Gbp7, Iigp1, Xaf1, Irf7, Eif2ak2, Stat1, Adar, Ddx58, Parp9, Igtp, Pml,






Ifit3, Ifit1


LV
G1
Toxoplasmosis
0.60505
Stat1, Irgm1, Lama3, Igtp


LV
G1
regulation of response to stress
0.63165
Pgap2, Irf7, Eif2ak2, Adar, Trim12c, Ddx58, Parp9, Dhx58, Cd47, Pml, Rsad2


LV
G1
regulation of response to stress
0.63165
Pgap2, Irf7, Eif2ak2, Adar, Trim12c, Ddx58, Parp9, Dhx58, Cd47, Pml, Rsad2


LV
G1
negative regulation of viral life cycle
0.74299
Eif2ak2, Adar, Pml, Rsad2


LV
G1
mRNA Editing
0.75953
Adar


LV
G1
NF-kB activation through FADD/RIP-1 pathway mediated
0.82806
Ifih1, Ddx58




by caspase-8 and -10


LV
G1
Ubiquitin mediated proteolysis | Cell cycle
0.8748
Ccnd2, Uba7, Shisa5, Pml


LV
G1
defense response to other organism
0.9983
Gbp7, Iigp1, Adar, Ddx58, Dhx58, Pml, Rsad2


LV
G2
cellular protein modification process
0.25047
Acer2, Thpo, Mylip, Gan, Nceh1, Plcb1, Tirap, Egfr, Clock, Ibtk, Ppap2a, Map3k3,






Nedd4l, Pdgfd, St5, Stk38l, Nlrp12, Gdf10, St3gal4, Braf, Garem, Metap2, Igfbp3,






Pofut2, Galnt2, Tbl1xr1, B3galt1, Fbxo31, Rlim


LV
G2
Metabolism of lipids and lipoproteins
0.25919
Acer2, Acox1, Crls1, Clock, Ppap2a, Slc25a20, Gpd2, Agpat3, Tbl1xr1, Insig2, Mgll,






Lpgat1


LV
G2
Vesicle-mediated transport
0.62121
Cd163, Ap2m1, Gja1, Tgoln1, Saa2, Saa1


LV
G2
O-linked glycosylation
0.90895
St3gal4, Pofut2, Galnt2, Galnt15


LV
G2
Fatty acid, triacylglycerol, and ketone body metabolism
0.98433
Clock, Slc25a20, Gpd2, Agpat3, Tbl1xr1


LV
G3
Innate Immune System
0.00399
Sirpa, Itgb2, Icam2, Traf2, Traf3, Rps6ka5, Clec4e, Kit, Cd300lb


LN
G1
immune system process
5.1E−07
Azi2, Pik3ap1, Itgb2, Coro1a, Tmem176a, Traf2, Traf3, Cd55, Clec4e, Kit, Cd300lb,






Trem3, Tbx1, Sh3pxd2a


LV
G3
Immune System
0.06852
Sirpa, Itgb2, Icam2, Traf2, Traf3, Rps6ka5, Clec4e, Kit, Cd300lb, Dync1li2


LV
G3
Innate Immune System | Adaptive Immune System |
0.07202
Sirpa, Itgb2, Coro1a, Icam2, Iqgap1, Traf2, Traf3, Rps6ka5, Clec4e, Kit, Pfn2,




Hemostasis | Pathways in cancer | Developmental

Cd300lb, Plek, Ptgir, Abcc1, Dync1li2, Mapre1




Biology | PI3K-Akt signaling pathway | Signaling




by Rho GTPases


LV
G3
immune effector process
0.11451
Cd93, Coro1a, Traf2, Traf3, Cd55, Clec4e, Kit, Cd300lb, Trem3


LV
G3
Cytokine-cytokine receptor interaction | Endocytosis |
0.1413
Itgb2, Coro1a, Icam2, Traf2, Traf3, Cd55, Kit, Dync1li2




Herpes simplex infection


LV
G3
cytoskeleton organization
0.16195
Sirpa, Coro1a, Kit, Pfn2, Plek, Spast, Dync1li2, Mapre1


LV
G3
cell adhesion
0.29629
Sirpa, Cc93, Itgb2, Coro1a, Icam2, Clec4e, Kit, Plek


LV
G3
leukocyte mediated immunity
0.32576
Coro1a, Traf2, Cd55, Kit, Cd300lb, Trem3


LV
G3
vesicle-mediated transport
0.48714
Sirpa, Pkdcc, Coro1a, Kit, Pfn2, Plek, Spast


LV
G3
cellular localization
0.70808
Itgb2, Pkdcc, Coro1a, Os9, Traf2, Ildr2, Clec4e, Slc25a37, Kit, Pfn2, Plek, Trem3,






Snx13, Spast, Dync1li2, Mepre1


LV
G4
Protein processing in endoplasmic reticulum
3.3E−24
Rnf185, Sec61b, Hsp90b1, Srp68, Sel1l, Syvn1, Fbxo6, Ssr1, Ssr2, Dnajc5, Mogs, Spcs2,






Spcs3, Ssr4, Atf6, Herpud1, Stt3a, Sec24d, Yod1, Rpn1, Derl1, Pdia3, Sar1a, Ube2j1,






Vcp, Rrbp1, Vimp, Serp1, Atf6b, Dnajc10, Sec23b, Gm10177, Wfs1, Srp9, Ddost, Hspa5,






Pdia6, Sec61a1, Dnajb11, Srp19, Pdia4, Srprb, Preb, Sec31a, Ckap4, Dnajc3, Txndc5,






Ufd1l, Edem2, Edem3, Edem1, Srp72, Hyou1, Srpr, Man1a, Sec13, Plaa, Hsp90aa1, Srp54a,






Ero1l, Calr, Ube2g2


LV
G4
Protein processing in endoplasmic reticulum
3.3E−20
Rnf185, Sec61b, Hsp90b1, Sel1l, Syvn1, Fbxo6, Ssr1, Ssr2, Dnajc5, Mogs, Ssr4, Atf6, Herpud1,






Stt3a, Sec24d, Yod1, Rpn1, Derl1, Pdia3, Sar1a, Ube2j1, Vcp, Rrbp1, Vimp, Atf6b, Dnajc10,






Sec23b, Gm10177, Wfs1, Ddost, Hspa5, Pdia6, Sec61a1, Dnajb11, Pdia4, Preb, Sec31a, Ckap4,






Dnajc3, Txndc5, Ufd1l, Edem2, Edem3, Edem1, Hyou1, Man1a, Sec13, Plaa, Hsp90aa1,






Ero1l, Calr, Ube2g2


LN
G1
response to stress
0.8518
Hopx, Aen, Mybbp1a, Tbl2, Cyp1b1, Selp, Pik3ap1, Bfar, Slamf8, Atg13, Cd28, Cebpg, Il1rn,






Arl6ip5, Hck, Ednra, Btg2, Map3k3, B4galt1, Il1r1, Rab12, Ints7, Qsox1, Hsp90b1, Fktn, Errfi1,






Cxcl9, Ung, Ctgf, Pdcd10, Sel1l, Actg1, Pnp, Hif1a, Eif4e, Dap, Marveld3, Lrrc8a, Lcn2, Syvn1,






Braf, Scly, Creb3, Senp2, Nod1, Apex1, Clec4d, Hilpda, Pfkp, Cd55, Serinc3, Psma1, Atp7a,






Adora1, Usp1, Tnfaip6, Irf8, Trib1, Grb2, Rbm18, Atf6, Atf3, Herpud1, Slc11a2, Slc11al, Exosc4,






Lbp, Skil, Polh, Uba5, Apc5, Ap1g1, Yod1, Fgr, Rcan2, Fgb, S100a8, Ppp4c, A2m, Slc35c1,






Fen1, Ifitm2, Ifitm1, Derl1, Atg9b, Pdia3, Itpr1, Fcer1g, Lnp, Srebf2, Ufm1, Cln8, H2afx, Vcp,






Dab2, Tnfrsf12a, Scfd1, Syk, Hmga2, Vimp, Adam17, Asns, Rraga, Hyal1, Ddx39, Cd44, Myh9,






Serp1, Ctla2a, Atf6b, Reg3b, Creb3l2, Dnajc10, F13a1, Plek, Sdf2l1, Gnl1, Wfs1, Supt5, Hipk3,






Txnl1, Gas6, Trim27, Il12rb1, Irak2, Iigp1, Sdc4, Itgam, Lig3, Gata6, Ncf1, Grina, Smarcad1,






Pld1, Rasgrp2, Tirap, Trp53inp2, Tlr1, Apoa4, Apoa5, Trip12, Gigyf2, Mt2, Mt1, Hnf4a, Adam9,






Hspa5, Jun, Pdia6, Nlrp12, Ubxn2b, Cox8a, Pdia4, Wipi1, Ifrd1, Ptpn2, Ptpn1, Ufl1, Casp4,






Parp2, Parp3, Il17ra, Il4ra, Ddx1, Thoc1, Zfp830, Fbxo31, Il1a, Nabp1, Eya3, Psen1, Ap5s1,






Erp44, Cdip1, Itgb3, Ccl17, Vav1, Ccl19, Wdr45b, Atg16l2, Eprs, Dnajc3, Trem3, Socs3, Txndc5,






Rps6ka3, Tnfrsf1b, Tnfrsf1a, Cpeb2, Rint1, Tmem173, Rab1, Ift20, Hyou1, Cdkn1a,






Gadd45b, Gadd45g, Sbno2, Sod2, Snap23, Pik3c2a, Coro1a, Pycard, Map1lc3b, Trp63, Fn1,






Clock, Alox12, Chmp4b, Ccr1, Txlna, Plaa, Clec4n, Seb1l, Cr1l, Fabp4, Hsp90aa1, Ero1l, Fcgr3,






Rab33b, Psmd14, Scara5, Calr, Polr3d, Nfkbi2, Prkce, Acot11, Crnkl1, Irg1, Myo1f, S100a9


LV
G4
response to endoplasmic reticulum stress
9.9E−12
Selk, Tbl2, Bfar, Sel1l, Syvn1, Creb3, Serinc3, Atf6, Atf3, Herpud1, Uba5, Yod1, Derl1, Pdia3, Itpr1,






Ufm1, Vimp, Serp1, Atf6b, Creb3l2, Dnajc10, Sdf2l1, Wfs1, Grina, Hspa5, Jun, Pdia6, Pdia4, Ptpn2,






Ptpn1, Ufl1, Casp4, Erp44, Dnajc3, Txndc5, Hyou1, Ero1l


LV
G4
macromolecule metabolic process
6.9E−08
Lars, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Bfar, Pdgfc, Mbd1, Sec61b, Prkag2,






Snx6, Utp6, Dhx30, Garem, Mlx, Cd28, Cebpg, Timp1, Cebpd, Ddx52, Mettl1, Mettl3, Ezr, Hnrnpa2b1,






Senp6, Hnrnpab, Polr1e, Pim3, Arl6ip5, Metap2, Hck, Etf1, Btg2, Ntmt1, Dhx40, Anapc4,






Wdr77, Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd, Tcf25, Rab12, Ints7, Ints5,






Rpf2, Qsox1, H5p90b1, Plcl1, Fktn, Pus3, Errfi1, Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1,






Mphosph8, Klf17, Pnp, Hif1a, Mphosph6, Smarce1, Eif4e, Dap, Marveld3, Sf3b4, Eif3d, Syvn1, Prg4,






Srsf2, Braf, Timp3, Creb3, Senp2, Efna1, Ell2, Nod1, Fbxo6, Ctif, Gmeb1, Ibtk, Apex1, Ube2s,






Trim39, Csnk1a1, Pcyox1, Npm3, Mmp8, Tasp1, Mmp13, Pcid2, Ddx21, Kdm6a, Cwc27, Rlim,






Dhx38, Cd55, Dok2, Psma1, Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Recql, Mrpl51, Mbnl2, Ell, Tbp,






Irf8, Trib1, Dhx35, Grb2, Cebp2, Atf6, Ints2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Tpp2,






Mmp9, Exosc1, Exosc4, Cad, Lbp, Serpina3k, Serpina3m, Serpina3n, Serpina3g, Snip1, Skil,






Stt3a, Lpin1, Ddx50, Polh, Gtf2f1, Nup98, Uba5, Apcs, Jmjd8, Lpar1, Foxk2, Yod1, Fgr, Arf4, Camkk2,






Ube2f, Fgb, Rpn1, Tgm2, Uspl1, S100a8, Tmsb4x, Ppp4c, A2m, Egf, Eif4g2, Fkbp11, Stag1,






Nedd4l, C1galt1, Fen1, Wdr61, Ebna1bp2, Trps1, Derl1, Spin1, Iars, Traf7, Pdia3, Acer2, Dpy19l1,






Saa3, Rars, Usp14, Tbl1xr1, Srebf2, Ufm1, Suco, Cul5, Cln8, Trappc2, H2afx, Ube2j1, Vcp, Scnm1,






Mcm2, Dab2, Dusp2, Asb4, Syk, St6gal1, Hmga2, Vimp, Adam17, Srgn, Hyal1, Eif4a3, Ddx39, Cd44,






Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1, Dzip3, Prdm1, Atf6b, Iqgap1, Pcsk5, Creb3l2,






Wdr12, Dnajc10, F13a1, Nelfa, Rbm39, Sox9, Nus1, Sdf2l1, Odc1, Wfs1, Supt5, Supt6, Hipk3,






Tmem165, Helb, Ngp, Txnl1, Gas6, Trim27, Rac2, Ppp2r1a, Il12rb1, Crem, Paip2b, Irak2, Hist1h3b,






Gemin8, Ngdn, Lyve1, Cdc73, Parn, Lig3, Gata6, Exog, Tspyl2, Smarcad1, Dis3, Gfpt1, Rasgrp1,






Nop58, Tirap, Trp53inp2, Fkbp5, Tlr1, Med17, Apoa4, Apoa5, Trip12, Hars, Zfp869, Ddost, Ick,






Gigyf2, Hnf4a, Adam9, Zfp160, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3, Jun, Pdia6, Dnajb11,






Nlrp12, Nudt21, Papola, Flot1, Crp, Ubxn2b, Denr, Cars2, Capn10, Farsb, Taf2, Nup62, Vars,






Pdia4, Wipi1, Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Ap1ar, Abca1, Ufl1, Ddx49, Casp4, Znrf1, Plaur,






Nceh1, Preb, Parp2, Parp3, Mppe1, Zbtb16, Zbtb21, Med25, Eif2b4, Ddx1, Thoc2, Thoc1, Elp3,






Inhbe, B3galt6, B3galt1, Rnps1, Tfb2m, Cdk7, Dph5, Exosc10, Zfp830, Brd8, Fbxo31, Il1a, Alg12,






Rps9, Nabp1, Kars, Stk17b, Son, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Sec22b, Psen1, Ap5s1,






Erp44, Exosc3, Hltf, Zfand2a, Cd38, Piga, Itgb3, Itgb2, Arid5a, Ccl19, Khsrp, Gan, Gtf2a2, Nfia,






Wdr45b, Atg16l2, Trmt61a, Kctd10, Eprs, D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1, Trem3,






Socs3, Dr1, Camk2b, Polr1a, Ubxn4, Trim3, Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2,






Edem3, Trib2, Edem1, Tmem173, Rab1, Cdkn1a, Taf4b, Nbas, Gadd45b, Gadd45g, Ppp1r11,






Magoh, Sbno2, Sod2, Msn, Pik3c2a, Nol9, Vegfc, Pycard, Trp63, Fn1, Stx5a, Clock, Bbs4, B3galnt2,






St3gal5, St3gal4, Pomgnt1, Chmp4b, Ppm1l, Ccr1, Cr1l, Fabp4, Hsp90aa1, Ero1l, Srsf1, Fcgr3,






Srsf3, Psmd14, Scara5, Zfp27, Serpina10, Calr, Pgm3, Rusc1, Nop56, Pofut2, Polr3k, Galnt2, Tbrg1,






Taf1, Pspc1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2, Lats2, Prkch, Ears2, Prkce, Klk1b24, Crnkl1,






Irg1, Rbm42, Larp7, Anapc16, Il6st, Ikbkap, Nif3l1, S100a9, Ube2g2


LV
G4
cellular macromolecule metabolic process
7.5E−08
Lars, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Bfar, Pdgfc, Mbd1, Sec61b, Prkag2,






Snx6, Utp6, Dhx30, Garem, Mlx, Cd28, Cebpg, Timp1, Cebpd, Ddx52, Mettl1, Mettl3, Ezr,






Hnrnpa2b1, Senp6, Hnrnpab, Polr1e, Pim3, Arl6ip5, Metap2, Hck, Etf1, Btg2, Ntmt1, Dhx40, Anapc4,






Wdr77, Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd, Tcf25, Rab12, Ints7, Ints5,






Rpf2, Qsox1, Hsp90b1, Plcl1, Fktn, Pus3, Errfi1, Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1,






Mphosph8, Klf17, Pnp, Hif1a, Mphosph6, Smarce1, Eif4e, Dap, Marveld3, Sf3b4, Eif3d, Syvn1, Srsf2,






Braf, Timp3, Creb3, Senp2, Efna1, Ell2, Nod1, Fbxo6, Ctif, Gmeb1, Ibtk, Apex1, Ube2s, Trim39,






Csnk1a1, Pcyox1, Npm3, Tasp1, Mmp13, Pcid2, Ddx21, Cwc27, Rlim, Dhx38, Dok2, Psma1,






Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Recql, Mrpl51, Mbnl2, Ell, Tbp, Irf8, Trib1, Dhx35, Grb2,






Cebp2, Atf6, Ints2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Mmp9, Exosc1, Exosc4, Cad,






Serpina3k, Serpina3m, Serpina3n, Serpina3g, Snip1, Skil, Stt3a, Lpin1, Ddx50, Polh, Gtf2f1,






Nup98, Uba5, Apcs, Jmjd6, Lpar1, Foxk2, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2, Uspl1,






S100a8, Tmsb4x, Ppp4c, A2m, Egf, Eif4g2, Fkbp11, Stag1, Nedd4l, C1galt1, Fen1, Wdr61, Ebna1bp2,






Trps1, Derl1, Spin1, Iars, Traf7, Pdia3, Acer2, Dpy19l1, Saa3, Rars, Usp14, Thl1xr1, Srebf2,






Ufm1, Cul5, Cln8, Trappc2, H2afx, Ube2j1, Vcp, Scnm1, Mcm2, Dab2, Dusp2, Asb4, Syk, St6gal1,






Hmga2, Vimp, Adam17, Eif4a3, Ddx39, Cd44, Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1, Dzip3,






Prdm1, Atf6b, Iqgap1, Pcsk5, Creb3l2, Wdr12, Dnajc10, F13a1, Nelfa, Rbm39, Sox9, Nus1,






Sdf2l1, Wfs1, Supt5, Supt6, Hipk3, Tmem165, Helb, Ngp, Txnl1, Gas6, Trim27, Rac2, Ppp2r1a,






Il12rb1, Crem, Paip2b, Irak2, Hist1h3b, Gemin8, Ngdn, Cdc73, Parn, Lig3, Gata6, Exog, Tspyl2,






Smarcad1, Dis3, Rasgrp1, Nop58, Tirap, Trp53inp2, Fkbp5, Tlr1, Med17, Trip12, Hars, Zfp869,






Ddost, Ick, Gigyf2, Hnf4a, Adam9, Zfp160, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3, Jun, Pdia6,






Dnajb11, Nlrp12, Nudt21, Papola, Flot1, Ubxn2b, Denr, Cars2, Farsb, Taf2, Nup62, Vars, Pdia4,






Wipi1, Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Ap1ar, Abca1, Ufl1, Ddx49, Znrf1, Plaur, Nceh1, Preb,






Parp2, Parp3, Mppe1, Zbtb15, Zbtb21, Med25, Eif2b4, Ddx1, Thoc1, Elp3, Inhbe, B3galt1, Rnps1,






Tfb2m, Cdk7, Dph5, Exosc10, Zfp830, Brd8, Fbxo31, Il1a, Alg12, Rps9, Nabp1, Kars, Stk17b,






Son, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Psen1, Ap5s1, Erp44, Exosc3, Hltf, Zfand2a, Cd38,






Piga, Itgb3, Itgb2, Arid5a, Ccl19, Khsrp, Gan, Gtf2a2, Nfia, Wdr45b, Atg16l2, Trmt61a, Kctd10,






Eprs, D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b, Polr1a, Ubxn4, Trim3,






Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2, Edem3, Trib2, Edem1, Tmem173, Rab1,






Cdkn1a, Taf4b, Nbas, Gadd45b, Gadd45g, Ppp1r11, Magoh, Sbno2, Sod2, Pik3c2a, Nol9, Vegfc,






Pycard, Trp63, Fn1, Clock, B3galnt2, St3gal4, Pomgnt1, Chmp4b, Ppm1l, Ccr1, Cr1l, Fabp4, Hsp90aa1,






Ero1l, Srsf1, Srsf3, Psmd14, Zfp27, Serpina10, Calr, Pgm3, Rusc1, Nop56, Pofut2, Polr3k,






Galnt2, Tbrg1, Taf1, Pspc1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2, Lats2, Prkch, Ears2, Prkce, Crnkl1,






Irg1, Rbm42, Larp7, Anapc16, Il6st, Ikbkap, Nif3l1, S100a9, Ube2g2


LV
G4
cellular response to stress
1.4E−07
Rad1, Selk, Mybbp1a, Tbl2, Cyp1b1, Bfar, Atg13, Cebpg, Arl6ip5, Btg2, Map3k3, Rab12, Ints7,






Qsox1, Fktn, Errfi1, Ung, Ctgf, Pdcd10, Sel1l, Pnp, Hif1a, Dap, Marveld3, Syvn1, Braf, Scly, Creb3,






Senp2, Nod1, Apex1, Pfkp, Serinc3, Atp7a, Usp1, Trib1, Grb2, Atf6, Atf3, Herpud1, Slc11a2, Skil,






Polh, Uba5, Yod1, Ppp4c, Slc35c1, Fen1, Derl1, Atg9b, Pdia3, Itpr1, Srebf2, Ufm1, H2afx, Vcp,






Dab2, Scfd1, Syk, Hmga2, Vimp, Asns, Rraga, Ddx39, Cd44, Serp1, Atf6b, Creb3l2, Dnajc10, Sdf2l1,






Gnl1, Wfs1, Supt5, Hipk3, Txnl1, Gas6, Lig3, Gata6, Grina, Smarcad1, Rasgrp1, Tirap, Trp53inp2,






Apoa4, Apoa5, Trip12, Gigyf2, Hspa5, Jun, Pdia6, Ubxn2b, Cox8a, Pdia4, Wipi1, Ptpn2, Ptpnl,






Ufl1, Casp4, Parp2, Parp3, Ddx1, Thoc1, Zfp830, Fbxo31, Il1a, Nabp1, Eya3, Psen1, Ap5s1, Erp44,






Cdip1, Ccl19, Wdr45b, Atg16l2, Dnajc3, Txndc5, Tnfrsf1b, Tnfrsf1a, Cpeb2, Rint1, Rab1, Ift20,






Hyou1, Cdkn1a, Gadd45b, Gadd45g, Sod2, Pycard, Map1lc3b, Trp63, Clock, Chmp4b, Txlna,






Ero1l, Rab33b, Psmd14, Scara5, Calr, Prkce, Crnkl1


LV
G4
Asparagine N-linked glycosylation
3.8E−07
B4galt1, Mvd, Mcfd2, Mogs, Nans,Sec24d, Pdia3, St6gal1, Gfpt1, Preb, Alg12, Sec31a, Gne,






St3gal3, Edem2, Edem3, Edem1, Gmppa, Man1a, Sec13, Dhdds, St3gal5, St3gal4, Uap1, Calr,






Pgm3, Gmppb


LV
G4
Metabolism of proteins
4.3E−07
Sec61b, Etf1, Pigm, B4galt1, Mvd, Srp68, Eif4e, Vbp1, Eif3d, Slc30a5, Timm21, Senp2, Fbxo6,






Mcfd2, Ssr1, Ssr2, Mogs, Nans, Spcs2, Spcs3, Ssr4, Atf6, Nup98, Sec24d, Gspt2, Rpn1, Cct3, Stag1,






C1galt1, Pdia3, Ranbp2, St6gal1, Serp1, Slc30a7, Nupl2, Gas6, Srp9, Gfpt1, Ddost, Kif5b, Sec61a1,






Nupl1, Srp19, Nup62, Srprb, Plaur, Preb, Eif2b4, Inhbe, Dph5, Alg12, Rps9, Sec31a, Plga, Gne,






StBgal3, Edem2, Edem3, Edem1, Srp72, Gmppa, Pfdn4, Srpr, Man1a, Sec13, Dhdds, St3gal5,






St3gal4, Seh1l, Srp54a, Ero1l, Uap1, Calr, Pgm3, Nop56, Pofut2, Galnt2, Gmppb, Klk1b24


LV
G4
vesicle-mediated transport
6.3E−07
Yipf5, Golga5, Snx6, Ezr, Rinl, Stx6, Cog6, Hck, B4galt1, Rab12, Stx12, Stx18, Arcn1, Uso1,






Krt18, Braf, Csnk1a1, Gosr2, Aak1, Marco, Irf8, Grb2, Blzf1, Slc11a1, Pdzd11, Lbp, Cd14,






Jmjd6, Ap1g1, Fgr, Fgb, Tgm2, Tmed9, Ap2a2, Sar1a, Vamp4, Rab35, Fcer1g, Dab2, Scfd1, Syk,






Ap4e1, Copb2, Myh9, Zw10, Rab5b, Creb3l2, Plek, Chic2, Gas6, Trim27, Rac2, Mfsd2a, Golga4,






Mon1b, Sdc4, Rasgrp1, Golph3, Flot1, Wipl1, Ptpn1, Ap1ar, Abca1, Mppe1, Il4ra, Bet1l, Ap3d1,






Syt12, Sec22b, Hook1, Psen1, Vav1, Ccl19, Snx10, S100a10, Cltb, Txndc5, Rint1, Rab1, C2cd5,






Snap23, Sec13, Pik3c2a, Coro1a, Pycard, Stx5a, Chmp4b, Ccr1, Fcgr3, Rab33b, Scara5, Calr,






Myo1f, Arfgap1


LV
G4
primary metabolic process
8.7E−07
Cers6, Lars, Sdsl, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Bfar, Pdgfc, Mbd1,






Sec61b, Prkag2, Snx6, Utp6, Dhx30, Garem, Tat, Mlx, Cd28, Cebpg, Timp1, Cebpd, Ddx52,






Mettl1, Mettl3, Hnrnpa2b1, Senp6, Hnrnpab, Polr1e, Il1rn, Pim3, Arl6ip5, Metap2, Cog6, Hck,






Etf1, Ednra, Btg2, Ntmt1, Dhx40, Anapc4, Wdr77, Mvp, Agk, Adcy1, Ppap2a, Map3k3, Krtcap2,






B4galt1, Pole4, Mvd, Bmper, Ficd, Tcf25, Rab12, Ints7, Ints5, Rpf2, Qsox1, Hsp90b1, Plcl1,






Atp6v0a1, Fktn, Pus3, Errfi1, Cxcl9, Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1, Mphosph8,






Klf17, Gpt2, Pnp, Hif1a, Mphosph6, Smarce1, Eif4e, Dap, Marveld3, Agpat9, Sf3b4, Eif3d,






Syvn1, Prg4, Srsf2, Braf, Scly, Timp3, Creb3, Senp2, Efna1, Ell2, Nod1, Fbxo6, Ctif, Gmeb1,






Cyp7b1, Ibtk, Apex1, Ube2s, Trim39, Csnk1a1, Pcyox1, Npm3, Mmp8, Tasp1, Aldh18a1, Mmp13,






Pcid2, Ddx21, Pfkp, Cwc27, Gls, Rlim, Dhx38, B4galnt1, Cd55, Dok2, Psma1, Atp7a, Usp8,






Entpd7, Usp1, Tnfaip1, Eogt, Aak1, Recql, Mrpl51, Acpp, Mbnl2, Golt1b, Ell, Tbp, Irf8, Trib1,






Dhx35, Grb2, Cebp2, Atf6, Ints2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Tpp2, Mmp9,






Exosc1, Exosc4, Cad, Lbp, Serpina3k, Serpina3m, Serpina3n, Serpina3g, Snip1, Skil, Stt3a,






Lpin1, Ddx50, Polh, Gtf2f1, Nup98, Uba5, Apcs, Jmjd6, Lpar1, Foxk2, Yod1, Fgr, Arf4, Camkk2,






Ube2f, Fgb, Rpn1,Tgm2, Uspl1, S100a8, Tmsb4x, , Ppp4c, A2m, Egf, Eif4g2, Slc35c1, Fkbp11,






Stag1, Fgl1, Nedd4l, C1galt1, Fen1, Wdr61, Ebna1bp2, Pkm, Trps1, Derl1, Spin1, Iars, Traf7,






Pdia3, Acer2, Dpy19l1, Saa3, Rars, Hdc, Usp14, Tbl1xr1, Pfkfb2, Srebf2, Ufm1, Cul5, Ranbp2,






Cln8, Trappc2, H2afx, Ube2j1, Vcp, Scnm1, Mcm2, Dab2, Sf1, Dusp2, Asb4, Svk, St6gal1,






Hmga2, Ppip5k1, Vimp, Adam17, Srgn, Asns, Eif4a3, Ddx39, Cd44, Myh9, Serp1, Neurl3, Ctla2a,






Ctla2b, Prmt1, Dzip3, Cyp51, Prdm1, Atf6b, Iqgap1, Pcsk5, Creb3l2, Wdr12, Dnajc10,






F13a1, Plek, Idi1, Nelfa, Rbm39, Sox9, Nus1, Sdf2l1, Odc1, Papss1, Wfs1, Supt5, Supt6, Hipk3,






Tmem165, Helb, Ngp, Txnl1, Gas6, Trim27, Rac2, Ppp2r1a, Il12rb1, Crem, Paip2b, Irak2, Hist1h3b,






Gemin8, Ngdn, Cdc73, Parn, Lig3, Gata6, Exog, Ncf1, Tspyl2, Smarcad1, Dis3, Pld1,






Tha1, Rasgrp1, Nop58, Tirap, Trp53inp2, Fkbp5, Tlr1, Med17, Apoa4, Apoa5, Trip12, Hars,






Zfp869, Ddost, Ick, Gigyf2, Hnf4a, Adam9, Zfp160, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3,






Jun, Pdia6, Dnajb11, Nlrp12, Nudt21, Papola, Ubxn2b, Denr, Cars2, Capn10, Farsb, Taf2, Ctps,






Nup62, Vars, Pdia4, Wipi1, Ppapdc1b, Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Abca1, Ufl1,






Ddx49, Casp4, Znrf1, Plaur, Nceh1, Preb, Parp2, Parp3, Mppe1, Zbtb16, Zbtb21, Med25, Eif2b4,






Ppip5k2, Ddx1, Thoc1, Elp3, Inhbe, B3galt1, Rnps1, Tfb2m, Cdk7, Dph5, Exosc10, Zfp830,






Brd8, Fbxo31, Il1a, Alg12, Rps9, Nabp1, Kars, Cda, Stk17b, Son, Pggt1b, Tiparp, Eya3, Slc9a3r1,






Prkrip1, Sec22b, Psen1, Ap5s1, Erp44, Exosc3, Hltf, Zfand2a, Cd38, Piga, Itgb3, Itgb2,






Arid5a, Ccl19, Khsrp, Gan, Gtf2a2, Nfia, Wdr45b, Atg16l2, Trmt61a, Kctd10, Eprs, D19Bwg1357e,






St3gal3, Dnajc3, Trim68, Rcor1, Trem3, Socs3, Dr1, Camk2b, Polr1a, Ubxn4, Trim3, Txndc5,






Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2, Edem3, Trib2, Edem1, Tmem173, Rab1, Cdkn1a,






Taf4b, Nbas, Hk2, Gadd45b, Sds, Gadd45g, Ppp1r11, Gale, Magoh, Sbno2, Sod2, Pik3c2a,






Nol9, Etnk2, Vegfc, Pycard, Adk, Trp63, Fn1, Stx5a, Clock, Bbs4, Alox12, B3galnt2, St3gal4,






Pomgnt1, Chmp4b, Ppm1l, Ccr1, Cr1l, Fabp4, Fabp5, Hsp90aa1, Ero1l, Lss, Srsf1, Fcgr3,






Srsf3, Psmd14, Zfp27, Serpina10, Calr, Pgm3, Rusc1, Nop56, Pofut2, Polr3k, Galnt2, Tbrg1,






Taf1, Pspc1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2, Lats2, Prkch, Ears2, Prkce, Acot11, Klk1b24,






Crnkl1, Irg1, Rbm42, Larp7, Anapc16, Il6st, Ikbkap, Nif3l1, S100a9, Ube2g2


LV
G4
metabolic process
5.3E−07
Cers6, Lars, Sdsl, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Abcc1, Nol8, Bfar, Slamf8,






Pdgfc, Mbd1, Sec61b, Prkag2, Snx6, Atg13, Utp6, 2700097O09Rik, Dhx30, Garem, Tat, Mlx,






Cd28, Cebpg, Timp1, Cebpd, Ddx52, Mettl1, Mettl3, Ezr, Rinl, Hnrnpa2b1, Senp6, Hnrnpab,






Polr1e, Il1rn, Pim3, Arl6ip5, Metap2, Cog6, Hck, Etf1, Ednra, Btg2, Ntmt1, Dhx40, Anapc4,






Wdr77, Mvp, Agk, Adcy1, Ppap2a, Map3k3, Krtcap2, B4galti, Pole4, Mvd, Bmper, Ficd, Tcf25,






Ldha, Rab12, Ints7, Ints5, Rpf2, Qsox1, Hsp90b1, Plcl1, Atp6v0a1, Fktn, Pus3, Errfi1, Cxcl9,






Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1, Mphosph8, Klf17, Gpt2, Pnp, Hif1a, Mphosph6,






Smarce1, Eif4e, Dap, Marveld3, Agpat9, Sf3b4, Eif3d, Syvn1, Prg4, Srsf2, Braf, Scly, Timp3,






Creb3, Senp2, Efna1, Rab13, Ell2, Nod1, Fbxo6, Ctif, Mcfd2, Gmeb1, Cyp7b1, Ibtk, Lrrc16a,






Apex1, Ube2s, Trim39, Csnk1a1, Pcyox1, Npm3, Mmp8, Tasp1, Aldh18a1, Mmp13, Pcid2,






Ddx2, Pfkp, Kdm6a, Cwc27, Gls, Rlim, B4galnt4, Dhx38, B4galnt1, Cd55, Dok2, Psma1, Atp7a,






Ubiad1, Usp8, Entpd7, Usp1, Tnfaip1, Eogt, Aak1, Recql, Mrpl51, Acpp, Mbnl2, Golt1b, Ell,






Tbp, Irf8, Trib1, Dhx35, Grb2, Cebp2, Atf6, Ints2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1,






Mthfr, Tpp2, Mmp9, Exosc1, Exosc4, Cad, Lbp, Serpina3k, Serpina3m, Serpina3n, Serpina3g,






Rbm26, Snip1, Skil, Stt3a, Lpin1, Ddx50, Polh, Dram1, Gtf2f1, Nup98, Kalrn, Uba5, Apcs,






Jmjd6, Lpar1, Foxk2, Yod1, Fgr, Arf4, Camkk2, Ube2f, Rcan2, Fgb, Rpn1, Tgm2, Uspl1, S100a8,






Tmsb4x, Ppp4c, Acot9, A2m, Egf, Eif4g2, Slc35c1, Fkbp15, Fkbp11, Stag1, Fgl1, Nedd4l, C1galt1,






Fen1, Wdr61, Ebna1bp2, Pkm, Trps1, Derl1, Spin1, Atg9b, Iars, Traf7, Pdia3, Acer2, Dpy19l1,






Saa3, Rab35, Rars, Hdc, Usp14, Tbl1xr1, Rrp1b, Pfkfb2, Srebf2, Ufm1, Suco, Far2, Cul5,






Ranbp2, Cln8, Trappc2, H2afx, Ube2j1, Vcp, Scnm1, Mcm2, Dab2, Sf1, Dusp2, Scfd1, Asb4,






Syk, St6gal1, Hmga2, Ppip5k1, Vimp, Adam17, Srgn, Asns, Rraga, Hyal1, Eif4a3, Ddx39, Cd44,






Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1, Dzip3, Cyp51, Prdm1, Atf6b, Iqgap1, Rab5b,






Pcsk5, Creb3l2, Wdr12, Dnajc10, F13a1, Tbc1d15, Plek, Steap4, Idi1, Nelfa, Rbm39, Sox9, Nus1,






Sdf2l1, Odc1, Papss1, Gnl2, Gnl1, Wfs1, Supt5, Supt6, Hipk3, Tmem165, Helb, Ngp, Txnl1,






Gas6, Trim27, Rac2, Zdhhc13, Ppp2r1a, Il12rb1, Crem, Paip2b, Irak2, Dynll1, Iigp1, Hist1h3b,






Gemin8, Ngdn, Lyve1, Cdc73, Parn, Gm21540, Lig3, Gata6, Exog, Ncf1, Tspyl2, Srm, Smarcad1,






Dis3, Gfpt1, Pld1, Tha1, Rasgrp1, Nop58, Tirap, Trp53inp2, Fkbp5, Tlr1, Med17, Apoa4,






Apoa5, Trip12, Hars, Zfp869, Ddost, Ick, Gigyf2, Mt2, Kif5b, Mt1, Hnf4a, Adam9, Zfp160,






Nnmt, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3, Jun, Pdia6, Dnajb11, Nlrp12, Nudt21, Papola,






Ralgapa2, Flot1, Crp, Ubxn2b, Denr, Cars2, Capn10, Cox8a, Farsb, Kif21a, Taf2, Ctps, Nup62,






Vars, Pdia4, Wipi1, Ppapdc1b, Rab18, Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Ap1ar, Abca1,






Ufl1, Ddx49, Casp4, Znrf1, Plaur, Nceh1, Preb, Parp2, Parp3, Mppe1, Zbtb16, Zbtb21,






Med25, Eif2b4, Ppip5k2, Ddx1, Thoc2, Thoc1, Elp3, Inhbe, B3galt6, B3galt1, Rnps1, Tfb2m, Cdk7,






Dph5, Exosc10, Zfp830, Brd8, Fbxo31, Il1a, Alg12, Rps9, Nabp1, Kars, Cda, Stk17b, Son,






Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Sec22b, Psen1, Ap5s1, Erp44, Exosc3, Hltf, Zfand2a,






Cd38, Piga, Itgb3, Itgb2, Arid5a, Vav1, Ccl19, Khsrp, Gan, Gtf2a2, Nfia, Wdr45b, Atg16l2,






Trmt61a, Gne, Kctd10, Eprs, D19Bwg1357e, S100a10, St3gal3, Dnajc3, Trim68, Rcor1, Trem3,






Socs3, Dr1, Camk2b, Polr1a, Ubxn4, Trim3, Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2,






Edem3, Trib2, Edem1, Tmem173, Rab1, Ift20, Cdkn1a, Taf4b, Nbas, Hk2, Gadd45b, Sds,






Gadd45g, Ppp1r11, Gale, Magoh, Sbno2, Sod2, Msn, Pik3c2a, Nol9, Etnk2, Vegfc, Pycard,






Map1lc3b, Adk, Trp63, Fn1, Stx5a, Clock, Bbs4, Dennd1b, Alox12, B3galnt2, St3gal5, St3gal4,






Pomgnt1, Chmp4b, Ppm1l, Ccr1, Txlna, Plaa, Gnai2, Cr1l, Fabp4, Fabp5, Hsp90aa1, Ero1l, Lss,






Srsf1, Fcgr3, Srsf3, Rab33b, Psmd14, Scara5, Zfp27, Serpina10, Calr, Pgm3, Rusc1, Nop56,






Pofut2, Polr3k, Galnt2, Tbrg1, Taf1, Pspc1, Atl1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2, Lats2,






Prkch, Ears2, Prkce, Acot11, Klk1b24, Crnkl1, Irg1, Rbm42, Larp7, Anapc16, Il6st, Rapgef5,






Ikbkap, Nif3l1, Arfgap1, S100a9, Ube2g2


LV
G4
organic substance metabolic process
1.1E−06
Cers6, Lars, Sdsl, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Bfar, Pdgfc, Mbd1, Sec61b,






Prkag2, Snx6, Utp6, Dhx30, Garem, Tat, Mlx, Cd28, Cebpg, Timp1, Cebpd, Ddx52, Mettl1, Mettl3,






Ezr, Hnrnpa2b1, Senp6, Hnrnpab, Polr1e, Il1rn, Pim3, Arl6ip5, Metap2, Cog6, Hck, Etf1, Ednra,






Btg2, Ntmt1, Dhx40, Anapc4, Wdr77, Mvp, Agk, Adcy1, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4,






Mvd, Bmper, Ficd, Tcf25, Ldha, Rab12, Ints7, Ints5, Rpf2, Qsox1, Hsp90b1, Plcl1, Atp6v0a1, Fktn,






Pus3, Errfi1, Cxcl9, Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1, Mphosph8, Klf17, Gpt2, Pnp, Hif1a,






Mphosph6, Smarce1, Eif4e, Dap, Marveld3, Agpat9, Sf3b4, Eif3d, Syvn1, Prg4, Srsf2, Braf, Scly,






Timp3, Creb3, Senp2, Efna1, Ell2, Nod1, Fbxo6, Ctif, Mcfd2, Gmeb1, Cyp7b1, Ibtk, Lrrc16a, Apex1,






Ube2s, Trim39, Csnk1a1, Pcyox1, Npm3, Mmp8, Tasp1, Aldh18a1, Mmp13, Pcid2, Ddx21, Pfkp,






Kdm6a, Cwc27, Gls, Rlim, Dhx38, B4galnt1, Cd55, Dok2, Psma1, Atp7a, Ubiad1, Usp8, Entpd7,






Usp1, Tnfaip1, Eogt, Aak1, Racql, Mrpl51, Acpp, Mbnl2, Golt1b, Ell, Tbp, Irf8, Trib1, Dhx35, Grb2,






Cebp2, Atf6, Ints2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Tpp2, Mmp9, Exosc1, Exosc4,






Cad, Lbp, Serpina3k, Serpina3m, Serpina3n, Serpina3g, Snip1, Skil, Stt3a, Lpin1, Ddx50, Polh,






Gtf2f1, Nup98, Uba5, Apcs, Jmjd6, Lpar1, Foxk2, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2,






Uspl1, S100a8, Tmsb4x, Ppp4c, Acot9, A2m, Egf, Eif4g2, Slc35c1, Fkbp11, Stag1, Fgl1, Nedd4l,






C1galt1, Fen1, Wdr61, Ebna1bp2, Pkm, Trps1, Derl1, Spin1, Iars, Traf7, Pdia3, Acer2, Dpy19l1, Saa3,






Rars, Hdc, Usp14, Tbl1xr1, Pfkfb2, Srebf2, Ufm1, Suco, Far2, Cul5, Ranbp2, Cln8, Trappc2, H2afx,






Ube2j1, Vcp, Scnm1, Mcm2, Dab2, Sf1, Dusp2, Asb4, Syk, St6gal1, Hmga2, Ppip5k1, Vimp, Adam17,






Srgn, Asns, Hyal1, Eif4a3, Ddx39, Cd44, Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1, Dzip3,






Cyp51, Prdm1, Atf6b, Iqgap1, Pcsk5, Creb3l2, Wdr12, Dnajc10, F13a1, Plek, Idi1, Nelfa, Rbm39, Sox9,






Nus1, Sdf2l1, Odc1, Papss1, Wfs1, Supt5, Supt6, Hipk3, Tmem165, Helb, Ngp, Txnl1, Gas6,






Trim27, Rac2, Ppp2r1a, Iil2rb1, Crem, Paip2b, Irak2, Hist1h3b, Gemin8, Ngdn, Lyve1, Cdc73, Parn,






Lig3, Gata6, Exog, Ncf1, Tspyl2, Srm, Smarcad1, Dis3, Gfpt1, Pld1, Tha1, Rasgrp1, Nop58, Tirap,






Trp53inp2, Fkbp5, Tlr1, Med17, Apoa4, Apoa5, Trip12, Hars, Zfp869, Ddost, Ick, Gigyf2, Hnf4a,






Adam9, Zfp160, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3, Jun, Pdia5, Dnajb11, Nlrp12, Nudt21,






Papola, Flot1, Crp, Ubxn2b, Denr, Cars2, Capn10, Farsb, Taf2, Ctps, Nup62, Vars, Pdia4, Wipi1,






Ppapdc1b, Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Ap1ar, Abca1, Ufl1, Ddx49, Casp4, Znrf1, Plaur, Nceh1,






Preb, Parp2, Parp3, Mppe1, Zbtb16, Zbtb21, Med25, Eif2b4, Ppip5k2, Ddx1, Thoc2, Thoc1, Elp3,






Inhbe, B3galt6, B3galt1, Rnps1, Tfb2m, Cdk7, Dph5, Exosc10, Zfp830, Brd8, Fbxo31, Il1a, Alg12,






Rps9, Nabp1, Kars, Cda, Stk17b, Son, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Sec22b, Psen1, Ap5s1,






Erp44, Exosc3, Hltf, Zfand2a, Cd38, Piga, Itgb3, Itgb2, Arid5a, Ccl19, Khsrp, Gan, Gtf2a2, Nfia,






Wdr45b, Atg16l2, Trmt61a, Gne, Kctd10, Eprs, D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1,






Trem3, Socs3, Dr1, Camk2b, Polr1a, Ubxn4, Trim3, Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7,






Cpeb2, Edem3, Trib2, Edem1, Tmem173, Rab1, Cdkn1a, Taf4b, Nbas, Hk2, Gadd45b, Sds, Gadd45g,






Ppp1r11, Gale, Magoh, Sbno2, Sod2, Msn, Pik3c2a, Nol9, Etnk2, Vegfc, Pycard, Adk, TrpB3, Fn1,






Stx5a, Clock, Bbs4, Alox12, B3galnt2, St3gal5, St3gal4, Pomgnt1, Chmp4b, Ppm1l, Ccr1, Cr1l,






Fabp4, Fabp5, Hsp90aa1, Ero1l, Lss, Srsf1, Fcgr3, Srsf3, Psmd14, Scara5, Zfp27, Serpina10, Calr,






Pgm3, Rusc1, Nop56, Pofut2, Polr3k, Galnt2, Tbrg1, Taf1, Pspc1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2,






Lats2, Prkch, Ears2, Prkce, Acot11, Klk1b24, Crnkl1, Irg1, Rbm42, Larp7, Anapc16, Il6st, Ikbkap,






Nif3l1, S100a9, Ube2g2


LV
G4
cellular metabolic process
3.5E−06
Cers6, Lars, Sdsl, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Bfar, Slamf8, Pdgfc, Mbd1,






Sec61b, Prkag2, Snx6, Atg13, Utp6, Dhx30, Garem, Tat, Mlx, Cd28, Cebpg, Timp1, Cebpd, Ddx52,






Mettl1, Mettl3, Ezr, Hnrnpa2b1, Senp6, Hnrnpab, Polr1e, Pim3, Arl6ip5, Metap2, Hck, Etf1,






Ednra, Btg2, Ntmt1, Dhx40, Anapc4, Wdr77, Mvp, Agk, Adcy1, Ppap2a, Map3k3, KrtcBp2, B4galt1,






Pole4, Mvd, Bmper, Ficd, Tcf25, Ldha, Rab12, Ints7, Ints5, Rpf2, Qsox1, Hsp90b1, Plcl1, Atp6v0a1,






Fktn, Pus3, Errfi1, Cxcl9, Ung, Ctgf, Nktr, Epm2aip1, Pdcd10, Trnt1, Mphosph8, Klf17, Gpt2, Pnp,






Hif1a, Mphosph6, Smarce1, Eif4e, Dap, MBrveld3, Agpat9, Sf3b4, Eif3d, Syvn1, Prg4, Srsf2, Braf,






Scly, Timp3, Creb3, Senp2, Efna1, Ell2, Nod1, Fbxo6, Ctif, Mcfd2, Gmeb1, Cyp7b1, Ibtk, Lrrc16a,






Apex1, Ube2s, Trim39, Csnk1a1, Pcyox1, Npm3, Tasp1, Aldh18a1, Mmp13, Pcid2, Ddx21, Pfkp,






Cwc27, Gls, Rlim, Dhx38, B4galnt1, Dok2, Psma1, Atp7a, Ubiad1, Usp8, Entpd7, Usp1, Tnfaip1,






Eogt, Aak1, Recql, Mrpl51, Acpp, Mbnl2, Ell, Tbp, Irf8, Trib1, Dhx35, Grb2, Cebp2, Atf6, Ints2, Atf3,






Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Mmp9, Exosc1, Exosc4, Cad, Lbp, Serpina3k, Serpina3m,






Serpina3n, Serpina3g, Rbm26, Snip1, Skil, Stt3a, Lpin1, Ddx50, Polh, Dram1, Gtf2f1, Nup98,






Uba5, Apcs, Jmjd6, Lpar1, Foxk2, Yod1, Fgr, Arf4, C3mkk2, Ube2f, Fgb, Rpn1, Tgm2, Uspl1, S100a8,






Tmsb4x, Ppp4c, Acot9, A2m, Egf, Eif4g2, Slc35c1, Fkbp15, Fkbp11, Stag1, Fgl1, Nedd4l, C1galt1,






Fen1, Wdr61, Ebna1bp2, Pkm, Trps1, Derl1, Spin1, Atg9b, lars, Traf7, Pdia3, Acer2, Dpy19l1,






Saa3, Rars, Hdc, Usp14, Tbl1xr1, Rrp1b, Pfkfb2, Srebf2, Ufm1, Far2, Cul5, Ranbp2, Cln8, Trappc2,






H2afx, Ube2j1, Vcp, Scnm1, Mcm2, Dab2, Dusp2, Scfd1, Asb4, Syk, St6gal1, Hmga2, Ppip5k1, Vimp,






Adam17, Asns, Rraga, Eif4a3, Ddx39, Cd44, Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1, Dzip3,






Prdm1, Atf6b, Iqgap1, Pcsk5, Creb3l2, Wdr12, Dnajc10, F13a1, Plek, Idi1, Nelfa, Rbm39, Sox9,






Nus1, Sdf2l1, Odc1, Papss1, Wfs1, Supt5, Supt6, Hipk3, Tmem165, Helb, Ngp, Txnl1, Gas6, Trim27,






Rac2, Ppp2r1a, Il12rb1, Crem, Paip2b, Irak2, Dynll1, Iigp1, Hist1h3b, Gemin8, Ngdn, Cdc73, Parn,






Lig3, Gata6, Exog, Ncf1, Tspyl2, Srm, Smarcad1, Dis3, Pld1, Tha1, Rasgrp1, Nop58, Tirap, Trp53inp2,






Fkbp5, Tlr1, Med17, Apoa4, Apoa5, Trip12, Hars, Zfp869, Ddost, Ick, Gigyf2, Hnf4a, Adam9,






Zfp160, Tra2b, Hspa5, Atf7ip, Csgalnact2, Golph3, Jun, Pdia6, Dnajb11, Nlrp12, Nudt21, Papola,






Flot1, Crp, Ubxn2b, Denr, Cars2, Cox8a, Farsb, Taf2, Ctps, Nup62, Vars, Pdia4, Wipi1, Ppapdc1b,






Ifrd1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Ap1ar, Abca1, Ufl1, Ddx49, Znrf1, Plaur, Nceh1, Preb, Parp2,






Parp3, Mppe1, Zbtb16, Zbtb21, Med25, Eif2b4, Ppip5k2, Ddx1, Thoc1, Elp3, Inhbe, B3galt1, Rnps1,






Tfb2m, Cdk7, Dph5, Exosc10, Zfp830, Brd8, Fbxo31, Il1a, Alg12, Rps9, Nabp1, Kars, Cda, Stk17b,






Son, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Psen1, Ap5s1, Erp44, Exosc3, Hltf, Zfand2a, Cd38,






Piga, Itgb3, Itgb2, Arid5a, Vav1, Ccl19, Khsrp, Gan, Gtf2a2, Nfia, Wdr45b, Atg16l2, Trmt61a, Gne,






Kctd10, Eprs, D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b, Polr1a, Ubxn4,






Trim3, Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2, Edem3, Trib2, Edem1, Tmem173,






Rab1, Ift20, Cdkn1a, Taf4b, Nbas, Hk2, Gadd45b, Sds, Gadd45g, Ppp1r11, Magoh, Sbno2, Sod2,






Pik3c2a, Nol9, Etnk2, Vegfc, Pycard, Map1lc3b, Adk, Trp63, Fn1, Clock, Alox12, B3galnt2, St3gal4,






Pomgnt1, Chmp4b, Ppm1l, Ccr1, Txlna, Cr1l, Fabp4, Fabp5, Hsp90aa1, Ero1l, Srsf1, Fcgr3, Srsf3,






Rab33b, Psmd14, Zfp27, Serpina10, Calr, Pgm3, Rusc1, Nop56, Pofut2, Polr3k, Galnt2, Tbrg1, Tafl,






Pspc1, Nfkbi2, Trmt10a, Enc1, Srrt, Josd2, Lats2, Prkch, Ears2, Prkce, Acot11, Crnkl1, Irg1, Rbm42,






Larp7, Anapc16, Il6st, Ikbkap, Nif3l1, S100a9, Ube2g2


LV
G4
glycosylation
4.2E−06
Cog6, Krtcap2, B4galt1, Fktn, Syvn1, Eogt, Stt3a, Arf4, Rpn1, Slc35c1, C1galt1, Acer2, Dpy19l1,






Ube2j1, Vcp, St6gal1, Nus1, Tmem165, Ddost, Parp3, B3galt1, Alg12, Tiparp, Psen1, St3gal3,






B3galnt2, St3gal4, Pomgnt1, Pgm3, Pofut2, Galnt2, Ube2g2


LV
G4
protein glycosylation
6.2E−06
Krtcap2, B4galt1, Fktn, Syvn1, Eogt, Stt3a, Arf4, Rpn1, C1galt1, Acer2, Dpy19l1, Ube2j1, Vcp,






St6gal1, Nus1, Tmem165, Ddost, Parp3, B3galt1, Alg12, Tiparp, Psen1, St3gal3, B3galnt2, St3gal4,






Pomgnt1, Pgm3, Pofut2, Galnt2, Ube2g2


LV
G4
Post-translational protein modification
7.7E−06
Pigm, B4galt1, Mvd, Senp2, Mcfd2, Mogs, Eogt, Nans, Stt3a, Nup98, Sec24d, Rpn1, Stag1, C1galt1,






Pdia3, Ranbp2, St6gal1, Galnt18, Nupl2, Gas6, Gfpt1, Ddost, Nupl1, Nup62, Plaur, Preb, B3galt1,






Dph5, Alg12, Sec31a, Piga, Gne, St3gal3, Edem2, Edem3, Edem1, Gmppa, Man1a, Sec13, Dhdds,






St3gal5, St3gal4, Pomgnt1, Seh1l, Uap1, Calr, Pgm3, Pofut2, Galnt2, Gmppb


LV
G4
Protein export
1.8E−05
Sec61b, Srp68, Spcs2, Spcs3, Gm10177, Srp9, Hspa5, Sec61a1, Srp19, Srprb, Srp72, Srpr, Srp54a


LV
G4
cellular protein metabolic process
1.96-05
Lars, Selk, Bfar, Pdgfc, Sec61b, Prkag2, Garem, Timp1, Mettl3, Senp6, Pim3, Arl6ip5, Metap2,






Hck, Etf1, Btg2, Ntmt1, Anapc4, Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd,






Rab12, Qsox1, Hsp90b1, Plcl1, Fktn, Errfl1, Ctgf, Nktr, Pdcd10, Eif4e, Dap, Marveld3, Elf3d,






Syvn1, Braf, Timp3, Senp2, Efna1, Nod1, Fbxo6, Ctif, Ibtk, Ube2s, Trim39, Csnk1a1, Pcyox1,






Mmp13, Pcid2, Cwc27, Rlim, Dok2, Psma1, Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Mrp151,






Trib1, Grb2, Atf3, Herpud1, Slc11a2, Gzma, Slc11a1, Mthfr, Mmp9, Cad, SerpinB3k, Serpina3m,






Serpina3n, Serpina3g, Stt3a, Lpin1, Uba5, Jmjd6, Lpar1, Yod1, Fgr, Arf4, Camkk2, Ube2f,






Fgb, Rpn1, Tgm2, Uspl1, S100a8, A2m, Egf, Eif4g2, Fkbp11, Nedd4l, C1galt1, Wdr61, Derl1,






Iars, Traf7, Pdia3, Acer2, Dpy19l1, Saa3, Rars, Usp14, Tbl1xr1, Ufm1, Cul5, Cln8, Ube2j1,






Vcp, Dab2, Dusp2, Asb4, Syk, St6gal1, Hmga2, Vimp, Adam17, Cd44, Myh9, Serp1, Neurl3,






Ctla2a, Ctla2b, Prmt1, Dzip3, Iqgap1, Pcsk5, Dnajc10, F13a1, Sox9, Nus1, Sdf2l1, Wfs1, Supt6,






Hipk3, Tmem165, Ngp, Txnl1, Gas6, Trim27, Rac2, Ppp2r1a, Il12rb1, Paip2b, Cdc73, Tspyl2,






Smarcad1, Rasgrp1, Tirap, Fkbp5, Tlr1, Trip12, Hars, Ddost, Ick, Gigyf2, Adam9, Hspa5,






Jun, Pdia6, Nlrp12, Ubxn2b, Denr, Cars2, Farsb, Nup62, Vars, Pdia4, Wipi1, Crtc2, Secisbp2,






Ptpn2, Ptpn1, Abca1, Ufl1, Znrf1, Plaur, Nceh1, Parp3, Mppe1, Eif2b4, Elp3, Inhbe, B3galt1,






Dph5, Brd8, Fbxo31, Il1a, Alg12, Rps9, Kars, Stk17b, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1,






Psen1, Erp44, Zfand2a, Piga, Itgb3, Itgb2, Arid5a, Ccl19, Gan, Wdr45b, Kctd10, Eprs,






D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b, Ubxn4, Trim3, Txndc5, Rps6ka3,






Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2, Edem3, Trib2, Edem1, Cdkn1a, Gadd45b, Gadd45g, Ppp1r11,






Pik3c2a, Vegfc, Pycard, Fn1, Clock, B3galnt2, St3gal4, Pomgnt1, Chmp4b, Ppm1l, Ccr1,






Cr1l, Fabp4, Hsp90aa1, Ero1l, Psmd14, Serptna10, Pgm3, Rusc1, Pofut2, Galnt2, Taf1, Enc1,






Josd2, Lats2, Prkch, Ears2, Prkce, Anapc16, Il6st, Ikbkap, S100a9, Ube2g2


LV
G4
Post-translational protein modification
2.1E−05
Pigm, B4galt1, Mvd, Senp2, Mcfd2, Mogs, Nans, Nup98, Sec24d, Stag1, C1galt1, Pdia3, Ranbp2,






St6gal1, Nupl2, Gas6, Gfpt1, Nupl1, Nup62, Plaur, Preb, Dph5, Alg12, Sec31a, Piga, Gne,






St3gal3, Edem2, Edem3, Edem1, Gmppa, Man1a, Sec13, Dhdds, St3gal5, St3gal4, Seh1l,






Uap1, Calr, Pgm3, Pofut2, Galnt2, Gmppb


LV
G4
protein metabolic process
3.1E−05
Lars, Selk, Bfar, Pdgfc, Sec61b, Prkag2, Garem, Cd28, Cebpg, Timp1, Mettl3, Senp6, Pim3,






Arl6ip5, Metap2, Hck, Etf1, Btg2, Ntmt1, Anapc4, Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1,






Pole4, Bmper, Ficd, Rab12, Qsox1, Hsp90b1, Plcl1, Fktn, Errfi1, Ctgf, Nktr, Pdcd10, Hif1a,






Eif4e, Dap, Marveld3, Eif3d, Syvn1, Prg4, Braf, Timp3, Senp2, Efna1, Nod1, Fbxo6, Ctif, Ibtk,






Ube2s, Trim39, Csnk1a1, Pcyox1, Mmp8, Tasp1, Mmp13, Pcid2, Cwc27, Rlim, Cd55, Dok2, Psma1,






Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Mrpl51, Trib1, Grb2, Atf3, Herpud1, Slc11a2,






Gzma, Slc11a1, Mthfr, Tpp2, Mmp9, Cad, Lbp, Serpina3k, Serpina3m, Serpina3n, Serpina3g,






Stt3a, Lpin1, Uba5, Apcs, Jmjd6, Lpar1, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2, Uspl1,






S100a8, A2m, Egf, Eif4g2, Fkbp11, Nedd4l, C1galt1, Wdr61, Derl1, Iars, Traf7, Pdia3, Acer2,






Dpy19l1, Saa3, Rars, Usp14, Tbl1xr1, Ufm1, Cul5, Cln8, Ube2j1, Vcp, Dab2, Dusp2, Asb4,






Syk, St6gal1, Hmga2, Vimp, Adam17, Srgn, Cd44, Myh9, Serp1, Neurl3, Ctla2a, Ctla2b, Prmt1,






Dzip3, Iqgap1, Pcsk5, Dnajc10, F13a1, Sox9, Nus1, Sdf2l1, Odc1, Wfs1, Supt6, Hipk3, Tmem165,






Ngp, Txnl1, Gas6, Trtm27, Rac2, Ppp2r1a, Il12rb1, Paip2b, Cdc73, Tspyl2, Smarcad1,






Rasgrp1, Tirap, Fkbp5, Tlr1, Apoa4, Apoa5, Trlp12, Hars, Ddost, Ick, Gigyf2, Adam9, Hspa5,






Csgalnact2, Golph3, Jun, Pdia6, Nlrp12, Ubxn2b, Denr, Cars2, Capn10, Farsb, Nup62, Vars,






Pdia4, Wipi1, Crtc2, Secisbp2, Ptpn2, Ptpn1, Abca1, Ufl1, Casp4, Znrf1, Plaur, Nceh1, Parp3,






Mppe1, Eif2b4, Elp3, Inhbe, B3galt1, Dph5, Brd8, Fbxo31, Il1a, Alg12, Rps9, Kars, Stk17b,






Pggt1b, Tiparp, 6ya3, Sk9a3r1, Prkrip1, Sec22b, Psen1, Erp44, Zfand2a, Piga, Itgb3, Itgb2,






Arid5a, Ccl19, Gan, Wdr45b, Kctd10, Eprs, D19Bwg1357e, St3gal3, Dnajc3, Trim68, Rcor1,






Trem3, Socs3, Dr1, CBmk2b, Ubxn4, Trim3, Txndc5, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Nek7, Cpeb2,






Edem3, Trib2, Edem1, Rab1, Cdkn1a, Gadd45b, Gadd45g, Ppp1r11, Plk3c2a, Vegfc, Pycard,






TrpB3, Fn1, Stx5a, Clock, B3galnt2, St3gal4, Pomgnt1, Chmp4b, Ppm1l, Ccrl, Cr1l, Fabp4,






Hsp90aa1, Ero1l, Fcgr3, Psmd14, Serpina10, Pgm3, Rusc1, Pofut2, Galnt2, Taf1, Enc1 Josd2,






Lats2, Prkch, Ears2, Prkce, Klk1b24, Anapc16, Il6st, Ikbkap, S100a9, Ube2g2


LV
G4
glycoprotein metabolic process
4.6E−05
Krtcap2, B4galt1, Fktn, Hif1a, Syvn1, Fbxo6, Atp7a, Eogt, Stt3a, Apcs, Arf4, Rpn1, C1galt1,






Acer2, Dpy19l1, Ube2j1, Vcp, St6gal1, Nus1, Tmem165, Ddost, Csgalnact2, Gdph3, Parp3,






B3galt1, Alg12, Tiparp, Psen1, Erp44, Ccl19, St3gal3, Edem3, Rab1, B3galnt2, St3gal4, Pomgnt1,






Pgm3, Pofut2, Galnt2, Ube2g2


LV
G4
regulation of response to stress
0.0001
Selp, Pik3ap1, Bfar, Cd28, Cebpg, Arl6ip5, Map3k3, Il1r1, Rab12, Qsox1, Fktn, Ctgf, Pdcd10,






Pnp, Hif1a, Marveld3, Syvn1, Braf, Scly, Creb3, Senp2, Nod1, Apex1, Hilpda, Cd55, Serinc3,






Psma1, Adora1, Usp1, Tnfaip6, Rbm18, Herpud1, Lbp, Skil, Ap1g1, Fgr, S100a8, Ppp4c, A2m,






Slc35c1, Fcer1g, Dab2, Tnfrsf12a, Scfd1, Syk, Hmga2, Vimp, Cd44, Ctla2a, Plek, Wfs1, Supt5,






Hipk3, Il12rb1, Irak2, Grina, Rasgrp1, Tirap, Trip12, Nlrp12, Cox8a, Ptpn2, Ptpn1, Casp4,






Il17ra, Thoc1, Il1a, Eya3, Vav1, Ccl19, Dnajc3, Socs3, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Rint1,






Tmem173, Ift20, Hyou1, Gadd45b, Gadd45g, Sbno2, Sod2, Pycard, Trp63, Clock, Alox12, Chmp4b,






Ccr1, Cr1l, Fabp4, Fcgr3, Rab33b, Scara5, Polr3d, Prkce, Crnkl1, Irg1, Myo1f, S100a9


LV
G4
Metal ion SLC transporters
0.00014
Slc41a1, Slc30a5, Slc39a6, Slc11a2, Slc11a1, Slc39a7, Slc39a10, Slc39a14, Slc30a7, Cp, Slc41a2


LV
G4
ncRNA metabolic process
0.00014
Lars, Nol8, Utp6, Ddx52, Mettl1, Polr1e, Ints7, Ints5, Rpf2, Pus3, Trnt1, Mphosph6, Ell2, Npm3,






Ell, Ints2, Exosc4, Ebna1bp2, Spin1, Iars, Rars, Wdr12, Ngdn, Dis3, Nop58, Har5, Cars2, Farsb,






Vars, Ddx1, Elp3, Exosc10, Kars, Exosc3, Khsrp, Trmt61a, Ncl9, Nop56, Trmt10a, Srrt, Ears2


LV
G4
ER-associated ubiquitin-dependent protein catabolic
0.00023
Sec61b, Hsp90b1, Syvn1, Fbxo6, Herpud1, Yod1, Derl1, Usp14, Ube2j1, Vcp, Vimp, Dnajc10,




process

Sdf2l1, Wfs1, Ubxn4, Edem1, Ube2g2


LV
G4
response to unfolded protein
0.00026
Tbl2, Bfar, Syvn1, Creb3, Atf3, Herpud1, Yod1, Derl1, Vimp, Serp1, Wfs1, Hspa5, Ptpn2, Ptpn1,






Erp44, Dnajc3, Hsp90aa1, Ero1l


LV
G4
glycoprotein biosynthetic process
0.00028
Krtcap2, B4galt1, Fktn, Syvn1, Atp7a, Eogt, Stt3a, Arf4, Rpn1, C1galt1, Acer2, Dpy19l1, Ube2j1,






Vcp, St6gal1, Nus1, Tmem165, Ddost, Csgalnact2, Golph3, Parp3, B3galt1, Alg12, Tiparp,






Psen1, Ccl19, St3gal3, B3galnt2, St3gal4, Pomgnt1, Pgm3, Pofut2, Galnt2, Ube2g2


LV
G4
carbohydrate metabolic process
0.00028
Prkag2, Cog6, Krtcap2, B4galt1, Fktn, Epm2aip1, Hif1a, Syvn1, Pfkp, Eogt, Atf3, Stt3a, Arf4,






Rpn1, Egf, Slc35c1, Fgl1, C1gBlt1, Pkm, Acer2, Dpy19l1, Pfkfb2, Ranbp2, Ube2j1, Vcp, St6gal1,






Ppip5k1, Serp1, Plek, Nus1, Tmem165, Ddost, Crtc2, Ptpn2, Parp3, Ppip5k2, B3galt1, Alg12,






Tiparp, Psen1, St3gal3, Hk2, Gale, B3galnt2, St3gal4, Pomgnt1, Fabp5, Pgm3, Pofut2, Galnt2,






Prkce, Il6st, Ube2g2


LV
G4
response to topologically incorrect protein
0.00046
Tbl2, Bfar, Syvn1, Creb3, Atf3, Herpud1, Yod1, Derl1, Vimp, Serp1, Sdf2l1, Wfs1, Hspa5, Ptpn2,






Ptpn1, Erp44, Dnajc3, Hsp90aa1, Ero1l


LV
G4
catabolic process
0.0006
Sdsl, Cyp1b1, Bfar, Sec61b, Prkag2, Atg13, Tat, Timp1, Btg2, Rab12, Qsox1, Hsp90b1, Pnp, Hif1a,






Dap, Syvn1, Timp3, Fbxo6, Ctlf, Ube2s, Trimm39, Pcyox1, Mmp13, PcId2, Pfkp, Gls, Rlim,






Psma1, Usp8, Entpd7, Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Mmp9, Exosc4, Lpin1, Dram1,






Yod1, S100a8, Egf, Slc35c1, Nedd4l, Fen1, Wdr61, Pkm, Derl1, Atg9b, Hdc, Usp14, Tbl1xr1,






Pfkfb2, Cul5, Cln8, Ube2j1, Vcp, Dab2, Scfd1, Vimp, Rraga, Hyal1, Cd44, Ctla2a, Ctla2b, Dnajc10,






Sox9, Sdf2l1, Odc1, Wfs1, Supt5, Iigp1, Lyve1, Parn, Exog, Ncf1, Dis3, Tha1, Trp53inp2,






Apoa4, Apoa5, Trip12, Adam9, Hspa5, Ubxn2b, Cox8a, Wipi1, Secisbp2, Ptpn1, Ufl1, Znrf1,






Rnps1, Exosc10, Fbxo31, Cda, Tiparp, Sec22b, Psen1, Exosc3, Zfand2a, Khsrp, Wdr45b, Atg16l2,






Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1, Rab1, Ift20, Nbas, Hk2, Sds, Gale,






Magoh, Pycard, Map1lc3b, Stx5a, Clock, Chmp4b, Txlna, Rab33b, Psmd14, TBf1, Enc1,






Prkce, Crnkl1, S100a9, Ube2g2


LV
G4
catabolic process
0.0006
Sdsl, Cyp1b1, Bfar, Sec61b, Prkag2, Atg13, Tat, Timp1, Btg2, Rab12, Qsox1, Hsp90b1, Pnp, Hif1a,






Dap, Syvn1, Timp3, Fbxo6, Ctif, Ube2s, Trim39, Pcyox1, Mmp13, Pcid2, Pfkp, Gls, Rlim, Psma1, Usp8,






Entpd7, Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Mmp9, Exosc4, Lpin1, Dram1, Yod1, S100a8, Egf, Slc35c1,






Nedd4l, Fen1, Wdr61, Pkm, Derl1, Atg9b, Hdc, Usp14, Tbl1xr1, Pfkfb2, Cul5, Cln8, Ube2j1, Vcp,






Dab2, Scfd1, Vimp, Rraga, Hyal1, Cd44, Ctla2a, Ctla2b, Dnajc10, Sox9, Sdf2l1, Odc1, Wfs1, Supt5,






Iigp1, Lyve1, Parn, Exog, Ncf1, Dis3, Tha1, Trp53inp2, Apoa4, Apoa5, Trip12, Adam9, Hspa5, Ubxn2b,






Cox8a, Wipi1, Secisbp2, Ptpn1, Ufl1, Znrf1, Rnps1, Exosc10, Fbxo31, Cda, Tiparp, Sec22b, Psen1,






Exosc3, Zfand2a, Khsrp, Wdr45b, Atg16l2, Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1,






Rab1, Ift20, Nbas, Hk2, Sds, Gale, Magoh, Pycard, Map1lc3b, Stx5a, Clock, Chmp4b, Txlna, Rab33b,






Psmd14, Taf1, Enc1, Prkce, Crnkl1, S100a9, Ube2g2


LV
G4
Golgi vesicle transport
0.00067
Yipf5, Golga5, Cog6, Stx18, Arcn1, Uso1, Krt18, Gosr2, Blzf1, Ap1g1, Tmed9, Sar1a, Vamp4, Rab35,






Copb2, Zw10, Creb3l2, Chic2, Golga4, Golph3, Wipi1, Ap1ar, Mppe1, Bet1l, Sec22b, Rint1, Rab1,






Sec13, Stx5a, Rab33b


LV
G4
tRNA metabolic process
0.00075
Lars, Mettl1, Pus3, Trnt1, Iars, Rars, Hars, Cars2, Farsb, Vars, Ddx1, Elp3, Kars, Exosc3, Trmt61a,






Trmt10a, Ears2


LV
G4
Gene Expression
0.0009
Cnot11, Sec61b, Prkag2, Mettl3, Hnrnpa2b1, Polr1e, Etf1, Wdr77, Srp68, Eif4e, Sf3b4, Eif3d, Gtf2e1,






Zfp143, Ssr1, Ssr2, Plrg1, Gls, Dhx38, Alyref, Psma1, Ell, Spcs2, Spcs3, Tbp, Ssr4, Exosc1, Exosc4,






Skil, Gtf2f1, Nup98, Gspt2, Snrnp40, Rpn1, Ccar1, Nedd4l, Ranbp2, H2afx, Casc3, Gtf2f2, Nupl2, Nelfa,






Supt5, Ppp2r1a, Srp9, Hist1h3b, Ssb, Parn, Dis3, Med15, Med17, Ddost, Hist1h2bp, Hnf4a, Hist1h2bf,






Sec61a1, Nudt21, Papola, Nupl1, Cox8a, Srp19, Taf2, Nup62, Srprb, Med25, Eif2b4, Rnps1,






Med20, TfB2m, Cdk7, Rps9, Exosc3, Khsrp, Gtf2a2, Nfia, Polr1a, Srp72, Taf4b, Magoh, Srpr, Seh1l,






Hsp90aa1, SrpS4a, Srsf1, Srsf3, Psmd14, Polr3d, Polr3k, Taf1


LV
G4
cellular protein modification process
0.00107
Selk, Bfar, Pdgfc, Prkag2, Garem, Senp6, Pim3, Arl6ip5, Metap2, Hck, Etf1, Btg2, Ntmt1, Anapc4,






Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd, Plcl1, Fktn, Errfi1, Ctgf, Nktr, Pdcd10,






Marveld3, Syvn1, Braf, Senp2, Efna1, Nod1, Ibtk, Ube2s, Trim39, Csnk1a1, Pcid2, Cwc27, Rlim, Dok2,






Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Trib1, Grb2, Atf3, Herpud1, Slc11a1, Mthfr, Mmp9, Cad,






Stt3a, Lpin1, Uba5, Jmjd6, Lpar1, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2, Uspl1, S100a8,






Egf, Fkbp11, Nedd4l, C1galt1, Wdr61, Traf7, Acer2, Dpy19l1, Saa3, Usp14, Tbl1xr1, Ufm1, Cul5, Ube2j1,






Vcp, Dab2, Dusp2, Asb4, Syk, St6gal1, Hmga2, Adam17, Cd44, Neurl3, Prmt1, Dzip3, Iqgap1, Dnajc10,






F13a1, Sox9, Nus1, Wfs1, Supt6, Hipk3, Tmem165, Gas6, Trim27, Rac2, Ppp2r1a, Il12rb1, Cdc73,






Tspyl2, Smarcad1, Rasgrp1, Tirap, Fkbp5, Tlr1, Trip12, Ddost, Ick, Adam9, Hspa5, Jun, Nlrp12,






Nup62, Wipi1, Crtc2, Ptpn2, Ptpn1, Abca1, Ufl1, Znrf1, Plaur, Nceh1, Parp3, Mppe1, Elp3, Inhbe,






B3galt1, Dph5, Brd8, Fbxo31, Il1a, Alg12, Stk17b, Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Psen1,






Piga, Itgb3, Itgb2, Arid5a, Ccl19, Gan, Wdr45b, St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b,






Trim3, Rps6ka3, Tnfrsf1a, Nek7, Trib2, Cdkn1a, Gadd45b, Gadd45g, Ppp1r11, Pik3c2a, Vegfc, Pycard,






Fn1, Clock, B3galnt2, St3gal4, Pomgnt1, Ppm1l, Ccr1, Fabp4, Psmd14, Pgm3, Rusc1, Pofut2, Galnt2,






Taf1, Enc1, Josd2, Lats2, Prkch, Prkce, Anapc16, Il6st, Ikbkap, S100a9, Ube2g2


LV
G4
transport
0.00108
Selk, Mybbp1a, Mfsd7b, Yipf5, Unc50, Sec61b, Golga5, Snx6, Atg13, Mlx, Ezr, Rinl, Hnrnpa2b1, Stx6,






Il1rn, Pim3, Arl6ip5, Cog6, Hck, Ednra, B4galt1, Rab12, Srp68, Stx12, Atp6v0a1, Stx18, Cxcl9, Arcn1,






Uso1, Hif1a, Slc37a3, Slc37a1, Lrrc8a, Lcn2, Krt18, Braf, Slc30a5, Timm21, Creb3, Rabl3, Slc39a6,






Ibtk, Csnk1a1, Slco3a1, Copa, Serinc3, Atp7a, Slc16a10, Adora1, Gosr2, Aak1, Marco, Irf8, Grb2,






Blzf1, Slc11a2, Slc11a1, Mmp9, Pdzd11, Lbp, Snip1, Atp11a, Wdr1, Cd14, Jmjd6, Ap1g1, Fgr, Fgb,






Tgm2, Cnnm2, S100a8, Tmed9, Egf, Slc35c1, Cct3, Ica1, Ap2a2, Derl1, Atp2a2, Litaf, Sar1a, Itpr1, Yrdc,






Ipo4, Vamp4, Rab35, Fcer1g, Zfyve16, Srebf2, Slc4a4, Cln8, Slc16a6, Vcp, Dab2, Actr2, Scfd1, Syk,






Hmga2, Vimp, Srgn, Ap4e1, Copb2, Ddx39, Myh9, Serp1, Zw10, Slc39a14, Golph3l, Rab5b, Mmgt1,






Creb3l2, Slc30a7, Plek, Steap4, Slc2a3, Nus1, Wfs1, Chlc2, Cklf, Supt6, Tmem165, Slc15a2, Gas6,






Trim27, Rac2, Zdhhc13, Mfsd2a, Golga4, Dynll1, Mon1b, Msr1, Sdc4, Gm21540, Dst, Ncf1, Slc10a6,






Rasgrp1, Slc10a2, Apoa4, Apoa5, Ick, Pcm1, Kif5b, Hnf4a, Adam9, Hspa5, Golph3, Jun, Sec61a1,






Nlrp12, Flot1, Crp, Emb, Capn10, Wipi1, Sidt2, Rab18, Crtc2, Ptpn1, Ap1ar, Abca1, Casp4, Slc38a10,






Scamp2, Mppe1, Il4ra, Antxr2, Thoc2, Thoc1, Il1a, Bet1l, Ap3d1, Slc38a2, Syt12, Slc9a3r1, Sec22b,






Hook1, Psen1, Ap5s1, Itgb3, Vav1, Ccl19, Slc7a6, Slc35a2, Snx10, S100a10, Trem3, Cltb, Camk2b,






Txndc5, Rint1, Srp72, Tmem173, Rab1, Ift20, C2cd5, Cdkn1a, Hk2, Snap23, Sec13, Pik3c2a, Coro1a,






Sybu, Pycard, Map1lc3b, Stx5a, Clock, Chmp4b, Nemf, Ccr1, Slc35e1, Clec4n, Slc13a3, Slc13a5,






Fabp5, Hsp90aa1, Ero1l, Fcgr3, Rab33b, Scara5, Calr, Lcp2, Pofut2, Tbrg1, Atp6v0e, Prkce, Myo1f,






Arfgap1, S100a9


LV
G4
RNA processing
0.00118
Nol8, Utp6, Dhx30, Ddx52, Mettl1, Mettt3, Hnrnpa2b1, Btg2, Dhx40, Ints7, Ints5, Rpf2, Pus3, Trnt1,






Mphosph6, Sf3b4, Srsf2, Npm3, Dhx38, Mbnl2, Dhx35, Ints2, Exosc1, Exosc4, Snip1, Jmjd6, Ebna1bp2,






Scnm1, Eif4a3, Ddx39, Wdr12, Supt5, Supt6, Gemin8, Ngdn, Cdc73, Dis3, Nop58, Tra2b, Nudt21,






Papola, Ddx1, Elp3, Rnps1, Exosc10, Son, Exosc3, Trmt61a, Magoh, Nol9, Srsf1, Srsf3, Nop56,






Trmt10a, Srrt, Crnkl1, Rbm42


LV
G4
endoplasmic reticulum unfolded protein response
0.00134
Tbl2, Bfar, Creb3, Atf3, Herpud1, Yod1, Derl1, Vimp, Serp1, Wfs1, Hspa5, Ptpn2, Ptpn1, Dnajc3, Ero1l


LV
G4
Vesicle-mediated transport
0.00151
Prkag2, Hsp90b1, Arcn1, Copa, Copb1, Marco, Sec24d, Ap1g1, Clint1, Copg1, Dab2, Ap4e1, Copb2,






Myh9, Msr1, Tbc1d8b, Ralgapa2, Preb, Sec31a, Tpd52, Cltb, Txndc5, C2cd5, Hyou1, Yipf6, Snap23,






Sec13, Pik3c2a, Chmp4b, Hsp90aa1, Scara5, Calr, Arfgap1


LV
G4
inflammatory response
0.00172
Selp, Pik3ap1, Cd28, Il1rn, B4galt1, Il1r1, Cxcl9, Hif1a, Cd55, Psma1, Adora1, Tnfaip6, Slc11a1, Lbp,






S100a8, A2m, Fcer1g, Syk, Vimp, Adam17, Hyal1, Cd44, Ctla2a, Itgam, Ncf1, Rasgrp1, Nlrp12, Ptpn2,






Casp4, Il17ra, Il7a, Socs3, Tnfrsf1b, Tnfrsf1a, Sbno2, Snap23, Pik3c2a, Pycard, Clock, Ccr1, Plaa,






Seh1l, Cr1l, Fabp4, Fcgr3, Nfkbi2, Irg1, S100a9


LV
G4
response to external stimulus
0.0019
Mybbp1a, Tbl2, Selp, Pik3ap1, Slamf8, Atg13, Cd28, Hck, Ldha, Il1r1, Rab12, Qsox1, Ppan, Cxcl9, Dap,






Lcn2, Braf, Creb3, Efna1, Nod1, Clec4d, Hilpda, Ddx21, Cd55, Psma1, Adora1, Tnfaip6, Irf8, Trib1,






Rbm18, Atf3, Slc11a1, Exosc4, Lbp, Cd14, Ap1g1, Fgr, Vasp, Fgb, S100a8, A2m, Slc35c1, Ifitm2, Ifitm1,






Atg9b, Litaf, Saa3, Usp14, Fcer1g, Tbl1xr1, Srebf2, Bag3, Fpr1, Scfd1, Syk, Vimp, Adam17, Asns,






Rraga, Hyal1, Ctla2a, Prdm1, Reg3b, Plek, Sox9, Supt5, Cklf, Gas6, Rac2, Il12rb1, Irak2, Iigp1, Cdc73,






Itgam, Ncf1, Pld1, Tirap, Trp53inp2, Tlr1, Kif5b, Adam9, Hspa5, Jun, Nlrp12, Flot1, Ubxn2b, Cox8a,






Wipi1, Ptpn2, Abca1, Casp4, Il17ra, Il4ra, Ddx1, Ap3d1, Psen1, Itgb3, Itgb2, Ccl17, Vav1, Ccl19,






Wdr45b, Atg16l2, Trem3, Socs3, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Tmem173, Rab1, Ift20, Cdkn1a, Sbno2,






Pik3c2a, Coro1a, Vegfc, Pycard, Map1lc3b, Clock, Bbs4, Alox12, Chmp4b, Ccr1, Clec4n, Seh1l, Cr1l,






Fabp4, Fcgr3, Rab33b, Scara5, Calr, Nfkbib, Prkce, Crnkl1, Irg1, Myo1f, S100a9


LV
G4
ribosome biogenesis
0.00196
Nol8, Utp6, Ddx52, Rpf2, Mphosph6, Npm3, Tsr1, Exosc4, Ebna1bp2, Ipo4, Wdr12, Gnl2, Gnl1,






Ngdn, Dis3, Nop58, Brix1, Exosc10, Exosc3, Nol9, Nop56, Eif6


LV
G4
protein N-linked glycosylation via asparagine
0.00263
Krtcap2, Syvn1, Stt3a, Rpn1, Ube2j1, Vcp, St6gal1, Ube2g2


LV
G4
cellular response to topologically incorrect protein
0.00267
Tbl2, Bfar, Creb3, Atf3, Herpud1, Yod1, Derl1, Vimp, Serp1, Sdf2l1, Wfs1, Hspa5, Ptpn2, Ptpn1






Dnajc3, Ero1l


LV
G4
defense response
0.00317
Selk, Selp, Pik3ap1, Slamf8, Cd28, Cebpg, Il1rn, Hck, B4galt1, Il1r1, Cxcl9, Hifle, Eif4e, Lcn2, Creb3,






Nod1, Clec4d, Hilpda, Cd55, Psma1, Adora1, Tnfaip6, Irf8, Rbm18, Slc11a1, Exosc4, Lbp, Apcs, Ap1g1,






Fgr, Fgb, S100a8, A2m, Slc35c1, Ifitm2, Ifitm1, Fcer1g, Syk, Vimp, Adam17, Hyal1, Cd44, Ctla2a,






Reg3b, Trim27, Il12rb1, Irak2, Iigp1, Itgam, Ncf1, Pld1, Rasgrp1, Tirap, Tlr1, Apoa4, Nirp12, Cox8a,






Ptpn2, Casp4, Il17ra, Il4ra, Il1a, Ccl17, Vav1, Eprs, Trem3, Socs3, Rps6ka3, Tnfrsf1b, Tnfrsf1a,






Tmem173, Rab1, Sbno2, Snap23, Pik3c2a, Coro1a, Pycard, Clock, Ccr1, Plaa, Clec4n, Seh1l, Cr1l, Fabp4,






Fcgr3, Polr3d, Nfkbiz, Prkce, Crnkl1, Irg1, Myo1f, S100a9


LV
G4
protein N-linked glycosylation
0.00792
Krtcap2, B4galt1, Syvn1, Stt3a, Rpn1, Ube2j1, Vcp, St6gal1, Tmem165, Ddost, Alg12, Pgm3,






Ube2g2


LV
G4
regulation of cellular response to stress
0.00836
Bfar, Cebpg, Arl6ip5, Map3k3, Rab12, Qsox1, Fktn, Ctgf, Pdcd10, Pnp, Hif1a, Marveld3, Syvn1,






Braf, Scly, Creb3, Senp2, Nod1, Apex1, Serinc3, Usp1, Herpud1, Skil, Ppp4c, Dab2, Scfd1,






Syk, Hmga2, Vimp, Cd44, Wfs1, Supt5, Hipk3, Grina, Rasgrp1, Tirap, Trip12, Ptpn2, Ptpn1,






Thoc1, Il1a, Eya3, Ccl19, Dnajc3, Rint1, Ift20, Hyou1, Gadd45b, Gadd45g, Sod2, Pycard, Trp63,






Chmp4b, Rab33b


LV
G4
negative regulation of response to endoplasmic reticulum
0.0089
Bfar, Syvn1, Creb3, Herpud1, Vimp, Wfs1, Grina, Ptpn1, Dnajc3, Hyou1




stress


LV
G4
cellular amino acid metabolic process
0.01264
Lars, Sdsl, Tat, Gpt2, Pcyox1, Aldh18a1, Gls, Atp7a, Mthfr, Cad, Iars, Rars, Hdc, Asns, Odc1,






Tha1, Hars, Hnf4a, Cars2, Farsb, Vars, Kars, Sds, Ero1l, Ears2


LV
G4
Synthesis of substrates in N-glycan biosythesis
0.01361
Mvd, Nans, St6gal1, Gfpt1, Gne, St3gal3, Gmppa, Dhdds, St3gal5, St3gal4, Uap1, Pgm3,






Gmppb


LV
G4
regulation of endoplasmic reticulum stress-induced
0.01401
Syvn1, Creb3, Serinc3, Herpud1, Vimp, Wfs1, Grina, Ptpn2, Ptpn1, Hyou1




intrinsic apoptotic signaling pathway


LV
G4
response to wounding
0.0142
Selp, Pik3ap1, Cd28, B4galt1, Il1r1, Pdcd10, Actg1, Hif1a, Braf, Cd55, Psma1, Adora1, Tnfaip6,






Slc11a1, Lbp, Fgb, S100a8, A2m, Fcer1g, Lnp, Tnfrsf12a, Syk, Vimp, Cd44, Myh9, Ctla2a,






F13a1, Plek, Gas6, Sdc4, Hnf4a, Jun, Pcia6, Nlrp12, Ifrd1, Ptpn2, Casp4, Il17ra, Il1a, Itgb3,






Socs3, Tnfrsf1b, Tnfrsf1a, Sbno2, Sod2, Pik3c2a, Pycard, Fn1, Clock, Alox12, Cr1l, Fabp4, Fcgr3,






Scara5, Prkce, Irg1, S100a9


LV
G4
macromolecule modification
0.01561
Selk, Bfar, Pdgfc, Mbd1, Prkag2, Garem, Mettl1, Mettl3, Senp6, Pim3, Arl6ip5, Metap2, Hck,






Etf1, Btg2, Ntmt1, Anapc4, Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd, Plcl1,






Fktn, Pus3, Errfi1, Ctgf, Nktr, Pdcd10, Mphosph8, Marveld3, Syvn1, Braf, Senp2, Efna1,






Nod1, Ibtk, Apex1, Ube2s, Trim39, Csnk1a1, Cwc27, Rlim, Dok2, Atp7a, Usp8, Usp1, Tnfaip1,






Eogt, Aak1, Trib1, Grb2, Atf3, Herpud1, Slc11a1, Mthfr, Mmp9, Exosc4, Cad, Stt3a, Lpin1,






Uba5, Jmjd6, Lpar1, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2, Uspl1, S100a8, Egf,






Fkbp11, Nedd4l, C1galt1, Wdr61, Traf7, Acer2, Dpy19l1, Saa3, Usp14, Tbl1xr1, Ufm1, Cul5,






Ube2j1, Vcp, Dab2, Dusp2, Asb4, Syk, St6gal1, Hmga2, Adam17, Cd44, Neurl3, Prmt1, Dzip3,






Iqgap1, Dnajc10, F13a1, Sox9, Nus1, Wfs1, Supt6, Hipk3, Tmem165, Gas6, Trim27, Rac2,






Ppp2r1a, Il12rb1, Cdc73, Tspyl2, Smarcad1, Rasgrp1, Nop58, Tirap, Fkbp5, Tlr1, Trip12, Ddost,






Ick, Adam9, Hspa5, Atf7ip, Jun, Dnajb11, Nlrp12, Nup62, Wipi1, Crtc2, Ptpn2, Ptpn1, Abca1,






Ufl1, Znrf1, Plaur, Nceh1, Parp3, Mppe1, Elp3, Inhbe, B3galt1, Dph5, Brd8, Il1a, Alg12, Stk17b,






Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Psen1, Exosc3, Piga, Itgb3, Itgb2, Arid5a, Ccl19,






Gan, Wdr45b, Trmt61a, St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b, Trim3, Rps6ka3,






Tnfrsf1a, Nek7, Trib2, Cdkn1a, Gadd45b, GBdd45g, Ppp1r11, Pik3c2a, Vegfc, Pycard, Fn1,






Clock, B3galnt2, St3gal5, St3gal4, Pomgnt1, Ppm1l, Ccr1, Fabp4, Psmd14, Pgm3, Rusc1,






Nop56, Pofut2, Galnt2, Taf1, Trmt10a, Enc1, Josd2, Lats2, Prkch, Prkce, Anapc16, Il6st, Ikbkap,






S100a9, Ube2g2


LV
G4
cellular response to starvation
0.01936
Mybbp1a, Tbl2, Atg13, Rab12, Qsox1, Dap, Atf3, Atg9b, Srebf2, Scfd1, Asns, Rraga, Supt5,






Gas6, Trp53inp2, Hspa5, Ubxn2b, Wipi1, Psen1, Wdr45b, Atg16l2, Rab1, Ift20, Map1lc3b,






Chmp4b, Rab33b


LV
G4
intrinsic apoptotic signaling pathway in response to
0.02034
Selk, Syvn1, Creb3, Serinc3, Herpud1, Itpr1, Vimp, Dnajc10, Wfs1, Grina, Ptpn2, Ptpn1, Casp4,




endoplasmic reticulum stress

Hyou1, Ero1l


LV
G4
RNA transport | Processing of Capped Intron-Containing
0.02073
Mettl3, Hnrnpa2b1, Etf1, Wdr77, Trnt1, Pnn, Eif4e, Sf3b4, Eif3d, Srsf2, Senp2, Plrg1, Gm10094,




Pre-mRNA

Dhx38, Alyref, Gtf2f1, Nup98, Gspt2, Snrnp40, Ccar1, Eif4g2, Ranbp2, Eif4a3, Casc3, Gtf2f2,






Nupl2, Ppp2r1a, Gemin8, Tra2b, Snrnp27, Nudt21, Papola, Nupl1,Nup62, Eif2b4, Thoc2,






Thoc1, Rnps1, Magoh, Sec13, Hbs1l, Seh1l, Srsf1, Srsf3, Crnkl1


LV
G4
Membrane Trafficking
0.02312
Prkag2, Arcn1, Copa, Copb1, Sec24d, Ap1g1, Clint1, Copg1, Dab2, Ap4e1, Copb2, Myh9,






Tbc1d8b, Ralgapa2, Preb, Sec31a, Tpd52, Cltb, Txndc5, C2cd5, Yipf6, Snap23, Sec13, Pik3c2a,






Chmp4b, Arfgap1


LV
G4
cell death
0.02705
Selk, Mybbp1a, Cyp1b1, Bfar, Cd28, Pim3, Arl6ip5, Hck, Btg2, B4galt1, Hsp90b1, Ctgf, Pdcd10,






Pnp, Hif1a, Dap, Lcn2, Krt18, Syvn1, Braf, Creb3, Nod1, Trim39, Dnajc5, Pcid2, Serinc3,






Atp7a, Atf3, Herpud1, Slc11a2, Gzma, Mmp9, Skil, Arf4, Fgb, Tgm2, S100a8, Ccar1, Pkm, Trps1,






Traf7, Pdia3, Acer2, Itpr1, Fcer1g, Cln8, Bag3, Vcp, Dab2, Tnfrsf12a, Hmga2, Vimp, Adam17,






Srgn, Asns, Rraga, Cd44, Qars, Ier3ip1, Dnajc10, Sox9, Sdf2l1, Wfs1, Hipk3, Gas6, Ppp2r1a,






Irak2, Tnfrsf22, Gata6, Grina, Mt1, Hspa5, Golph3, Jun, Nlrp12, Capn10, Nup62, Ptpn2, Ptpn1,






Casp4, Plaur, Parp2, Zbtb16, Thoc1, Inhbe, Rnps1, Dcun1d3, Il1a, Stk17b, Son, Slc9a3r1,






Psen1, Cdip1, Cd38, Ccl19, Dnajc3, Camk2b, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Hyou1, Cdkn1a, Hk2,






Gadd45b, Gadd45g, Sod2, Coro1a, Pycard, Trp63, Ivns1abp, Alox12, Chmp4b, Ero1l, Prkch,






S100a9


LV
G4
Golgi organization
0.03053
Golga5, Surf4, Stx6, Stx18, Pdcd10, Blzf1, Tmed9, Vamp4, Zw10, Golph3l, Gm21540, Golph3,






Ubxn2b, Rab1, Stx5a, Rab33b, Atl2


LV
G4
Goigi to ER Retrograde Transport
0.03083
Arcn1, Copa, Copb1, Copg1, Copb2, Arfgap1


LV
G4
RNA Polymerase II Transcription
0.0314
Gtf2e1, Dhx38, Alyref, Ell, Tbp, Gtf2f1, Gtf2f2, Nelfa, Supt5, Nudt21, Papola, Taf2, Rnps1,






Cdk7, Gtf2a2, Taf4b, Magoh, Srsf1, Srsf3, Taf1


LV
G4
cellular response to external stimulus
0.03307
Mybbp1a, Tbl2, Atg13, Ldha, Rab12, Qsox1, Ppan, Dap, Atf3, Atg9b, Srebf2, Bag3, Scfd1,






Asns, Rraga, Sox9, Supt5, Gas6, Trp53inp2, Hspa5, Ubxn2b, Wipi1, Psen1, Wdr45b, Atg16l2,






Rab1, Ift20, Cdkn1a, Map1lc3b, Chmp4b, Rab33b


LV
G4
intracellular transport
0.03565
Mybbp1a, Mfsd7b, Yipf5, Sec61b, Golga5, Snx6, Atg13, Mlx, Stx6, Cog6, Rab12, Srp68, Stx12,






Stx18, Cxcl9, Arcn1, Uso1, Hif1a, Krt18, Timm21, Creb3, Rabl3, Ibtk, Gosr2, Blzf1, Slc11a1,






Snip1, Ap1g1, Fgr, Tmed9, Egf, Ap2a2, Derl1, Atp2a2, Litaf, Sar1a, Itpr1, Ipo4, Vamp4, Rab35,






Fcer1g, Zfyve16, Vcp, Dab2, Actr2, Syk, Vimp, Ap4e1, Copb2, Ddx39, Zw10, Rab5b, Creb3l2,






Nus1, Chic2, Supt6, Tmem165, Gas6, Trim27, Rac2, Golga4, Dynll1, Gm21540, Dst, Ncf1,






Rasgrp1, Ick, Pcm1, Kif5b, Golph3, Jun, Sec61a1, Nlrp12, Capn10, Wipi1, Rab18, Ptpn1, Ap1ar,






Abca1, Mppe1, Il4ra, Thoc2, Thoc1, Bet1l, Ap3d1, Sec22b, Hook1, Psen1, Ap5s1, Ccl19, Rint1,






Srp72, Tmem173, Rab1, Ift20, C2cd5, Cdkn1a, Hk2, Snap23, Sec13, Coro1a, Stx5a, Chmp4b,






Nemf, Hsp90aa1, Ero1l, Rab33b, Calr, Tbrg1, Prkce


LV
G4
biosynthetic process
0.03959
Cers6, Lars, Selk, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Pdgfc, Snx6, Mlx, Cd28, Cebpg,






Cebpd, Mettl3, Hnrnpa2b1, Hnrnpab, Polr1e, Hck, Etf1, Ednra, Btg2, Wdr77, Agk, Adcy1,






Ppap2a, Krtcap2, B4galt1, Mvd, Bmper, Tcf25, Ldha, Atp6v0a1, Fktn, Epm2aip1, Mphosph8,






Klf17, Pnp, Hif1a, Smarce1, Eif4e, Dap, Agpat9, Eif3d, Syvn1, Prg4, Creb3, Senp2, Efna1, Ell2,






Nod1, Ctif, Gmeb1, Cyp7b1, Npm3, Tasp1, Aldh18a1, Pcid2, Ddx21, Gls, Rlim, B4galnt1, Atp7a,






Ubiad1, Tnfaip1, Eogt, Mrpl51, Ell, Tbp, Irf8, Trib1, Cebp2, Atf6, Atf3, Slc11a2, Slc11a1,






Mthfr, Cad, Lbp, Snip1, Skil, Stt3a, Lpin1, Polh, Gtf2f1, Mup98, Foxk2, Arf4, Rpn1, Tmsb4x,






Egf, Eif4g2, Stag1, C1galt1, Fen1, Wdr61, Trps1, Spin1, Iars, Traf7, Acer2, Dpy19l1, Rars, Hdc,






Tbl1xr1, Srebf2, Suco, Far2, Ranbp2, Trappc2, Ube2j1, Vcp, Mcm2, Dab2, Sf1, Syk, St6gal1,






Hmga2, Vimp, Asns, Hyal1, Serp1, Prmt1, Cyp51, Prdm1, Atf6b, Pcsk5, Creb3l2, Plek, Idi1,






Nelfa, Rbm39, Sox9, Nus1, Odc1, Papss1, Wfs1, Supt5, Supt6, Hipk3, Tmem165, Helb, Gas6,






Trim27, Crem, Paip2b, Irak2, Dynll1, Hist1h3b, Cdc73, Lig3, Gata6, Ncf1, Tspyl2, Srm, Pld1,






Tha1, Tirap, Trp53inp2, Tlr1, Med17, Apoa4, Apoa5, Hars, Zfp869, Ddost, Gigyf2, Hnf4a, Zfp160,






Atf7ip, Csgalnact2, Golph3, Jun, Nlrp12, Denr, Cars2, Farsb, Taf2, Ctps, Nup62, Vars, Wipi1,






Ifrd1, Crtc2, Secisbp2, Ptpn2, Abca1, Ufl1, Preb, Parp3, Mppe1, Zbtb16, Zbtb21, Med25,






Eif2b4, Thoc1, Elp3, B3galt6, B3galt1, Tfb2m, Cdk7, Dph5, Zfp830, Il1a, Alg12, Rps9, Kars,






Tiparp, Psen1, Hltf, Cd38, Piga, Itgb3, Arid5a, Ccl19, Gtf2a2, Nfia, Wdr45b, Atg16l2, Eprs,






D198wg1357e, Spg20, St3gal3, Dnajc3, Rcor1, Polr1a, Rps6ka3, Tnfrsf1a, Cpeb2, Trib2, Tmem173,






Cdkn1a, Taf4b, Sds, Sbno2, Sod2, Pik3c2a, Etnk2, Pycard, Adk, Trp63, Clock, Alox12, B3galnt2,






St3gal4, Pomgnt1, Fabp4, Fabp5, Hsp90aa1, Lss, Fcgr3, Zfp27, Calr, Pgm3, Pofut2, Polr3k,






Galnt2, Tbrg1, Tal, Pspc1, Nfkbiz, Enc1, Srrt, Prkch, Ears2, Irg1, Larp7, Ikbkap, Nif3l1,






S100a9, Ube2g2


LV
G4
negative regulation of endoplasmic reticulum stress-induced
0.03966
Syvn1, Creb3, Herpud1, Vimp, Wfs1, Grins, Ptpn1, Hyou1




intrinsic apoptotic signaling pathway


LV
G4
Biosynthesis of the N-glycan precursor (dolichol lipid-linked
0.04085
Mvd, Nans, St6gal1, Gfpt1, Alg12, Gne, St3gal3, Gmppa, Dhdds, St3gal5, St3gal4, Uap1,




oligosaccharide, LLO) and transfer to a nascent protein

Pgm3, Gmppb


LV
G4
Transcription
0.04772
Polr1e, Gtf2e1, Zfp143, Dhx38, Alyref, Ell, Tbp, Gtf2f1, Gtf2f2, Nelfa, Supt5, Hist1h3b, Ssb,






Nudt21, Papola, Taf2, Rnps1, Tfb2m, Cdk7, Gtf2a2, Nfia, Polr1a, Taf4b, Magoh, Srsf1, Srsf3,






Polr3d, Polr3k, Taf1


LV
G4
Aminoacyl-tRNA biosynthesis
0.05146
Lars, Scly, Mars, Iars, Rars, Qars, Papss1, Hars, Cars2, Farsb, Vars, Kars, Eprs, Ears2


LV
G4
single-organism intracellular transport
0.05511
Mfsd7b, Yipf5, Sec61b, Golga5, Snx6, Atg13, Stx6, Cog6, Rab12, Srp68, Stx18, Cxcl9, Arcn1,






Uso1, Hif1a, Krt18, Timm21, Creb3, Ibtk, Gosr2, Blzf1, Slc11a1, Snip1, Ap1g1, Fgr, Tmed9,






Egf, Derl1, Atp2a2, Litaf, Sar1a, Itpr1, Ipo4, Vamp4, Rab35, Fcer1g, Zfyve16, Vcp, Dab2, Syk,






Vimp, Copb2, Ddx39, Zw10, Rab5b, Creb3l2, Nus1, Chic2, Supt6, Tmem165, Gas6, Trim27, Rac2,






Golga4, Dynll1, Gm21540, Dst, Ncf1, Rasgrp1, Ick, Pcm1, Kif5b, Golph3, Jun, Sec61a1, Nlrp12,






Wipi1, Ap1ar, Abca1, Mppe1, Il4ra, Thoc2, Thoc1, Bet1l, Ap3d1, Sec22b, Hook1, Psen1,






Ap5s1, Ccl19, Rint1, Srp72, Tmem173, Rab1, Ift20, C2cd5, Cdkn1a, Hk2, Snap23, Sec13,






Coro1a, Stx5a, Chmp4b, Hsp90aa1, Ero1l, Rab33b, Calr, Tbrg1, Prkce


LV
G4
intrinsic apoptotic signaling pathway
0.0602
Selk, Mybbp1a, Cyp1b1, Arl6ip5, Pdcd10, Pnp, Hif1a, Syvn1, Creb3, Serinc3, Herpud1, Mmp9,






Skil, S100a8, Itpr1, Vimp, Cd44, Dnajc10, Wfs1, Grina, Ptpn2, Ptpn1, C3sp4, Plaur, Slc9a3r1,






Cdip1, Tnfrsf1b, Tnfrsf1a, Hyou1, Cdkn1a, Sod2, Pycard, Trp63, Ivns1abp, Ero1l, S100a9


LV
G4
Processing of Capped Intron-Containing Pre-mRNA
0.06182
Mettl3, Hnrnpa2b1, Eif4e, Sf3b4, Plrg1, Dhx38, Alyref, Gtf2f1, Nup98, Snrnp40, Ccar1,






Ranbp2, Gtf2f2, Nupl2, Nudt21, Papola, Nupl1, Nup62, Rnps1, Magoh, Seh1l, Srsf1, Srsf3


LV
G4
SNARE interactions in vesicular transport
0.07286
Stx6, Stx18, Gosr2, Vamp4, Bet1l, Sec22b, Snap23, Stx5a


LV
G4
phagocytosis
0.073
Hck, Csnk1a1, Marco, Irf8, Slc11a1, Lbp, Jmjd6, Fgr, Tgm2, Fcer1g, Scfd1, Syk, Myh9, Gas6,






Abca1, Vav1, Txndc5, Coro1a, Pycard, Fcgr3, Calr


LV
G4
cellular response to extracellular stimulus
0.07784
Mybbp1a, Tbl2, Atg13, Ldha, Rab12, Qsox1, Ppan, Dap, Atf3, Atg9b, Srebf2, Scfd1, Asns,






Rrag8, Supt5, Gas6, Trp53inp2, Hspa5, Ubxn2b, Wipi1, Psen1, Wdr45b, Atg16l2, Rab1, Ift20,






Cdkn1a, Map1lc3b, Chmp4b, Rab33b


LV
G4
Platelet activation, signaling and aggregation
0.08181
Selp, Timp1, Grb2, Wdr1, Fgb, Tmsb4x, A2m, Egf, Itpr1, Fcer1g, Syk, Srgn, F13a1, Plek, Csf2rb,






Gas6, Rac2, Rasgrp1, Hspa5, Ptpn1, Itgb3, Vav1, Vegfc, Fn1, Gnai2, Lcp2, Prkch, Prkce


LV
G4
ER to Golgi vesicle-mediated transport
0.08751
Yipf5, Stx18, Uso1, Gosr2, Sar1a, Rab35, Zw10, Creb3l2, Mppe1, Sec22b, Rint1, Rab1, Sec13,






Stx5a


LN
G1
regulation of immune system process
0.93881
Pik3ap1, Trim30a, Pik3ap1, Mfsd7b, Cd28, Cebpg, Cebpd, Hck, B4galt1, Cxcl9, Ung, Pnp, Hif1a,






Lrrc8a, Lcn2, Prg4, Braf, Creb3, Nod1, Clec4d, Hilpda, Pcid2, Cd55, Psma1, Atp7a, Adora1,






Tnfaip1, Irf8, Trib1, Rbm18, Slc11a2, Slc11a1, Exosc4, Lbp, Skil, Apcs, Jmjd6, Ap1g1, Fgr,






Fgb, S100a8, A2m, Slc35c1, Wdr61, Ifitm2, Ifitm1, Ap2a2, Gpatch4, Rab35, Fcer1g, Dab2, Syk,






Vimp, Adam17, Cd44, Myh9, Ctla2a, Prmt1, Prdm1, Rab5b, H2-D1, Sox9, Cklf, Supt6, Gas6, Trim27,






Rac2, Il12rb1, Irak2, Cdc73, Itgal, Itgam, Ncf1, Rasgrp1, Tirap, Tlr1, Apoa4, Ddost, Adam9,






Golph3, Jun, Nlrp12, Cox8a, Ctps, Ptpn2, Zbtb16, Il4ra, Thoc1, Ap3d1, Tpd52, Tiparp, Psen1,






Exosc3, Cd38, Itgb3, Itgb2, Vav1, Ccl19, Eprs, Snx10, Rcor1, Trem3, Rps6ka3, Tmem173, Cdkn1a,






Gadd45g, Sbno2, Sod2, Snap23, Msn, Pik3c2a, Coro1a, Vegfc, Pycard, Ccr1, Txlna, Clec4n, Cd48,






Cr1l, Hsp90aa1, Fcgr3, Calr, Pgm3, Polr3d, Lcp2, Galnt2, Clptm1, Prkch, Prkce, Crnkl1, Irg1,






Myo1f, Il6st, S100a9


LV
G4
macromolecule catabolic process
0.09361
Bfar, Sec61b, Timp1, Btg2, Rab12, Hsp90b1, Syvn1, Timp3, Fbxo6, Ctif, Ube2s, Trim39, Pcyox1,






Rlim, Psma1, Usp8, Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Exosc4, Yod1, Egf, Nedd4l, Fen1,






Wdr61, Derl1, Usp14, Tbl1xr1, Cul5, Cln8, Ube2j1, Vcp, Dab2, Vimp, Hyal1, Cd44, Ctla2a,






Ctla2b, Dnajc10, Sox9, Sdf2l, Odc1, Wfs1, Lyve1, Parn, Exog, Dis3, Trip12, Adam9, Hspa5,






Ubxn2b, Secisbp2, Ptpn1, Ufl1, Znrf1, Rnps1, Exosc10, Fbxo31, Tiparp, Sec22b, Psen1, Exosc3,






Zfand2a, Khsrp, Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1, Nbas, Magoh,






Stx5a, Clock, Chmp4b, Psmd14, Taf1, Enc1, Ube2g2


LV
G4
organic substance transport
0.10013
Selk, Mfsd7b, Sec61b, Snx6, Atg13, Ezr, Hnrnpa2b1, Stx6, Il1rn, Pim3, Arl6ip5, Ednra, Rab12,






Srp68, Stx12, Uso1, Hif1a, Krt18, Braf, Timm21, Creb3, Rabl3, Slco3a1, Serinc3, Gosr2, Blzf1,






Slc11a1, Lbp, Snip1,Atp11a, Ap1g1, Fgr, Fgb, S100a8, Egf, Ap2a2, Derl1, Litaf, Ipo4, Vamp4,






Rab35, Fcer1g, Zfyve16, Srebf2, Slc4a4, Cln8, Slc16a6,Vcp, Dab2, Scfd1, Syk, Hmga2, Vimp,






Srgn, Ap4e1, Ddx39, Myh9, Serp1, Golph3l, Rab5b, Plek, Slc2a3, Nus1, Supt6, Slc15a2,






Gas6, Trim27, Rac2, Mfsd2a, Golga4, Msr1, Gm21540, Ncf1, Slc10a6, Rasgrp1, Slc10a2, Apoa4,






Apoa5, Pcm1, Hnf4a, Adam9, Hspa5, Golph3, Jun, Sec61a1, Nlrp12, Crp, Emb, Capn10, Sidt2,






Rab18, Ptpn1, Abca1, Casp4, Slc38a10, Scamp2, Il4ra, Thoc2, Thoc1, Il1a, Ap3d1, Slc38a2,






Slc9a3r1, Psen1, Itgb3, Ccl19, Slc7a6, Slc35a2, Trem3, Srp72, Tmem173, Rab1, Ift20, C2cd5,






Cdkn1a, Hk2, Snap23, Sybu, Pycard, Stx5a, Clock, Chmp4b, Slc35e1, Clec4n, Slc13a3,






Slc13a5, Fabp5, Hsp90aa1, Fcgr3, Rab33b, Calr, Lcp2, Prkce, S100a9


LV
G4
N-glycan trimming in the ER and Calnexin/Calreticulin
0.1026
Mogs, Pdia3, Edem2, Edem3, Edem1, Calr




cycle


LV
G4
ncRNA processing
0.14837
Nol8, Utp6, Ddx52, Mettl1, Ints7, Ints5, Rpf2, Pus3, Trnt1, Mphosph6, Npm3, Ints2, Exosc4,






Ebna1bp2, Wdr12, Ngdn, Dis3, Nop58, Ddx1, Eip3, Exosc10, Exosc3, Trmt61a, Nol9, Nop56,






Trmt10a, Srrt


LV
G4
Transport of Mature mRNA derived from an Intron-Containing
0.15115
Eif4e, Dhx38, Alyref, Nup98, Ranbp2, Nupl2, Nupl1, Nup62, Rnps1, Magoh, Seh1l, Srsf1, Srsf3




Transcript


LV
G4
catabolic process
0.15182
Sdsl, Cyp1b1, Bfar, Sec61b, Prkag2, Atg13, Tat, Timp1, Btg2, Rab12, Qsox1, Hsp90b1, Pnp,






Hif1a, Dap, Syvn1, Timp3, Fbxo6, Ctif, Ube2s, Trim39, Pcyox1, Mmp13, Pfkp, Gls, Rlim, Psma1,






Usp8, Entpd7, Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Mmp9, Exosc4, Lpin1, Dram1, Yod1,






S100a8, Egf, Slc35c1, Nedd4l, Fen1, Wdr61, Pkm, Derl1, Atg9b, Hdc, Usp14, Tbl1xr1, Pfkfb2,






Cul5, Cln8, Ube2j1, Vcp, Dab2, Scfd1, Vimp, Rraga, Hyal1, Cd44, Ctla2a, Ctla2b, Dnajc10,






Sox9, Sdf2l1, Odc1, Wfs1, Supt5, Ilgp1, Lyve1, Parn, Exog, Ncf1, Dis3, Tha1, Trp53inp2, Apoa4,






Apoa5, Trip12, Adam9, Hspa5, Ubxn2b, Cox8a, Wipi1, Secisbp2, Ptpn1, Ufl1, Znrf1, Rnps1,






Exosc10, Fbxo31, Cda, Tiparp, Sec22b, Psen1, Exosc3, Zfand2a, Khsrp, Wdr45b, Atg16l2,






Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1, Rab1, Ift20, Nbas, Hk2, Sds, Gale, Magoh,






Pycard, Map1lc3b, Stx5a, Clock, Chmp4b, Txlna, Rab33b, Psmd14, Taf1, Enc1, Prkce, Crnkl1,






S100a9, Ube2g2


LV
G4
regulation of response to external stimulus
0.15254
Selp, Pik3ap1, Cd28, Il1r1, Rab12, Qsox1, Cxcl9, Braf, Creb3, Hilpda, Cd55, Psma1, Adora1,






Tnfaip6, Trib1, Rbm18, Lbp, S100a8, A2m, Slc35c1, Usp14, Fcer1g, Scfd1, Syk, Vimp, Adam17,






Ctla2a, Prdm1, Plek, Supt5, Gas6, Rac2, Il12rb1, Tirap, Nlrp12, Cox8a, Ptpn2, Casp4, Il17ra,






Ccl19, Socs3, Tnfrsf1b, Tnfrsf1a, Tmem173, Ift20, Sbno2, Vegfc, Pycard, Clock, Bbs4, Alox12,






Chmp4b, Ccr1, Cr1l, Fabp4, Fcgr3, Rab33b, Scara5, Calr, Crnkl1, Irg1, S100a9


LV
G4
cellular macromolecule catabolic process
0.16017
Bfar, Sec61b, Btg2, Rab12, Hsp90b1, Syvn1, Fbxo6, Ctif, Ube2s, Trim39, Pcyox1, Rlim, Psma1,






Usp8, Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Exosc4, Yod1, Egf, Nedd4l, Fen1, Wdr61, Derl1,






Usp14, Tbl1xr1, Cul5, Cln8, Ube2j1, Vcp, Dab2, Vimp, Ctla2a, Ctla2b, Dnajc10, Sdf2l1, Wfs1,






Parn, Exog, Dis3, Trip12, Hspa5, Ubxn2b, Secisbp2, Ufl1, Znrf1, Rnps1, Exosc10, Fbxo31,






Psen1, Exosc3, Zfand2a, Khsrp, Kctd10, Dnajc3, Ubxn4, Edem3, Trib2, Edem1, Nbas, Magoh,






Clock, Chmp4b, Psmd14, Taf1, Enc1, Ube2g2


LV
G4
SLC-mediated transmembrane transport
0.16306
Slc41a1, Slc30a5, Slc39a6, Slco3a1, Slc20a1, Slc16a10, Slc11a2, Slc11a1, Pdzd11, Nup98,






Slc35c1, Slc39a7, Slc4a4, Ranbp2, Slc39a10, Slc39a14, Slc30a7, Nupl2, Slc2a3, Slc15a2,






Slc10a6, Nupl1, Nup62, Cp, Slc38a2, Slc7a6, Slc35a2, Slc41a2, Hk2, Seh1l, Slc13a3, Slc13a5


LV
G4
autophagy
0.16436
Atg13, Rab12, Qsox1, Hif1a, Dap, Pfkp, Dram1, S100a8, Slc35c1, Atg9b, Scfd1, Rraga, Supt5,






Iigp1, Trp53lnp2, Ubxn2b, Cox8a, Wlpi1, Psen1, Wdr45b, Atg16l2, Rab1, Ift20, Pycard,






Map1lc3b, Chmp4b, Txlna, Rab33b, Crnkl1, S100a9


LV
G4
neutrophil chemotaxis
0.16581
Lbp, S100a8, Fcer1g, Syk, Cklf, Rac2, Itgam, Tirap, Itgb2, Vav1, Ccl19, Trem3, Fcgr3, S100a9


LV
G4
carbohydrate derivative biosynthetic process
0.16801
Ednra, Adcy1, Krtcap2, B4galt1, Atp6v0a1, Fktn, Pnp, Syvn1, B4galnt1, Atp7a, Eogt, Stt3a,






Arf4, Rpn1, Egf, C1galt1, Acer2, Dpy19l1, Ube2j1, Vcp, St6gal1, Hyal1, Nus1, Papss1, Wfs1,






Tmem165, Ddost, Csgalnact2, Golph3, Ctps, Abca1, Parp3, Mppe1, B3galt6, B3galt1, Alg12,






Tiparp, Psen1, Piga, Ccl19, St3gal3, Adk, B3galnt2, St3gal4, Pomgnt1, Pgm3, Pofut2, Galnt2,






Ube2g2


LV
G4
leukocyte migration
0.18595
Selk, Selp, B4galt1, Cxcl9, Creb3, Adora1, Lbp, S100a8, Fcer1g, Syk, Adam17, Cklf, Gas6, Rac2,






Itgam, Tirap, Golph3, Nlrp12, Itgb2, Vav1, Ccl19, Trem3, Msn, Coro1a, Vegfc, Pycard, Ccr1,






Fcgr3, Calr, S100a9


LV
G4
regulation of inflammatory response
0.19888
Pik3ap1, Cd28, Il1r1, Cd55, Psma1, Adora1, Tnfaip6, Lbp, S100a8, A2m, Fcer1g, Vimp, Ctla2a,






Nlrp12, Ptpn2, Casp4, Il17ra, Socs3, Tnfrsf1b, Tnfrsf1a, Sbno2, Pycard, Clock, Cr1l, Fabp4,






Fcgr3, Irg1, S100a9


LV
G4
autophagosome assembly
0.20309
Atg13, Atg9b, Scfd1, Trp53inp2, Ubxn2b, Wipi1, Psen1, Wdr45b, Atg16l2, Rab1, Ift20, Map1lc3b,






Chmp4b, Rab33b


LV
G4
translation
0.20313
Lars, Mettl3, Etf1, Btg2, Eif4e, Eif3d, Ctif, Mrpl51, Eif4g2, Iars, Rars, Serp1, Paip2b, Hars,






Gigyf2, Denr, Cars2, Farsb, Vars, Secisbp2, Eif2b4, Rps9, Kars, Eprs, D19Bwg1357e, Dnajc3,






Rps6ka3, Cpeb2, Enc1, Ears2, S100a9


LV
G4
Platelet degranulation
0.25253
Selp, Timp1, Wdr1, Fgb, Tm5b4x, A2m, Egf, Srgn, F13a1, Plek, Gas6, Hspa5, Itgb3, Vegfc, Fn1


LV
G4
single-organism metabolic process
0.25861
Cers6, Lars, Sdsl, Rad1, Selk, Mybbp1a, Nampt, Cyp1b1, Prkag2, Atg13, Garem, Tat, Cd28,






Cebpg, Ezr, Il1rn, Arl6ip5, Cog6, Ednra, Agk, Adcy1, Ppap2a, Map3k3, Krtxap2, B4galt1, Pole4,






Mvd, Bmper, Ldha, Rab12, Qsox1, Atp6v0a1, Fktn, Cxcl9, Ung, Ctgf, Epm2aip1, Pdcd10, Gpt2,






Pnp, Hif1a, Marveld3, Agpat9, Syvn1, Br3f, Scly, Senp2, Efna1, Nodl, Mcfd2, Cyp7b1, Lrrc16a,






Apex1, Pcyox1, Aldh18a1, Mmp13, Pfkp, Gls, B4galnt1, Cd55, Dok2, Atp7a, Ubiad1, Entpd7,






Usp1, Eogt, Acpp, Golt1b, Trib1, Grb2, Atf3, Slc11a2, Slc11a1, Mthfr, Mmp9, Cad, Lbp,






Snip1, Stt3a, Lpin1, Polh, Jmjd6, Lpar1, Fgr, Arf4, Fgb, Rpn1, Ppp4c, A2m, Egf, Slc35c1, Fgl1,






C1galt1, Fen1, Wdr61, Pkm, Atg9b, Iars, Traf7, Acer2, Dpy19l1, Rars, Hdc, Tbl1xr1, Pfkfb2,






Suco, Ranbp2, Cln8, H2afx, Ube2j1, Vcp, Dab2, Sf1, Scfd1, Syk, St6gal1, Hmga2, Ppip5k1, Asns,






Hyal1, Cd44, Serp1, Prmt1, Cyp51, Plek, Steap4, Idi1, Sox9, Nus1, Sdf2l1, Odc1, Papss1,






Wfs1, Supt5, Supt6, Hipk3, Tmem165, Txnl1, Gas6, Rac2, Crem, Cdc73, Lig3, GBta6, Ncf1,






Smarcad1, Pld1, Tha1, Rasgrp1, Tirap, Trp53inp2, Apoa4, Apoa5, Trip12, Hars, Zfp869, Ddost,






Hnf4a, Adam9, Csgalnact2, Nlrp12, Flot1, Ubxn2b, Cars2, Cox8a, Farsb, Ctps, Nup62, Vars,






Wipi1, Ppapdc1b, Crtc2, Ptpn2, Ptpn1, Abca1, Parp2, Parp3, Mppe1, Ppip5k2, Ddx1, Thoc1,






Elp3, Inhbe, B3galt6, B3galt1, Tfb2rn, Dph5, Zfp830, Brd8, Il1a, Alg12, Nabp1, Kars, Cda, Tiparp,






Eya3, Slc9a3r1, Psen1, Ap5s1, Exosc3, Piga, Arid5a, Ccl19, Wdr45b, Atg16l2, Gne, St3gal3,






Dnajc3, Rcor1, Dr1, Tnfrsf1a, Trib2, Rab1, Ift20, Hk2, Gadd45b, Sds, Gadd45g, Gale, Sod2,






Pik3c2a, Etnk2, Pycard, Map1lc3b, Adk, Clock, Bbs4, Alox12, B3galnt2, St3gal4, Pomgnt1,






Chmp4b, Ppm1l, Ccr1, Txlna, Cr1l, Fabp5, Ero1l, Lss, Rab33b, Psmd14, Pgm3, Pofut2, Galnt2,






Taf1, Srrt, Ears2, Prkce, Acot11, Crnkl1, Il6st, Ube2g2


LV
G4
positive regulation of DNA-templated transcription,
0.2655
Ell2, Ell, Wdr61, Dab2, Supt5, Supt6, Cdc73, Thoc1




elongation


LV
G4
tRNA aminoacylation for protein translation
0.27988
Lars, Iars, Rars, Hars, Cars2, Farsb, Vars, Kars, Ears2


LV
G4
cellular nitrogen compound metabolic process
0.28514
Cers6, Lars, Rad1, Mybbp1a, Nampt, Cyp1b1, Armcx3, Nol8, Pdgfc, Mbd1, Prkag2, Snx6, Utp6,






Dhx30, Mlx, Cd28, Cebpg, Cebpd, Ddx52, Mettl1, Mettl3, Hnrnpa2b1, Hnrnpab, Polr1e, Hck,






Ednra, Btg2, Dhx40, Wdr77, Agk, Adcy1, Ppap2a, Bmper, Tcf25, Ints7, Ints5, Rpf2, Atp6v0a1,






Pus3, Cxcl9, Ung, Trnt1, Mphosph8, Klf17, Pnp, Hif1a, Mphosph6, Smarce1, Dap, Sf3b4, Srsf2,






Creb3, Senp2, Efna1, Ell2, Nod1, Ctif, Gmeb1, Lrrc16a, Apex1, Npm3, Tasp1, Pcid2, Ddx21,






Pfkp, Rlim, Dhx38, B4galnt1, Atp7a, Entpd7, Usp1, Tnfaip1, Recql, Acpp, Mbnl2, Ell, Tbp, Irf8,






Trib1, Dhx35, Cebp2, Atf6, Ints2, Atf3, Slc11a2, Gzma, Slc11a1, Mthfr, Exosc1, Exosc4, Cad,






Snip1, Skil, Lpin1, Ddx50, Polh, Gtf2f1, Nup98, Jmjd6, Foxk2, Arf4, Tmsb4x, Ppp4c, Egf, Stag1,






Fen1, Wdr61, Ebna1bp2, Pkm, Trps1, Spin1, Iars, Traf7, Acer2, Rars, Hdc, Tbl1xr1, Pfkfb2, Srebf2,






Cln8, Trappc2, H2afx, Vcp, Scnm1, Mcm2, Dab2, Syk, Hmga2, Asns, Eif4a3, Ddx39, Prmt1,






Prdm1, Atf6b, Pcsk5, Creb3l2, Wdr12, Nelfa, Rbm39, Sox9, Odc1, Papss1, Wfs1, Supt5, Supt6,






Hipk3, Helb, Gas6, Trim27, Crem, Irak2, Dynll1, Hist1h3b, Gemin8, Ngdn, Cdc73, Parn, Lig3,






Gata6, Exog, Ncf1, Tspyl2, Srm, Smarcad1, Dis3, Nop58, Tirap, Trp53inp2, Med17, Apoa4, Apoa5,






Trip12, Hars, Zfp869, Hnf4a, Zfp160, Tra2b, Hspa5, Atf7ip, Jun, Dnajb11, Nlrp12, Nudt21,






Papola, Cars2, Farsb, Taf2, Ctps, Nup62, Vars, Ifrd1, Crtc2, Secisbp2, Abca1, Ufl1, Ddx49, Preb,






Parp2, Parp3, Zbtb16, Zbtb21, Med25, Ddx1, Thoc1, Elp3, Rnps1, Tfb2m, Cdk7, Exosc10, Zfp830,






Il1a, Nabp1, Kars, Cda, Son, Eya3, Psen1, Ap5s1, Exosc3, Hltf, Cd38, Arid5a, Khsrp, Gtf2a2,






Nfia, Atg16l2, Trmt61a, Spg20, Rcor1, Polr1a, Rps6ka3, Tnfrsf1a, Trib2, Tmem173, Cdkn1a,






Taf4b, Nbas, Hk2, Magoh, Sbno2, Sod2, Nol9, Pycard, Adk, Trp63, Clock, B3galnt2, Fabp4,






Fahp5, Hsp90aa1, Srsf1, Srsf3, Psmd14, Zfp27, Calr, Pgm3, Nop56, Polr3k, Tbrg1, Taf1, Pspc1,






Nfkbiz, Trmt10a, Srrt, Prkch, Ears2, Crnkl1, Irg1, Rbm42, Larp7, Ikbkap, Nif3l1


LV
G4
macroautophagy
0.29538
Atg13, Rab12, Qsox1, Atg9b, Scfd1, Supt5, Trp53inp2, Ubxn2b, Wipi1, Psen1, Wdr45b, Atg16l2,






Rab1, Ift20, Map1lc3b, Chmp4b, Rab33b


LV
G4
Translation
0.29607
Sec61b, Etf1, Srp68, Eif4e, Eif3d, Ssr1, Ssr2, Spcs2, Spcs3, Ssr4, Gspt2, Rpn1, Srp9, Ddost,






Sec61a1, Srp19, Srprb, Eif2b4, Rps9, Srp72, Srpr, Srp54a


LV
G4
response to bacterium
0.32759
Slamf8, Hck, Cxcl9, Nod1, Clec4d, Irf8, Trib1, Slc11a1, Lbp, Cd14, Fgr, Fgb, Litaf, Fcer1g, Syk,






Vimp, Adam17, Prdm1, Reg3b, Irak2, Iigp1, Cdc73, Ncf1, Pld1, Tirap, Tlr1, Adam9, Jun, Abca1,






Trem3, Rps6ka3, Tnfrsf1b, Tnfrsf1a, Rab1, Pycard, Seh1l, Nfkbib, Prkce, Irg1, Myo1f


LV
G4
myeloid cell activation involved in immune response
0.3279
Lbp, Fgr, Fcer1g, Syk, Rac2, Rasgrp1, Il4ra, Sbno2, Snap23, Pycard, Prkce, Myo1f


LV
G4
leukocyte chemotaxis
0.33499
Cxcl9, Creb3, Lbp, S100a8, Fcer1g, Syk, Adam17, Cklf, Gas6, Rac2, ltgam, Tirap, Itgb2, Vav1,






Ccl19, Trem3, Coro1a, Vegfc, Ccr1, Fcgr3, Calr, S100a9


LV
G4
cellular catabolic process
0.33919
Sdsl, Cyp1b1, Bfar, Sec61b, Prkag2, Atg13, Tat, Timp1, Btg2, Rab12, Qsox1, Hsp90b1, Pnp, Hif1a,






Dap, Syvn1, Timp3, Fbxo6, Ctif, Ube2s, Trim39, Pcyox1, Pfkp, Gls, Rlim, Psma1, Usp8, Entpd7,






Usp1, Tnfaip1, Trib1, Herpud1, Gzma, Exosc4, Lpin1, Dram1, Yod1, S100a8, Egf, Slc35c1,






Nedd4l, Fen1, Wdr61, Derl1, Atg9b, Hdc, Usp14, Tbl1xr1, Cul5, Cln8, Ube2j1, Vcp, Dab2, Scfd1,






Vimp, Rraga, Ctla2a, Ctla2b, Dnajc10, Sdf2l1, Wfs1, Supt5, Iigp1, Parn, Exog, Ncf1, Dis3, Tha1,






Trp53inp2, Apoa4, Apoa5, Trip12, Adam9, Hspa5, Ubxn2b, Cox8a, Wipi1, Secisbp2, Ptpn1,






Ufl1, Znrf1, Rnps1, Exosc10, Fbxo31, Cda, Psen1, Exosc3, Zfand2a, Khsrp, Wdr45b, Atg16l2,






Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1, Rab1, Ift20, Nbas, Sds, Magoh, Pycard,






Map1lc3b, Clock, Chmp4b, Txlna, Rab33b, Psmd14, Taf1, Enc1, Crnkl1, S100a9, Ube2g2


LV
G4
mRNA processing
0.38074
Mettl3, Hnrnpa2b1, Btg2, Sf3b4, Srsf2, Dhx38, Mbnl2, Jmjd6, Eif4a3, Ddx39, Supt5, Supt6,






Gemin8, Cdc73, Tra2b, Nudt21, Papola, Rnps1, Son, Magoh, Srsf1, Crnkl1, Rbm42


LV
G4
mRNA processing
0.38074
Mettl3, Hnrnpa2b1, Btg2, Sf3b4, Srsf2, Dhx38, Mbnl2, Jmjd6, Eif4a3, Ddx39, Supt5, Supt6,






Gemin8, Cdc73, Tra2b, Nudt21, Papola, Rnps1, Son, Magoh, Srsf1, Crnkl1, Rbm42


LV
G4
cell adhesion
0.38488
Selk, Cyp1b1, Selp, Cd28, Il1rn, B4galt1, Ctgf, Actg1, Pnp, Pnn, Braf, Efna1, Lrrc16a, Icam2,






Clec4d, Atp7a, Cytip, Slc11a1, Jmjd6, Hsd17b12, Fgb, Tgm2, S100a8, Acer2, Fcer1g, Pvr, Fndc3b,






Dab2, Tnfrsf12a, Syk, Adam17, Hyal1, Col13a1, Hapln4, Cd44, Myh9, Ctla2a, Plek, Sox9, Gas6,






Rac2, Il12rb1, Itgal, Itgam, Rasgrp1, Apoa4, Ddost, Adam9, Golph3, Pdia6, Flot1, Ctps, Ptpn2,






Ap1ar, Zbtb16, Il4ra, Ap3d1, Psen1, Itgb3, Itgb2, Vav1, Ccl19, S100a10, Cldn14, Rab1, Gadd45g,






Coro1a, Vegfc, Pycard, Fn1, Alox12, Cd48, Hsp90aa1, Calr, Clptm1, Prkce, Myo1f, Il6st, S100a9


LV
G4
macromolecular complex assembly
0.38547
Selp, Pdgfc, Senp6, Polr1e, Hck, Rpf2, Atp6v0a1, Eif3d, Timm21, 2610034B18Rik, Lrrc16a, Ube2s,






Gls, Capza1, Mpp7, Irf8, Grb2, Tsr1, Skil, Nup98, Vasp, Fgb, Pde4dip, Cct3, Foxred1, Arpc1b,






Sar1a, Ipo4, Vamp4, Mapre1, Vcp, Mcm2, Dab2, Actr2, Hmga2, Zw10, Plek, Sox9, Trim27,






Gemin8, Rasgrp1, Apoa4, Apoa5, Hist1h2bp, Atf7tp, Jun, Nudt21, Denr, Ap1ar, Abca1, Med25,






Brix1, Cda, Pdcl, Gtf2a2, S100a10, Arfip2, Trim6, Rab1, Sod2, Snap23, Pik3c2a, Coro1a,






Pycard, Trp63, Fn1, Bbs4, Aspm, Chmp4b, Hsp90aa1, Srsf1, Scara5, Calr, Atl2, Atl1, Prkce, Eif6,






Crnkl1


LV
G4
cell activation
0.39267
Selk, Selp, Cd28, Cebpg, Bmper, Actg1, Pnp, Lrrc8a, Braf, Clec4d, Pcid2, Atp7a, Slc11a1, Lbp,






Jmjd6, Ap1g1, Fgr, Fgb, Fcer1g, Syk, Adam17, Myh9, Ctla2a, Prdm1, Plek, Supt6, Gas6, Rac2,






Il12rb1, Itgal, Itgam, Rasgrp1, Tirap, Tlr1, Ddost, Adam9, Jun, Pdia6, Ctps, Ptpn2, Il17ra,






Zbtb16, Il4ra, Thoc1 , Ap3d1, Tpd52, Psen1, Exosc3, Cd38, Itgb3, Itgb2, Vav1, Ccl19, Camk2b,






Cdkn1a, Gadd45g, Sbno2, Snap23, Pik3c2a, Coro1a, Pycard, Alox12, Txlna, Cd48, Hsp90aa1, Fcgr3,






Lcp2, Clptm1, Prkce, Myo1f, Il6st


LV
G4
cellular protein modification process
0.39691
Selk, Bfar, Pdgfc, Prkag2, Garem, Senp6, Pim3, Arl6ip5, Metap2, Hck, Etf1, Btg2, Ntmt1, Anapc4,






Mvp, Ppap2a, Map3k3, Krtcap2, B4galt1, Pole4, Bmper, Ficd, Plcl1, Fktn, Errfi1, Ctgf, Nktr,






Pdcd10, Marveld3, Syvn1, Braf, Senp2, Efna1, Nod1, Ibtk, Ube2s, Trim39, Csnk1a1, Cwc27, Rlim,






Dok2, Atp7a, Usp8, Usp1, Tnfaip1, Eogt, Aak1, Trib1, Grb2, Atf3, Herpud1, Slc11a1, Mthfr,






Mmp9, Cad, Stt3a, Lpin1, Uba5, Jmjd6, Lpar1, Yod1, Fgr, Arf4, Camkk2, Ube2f, Fgb, Rpn1, Tgm2,






Uspl1, S100a8, Egf, Fkbp11, Nedd4l, C1galt1, Wdr61, Traf7, Acer2, Dpy19l1, Saa3, Usp14,






Tbl1xr1, Ufm1, Cul5, Ube2j1, Vcp, Dab2, Dusp2, Asb4, Syk, St6gal1, Hmga2, Adam17, Cd44,






Neurl3, Prmt1, Dzip3, Iqgap1, Dnajc10, F13a1, Sox9, Nus1, Wfs1, Supt6, Hipk3, Tmem165,






Gas6, Trim27, Rac2, Ppp2r1a, Il12rb1, Cdc73, Tspyl2, Smarcad1, Rasgrp1, Tirap, Fkbp5, Tlr1,






Trip12, Ddost, Ick, Adam9, Hspa5, Jun, Nlrp12, Nup62, Wipi1, Crtc2, Ptpn2, Ptpn1, Abca1, Ufl1,






Znrf1, Plaur, Nceh1, Parp3, Mppe1, Elp3, Inhbe, B3galt1, Dph5, Brd8, Il1a, Alg12, Stk17b,






Pggt1b, Tiparp, Eya3, Slc9a3r1, Prkrip1, Psen1, Piga, Itgb3, Itgb2, Arid5a, Ccl19, Gan, Wdr4db,






St3gal3, Dnajc3, Trim68, Rcor1, Socs3, Dr1, Camk2b, Trim3, Rps6ka3, Tnfrsf1a, Nek7, Trib2,






Cdkn1a, Gadd45b, Gadd45g, Ppp1r11, Pik3c2a, Vegfc, Pycard, Fn1, Clock, B3galnt2, St3gal4,






Pomgnt1, Ppm1l, Ccr1, Fabp4, Psmd14, Pgm3, Rusc1, Pofut2, Galnt2, Taf1, Enc1, Josd2, Lats2,






Prkch, Prkce, Anapc16, Il6st, Ikbkap, S100a9, Ube2g2


LV
G4
regulation of response to wounding
0.39813
Selp, Pik3ap1, Cd28, Il1r1, Braf, Cd55, Psma1, Adora1, Tnfaip6, Lbp, S100a8, A2m, Fcer1g,






Tnfrsf12a, Syk, Vimp, Ctla2a, Plek, Nlrp12, Ptpn2, Casp4, Il17ra, Socs3, Tnfrsf1b, Tnfrsf1a,






Sbno2, Pycard, Clock, Alox12, Cr1l, Fabp4, Fcgr3, Scara5, Prkce, Irg1, S100B9


LV
G4
Transport to the Golgi and subsequent modification
0.40154
B4galt1, Mcfd2, Sec24d, St6gal1, Preb, Sec31a, Man1a, Sec13, St3gal4


LV
G4
KSRP destabilizes mRNA
0.4071
Exosc1, Exosc4, Parn, Dis3, Exosc3, Khsrp


LV
G4
RNA Polymerase II Pre-transcription Events
0.45004
Gtf2e1, Ell, Tbp, Gtf2f1, Gtf2f2, Nelfa, Supt5, Taf2, Cdk7, Gtf2a2, Taf4b, Taf1


LV
G4
cellular localization
0.47989
Mybbp1a, Mfsd7b, Yipf5, Armcx3, Sec61b, Golga5, Snx6, Tacc2, Atg13, Mlx, Ezr, Stx6, Il1rn, Pim3,






Cog6, Tbccd1, B4galt1, Rab12, Srp68, Stx12, Stx18, Cxcl9, Arcn1, Pdcd10, Uso1, Hif1a, Lcn2,






Krt18, Braf, Timm21, Creb3, Rabl3, Ibtk, Plrg1, Gosr2, Blzf1, Slc11a1, Pdzd11, Snip1, Ap1g1,






Fgr, Arf4, Fgb, Tmed9, Egf, Ica1, Ap2a2, Tacc1, Derl1, Atg9b, Atp2a2, Litaf, Sar1a, Itpr1, Ipo4,






Vamp4, Rab35, Fcer1g, Zfyve16, Mapre1, Vcp, Dab2, Actr2, Syk, Hmga2, Vimp, Srgn, Rraga, Ap4e1,






Copb2, Ddx39, Myh9, Casc3, Serp1, Zw10, Golph3l, Rab5b, Creb3l2, Plek, Sox9, Nus1, Chic2,






Cklf, Supt6, Tmem165, Gas6, Trim27, Rac2, Golga4, Dynll1, Sdc4, Gm21540, Itgal, Dst, Ncf1,






Rasgrp1, Ick, Pcm1, Kif5b, Hnf4a, Adam9, Hspa5, Golph3, Jun, Sec61a1, Nlrp12, Ralgapa2, Flot1,






Crp, Capn10, Wipi1, Sidt2, Rab18, Ptpn1, Ap1ar, Abca1, Casp4, Parp3, Mppe1, Il4ra, Thoc2,






Thoc1, Exosc10, Il1a, Bet1l, Ap3d1, Syt12, Slc9a3r1, Sec22b, Hook1, Psen1, Ap5s1, Exosc3, Itgb2,






Ccl19, Wdr45b, Asun, Snx10, S100a10, Trem3, Rint1, Srp72, Tmem173, Rab1, Ift20, C2cd5, Cdkn1a,






Hk2, Snap23, Sec13, Coro1a, Sybu, Pycard, Stx5a, Clock, Bbs4, Aspm, Chmp4b, Nemf, Ccr1,






Clec4n, Seh1l, Hsp90aa1, Ero1l, Fcgr3, Rab33b, Calr, Lcp2, Tbrg1, Lats2, Prkce, Myo1f


LV
G4
Calnexin/calreticulin cycle
0.48294
Pdia3, Edem2, Edem3, Edem1, Calr


LV
G4
RNA degradation
0.49399
Cnot11, Btg2, Mphosph6, Eif4e, Pfkp, Exosc1, Exosc4, Wdr61, Ttc37, Parn, Dis3, Exosc10, Exosc3


LV
G4
Carbohydrate metabolism
0.51204
Pcx, B4galt1, Pfkp, Nans, Nup98, Pkm, Pfkfb2, Ranbp2, Hyal1, Cd44, Nupl2, Slc2a3, Papss1,






Ppp2r1a, Lyve1, Sdc4, Gfpt1, Csgalnact2, Nupl1, Nup62, B3galt6, Gne, St3gal3, Gmppa, Hk2,






Gale, St3gal4, Seh1l, Uap1, Pgm3, Gmppb


LV
G4
RNA transport
0.51898
Trnt1, Pnn, Eif4e, Eif3d, Senp2, Gm10094, Alyref, Nup98, Eif4g2, Ranbp2, Eif4a3, Casc3, Nupl2,






Gemin8, Nupl1, Nup62, Eif2b4, Thoc2, Thoc1, Rnps1, Magoh, Sec13, Seh1l


LV
G4
Ribosome biogenesis in eukaryotes
0.52099
Utp6, Wdr75, Gar1, Riok1, Rbm28, Gnl2, Nop58, Utp14b, Utp14a, Wdr43, Nop56, Eif6


LV
G4
protein folding
0.53287
Qsox1, Fkbp11, Pdia3, Dnajc10, Txnl1, Fkbp5, Pdia6, Pdia4, Erp44, Itgb3, Txndc5, Hsp90aa1,






Ero1l


LV
G4
apoptotic signaling pathway
0.54199
Selk, Mybbp1a, Cyp1b1, Cd28, Arl6ip5, Pdcd10, Pnp, Hif1a, Dap, Lcn2, Krt18, Syvn1, Creb3,






Trim39, Serinc3, Atp7a, Atf3, Herpud1, Mmp9, Skil, Fgb, S100a8, Trps1, Traf7, Pdia3, Itpr1, Bag3,






Dab2, Tnfrsf12a, Vimp, Srgn, Cd44, Dnajc10, Wfs1, Ppp2r1a, Tnfrsf22, Grina, Ptpn2, Ptpn1,






Casp4, Plaur, Parp2, Il1a, Slc9a3r1, Psen1, Cdip1, Camk2b, Tnfrsf1b, Tnfrsf1a, Hyou1, Cdkn1a,






Sod2, Pycard, Trp63, Ivns1abp, Ero1l, S100a9


LV
G4
Immune System
0.56558
Pik3ap1, Sec61b, Cd28, Il1rn, Hck, Ap2b1, Anapc4, Adcy1, Map3k3, Il1r1, Hsp90b1, Actg1, Eif4e,






Nod1, Fbxo6, Icam2, Clec4d, Tank, Capza1, Psma1, Irf8, Grb2, Tpp2, Lbp, Cd14, Uba5, Sec24d,






Ap1g1, Fgr, Vasp, Egf, Nedd4l, Ifitm2, Ifitm1, Ap2a2, Pdia3, Itpr1, Dapp1, Fcer1g, Pvr, Cul5,






Nrg4, Actr2, Asb3, Asb4, Syk, Adam17, Cd44, Dzip3, Csf2rb, H2-D1, Ppp2r1a, Irak2, Dynll1, Ncf1,






Rasgrp1, Tirap, Trip12, Ddost, Mt2, Jun, Sec61a1, Crp, Ptpn2, Ptpn1, Zbtb16, Il1a, Sec31a, Itgb2,






Vav1, Gan, Trim63, Socs3, Camk2b, Rps6ka3, Tmem173, Cdkn1a, Sec13, Pycard, Clec4n, Cr1l, Hsp90aa1,






Fcgr3, Psmd14, Calr, Polr3d, Lcp2, Polr3k, Nfkbib, Sugt1, Prkce, Il6st


LV
G4
symbiosis, encompassing mutualism through parasitism
0.56648
Creb3, Cd55, Irf8, Lbp, Apcs, Osbp, Ifitm2, Ifitm1, Vcp, Hmga2, Rraga, Pcsk5, Gas6, Trim27, Ncf1,






Tirap, Jun, Thoc2, Thoc1, Itgb3, Atg16l2, Trem3, Rab1, Chmp4b


LV
G4
Zinc transporters
0.59484
Slc30a5, Slc39a6, Slc39a7, Slc39a10, Slc39a14, Slc30a7


LV
G4
cellular component assembly
0.64457
Selp, Pdgfc, Atg13, Ezr, Senp6, Polr1e, Hck, Capg, Rpf2, Hsp90b1, Atp6v0a1, Stx18, Ctgf, Ccp110,






Pdcd10, Arhgef26, Actg1, Marveld3, Eif3d, Br3f, Timm21, 2610034B18Rik, Lrrc16a, Ube2s,






Gls, Capza1, Mpp7, Atp7a, Tnfaip1, Irf8, Grb2, Tsr1, Skil, Wdr1, Nup98, Lpar1, Vasp, Fgb, Pde4dip,






Cct3, Foxred1, Atg9b, Arpc1b, Sar1a, Ipo4, Vamp4, Mapre1, Vcp, Mcm2, Dab2, Actr2, Scfd1,






Hmga2, Hyal1, Zw10, Plec, Plek, Sox9, Gas6, Trim27, Rac2, Gemin8, Sdc4, Pld1, Rasgrp1, Trp53inp2,






Apoa4, Apoa5, Ick, Hist1h2bp, Pcm1, Atf7ip, Golph3, Jun, Nudt21, Flot1, Ubxn2b, Denr,






Wipi1, Intu, Ap1ar, Abca1, Med25, Brix1, Cda, Slc9a3r1, Psen1, Itgb3, Ccl19, Pdcl, Gtf2a2, Wdr45b,






Atg16l2, Snx10, S100a10, Arfip2, Trim6, Rab1, Ift20, Sod2, Snap23, Pik3c2a, Coro1a, Pycard,






Map1lc3b, Trp63, Fn1, Bbs4, Aspm, Chmp4b, Hsp90aa1, Srsf1, Rab33b, Scara5, Calr, Atl2, Atl1,






Prkch, Prkce, Eif6, Crnkl1


LV
G4
myeloid leukocyte activation
0.70771
Slc11a1, Lbp, Jmjd6, Fgr, Fcer1g, Syk, Rac2, Rasgrp1, Tlr1, Adam9, Jun, Il4ra, Psen1, Sbno2, Snap23,






Pycard, Fcgr3, Lcp2, Prkce, Myo1f


LV
G4
Translation
0.71134
Sec61b, Etf1, Srp68, Eif4e, Eif3d, Ssr1, Ssr2, Spcs2, Spcs3, Ssr4, Gspt2, Rpn1, Casc3, Ppp2r1a, Srp9,






Ddost, Sec61a1, Srp19, Srprb, Eif2b4, Rnps1, Rps9, Srp72, Magoh, Srpr, Srp54a


LV
G4
Cholesterol biosynthesis
0.71943
Mvd, Cyp51, Idi1, Lbr, Sqle, Lss


LV
G4
cytoplasmic transport
0.73544
Mybbp1a, Yipf5, Sec61b, Atg13, Mlx, Rab12, Srp68, Stx18, Cxcl9, Uso1, Krt18, Timm21, Creb3,






Ibtk, Gosr2, Bizf1, Slc11a1, Snip1, Ap1g1, Egf, Derl1, Litaf, Sar1a, Itpr1, Ipo4, Vamp4, Rab35,






Zfyve16, Vcp, Dab2, Actr2, Syk, Vimp, Ddx39, Zw10, Rab5b, Creb3l2, Chic2, Supt6, Gas6, Rac2,






Golga4, Golph3, Jun, Sec61a1, Nlrp12, Wipi1, Ap1ar, Mppe1, Thoc2, Thoc1, Ap3d1, Sec22b, Hook1,






Ccl19, Rint1, Srp72, Tmem173, Rab1, Cdkn1a, Sec13, Coro1a, Stx5a, Chmp4b, Nemf, Hsp90aa1,






Ero1l, Rab33b, Calr, Prkce


LV
G4
cellular zinc ion homeostasis
0.76449
Slc30a5, Slc39a6, Slc39a14, Slc30a7, Mt2, Mt1, Ap3d1


LV
G4
establishment of localization in cell
0.82981
Mybbp1a, Mfsd7b, Vipf5, Sec61b, Golga5, Snx6, Atg13, Mlx, Ezr, Stx6, Il1rn, Pim3, Cog6, B4galt1,






Rab12, Srp68, Stx12, Stx18, Cxcl9, Arcn1, Pdcd10, Uso1, Hif1a, Lcn2, Krt18, Braf, Timm21, Creb3,






Rabl3, Ibtk, Gosr2, Blzf1, Slc11a1, Pdzd11, Snip1, Ap1g1, Fgr, Fgb, Tmed9, Egf, Ica1, Ap2a2,






Derl1, Atp2a2, Litaf, Sar1a, Itpr1, Ipo4, Vamp4, Rab35, Fcer1g, Zfyve16, Vcp, Dab2, Actr2, Syk,






Hmga2, Vimp, Srgn, Ap4e1, Copb2, Ddx39, Myh9, Serp1, Zw10, Golph3l, Rab5b, Creb3l2, Plek,






Nus1, Chic2, Cklf, Supt6, Tmem165, Gas6, Trim27, Rac2, Golga4, Dynll1, Sdc4, Gm21540, Dst,






Ncf1, Rasgrp1, Ick, Pcm1, Kif5b, Hnf4a, Adam9, Golph3, Jun, Sec61a1, Nlrp12, Crp, Capn10, Wipi1,






Sidt2, Rab18, Ptpn1, Ap1ar, Abca1, Casp4, Mppe1, Il4ra, Thoc2, Thoc1, Il1a, Bet1l, Ap3d1,






Syt12, Sec22b, Hook1, Psen1, Ap5s1, Ccl19, Trem3, Rint1, Srp72, Tmem173, Rab1, Ift20, C2cd5,






Cdkn1a, Hk2, Snap23, Sec13, Coro1a, Sybu, Pycard, Stx5a, Clock, Chmp4b, Nemf, Ccr1, Clec4n,






Seh1l, Hsp90aa1, Ero1l, Fcgr3, Rab33b, Calr, Lcp2, Tbrg1, Prkce, Myo1f


LV
G4
organic substance catabolic process
0.84797
Sdsl, Cyp1b1, Bfar, Sec61b, Prkag2, Tat, Timp1, Btg2, Rab12, Hsp90b1, Pnp, Hif1a, Syvn1, Timp3,






Fbxo6, Ctif, Ube2s, Trim39, Pcyox1, Pfkp, Gls, Rlim, Psma1, Usp8, Entpd7, Usp1, Tnfaip1, Trib1,






Herpud1, Gzma, Exosc4, Lpin1, Yod1, Egf, Nedd4l, Fen1, Wdr61, Pkm, Derl1, Hdc, Usp14, Tbl1xr1,






Pfkfb2, Cul5, Cln8, Ube2j1, Vcp, Dab2, Vimp, Hyal1, Cd44, Ctla2a, Ctla2b, Dnajc10, Sox9,






Sdf2l1, Odc1, Wfs1, Lyve1, Parn, Exog, Ncf1, Dis3, Tha1, Apoa4, Apoa5, Trip12, Adam9, Hspa5,






Ubxn2b, Secisbp2, Ptpn1, Ufl1, Znrf1, Rnps1, Exosc10, Fbxo31, Cda, Tiparp, Sec22b, Psen1,






Exosc3, Zfand2a, Khsrp, Kctd10, Dnajc3, Ubxn4, Tnfrsf1b, Edem3, Trib2, Edem1, Nbas, Hk2, Sds,






Gale, Magoh, Stx5a, Clock, Chmp4b, Psmd14, Taf1, Enc1, Prkce, Ube2g2


LV
G4
retrograde protein transport, ER to cytosol
0.90417
Sec61b, Derl1, Vcp, Vimp


SP
G1
Interferon Signaling
1.4E−08
Uba7, H2-T24, Ube2l6, Oas2, Trim12a, Trim12c, Oas1a, Fcgr1


SP
G1
Interferon gamma signaling
6.9E−06
H2-T24, Oas2, Trim12a, Trim12c, Oas1a, Fcgr1


SP
G1
response to virus
0.12414
Oas2, Ifi27l2a, Adar, Samhd1, Oas1a


SP
G1
ISG15-protein conjugation
0.20952
Uba7, Ube2l6


SP
G1
mRNA Editing
0.39433
Adar


SP
G1
Herpes simplex infection
0.39469
H2-T24, Oas2, Sp100, Oas1a


SP
G1
synaptic growth at neuromuscular junction
0.43929
Lrp4, Agrn


SP
G1
response to other organism
0.71946
Oas2, Ifi27l2a, Adar, Samhd1, Oas1a, Fcgr1


SP
G2
Cytokine-cytokine receptor interaction | Endocytosis |
0.02507
H2-Q10, Cxcr2, Flt3, Lepr, Mrc1, Igf1r, Ctsb, Pdgfra, Maf, Il13ra1, Ccl6, Ccr1, Cxcl12, Fcgr3




Herpes simplex infection


SP
G2
Cytokine-cytokine receptor interaction
0.03758
Cxcr2, Flt3, Lepr, Pdgfra, Il13ra1, Ccl6, Ccr1, Cxcl12


SP
G2
positive regulation of monocyte chemotaxis
0.23286
Pla2g7, Ccr1, Cxcl12


SP
G2
Glutathione metabolism
0.23841
Gsr, Gsta3, Ggt5


SP
G2
cell motility
0.4525
Cxcr2, Igf1r, Gcnt2, Pdgfra, Nr2f2, Tgfbr3, Nlrp12, Pla2g7, Ccr1, Cxcl12, Fcgr3


SP
G2
locomotion
0.53194
Cxcr2, Flot1, Igf1r, Gcnt2, Pdgfra, Nr2f2, Tgfbr3, Nlrp12, Pla2g7, Ccr1, Cxcl12, Fcgr3


SP
G2
immune system process
0.58417
Rab27a, H2-Q10, Cxcr2, Flt3, Mrc1, Igf1r, Pdgfra, Tgfbr3, Nlrp12, Pla2g7, Ccr1, Mertk, Mafb,






Cxcl12, Fcgf3


SP
G2
lipid metabolic process
0.6968
Lepr, Hsd17b11, Pdgfra, Cyp2d22, Pla2g7, Ggt5, Enpp6, Alox5ap, Galc


SP
G2
sulfur compound metabolic process
0.71823
Acpp, Ctns, Gsr, Gsta3


SP
G2
Lysosome
0.97588
Ap1s2, Ctsb, Ctns, Galc


LN
G1
response to stress
1.9E−05
Rbbp8, Pik3ap1, Ier3, C5ar1, Clec4n, Alox5, Prkd1, Nlrp12, Plek, Lgr4, Hmox1, Tbx3,






S100a8, Il1r1, Clec5a, Trem1, Trem3, Cd44


LV
G3
immune system process
0.00642
Sirpa, Cd93, Clec4n, Nlrp12, Lgr4, Hmox1, Fam20c, S100a8, Clec5a, Cebpd, Trem1, Trem3,






Cd44, Pawr


SP
G3
cytokine secretion
0.00643
Clec4n, Nlrp12, Lgr4, Clec5a, Trem1, Trem3


SP
G3
defense response
0.01338
Ier3, C5ar1, Clec4n, Alox5, Nlrp12, Hmox1, S100a8, Il1r1, Clec5a, Trem1, Trem3, Cd44


SP
G3
locomotion
0.01413
Dpysl3, C5ar1, Prkd1, Nlrp12, Myo10, Mmp9, Gcnt2, S100a8, Clec5a, Trem1, Trem3


SP
G3
cell motility
0.01771
Dpysl3, C5ar1, Prkd1, Nlrp12, Myo10, Mmp9, Gcnt2, S100a8, Trem1, Trem3


SP
G3
protein secretion
0.02786
Clec4n, Nlrp12, Plek, Lgr4, Clec5a, Trem1, Trem3, Rab15


SP
G3
osteoblast differentiation
0.09015
Id2, Prkd1, Lgr4, Fam20c, Clec5a, Cebpd


SP
G3
secretion
0.13843
Clec4n, Nlrp12, Plek, Lgr4, Tbx3, S100a8, Clec5a, Trem1, Trem3, Rab15


SP
G3
cell death
0.18627
Ier3, Prkd1, Nlrp12, Mmp9, Hmox1, Tbx3, S100a8, Clec5a, Cd44, Pawr


SP
G3
neutrophil chemotaxis
0.21941
C5ar1, S100a8, Trem1, Trem3


LN
G1
response to stress
0.02261
Rbbp8, Pik3ap1, Ier3, C5ar1, Clec4n, Alox5, Prkd1, Nlrp12, Plek, Hmox1, Tbx3, S100a8,






Il1r1, Clec5a, Trem1, Trem3, Cd44


SP
G3
positive regulation of multicellular organismal process
0.25448
Dpysl3, Id2, C5ar1, Clec4n, Prkd1, Plek, Lgr4, Mmp9, Gcnt2, Fam20c, Clec5a, Cebpd


SP
G3
response to wounding
0.30016
Dpysl3, Ier3, Nlrp12, Plek, Hmox1, S100a8, If1r1, Cd44


SP
G3
secretion by cell
0.32505
Clec4n, Nlrp12, Plek, Lgr4, Tbx3, Clec5a, Trem1, Trem3, Rab15


SP
G3
inflammatory response
0.34004
Ier3, Alox5, Nlrp12, Hmox1, S100a8, Il1r1, Cd44


SP
G3
transport
0.34214
Ier3, Clec4n, Prkd1, Nlrp12, Plek, Myo10, Lgr4, Mmp9, Tbx3, S100a8, Clec5a, Slc25a13,






Trem1, Trem3, Rab1S


SP
G3
cytokine production
0.36944
C5ar1, Clec4n, Nlrp12, Lgr4, Clec5a, Treml, Trem3, Pawr


SP
G3
Innate Immune System | Adaptive Immune System |
0.38688
Dpysl3, Id2, Fosl2, Clec4n, Ppp1r3b, Afox5, Prkd1, Cd244, Plek, Myo10, Mmp9, Hmox1,




Hemostasis | Pathways in cancer | Developmental

Tbx3, Il1r1, Clec5a, Cebpd, Trem1, Cd44




Biology | PI3K-Akt signaling pathway | Signaling




by Rho GTPases


SP
G3
neutrophil extravasation
0.51755
Trem1, Trem3


SP
G3
bundle of His development
0.51755
Id2, Tbx3


SP
G3
cell proliferation
0.80295
Id2, C5ar1, Fosl2, Prkd1, Lgr4, Gcnt2, Hmox1, Tbx3, Pawr


SP
G3
cell adhesion
0.84305
Mcam, Plek, Myo10, Gcnt2, S100a8, Cd44, Pawr


SP
G3
generation of precursor metabolites
0.96858
Ier3, Ppp1r3b, Slc25a13




and energy


SP
G4
Innate Immune System | Adaptive Immune System |
0.02903
Cdkn2b, Cxcl2, Socs2, Atp6v0c, Insm1, Dusp5, Scin, Anxa2, Tnfrsf1b, Ccl22, Plek, Mmp14,




Hemostasis | Pathways in cancer | Developmental

Ccl3, Sgpl1, Slc7a11, F10, Pdgfrb, Gadd45b, Prok2, Cacnb3




Biology | PI3K-Akt signaling pathway | Signaling




by Rho GTPases


SP
G4
locomotion
0.0655
Cxcl2, Insm1, Anxa3, Rap2b, Mmp14, Ccl3, Sgpl1, Pdgfrb, Strip2, Prok2


SP
G4
cell motility
0.03528
Cxcl2, Insm1, Anxa3, Rap2b, Mmp14, Cd3, Sgpl1, Pdgfrb, Strip2


SP
G4
response to wounding
0.3771
Rap2b, Anxa2, Tnfrsf1b, Plek, Ccl3, Slc7a11, Papss2, Myof


SP
G4
blood coagulation
0.42852
Rap2b, Anxa2, Plek, Slc7a11, Papss2


SP
G4
regulation of body fluid levels
0.52026
Socs2, Rap2b, Anxa2, Plek, Slc7a11, Papss2


SP
G4
wound healing
0.7751
Rap2b, Anxa2, Plek, Slc7a11, Papss2, Myof


SP
G3
immune system process
0.00433
Id2, C5ar1, Anxa3, Samsn1, Scin, Anxa2, Ccl3, Sgpl1, Gpr183, Cacnb3


SP
G4
Chemokine receptors bind chemokines
0.8244
Cxd2, Cd22, Ccl3


SP
G4
cell proliferation
0.8418
Cdkn2b, Cxcl2, Insm1, Scin, Tnfrsf1b, Mmp14, Pdgfrb, Gpr183, Prok2


SP
G4
Cytokine-cytokine receptor interaction
0.95292
Cxcl2, Tnfrsf1b, Ccl22, Cd3, Pdgfrb


LN
G1
cellular nitrogen compound metabolic process
0.09306
Hopx, Dnajb6, Aen, Trdmt1, Tlr9, Hck, Scaf11, Raf1, Bcl9, Nabp1, Wdr33, Thrap3, Timeless,






Cask, Rad54l2, Smc1a, Cebpd, Tox4, Fkbp8


SP
G3
response to stress
0.0005
Dpysl3, Id2, Tlr9, Hck, Tnfrsf1b, Bcl9, Nabp1, Timeless, Cask, Rnf34, Smc1a, S1pr3


LN
G1
Immune System
1.1E−10
Pik3ap1, Trim30a, Trim30d, Sos1, Irf9, Ctss, Gm14446, Dtx3l, Ifih1, Ifi204, Ifi205, Asb13, Uba7,






Lgals9, Psmb9, Psmb8, Hck, Psmb10, Stat2, Stat1, Klrk1, Trim12c, Oas1a, Ywhaz, Trip12, H2-M3,






Ifitm3, Raf1, Blnk, Capza2, Keap1, Psme4, Trim21, Cybb, Nod1, Pml, Casp8, Actr2, Tapbp, Phlpp1,






Mndal


LV
G4
immune system process
0.09263
Selk, Selp, Trim30a, Sos1, Ctsc, Slamf7, Ctss, Adar, Tgtp1, Ifih1, Ifi204, Cxcl10, Psmb9, Psmb8,






Hck, Trafd1, Psmb10, Nmi, Stat2, Stat1, Klrk1, Trim12c, Ankrd17, Samd9l, Parp9, Dhx58, Prkx, H2-M3,






Ifitm3, Gbp3, Gbp7, Pnp, Blnk, Samhd1, Trim21, Cybb, Nod1, Pml, Casp8, Tapbp, Herc6, Elmod2,






Cd47, Dpp4


LN
G1
Cytokine Signaling in Immune system
1.1E−06
Trim30a, Trim30d, Sos1, Irf9, Gm14446, Uba7, Lgals9, Stat2, Stat1, Trim12c, Oas1a, Ywhaz,






H2-M3, Raf1, Blnk, Trim21, Nod1, Pml


LN
G1
innate immune response
5.4E−06
Pik3ap1, Adar, Ifih1, Hck, Trafd1, Nmi, Stat1, Klrk1, Trim12c, Ankrd17, Parp9, Dhx58, H2-M3,






Ifrtm3, Gbp3, Gbp7, Samhd1, Trim21, Cybb, Pml


SP
G3
response to stress
0.25294
Dpysl3, Id2, Trim30a, Shisa5, Bfar, Ctss, Adar, Dtx3l, Ifih1, Pdk3, Cxcl10, Hck, Nono, Dusp28,






Trafd1, Ifi47, Nmi, Stat2, Stat1, Klrk1, Trim12c, Ankrd17, Parp9, Dhx58, Trip12, H2-M3,






Ifitm3, Gbp3, Gbp7, Pdcd10, Pnp, Chmp4b, Sh3glb1, Dek, Irgm1, Samhd1, Tlk2, Psme4, Trim21,






Cybb, Nod1, Pml, Hmox2, Tapbp, Xrn2, Zfyve26, Elmod2, Cd47, Aida, Dpp4


LN
G1
Interferon Signaling
5.9E−05
Trim30a, Trim30d, Irf9, Gm14446, Uba7, Stat2, Stat1, Trim12c, Oas1a, H2-M3, Trim2i, Pml


LN
G1
Adaptive Immune System
0.00013
Pik3ap1, Sos1, Ctss, Dtx3l, Asb13, Uba7, Psmb9, Psmb8, Psmb10, Klrk1, Ywhaz, Trip12, H2-M3,






Ifitm3, Raf1, Blnk, Keap1, Psme4, Trim21, Cybb, Tapbp, Phlpp1


LN
G1
defense response
0.0002
Pik3ap1, Ctss, Adar, Ifih1, Cxcl10, Hck, Trafd1, Ifi47, Nmi, Stat2, Stat1, Klrk1, Trim12c, Ankrd17,






Parp9, Dhx58, H2-M3, Ifitm3, Gbp3, Gbp7, Irgm1, Samhd1, Trim21, Cybb, Nod1, Pml, Tapbp, Elmod2,






Cd47, Dpp4


LN
G1
Class | MHC mediated antigen processing & presentation
0.00045
Ctss, Dtx3l, Asb13, Uba7, Psmb9, Psmb8, Psmb10, Trip12, H2-M3, Keap1, Psme4, Trim21, Cybb, Tapbp


LN
G1
immune response
0.00075
Pik3ap1, Ctsc, Ctss, Adar, Tgtp1, Ifih1, Cxcl10, Hck, Trafd1, Nmi, Stat1, Klrk1, Trim12c, Ankrd17,






Parp9, Dhx58, H2-M3, Ifitm3, Gbp3, Gbp7, Pnp, Blnk, Samhd1, Trim21, Cybb, Pml


LN
G1
Interferon gamma signaling
0.00191
Trim30a, Trim30d, Irf9, Stat1, Trim12c, Oas1a, H2-M3, Trim21, Pml


LN
G1
response to other organism
0.00301
Adar, Tgtp1, Ifih1, Plscr3, Cxcl10, Hck, Stat2, Stat1, Klrk1, Oas1a, Ankrd17, Dhx58, H2-M3,






Ifitm3, Gbp3, Gbp7, Samhd1, Nod1, Pml, Ifit2, Elmod2, Cd47


LN
G1
Influenza A
0.00378
Irf9, Adar, Akt3, Ifih1, Cxcl10, Stat2, Stat1, Oas1a, Raf1, Tnfsf10, Pml


LN
G1
Herpes simplex infection
0.00465
Daxx, Irf9, Gm14446, Ifih1, Stat2, Stat1, Oas1a, H2-T10, H2-M3, Pml, Casp8, Sp100


LN
G1
regulation of innate immune response
0.00467
Pik3ap1, Trim30a, Adar, Trafd1, Nmi, Klrk1, Trim12c, Ankrd17, Parp9, Dhx58, H2-M3, Samhd1


LN
G1
response to interferon-beta
0.0049
Ifi205, Stat1, Ifitm3, Gbp3, Xaf1, Pyhin1


LN
G1
Innate Immune System | Adaptive Immune System |
0.00504
Cers6, Daxx, Azi2, Pik3ap1, Trim30a, Trim30d, Sos1, Irf9, Helz2, Ctss, Gm14446, Adar, Cd2ap,




Hemostasis | Pathways in cancer | Developmental

Akt3, Dtx3l, Ifih1, Ifi204, Ifi205, Cxcl10, Asb13, Rtn4, Uba7, Lgals9, Psmb9, Psmb8, Hck, Psmb10,




Biology | PI3K-Akt signaling pathway | Signaling

Gm11787, Ifi47, Stat2, Stat1, Klrk1, Trim12c, Oas1a, H2-T10, Ywhaz, Dhx58, Trip12, Prkx,




by Rho GTPases

H2-M3, Ifitm3, Arhgap30, Raf1, Blnk, Tnfsf10, Capza2, Keap1, Pttg1, Irgm1, Psme4, Trim21, Cybb,






Nod1, Pml, Ranbp2, Casp8, Cab39l, Actr2, Tapbp, Max, Rbl1, Phlpp1, Sp100, Cd47, Atp1b3,






Mndal


LN
G1
symbiosis, encompassing mutualism through parasitism
0.01421
Trim30a, Adar, Stat1, Ifitm3, Gbp3, Gbp7, Chmp4b, Trtm21, Pml, Dpp4


LN
G1
Antigen processing-Cross presentation
0.01876
Ctss, Psmb9, Psmb8, Psmb10, H2-M3, Psme4, Cybb, Tapbp


LN
G1
regulation of response to stress
0.02164
Pik3ap1, Trim30a, Bfar, Ctss, Adar, Nono, Trafd1, Nmi, Stat2, Klrk1, Trim12c, Ankrd17,






Parp9, Dhx58, Trip12, H2-M3, Pdcd10, Pnp, Chmp4b, Sh3glb1, Dek, Samhd1, Nod1, Pml, Elmod2,






Cd47, Aida


LN
G3
response to stress
0.00102
Rbm4, Cyp1b1, Shisa5, Bfar, Ctss, Adar, Dtx3l, Ifih1, Pdk3, Cxcl10, Hck, Nono, Dusp28,






Trafd1, Ifi47, Nmi, Stat2, Stat1, Klrk1, Trim12c, Ankrd17, Parp9, Dhx58, Trip12, H2-M3,






Ifitm3, Gbp3, Gbp7, Pdcd10, Pnp, Chmp4b, Sh3glb1, Dek, Irgm1, Samhd1, Tlk2, Psme4,






Trim21, Cybb, Nod1, Pml, Hmox2, Tapbp, Xrn2, Zfyve26, Elmod2, Cd47, Aida, Dpp4


LN
G1
response to virus
0.02368
Adar, Tgtp1, Ifih1, Cxcl10, Stat2, Oas1a, Ankrd17, Dhx58, Ifitm3, Samhd1, Pml, Ifit2, Elmod2


LN
G1
Innate Immune System
0.02895
Sos1, Ctss, Ifih1, Ifi204, Ifi205, Psmb9, Psmb8, Hck, Psmb10, Klrk1, Raf1, Capza2, Psme4,






Trim21, Nod1, Casp8, Actr2, Phlpp1, Mndal


LN
G1
Antigen processing: Ubiquitination & Proteasome
0.03511
Dtx3l, Asb13, Uba7, Psmb9, Psmb8, Psmb10, Trip12, Keap1, Psme4, Trim21




degradation


LN
G1
proteasomal ubiquitin-independent protein catabolic
0.05458
Psmb9, Psmb8, Psmb10, Keap1, Psme4




process


LN
G1
regulation of defense response
0.08271
Pik3ap1, Trim30a, Ctss, Adar, Trafd1, Nmi, Stat2, Klrk1, Trim12c, Ankrd17, Parp9, Dhx58,






H2-M3, Samhd1, Pml, Elmod2, Cd47


LN
G1
catabolic process
0.10447
Trim30a, Ctsc, Bfar, Ctss, Uba7, Synj1, Psmb9, Psmb8, Psmb10, Mycbp2, Stat2, Cnot6l,






Rnf139, Samd9l, Trip12, Pnp, Phyh, Chmp4b, Sh3glb1, Keap1, Samhd1, Tlk2, Psme4, Pml,






Casp8, Usp25, Xrn2, Herc6


LN
G1
defense response to other organism
0.10585
Adar, Cxcl10, Hck, Stat2, Klrk1, Ankrd17, Dhx58, H2-M3, Ifitm3, Gbp3, Gbp7, Samhd1, Nod1,






Pml, Efmod2


LN
G1
response to interferon-alpha
0.16494
Adar, Ifi204, Ifitm3, Ifit2


LN
G1
Hepatitis C
0.18519
Sos1, Irf9, Gm14446, Akt3, Stat2, Stat1, Oas1a, Raf1


LN
G1
Measles
0.19482
Irf9, Adar, Akt3, Ifih1, Stat2, Stat1, Oas1a, Tnfsf10


LN
G1
STING mediated induction of host immune responses
0.20015
Ifi204, Ifi205, Trim21, Mndal


LN
G1
B cell receptor signaling pathway
0.2258
Pik3ap1, Sos1, Akt3, Gm11787, Raf1, Blnk


LN
G1
Signaling by Interleukins
0.23245
Sos1, Lgals9, Stat1, Ywhaz, Raf1, Blnk, Nod1


LN
G1
protein catabolic process
0.29429
Trim30a, Ctsc, Bfar, Ctss, Uba7, Psmb9, Psmb8, Psmb10, Mycbp2, Rnf139, Samd9l, Trip12,






Chmp4b, Keap1, Tlk2, Psme4, Pml, Casp8, Usp25, Herc6


LN
G1
macromolecule catabolic process
0.32228
Trim30a, Ctsc, Bfar, Ctss, Uba7, Psmb9, Psmb8, Psmb10, Mycbp2, Cnot6l, Rnf139, Samd9l,






Trip12, Chmp4b, Sh3glb1, Keap1, Tlk2, Psme4, Pml, Casp8, Usp25, Xrn2, Herc6


LN
G1
response to cytokine
0.35197
Adar, Ifi204, lfi205, Serpina3g, Cxcl10, Stat1, Parp9, Ifitm3, Gbp3, Gbp7, Keap1, Pml, Ifit2,






Casp8, Xaf1, Pyhin1


LN
G1
proteolysis involved in cellular protein catabolic
0.41796
Ctsc, Bfar, Ctss, Uba7, Psmb9, Psmb8, Psmb10, Rnf139, Trip12, Chmp4b, Keap1, Tlk2, Psme4,




process

Pml, Casp8, Usp25, Herc6


LN
G1
Ubiquitin mediated proteolysis | Cell cycle
0.48008
Shisa5, Uba7, Ywhaz, Trip12, Keap1, Pttg1,Pml, Casp8, Rbl1


LN
G1
Chemokine signaling pathway
0.50409
Sos1, Akt3, Cxcl10, Hck, Gm11787, Stat2, Stat1, Prkx, Raf1


LN
G1
Lysosome
0.54614
Cers6, Acer3, Ctsc, Ctss, Gns, Scarb2, Laptm4a


LN
G1
ER-Phagosome pathway
0.59186
Psmb9, Psmb8, Psmb10, H2-M3, Psme4, Tapbp


LN
G1
Cytokine-cytokine receptor interaction | Endocytosis |
0.65657
Daxx, Irf9, Ctss, Gm14446, Sgcb, , Ifih1, Cxcl10, Stat2, Stat1, Oas1a, H2-T10, H2-M3,




Herpes simplex infection

Blnk, Tnfsf10, Chmp4b, Sh3glb1, Cybb, Pml, Casp8, Tapbp, Sp100


LN
G1
Mitotic Anaphase
0.72336
Psmb9, Psmb8, Ankle2, Psmb10, Pttg1, Psme4, Ranbp2, Vrk1


LN
G1
APC/C:Cdc20 mediated degradation of Securin
0.91491
Psmb9, Psmb8, Psmb10, Pttg1, Psme4


LN
G1
multi-organism cellular process
0.91725
Trim30a, Adar, Stat1, Ankrd17, Dhx58, Ifitm3, Chmp4b, Trim21, Pml, Dpp4


LN
G1
cellular response to interferon-beta
0.9289
Ifi205, Stat1, Gbp3, Pyhin1


SP
G4
immune system process
0.8014
Cdkn2b, Cxcl2, Sos1, Slamf7, Adar, Ifi204, Cxcl10, Trafd1, Nmi, Stat2, Klrk1, Trim12c,






Ankrd17, Parp9, Dhx58, H2-M3, Pnp, Samhd1, Nod1, Pml, Casp8, Elmod2, Cd47, Dpp4


LN
G2
Metabolism of lipids and lipoproteins
0.03291
Hsd3b7, Ptgs1, Scarb1, Elovl6, Pla2g15, Abca1, Acox3, Slc44a2, Pik3cg, Sgms1, Mgll, Gpam


LN
G2
membrane organization
0.05633
Zdhhc23, Fcho1, Abca1, Reep1, Rhoq, Chchd3, Rap2a, Vamp4, Scn3b, Dab2, Myo1c, Nsf


LN
G2
plasma membrane organization
0.05711
Zdhhc23, Rhoq, Rap2a, Vamp4, Scn3b, Dab2, Nsf


LN
G2
Lysosome
0.09937
Ctsb, Tpp1, Cltb, Pla2g15, Sgms1, Ap1s2


LN
G2
Vesicle-mediated transport
0.1992
Scarb1, Cltb, Rhoq, Apls2, Dab2, Myo1c


LN
G2
biosynthetic process
0.23956
Dnmt3b, Pcgf6, Nr2f2, Flcn, Tfec, Zfhx3, Ptgs1, Zdhhc23, Pbx2, Lbp, Cux1, Zdhhc9, Scarb1, Gxylt1,






Elovl6, Abca1, Hdac4, Rhoq, Sbno2, Trps1, Scd1, Ldb2, Chchd3, Pik3cg, Insr, Sgms1, Esrra, Osr1,






Prg4, Nos3, Cdkn2d, Dab2, Nars2, Gpam, Brwd1, Hfe, Cd1d1


LN
G2
Metabolism of lipids and lipoproteins
0.36512
Hsd3b7, Ptgs1, Scarb1, Elovl6, Abca1, Acox3, Slc4432, Pik3cg, Sgms1, Mgll, Gpam


LN
G2
cell motility
0.50891
Gab1, Nr2f2, Flcn, Lbp, Scarb1, Rap2a, Pik3cg, Insr, Matn2, Nos3, Dab2, Mtus1, Eps8, Myo1c


LN
G2
lipopolysaccharide transport
0.56442
Lbp, Scarb1


LN
G2
vesicle-mediated transport
0.61791
Lbp, Scarb1, Fcho1, Cltb, Abca1, Trpc2, Vamp4, Apls2, Clcn5, Dab2, Map4k2, Nsf


LN
G2
locomotion
0.81552
Gab1, Nr2f2, Flcn, Lbp, Scarb1, Ceacam1, Rap2a, Pik3cg, Insr, Matn2, Nos3, Dab2, Mtus1, Eps8,






Myo1c


LN
G2
Biosynthesis of unsaturated fatty acids
0.87766
Elovl6, Acox3, Scd1


LN
G2
homeostatic process
0.96158
Flcn, Tpp1, Scarb1, Ubash3b, Ceacam1, Abca1, Ldb2, Trpc2, Insr, Scn3b, Cib2, Gpam, Hfe, Ccdc47


LN
G3
response to stress
0.20788
Rbm4, Cyp1b1, Atg4c, Ret, Pten, Gprc5b, Map3k6, Fktn, Pdcd4, Rad1, Serpine2, Glul, Errfi1, Apex1,






Tsc22d3, Herc2, Hamp, Birc3, Nfkb2, Prep, Scamp5, Thbs4, Ddit4, Cdc7, Per2, Bbc3, Hyal1,






Pdia6, Pdia4, Acp5, Upf3b, Spred2, Kit, Fads1, Rad51d, Bid, Large, Lyst, Swsap1, Seh1l, Pdk4,






Cd160, Mrc1, Ryr1, Nfe2l2, Tcf7l2, Stc2, Adam9, Acadm, Pik3cg, Pdgfra, Wipi1, Dab2, Agtr1a, Il27ra,






Rps3, Uchl1, Clec10a, Cd36, Cxcl13, Rps6ka6, Dpep1, Bok, Comt, Bcl2l11, Tnk2, St8sia1, Fabp1,






Srsf6, Polr3g, Scara5, Smc6, Ndufs8, Trpv1, Atp1b1, Fancc


LN
G3
organic substance metabolic process
0.00104
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Ret, Pten, Chchd10, Gprc5b,






Btg1, Prmt3, Tox2, Map3k6, Lss, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4, Ahcyl2,






Errfi1, Apex1, Ddx23, Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Gls, Rlim, Hamp,






Birc3, Mcf2l, Nfkb2, Prcp, Ip6k2, Zbtb16, Thbs4, Ptgs1, Ddit4, Serpina3m, Serpina3n, Serpina3c,






Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7, Msc, Mbd3, Aebp2, Per3, Per2, Trmt1, Bbc3, Scd2, Hyal1,






Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Upf3b, Tbl1xr1, Dis3l2, Fmo2, Tcf4, Rspo1, Spred2, Dusp4,






Dusp6, Dusp1, Dapk2, Otud3, Kit, Fads1, Rad51d, Mthfd1, Acer2, Alg8, Alg3, Hoxd8, Bid, Wdr12,






Pank1, Nrip1, Large, Lyst, Swsap1, Pdk4, Pik3ip1, Mafb, Mafg, Vars2, Pecr, Sae1, Plce1, Nfe2l2,






Tcf7l1, Tcf7l2, Zmiz1, Fkbp5, Cdk4, Mvd, Stc2, Adam9, Acadm, Fcer2a, Pik3cg, Crp, Psmd11,






Pdgfra, Vars, Zfp322a, Wipi1, Secisbp2, Ptpn3, Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7,






Uchl1, Suv420h1, Dhx29, Cd36, Pinx1, Abcg2, Npy1r, Ccdc22, Pkig, Rps6ka6, Zfp52, Zfp53,






Dpep1, Angpt4, Rprd1a, Bok, Gusb, Galc, Hpgd, Deptor, Dync2h1, Adk, Lrpprc, Comt, Vcam1, Glce,






Ptpn22, Bcl2l11, Heatr1, Hagh, Mecr, Aldh1a1, Tnk2, St8sia6, Mmachc, Fabp1, Il7r, Srsf6,






Zfp612, Polr3g, Nrp1, Scara5, Smc6, Maf, Adh1, Trpv1, Anapc16, Atp1b1, Fancc, Baz2a, Chst4,






Fbl


LN
G3
metabolic process
0.01424
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Evi5, Ret, Pten, Chchd10,






Gprc5b, Btg1, Prmt3, Tox2, Map3k6, Lss, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4,






Ahcyl2, Errfi1, Apex1, Myo7a, Ddx23, Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Gls,






Rlim, Hamp, Birc3, Mcf2l, Nfkb2, Prcp, Ip6k2, Zbtb16, Thbs4, Ptgs1, Ddit4, Serpina3m, Serpina3n,






Serhl, Serpina3c, Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7, Msc, Mbd3, Aebp2, Per3, Per2, Trmt1,






Bbc3, Scd2, Hyal1, Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Acp5, Upf3b, Tbl1xr1, Dis3l2, Fmo2,






Tcf4, Rspo1, Spred2, Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Fads1, Rad51d, Mthfd1, Acer2,






Alg8, Alg3, Hoxd8, Bid, Wdr12, Pank1, Nrip1, Large, Lyst, Swsap1, Pdk4, Pik3ip1, Mafb, Mafg,






Vars2, Pecr, Sae1, Plce1, Nfe2l2, Tcf7l1, Tcf7l2, Zmi21, Fkbp5, Cdk4, Mvd, Stc2, Pde1c, Dnajc24,






Adam9, Kif13a, Acadm, Fcer2a, Pik3cg, Crp, Psmd11, Pdgfra, Vars, Zfp322a, Wipi1, Secisbp2,






Ptpn3, Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7, Uchl1, Suv420h1, Dhx29, Vmp1, Tbrg4,






Cd36, Pinx1, Abcg2, Npy1r, Cxcl13, Ccdc22, Pkig, Rps6ka6, Zfp52, Zfp53, Dpep1, Angpt4, Rprd1a,






Bok, Gusb, Galc, Hpgd, Deptor, Dync2h1, Adk, Lrpprc, Comt, Vcam1, Glce, Ptpn22, Bcl2l11,






Heatr1, Hagh, Mecr, Aldh1a1, Tnk2, St8sia6, Mmachc, Fabp1, Il7r, Srsf6, Zfp612, Polr3g, Nrp1,






Plxnb1, Scara5, Smc6, Maf, Adh1, Trpv1, Anapc16, Atp1b1, Fancc, Baz2a, Chst4, Fbl


LN
G3
Fatty acid metabolism
0.01582
Hsd17b12, Scd2, Fads1, Pecr, Acadm, Fads2, Mecr


LN
G3
Biosynthesis of unsaturated fatty acids
0.02407
Hsd17b12, Scd2, Fads1, Pecr, Fads2


LN
G3
PPAR signaling pathway | Peroxisome
0.03946
Hmgcs2, Hsd17b12, Scd2, Fads1, Pecr, Pex11a, Acadm, Cd36, Fads2, Mecr, Fabp1, Adh1


LN
G3
primary metabolic process
0.04541
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Ret, Pten, Chchd10,






Gprc5b, Btg1, Prmt3, Tox2, Map3k6, Lss, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4,






Ahcyl2, Errfi1, Apex1, Ddx23, Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Gls, Rlim,






Hamp, Birc3, Mcf2l, Nfkb2, Prcp, Ip6k2, Zbtb16, Thbs4, Ptgs1, Ddit4, Serpina3m, Serpina3n,






Serpina3c, Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7, Msc, Mbd3, Aebp2, Per3, Per2, Trmt1, Bbc3,






Scd2, Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Upf3b, Tbl1xr1, Dis3l2, Fmo2, Tcf4, Rspo1, Spred2,






Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Fads1, Rad51d, Mthfd1, Acer2, Alg8, Alg3, Hoxd8, Bid,






Wdr12, Pank1, Nrip1, Large, Lyst, Swsap1, Pdk4, Pik3ip1, Mafb, Vars2, Pecr, Sae1, Plce1, Nfe2l2,






Tcf7l1, Tcf7l2, Zmiz1, Fkbp5, Cdk4, Mvd, Adam9, Acadm, Pik3cg, Psmd11, Pdgfra, Vars, Zfp322a,






Wipi1, Secisbp2, Ptpn3, Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7, Uchl1, Suv420h1,






Dhx29, Cd36, Pinx1, Npy1r, Ccdc22, Pkig, Rps6ka6, Zfp52, Zfp53, Dpep1, Angpt4, Rprd1a,






Bok, Gusb, Galc, Hpgd, Deptor, Dync2h1, Adk, Lrpprc, Glce, Ptpn22, Bcl2l11, Heatr1, Mecr,






Aldh1a1, Tnk2, St8sia6, Fabp1, Srsf6, Zfp612, Polr3g, Nrp1, Smc6, Maf, Adh1, Trpv1, Anapc16,






Fancc, Baz2a, Chst4, Fbl


LN
G3
cellular metabolic process
0.06094
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Ret, Pten, Chchd10,






Gprc5b, Btg1, Prmt3, Tox2, Map3k6, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4, Ahcyl2,






Errfi1, Apex1, Ddx23, Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Gls, Rlim, Hamp,






Birc3, Mcf2l, Nfkb2, Prcp, Ip6k2, Zbtb16, Thbs4, Ptgs1, Ddit4, Serpina3m, Serpina3n, Serpina3c,






Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7, Msc, Mbd3, Aebp2, Per3, Per2, Trmt1, Bbc3, Scd2,






Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Acp5, Upf3b, Tbl1xr1, Dis3l2, Fmo2, Tcf4, Rspo1, Spred2,






Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Fads1, Rad51d, Mthfd1, Acer2, Alg8, Hoxd8, Bid, Wdr12,






Pank1, Nrip1, Large, Lyst, Swsap1, Pdk4, Pik3ip1, Mafb, Vars2, Pecr, Sae1, Plce1, Nfe2l2,






Tcf7l1, Tcf7I2, Zmiz1, Fkbp5, Cdk4, Mvd, Stc2, Adam9, Acadm, Pik3cg, Crp, Psmd11, Pdgfr3,






Vars, Zfp322a, Wipi1, Secisbp2, Ptpn3, Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7, Uchl1,






Suv420h1, Dhx25, Vmp1, Tbrg4, Cd36, Pinx1, Abcg2, Ccdc22, Pkig, Rps6ka6, Zfp52, Zfp53,






Dpep1, Angpt4, Rprd1a, Bok, Galc, Hpgd, Deptor, Adk, Lrpprc, Comt, Glce, Ptpn22, Bcl2l11,






Heatr1, Hagh, Mecr, Aldh1a1, Tnk2, St8sia6, Mmachc, Fabp1, Srsf6, Zfp612, Polr3g, Nrp1, Smc6,






Maf, Adh1, Anapc16, Atp1b1, Fancc, Baz2a, Chst4, Fbl


LN
G3
lipid metabolic process
0.07061
Cyp1b1, Pten, Lss, Ip6k2, Ptgs1, Per2, Scd2, Tbl1xr1, Kit, Fads1, Acer2, Alg8, Lyst, Pdk4, Pik3ip1,






Pecr, Tcf7l2, Mvd, Acadm, Pik3cg, Pdgfra, Agtr1a, Galc, Hpgd, Mecr, Aldh1a1, St8sia6, Adh1,






Trpv1


LN
G3
cellular protein modification process
0.09189
Sorl1, Rbm4, Atg4c, Smurf2, Cops3, Ret, Pten, Gprc5b, Btg1, Prmt3, Map3k6, Fktn, Pdcd4, Errfi1,






Skp2, Trim37, Herc2, Klhl2, Ndrg2, Rlim, Birc3, Thbs4, Ddit4, Ncor2, Cdc7, Mbd3, Per2, Ttll4,






Ttll7, Tbl1xr1, Rspo1, Spred2, Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Acer2, Alg8, Large,






Pdk4, Sae1, Plce1, Nfe2l2, Fkbp5, Cdk4, Adam9, Pik3cg, Pdgfra, Wipi1, Ptpn3, Dab2, Brd7, Uchl1,






Suv420h1, Cd36, Ccdc22, Pkig, Rps6ka6, Angpt4, Rprd1a, Deptor, Ptpn22, Tnk2, St8sia6,






Nrp1, Anapc16, Baz2a, Chst4, Fbl


LN
G3
cellular protein metabolic process
0.19382
Sorl1, Rbm4, Atg4c, Smurf2, Cops3, Ret, Pten, Gprc5b, Btg1, Prmt3, Map3k6, Fktn, Pdcd4, Mrpl11,






Serpine2, Timp4, Errfi1, Skp2, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Rlim, Hamp, Birc3, Thbs4,






Ddit4, Serpina3m, Serpina3n, Serpina3c, Ncor2, Cdc7, Mbd3, Per2, Bbc3, Foxred2, Ttll4, Ttll7,






Pdia6, Pdia4, Upf3b, Tbl1xr1, Rspo1, Spred2, Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Acer2,






Alg8, Bid, Large, Pdk4, Vars2, Sae1, Plce1, Nfe2l2, Fkbp5, Cdk4, Adam9, Pik3cg, Psmd11, Pdgfra,






Vars, Wipi1, Secisbp2, Ptpn3, Dab2, Agtr1a, Brd7, Uchl1, Suv420h1, Cd36, Ccdc22, Pkig, Rps6ka6,






Dpep1, Angpt4, Rprd1a, Bok, Deptor, Lrpprc, Ptpn22, Bcl2l11, Tnk2, St8sia6, Fabp1, Nrp1,






Anapc16, Baz2a, Chst4, Fbl


LN
G3
cell proliferation
0.20172
Kcnh1, Cyp1b1, Pten, Btg1, Fktn, Serpine2, Glul, Ndrg2, Trnp1, Zbtb16, Thbs4, Ptgs1, Ddit4, Ncor2,






Cdc7, Per2, Hyal1, Dis3l2, Rspo1, Kit, Vpreb1, Acer2, Bid, Wdr12, Mafg, Btla, Tcf7l2, Zmiz1,






Cdk4, Mvd, Cr2, Pdgfra, Agtr1a, Brd7, Uchl1, Cxcr4, Cxcl13, Vcam1, Ptpn22, Tnk2, St8sia1, Il7r,






Srsf6, Polr3g, Plxnb1


LV
G4
response to stress
4.1E−13
Rad1, Selk, Atg4c, Ret, Pten, Gprc5b, Map3k6, Fktn, Pdcd4, Rad1, Serpine2, Glul, Errfi1, Apex1,






Tsc22d3, Herc2, Hamp, Nfkb2, Prcp, Scamp5, Thbs4, Ddit4, Cdc7, Per2, Bbc3, Hyal1, Pdia6, Pdia4,






Acp5, Upf3b, Spred2, Kit, Fads1, Rad51d, Bid, Large, Lyst, Swsap1, Seh1l, Pdk4, Cd160, Mrc1,






Ryr1, Nfe2l2, Tcf7l2, Stc2, Adam9, Acadm, Pik3cg, Pdgfra, Wipi1, Dab2, Agtr1a, Il27ra, Rps3,






Uchl1, Clec10a, Cd36, Cxcl13, Rps6ka6, Dpep1, Bok, Comt, Bcl2l11, Tnk2, St8sia1, Fabp1, Srsf6,






Polr3g, Scara5, Smc6, Ndufs8, Trpv1, Atp1b1, Fancc


LN
G3
homeostatic process
0.27783
Gprc5b, Plcg1, Apex1, Tsc22d3, Rps17, Hamp, Prcp, Ncor2, Bbc3, Acp5, Kit, Rad51d, Vpreb1,






Mthfd1, Large, Lyst, Pdk4, Mafb, Ryr1, Nfe2l2, Tcf7l2, Stc2, Pdgfra, Ptpn3, Agtr1a, Pinx1,






Cxcl13, Ccdc22, Vcam1, Bcl2l11, Il7r, Scara5, Smc6, Trpv1, Atp1b1, Fancc


LN
G3
macromolecule metabolic process
0.42101
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Ret, Pten, Gprc5b, Btg1,






Prmt3, Tox2, Map3k6, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4, Errfi1, Apex1, Ddx23,






Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Rlim, Hamp, Birc3, Mcf2l, Nfkb2, Prcp,






Zbtb16, Thbs4, Ddit4, Serpina3m, Serpina3n, Serpina3c, Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7,






Msc, Mbd3, Aebp2, Per3, Per2, Trmt1, Bbc3, Hyal1, Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Upf3b,






Tbl1xr1, Dis3l2, Tcf4, Rspo1, Spred2, Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Rad51d, Acer2,






Alg8, Hoxd8, Bid, Wdr12, Nrip1, Large, Swsap1, Pdk4, Mafb, Mafg, Vars2, Sae1, Plce1, Nfe2l2,






Tcf7l1, Tcf7l2, Zmiz1, Fkbp5, Cdk4, Stc2, Adam9, Acadm, Fcer2a, Pik3cg, Crp, Psmd11, Pdgfra,






Vars, Zfp322a, Wipi1, Secisbp2, Ptpn3, Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7, Uchl1,






Suv420h1, Dhx29, Cd36, Pinx1, Ccdc22, Pkig, Rps6ka6, Zfp52, Zfp53, Dpep1, Angpt4, Rprd1a, Bok,






Deptor, Dync2h1, Lrpprc, Glce, Ptpn22, Bcl2l11, Heatr1, Tnk2, St8sia6, Fabp1, Il7r, Sr5f6,






Zfp612, Polr3g, Nrp1, Scara5, Smc6, Maf, Anapc16, Atp1b1, Fancc, Baz2a, Chst4, Fbl


LN
G3
cell motility
0.42249
Sorl1, Cyp1b1, Smurf2, Ret, Pten, Fktn, Plcg1, Tns1, Tmem201, Prcp, Thbs4, Ddit4, Hyal1, Kit,






Lyst, Strip2, Adam9, Pik3cg, Pdgfra, Dab2, Shroom2, Elp6, Agtr1a, Cxcr4, Cxcl13, Dpep1, Angpt4,






Sema6d, Vcam1, Tnk2, Nrp1, Chst4


LN
G3
pigmentation
0.54296
Myo7a, Kit, Lyst, Kif13a, Shroom2, Bcl2l11


LN
G3
cellular macromolecule metabolic process
0.56618
Sorl1, Pabpn1, Rbm4, Kpna6, Cyp1b1, Hnrnpa1, Atg4c, Smurf2, Cops3, Ret, Pten, Gprc5b, Btg1,






Prmt3, Tox2, Map3k6, Fktn, Pdcd4, Mrpl11, H3f3b, Rad1, Serpine2, Timp4, Errfi1, Apex1, Ddx23,






Skp2, Tsc22d3, Trim37, Herc2, Klhl2, Rps17, Ndrg2, Rlim, Hamp, Birc3, Mcf2l, Nfkb2, Zbtb16,






Thbs4, Ddit4, Serpina3m, Serpina3n, Serpina3c, Zfp36l2, Mettl1, Ncor2, Thrb, Cdc7, Msc,






Mbd3, Aebp2, Per3, Per2, Trmt1, Bbc3, Foxred2, Ttll4, Ttll7, Pdia6, Pdia4, Upf3b, Tbl1xr1, Dis3l2,






Tcf4, Rspo1, Spred2, Dusp4, Dusp6, Dusp1, Dapk2, Otud3, Kit, Rad51d, Acer2, Alg8, Hoxd8,






Bid, Wdr12, Nrip1, Large, Swsap1, Pdk4, Mafb, Vars2, Sae1, Plce1, Nfe2l2, Tcf7l1, Tcf7l2, Zmiz1,






Fkbp5, Cdk4, Adam9, Acadm, Pik3cg, Psmd11, Pdgfra, Vars, Zfp322a, Wipi1, Secisbp2, Ptpn3,






Dab2, Agtr1a, Zfp932, Il27ra, Med24, Rps3, Brd7, Uchl1, Suv420h1, Dhx29, Cd36, Pinx1, Ccdc22,






Pkig, Rps6ka6, Zfp52, Zfp53, Dpep1, Angpt4, Rprd1a, Bok, Deptor, Lrpprc, Glce, Ptpn22, Bcl2l11,






Heatr1, Tnk2, St8sia6, Fabp1, Srsf6, Zfp612, Polr3g, Nrp1, Srnc6, Maf, Anapc16, Fancc,






Baz2a, Chst4, Fbl


LN
G3
single-organism metabolic process
0.72953
Sorl1, Rbm4, Cyp1b1, Atg4c, Ret, Pten, Chchd10, Prmt3, Map3k6, Lss, Fktn, Pdcd4, Rad1, Ahcyl2,






Apex1, Trim37, Herc2, Ndrg2, Gls, Prcp, Ip6k2, Ptgs1, Ddit4, Ncor2, Cdc7, Mbd3, Per2, Scd2,






Hyal1, Foxred2, Tbl1xr1, Fmo2, Rspo1, Spred2, Dusp6, Dusp1, Kit, Fads1, Rad51d, Mthfd1, Acer2,






Alg8, Alg3, Pank1, Large, Lyst, Swsap1, Pdk4, Pik3ip1, Vars2, Pecr, Plce1, Nfe2l2, Tcf7l2,






Mvd, Adam9, Acadm, Pik3cg, Pdgfra, Vars, Wipi1, Dab2, Agtr1a, Il27ra, Rps3, Brd7, Uchl1, Suv420h1,






Tbrg4, Cd36, Pinx1, Abcg2, Npy1r, Rps6ka6, Dpep1, Galc, Hpgd, Adk, Lrpprc, Vcam1, Glce,






Mecr, Aldh1a1, St8siB6, Mmachc, Fabp1, Nrp1, Smc6, Adh1, Trpv1, Atp1b1, Fancc, Baz2a, Fbl


LN
G3
embryo development
0.73052
Tmie, Cops3, Ret, Stox2, Plcg1, Myo7a, Zbtb16, Ncor2, Mbd3, Hyal1, Dusp4, Dusp6, Dusp1, Kit,






Mthfd1, Mafb, Mafg, Nfe2l2, Tcf7l1, Tcf7l2, Zmiz1, Pdgfra, Dab2, Hoxc4, Rps6ka6, Epn2, Dync2h1,






Vcam1, Bcl2l11, Aldh1a1, Nrp1


LN
G3
cellular lipid metabolic process
0.83984
Cyp1b1, Pten, Ip6k2, Ptgs1, Per2, Scd2, Tbl1xr1, Kit, Fads1, Acer2, Alg8, Lyst, Pdk4, Pik3ip1,






Pecr, Tcf7l2, Mvd, Acadm, Pik3cg, Pdgfra, Agtr1a, Galc, Hpgd, Mecr, Aldh1a1, St8sia6, Adh1


LN
G3
protein maturation
0.88005
Sorl1, Atg4c, Ret, Serpine2, Birc3, Bbc3, Acer2, Bid, Bok, Dync2h1, Bcl2l11


LN
G3
Ubiquitin mediated proteolysis | Cell cycle
0.89005
Smurf2, Pten, Skp2, Sesn1, Trim37, Herc2, Birc3, Cdc7, Bbc3, Zmat3, Bid, Sae1, Cdk4


LN
G3
cell adhesion
0.94449
Cyp1b1, Ret, Pten, Serpine2, Zbtb16, Thbs4, Zfp36l2, Hsd17b12, Hyal1, Pdia6, Kit, Acer2, Mafb,






Btla, Adam9, Pik3cg, Pdgfra, Dab2, Vmp1, Cd36, Cxcr4, Cxcl13, Vcam1, Ptpn22, Bcl2l11, Il7r,






Plxnb1, Dtx1, Chst4


LN
G3
sulfur compound metabolic process
0.95768
Ahcyl2, Mthfd1, Pdk4, Nfe2l2, Tcf7l2, Dpep1, Glce, Hagh, Chst4


LN
G5
cofactor metabolic process
0.01335
Cyp4f18, Prkag2, Adck3, Nudt7, Fech, Ppcdc, Entpd5, Ogt, Pfkfb2


LN
G5
Integrin alphaIIb beta3 signaling
0.12096
Rasgrp2, Rasgrp1, Syk, Rapgef4


LN
G5
cellular protein modification process
0.18764
Prkag2, Ssh1, Flcn, Agbl3, Rnf180, Caprin2, Zdhhc20, Sox4, Eogt, Zdhhc9, Cdc25b, Fgf14, Camk2a,






Srpk2, Map3k9, Tinf2, Rprd1a, Rasgrp1, Ttn, Stox1, Fech, Entpd5, Ttll5, Nek1, Ogt, Ctdsp2,






Nlrc3, Clcf1, Ick, Ttc3, Insm1, Stk38, Map4k2, Ubash3b, Syk, Tnik, Cdon, Man2b1


LN
G5
phosphate-containing compound metabolic process
0.26397
Prkag2, Ssh1, Flcn, Gucy1a3, Caprin2, Adck3, Cdc25b, Nudt7, Fgf14, Camk2a, Adcy7, Srpk2,






Map3k9, Rprd1a, Rasgrp1, Ttn, Stox1, Fech, Ppcdc, Entpd5, Nek1, Ogt, Ctdsp2, Pik3r2, Syt12,






Pfkfb2, Clcf1, Ick, Ak3, Insm1, Stk38, MBp4k2, Ubash3b, Syk, Tnik, Akap5, Rbl2, Cdon


LN
G5
nucleobase-containing compound catabolic process
0.31009
Prkag2, Pan3, Nudt7, Entpd5, Ogt, Ago4, Upf3b


LN
G5
Rap1 signalling
0.41749
Rasgrp2, Rasgrp1, Rapgef4


LN
G5
Innate Immune System | Adaptive Immune System |
0.54744
Plekhg2, Prkag2, Cers4, Ssh1, Flcn, Cldn11, Gucy1a3, Rgs3, Il13ra2, Amph, Thbs2, Agtr1a,




Hemostasis | Pathways in cancer | Developmental

Cd28, Cdc25b, Ciita, Fgf14, Camk2a, Adcy7, Scai, S100B10, H2-Ob, Ppp1r3c, Rasgrp2, Rasgrp1,




Biology | PI3K-Akt signaling pathway | Signaling

Sike1, Coro1a, Sh3kbp1, Itga8, E2f2, Pik3r2, Pfkfb2, Habp4, Clcf1, Cacna1b, Ak3, Insm1,




by Rho GTPases

Map4k2, Syk, Arhgap9, Arhgap4, Atg16l2, Depdc7, Rbl2, Cdon, Atp6v0e2, Rapgef4, Pde10a


LN
G5
generation of precursor metabolites and energy
0.85945
Prkag2, Flcn, Ppp1r3c, Entpd5, Ogt, Pfkfb2


LN
G5
purine ribonucleotide metabolic process
0.86597
Prkag2, Flcn, Gucy1a3, Nudt7, Adcy7, Ppcdc, Entpd5, Ogt, Pfkfb2, Ak3, Akap5


LN
G5
cellular component assembly
0.90128
Ssh1, Flcn, Anxa6, Pan3, Cep164, S100a10, Srpk2, Clec2i, Tinf2, Rasgrp1, Ttn, Fech, Hist1h1c,






Haus8, Coro1a, Trtll5, Nek1, Rfx3, Ago4, Pik3r2, Ick, Insm1, Tnik, Atg16l2, Cryz


LN
G6
immune system process
0.00683
Lyl1, Klf2, Rgcc, C5ar1, Gprc5b, H2-DMb2, Syk, Hhex, Hif1a, Clec4e, Sox4, H2-Ab1,






Fgr, Kcnab2, Adora2b, Clec7a, Sirpa, Calr, Dusp3, Lat2, Cd79b, Matk, Tfeb, Inpp4b, Trim59,






Meis2, Lgals3, Itgax, Nfe2l2, Hist2h2be, Fcer2a, Itga9, Pla2g2d, Ednrb, Itgb1, Hand2,






Cxcl12, Gbf1, Slc40a1, Fam20c, Ceacam1, Rab4a, Egr3, Vegfc, 5430435G22Rlk, Cd300lb, H2-DMa,






Itpkb, Stap1, Myo1f, Mtss1, Jak3


LN
G6
locomotion
0.03084
Rgcc, Hexb, C5ar1, Pfn2, Syk, Hif1a, Mmp12, Top2b, Flrt3, Fgr, Pdgfa, Sh3kbp1, Calr, Ptp4a3,






Hyal1, Matk, Trim59, Npc1, Lgals3, Itga9, Ntrk3, Ednrb, Spns3, Itgb1, Hand2, Cxcl12, Zfand5,






Gbf1, Ceacam1, Scai, Egr2, Egr3, Vegfc, Vcan, Stap1, Myo1f, Myo18a, Jak3


LN
G6
transport
0.04636
Rbm4, Rgcc, Apoc2, Cltb, Pfn2, Slc36a4, Syk, Stim1, Hhex, Hif1a, Agpat6, Slc39a6, Slc35f6,






Soat1, Clec4e, Atp6v1c1, Sox4, Arf2, Eif2ak3, Fgr, Cacna2d4, Abca13, Kcnab2, Scn4a, Adora2b,






Itpr3, Clec7a, Sirpa, Calr, Gnpda1, Slc16a5, Arhgap17, Kif1c, Inpp4b, Slc30a1, Slc16a12,






Npc1, Lgals3, Msr1, Itgax, Ibtk, Fabp5, Nfe2l2, Pea15a, Tpcn1, Slc35d2, Trim16, Kcnj10, Scrn3,






Rab30, Pdzd11, Clec4b1, Sec22c, Ptpn14, Zfand2b, Sgk1, Plin2, Cenpf, Ccdc93, Txndc5, Gbf1,






Fam21, Slc40a1, Ceacam1, Fam89b, Rab4a, Egr2, 5430435G22Rik, H2-DMa, Exoc8, Stap1, Xpo7,






Myo1f, Rapgef4, Myo18a, Jak3


LN
G6
cell motility
0.09897
Rgcc, Hexb, C5ar1, Pfn2, Syk, Hif1a, Mmp12, Top2b, Fgr, Pdgfa, Sh3kbp1, Calr, Ptp4a3, Hyal1,






Matk, Lgals3, Itga9, Ntrk3, Ednrb, Itgb1, Hand2, Cxcl12, Zfand5, Gbf1, Scai, Egr3, Vegfc,






Vcan, Stap1, Myo1f, Myo18a, Jak3


LN
G6
Phagosome
0.10643
H2-DMb2, Atp6v1c1, Atp6v0d2, H2-Ab1, Atp6v1a, Clec7a, Calr, Msr1, Itgb1, 5430435G22Rik,






H2-DMa, Tubb4b


LN
G6
vesicle-mediated transport
0.13082
Apoc2, Cltb, Pfn2, Syk, Fgr, Adora2b, Clec7a, Sirpa, Calr, Gnpda1, Arhgap17, Kif1c, Npc1,






Lgals3, Scrn3, Pdzd11, Sec22c, Ccdc93, Txndc5, Gbf1, Fam21, Rab4a, 5430435G22Rik, Exoc8,






Stap1, Myo1f, Rapgef4, Myo18a


LN
G6
Protein processing in endoplasmic reticulum
0.30308
Ssr1, Eif2ak3, Calr, Nfe2l2, Dnajbl2, Preb, Rrbp1, Ckap4, Txndc5, Hyou1


LN
G6
Lysosome
0.30308
Cers4, Hexb, Cltb, Ppap2a, Atp6v0d2, Pla2g15, Hyal1, Npc1, Ctsb, Cd68


LN
G6
Cytokine-cytokine receptor interaction | Enclocytosis |
0.31884
C5ar1, Cltb, Fgfr3, H2-DMb2, Skp2, Atp6v1c1, Asap1, Atp6v0d2, H2-Ab1, Atp6v1a, Eif2ak3,




Herpes simplex infection

Pdgfa, Sh3kbp1, Clec7a, Calr, Ccr6, Pip5k1b, Msr1, Fter2a, Itga9, Itgb1, Ctsb, Mpzl1,






Cxcl12, Gbf1, Reb4a, Vegfc, 5430435G22Rik, H2-DMa, Tubb4b, Vcan, Jak3


LN
G6
Steroid biosynthesis
0.32098
Dhcr24, Lss, Soat1


LN
G6
localization
0.33692
Rbm4, Rgcc, Apoc2, Dhcr24, Hexb, C5ar1, Cltb, Pfn2, Slc36a4, Syk, Stim1, Hhex, Hif1a, Agpat6,






Slc39a5, Slc35f6, Soat1, Clec4e, Mmp12, Top2b, Sox4, Arf2, Eif2ak3, Gpr158, Fgr, Cacna2d4,






Abca13, Kcnab2, Scn4a, Pdgfa, Adora2b, Itpr3, Sh3kbp1, Clec7a, Sirpa, Calr, Gnpda1, Slc16a5,






Arhgap17, Ptp4a3, Hyal1, Kif1c, Matk, Inpp4b, Slc30a1, Bub3, Slc16a12, Npc1, Lgals3, Msr1,






Itgax, Ibtk, Fabp5, Nfe2l2, Pea15a, Tmem106b, Tpcn1, Slc35d2, Trim16, Itga9, Ntrk3, Kcnj10,






Scrn3, Rab30, Pdzd11, Ednrb, Clec4b1, Stk11ip, Sec22c, Ptpn14, Zfand2b, Itgb11, Sgk1, Msx1,






Plin2, Cenpf, Hand2, Cxcl12, Ccdc93, Zfand5, Txndc5, Gbf1, Fam21, Slc40a1, Ceacam1, Fam89b,






Scai, Rab4a, Egr2, Egr3, Smyd3, Vegfc, 5430435G22Rik, Tspan10, H2-DMa, Exoc8, Vcan, Stap1,






Xpo7, Myo1f, Rapgef4, Myo18a, Jak3


LN
G6
Antigen activates B Cell Receptor (BCR) leading to generation
0.42632
Syk, Stim1, Itpr3, Sh3kbp1, Cd79b




of second messengers


LN
G6
lipid metabolic process
0.52612
Cers4, Apoc2, Dhcr24, Hexb, Fgfr3, Ppap2a, Syk, Lss, Agpat6, Soat1, Mgll, Stard4, Eif2ak3,






Fgr, Pla2g15, Pdgfa, Tbl1xr1, Pip5k1b, Inpp4b, Npc1, Fabp5, Hpgds, Elovl7, Rbl2, Decr2


LN
G6
regulation of developmental process
0.66389
Rbm4, Rgcc, Hexb, C5ar1, Gprc5b, Fgfr3, Syk, Hhex, Runx1t1, Hif1a, Agpat6, Timp2, Vat1,






Mgll, Asap1, Flrt3, Sox4, Setx, Ttbk1, Sostdc1, Eif2ak3, Fgr, Pdgfa, Ldb2, Sh3kbp1, Calr,






Metrnl, Hyal1, Inpp4b, Meis2, Msr1, Mapk11, Nfe2l2, Trim16, Ntrk3, Ednrb, Brwd1, Frzb, Itgb1,






Msx1, Cenpf, Arhgef15, Hand2, Cxcl12, Foxp2, Fam20c, Egr3, Vegfc, 5430435G22Rik, H2-DMa,






Zfp157, Ankrd6, Itpkb, Odf2, Stap1, Zfpm2, Jak3


LN
G6
anatomical structure development
0.79035
Rbm4, Lyl1, Klf2, Hes6, Endog, Rgcc, Dhcr24, Hexh, C5ar1, Gprc5b, Fgfr3, Syk, Stim1, Hhex,






Runx1t1, Pmp22, Hif1a, Agpat6, Timp2, Vat1, Clec4e, Mmp12, Mgll, Dok4, Top2b, Asap1, Flrt3,






Sox4, Setx, H2-Ab1, Ttbk1, Sostdc1, Eif2ak3, Fgr, Phf8, Kcnab2, Pdgfa, Ldb2, Sh3kbp1, Tbl1xr1,






Sirpa, Calr, Hyal1, Cml1, Matk, Tfeb, Inpp4b, Slc30a1, Zfp191, Edaradd, Meis2, Lgals3, Mapk11,






Mapk12, Srpk3, Nfe2l2, Tpra1, Tmem106b, Trim16, Aes, Ntrk3, Pla2g2d, Kcnj10, Ednrb, Brwd1,






Frzb, Ptpn14, Itgb1, Ctsb, Msx1, Cenpf, Arhgef15, Hand2, Cxcl12, Zfand5, Foxp2, Slc40a1, Fam20c,






Ceacam1, Egr2, Egr3, Smyd3, Vegfc, 5430435G22Rik, H2-DMa, Zfp157, Ankrd6, Vcan, Itpkb, Odf2,






Stap1, Zfpm2, Mtss1, Jak3


LN
G6
cell adhesion
0.89686
Rgcc, Syk, Pnn, Clec4e, Sox4, H2-Ab1, Clec7a, Sirpa, Calr, Dusp3, Hyal1, Cml1, Lgals3, Itgax, Itga9,






Pla2g2d, Dsc2, Itgb1, Cxcl12, Gbf1, Ceacam1, Egr3, Vegfc, H2-DMa, Vcan, Itpkb, Myo1f , Jak3


LN
G6
Rheumatoid arthritis
0.96573
H2-DMb2, Atp6v1c1, Atp6v0d2, H2-Ab1, Atp6v1a, Cxcl12, H2-DMa



















TABLE 19





Upstream
Molecule
p-value



Regulator
Type
of overlap
Target molecules in dataset















UpStream_LN G1(8adj)










IL6
cytokine
1.64E−02
CEBPD, CP, GLRX, TLR9, TNFRSF1B


TNFSF11
cytokine
1.69E−02
GLRX, HCK, TNFRSF1B


IFNG
cytokine
1.78E−02
CEBPD, CP, HCK, S1PR3, SLC15A3, TLR9, TNFRSF1B


IL2
cytokine
2.29E−02
HCK, RAF1, SOS1, TNFRSF1B







UpStream_LN G1(5adj)










IFNA2
cytokine
3.84E−24
CORO2A, CXCL10, DPP4, HERC6, HLA-E, IFI16, IFI35, IFIH1,





IFIT1, IFIT2, IFITM3, IRF9, LY6E, LYN, OAS1, PARP9, PHF11, PML, PTTG1, RBL1,





SAMHD1, STAT1, TNFSF10, TREX1, TRIM21, UBA7, XAF1


IFNB1
cytokine
4.46E−23
CASP8, CXCL10, DAXX, DHX58, GBP4, GBP7, Gvin1 (includes





others), IFI16, Ifi47, IFIH1, IFIT1, IFIT2, IRF9, Irgm1, NMI, NOD1, NT5C3A, OAS1, PML,





SLFN13, STAT1, STAT2, Tgtp1/Tgtp2, TNFSF10, TRIM21, Trim30a/Trim30d, UBA7, XAF1


IFNG
cytokine
9.98E−21
BLNK, CASP8, CERS6, CLIC4, CTSC, CTSS, CXCL10, CYBB, DPP4, DTX3L, FGL2, GBP4, GBP7, Gvin1





(includes others), HCK, HERC6, HLA-E, HLA-G, IFI16, IFI35,





Ifi47, IFIH1, IFIT1, IFIT2, IFITM3, IRF9, Irgm1, LGALS9B, LY6E, LYN, MAX, Ms4a4b





(includes others), NMI, OAS1, PARP9, PML, PSMB10, PSMB8, PSMB9, SAMHD1, Serpina3g





(includes others), STAT1, STAT2, TAPBP, Tgtp1/Tgtp2, TNFSF10, TRAFD1, TRIM21, UBE2E1


IFNL1
cytokine
3.20E−14
CXCL10, HERC6, IFI35, IFIH1, IFIT1, IFIT2, IFITM3, IRF9, OAS1, PHF11, PML, PSMB9, STAT1


IFNA1/
cytokine
2.21E−11
CXCL10, DHX58, IFIH1, IFIT1, IFIT2, OAS1, STAT1, STAT2, Tgtp1/Tgtp2, UBE2E1


IFNA13


IL15
cytokine
1.79E−06
CXCL10, DPP4, IFI35, IFIT1, Klrk1, LYN, PNP, PSMB10, PSMB8, PSMB9, RBBP8, SOS1,





TNFSF10, TRIM5


IL4
cytokine
5.49E−06
BBX, CTSC, CXCL10, DPP4, GBP7, HCK, IFI16, Klrk1, Ms4a4b (includes





others), PNP, PSMB10, PSMB8, Serpina3g (includes





others), SLAMF7, SMG7, STAT1, STAT2, TAPBP, Tgtp1/Tgtp2, Trim30a/Trim30d


IL1RN
cytokine
6.98E−06
CTSS, HERC6, IFIH1, IRF9, OAS1, PML, STAT2, TNFSF10


IFNK
cytokine
3.38E−05
CXCL10, IFIH1, OAS1, STAT1


IFNA4
cytokine
4.03E−05
CXCL10, IFIH1, IFIT1, IFIT2


IL1B
cytokine
5.73E−05
CTSS, CXCL10, CYBB, DPP4, HELZ2, HLA-E, IFI16, Ifi47, IFIT1, LGALS9B,





NMI, PSMB10, PSMB8, PSMB9, Slfn2, STAT1, Tgtp1/Tgtp2, TNFSF10, TRAFD1


IL12B
cytokine
7.88E−05
CXCL10, IRF9, PIK3AP1, STAT1, STAT2


TNFSF10
cytokine
7.99E−05
CASP8, IFI16, IFIT1, IRF9, STAT1, TNFSF10


TNF
cytokine
1.53E−04
CASP8, CD47, CLIC4, CTSC, CTSS, CXCL10, CYBB, DAXX, DPP4, HLA-E, IFI16,





IFIH1, IFIT1, LGALS9B, LYN, OAS1, PDK3, PML, PSMB10, PSMB8, PSMB9, Slfn2, STAT1,





TAPBP, Tgtp1/Tgtp2, TNFSF10, TRAFD1


IFNA10
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNA21
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNA5
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNA7
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNA14
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNA6
cytokine
1.83E−04
CXCL10, IFIH1, IFIT1


IFNE
cytokine
2.07E−04
CXCL10, IFIH1, IFIT2, STAT1


IFNA8
cytokine
2.27E−04
CXCL10, IFIH1, IFIT1


IFNA16
cytokine
2.27E−04
CXCL10, IFIH1, IFIT1


CD40LG
cytokine
3.50E−04
CTSC, CXCL10, IFIT1, IFIT2, PML, PSMB10, PSMB8, PSMB9, SMG7, STAT1, TNFSF10


IL6
cytokine
5.19E−04
CTSC, CXCL10, CYBB, IFI16, IFIT1, IFIT2, IFITM3, IRF9, PSMB10, P5MB8, PSMB9, PTTG1,





SH3GLB1, STAT1, TNFSF10


IL2
cytokine
8.16E−04
CTSC, CXCL10, DPP4, HCK, LY6E, NMI, PNP, PRKX, RAF1, RGPD4 (includes others), SOS1,





TNFSF10


IL12A
cytokine
9.91E−04
CXCL10, IRF9, STAT1, STAT2


IL27
cytokine
3.67E−03
CXCL10, HLA-E, STAT1, STAT2, TNFSF10


OSM
cytokine
3.83E−03
CXCL10, GCA, GNS, IFI35, IRF9, MYCBP2, OAS1, PSMB8, PSMB9, STAT1, TAPBP


IFNA17
cytokine
5.66E−03
CXCL10, IFIH1


EBI3
cytokine
5.91E−03
STAT1, STAT2, TNFSF10


IL10
cytokine
8.02E−03
CASP8, CTSS, CXCL10, HLA-G, IFIT2, PSMB9, STAT1, TNFSF10


IFNW1
cytokine
9.32E−03
CXCL10, IFIH1


TNFSF11
cytokine
1.65E−02
HCK, KEAP1, Slfn2, STAT1, VRK1, YWHAZ


IL7
cytokine
2.49E−02
BLNK, DPP4, LYN, TNFSF10







UpStream_LN G2










IL33
cytokine
9.70E−03
ABCA1, SCARB1, TFEC


TNF
cytokine
1.18E−02
ABCA1, CIB2, CSF2RB, CTSB, GAB1, GPAM, INSR, LBP, NOS3, PRSS23, PTGS1, PTPN12, RGS4,





SCARB1, SCD, SLC12A6


IL10
cytokine
1.32E−02
CDKN2D, CEACAM1, CSF2RB, CTSB, NOS3, PIK3CG


IL6
cytokine
1.41E−02
ABCA1, CDKN2D, CEACAM1, CIB2, CSF2RB, HFE, LBP, NOS3, SBNO2


IL5
cytokine
1.58E−02
CEACAM1, CSF2RB, EPS8, GPAM, KIAA1147


OSM
cytokine
2.63E−02
ABCA1, GAB1, GLUL, GMPR, LBP, MGLL, RAP2A


CRH
cytokine
4.52E−02
NOS3, SCARB1







UpStream_LN G3










TNF
cytokine
6.56E−10
ABCG2, ACADM, AGTR1, AK2, BBC3, BCL2L11, BID, BIRC3, BTG1, C10orf10, Ccl9, CCR6, CD163,





CD209, CD36, CDK4, CHST4, CRP, CXCL13, CXCR4, CYP1B1, DUSP1, DUSP6, FABP1, FADS1, FBL,





FCER2, GLS, GUSB, Hamp/Hamp2, HPGD, IFNGR2, IL7R, KIT, LSS, MSC, NCOR2, NFE2L2, NFKB2,





NRIP1, NRP1, PCP4, PDGFRA, PDIA4, PER2, PEX11A, PPP5C, PTEN, PTGS1, RPS3, SERPINA3,





SERPINE2, SMURF2, SND1, TIMP4, TSC22D3, VCAM1, VMP1


IL10
cytokine
3.13E−06
ARAP2, C10orf10, CCR6, CD163, CD209, CXCL13, CXCR4, DUSP1, FCER2, FKBP5, GPRC5B, HPGD,





IL7R, ITGAE, MAF, MRC1, PIK3CG, TSC22D3, VCAM1


IL4
cytokine
5.79E−06
Acp5, ALG3, CCR6, CD163, CD209, CD36, CLEC10A, CR2, CXCR4, EVI5, FBL, FCER2, FKBP5, HPGD,





IFNGR2, IL27RA, IL7R, ITGAE, KIT, KLHDC2, MAF, MAFB, MRC1, NFKB2, NRP1, PHB, PPP5C,





Scd2, VCAM1, Zfp53


IL2
cytokine
4.56E−05
BBC3, BIRC3, CCR6, CD160, CDK4, CXCR4, DAPK2, DUSP4, DUSP6, IFNGR2, IL27RA, IL7R, IP6K2,





ITGAE, KIT, MAF, PDCD4, PHB, PINX1, SESN1, SPRED2


IFNG
cytokine
7.84E−05
AGTR1, ARAP2, ATP1B1, BBC3, BCL2L11, BID, BTG1, C10orf10, CCR6, CD163, CD209, CD36, CDK4,





CLEC10A, CXCR4, CYB561, DUSP1, FAM107A, FCER2, FKBP5, GLS, GLUL, GPR64, GPRC5B, GUSB,





IFNGR2, IL7R, LARGE, MRC1, NFKB2, PCDH17, PLCG1, PPP5C, TCF7L2, TIMP4, TSC22D3, VCAM1,





ZBTB16


IL6
cytokine
2.42E−04
ABCG2, Acp5, BBC3, 8CL2L11, CCR6, CD163, CD209, CD36, CRP, CXCL13, CXCR4, CYP1B1, DUSP1,





DUSP6, FCER2, Hamp/Hamp2, HPGD, IL7R, KIT, LARGE, MAF, PHB, SERPINA3, VCAM1


IL13
cytokine
3.23E−04
Acp5, BID, CD163, CD209, CD36, CXCR4, FADS1, FADS2, FCER2, IFNGR2, MAF, MRC1, TIMP4, TNS1,





TRPV1, VCAM1


CSF1
cytokine
3.32E−04
Acp5, BCL2L11, CD163, CD209, CDK4, DUSP1, MAFB, MRC1, NFKB2, VCAM1


EPO
cytokine
6.19E−04
ABCA13, BCL2L11, BTG1, CD36, CXCR4, FABP1, GLUL, KIT, N4BP2L1, PDCD4, PIK3IP1, SKP2, TSC2





2D3


IL1B
cytokine
8.20E−04
ABCG2, AGTR1, BIRC3, Ccl9, CCR6, CRP, CXCR4, DAB2, DDIT4, DU5P1, ERRFI1, FABP1, FCER2,





FKBP5, GUSB, Hamp/Hamp2, NFE2L2, NFKB2, NRP1, PDGFRA, PTGS1, SERPINA3, SESN1, TIMP4,





TSC22D3, VCAM1


PRL
cytokine
1.99E−03
ABCG2, BOK, CDK4, MAFG, PDGFRA, PDIA4, PDK4, PKIG, SERPINA3, SND1


IL3
cytokine
2.38E−03
BCL2L11, BIRC3, CXCR4, FCER2, KLF9, NRP1, PTPN22, RPL3, RPS17, RPS20, RPS3, SERPINA3,





VCAM1


LTB
cytokine
4.79E−03
CHST4, CRP, CXCL13


IL15
cytokine
5.19E−03
BBC3, BCL2L11, BTG1, CD160, CDK4, CXCR4, IL7R, KIT, MAF, PDCD4, PDIA4, SORL1, VCAM1


CCL5
cytokine
5.27E−03
CD163, CYP1B1, DUSP1, DUSP6, NCOR2


TNFSF13B
cytokine
7.37E−03
BCL2L11, CDK4, CR2, NFKB2


CD40LG
cytokine
8.69E−03
ACY1, ALG3, BIRC3, BTG1, CXCL13, CXCR4, CYP1B1, DUSP1, DUSP4, FBL, IL7R, NFKB2, VCAM1


IL17A
cytokine
1.02E−02
CD163, CRP, CXCL13, GUSB, MRC1, NRP1, TIMP4, VCAM1


SPP1
cytokine
1.34E−02
ABCG2, Acp5, BCL2L11, MECR, NDRG2, NDUFS8


IL27
cytokine
1.44E−02
C10orf10, CD163, CDK4, HPGD, MAF, MRC1


CSF3
cytokine
1.50E−02
AGTR1, BIRC3, CDK4, CXCR4, DUSP6, KIT, VCAM1


TNFSF11
cytokine
1.74E−02
Acp5, AEBP2, Ccl9, DAB2, DUSP1, MAFB, NFKB2, SIGMAR1, VCAM1


IL1A
cytokine
1.92E−02
ALDH1A1, BIRC3, CCR6, Hamp/Hamp2, IFNGR2, KIT, 5ERPINA3, VCAM1


IL7
cytokine
2.03E−02
BCL2L11, BTLA, CXCR4, IL7R, MAF, SMURF2


CSF2
cytokine
2.40E−02
BBC3, BCL2L11, BID, BIRC3, CD209, CXCR4, DUSP6, ITGAE, MRC1, NFKB2, NRP1, PIK3CG, TCF4


WNT5A
cytokine
2.64E−02
CXCR4, DAB2, KIT, PTEN, RET


LTA
cytokine
3.20E−02
CHST4, CRP, CXCL13


OSM
cytokine
3.79E−02
CRP, CXCL13, CYP1B1, GLUL, Hamp/Hamp2, LARGE, MRC1, MSC, PDCD4, PKIG, PTEN, SERPINA3,





ST8SIA1, VCAM1


IL11
cytokine
4.42E−02
CRP, SERPINA3, VCAM1


NAMPT
cytokine
4.65E−02
ATXN10, NPY1R







UpStream_LN G5










IL5
cytokine
1.67E−02
AK3, ANXA6, CD55, CIITA, Crisp1/Crisp3, RASGRP2


IK
cytokine
3.06E−02
CIITA


EBI3
cytokine
4.74E−02
CIITA, HLA-DOB







UpStream_LN G6










IL4
cytokine
6.27E−06
AUH, BPGM, C5AR1, CAPG, CCR6, CLEC4C, CLEC7A, Cmah, DDAH1, EGR2, FCER2, GNA15, HLA-





DMA, ITGAX, ITGB1, JAK3, KCNAB2, LGALS3, LILRB4, LYL1, MATK, MMP12, MSR1, PLIN2,





PMP22, SIRPA, SYK, TIMP2, XPO7


IFNG
cytokine
2.84E−04
ADORA2B, C5AR1, CCR6, CEACAM1, CLEC4E, CTSB, CXCL12, E2F2, EDNRB, EGR2, EGR3,





FABP5, FCER2, GFM1, GPR158, GPR83, GPRC5B, HIF1A, HLA-DMA, HLA-DMB, HLA-





DQB1, ITGB1, ITPKB, JAK3, KLF2, LAT2, LGALS3, MMP12, MSR1, PDGFA, PEA15, SLC40A1,





SOAT1, SPI1, VEGFC


TNF
cytokine
5.79E−04
ADAMTS5, ADORA2B, APOC2, C5AR1, CALR, CCR6, CLEC4E, CTSB, CXCL12, DSC2, EDNRB,





EGR2, ENTPD5, FABP5, FBXO32, FCER2, FRZB, HEXB, HIF1A, HPGDS, ITGB1, KLF2, LGALS12,





LGALS3, LSS, MMP12, MSR1, MTHFD2L, NFE2L2, NME1, PDGFA, PLIN2, PLK2, SGK1, SLC16A5,





SNN, SOAT1, SOX4, TIMP2, VEGFC


IL6
cytokine
7.27E−04
ADAMTS5, BUB3, C5AR1, CCR6, CD68, CEACAM1, CLEC4E, E2F2, EGR2, FCER2, HIF1A, HLA-





DMA, ITGB1, MMP12, MSR1, PDGFA, PLA2G2D, POLD1, PTP4A3, SGK1, SLC40A1, SPI1


IL1A
cytokine
1.40E−03
ADAMTS5, ADORA2B, CCR6, CXCL12, FAM89B, ITGB1, MMP12, PDGFA, RBL2, VEGFC


IL13
cytokine
1.69E−03
ADORA2B, CHN2, CLEC4E, CLEC7A, CTSB, DHCR24, FCER2, LILRB3, MATK, MMP12, MTSS1,





PDGFA, PLA2G2D, TIMP2


IL5
cytokine
2.22E−03
CEACAM1, CKAP4, EGR2, EGR3, ENDOG, FAM65B, HIF1A, KCNAB2, PMP22, RASGRP2, SGK1


IL10
cytokine
2.49E−03
CCR6, CD68, CEACAM1, CLEC7A, CTSB, CXCL12, FCER2, GPRC5B, LILRB4, MSR1, SLC9A3R2,





TUBB4B, VCAN


CSF2
cytokine
3.01E−03
ADORA2B, C5AR1, CEACAM1, CLEC4C, CLEC7A, EGR2, EGR3, FAM65B, HLA-





DQB1, ITGAX, PLA2G15, PMP22, POLD1, SGK1, SPI1


TNFSF11
cytokine
4.48E−03
ADORA2B, AKAP13, ATP6V0D2, CLEC4E, EGR2, FGR, ITPKB, LDB2, PDGFA, POR


IL33
cytokine
6.77E−03
ADORA2B, CLEC4E, EGR2, MSR1, PLIN2


CCL11
cytokine
7.69E−03
CXCL12, ITGB1, PDGFA, VEGFC


TNFSF12
cytokine
9.16E−03
CD68, CLEC4E, FBXO32, HES6, MMP12


IL3
cytokine
1.17E−02
CALR, CLEC4C, EGR2, EGR3, EMP3, FCER2, ITGAX, LILRB4, SOX4, SPI1, ZFAND5


CCL5
cytokine
2.21E−02
C5AR1, DDAH1, SGK1, TUBB4B


IL17A
cytokine
2.39E−02
CD68, CXCL12, HEXB, KLF2, KLF3, TIMP2, VEGFC


EDN1
cytokine
2.71E−02
EDNRB, HAND2, HIF1A, ITGB1, PDGFA, VCAN, VEGFC


CSF3
cytokine
3.69E−02
CAPG, CXCL12, EGR2, EGR3, JAK3, SPI1


CSF1
cytokine
4.08E−02
CD68, EGR3, ITGB1, MMP12, MSR1, SPI1


CX3CL1
cytokine
4.63E−02
HIF1A, MSR1


IL1B
cytokine
4.87E−02
ADAMTS5, APOC2, CCR6, CTSB, CXCL12, E2F2, FABPS, FCER2, FGFR3, HEXB, HIF1A,





HPGDS, ITGB1, ITPKB, MMP12, NFE2L2, TIMP2, VCAN, VEGFC







UpStream_SP G1










IFNA2
cytokine
2.68E−09
IFI16, OAS1, OAS2, OGFR, SAMHD1, UBA7, UBE2L6


IFNG
cytokine
3.71E−06
AGRN, FCGR1A, IFI16, OAS1, OAS2, RNF31, SAMHD1, SLFN12L, UBE2L6


IFNB1
cytokine
8.79E−06
IFI16, Ifi27l2a/Ifi27l2b, OAS1, OAS2, UBA7


IFNA1/
cytokine
2.91E−05
OAS1, OAS2, UBE2L6


IFNA13


IFNL1
cytokine
7.08E−05
OAS1, OAS2, UBE2L6


IL1RN
cytokine
7.67E−03
OAS1, OAS2


IFNK
cytokine
2.48E−02
OAS1







UpStream_SP G2










IL13
cytokine
1.37E−09
ALOX5AP, ARG2, CASP6, Ccl6, CTSB, CXCR2, DMXL2, FCGR2A, FLOT1, IL13RA1, MAF, MRC1


IL10
cytokine
1.02E−07
ALOX5AP, ARG2, Ccl6, CCR1, CTSB, CXCL12, FCGR2A, MAF, MERTK, MRC1


IFNG
cytokine
1.43E−07
ALOX5AP, ARG2, CASP6, Ccl6, CCR1, CTSB, CXCL12, FCGR2A, GGT5, GLUL, HLA-





A, IGF1R, MERTK, MRC1, PLA2G7, RAB27A, TGFBR3


IL4
cytokine
5.22E−07
ALOX5AP, ARG2, CASP6, Ccl6, CTNS, FCGR2A, IGF1R, IL13RA1, MAF, MAFB, MERTK, MRC1,





TGFBI


CSF1
cytokine
1.60E−04
FCGR2A, IGF1R, MAFB, MERTK, MRC1


TNF
cytokine
7.42E−04
ADAMTS5, ALOX5AP, CASP6, Ccl6, CCR1, CTSB, CXCL12, CXCR2, HLA-





A, IGF1R, NLRP12, PDGFRA, SLC43A2


OSM
cytokine
1.06E−03
ADAMTS5, CXCL12, GLUL, HLA-A, IL13RA1, MRC1, PYGL


CXCL10
cytokine
2.64E−03
Ccl6, CXCR2


IL3
cytokine
3.40E−03
ALOX5AP, Ccl6, FCGR2A, HLA-A, PLA2G7


IL15
cytokine
5.02E−03
CCR1, CXCL12, GGT5, MAF, PYGL


IL27
cytokine
6.66E−03
HLA-A, MAF, MRC1


IL1B
cytokine
1.37E−02
ACPP, ADAMTS5, CCR1, CTSB, CXCL12, HLA-A, PDGFRA


IL6
cytokine
1.81E−02
ADAMTS5, CCR1, HLA-A, MAF, MERTK, RAB27A


IL17A
cytokine
2.00E−02
CXCL12, MERTK, MRC1


IL33
cytokine
2.44E−02
CXCR2, LEPR


PRL
cytokine
2.69E−02
CPD, CTSB, PDGFRA


IL1A
cytokine
2.69E−02
ADAMTS5, CXCL12, NR2F2


MYDGF
cytokine
2.72E−02
MAF


CXCL1
cytokine
2.72E−02
CXCR2


TNFSF10
cytokine
2.92E−02
HLA-A, IGF1R


PPBP
cytokine
3.31E−02
CXCR2


TNFSF11
cytokine
4.04E−02
CCR1, IL13RA1, MAFB


CXCL2
cytokine
4.78E−02
CXCR2







UpStream_SP G3










TNF
cytokine
2.11E−09
ALOX5, C5AR1, CD44, CEBPD, CH25H, CLEC5A, FOSL2, HMOX1, IER3, IL1R1, MCAM,





MMP9, NLRP12, PTPN12, S100A8, TREM1, TREM3


IFNG
cytokine
4.83E−07
C5AR1, CD44, CEBPD, CH25H, CLEC5A, HMOX1, IER3, IL1R1, MMP9, PLEK, S100A8, TREM1,





TREM3


IL13
cytokine
7.80E−06
CD44, HMOX1, IL1R1, MMP9, S100A8, SLA, SPINT1


CSF2
cytokine
2.29E−05
ALOX5, C5AR1, ID2, IER3, IL1R1, MMP9, TREM1


IL1B
cytokine
2.97E−05
CD44, CEBPD, HMOX1, IER3, IL1R1, MMP9, S100A8, TREM1, TREM3


IL6
cytokine
4.94E−05
C5AR1, CD44, CEBPD, HMOX1, ID2, IL1R1, MMP9, STAP2


IL1A
cytokine
5.74E−05
CD44, HMOX1, MCAM, MMP9, S100A8


IL10
cytokine
5.83E−05
CD44, HMOX1, IL1R1, MMP9, S100A8, TREM1


CD40LG
cytokine
1.03E−04
CD44, FOSL2, ID2, PAWR, PLEK, PTPN12


IL4
cytokine
1.14E−04
ALOX5, C5AR1, CD44, IER3, IL1R1, MMP9, PAWR, S100A8


CCL5
cytokine
3.69E−04
C5AR1, CD44, MMP9


IL19
cytokine
5.04E−04
HMOX1, MMP9


OSM
cytokine
5.17E−04
CEBPD, CH25H, HMOX1, ID2, MMP9, S100A8


IL22
cytokine
7.16E−04
HMOX1, MMP9, S100A8


CX3CL1
cytokine
9.31E−04
HMOX1, MMP9


TNFSF11
cytokine
1.41E−03
CD44, HMOX1, IER3, MMP9


TIMP1
cytokine
1.48E−03
CD44, MMP9


IL21
cytokine
1.54E−03
CD244, ID2, MMP9


SPP1
cytokine
1.82E−03
CD44, HMOX1, MMP9


IL18
cytokine
2.42E−03
CD244, CD44, MMP9


IL2
cytokine
2.80E−03
CD244, CD44, IER3, IL1R1, MMP9


CSF3
cytokine
4.04E−03
CD44, HMOX1, MMP9


IL12B
cytokine
5.07E−03
CEBPD, S100A8


CXCL11
cytokine
5.88E−03
MMP9


IL17A
cytokine
6.00E−03
CEBPD, MMP9, S100A8


IL17B
cytokine
7.84E−03
MMP9


CXCL9
cytokine
7.84E−03
MMP9


MIF
cytokine
8.38E−03
CD44, MMP9


CXCL8
cytokine
8.85E−03
CD44, MMP9


CXCL6
cytokine
9.79E−03
MMP9


TNFSF12
cytokine
1.22E−02
MMP9, S100A8


CCL18
cytokine
1.37E−02
MMP9


IL5
cytokine
1.49E−02
FOSL2, IER3, MMP9


IFNB1
cytokine
1.52E−02
CH25H, IL1R1, MMP9


Ccl6
cytokine
1.76E−02
MMP9


IL20
cytokine
1.95E−02
MMP9


C70
cytokine
2.14E−02
CD44


TNFSF14
cytokine
3.29E−02
MMP9


IL15
cytokine
3.41E−02
CD244, CD44, PLEK


NAMPT
cytokine
4.24E−02
MMP9


FAM3B
cytokine
4.43E−02
IER3


CXCL12
cytokine
4.69E−02
CD44, MMP9


CXCL10
cytokine
4.80E−02
MMP9


TNFSF15
cytokine
4.80E−02
MMP9







UpStream_LV G1










IFNB1
cytokine
1.85E−24
DDX58, DHX58, EIF2AK2, GBP7, IFI16, IFIH1, IFIT1B, IFIT3, Igtp, IRF7, IRF9, Irgm1, Oas1d (includes





others), PML, RSAD2, STAT1, Trim30a/Trim30d, UBA7, XAF1


IFNA2
cytokine
2.29E−17
DDX58, EIF2AK2, IFI16, IFIH1, IFIT3, IRF7, IRF9, PARP9, PML, RSAD2, STAT1, TDRD7, UBA7, XAF1


IFNL1
cytokine
5.24E−14
DDX58, EIF2AK2, IFIH1, IFIT3, IRF9, PML, RSAD2, STAT1, TDRD7


IFNG
cytokine
4.47E−09
CCND2, DDX58, EIF2AK2, GBP7, IFI16, IFIH1, IFIT3, Igtp, Iigp1, IRF7, IRF9, Irgm1, PARP9, PML,





RSAD2, STAT1


IL1RN
cytokine
7.99E−09
DDX58, IFIH1, IFIT3, IRF7, IRF9, PML, RSAD2


IFNA1/
cytokine
1.30E−07
DHX58, EIF2AK2, IFIH1, RSAD2, STAT1


IFNA13


IL4
cytokine
6.62E−07
ASCC3, CCND2, GBP7, IFI16, IFIT1B, IFIT3, Iigp1, IRF7, SMG7, STAT1, Trim30a/Trim30d


IFNK
cytokine
1.68E−05
EIF2AK2, IFIH1, STAT1


TNF
cytokine
1.96E−05
CCND2, CD47, DDX58, EIF2AK2, IFI16, IFIH1, IFIT18, IFIT3, IRF7, LAMA3, PML, 5TAT1, TDRD7


TNFSF10
cytokine
3.92E−05
EIF2AK2, IFI16, IRF9, STAT1


CD40LG
cytokine
1.88E−04
ASCC3, EIF2AK2, IFIT3, PML, SMG7, STAT1


IFNA17
cytokine
4.18E−04
EIF2AK2, IFIH1


CSF1
cytokine
4.84E−04
CCND2, Iigp1, IRF7, STAT1


IL1B
cytokine
2.13E−03
IfI16, IFIT1B, IFIT3, IRF7, LAMA3, RSAD2, STAT1


IFNE
cytokine
2.81E−03
IFIH1, STAT1


EBI3
cytokine
4.36E−03
CCND2, STAT1


IL12A
cytokine
6.22E−03
IRF9, STAT1


IL12B
cytokine
6.22E−03
IRF9, STAT1


IL10
cytokine
6.97E−03
CCND2, Igtp, Iigp1, STAT1


IL5
cytokine
1.97E−02
CCND2, GBP7, IRF7


WNT5A
cytokine
2.25E−02
LAMA3, STAT1


OSM
cytokine
2.68E−02
CCND2, IRF7, IRF9, STAT1


IFNA10
cytokine
3.02E−02
IFIH1


IFNA21
cytokine
3.02E−02
IFIH1


IFNA5
cytokine
3.02E−02
IFIH1


IFNA7
cytokine
3.02E−02
IFIH1


IFNA14
cytokine
3.02E−02
IFIH1


IFNA6
cytokine
3.02E−02
IFIH1


IL27
cytokine
3.05E−02
CCND2, STAT1


IFNA8
cytokine
3.23E−02
IFIH1


IFNA16
cytokine
3.23E−02
IFIH1


IFNW1
cytokine
3.86E−02
IFIH1







UpStream_LV G2










IL6
cytokine
1.41E−06
ACOX1, CD163, CXCL2, EGFR, Hamp/Hamp2, ID2, IGFBP3, IL1R1, LRG1, NR1I2, PPRC1, SAA1,





SERPINA3, SLC39A14, STAT3, THPO


OSM
cytokine
3.69E−05
ACOT2, CXCL2, FMO5, Hamp/Hamp2, ID2, MGLL, NEDD4L, PTP4A1, QSOX1, SAA1, SERPINA3,





STAT3


FAM3B
cytokine
2.78E−04
GJA1, IGFBP3, LDHA


TNF
cytokine
3.73E−04
ACOX1, BACE1, CASP8, CD163, CXCL2, EGFR, GJA1, GPD2, Hamp/Hamp2, IGFBP3, IL1R1, LDHA,





LRG1, MFSD2A, NLRP12, NR1I2, PLIN2, SAA1, SERPINA3, ST3GAL5


IL13
cytokine
1.23E−03
ACOX1, CASP8, CD163, CXCL2, EGFR, IL1R1, PLXNC1, QSOX1


WNT5A
cytokine
2.98E−03
CXCL2, EGFR, ID2, STAT3


SPP1
cytokine
5.07E−03
CXCL2, EGFR, FOXRED1, POR


IL1A
cytokine
6.72E−03
CXCL2, Hamp/Hamp2, LDHA, SAA1, 5ERPINA3


IL1B
cytokine
7.23E−03
CXCL2, GJA1, Hamp/Hamp2, IGFBP3, IL1R1, LDHA, PTP4A1, SAA1, SAA2-





SAA4, SERPINA3, STAT3


CCL5
cytokine
7.40E−03
CD163, CXCL2, STAT3


CNTF
cytokine
1.06E−02
GJA1, SERPINA3, STAT3


IL22
cytokine
1.37E−02
CXCL2, SAA1, SERPINA3


IFNB1
cytokine
1.67E−02
CASP8, CXCL2, Hamp/Hamp2, IL1R1, SLC39A14


IL17A
cytokine
2.14E−02
CD163, CXCL2, HSPB8, STAT3


CCL22
cytokine
2.22E−02
CXCL2


IL21
cytokine
2.74E−02
ID2, STAT3, TIRAP


PRL
cytokine
3.09E−02
EGFR, ID2, IGFBP3, SERPINA3


IL10
cytokine
4.60E−02
CASP8, CD163, CXCL2, IL1R1, STAT3







UpStream_LV G3


UpStream_LV G3










IL15
cytokine
5.58E−05
CD55, CORO1A, ICAM2, KIT, PLEK, TXNRD1


TNF
cytokine
4.74E−04
ABCC1, CD55, CLEC4E, ICAM2, ITGB2, KIT, TRAF2, TRAF3, TREM3, TXNRD1


IL4
cytokine
3.01E−03
CD55, ITGB2, KIT, LILRB4, SIRPA, TRAF3


IFNG
cytokine
7.95E−03
CD55, CLEC4E, CORO1A, ITGB2, PLEK, TRAF2, TREM3


CCL18
cytokine
1.31E−02
S100A4


112
cytokine
1.38E−02
ABCC1, KIT, S100A4, TRAF2


C5
cytokine
1.79E−02
CD55, ITGB2


CCL1
cytokine
1.86E−02
ITGB2


SCGB1A1
cytokine
2.96E−02
ITGB2


CD40LG
cytokine
3.57E−02
PLEK, TRAF2, TRAF3


CSF3
cytokine
3.76E−02
ITGB2, KIT


IL6
cytokine
3.89E−02
ABCC1, CLEC4E, KIT, TRAF3







UpStream_LV G4










IL1B
cytokine
3.41E−16
A2M, ACPP, APC5, ARL6IP5, ATF3, ATP2A2, BTG2, CASP4, CCL17, CCL22, CCR1, CD14, CD44,





CD55, CDKN1A, CEBPD, CEBPG, CP, CREM, CRP, CSF2RB, CTGF, Cxcl9, CYTIP, DAB2, DR1, EFNA1,





EIF4E, ERRFI1, F13A1, FABP5, FGB, FKBP5, FN1, FPR2, GADD45B, GA56, GNL1, GRN, GTF2F1,





HIF1A, HK2, HLA-A, HNF4A, IFI16, IFRD1, IL1A, IL1R1, IL1RN, IRAK2, IRG1, ITGAM, ITGB3,





JUN, LBP, LCN2, LDHA, MMP13, MMP8, MMP9, Mt1, Mt2, NAMPT, NFKBIB, NFKBIZ, ODC1, PFKP,





PIM3, PLAA, PLD1, PSEN1, PTP4A1, RAC2, RAN, S100A10, S100A8, S100A9, Saa3, SCLY, SDC4,





SERPINA3, Sf1, SLC10A2, SLC11A2, SLC20A1, SOCS3, SOD2, SOX9, SYVN1, TBP, TGM2, TIMP1,





TIMP3, TNFAIP6, TNFRSF1B, TREM3, UAP1, VASP, VEGFC


TNF
cytokine
2.76E−14
ABCA1, ABCC1, ADAM17, ADORA1, AIMP1, APCS, ARF4, ARL6IP5, ATF3, ATP2A2, B4GALNT1,





B4GALT1, BMPER, BTG2, CALR, CASP4, CCL17, CCL19, CCL22, CCR1, CD14, CD28, CD38, CD44,





CD55, CDKN1A, CEBPD, CEBPG, CP, CREB3, CREM, CRP, CSF2RB, CTGF, Cxcl9, CYP1B1, CYP7B1,





CYTIP, DUSP2, EFNA1, FABP4, FABP5, FCER1G, FN1, FPR1, FPR2, GADD45B, GADD45G, GLS,





GNAI2, GNL1, GOSR2, GRN, HDC, HIF1A, HIPK3, HK2, HLA-A, HNF4A, ICAM2, IDE, IFI16,





IL1A, IL1R1, IL1RN, IL4R, IRAK2, IRF8, IRG1, ITGAL, ITGAM, ITGB2, ITGB3,





ITPR1, JUN, KLF6, KLK3, LBP, LCN2, LDHA, LITAF, LRG1, LSS, LYVE1, MFSD2A, MMP13, MMP8,





MMP9, MSR1, Mt1, Mt2, MVP, MYH9, NAMPT, NCF1, NFKBIB, NFKBIZ, NLRP12, NNMT, NPM3,





NUCB2, NUP98, ODC1, PC, PDIA4, PIM3, PLAA, PLAUR, PRDM1, PSEN1, PYCARD, RAPGEF5,





RCAN2, S100A8, S100A9, Saa3, SDC4, SEC228, SELP, SERPINA3, Sf1, SLC11A2, SLC20A1,





SOCS3, SOD2, SOX9, SQLE, ST3GAL3, ST3GAL5, ST6GAL1, STEAP4, SYNPO, SYVN1, TGM2,





TIFA, TIMP1, TIMP3, TNFAIP6, TNFRSF1A, TNFRSF1B, TPP2, TREM3, VASP, VEGFC, ZNF330


IFNG
cytokine
3.79E−13
A2M, ABCA1, ADORA1, ALDH1L1, ARG2, ARL6IP5, ATF3, ATP2A2, CASP4, CCL17, CCL19, CCL22,





CCR1, CD14, CD44, CD55, CDKN1A, CEBPD, CERS6, CORO1A, CP, CREM, CSF2RB, CTGF, Cxcl9,





CYB561, DRG1, EDNRA, EGF, FABP5, FCER1G, FCGR2A, FKBP5, FN1, FPR2, GAS6, GLS, GNAI2,





GNL1, HCK, HIF1A, HK2, HLA-A, H5P90AA1, IDE, IDI1, IFI16,





IFITM2, Iigp1, IL12RB1, IL17RA, IL1A, IL1R1, IL1RN, IL4R, IRAK2, IRF8,





IRG1, ITGAL, ITGAM, ITGB2, ITGB3, ITPR1, JUN, KARS, KLF6, LCN2, LCP2, MED15, METAP2,





MMP13, MMP9, MSR1, Mt1, MYH9, NAMPT, NEURL3, NFKBIB, NFKBIZ, NUP98, ODC1, PIM3,





PLAUR, PLD1, PLEK, PRDM1, PTPN1, RAB12, RAC2, S100A10, S100A8, S100A9, Saa3, SBNO2,





SCLY, SDC4, SELP, SERP1, Serpina3g (includes others), SLC11A1,





SLC11A2, SOCS3, SOD2, SQLE, SREBF2, SRP54, TIMP1, TIMP3, TLR1, TNFAIP6, TNFRSF12A,





TNFRSF1A, TNFRSF1B, TP63, TREM3, TRIB2, VEGFC, WARS, Wfdc17, ZBTB16, ZKSCAN1


IL6
cytokine
6.89E−11
A2M, ABCA1, ABCC1, APCS, ATF3, ATP2A2, BTG2, CCR1, CD14, CD44, CD48, CD97, CDKN1A,





CEBPD, Chil3/Chil4, CP, CRP, CSF2RB, CTGF, Cxcl9, CYP1B1, CYTIP, FGB, FGL1, FN1,





FPR2, GADD45B, GADD45G, GALE, HIF1A, HLA-A, HSPA5, IFI16, IK,





IL1R1, IL1RN, IL4R, IL6ST, ITGAM, ITGB3, ITPR1, JUN, KLK3, KRT18, LBP, LCN2,





LRG1, MMP13, MMP8, MMP9, MSR1, Mt1, Mt2, NAMPT, NUCB2, Orm1 (includes





others), S100A9, Saa3, SBNO2, SENP2, SERPINA3, SLC39A14, SNX10, SOCS3, SOD2, STAP2,





TGM2, TIMP1, TLR1, TNFRSF1A, TNFRSF1B, TRAF7, VASP, WARS, ZW10


IL13
cytokine
4.30E−10
ABCA1, ADORA1, ARG2, ATF3, BZW2, CCL17, CCL22, CD14, CD44, CD48, Chil3/Chil4, CLEC4A,





CTGF, EZR, F13A1, FABP4, FCGR2A, FLOT1, FPR2, GAS6, GNAI2, HDC, IARS, IL13RA1, IL1R1,





IL1RN, KLF6, LILRB3, MMP13, MMP9, MSMO1, PAPSS1, PCM1, PDGFC, PFKP, QSOX1, S100A8,





SELP, SEPT11, SLA, SLC16A6, SLC43A3, SOCS3, TGM2, TIMP1, TLR1, TNFRSF1A,





TNFRSF1B, TRPS1


IL5
cytokine
7.49E−10
A2M, ASNS, CASP4, CCL22, CCR1, CD55, CDKN1A, CKAP4, CRELD2, CSF2RB, DDX21, DNTTIP2,





ELL2, ERO1L, GADD45G, HIF1A, HSP90B1, HSPA5, IDI1, IL4R, ITGAM, LCP2, MANF, MMP9,





NABP1, PDIA6, PFKP, PKM, PRDM1, QSOX1, RBM3, RPN1, SDF2L1, Serpina3g (includes





others), SNAP23, SPCS2, SUPT6H, TLR1, ZNF25


OSM
cytokine
8.85E−10
A2M, ABCA1, ABCC1, ADAM17, ASNS, BRD8, CASP4, CDA, CDKN1A, CEBPD, CRP, CYP1B1,





DNAJC3, EXOSC10, FGB, FN1, GADD45G, GFPT1, HIF1A, HIPK3, HK2, HLA-A, HSPA5,





IL13RA1, IL4R, IL6ST, ITGAL, JMJD1C, JUN, KIF5B, LBP, LCN2, LITAF, MMP13, MMP8,





MMP9, MOAP1, NAMPT, NEDD4L, NOMO1 (includes others), PFKFB2,





PGGT1B, PRDM1, PTP4A1, QSOX1, S100A8, S100A9, SEL1L, SELP, SERPINA3, SLC16A6,





SLC39A7, SOCS3, SON, TAT, TIMP1, TIMP3, UAP1, ZNF266, ZNF330


IL4
cytokine
1.27E−09
ACOT9, ALOX12, ARG2, B4GALNT1, CAPG, CCDC86, CCL17, CCL22, CCT3, CD14, CD38, CD44,





CD55, CDKN1A, Chil3/Chil4, CSF2RB, CTGF, Ctla2a/Ctla2b, Cxcl9, EBNA1BP2, F13A1, FABP4,





FCER1G, FCGR2A, FKBP5, FN1, FPR2, GADD45G, G2MA, HCK, HK2, IDE, IFI16, Iigp1, IL12RB1,





IL13RA1, IL1A, IL1R1, IL1RN, IL4R, IL6ST, IRF8, ITGAL, ITGB2, ITGB3, JUN, KLF6, KLK3,





LGALSL, LIG3, MARCO, MMP9, MSR1, NABP1, NAP1L1, PFKP, PLD1, PNP, PRDM1, PRMT1, RNPS1,





S100A10, S100A8, S100A9, Saa3, SDF2L1, SELP, Serpina3g (includes others), Snrpa (includes





others), SOCS3, SRM, ST6GAL1, SYK, SYT11, TGM2, TIMP1, TIMP3, TXLNA


TNFSF11
cytokine
1.71E−09
CCR1, CD14, CD44, CDKN1A, CLOCK, DAB2, DOK2, FGR, FPR1, GADD45B, HCK, IL13RA1, IL1A,





IL1RN, IRF8, IRG1, ITGAL, ITGAM, ITGB3, JUN, MMP9, NFKBIB, NFKBIZ, NME6, PLAUR, PLD1,





PRDM1, Saa3, SDC4, SEL1L, SENP2, SLC11A2, SLC20A1, SMARCAD1, SOCS3, SOD2, TNFRSF1B


IL3
cytokine
1.93E−09
BTG2, CALR, CAMKK2, CCL19, CD14, CD48, CD97, CDKN1A, CEBPG, CEBPZ, CREM, CSNK1A1,





DOK2, FCGR2A, GADD45B, GADD45G, GZMA, HK2, HLA-A, HNF4A,





HNRNPA2B1, HSP90B1, HSPA5, IK, ITGAM, ITGB3, JUN, LCN2, Mt1, NARS, NCF1, NIFK,





ODC1, RAN, RANBP1, RARS, RBM3, SERPINA3, Serpina3g (includes





others), SLC2A3, SOCS3, STT3A, TPD52, VASP


IL10
cytokine
2.24E−07
APCS, ARG2, CCL17, CCL19, CCL22, CCR1, CD14, CD28, CD44, CD55, CDKN1A, CR1L, CSF2RB,





Cxcl9, FCER1G, FCGR2A, FKBP5, FPR1, GZMA, Iigp1, IL17RA, IL1A, IL1R1, IL1RN, IL4R,





IL6ST, ITGAL, JUN, MMP8, MMP9, MSR1, PRDM1, PTPN1, PTPN2, REG3A, S100A8, SNAP23, SOCS3,





TIMP1, TNFRSF1A, TNFRSF1B


EDN1
cytokine
2.89E−07
ATF6, ATP2A2, CDC25A, CTGF, EDNRA, ERRFI1, EZR, FN1, GRN, HIF1A, HSPA5, ITGAM, ITGB3,





JUN, MMP13, MMP9, MSN, NCF1, ODC1, PDIA6, PLAUR, PRKCE, SOCS3, TGM2, TIMP1, TIMP3,





TPM3, VEGFC


IL17A
cytokine
1.92E−06
CCL17, CCL22, CD14, CEBPD, CRP, CTGF, CYP7B1, HSPB8, IL17RA, IL1A, IL1RN, ITPR1, JUN,





KLF3, LCN2, MMP13, MMP8, MMP9, NFKBIZ, REG3A, S100A8, Saa3, SELP, SOCS3, TIMP1, VEGFC


CTF1
cytokine
6.73E−06
A2M, CEBPD, CRP, IL6ST, LBP, REG3A, SERPINA3, SOCS3, TIMP1


CSF1
cytokine
7.91E−06
CD97, CDKN1A, DOK2, FCER1G, FCGR2A, FN1, HSP90B1, HSPA5, Iigp1, IL1A, IL6ST, ITGAM,





ITGB3, JUN, MAP3K3, MARCO, MMP9, MSR1, STX12, STX6, TLR1, TNFRSF1A, TNFRSF1B


IL2
cytokine
1.45E−05
ABCC1, CD244, CD28, CD38, CD44, CDC25A, CDKN1A, CSF2RB, CTPS1, Cxcl9, CYTIP, DAP,





DDX21, DUSP2, FCER1G, GADD45B, GADD456, GZMA, HCK, HK2, HSP90B1, HSPA5, IDI1, IL12RB1,





IL1A, IL1R1, IL4R, JUN, KLF6, LCP2, MMP9, NIFK, PDGFC, PDIA3, PNP, POLR3K, PRDM1,





RGPD4 (includes others), SLC2A3, SNAP23, SOCS3, STK17B, TIMP1, TNFRSF12A, TNFRSF1A,





TNFRSF1B, TRIB2


CSF3
cytokine
1.71E−05
CAPG, CD14, CD44, FGB, FPR1, GADD45B, GADD45G, HDC, IK, IL1RN, ITGAM, ITGB2, JUN, LBP,





LCN2, MMP8, MMP9, ODC1, PRKCE, SOCS3, TNFRSF1A, TNFRSF1B


IL11
cytokine
2.49E−05
A2M, CEBPD, CRP, EZR, FABP4, IL6ST, LBP, MMP13, SERPINA3, SOCS3, TIMP1


CCL5
cytokine
3.77E−05
CCR1, CD44, CD97, CYP1B1, EGF, HDC, MMP9, NAMPT, PLAUR, PLEC, PNP, SQLE, VASP


CSF2
cytokine
3.81E−05
ABCA1, CASC3, CCL17, CCR1, CD14, CD28, CD38, CD97, CDKN1A, CSF2RB, Cxcl9, DOK2, FPR2,





HDC, IK, IL1A, IL1R1, IL1RN, ITGAM, ITGB3, MARCO, MFSD2A, MMP9, ODC1, PIM3, PSEN1,





QSOX1, RBM3, RE63A, RINT1, Saa3, SLC11A2, SLC30A7, SNAP23, SOCS3, SOD2, TGM2, TIFA,





TLR1, TNFRSF1A, TNFRSF1B, ZNF25


CD40LG
cytokine
4.12E−05
ADAM17, AKAP2, ATF3, CCL17, CCL13, CCL22, CCR1, CD38, CD44, CDKN1A, Chil3/Chil4, CTGF,





CYP1B1, CYTIP, DAPP1, DUSP2, HIF1A, HK2, HSP90AA1, IL12RB1, IL13RA1, IL1A, ITGAM, JUN,





LIG3, NAMPT, NAP1L1, NFKBIB, PLAUR, PLEK, PRDM1, PTPN1, SELP, SERPINA10, SOD2, TANK,





TGM2, TNFAIP6


LIF
cytokine
1.03E−04
A2M, CDKN1A, CEBPD, CRP, FGB, FN1, GATA6, HIF1A, HK2, IFI16, IL6ST, JUN, LBP, MMP13,





PIM3, PLD1, PTPN1, REG3A, SERPINA3, SNAP23, SOCS3, SOD2, TIMP1


CX3CL1
cytokine
6.88E−04
HIF1A, IL1A, MMP9, MSR1, SELP, TIMP1


TNFSF10
cytokine
1.12E−03
CD14, HLA-A, IFI16, IFITM2, IL1RN, ITGAM, JUN, MMP9, PRKCE, TIMP1, Tnfrsf22/Tnfrsf23,





TP63


PRL
cytokine
1.77E−03
A2M, CDKN1A, CEBPD, EGF, FN1, HERPUD1, HIPK3, IDE, JUN, LYVE1, MSN, ODC1, PC, PDIA4,





PIM3, SERPINA3, SOCS3, TIMP1, Tmsb4x (includes others), TP63, TPM3


TIMP1
cytokine
2.51E−03
CD38, CD44, CDKN1A, MMP9, PLAUR, TIMP3


IL22
cytokine
4.25E−03
CCL17, CCL22, IL1A, LBP, LCN2, MMP9, REG3A, S100A8, S100A9, SERPINA3, SOCS3


FAM3B
cytokine
4.59E−03
CASP4, CDKN1A, FN1, LCN2, LDHA


LTA
cytokine
5.34E−03
APCS, CCL19, CRP, HDC, ITGB3, LYVE1, ODC1


CNTF
cytokine
6.31E−03
A2M, CEBPD, CRP, IL1A, IL6ST, LBP, REG3A, SERPINA3, SOCS3, TIMP1


II3
cytokine
6.54E−03
CD14, CDKN1A, ITGAM, LCN2, PRKCE, SNAP23, SRP9, TNFRSF1A, TNFRSF1B


IL15
cytokine
7.11E−03
CCL17, CCL19, CCR1, CD244, CD28, CD38, CD44, CD55, CDKN1A, COPB2, CORO1A, CTGF, GNL2,





HNRNPA2B1, ICAM2, IL12RB1, JUN, PDIA3, PDIA4, PFDN4, PLD1, PLEK, PNP, PTP4A1, RAC2,





SYNPO, TIMP1, TNFRSF1A, VBP1


IL1A
cytokine
8.35E−03
CD44, CDKN1A, HDC, IK, IL1A, IL1RN, ITGB3, JUN, LCN2, LDHA, MMP13, MMP9, NFKBIZ,





S100A8, S100A9, Saa3, SERPINA3, SOD2, VEGFC


CCL2
cytokine
8.62E−03
ABCA1, HDC, IL1A, ITGAL, LCN2, MMP9, NCF1, SLC11A1, TIMP1


TNFSF12
cytokine
8.82E−03
CCL19, CCR1, CLEC40, ITGAM, MMP13, MMP9, S100A8, S100A9, TIMP1, TNFRSF12A


CXCL12
cytokine
1.35E−02
CD44, FN1, HIPK3, IL1A, ITGAL, ITGB3, JMJD1C, JUN, LCP2, MMP13, MMP9, RPS6KA3, SOCS3,





TDG, TNFRSF1B, ZBTB16


IL17B
cytokine
1.36E−02
CDKN1A, MMP9


WNT5A
cytokine
1.53E−02
CD14, CD38, DAB2, DOK2, FABP4, FN1, IL1A, IRF8, MMP13, SOCS3, TLR1


CXCL5
cytokine
3.18E−02
ITGAL, ITGAM


IL17C
cytokine
3.38E−02
IL1A, LCN2, PRDM1, S100A8, S100A9


IL18
cytokine
3.63E−02
CD244, CD44, GADD45B, GADD45G, IL12RB1, IL1A, ITGAM, JUN, MMP13, MMP9, NFKBIZ, TIMP1


C5
cytokine
4.24E−02
APCS, ATF3, CD55, CRP, FCGR2A, IL1A, ITGAM, ITGB2, ST6GAL1, VEGFC


IL12B
cytokine
4.86E−02
CEBPD, IL1A, PIK3AP1, S100A8, S100A9, SOCS3


Ctf2
cytokine
4.92E−02
SOCS3









As expected, the G1 adjuvants were characterized by interferon responses. G2 to G6 adjuvants were associated with inflammatory cytokines such as IL6 and TNF. G2 adjuvants had weaker associations with lipid metabolism, and IPA analysis suggested oncostatin M and IL10 association. It was difficult to retrieve preferential associations for the G3-G5 adjuvants because these groups contained a limited number of adjuvants and organs. Although conducted under limited conditions, the analysis suggested that the G3 adjuvants might be associated with T and NK cell cytokines such as IL2, IL4 and IL15. G4 adjuvants had broader profiles than the other adjuvants and contained many cytokines. However, the preferential M32SP module association suggested that G4 might be characterized by TNF responses (FIG. 6B). G5 adjuvants were associated with phosphate-containing compounds with metabolic processes suggestive of nucleotide metabolic processes, a finding consistent with G5 being a CpG nucleic acid adjuvant group. G6 adjuvants such as AddaVax, an MF59 equivalent oil adjuvant, were associated with the phagosome, and IL1A and IL33 were suggested as the cytokines for them by IPA upstream cytokine analysis.


(Adjuvant Group Analysis Predicts the Modes of Action of ADX, bCD and ENDCN)


The G1 adjuvants (cdiGMP, cGAMP, DMXAA, PolyIC, and R848), which were RNA-related adjuvants or STING ligands, were strongly associated with type I and type II interferon responses, making them clearly distinct from the G2-G6 adjuvants. G2 to G6 adjuvants appear to be associated with inflammatory responses. G2 includes ALM and bCD, which are both expected to work through DAMPs (Marichal, T. et al., Nat Med 17, 996-1002 (2011) and Onishi, M. et al., Journal of immunology 194, 2673-2682 (2015)). Intriguingly, the strongly responding D35 and K3 samples (D35_ID_x2 and K3_ID_x3 in LV), which were mentioned above as clustering exceptions, are indicated in red in both clustered in G2LV. Hence, it is possible that in certain situations D35 and K3 can mimic the DNA released from damaged cells to induce host immune responses (Marichal, T. et al., Nat Med 17, 996-1002 (2011) and Onishi, M. et al., Journal of immunology 194, 2673-2682 (2015)). G3 and G4 consisted mainly of adjuvants derived from bacterial cell wall-derived PAMPs. G5 consisted of CpG adjuvants. Although G5 adjuvants such as D35, K3, and K3SPG are recognized as good interferon inducers in vitro, the analysis suggested that the CpG adjuvants differ biologically from the typical G1-type TLR and RLR ligands in vivo. G6 consisted of AddaVax, a MF59 equivalent. MF59's mechanism of action has been extensively examined. It was suggested that ATP (another DAMP) released from muscle acts as a mediator of the adjuvant effect (Vono, M. et al., Proceedings of the National Academy of Sciences of the United States of America 110, 21095-21100 (2013)). The adjuvant clustering results may support this interpretation, as G2 and G6 formed related but separate clusters in the samples from LNs.


Taken together, the resulting data strongly suggest that the adjuvants investigated were grouped according to shared similarities in their modes of action. Hence, it is possible to predict the mode of the action of a new adjuvant with knowledge about its grouping. To verify this hypothesis, two adjuvants were selected for testing. ENDCN is a novel lipid-based nasal adjuvant, currently in phase I/II clinical studies as part of an influenza vaccine (Falkeborn, T. et al., PloS one 8, e70527 (2013) and Maltais, A. K. et al., Vaccine 32, 3307-3315 (2014)). ENDCN was categorized in G2, the same group as bCD in both LV and SP. It has been previously showed that the adjuvanticity of bCD is likely to be associated with host cell-derived dsDNA via DAMPs (Onishi, M. et al., Journal of immunology 194, 2673-2682 (2015)). G2 categorization of ENDCN strongly suggested that ENDCN utilizes host-derived factors for its adjuvanticity. This hypothesis was further tested, and detailed immunological analyses of ENDCN in vivo have revealed that host cell releasing RNA and TBK1 are involved in the adjuvanticity (Hayashi, M. et al., Scientific Reports, 6, Article number: 29165 (2016)), further supporting ENDCN as a G2 adjuvant. Another example is ADX (Honda-Okubo, Y. et al., Vaccine 30, 5373-5381 (2012) and Saade, F. et al., Vaccine 31, 1999-2007 (2013)), which is an inulin-based particulate adjuvant with an unknown mode of action. G3LV/G4SP but not G2 clustering suggests that ADX works through unidentified PAMPs receptors, instead of DAMPs mediators. More detailed analysis of ADX is currently on going (Hayashi et al. Scientific Reports 6, Article number: 29165 (2016)).


(Adjuvant Gene Space Analysis Reveals Differences Among the Same Receptor-Targeted Adjuvants)


D35, K3 and K3SPG all act on TLR9, and cdiGMP, cGAMP, and the DMXAA target STING (Gao, P. et al., Cell 154, 748-762(2013)). Correspondingly, these adjuvants clustered in the same adjuvant group by the method of the invention. All TLR9 ligands were clustered in G5, and all STING ligands were clustered in G1. D35 and K3 have been known to induce overlapping but substantially different biological responses (Steinhagen, F. et al., Journal of Leukocyte Biology 92, 775-785 (2012)). With STING ligands, no direct comparison has been reported yet, but cdiGMP appears to induce Th1 responses (Madhun, A. S. et al., Vaccine 29, 4973-4982 (2011)), whereas DMXAA induces Th2 responses (Tang, C. K. et al., PloS one 8, e60038(2013)). Although the three STING ligands (cdiGMP, cGAMP, and DMXAA) share similar chemical structures, each of them is derived from bacteria, host cells, and synthetic chemicals, respectively (Gao, P. et al., Cell 154, 748-762 (2013)). Simple Venn analysis of the sDEGs from CpG (FIG. 7) and STING ligands (FIG. 8) in LNs only confirmed the well-known fact that these adjuvants induce interferon responses, and their individual differences were difficult to retrieve. However, use of z-scores and 40 module mapping (FIG. 6) analysis in the adjuvant gene space enabled identification of more detailed differences among them. D35 was a relatively stronger interferon inducer than K3 or K3SPG in vivo under the experimental conditions (FIGS. 2A and Table 20).
















TABLE 20








Module#
(5)D35.
(5)D35.
(5)D35.
(3)K3.
(3)K3.
(3)K3.


ProbeGenes
(LN)
ID.LN.x2
ID.LN.x1
ID.LN.x3
ID.LN.x1
ID.LN.x2
ID.LN.x3





1421009_at Rsad2
37
−0.05
1.735
1.521
−0.63
−0.36
0.231


1436058_at Rsad2
37
0.104
1.354
1.822
−0.57
−0.44
0.303


1421008_at Rsad2
37
−0.14
1.446
1.916
−0.83
−0.44
−0.15


1418293_at Ifit2
40
−0.13
1.614
1.531
−0.65
−0.28
0.445


1419603_at Ifi204
40
1.185
1.269
1.205
−1.05
−0.47
0.239


1423555_a_at Ifi44
40
1.105
0.679
1.768
−0.79
−0.51
−0.13


1453196_a_at Oasl2
40
1.943
0.714
0.467
−1.26
−0.27
0.262


1440371_at ---
8
1.485
1.31
0.495
−0.52
−0.37
−0.36


1418580_at Rtp4
40
0.809
1.609
0.611
−1.12
0.119
−0


1460069_at Smc6
7
0.716
1.173
1.385
−1.3
−1.14
−1.08


1418191_at Usp18
40
0.39
0.966
1.679
−0.63
0.534
0.071


1424339_at Oasl1
37
0.62
1.742
0.836
−0.81
0.112
0.196


1457035_at Al607873
40
0.908
1.231
1.239
−0.55
0.616
−0.32


1452349_x_at
40
0.51
1.197
1.416
−1.27
−0.1
−0.11


Ifi205///Mnda


1422006_at Eif2ak2
40
1.174
1.037
1.068
−1.75
0.425
−0.62


1450403_at Stat2
40
2.023
0.499
0.867
−0.99
0.138
−0.21


1440475_at AW011738
36
0.863
0.622
1.554
−1.4
−0.73
−0.23


1443302_at ---
12
1.287
0.399
1.721
−1.23
−0.42
−1.13


1422141_s_at


Csprs///Gm15433///
40
1.776
0.507
0.791
−0.17
−0.74
−0.45


Gm2666///Gm7609///


LOC100041903///


LOC100503923


1453757_at Herc6
40
0.97
1.805
0.409
−0.43
0.015
0.213


1421911_at Stat2
40
0.612
1.757
0.768
−0.53
−0.24
0.338


1441344_at Erlin1
21
1.36
0.052
1.758
−1.03
−0.98
−0.61


1431095_a_at Herc6
40
1.937
0.426
0.702
−0.94
0.033
0.178


1450377_at Thbs1
28
1.225
0.185
1.593
−0.92
−0.96
−0.98


1456164_at AW011738
40
1.139
0.14
2.068
−0.91
−0.18
−0.99


1425105_at Rbp3
10
1.968
0.568
0.796
−1.04
−0.86
−0.21


1432417_a_at Tspan2
40
1.133
1.404
1.369
−0.99
−0.72
−0.43


1442518_at
14
0.953
1.161
1.432
−0.25
0.036
−1.37


C030044O21Rik


1436472_at Slfn9
37
1.143
1.047
1.405
−0.56
−0.79
−0.65


1419486_at Foxc1
19
1.628
0.676
1.04
−0.77
0.046
−0.03


1443853_x_at Sfmbt1
32
1.228
1.862
0.419
−0.91
−0.9
−0.79


1448179_at Usmg5
24
0.103
1.297
2.062
−0.66
−0.66
−0.7


1451777_at Ddx60
40
0.83
1.215
0.975
−0.5
0.042
0.618


1447241_at ---
37
0.048
2.27
0.715
−0.88
−0.82
−0.89


1416380_at Mov10
37
1.449
0.681
1.147
−1.16
−1.48
0.219


1443392_at Trpv1
33
1.731
0.59
0.863
0.024
0.219
−1.28


1455796_x_at Olfm1
11
0.86
0.592
1.811
−0.89
−0.11
−0.44


1426013_s_at Plekha4
37
0.54
1.48
1.208
−0.2
0.205
−1.53


1416039_x_at Cyr61
39
1.724
0.461
0.83
0.035
−0.72
−0.89


1453299_a_at Pnp///Pnp2
40
1.476
0.696
0.948
−0.96
−0.05
0.23


1424609_a_at
40
1.178
1.294
0.958
−1.21
−1.2
−0.36


Cwc22///Gm4354///Xdh


1424444_a_at
40
0.904
0.235
1.969
−0.22
−0.65
0.015


1600014C10Rik


1439886_at ---
40
0.159
1.149
1.843
−0.46
0.3
−1.03


1424607_a_at Gm4354
40
1.005
1.314
1.103
−0.62
−0.48
0.206


1423611_at Alpl
24
0.707
1.244
1.244
−1.13
−0
0.131


1456346_at ---
9
0.271
1.15
2.034
−0.37
−0.96
−0.81


1420894_at Tgfbr1
3
0.691
0.803
1.839
0.157
−1.17
−1.13


1444615_x_at Runx1t1
1
0.71
1.504
1.304
−0.02
−1.32
−0.77


1458457_at ---
26
0.123
1.673
1.351
−0.58
0.355
−0.11


















(5)K3SPG.
(5)K3SPG.
(5)K3SPG.
D35(z
K3(z
K35PG(z
Prefer-


ProbeGenes
ID.LN.x3
ID.LN.x1
ID.LN.x2
sum)
sum)
sum)
ence





1421009_at Rsad2
−0.57
−0.99
−0.89
3.209
−0.76
−2.45
D35


1436058_at Rsad2
−0.76
−0.93
−0.88
3.281
−0.71
−2.57
D35


1421008_at Rsad2
−0.21
−0.82
−0.77
3.221
−1.42
−1.81
D35


1418293_at Ifit2
−0.5
−1.12
−0.9
3.02
−0.49
−2.53
D35


1419603_at Ifi204
−0.75
−1.18
−0.45
3.659
−1.28
−2.38
D35


1423555_a_at Ifi44
−0.11
−0.64
−1.37
3.551
−1.43
−2.12
D35


1453196_a_at Oasl2
−0.01
−0.75
−1.09
3.123
−1.27
−1.85
D35


1440371_at ---
−0.52
0.197
−1.73
3.29
−1.24
−2.05
D35


1418580_at Rtp4
0.349
−1.48
−0.89
3.028
−1
−2.03
D35


1460069_at Smc6
0.094
0.098
0.059
3.274
−3.53
0.251
D35


1418191_at Usp18
−0.56
−1.45
−0.99
3.035
−0.03
−3.01
D35


1424339_at Oasl1
−0.37
−0.76
−1.57
3.198
−0.5
−2.7
D35


1457035_at Al607873
−1.27
−0.83
−1.02
3.377
−0.26
−3.12
D35


1452349_x_at
0.515
−0.9
−1.25
3.123
−1.48
−1.64
D35


Ifi205///Mnda


1422006_at Eif2ak2
−0.63
−0.09
−0.62
3.279
−1.95
−1.33
D35


1450403_at Stat2
−0.75
−0.62
−0.95
3.389
−1.07
−2.32
D35


1440475_at AW011738
−1.31
0.286
0.341
3.038
−2.36
−0.68
D35


1443302_at ---
−0.11
−0.5
−0.02
3.407
−2.78
−0.63
D35


1422141_s_at


Csprs///Gm15433///
−1.28
0.634
−1.07
3.074
−1.36
−1.72
D35


Gm2666///Gm7609///


LOC100041903///


LOC100503923


1453757_at Herc6
−0.51
−1.01
−1.45
3.183
−0.2
−2.98
D35


1421911_at Stat2
−0.53
−0.44
−1.74
3.137
−0.43
−2.71
D35


1441344_at Erlin1
−0.78
0.047
0.191
3.171
−2.63
−0.54
D35


1431095_a_at Herc6
−0.06
−1.31
−0.97
3.065
−0.73
−2.34
D35


1450377_at Thbs1
0.661
−0.86
0.068
3.002
−2.87
−0.14
D35


1456164_at AW011738
−0.36
−0.55
−0.35
3.347
−2.08
−1.27
D35


1425105_at Rbp3
0.173
−0.31
−1.09
3.333
−2.11
−1.23
D35


1432417_a_at Tspan2
−0.27
−0.74
−0.74
3.906
−2.15
−1.76
D35


1442518_at
−0.52
−0.25
−1.2
3.546
−1.58
−1.97
D35


C030044O21Rik


1436472_at Slfn9
0.326
−0.56
−1.36
3.595
−2
−1.59
D35


1419486_at Foxc1
−1.67
−0.49
−0.43
3.344
−0.76
−2.58
D35


1443853_x_at Sfmbt1
−0.77
−0.03
−0.12
3.51
−2.59
−0.92
D35


1448179_at Usmg5
−0.48
−0.46
−0.5
3.461
−2.02
−1.44
D35


1451777_at Ddx60
−0.61
−1.82
−0.75
3.02
0.163
−3.18
D35


1447241_at ---
−0.03
−0.18
−0.23
3.033
−2.6
−0.44
D35


1416380_at Mov10
−0.65
0.088
−0.28
3.277
−2.43
−0.85
D35


1443392_at Trpv1
−0.68
−1.32
−0.15
3.184
−1.04
−2.15
D35


1455796_x_at Olfm1
−0.46
0.18
−1.55
3.262
−1.44
−1.83
D35


1426013_s_at Plekha4
−0.18
−1.23
−0.3
3.228
−1.53
−1.7
D35


1416039_x_at Cyr61
−0.18
−1.63
0.372
3.015
−1.58
−1.44
D35


1453299_a_at Pnp///Pnp2
0.266
−1.27
−1.33
3.12
−0.78
−2.34
D35


1424609_a_at
−0.96
0.293
0
3.429
−2.76
−0.67
D35


Cwc22///Gm4354///Xdh


1424444_a_at
−0.61
−1.55
−0.09
3.108
−0.85
−2.25
D35


1600014C10Rik


1439886_at ---
−1.2
−0.09
−0.67
3.151
−1.19
−1.96
D35


1424607_a_at Gm4354
−1.72
−0.19
−0.62
3.422
−0.89
−2.53
D35


1423611_at Alpl
0.066
−1.66
−0.61
3.194
−1
−2.2
D35


1456346_at ---
−0.19
−0.78
−0.35
3.455
−2.13
−1.32
D35


1420894_at Tgfbr1
0.079
−0.72
−0.55
3.333
−2.14
−1.19
D35


1444615_x_at Runx1t1
−0.57
−0.93
0.09
3.519
−2.11
−1.4
D35


1458457_at ---
−1.23
−1.04
−0.54
3.147
−0.33
−2.81
D35









K3 preferentially induced RNA biogenesis, which was enriched in M26LN, compared with D35 or K3SPG (FIG. 2A). Similar analysis (FIGS. 2B and Table 21) revealed that cdiGMP was the strongest interferon inducer, and it was associated with preferential gene mapping in M37LN and M40LN more than cGAMP and DMXAA (FIG. 2B).
















TABLE 21









(8)cdiGMP.
(8)cdiGMP.
(8)cdiGMP.
(9)cGAMP.
(9)cGAMP.
(9)cGAMP.


ProbeGenes
Modules
ID.LN.x2
ID.LN.x1
ID.LN.x3
ID.LN.x1
ID.LN.x2
ID.LN.x3





1421578_at Ccl4
37
1.402
1.22
1.368
−0.76
−0.67
−0.72


1457404_at Nfkbiz
37
1.371
1.232
1.386
−0.73
−0.57
−0.63


1442556_at ---
35
1.421
1.315
1.245
−0.74
−0.5
−0.71


1421679_a_at Cdkn1a
37
1.429
1.421
1.13
−0.55
−0.71
−0.72


1455197_at Rnd1
29
1.54
1.27
1.166
−0.77
−0.65
−0.7


1453636_at Pcgf5
36
1.332
1.143
1.496
−0.76
−0.48
−0.62


1425687_at Cflar
36
1.199
1.3
1.469
−0.81
−0.59
−0.81


1441843_s_at
35
1.442
1.354
1.171
−0.71
−0.47
−0.54


5230400M03Rik


1436387_at
29
1.372
1.437
1.158
−0.43
−0.59
−0.71


C330006P03Rik///


Homer1


1423028_at Ifna2
29
1.076
1.31
1.58
−0.68
−0.59
−0.65


1434350_at Csrnp1
37
1.353
1.305
1.308
−0.59
−0.54
−0.85


1459144_at ---
35
1.275
1.199
1.493
−0.62
−0.57
−0.72


1458376_at
35
1.188
1.502
1.273
−0.6
−0.61
−0.64


B930025B16Rik


1453851_a_at Gadd45g
37
1.368
1.383
1.21
−0.46
−0.65
−0.85


1418424_at Tnfaip6
29
1.141
1.631
1.189
−0.66
−0.69
−0.7


1425837_a_at Ccrn4l
29
1.34
1.091
1.529
−0.79
−0.65
−0.67


1421473_at Il1a
34
1.525
1.178
1.257
−0.76
−0.72
−0.76


1422305_at Ifnb1
29
0.997
1.431
1.532
−0.67
−0.66
−0.55


1427736_a_at Ccrl2
36
1.328
1.075
1.554
−0.48
−0.63
−0.62


1448325_at Ppp1r15a
29
1.314
1.203
1.441
−0.74
−0.78
−0.81


1450297_at Il6
29
1.437
0.989
1.53
−0.69
−0.69
−0.69


1451340_at Arid5a
37
1.5
1.292
1.161
−0.57
−0.61
−0.49


1417252_at Ptgs2
34
1.507
1.214
1.232
−0.83
−0.79
−0.79


1419534_at Olr1
29
1.484
1.094
1.375
−0.81
−0.75
−0.79


1439981_at Tespa1
35
1.343
1.427
1.18
−0.76
−0.81
−0.58


1419132_at Tlr2
34
1.576
1.128
1.246
−0.42
−0.66
−0.78


1433508_at Klf6
37
1.271
1.413
1.263
−0.87
−0.64
−0.91


1458589_at ---
37
1.137
1.339
1.469
−0.76
−0.65
−0.95


1417263_at Ptgs2
34
1.51
0.965
1.47
−0.73
−0.73
−0.71


1428027_at
26
1.302
1.534
1.106
−0.51
−0.4
−0.81


LOC100653389


1428735_at Cd69
40
1.22
1.344
1.378
−0.68
−0.43
−0.41


1457944_at ---
35
1.366
1.428
1.146
−0.83
−0.54
−0.41


1439349_at Sbno2
29
1.367
1.407
1.166
−0.45
−0.8
−0.85


1448724_at Cish
37
1.476
1.357
1.106
−0.69
−0.91
−0.79


1419508_at Ripk1
37
1.035
1.437
1.467
−0.54
−0.5
−0.85


1445534_at ---
35
1.345
1.515
1.078
−0.35
−0.71
−0.87


1444010_at Eif4e
29
1.659
1.049
1.227
−0.79
−0.61
−0.6


1424638_at Cdkn1a
37
1.624
1.232
1.074
−0.62
−0.81
−0.78


1444598_at ---
35
1.559
1.318
1.053
−0.84
−0.43
−0.67


1455899_x_at Socs3
29
1.206
1.544
1.18
−0.71
−0.6
−0.83


1439680_at Tnfsf10
37
1.169
1.36
1.399
−0.59
−0.29
−0.78


1421457_a_at Samsn1
29
1.437
1.192
1.298
−0.39
−0.59
−0.9


1457888_at ---
36
1.466
1.08
1.38
−0.81
−0.3
−0.79


1419721_at Niacr1
27
1.493
1.403
1.03
−0.43
−0.74
−0.87


1449311_at Bach1
29
1.178
1.558
1.189
−0.83
−0.72
−0.41


1443414_at C78513
35
1.402
0.963
1.557
−0.7
−0.77
−0.44


1449169_at Has2
36
1.306
1.158
1.458
−0.47
−0.39
−0.81


1422054_a_at 5kil
40
1.491
1.218
1.211
−0.38
−0.83
−1.02


1446245_at ---
35
1.239
1.22
1.46
−0.6
−0.31
−0.67


















(10)DMXAA.
(10)DMXAA.
(10)DMXAA.
cdiGMP(z
cGAMP(z
DMXAA(z
prefer-


ProbeGenes
ID.LN.x2
ID.LN.x1
ID.LN.x3
sum)
sum)
sum)
ence





1421578_at Ccl4
−0.64
−0.64
−0.57
3.99
−2.14
−1.85
cdiGMP


1457404_at Nfkbiz
−0.75
−0.72
−0.59
3.989
−1.92
−2.06
cdiGMP


1442556_at ---
−0.64
−0.59
−0.8
3.981
−1.95
−2.03
cdiGMP


1421679_a_at Cdkn1a
−0.71
−0.62
−0.66
3.98
−1.99
−1.99
cdiGMP


1455197_at Rnd1
−0.66
−0.62
−0.58
3.976
−2.12
−1.86
cdiGMP


1453636_at Pcgf5
−0.69
−0.72
−0.7
3.972
−1.86
−2.11
cdiGMP


1425687_at Cflar
−0.58
−0.48
−0.7
3.968
−2.21
−1.76
cdiGMP


1441843_s_at
−0.84
−0.73
−0.68
3.967
−1.71
−2.25
cdiGMP


5230400M03Rik


1436387_at
−0.79
−0.71
−0.73
3.967
−1.73
−2.24
cdiGMP


C330006P03Rik///


Homer1


1423028_at Ifna2
−0.69
−0.68
−0.67
3.966
−1.92
−2.04
cdiGMP


1434350_at Csrnp1
−0.83
−0.72
−0.45
3.966
−1.97
−1.99
cdiGMP


1459144_at ---
−0.52
−0.89
−0.65
3.966
−1.91
−2.06
cdiGMP


1458376_at
−0.9
−0.72
−0.49
3.963
−1.85
−2.11
cdiGMP


B930025B16Rik


1453851_a_at Gadd45g
−0.84
−0.5
−0.66
3.961
−1.96
−2
cdiGMP


1418424_at Tnfaip6
−0.66
−0.67
−0.58
3.961
−2.05
−1.91
cdiGMP


1425837_a_at Ccrn4l
−0.65
−0.73
−0.47
3.961
−2.11
−1.85
cdiGMP


1421473_at Il1a
−0.7
−0.41
−0.6
3.96
−2.25
−1.71
cdiGMP


1422305_at Ifnb1
−0.66
−0.67
−0.66
3.959
−1.97
−1.99
cdiGMP


1427736_a_at Ccrl2
−0.75
−0.75
−0.72
3.958
−1.74
−2.22
cdiGMP


1448325_at Ppp1r15a
−0.66
−0.64
−0.34
3.957
−2.32
−1.63
cdiGMP


1450297_at Il6
−0.65
−0.64
−0.59
3.956
−2.07
−1.88
cdiGMP


1451340_at Arid5a
−0.84
−0.56
−0.87
3.953
−1.67
−2.28
cdiGMP


1417252_at Ptgs2
−0.57
−0.53
−0.44
3.953
−2.41
−1.55
cdiGMP


1419534_at Olr1
−0.53
−0.46
−0.6
3.953
−2.35
−1.6
cdiGMP


1439981_at Tespa1
−0.38
−0.57
−0.85
3.95
−2.15
−1.8
cdiGMP


1419132_at Tfr2
−0.74
−0.6
−0.75
3.95
−1.86
−2.09
cdiGMP


1433508_at Klf6
−0.54
−0.6
−0.39
3.947
−2.41
−1.54
cdiGMP


1458589_at ---
−0.61
−0.43
−0.55
3.945
−2.36
−1.59
cdiGMP


1417263_at Ptgs2
−0.64
−0.59
−0.53
3.945
−2.18
−1.77
cdiGMP


1428027_at
−0.7
−0.78
−0.74
3.943
−1.72
−2.22
cdiGMP


LOC100653389


1428735_at Cd69
−0.93
−0.66
−0.83
3.941
−1.52
−2.42
cdiGMP


1457944_at ---
−0.95
−0.57
−0.65
3.94
−1.78
−2.16
cdiGMP


1439349_at Sbno2
−0.86
−0.48
−0.49
3.94
−2.1
−1.84
cdiGMP


1448724_at Cish
−0.48
−0.65
−0.42
3.94
−2.38
−1.56
cdiGMP


1419508_at Ripk1
−0.69
−0.51
−0.84
3.938
−1.9
−2.04
cdiGMP


1445534_at ---
−0.61
−0.67
−0.73
3.938
−1.93
−2
cdiGMP


1444010_at Eif4e
−0.78
−0.51
−0.66
3.936
−1.99
−1.94
cdiGMP


1424638_at Cdkn1a
−0.76
−0.46
−0.5
3.93
−2.22
−1.71
cdiGMP


1444598_at ---
−0.49
−0.84
−0.67
3.93
−1.93
−2
cdiGMP


1455899_x_at Socs3
−0.89
−0.34
−0.56
3.93
−2.14
−1.79
cdiGMP


1439680_at Tnfsf10
−0.63
−0.68
−0.97
3.927
−1.65
−2.27
cdiGMP


1421457_a_at Samsn1
−0.87
−0.4
−0.77
3.927
−1.88
−2.04
cdiGMP


1457888_at ---
−0.63
−0.84
−0.54
3.925
−1.91
−2.02
cdiGMP


1419721_at Niacr1
−0.54
−0.49
−0.84
3.925
−2.05
−1.88
cdiGMP


1449311_at Bach1
−0.78
−0.8
−0.39
3.924
−1.95
−1.97
cdiGMP


1443414_at C78513
−0.5
−0.83
−0.69
3.922
−1.91
−2.02
cdiGMP


1449169_at Has2
−0.99
−0.52
−0.75
3.922
−1.67
−2.25
cdiGMP


1422054_a_at 5kil
−0.51
−0.59
−0.59
3.92
−2.23
−1.69
cdiGMP


1446245_at ---
−0.56
−1.04
−0.75
3.919
−1.58
−2.34
cdiGMP









DMXAA preferentially induced RNA processing and gene expression responses more than cdiGMP or cGAMP (FIG. 2B). Analyses indicated that cdiGMP induced more interferon responses in vivo than the other sting ligands. This may be related to the fact that cdiGMP is produced by pathogenic bacteria (Woodward, J. J. et al., Science 328, 1703-1705 (2010)).


(Systemic Gene Responses are Associated with Adjuvant-Induced Hematological Changes)


Many adjuvants affect the numbers of a variety of circulating blood cells. Granulocytosis was the most common event after administrating any of the 21 adjuvants (FIG. 3A), a finding consistent with the fact that neutrophils were the cells that responded most to the adjuvant administration. Monocytosis was the next most common event, and was clearly induced by bCD, cdiGMP, cGAMP, MPLA, and Pam3CSK4 (FIG. 3A). Lymphopenia was less common but strongly induced by FK565, MALP2s, PolyIC, and R848 (FIG. 3A), and these adjuvants also caused leukopenia (FIG. 3A). Interestingly, FK565 caused granulocytosis (as was observed in a previous study (Tanaka, M. et al., Bioscience, Biotechnology, and Biochemistry 57, 1602-1603 (1993))) and lymphopenia at the same time (FIG. 3A). Next, correlations between these hematological parameters and the gene expression changes in the organs were examined using a linear model (see http://sysimg.ifrec.osaka324u.ac.jp/adjvdb/methodologies/hema.html for the scheme). A variety of genes were retrieved for each blood cell type in each organ by this approach (FIG. 3B and Table 22). Based on the correlated gene numbers, LVs exhibited the most hematological changes (FIG. 3B). As a representative example, Cxcl9 upregulation in LVs, which is strongly induced by interferons and has been reported as an influenza vaccine safety marker (Mizukami, T. et al., Vaccine 26, 2270-2283 (2008) and Mizukami, T. et al., PloS one 9, e101835 (2014))) was clearly correlated with leukopenia. Downregulated examples include 117 and S1pr5 in SP. These were also correlated with lymphopenia (FIG. 3B). Contrastingly, no correlation was found for genes involved in monocytosis and granulocytosis (FIG. 3B and Table 22), suggesting that multiple genes are involved in these processes. These results suggest that the number of granulocytes, monocytes, and lymphocytes in the blood is regulated by different mechanisms, and gene changes in the systemic organs may lead to adjuvant-induced leukopenia and lymphopenia, possibly through interferons, 117, and S1P related mechanisms.
















TABLE 22





Probe_set_id
Gene.Symbol
Gene.Title
pos.bd
coef.sig
incline
ttest
org






















1455320_at
Nampt
nicotinamide phosphoribosyitransferase
WBC
−0.9402
−1.1902
0.03066
LV


1433883_at
Tpm4
tropomyosin 4
WBC
−0.8228
−1.1881
0.00937
LV


1452352_at
Ctla2b
cytotoxic T lymphocyte-associated protein 2 beta
WBC
−0.9913
−1.1869
0.01334
LV


1434512_x_at
Srsf3
serine/arginine-rich splicing factor 3
WBC
−0.9445
−1.1848
0.03175
LV


1426324_at
H2-D1 ///
histocompatibility 2, D region locus 1 ///
WBC
−0.8181
−1.1798
0.01491
LV



H2-L
histocompatibility 2, D region locus L


1416927_at
Trp53inp1
transformation related protein 53 inducible
WBC
−0.9651
−1.1756
0.04645
LV




nuclear protein 1


1460197_a_at
Steap4
STEAP family member 4
WBC
−0.9749
−1.1602
0.0008
LV


1415993_at
Sqle
squalene epoxidase
WBC
−0.9363
−1.1598
0.00334
LV


1439448_x_at
Tmed9
transmembrane emp24 protein transport domain
WBC
−0.9289
−1.1579
0.02781
LV




containing 9


1418536_at
H2-Q7 ///
histocompatibility 2, Q region locus 7 ///
WBC
−0.9853
−1.1574
0.04534
LV



H2-Q9
histocompatibility 2, Q region locus 9


1426708_at
Antxr2
anthrax toxin receptor 2
WBC
−0.8664
−1.1562
0.0269
LV


1428070_at
Syvn1
synovial apoptosis inhibitor 1, synoviolin
WBC
−0.9785
−1.1542
0.04024
LV


1416234_at
Lrrc59
leucine rich repeat containing 59
WBC
−0.9545
−1.1527
0.02457
LV


1449111_a_at
Grb2
growth factor receptor bound protein 2
WBC
−0.8924
−1.1491
0.03008
LV


1434372_at
AW112010
expressed sequence AW112010
WBC
−0.9301
−1.1422
0.02126
LV


1431295_a_at
Stx18
syntaxin 18
WBC
−0.9956
−1.1413
0.0306
LV


1431644_a_at
Ica1
islet cell autoantigen 1
WBC
−0.9308
−1.138
0.02401
LV


1437495_at
Mbtps2 ///
membrane-bound transcription factor peptidase,
WBC
−0.9064
−1.1365
0.04603
LV



Yy2
site 2 /// Yy2 transcription factor


1450697_at
Slc30a7
solute carrier family 30 (zinc transporter), member
WBC
−0.8352
−1.1314
0.0456
LV




7


1416250_at
Btg2
B cell translocation gene 2, anti-proliferative
WBC
−0.9746
−1.118
0.00496
LV


1455133_s_at
Suco
SUN domain containing ossification factor
WBC
−0.8811
−1.117
0.01699
LV


1423192_at
Pspc1
paraspeckle protein 1
WBC
−0.8284
−1.1079
0.04267
LV


1455892_x_at
---
---
WBC
−0.8722
−1.1071
0.03373
LV


1428579_at
Fmnl2
formin-like 2
WBC
−0.9752
−1.1023
0.0179
LV


1431146_a_at
Cpne8
copine VIII
WBC
−0.9789
−1.0864
0.0198
LV


1431339_a_at
Efhd2
EF hand domain containing 2
WBC
−0.8356
−1.0829
0.038
LV


1416530_a_at
Pnp
purine-nucleostde phosphorylase
WBC
−0.9973
−1.0796
0.0271
LV


1449328_at
Ly75
lymphocyte antigen 75
WBC
−0.9881
−1.0789
0.01909
LV


1417612_at
Ier5
immediate early response 5
WBC
−0.8485
−1.0647
0.01377
LV


1460351_at
Gm12854 ///
predicted gene 12854 /// predicted gene 5068 ///
WBC
−0.9532
−1.0386
0.00772
LV



Gm5068 ///
S100 calcium binding protein A11 (calgizzarin)



S100a11


1433897_at
Al597468
expressed sequence Al597468
WBC
−0.9123
−1.0322
0.04206
LV


1416926_at
Trp53inp1
transformation related protein 53 inducible
WBC
−0.9777
−1.0303
0.02814
LV




nuclear protein 1


1448550_at
Lbp
lipopolysaccharide binding protein
WBC
−0.8689
−1.0246
0.01525
LV


1451335_at
Plac8
placenta-specific 8
WBC
−0.8843
−1.0201
0.04035
LV


1418674_at
Osmr
oncostatin M receptor
WBC
−0.9967
−1.0195
0.00976
LV


1417190_at
Nampt
nicotinamide phosphoribosyltransferase
WBC
−0.9854
−1.0187
0.02931
LV


1453128_at
Lyve1
lymphatic vessel endothelial
WBC
−0.9471
−1.0159
0.01434
LV




hyaluronan receptor 1


1417303_at
Mvd
mevalonate (diphospho) decarboxylase
WBC
−0.8166
−1.0028
0.02977
LV


1451776_s_at
Hopx
HOP homeobox
WBC
−0.9922
−1.0017
0.04008
LV


1429502_at
Hspa13
heat shock protein 70 family, member 13
WBC
−0.8317
−0.9964
0.04005
LV


1427578_a_at
Eif6
eukaryotic translation initiation factor 6
WBC
−0.8366
−0.9859
0.04884
LV


1443609_s_at
Syvn1
synovial apoptosis inhibitor 1, synoviolin
WBC
−0.9848
−0.9838
0.04002
LV


1451532_s_at
Steap1
six transmembrane epithelial antigen of the
WBC
−0.9926
−0.9769
0.02028
LV




prostate 1


1425106_a_at
LOC101056688 ///
tryptophan--tRNA ligase, cytoplasmic-like ///
WBC
−1
−0.9743
0.042
LV



Wars
tryptophanyl-tRNA synthetase


1457035_at
Al607873
expressed sequence Al607873
WBC
−0.9246
−0.9626
0.03115
LV


1429379_at
Lyve1
lymphatic vessel endothelial
WBC
−0.9865
−0.9603
0.01806
LV




hyaluronan receptor 1


1448632_at
Psmb10
proteasome (prosome, macropain) subunit, beta
WBC
−0.9753
−0.9536
0.02755
LV




type 10


1434813_x_at
LOC101056688 ///
tryptophan--tRNA ligase, cytoplasmic-like ///
WBC
−0.9831
−0.9257
0.04759
LV



Wars
tryptophanyl-tRNA synthetase


1433428_x_at
Tgm2
transglutaminase 2, C polypeptide
WBC
−0.8966
−0.9232
0.01276
LV









(AS04 Characterization in the Adjuvant Gene Space)


Finally, the flexibility of the adjuvant gene space approach was tested by investigating another test adjuvant, AS04 (Didierlaurent, A. M. et al., Journal of immunology 183, 6186-6197 (2009)). AS04 consists of aluminum hydroxide and 3-O-desacyl-4′-monophosphoryl lipid A, which is utilized in Cervarix, a vaccine against human papillomavirus 16/18. AS04, the buffer control, and alum-only control were administered i.d. to the mice, and their hematological parameters and organ gene expressions were examined (see Experiment 11).


Experiment 11 is the following.









TABLE 23







Substance used in the test and dosage thereof












Group
Test
Route of


Volume


No.
substance
administration
Dose
Concentration
dosage
















1
AS04Buffer
i.d.
0
mg
150 mM NaCl
100 μl


(Control)
(GSK)












2
AS04 (GSK)
i.d.
0.1 mg +
1 mg/mL of alum,
100 μl





0.01 mg
100 μg/mL of MPL













3
Alum (GSK)
i.d.
0.1
mg
1 mg/mL
100 μl





*All groups included three mice.


*Samples were collected after 6 hours from administration.






(Hematology (Associated with FIG. 3A))


The effect of adjuvant administration with respect to the changes in several hematological parameters was investigated. The following parameters were studied: white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA), relative (%) content of lymphocytes (LY %), relative (%) content of monocytes (MO %), relative (%) content of granulocytes (GR %), red blood cells (RBC), hemoglobins (Hb, HGB), hematocrits (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobins (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT), platelet concentration (PCT), mean platelet volume (MPV), and platelet distribution width (PDW). Data was obtained for the items shown in FIG. 3.


The volcano plot: format is explained. The x and y axes correspond to the log(fold change) and log(p-value), respectively.


Venn diagram for three mice: format is explained. Upregulated gene probes are determined from three individual mice. The three individuals are denoted as x1, x2 and x3, respectively.


Analysis Using sDEGs (FC>1.5 or <0.67, p<0.01, cPA=1)


a. The Number of sDEGs is the Following.












TABLE 24









Up
Down














LV
SP
LN
LV
SP
LN



















ALM_gsk
35
13
10
20
29
20



AS04
88
71
53
33
16
43











b. Gene List of sDEGs


The gene differential expression is represented as log 2(fold change). Gene count with non-zero values corresponds to the counts in the table above.


c. Lists of Biological Process Given by sDEGs


The functional analysis is scored with −log(p-value). Thus, a function with a high score suggests that it is more significant than functions with a lower score.


Hematological assessment showed that AS04 increased monocyte and granulocyte levels in the blood which is similar to the MPLA hematological profile (FIG. 3A). After cluster analysis, the AS04-derived individual samples were closely connected to the FCA samples in G2LV, the same group as that of bCD, ALM_IP and ENDCN. In SP, AS04 formed a new cluster neighboring G2, which again included bCD, ALM_IP, and ENDCN. AS04 was categorized in G1LN, the group that represents most of the PAMP ligands besides CpG oligodeoxynucleotides. These data suggest that AS04-induced local responses are similar to many of the PAMP ligands categorized in G1LN, but relatively mild systemic responses similar to G2 adjuvants were observed, including bCD, ALM_IP and ENDCN, which is consistent with the fact that AS04 is a combination adjuvant of aluminum hydroxide and 3-O-desacyl-4′-monophosphoryl lipid A. The adjuvant groupings including AS04 are summarized in FIG. 9. Taken together, these results suggest that the adjuvant gene space provides a flexible but reliable and insightful profile of many different adjuvants.


DISCUSSION

The results are discussed below. This Example studied a comprehensive organ transcriptome analysis of 21 different adjuvants administered to mice. The results showed that the adjuvant induced gene responses were influenced by many factors including the adjuvant type, the administration route, and the examined organ. Surprisingly, organs such as LV and SP, which are remote from the injection site and also considered as systemic organs, were also useful organs for characterizing the general mode of action of a given adjuvant. The data collected from LVs and SPs augmented or compensated local LN-derived information. Currently, many clinical vaccines are administered intramuscularly and subcutaneously. In addition, mucosal administration via the intranasal route is also practiced. With the nonparenteral administration route, it is sometimes difficult to sample the directly draining lymphoid tissues, and with ENDCN (Falkeborn, T. et al., PloS one 8, e70527 (2013) and Maltais, A. K. et al., Vaccine 32, 3307-3315 (2014)), that proved to be the case. Nevertheless, the transcriptomes of LV and SP revealed ENDCN as a potential DAMP-releasing adjuvant. ENDCN shared some similarity with bCD25 (Onishi, M. et al., Journal of immunology 194, 2673-2682 (2015)) with regard to its mode of action. Furthermore, remote organ transcriptome examination provided the biodistribution of the “adjuvant effect” in vivo (FIG. 9). Although organ transcriptome analysis cannot verify whether the adjuvant itself reached the organ or not, it can confirm that the organ responded to its administration. Consequently, the results indicated that many locally administered adjuvants have the potential to cause gene expression change in systemic organs that are remote from the injection site. However, remote organ gene expression after adjuvant administration does not necessarily imply organ toxicities, and the doses used for mice were generally overdosed for human applications on a per weight basis. Therefore, direct extrapolation of the results to humans also has a limitation. These points need be examined in future studies.


Another important observation with this approach involved the batch effect. It has been reported that many array- and Next Generation Sequencer-based comprehensive gene analysis methods can be disturbed by the batch effect (Leek, J. T. et al., Nat Rev Genet 11, 733-739 (2010)). A batch effect was observed in the data, but it turned out to be beneficial and a useful internal threshold in this approach. The batch effect discriminated whether the organs responded substantially or did not respond to adjuvant administration. If necessary and sufficient amount of microarray data could be obtained, batch effects would simply be reduced to “background noise” such as gene expression fluctuations. However, the 330 microarray data in this study were still not enough to determine the background noise levels without batch effect signatures. In this sense, this approach of examining three different adjuvants in one experiment is a simple and effective method to monitor the batch effect, which worked as a sensitive internal threshold for determining whether the gene expression was adjuvant-specific or not. Of all the adjuvants examined in this study, ALM was one of the least gene response-inducing adjuvants when it was administered locally. Similarly, MPLA, D35, K3, K3SPG and AddaVax acted on local LNs but not in the systemic remote organs, suggesting their local action tendencies (FIG. 9). Other adjuvants induced detectable levels of gene responses in LV and SP (FIG. 9), but, as mentioned above, this does not directly mean the organ toxicities, and further study is required for their interpretations.


To construct the adjuvant gene space, strict data Quality Check (QC) protocol was followed (standard procedure 3). This strict protocol forced discarding of some data and necessitated the creation of a virtual control as a data replacement for QC failed samples. However, concurrently, this strict data processing policy enabled observation of a clear batch effect and, more importantly, “self-organizing” adjuvant clusters, which consisted of the batch effect and host response-based clusters without any tagging or subjective instructions. Furthermore, the addition of AS04 as a test adjuvant to the data analysis did not change the overall adjuvant cluster structures, thereby enabling a relative comparison among the other adjuvants, and confirming the dynamic flexibility for characterizing additional new adjuvants of the inventor's database.


Taken together, the results suggest that this approach can provide a useful platform for evaluating preclinical and clinical adjuvants in a comprehensive and objective manner. The flexibility of this approach also allows for further improvement in the future by depositing data sets from new adjuvants, additional variations in administration, or by including antigen data. In this study, 21 adjuvants were examined at only 6-hour time-points without antigens. Future studies with other time points and antigens, and in silico integration of the publicly available vaccine adjuvant related immunological signatures (Knudsen, N. P. et al., Scientific reports 6, 19570(2016)) are required to understand adjuvanted vaccine mechanisms in more detail. In parallel, the inventors acquired analogous microarray data in rats to evaluate toxicological aspects of adjuvants (see Example 4). The rat transcriptome data can be integrated directly with the toxicogenomics datasets from open TG-GATEs (Igarashi, Y. et al., Nucleic acids research 43, D921-927 (2015)) (which is also in rats). Furthermore, the inventors collected micro RNA expression profiles from human clinical samples (see Example 4). Integration of all the aforementioned datasets demonstrated that the databases enable more integrated and comprehensive analyses on adjuvant-induced gene expression signatures from different species under different experimental settings.


Example 2

This Example carries out a method of classifying substances with an unknown adjuvant function using the method of the invention.


Appropriate candidate substances are provided as the substances.


Adjuvant administration, gene marker expression analysis, clustering, other data analysis and the like are performed in accordance with Example 1.


Candidate substances and reference adjuvants (G1) dciGMP, cGAMP, DMXAA, PolyIC, and R848; (G2) bCD; (G3) FK565; (G4) MALP2s; (G5) D35, K3, and K3SPG; and (G6) AddaVax) are clustered.


(Results)


The transcriptome of candidate substances when administered to murine spleen, liver, or the like and the transcriptome of reference adjuvants of G1 to G6 are compared, and substances classified to the same cluster are each classified to G1 to G6.


Example 3: Adjuvant of Adjuvant

(Materials and Methods)


(Mice)


Six-week-old female C57BL/6J mice were purchased from CLEA Japan. Tlr7−/− or Il-1ra−/− mice were purchased from Oriental BioService and the Jackson Laboratory, respectively. Card9−/− (Hara et al., 2007, Nature immunology 8, 619-629.), Fcrg−/− (Arase et al., 1997, J Exp Med 186, 1957-1963.) or Dap12−/− (Takai et al., 1994, Cell 76, 519-529.) mice were donated by Dr. Hara, Dr. Saito, or Dr. Takai, respectively. Tnfa−/− mice were described previously ((Marichal et al., 2011, Nature medicine 17, 996-1002.). All animal experiments were approved by the Institutional Animal Care and Use Committee, and performed in accordance with institutional guidelines for the National Institute of Biomedical Innovation, Health and Nutrition animal facility.


(Antigens, Antibodies, Adjuvants and Peptides)


Ovalbumin was purchased from Seikagaku-kogyo. SV and inactivated WV derived from A/New Caledonia/20/99 strain were a gift from the Institute of Microbial Chemistry (Osaka, Japan). CpG-ODN (5′-ATCGACTCTCGAGCGTTCTC-3′=SEQ ID NO: 1) was synthesized by GeneDesign (Osaka, Japan). CpG-SPG was prepared as previously reported Kobiyama et al., 2014, Proceedings of the National Academy of Sciences of the United States of America 111, 3086). Alum was purchased from Sigma. Advax™ and hepatitis B surface antigen (HBs) were provided by Vaxine Pty Ltd. LPS was purchased from Sigma. MHC Class I (ASNENMETM=SEQ ID NO: 2) and class II (ARSALILRGSVAHKSCLPACVYGP=SEQ ID NO: 3) epitope peptides of nucleoprotein were synthesized by Operon Biotechnologies. Cytokine ELISA kits for IFN-γ, IL-13, IL-17, TNF-α and IL-1β were purchased from R&D Systems.


(Immunization)


C57BL/6J mice were immunized twice either intramuscularly (i.m.) or i.d. with 2 week intervals (days 0 and 14). For antigen-specific ELISA, blood samples were taken on days 14 and 28. During vaccination and blood collection, mice were anesthetized with ketamine. Alum-adjuvanted antigen was rotated for more than one hour before immunization. Advax™, alum, and CpG-SPG were used for immunization at 1 mg per mouse, 0.67 mg per mouse, and 10 μg per mouse, respectively.


(Antibody Titers)


For ELISA, 96-well plates were coated with 1 μg/ml SV in carbonate buffer (pH 9.6) for SV- and WV-vaccinated groups, 10 μg/ml OVA for OVA-vaccinated groups, and 1 μg/ml HB for HB-vaccinated groups. Wells were blocked with PBS containing 1% bovine serum albumin and diluted sera from immunized mice were incubated on the antigen-coated plate. After washing, goat anti-mouse total IgG, IgG1 or IgG2c conjugated with horseradish peroxidase (Southern Biotech) was added and incubated for 1 h at room temperature. After additional washing, the plates were incubated with TMB substrate for 30 min, the reaction was stopped with 1N H2SO4, and then the absorbance was measured. Antibody titers were calculated. OD of 0.2 was set as the cut-off value for positive samples. The concentration of total IgE in serum was measured by a total IgE ELISA kit (Bethyl).


(Measurement of Antigen-Specific Cytokine Responses)


Two weeks after the second immunization, spleens were collected from mice. 1×106 splenocytes were plated on 96-well plates and stimulated with MHC class I or II epitope peptides of nucleoprotein. Two days after stimulation, IFN-γ, IL-13, and IL-17 in supernatant were measured by ELISA.


(Cytokine Production Profile)


At several time points after i.p. injection of adjuvant, mice were sacrificed and peritoneal lavage fluids were collected. The cytokines in the fluids were measured by Bio-plex (BioRad).


(Activation of DCs)


For in vitro experiments, bone marrow-derived DCs were generated by cultivation of bone marrow cells in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin solution (Naclai Tesque) and 100 ng/ml of human fms-like tyrosine kinase 3 ligand (Flt3L) (PeproTech) for 7 days, stimulated with 1 mg/ml alum, 1 mg/ml Advax™, or 50 ng/ml LPS (Sigma) for 15 h and then CD40 expression on plasmacytoid DCs (pDCs) was evaluated by FACS. We defined pDC as CD11c+/SiglecH+ cells.


In vivo experiments were performed as described previously (Kobiyama et al., 2014, Proceedings of the National Academy of Sciences of the United States of America 111, 3086.). Briefly, C57BL/6J mice were injected with 0.67 mg alum, 1 mg Advax™, or 50 ng LPS at the base of tail. Twenty-four hours after the injection, draining lymph nodes were removed, treated with DNaseI and collagenase for 30 min, then stained with anti-mCD11c (N418), mCD8a (56-6.7), mPDCA-1 (JF05-1C2.4.1), mCD40 (3/23) antibodies and 7AAD, and analysed by FACS. pDC was defined as CD11c+/mPDCA-1+ cells, CD8α+DC as CD11c+/CD8α+ cells, and CD8αDC as CD11c+/CD8α/mPDCA-1 cells.


(In Vitro Stimulation of Macrophages and GM-DCs)


For macrophage preparation, mice were i.p. injected with 3 ml of 4% (w/v) thioglycolate (Sigma) solution. Four days later, macrophages were collected from the peritoneal cavity and plated on 96-well plates. Macrophages were primed with 50 ng/ml LPS for 18 h, and stimulated with adjuvants for 8 h. IL-1β in supernatants was measured by ELISA. TNF-α in supernatants was measured by ELISA after stimulation with Advax™ or alum without priming by LPS. For GM-DC preparation, mouse bone marrow cells were cultured in RPMI 1640 supplemented with 10% FBS, 1% Penicillin/Streptomycin solution, and 20 ng/ml mouse GM-CSF (PeproTech) for 7 days.


GM-DCs were collected, plated on 96-well plates, primed with 50 ng/ml LPS for 18 h and then stimulated with adjuvants for 8 h. IL-1β in supernatants was measured by ELISA.


(Two Photon Microscopy Analysis)


Biotinylated delta inulin particles (1 mg) were pre-mixed with Brilliant Violet 421 Streptavidin (BioLegend), and then administered i.d. at the tail base of mice. At 30 min before inguinal LN removal, mice were i.d. administered anti-MARCO-phycoerythrin or anti-CD169-FITC antibodies. Distributions of Advax™ particles in the inguinal lymph nodes were examined by two-photon excitation microscopy (FV1000MPE; Olympus, Tokyo, Japan).


(Clodronate Liposome Injection)


Clodronate liposome (FormMax) was administered to the base of the tail of mice either 7 or 2 days before immunization. Mice were immunized with WV (1.5 μg)+adjuvant at the base of the tail on days 0 and 14. The day −2 clodronate treatment depleted both macrophages and DCs on day 0. Day −7 treatment depleted macrophages, but DCs were already recovered on day 0. Blood samples were taken on days 14 and 28, and serum antibody titers were measured by ELISA.


(Microarray Analysis)


Six hours after administration of adjuvant, the spleen, lung, kidney, and liver were removed (n=3), and total RNAs were extracted as described previously Onishi et al., 2015, Journal of immunology 194, 2673-2682). After total RNA preparation, the gene expression profiles were obtained using 3′ IVT Express Kit and GeneChip Mouse Genome 430 2.0 Array (Affymetrix). The expression values were normalized by the median value of each GeneChip. The resulting digital image files were preprocessed using the Affymetrix Microarray Suite version 5.0 algorithm (MAS5.0). The differential expression was computed as the ratio between the mean of the treated samples and control samples. The presence and absence (PA) call in MAS5.0 was further customized as follows. When the ratio is >1, the PA call is dependent on treated samples. When the ratio is <1 or =1, the call is dependent on control vehicle samples. Dominant calls (over half) were applied to a set of samples (e.g. when the ratio <1 and PA call of control samples are “P”, “P”, and “A”, then the customized PA call of the set is “1”). The MAS5.0 and PA calls analysis were conducted using the Bioconductor Affy package for R (http://www.bioconductor.org). The p-values for the significance of differentially expressed genes were calculated by using t-test between the normalized treated samples and the normalized vehicle samples. For subsequent analyses, only probes where the fold-change between control and stimulated samples was >2 were used. Probes that were Absent-flagged, i.e., PA call was “0”, were excluded.


(Cell Population Analysis)


The cell population analysis was carried out as follows. The gene expression profile of various immune cell types from a steady state condition were obtained directly from the ImmGen database (http://www.immgen.org/). These expression profiles were used to estimate the cell type origin of the genes. Each gene (i) in all ten immune cell types (j) was first weighted as:










ω
ij

=


e
ij





j
=
1

10


e
ij







[

Numeral


3

]







It is assumed that the weight of a cell type of a given gene depended only on the expression level of that gene in that particular cell type, independent from the expression in other cell types. Given the expression profile of each adjuvant sample, the genes were determined based on the fold change (FC>2) and PA-call threshold (PA=1). Finally, the cell type contribution for sample (k) for cell type (j) was calculated as:










S
jk

=





i



S
ik



ω
ij







i



S
i



ω
ij



+
C


.





[

Numeral


4

]







where c is the pseudocount.


(Upstream Regulator Analysis in IPA)


Upstream analysis was performed using the IPA Regulator Effects feature. The main purpose was to elucidate the upstream regulatory mechanism and their connection to downstream functional impact. For this analysis, genes with fold change >2 and PA-call=1 were selected. Finally reported networks had a P-value <0.001.


(Statistical Analysis)


Statistical significance (P<0.05) between groups was determined by Dunnett's multiple comparison test or Student t-test.


(Results)


(Advax™ adjuvant enhances Th2 responses when combined with a Th2-type antigen)


To understand the adjuvant effect of Advax™, immune responses in mice immunized with Advax™ plus influenza split vaccine (SV), an antigen previously shown to elicit Th2 immune responses, were first examined (Kistner et al., 2010, PloS one 5, e9349). SV immunization alone elicited IgG1 production (a Th2-type IgG subclass) and at higher immunization doses induced IL-13 (Th2-type cytokine) production, consistent with it being a Th2-inducing antigen (FIGS. 10A to 10B). The addition of Advax™ to SV enhanced IgG1 but not IgG2c antibody production, which is prominent with lower (0.015 and 0.15) antigen dose (FIGS. 10A to 10B). Alum, a typical Th2 type adjuvant commonly used for human vaccination also enhanced IgG1 dominant antibody responses. Advax™, similar to alum, induced Th2 responses to SV antigen. Alum is also known to induce IgE production, potentially increasing the risk of vaccine allergy (Nordvall, 1982, Allergy 37, 259-264). Therefore, IgE-levels in sera were measured after SV immunization with either Advax™ or alum. While alum significantly increased IgE production in a dose dependent manner, Advax™ did not induce IgE production (FIG. 10B), consistent with the observation that Advax™ only magnifies the antibody subtype response induced by the SV alone.


T-cell responses were examined by stimulating splenocytes from immunized mice with MHC class I (CD8 T cell) or class II (CD4 T cell) peptides from the influenza virus nucleoprotein. CD4 T cell IL-13 production was not significantly different in mice immunized with SV+Advax™ versus controls, although Advax™ enhanced the ability of SV to produce IgG1 subclass antibody. Then, alum exhibited significantly increased IL-13 production (FIG. 10C). These results demonstrated that Advax™ functions as a Th2-type adjuvant when combined with SV, but Advax™ did not increased IL-13 and IgE compared with alum.


(Advax™ adjuvant enhances Th1 responses when combined with a Th1-type antigen) Next, immune responses in mice immunized with inactivated whole virion influenza vaccine (WV) plus either Advax™ or alum were examined. This WV contains viral RNA that induces TLR7 activation and thereby acts as an endogenous adjuvant that induces Th1 responses (Koyama et al., 2010, Science translational medicine 2, 25ra24). Advax™ adjuvant only enhanced IgG2c (Th1-type subclass) production with minimal effects on IgG1 production, whereas alum suppressed the WV-induced IgG2c response and significantly increased IgG1 levels (FIGS. 11A to 11B). At the cytokine level, Advax™ enhanced IFN-γ (Th1-type cytokine) production by CD4 and CD8 T cells when compared to mice immunized with WV alone. By contrast, alum suppressed IFN-γ production, while significantly increasing IL-13 production by CD4 T cells (FIG. 11C). These results demonstrated that Advax™ functioned as a Th1-type adjuvant when combined with WV, whereas alum functioned as a Th2-type adjuvant for both SV and WV.


(Advax™ Adjuvant Effect is Shaped by Endogenous Adjuvant in the Vaccine Antigen)


It is understood from the above findings that the combination of Advax™ with a Th2-type antigen enhanced Th2 immunity, whereas its combination with a Th1-type antigen enhanced Th1 immunity. This led to a hypothesis that the adjuvant effect of Advax™ might be mediated by an effect on potentiating the inherent adjuvanticity encapsulated within each antigen. By contrast, other adjuvants have a fixed immune bias effect whereby, for example, alum always biases to Th2 responses and CpG-ODN always biases to Th1 responses, regardless of the innate properties of the antigen with which they are co-administered. To test this hypothesis, the adjuvant effect of Advax™ on ovalbumin (OVA) antigen, which is considered a neutral Th0-type antigen, was tested. Advax™ failed to enhance the OVA-specific antibody response, whereas alum, as expected, solely enhanced IgG1 production (FIG. 12).


It was previously shown that the endogenous built-in adjuvant effect of WV was abolished in Tlr7-deficient mice, establishing the importance of TLR7 signalling in WV immunogenicity (Koyama et al., 2010, Science translational medicine 2, 25ra24.) Finally, the adjuvant effect of Advax™ on WV in Tlr7-deficient mice was tested. Notably, the enhanced WV-antibody response seen with Advax™ in wild type mice was lost in Tlr7-deficient mice, whereas the enhanced IgG1 response induced by alum was preserved in Tlr7-deficient mice (FIG. 12). Taken together, these findings suggest that Advax™ belongs to a new additional class of adjuvant that functions as an amplifier of endogenous built-in adjuvants contained within vaccine antigens without changing their inherent immune-polarizing properties.


(Advax Adjuvant Activates Dendritic Cells In Vivo but not In Vitro)


Since dendritic cells (DCs) play a central role in adjuvant-induced immune responses, the ability of Advax™ to activate DCs, in vitro and in vivo, was investigated. Mouse bone marrow-derived DCs were stimulated with Advax™, alum or lipopolysaccharide (LPS) for 15 h in vitro and the expression of CD40, an activation marker on DCs, was evaluated by flow cytometry. While LPS expectedly increased CD40 expression on DCs in vitro, neither Advax™ nor alum influenced CD40 expression on DCs (FIG. 13). Next, the ability of Advax™ or alum to activate DC CD40 expression, in vivo, was investigated by measuring CD40 expression on DCs from draining lymph nodes following adjuvant administration. In contrast to the in vitro findings, both Advax™ and alum increased the frequency of activated DCs in draining lymph nodes when administered in vivo (FIG. 13). As alum induces extracellular DNA release from dead cells at the injection site (Marichal et al., 2011, Nature medicine 17, 996-1002.), this extracellular DNA via binding DAMP receptors likely explains how alum induced DC activation in vivo but not in vitro. Therefore, it was asked whether Advax™ might similarly be inducing cell death at the injection site and thereby indirectly activating DCs via DAMP receptors. To test this possibility, cytotoxicity and host DNA/RNA release at the injection site of Advax™ or alum were evaluated in vivo. After the intraperitoneal (i.p.) administration of Advax™ or alum, peritoneal lavage fluids were collected, and the number of dead cells and the concentration of DNA/RNA in these fluids were measured. Whereas alum induced cell death and nucleic acid release as expected, injection of Advax™ induced no cytotoxicity or nucleic acid release, indicating that DAMP signalling pathways are not involved in the ability of Advax™ to induce DC activation and CD40 expression, in vivo.


(Phagocytic Macrophages are Cellular Mediator of the Adjuvant Effect of Advax™)


Although the immune complex and inactivated influenza virus are captured by CD169+ (also called Siglec-1 or MOMA-1) macrophages in draining lymph node (DLN) to induce humoral immune responses (Gonzalez et al., 2010, Nature immunology 11, 427-434.; Suzuki et al., 2009, J Exp Med 206, 1485-1493.), some particulate adjuvants are efficiently taken up by MARCO+ macrophages (Aoshi et al., 2009, European journal of immunology 39, 417-425.; Kobiyama et al., 2014, Proceedings of the National Academy of Sciences of the United States of America 111, 3086.). Thus, the behaviour of Advax™ in DLN was analyzed in vivo using fluorescent-labelled Advax™ particles. One hour after intradermal (i.d.) administration, a weak Advax™ signal co-localized with MARCO+ macrophages in the DLN, with this co-localization signal being much higher 24 h later. Almost no co-localization of Advax™ with CD169+ macrophages was observed in the DLN (FIG. 14A), suggesting i.d. administered Advax™ is taken up by MARCO+ macrophages. Next, to investigate the requirement of Advax™ uptake into macrophages for its adjuvant effect, the different recovery kinetics of macrophages and DCs following clodronate liposome injection was utilized. This depletes both macrophages and DCs completely by day 2 but subsequently, macrophages do not recover for at least 7 days whereas DCs are mostly recovered by this time (Aoshi et al., 2008, Immunity 29, 476-486.; Kobiyama et al., 2014, Proceedings of the National Academy of Sciences of the United States of America 111, 3086.). Interestingly, the adjuvant effect of Advax™ was significantly reduced with clodronate liposome treatment, irrespective of the time point tested (day 2 or 7) (FIG. 14B), suggesting Advax™ adjuvanticity is dependent on the presence of macrophages. By contrast, the adjuvanticity of CpG-SPG was significantly reduced on day 2 post-clodronate treatment but not on day 7, indicating that the adjuvanticity of CpG-SPG was mostly dependent on DCs but not macrophages, as previously reported (Kobiyama et al., 2014, Proceedings of the National Academy of Sciences of the United States of America 111, 3086.)


(Advax™ Alters the Gene Expression of IL-1β-, C-Type Lectin Receptors- and TNF-α-Related Signalling Pathways)


To understand the biological properties of Advax™, its effect on cytokine production was investigated. At several time points after i.p. administration of Advax™ or alum, peritoneal lavage fluids were collected, and cytokines in the fluids were analysed. Alum induced the production of various cytokines including interleukin (IL)-5, IL-10, IL-12, tumor necrosis factor (TNF)-α, and granulocyte-colony stimulating factor (G-CSF), whereas limited cytokine response such as IL-10, G-CSF, or macrophage inflammatory protein (MIP)-1 was observed by Advax™ administration.


To further explore the predominated biological effect of Advax™ in vivo, gene expression profiles were examined in tissues after local (i.d.) or systemic (i.p.) administration of Advax™. Advax™ administration via i.d. conferred limited gene expression changes, whereas i.p. administration altered gene expression in several tissues. Administration via i.p. resulted in differential regulation of genes associated with the acute phase response, inflammation, chemokines, complement/platelet, and C-type lectin receptor (CLRs)-related responses. Furthermore, analysis of the responding cell population revealed Advax™ induced neutrophil and macrophage responses in vivo. Additionally, upstream regulator analysis in Ingenuity pathway analysis (IPA) suggested that four upstream regulators: NF-κB (complex), IL-1β, IFN-γ, and TNF-α were affected by i.p. administration of Advax™. This drives the expression of genes that enhance phagocyte adhesion, neutrophil chemotaxis and movement of hematopoietic and natural killer cells (FIG. 15A).


To examine the contribution of these biological factors to the adjuvanticity of Advax™, the ability of Advax™ to induce IL-1β production was first investigated. Peritoneal macrophages or granulocyte-macrophage colony stimulating factor (GM-CSF)-induced bone marrow-derived DCs (GM-DCs) were stimulated with Advax™ or alum following LPS-priming, and then IL-1β production was examined. However, IL-1β production was not detected in these cells stimulated with Advax™ in vitro, whereas alum significantly induced IL-1β production. Because some particulate adjuvants require NLRP3 inflammasome activation and subsequent IL-1β production for their adjuvanticity (Eisenbarth et al., 2008, Nature 453, 1122-1126.; Kuroda et al., 2013, Int Rev Immunol 32, 209-220.), the effect of the absence of the NLPR3 inflammasome components, Nlrp3, Caspasel, or IL-1r on Advax™ adjuvanticity was examined in vivo. Advax™ conferred significant adjuvant effects in each of these NLPR3 inflammasome deficient mice, indicating the adjuvant effect of Advax™ is independent of the NLPR3 inflammasome/IL-1β signalling pathway. Next, to examine the involvement of CLR-related signalling pathways in Advax™ adjuvanticity, its adjuvant effect was examined in mice lacking the CLRs signalling pathway related genes, Fcrg−/−, Card9−/−, or Dap12−/−. Absence of these genes did not affect the ability of Advax™ to enhance antibody responses, indicating that Advax™'s adjuvant effect is independent of these CLR-related signalling pathways.


(TNF-α is Essential for the Adjuvant Effect of Advax™)


Gene expression analysis also indicated that i.p. administration of Advax™ affected TNF-α-related signalling pathways (FIG. 15A). Thus, the ability of Advax™ to generate TNF-α production was examined. Macrophages were stimulated with Advax™ in vitro and TNF-α in the culture supernatant was measured. Advax™ did not induce TNF-α production, whereas stimulation with alum significantly induced macrophage TNF-α production in vitro (FIG. 16A). However, i.p. injection of Advax™ did result in significantly increased serum TNF-α levels (FIG. 16A), whereas paradoxically i.p. alum did not affect serum TNF-α levels. Because i.p. injection of Advax™ influenced gene expression including TNF-α-signalling pathway-related genes in remote tissues such as the lung and spleen, TNF-α in the serum might be derived from these tissues. Finally, to examine the role of TNF-α in Advax™'s adjuvant effect, Tnfa−/− mice were immunized with Advax™-adjuvanted WV or hepatitis B surface antigen, and the resulting antibody responses were examined (FIG. 16B). The absence of Tnfa significantly decreased the adjuvant effect of Advax™ on antibody responses, suggesting that intact TNF-α signalling is important for Advax™'s adjuvant effect on antibody responses.


Example 4: Safety and Efficacy Model Construction

Next, this Example demonstrates that safety and efficacy databases and models can be constructed using the present invention.


Use of a toxicogenomic database (GATE) in addition to the adjuvant databases shown in Examples 1 to 2 enables transcriptome based toxicity and safety prediction by machine learning or the like.


(Toxicity Prediction at 6 Hours and 24 Hours in Rat Liver Transcriptome)


A “toxic” group” including 10 compounds and a “non-toxic” group including 10 compounds were each created from a toxicogenomic database. The compounds in the “toxic” group were compounds exhibiting pathological findings within 4 administrations. The compounds in the “non-toxic” group were compounds with no toxicity related feature observed (FIG. 17) (these data are available from publicly open TG-GATE (Igarashi, Y. et al., Nucleic acids research 43, D921-927 (2015)).


A pattern of differentially expressed genes by administration of a compound in the “toxic” group and a pattern of differentially expressed genes by administration of a compound in the “non-toxic” group were obtained based on results of transcriptome analysis after 6 hours and 24 hours from administration of the compound in the “toxic” group and the compound in the “non-toxic” group (these data are available from publicly open TG-GATE (Igarashi, Y. et al., Nucleic acids research 43, D921-927 (2015)). Probes (genes) selected for a necrosis prediction model were concentrated into 5 routes associated with immune response (1) and metabolism (4) (FIG. 18A).


The gene variation patterns as of 6 and 24 hours after each adjuvant administration in the adjuvant database shown in Examples 1 to 2 were compared to the “toxic” gene pattern and “non-toxic” gene pattern as of 6 and 24 hours after administration of the known compounds to calculate the degree of “toxicity” score of each adjuvant (FIG. 18B). See, for example, Igarashi, Y. et al., Nucleic acids research 43, D921-927 (2015) and Uehara, T. et al., Toxicol Appl Pharmacol 2011 Sep. 15; 255(3): 297-306 for “toxicity” score calculation methods.


Activity of ALT after 6 hours and 24 hours was measured by actually administering each adjuvant (FIG. 19). AST and ALT activities were measured by an approach known in the art (e.g., colorimetric measurement using a kinetics assay or endpoint assay). The data was commissioned to and measured by LSI Medience.


Toxicity of adjuvant X (FK565) expected to have high toxicity was actually examined in mice.


PBS and FK565 (1 μg/kg, 10 μg/kg, or 100 μg/kg) or LPS (1 mg/kg) were intraperitoneally administered to mice. After 3 hours/6 hours, blood and liver were collected on day 1, day 2, day 3, and day 5. Hematoxylin and eosin staining was applied to the collected liver and histologically analyzed (FIG. 20). Further, the liver was stained with TUNEL to examine apoptosis (FIG. 21). The serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were biochemically analyzed (FIG. 22).


The suitability of a toxicity prediction model was demonstrated from a ROC curve. Suitability was also demonstrated from PCA analysis of a selected probe for an adjuvanticity prediction model (adjuvant database+toxicogenomic database (GATE)). A heat map of routes centered around Osmr was created (not shown).


A gene cluster centered around Osmr was obtained from analysis of the database described above (FIG. 23). Gene Y (Osmr), which was predicted to have a strong relationship with toxicity, was investigated. The top row of FIG. 24 shows a change in expression (fold change) of gene Y in the liver after 6 hours from administering each adjuvant to rats. Osmr knockout mice obtained from Jackson Laboratories were used as the base.


PBS, FK565 (1 μg/kg, 10 μg/kg, or 100 μg/kg), or LPS (1 mg/kg) was intraperitoneally administered to wild-type and Osmr knockout mice. The blood and liver were collected after one day. DRI-CHEM 7000V (Fujifilm, Tokyo, Japan) was used to analyze the serum AST and ALT levels (FIG. 25). Further, the liver was stained with TUNEL to examine apoptosis (bottom row of FIG. 24). AST and ALT activities were measured by an approach known in the art (e.g., colorimetric measurement using a kinetics assay or endpoint assay).


(Examination of Liver Virulence Gene in Mice)


For example, a gene suggested to be related to toxicity in rats can be selected as a knockout candidate in mice by referring to the following table (e.g., NOD1 to a NOD1 ligand). In this Example, Osmr was knocked out.












TABLE 25







Name
Receptor


















1
AddaVax
Unknown


2
ADX
Unknown


3
ALM
Unknown


4
bCD
Unknown


5
cdiGMP
STING


6
cGAMP
STING


7
D35
TLR9


8
DMXAA
STING


9
ENDCN
Unknown


10
FCA
CLR and unkown


11
FK565
NOD1


12
ISA51VG
Unknown


13
K3
TLR9


14
K3SPG
TLR9


15
MALP2s
TLR2/6


16
MBT
NOD2


17
MPLA
TLR4


18
Pam3CSK4
TLR1/2


19
PolyIC
TLR3 and MDA5


20
R848
TLR7


21
sHZ
Unknown









(Prediction of Adjuvanticity in Rat Liver Model)


As shown in FIG. 26, a feature of a safe adjuvant is searched from an adjuvant database, and a compound that acts as an adjuvant is searched from a public toxicogenomic database.


Many drugs were suggested as a potential adjuvant by an adjuvanticity prediction model. This was investigated by an experiment using mice.


Probes (gene) selected from an adjuvanticity prediction model were concentrated to 42 routes related to cell death (4), immune response (2), and metabolism (36). In the Venn diagram of genes constituting a route related to cell death (4) and immune response (2), 7 genes were shared therebetween (FIG. 27).


Many drugs, immunostimulants, LPS, and TNF had a high score from an adjuvanticity prediction model (FIG. 28, top). Drugs indicated by colored letters were purchased and then tested in mice. Suitability of the adjuvanticity prediction model was confirmed by a ROC curve (FIG. 28, bottom).


Ovalbumin as well as alum, CpGk3, or five types of drugs (ACAP, BOR, CHX, COL, and PHA) were intradermally administered to mice on day 0 and day 14 at 2 to 3 different doses. Blood and spleen were collected on day 21. The anti-ovalbumin (ova) antibody titers (IgG1 and IgG2) were measured on day 21. Different adjuvant properties of IgG1 and IgG2 titers were observed depending on the drug (FIG. 29). Each drug, ovalbumin, and the like in the above example were obtained from Japan Biochemical or WAKO. In addition to drugs from these suppliers, drugs available from other supplies can also be used.


Furthermore, supernatant was collected after adding ovalbumin (ova), 257-264 peptide (OVA-MHC1), ova 323-339 peptide (OVA-MHC2), or ova protein (OVA-whole) in addition to each drug in vitro and stimulating, or treating without additional stimulation, spleen cells (cells from organ collected from immunized mice). Th1 (IL-2 and IFN-γ) (FIG. 30)- and Th2 (IL-4 and IL-5) (FIG. 31)-type cytokines in the supernatant were measured by ELISA.


Ovalbumin as well as alum, CpGk3, or five types of drugs (ACAP, BOR, CHX, COL, and PHA) were administered to mice. Blood was collected after three hours (day 0). DRI-CHEM 7000V (Fujifilm, Tokyo, Japan) was used for biochemical analysis on the serum aspartate transaminase (AST) and alanine transaminase (ALT) levels (FIG. 32).


Each drug was intraperitoneally administered to mice. Blood was collected after 6 hours and 24 hours to analyze miRNA in the circulating blood (FIG. 33).


(Results)


In view of the above results, it can be understood that drug components such as adjuvants can be classified by the transcriptome analysis of the invention, and efficacy and safety can be tested.


In this Example, the inventors obtained microarray data similar to that of Examples 1 for rats in order to evaluate toxicological properties of adjuvants. Since transcriptome data of rats can be directly incorporated into a toxicogenomic data set of the public TG-GATE (Igarashi, Y. et al., Nucleic acids research 43, D921-927 (2015)) (this is also for rats), analysis was performed based thereon, which demonstrated that toxicity can be analyzed. Furthermore, knockout experiments were conducted with mice based on the findings from transcriptome analysis. It was substantiated to be a gene involved with toxicity. Therefore, it was substantiated that the results in this Example can be demonstrated across species, and the present invention and the findings obtained in the present invention can be utilized in verification of toxicity experiments in animal models. Furthermore, the inventors collected microRNA expression profiles from human clinical samples to analyze miRNA therewith. Integration of all of the aforementioned datasets proved that a more integrated and comprehensive analysis of adjuvant induced gene expression signatures of different species under different experimental conditions can be performed with such databases, and a test based on findings from transcriptome analysis can also be conducted for efficacy.


For toxicity and efficacy, those skilled in the art understand that the same analysis is possible in principle for not only adjuvants but also for all drug components. Especially for toxicity, the same test can be conducted on not only active ingredients, but also additives (excipient, carrier, and the like), and the same test can be conducted on a final formulation of mixture prepared by mixing a plurality of the same or different drug components. Those skilled in the art also understand that the same test using transcriptome analysis is possible for active ingredients based on an indicator of active ingredients.


As described above, the present invention is exemplified by the use of its preferred embodiments. However, it is understood that the scope of the present invention should be interpreted solely based on the Claims. It is also understood that any patent, any patent application, and any references cited herein should be incorporated herein by reference in the same manner as the contents are specifically described herein. The present application claims priority to Japanese Patent Application No. 2016-256270 and Japanese Patent Application No. 2016-256278 filed on Dec. 28, 2016 In Japan. The entire content of these applications is incorporated herein by reference in the same manner as the contents are specifically described herein.


INDUSTRIAL APPLICABILITY

Clinical application with high precision including classification of adjuvants is possible for immune related diseases.











[Sequence Listing Free Text]



SEQ ID NO: 1



CpG-ODN(5′-ATCGACTCTCGAGCGTTCTC-3′)







SEQ ID NO: 2



MHC class I (ASNENMETM)







SEQ ID NO: 3



MHC class II (ARSALILRGSVAHKSCLPACVYGP)





Claims
  • 1. A method of classifying a drug component, the method comprising: (a) providing a candidate drug component;(b) obtaining gene expression data by performing transcriptome analysis on the candidate drug component;(c) clustering the gene expression data by combined use of adjuvant database created by performing transcriptome analysis for efficacy as an adjuvant and toxicity genome data obtained from toxicity database created by performing transcriptome analysis for toxicity of drugs; and(d) determining that the candidate drug component has efficacy as an adjuvant and/or toxicity similar to drugs having efficacy as an adjuvant and/or toxicity in the databases if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one of the drugs.
  • 2. The method of claim 1, wherein the combination of the adjuvant database and the toxicity genome data comprises gene expression data from heterogeneous animals.
  • 3. The method of claim 1, wherein the combination of the adjuvant database and the toxicity genome data comprises human gene expression data.
  • 4. The method of claim 3, which determines efficacy as an adjuvant and/or toxicity in human for the candidate drug component.
  • 5. The method of claim 1, wherein the toxicity genome data comprises genome data for drug components in toxicity group and drug components in non-toxicity group.
  • 6. The method of claim 1, wherein the clustering comprises creating a cluster for efficacy as an adjuvant and creating a cluster for toxicity.
  • 7. The method of claim 6, which determines efficacy as an adjuvant and toxicity for the candidate drug component.
  • 8. The method of claim 1, comprising extracting a characteristic gene from a gene expression profile with machine learning.
  • 9. The method of claim 1, comprising creating a prediction model which determines efficacy as an adjuvant and/or toxicity with machine learning.
  • 10. The method of claim 8, wherein the machine learning comprises neural networking method, support vector machine, random forest, linear regression, logistic regression, support vector machine or cross validation.
  • 11. The method of claim 8, using data from the adjuvant database and/or the toxicity database as training data for the machine learning.
  • 12. The method of claim 1, comprising determining a candidate gene of toxicity bottleneck gene by the transcriptome analysis.
  • 13. The method of claim 12, wherein the candidate gene of toxicity bottleneck gene is a significantly differentially expressed gene.
  • 14. The method of claim 1, wherein the classification further comprises classification by at least one feature selected from the group consisting of classification based on a host response, classification based on a mechanism, classification by application based on a mechanism or cells (liver, lymph node, or spleen), and module classification.
  • 15. A program for implementing a drug component classification method comprising classifying a drug component based on the method of claim 1.
  • 16. A recording medium storing a program for implementing a drug component classification method comprising classifying a drug component based on the method of claim 1.
  • 17. A system for classifying a drug component comprising a classification unit which classifies a drug component based on the method of claim 1.
  • 18. A computer system for classifying a drug component, the system comprising: (a) a storing unit for adjuvant database created by performing transcriptome analysis for efficacy as an adjuvant;(b) a storing unit for toxicity database created by performing transcriptome analysis for toxicity of drugs;(c) a transcriptome clustering analysis unit for obtaining gene expression data by performing transcriptome analysis on the candidate drug component and clustering the gene expression data by combined use of the adjuvant database and toxicity genome data obtained from the toxicity database; and(d) a determination unit for determining that the candidate drug component has efficacy as an adjuvant and/or toxicity similar to drugs having efficacy as an adjuvant and/or toxicity in the databases if a cluster to which the candidate drug component belongs is classified to the same cluster as at least one of the drugs.
  • 19. A gene analysis panel comprising means to detect nucleic acids or proteins for use in classification specified by the method of claim 1.
Priority Claims (2)
Number Date Country Kind
2016-256270 Dec 2016 JP national
2016-256278 Dec 2016 JP national
Continuations (1)
Number Date Country
Parent 16475038 Jun 2019 US
Child 17724335 US