Synergistic Combination of Alum and Non-Liposome, Non-Micelle Particle Vaccine Adjuvants

Information

  • Patent Application
  • 20230109193
  • Publication Number
    20230109193
  • Date Filed
    July 29, 2022
    2 years ago
  • Date Published
    April 06, 2023
    a year ago
Abstract
Compositions are disclosed that include alum and a non-liposome, non-micelle particle, where the particle comprises a lipid, a sterol, a saponin, and an optional additional non-alum adjuvant, wherein the particle is optionally bound to the alum, and the use of the compositions as vaccine adjuvants and methods for eliciting immune responses.
Description
SEQUENCE LISTING STATEMENT

A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Aug. 9, 2022 having the file name “21-1148-US.xml” and is 9 kb in size.


BACKGROUND

Alum remains an adjuvant that does not stimulate many of the innate immune recognition pathways that might be exploited to drive robust immune responses, and improved compositions and methods for greater immune stimulation are required.


SUMMARY

In a first aspect, the disclosure provides compositions, comprising:


(a) alum; and


(b) a non-liposome, non-micelle particle, wherein the particle comprises a lipid, a sterol, a saponin, and an optional additional non-alum adjuvant, wherein the particle is optionally bound to the alum. In one embodiment, the particle may be a porous, cage-like nanoparticle, and may be about 30 nm to about 60 nm in diameter. In one embodiment, the lipid may be a phospholipid; in one such embodiment, the phospholipid is 2-Dipalmitoyl-snglycero-3-phosphocholine (DPPC). In another embodiment, the sterol may comprise cholesterol or a derivative thereof. In a further embodiment, the saponin may be a natural or synthetic Saponin; in exemplary such embodiments, the saponin may comprise Quil A® or a submixture or pure saponin separated therefrom. In one embodiment, the saponin may comprises a natural or synthetic Q-21, or an analog thereof. In another embodiment, the lipid is DPPC, the additional adjuvant is a natural or synthetic MPLA, the sterol is cholesterol, and the saponin is Quil A®.


In one embodiment, the additional adjuvant is present and may comprise a TLR4 agonist. In various such embodiment, the TLR4 agonist may be a lipopolysaccharide (LPS) or a lipid A derivative thereof, or a natural or synthetic monophosphoryl lipid A (MPLA) or a derivative thereof. In one embodiment, the MPLA or derivative thereof may be natural or synthetic 4′-monophosphoryl lipid A (MPLA) or 3-O-deacylated monophosphoryl lipid A (3D-MPLA). In another embodiment, the additional adjuvant may comprise a pathogen-associated molecular pattern (PAMP). In various embodiments, the PAMP may comprise a lipid, a TLR ligand, a NOD ligand, an RLR ligand, a CLR ligand, an inflammasome inducer, a STING ligand, or a combination thereof.


In one embodiment, the compositions may comprise a lipid:additional adjuvant:sterol:saponin molar ratio of 2.5:1:10:10, or a variation thereof wherein the molar ratio of lipid, additional adjuvant, sterol, saponin or any combination thereof is increased or decreased by any value between about 0 and about 3. In one embodiment, the composition may comprise DPPC:MPLA:cholesterol:Quil A® in a molar ratio of 2.5:1:10:10. In another embodiment, the composition may comprise Quil-A:chol:DPPC:MPLA in a mass ratio of 10:2:1:1.


In one embodiment, the alum may comprise a salt of aluminum, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or combinations thereof. In another embodiment, the alum and the particle are bound. In one such embodiment, the particle is covalently bound to the alum via phosphate residues in the particle.


In another embodiment, the composition may further comprise an antigen bound to the alum and/or the particle, including but not limited to an antigenic polypeptide. In one embodiment, the antigen or the antigenic polypeptide is covalently bound to the alum and/or the particle. In one embodiment, the antigen or the antigenic polypeptide may comprise at least one linker comprising 2-12 phosphoserine residues, wherein the antigen is covalently bound to the alum via the phosphoserine residues. In one embodiment, the at least one linker may be present at the N-terminus or C-terminus of the antigenic polypeptide. In other embodiments, the antigen or the antigenic polypeptide may comprise a cancer antigen, a viral antigen, a bacterial antigen, a parasite antigen, or a fungal antigen.


In one embodiment, a molar ratio of alum:particle may be between about 1:500 and about 500:1. In another embodiment, a molar ratio of alum:antigenic or antigenic polypeptide may be between about 1:500 and about 500:1.


In another embodiment, the disclosure provides pharmaceutical compositions comprising the composition of any embodiment or combination of embodiments of the disclosure, and a pharmaceutically acceptable carrier. In one embodiment, the disclosure provides vaccines comprising the composition of any embodiment or combination of embodiments of the disclosure that include an antigen.


In another aspect, the disclosure provides methods for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of


(a) the composition of any embodiment or combination of embodiments of the disclosure that do not include an antigen; and


(b) an antigen.


In a further aspect, the disclosure provides methods for generating an immune response against an antigen, or for treating a subject in need thereof, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition of any embodiment or combination of embodiments of the disclosure that does include an antigen





DESCRIPTION OF THE FIGURES


FIG. 1. pSer-modification of SARS-CoV-2 RBD immunogens facilitates binding to alum with retention of key structural epitopes. (A) RBD antigens with phosphoserine peptides conjugated at the N-(pSer4-RBD) (SEQ ID NO: 4-RBD) or C-(RBD-pSer4) (RBD-SEQ ID NO: 4) terminus were assayed for phosphates per protein by a malachite green assay. (B) pSer-conjugated or unmodified RBD were mixed with alum, and the fraction of protein bound to alum was assessed before (“Loading”) and after incubation for 24 hours in 10% mouse serum at 37° C. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (C) Schematic of modified sandwich ELISA to analyze the antigenicity profile of free RBD (left) or RBD bound to alum-coated plates (right). (D-F) Shown are binding profiles of hACE2-Fc (D), CR3022 (E), H4 (F), and B38 (G) to RBDs captured on anti-histag or alum-coated plates (n=3 replicates), and the area under individual binding curves normalized to unmodified RBD (H). Dashed line indicates signal equivalent to unmodified RBD. Statistical significance was determined by Mann-Whitney test. Values plotted are means±standard deviation. ns p>0.05, **** p<0.0001.



FIG. 2. pSer modification enhances the immunogenicity of alum-adsorbed RBD in mice. BALB/c mice (n=5 animals/group) were immunized with 10 μg unmodified or N- or C-terminal pSer4-conjugated RBD (SEQ ID NO: 4 N- or C-conjugated RBD) in 50 μg Alhydrogel and boosted at 6 weeks. (A) Serum IgG responses were assessed longitudinally by ELISA. Arrows indicate immunization time points. Values plotted are geometric means±geometric standard deviation. (B) Individual mouse IgG responses from selected time points. Values plotted are geometric means±geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. SARS-CoV-2 pseudovirus neutralizing titer (half-maximal, PSV NT50) (C) and NT80 (D) were assessed for serum collected at day 21 and day 56. The dashed line indicates the limit of detection. Values plotted are means standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (E) RBD-specific antibody secreting cells (ASCs) in the bone marrow were assessed by ELISPOT at day 112. Representative ELISPOT plate images are shown. Values plotted are means±standard deviation. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01.



FIG. 3. pSer-conjugated mutant RBDs elicit potent germinal center responses and neutralizing antibodies in mice. (A, B) Mice (n=4/group) were immunized with 10 μg labeled unmodified or pSer-conjugated RBDJ plus 100 μg alum and injection site fluorescence was tracked longitudinally by IVIS imaging. Shown are whole-animal images (A) and fluorescence quantification (B, means±SD). (C-G) Mice (n=5/group) were immunized and GC and TFH responses in dLNs were analyzed by flow cytometry. Shown are representative gating of RBD-specific GC B cells (C), total GC B cell counts (D), RBD-specific GC B cell counts (E), percent RBD-specific GC B cells (F), and TFH enumeration at day 14 (G). Shown are means±SEM. (H) Mice (n=5/group) were immunized twice (indicated by arrows), and serum antibody responses (geometric means±geometric SD) were tracked by ELISA. (I) Mice (n=5/group) were immunized with varying antigen densities on alum. pSer4-RBDJ (SEQ ID NO: 4-RBDJ) was loaded on alum at the indicated ratios; all groups received 200 μg alum. Shown are half-maximal pseudovirus neutralization titers (PSV NT50); dashed line indicates LOD. Shown are means±SD. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test (B, D-G) or Sidak's multiple comparisons test (I). ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 4. Combining pSer-RBD with alum-binding co-adjuvants enhances humoral immunity. (A) CpG or SMNP were added to alum for 30 min and the fraction of alum-bound adjuvant was measured. (B) The fraction of pSer4-RBDJ (SEQ ID NO: 4-RBDJ) binding to alum co-loaded with CpG or SMNP was assessed before (“Loading”) and after 24 hours incubation (10% mouse serum, 37° C.). (C-D) Mice (n=3/group) were immunized with 30 μg labeled CpG (C) or 5 μg labeled SMNP (D) with 10 μg RBDJ±100 μg alum and injection site fluorescence was assessed by IVIS. (E-J) Mice (n=5/group) were immunized twice (indicated by arrows) with RBDJ combined with the indicated adjuvants. Shown are serum IgG titers over time (E, G), total IgG from individual animals on day 42 (F), antibody titers by isotype on indicated days (H), Half-maximal inhibitory titers (ID50) values assessed for hACE2-RBD binding in the presence of indicated sera (I), and half-maximal pseudovirus neutralizing titers (PSV NT50, J). Dashed line indicates the LOD. Shown are means±SD (A-D,J) or geometric means±geometric SD (E-I). Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test (A,B,E,F,H,I) or Sidak's multiple



FIG. 5. BALB/c mice (n=5 animals/group) were immunized with 5 μg pSer-conjugated HIV envelope trimer antigen, MD39, in 50 μg Alhydrogel (alum)±varying doses of SMNP and boosted at 6 weeks. (A) Serum IgG responses were assessed longitudinally by ELISA. Arrows indicate immunization time points. (B) Responses are plotted for individual mice. Values plotted are geometric means±geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001.



FIG. 6. pSer-modification of RBD antigens facilitates anchoring to alum. (A) RBD antigens were expressed with terminal cysteines which can be coupled to short peptide linkers consisting of an N-terminal maleimide group and C-terminal pSer residues separated by a 6-unit poly(ethylene glycol) spacer. (B) pSer-modified RBD antigens are anchored to alum via ligand exchange between the phosphates in the pSer residues and hydroxyls on the surface of alum.



FIG. 7. pSer valency enables tuning of antigen-alum binding and influences humoral immune responses. (A) RBDJ antigens with pSer4 (SEQ ID NO: 4) or pSer8 (SEQ ID NO: 5) peptides conjugated at the N-terminus were assayed for phosphates per protein by a malachite green assay. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (B) Unmodified, pSer4-, or pSer8-conjugated RBDJ (SEQ ID NOs: 4 or 5 conjugated RBDJ) were mixed with alum, and the fraction of protein bound to alum was assessed before (“Loading”) and after incubation for 24 hours in 10% mouse serum at 37° C. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (C) Unmodified, pSer4-, or pSer8-conjugated RBDJ (SEQ ID NOs: 4 or 5 conjugated RBDJ) were mixed with alum and incubated in 10% mouse serum at 37° C. The fraction of protein bound to alum was assessed longitudinally. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (D) A modified sandwich ELISA approach was used to analyze the antigenicity profile of pSer-modified RBDJ. Shown are binding profiles of hACE2-Fc (top left), CR3022 (top right), H4 (bottom left), and B38 (bottom right) to RBDs captured on alum-coated plates (n=3 replicates), and the area under individual binding curves (E). Statistical significance was determined by two-way ANOVA followed by Sidak's multiple comparison test. Values plotted are means±standard deviation. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 8. pSer valency influences germinal center responses. Representative flow cytometry gating plots of (A) RBD-specific germinal center (GC) B cell, and (B) T follicular helper (TFH) cell staining. (C) BALB/c mice (n=5 animals/group) were immunized with 10 μg unmodified, pSer4-, or pSer8-conjugated RBDJ (SEQ ID NOs: 4 or 5 conjugated RBDJ) and 100 μg alum, and germinal center (GC) responses in dLNs were analyzed by flow cytometry over time for total GC B cell counts (top), RBD-specific GC B cell counts (middle), and percent RBD-specific GC B cells (bottom) at 9, 14, 21, and 28 days post-immunization. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. Values plotted are means±standard deviation. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, p<0.0001.



FIG. 9. pSer-RBDJ drainage is a combination of antigen-alum complex trafficking and release of antigen from alum at the injection site. (A) BALB/c mice (n=5 animals/group) were immunized with 10 μg unmodified, pSer4-, or pSer8-conjugated RBDJ (SEQ ID NOs: 4 or 5 conjugated RBDJ) and 100 μg alum and boosted at 6 weeks. Half-maximal inhibitory titers (ID50) values were assessed for hACE2-RBD interactions at day 35 and day 70. The dashed line indicates the limit of detection. Values plotted are geometric means±geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Sidak's multiple comparisons test. (B) Mice were immunized with 10 μg fluorescently labeled RBDJ plus alum and the fluorescence at the injection site was quantified longitudinally (n=4 animals/group), as in FIG. 3A-B. The signal remaining at the injection site at day 49 for pSer8-RBDJ (SEQ ID NO: 5-RBDJ) was subtracted from the longitudinal pSer8-RBDJ (SEQ ID NO: 5-RBDJ) signal and plotted for comparison to pSer4-RBDJ (SEQ ID NO: 4-RBDJ). Values plotted are means±standard deviation. Statistical significance between pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and pSer8-RBDJ (SEQ ID NO: 5-RBDJ) was determined by two-way ANOVA followed by Tukey's post-hoc test. (C) Mice were immunized with 10 μg RBDJ plus 100 μg of labeled alum and the fluorescence at the injection site was quantified longitudinally (n=3 animals/group). Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 10. Average antigen density of pSer-RBDJ on alum does not significantly alter humoral responses. (A) pSer4-RBDJ (SEQ ID NO: 4-RBDJ) was mixed with alum in varying ratios, and the fraction of protein bound to alum was assessed before (“Loading”) and after incubation for 24 hours in 10% mouse serum at 37° C. Values plotted are means±standard deviation. Arrows indicate ratios selected for further evaluation. (B) BALB/c mice (n=5 animals/group) were immunized with 10 μg pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and varying amounts of alum and boosted at 6 weeks. Serum IgG antibody responses were assessed longitudinally by ELISA. Arrows indicate immunization time points. Values plotted are geometric means±geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (C) Mice (n=5 animals/group) were immunized with 10 μg unmodified or pSer4-conjugated RBDJ (SEQ ID NO: 4-RBDJ) and varying amounts of alum, and germinal center (GC) responses in dLNs were analyzed by flow cytometry at day 14 post-immunization. Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (D) Alum loaded with AlexaFluor™ 647-labeled pSer4-RBDJ (SEQ ID NO: 4-RBDJ) were mixed with alum labeled with pSer4-AlexaFluor™ 488 (labeled SEQ ID NO: 4-AlexaFluor™ 488) at low density and incubated together for 2 days prior to imaging (left). Representative image shown. The fluorescence overlap was assessed (right) and (E) the fraction of alum particles with fluorescent signal colocalization was measured. Values plotted are means±standard deviation. (F) Mice (n=5 animals/group) were immunized with 10 μg pSer4-RBDJ (SEQ ID NO: 4-RBDJ) plus 200 μg alum at varying average antigen densities. Immunizations were prepared by first loading pSer4-RBDJ (SEQ ID NO: 4-RBDJ) on alum at the indicated ratios and then supplementing alum just prior to immunization such that all groups received an equal alum dose. GC responses in dLNs were analyzed by flow cytometry at day 14 post-immunization. Values plotted are means standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (G) Mice (n=5 animals/group) were immunized with 10 μg pSer4-RBDJ (SEQ ID NO: 4-RBDJ) plus 200 μg alum at varying average antigen densities. Serum IgG antibody responses were assessed longitudinally by ELISA. Arrows indicate immunization time points. Values plotted are geometric means±geometric standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. Statistical comparisons between all pSer4-RBDJ (SEQ ID NO: 4-RBDJ) groups are denoted above plot, while statistical comparison between each pSer4-RBDJ (SEQ ID NO: 4-RBDJ) group against the RBDJ group is denoted between pSer4-RBDJ (SEQ ID NO: 4-RBDJ) groups and RBDJ group. (H) Serum SARS-CoV-2 pseudovirus neutralizing titer ID80 (PSV NT80) were assessed for serum collected at day 28 and day 56. The dashed line indicates the limit of detection. Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 11. Co-adjuvants SMNP and CpG promote balanced antibody isotype responses and enhance humoral responses. (A) BALB/c mice were immunized with 10 μg fluorescently labeled RBDJ with or without alum plus co-adjuvants CpG or SMNP, and the fluorescence at the injection site was quantified longitudinally (n=3 animals/group). Values plotted are means±standard deviation. Statistical significance between pSer4-RBDJ (SEQ ID NO: 4-RBDJ) groups was determined by one-way ANOVA followed by Tukey's post-hoc test. (B) Mice (n=5 animals/group) were immunized with 10 μg unmodified RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and 100 μg alum and/or 30 μg CpG or 5 μg SMNP and boosted at 6 weeks, as in FIG. 4E-G. The ratio of IgG2a to IgG1 (left) and IgG2b to IgG1 (right) were calculated at day 35 and day 70. Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (C) Mice (n=5 animals/group) were immunized with 10 μg unmodified RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and 100 μg alum and/or 5 μg SMNP and boosted at 6 weeks, as in FIG. 4G. Serum SARS-CoV-2 pseudovirus neutralizing titer ID80 (PSV NT80) were assessed for serum collected at day 42 and day 84. The dashed line indicates the limit of detection. Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. (D) Mice (n=5 animals/group) were immunized with 10 μg unmodified RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and 100 μg alum and/or 5 μg SMNP and boosted at 6 weeks, as in FIG. 4G. Plotted is the binding titer versus the neutralizing titer for both day 42 and 84. The corresponding linear fit is plotted as a dashed line, and the Pearson correlation was assessed for each timepoint. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 12. Co-adjuvants enhance antigen uptake and germinal center responses BALB/c mice (n=5 animals/group) were immunized with 10 μg AlexaFluor™ 555 labeled antigen and 100 μg alum and 5 μg SMNP or 30 μg CpG, and the inguinal lymph nodes were collected 7 days post-immunization. (A) Representative flow cytometry gating plots. (B) The number of cells positive for AlexaFluor™ 555 labeled antigen is plotted for B cells, monocytes, neutrophils, subcapsular sinus macrophages, medullary macrophages, and dendritic cells. Values plotted are means±standard deviation. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. (C) Mice (n=5 animals/group) were immunized with 10 μg unmodified RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and 100 μg alum and/or 5 μg SMNP and the RBD-specific germinal center (GC) B cell responses in the dLNs were analyzed by flow cytometry at day 14 post-immunization. Values plotted are means±standard error of the mean. Statistical significance was determined by one-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.



FIG. 13. Overview of immunization platform and optimization strategy. (A) Outline of the iterations of the immunization platform. From left to right, we first investigated the impact of N- versus C-terminus pSer conjugation to RBD, moving forward with the N-terminal pSer conjugation approach. Next, we assessed the role of pSer valency with an engineered RBD protein called RBDJ and tested the impact of antigen density on alum. Finally, we added alum-binding co-adjuvants to investigate synergistic enhancement of responses. (B) The corresponding post-boost serum SARS-CoV-2 pseudovirus neutralizing titer ID50 (PSV NT50) for relevant groups, reproduced from FIG. 2C, FIG. 3I, and FIG. 4J. The dashed line indicates the limit of detection. Values plotted are means±standard deviation. Statistical significance was determined by two-way ANOVA followed by Tukey's post-hoc test. ns p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.





DETAILED DESCRIPTION

As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.


Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural or singular number, respectively. Additionally, the words “herein,” “above” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.


As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).


All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.


In all embodiments of polypeptides disclosed herein, any N-terminal methionine residues are optional (i.e.: the N-terminal methionine residue may be present or may be absent, and may be included or excluded when determining percent amino acid sequence identity compared to another polypeptide).


In all embodiments of polypeptides disclosed herein, 1, 2, 3, 4, or 5 amino acids may be deleted from the N-terminus and/or the C-terminus so long as function is maintained, and not be considered when determining percent identity.


As used herein, the term “nanoparticle” refers to submicron particles less 100 nm in dimension. In some embodiments, when nanoparticles form aggregates, the size of the aggregates may exceed 100 nm.


As used herein, “about” will mean up to plus or minus 5% of the particular value.


As used herein, the term “adjuvant” refers to any substance that acts to augment and/or direct antigen-specific immune responses when used in combination with specific antigens. When combined with a vaccine antigen, adjuvant increases the immune response to the vaccine antigen as compared to the response induced by the vaccine antigen alone. Adjuvants help drive immunological mechanisms and shape the output immune response to vaccine antigens.


In a first aspect, the disclosure provides compositions, comprising:


(a) alum; and


(b) a non-liposome, non-micelle particle, wherein the particle comprises a lipid, a sterol, a saponin, and an optional additional non-alum adjuvant, wherein the particle is optionally bound to the alum.


As shown in the studies described herein, compositions of the disclosure provided synergistic enhancements in vaccine immunogenicity when complexed with an antigen (exemplified by SARS-CoV-2 and HIV antigens). Thus, the compositions can be used, for example, as co-adjuvants for significantly improved immune response stimulation.


As used herein, alum is any salt of aluminum. In one embodiment, the alum comprises aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or combinations thereof. In another embodiment, the alum comprises aluminum hydroxide.


The particle is optionally bound to the alum. In one embodiment, the alum and the particle are not bound. In another embodiment, the alum and particle are bound. When bound, the alum and particle may be covalently or non-covalently bound. In one embodiment, the particle is covalently bound to the alum via phosphate residues in the particle.


The particle is a non-liposome, non-micelle particle, wherein the particle comprises a lipid, a sterol, a saponin, and an optional additional non-alum adjuvant. Such particles are described, for example, in published U.S. patent application 20200085756, incorporated by reference herein in its entirety.


In one embodiment the particle is a porous, cage-like nanoparticle comprising saponin, sterol, lipid, and an optional additional adjuvant. Exemplary saponins, sterols, lipids, additional adjuvants including TLR4 agonists, and antigens are discussed in more detail below.


Generally, the nanocage particle is formed by mixing the components together in the presence of a detergent in a suitable ratio such that when the detergent is removed (e.g., by dialysis), the components self-assemble into nanocages. The size of the nanocages is typically dictated by the properties of the components and the self-assembly process. The disclosed compositions and methods typically yield nanocages in the range of about 30 nm and about 60 nm, or about 40 nm to about 50 nm, with an exemplary size being about 40 nm. The nanocages generally assume a distinctive porous morphology that can be structurally distinguished by transmission electronic microscope (TEM) from lipid monolayer (micelle) and lipid bilayer (liposome) particles. The particles are not micelles or liposomes.


A. Saponin

The particles include one or more saponins. A suitable saponin is one that can induce or enhance an immune response. Saponins from plants have proven to be very effective as adjuvants. Saponins are triterpene and steroid glycosides widely distributed in the plant kingdom. Structurally, saponins are amphiphilic surfactants, which explains their surfactant properties, ability to form colloidal solutions, hemolytic activity and ability to form mixed micelles with lipids and sterols. The saponins most studied and used as adjuvants are those from Chilean tree Quillaja saponaria, which have cellular and humoral adjuvant activity. Saponins extracts from Quillaja saponaria with adjuvant activity are known and employed in commercial or experimental vaccines formulation.


A particular saponin preparation is called Quil A®, a saponin preparation isolated from the South American tree Quillaja Saponaria Molina and was first described by Dalsgaard et al. in 1974 (“Saponin adjuvants,” Archiv. für die gesamte Virus forschung, Vol. 44, Springer Verlag, Berlin, p 243-254) to have adjuvant activity. The isolation of pure saponins or better defined mixtures from the Quil A® product having adjuvant activity and lower toxicity than Quil A® have also been described. Purified fragments of Quil A® that retain adjuvant activity without the toxicity associated with Quil A® (EP 0362 278), for example QS7 and QS21 (also known as QA7 and QA21), have been isolated by HPLC. QS-21 is a natural saponin derived from the bark of Quillaja Saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response. QS-21 has been used or is being studied as an adjuvant for various types of vaccines. See also EP 0 362 279 B1 and U.S. Pat. No. 5,057,540.


The isolation and adjuvant activity of other isolated Quil A® saponins, including those called QS-17, and 18 have also been reported, and can also be used in the disclosed nanocages


In other embodiments, the saponin is from Quillaja brasiliensis (A. St.-Hil. et Tul.) Mart., which is native to southern Brazil and Uruguay and has saponins that have proven to be effective as adjuvants with a similar activity against viral antigens as Quil A® (Silveira et al., Vaccine 29 (2011), 9177-9182).


Other useful saponins are derived from the plants Aesculus hippocastanum or Gypsophila struthium. Other saponins which have been described in the literature include escin, which has been described in the Merck index (12th ed: entry 3737) as a mixture of saponins occurring in the seed of the horse chestnut tree, Lat: Aesculus hippocastanum. Its isolation by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and by ion exchange resins (Erbring et al., U.S. Pat. No. 3,238,190) has been described. Fractions of escin have been purified and shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) August 1996; 44(8): 1454-1464)). Sapoalbin from Gypsophila struthium (R. Vochten et al., 1968, J. Pharm. Belg., 42, 213-226) has also been described.


In other embodiments, the saponin is a synthetic saponin. See, e.g., U.S. Published Application No. 2011/0300177 and U.S. Pat. No. 8,283,456, which describe the Triterpene Saponin Synthesis Technology (TriSST) platform, a convergent synthetic approach in which the four domains in QS-21 (branched trisaccharide+triterpene+linear tetrasaccharide+fatty acyl chain) are synthesized separately and then assembled to produce the target molecule. Each of the domains can be modified independently and then combined to produce a virtually infinite number of rationally designed QS-21 analogs. Initially, fully synthetic QS-21 (SQS-21) was shown to be safe and immunologically active in a Phase 1 clinical trial, and later over 100 analogues were prepared and tested in a systematic sequential series of studies. See, e.g., Ragupathi, et al., Expert Rev Vaccines. 2011 April; 10(4): 463-470. See also Zu, et al., Journal of Carbohydrate Chemistry, Volume 33, 2014—Issue 6, pages 269-97.


Preferably the saponin component is in a substantially pure form, for example, at least 90% pure, preferably at least 95% pure and most preferably at least 98% pure.


B. Sterol

The particles include one or more sterols. Sterols include p-sitosterol, stigmasterol, ergosterol, ergocalciferol, campesterol, and cholesterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11th Ed., page 341, as a naturally occurring sterol found in animal fat. In preferred embodiments, the sterol is cholesterol or a derivative thereof e.g., ergosterol or cholesterylhemisuccinate.


C. Lipid

The particles include one or more lipids, such as one or more phospholipids. The lipid can be neutral, anionic, or cationic at physiologic pH. Phospholipids include, but are not limited to, diacylglycerides such as phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin) (PC), phosphatidylserine (PS), and phosphoinositides, e.g., phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol trisphosphate (PIP3), as well as phosphoshingolipids such as ceramide phosphorylcholine (Sphingomyelin) (SPH), ceramide phosphorylethanolamine (Sphingomyelin) (Cer-PE), and ceramide phosphoryllipid, and natural and synthetic phospholipid derivatives such as egg PC (Egg lecithin), egg PG, soy PC, hydrogenated soy PC, sphingomyelin, phosphatidic acid (DMPA, DPPA, DSPA), phosphatidylcholine (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, DEPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), phosphatidylserine (DOPS), and PEG phospholipid (mPEG-phospholipid, polyglycerin-phospholipid, functionalized-phospholipid, terminal activated-phospholipid). Thus, particles can include any one of more of 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-Dierucoyl-sn-glycero-3-phosphate (Sodium Salt) (DEPA-NA), 1,2-Dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) 1,2-Dierucoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DEPG-NA), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-Dilauroyl-sn-glycero-3-phosphate (Sodium Salt) (DLPA-NA) 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC) 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DLPG-NA), 1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Ammonium Salt) (DLPG-NH4), 1,2-Dilauroyl-sn-glycero-3-phosphoserine (Sodium Salt) (DLPS-NA), 1,2-Dimyristoyl-sn-glycero-3-phosphate (Sodium Salt) (DMPA-NA), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DMPG-NA), 1,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Ammonium Salt) (DMPG-NH4), 1,2-Dimyristoyl-sn-glycero-3 [Phospho-rac-(1-glycerol) (Sodium/Ammonium Salt) (DMPG-NH4/NA), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (Sodium Salt) (DMPS-NA), 1,2-Dioleoyl-sn-glycero-3-phosphate (Sodium Salt) (DOPA-NA), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DOPG-NA), 1,2-Dioleoyl-sn-glycero-3-phosphoserine (Sodium Salt) (DOPS-NA), 1,2-Dipalmitoyl-sn-glycero-3-phosphate (Sodium Salt) (DPPA-NA), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DPPG-NA), 1,2-Dipalmitoyl-sn-glycero-3 [Phospho-rac-(1-glycerol) (Ammonium Salt) (DPPG-NH4), 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (Sodium Salt) (DPPS-NA), 1,2-Distearoyl-sn-glycero-3-phosphate (Sodium Salt) (DSPA-NA), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt) (DSPG-NA), 1,2-Distearoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Ammonium Salt) (DSPG-NH4), 1,2-Distearoyl-sn-glycero-3-phosphoserine (Sodium Salt) (DSPS-NA), Egg-PC (EPC), Hydrogenated Egg PC (HEPC), Hydrogenated Soy PC (HSPC), 1-Myristoyl-sn-glycero-3-phosphocholine (LYS OPC MYRISTIC), 1-Palmitoyl-sn-glycero-3-phosphocholine (LYS OPC PALMITIC), 1-Stearoyl-sn-glycero-3-phosphocholine (LYS OPC STEARIC), 1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (Milk Sphingomyelin MPPC), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol) . . . ] (Sodium Salt) (POPG-NA), 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC). Any of the lipids can be PEGylated lipids, for example PEG-DSPE. In a specific embodiment, the phospholipid is 2-Dipalmitoyl-snglycero-3-phosphocholine (DPPC).


D. Adjuvant

The particles may optionally include one or more additional adjuvants. In one embodiment, the particle comprises an additional adjuvant. The additional adjuvant typically has physical and biochemical properties compatible with its incorporation into structure of the particle and that do not prevent particle self-assembly. The additional adjuvant also typically increases at least one immune response relative to the same nanocage formulation in the absence of the additional adjuvant. Immune responses include, but are not limited to, an increase in an antigen-specific antibody response (e.g., IgG, IgG2a, IgG1, or a combination thereof), an increase in a response in germinal centers (e.g., increase in the frequency of germinal center B cells, an increase in frequencies and/or activation of T follicular helper (Tfh) cells, an increase in B cell presence or residence in dark zone of germinal center or a combination thereof), an increase in plasmablast frequency, an increase in inflammatory cytokine expression (e.g., IL-6, IFN-γ, IFN-α, IL-1β, TNF-α, CXCL10 (IP-10), or a combination thereof), an increase in drainage of antigen from the injection site, an in increase in antigen accumulation in the lymph nodes, an increase in lymph node permeability, an increase in lymph flow, an increase in antigen-specific B cell antigen uptake in lymph nodes, an increase in humoral responses beyond the proximal lymph node, increased diffusion of antigen into B cell follicles, or a combination thereof, when the nanocages are administered to a subject, preferably in combination with an antigen.


1. TLR4 Agonists


In some embodiments, the additional adjuvant is a TLR agonist. TLR4 is a transmembrane protein member of the toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signaling pathway NF-κB and inflammatory cytokine production responsible for activating the innate immune system. Classes of TLR agonists include, but are not limited to, viral proteins, polysaccharides, and a variety of endogenous proteins such as low-density lipoprotein, beta-defensins, and heat shock protein.


Exemplary TLR4 agonist include without limitation derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland).


In another embodiment, the TLR4 agonist is a natural or synthetic lipopolysaccharide (LPS), or a lipid A derivative thereof such as MPLA or 3D-MPLA. Lipopolysaccharides are the major surface molecule of, and occur exclusively in, the external leaflet of the outer membrane of gram-negative bacteria. LPS impede destruction of bacteria by serum complements and phagocytic cells, and are involved in adherence for colonization. LPS are a group of structurally related complex molecules of approximately 10,000 Daltons in size and contain three covalently linked regions: (i) an O-specific polysaccharide chain (O-antigen) at the outer region (ii) a core oligosaccharide central region (iii) lipid A—the innermost region which serves as the hydrophobic anchor, it includes glucosamine disaccharide units which carry long chain fatty acids.


The biological activities of LPS, such as lethal toxicity, pyrogenicity and adjuvanticity, have been shown to be related to the lipid A moiety. In contrast, immunogenicity is associated with the 0-specific polysaccharide component (O-antigen). Both LPS and lipid A have long been known for their strong adjuvant effects, but the high toxicity of these molecules has precluded their use in vaccine formulations. Significant effort has therefore been made towards reducing the toxicity of LPS or lipid A while maintaining their adjuvanticity.


The Salmonella minnesota mutant R595 was isolated in 1966 from a culture of the parent (smooth) strain (Luderitz et al. 1966 Ann. N. Y. Acad. Sci. 133:349-374). The colonies selected were screened for their susceptibility to lysis by a panel of phages, and only those colonies that displayed a narrow range of sensitivity (susceptible to one or two phages only) were selected for further study. This effort led to the isolation of a deep rough mutant strain which is defective in LPS biosynthesis and referred to as S. minnesota R595.


In comparison to other LPS, those produced by the mutant S. minnesota R595 have a relatively simple structure. (i) they contain no O-specific region—a characteristic which is responsible for the shift from the wild type smooth phenotype to the mutant rough phenotype and results in a loss of virulence (ii) the core region is very short—this characteristic increases the strain susceptibility to a variety of chemicals (iii) the lipid A moiety is highly acylated with up to 7 fatty acids. 4′-monophosphoryl lipid A (MPLA), which may be obtained by the acid hydrolysis of LPS extracted from a deep rough mutant strain of gram-negative bacteria, retains the adjuvant properties of LPS while demonstrating a toxicity which is reduced by a factor of more than 1000 (as measured by lethal dose in chick embryo eggs) (Johnson et al. 1987 Rev. Infect. Dis. 9 Suppl:S512-S516). LPS is typically refluxed in mineral acid solutions of moderate strength (e.g. 0.1 M HCl) for a period of approximately 30 minutes. This process results in dephosphorylation at the 1 position, and decarbohydration at the 6′ position, yielding MPLA. In some embodiments, the TLR4 agonist is MPLA.


3-O-deacylated monophosphoryl lipid A (3D-MPLA), which can be obtained by mild alkaline hydrolysis of MPLA, has a further reduced toxicity while again maintaining adjuvanticity, see U.S. Pat. No. 4,912,094 (Ribi Immunochemicals). Alkaline hydrolysis is typically performed in organic solvent, such as a mixture of chloroform/methanol, by saturation with an aqueous solution of weak base, such as 0.5 M sodium carbonate at pH 10.5. In some embodiments, the TLR4 agonist is 3D-MPLA.


In some embodiments, the MPLA is a fully synthetic MPLA such as Phosphorylated HexaAcyl Disaccharide (PHAD®), the first fully synthetic monophosphoryl Lipid A available for use as an adjuvant in human vaccines, or Monophosphoryl 3-Deacyl Lipid A (Synthetic) (3D-PHAD®). See also U.S. Pat. No. 9,241,988.


2. Other Exemplary Adjuvants


As introduced above, the additional adjuvant typically has physical and biochemical properties compatible with its incorporation into the structure of the particle and that do not prevented particle self-assembly and increase an immune response. Thus, other suitable adjuvants immunostimulators include those that include a lipid tail, or can be modified to contain a lipid tail. Examples of molecules that include a lipid tail, or can be modified to include one, can be, for example, pathogen-associated molecular patterns (PAMPs). PAMPS are recognized by pattern recognition receptors (PRRs). Five families of PRRs have been shown to initiate pro-inflammatory signaling pathways: Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs). Also, some NLRs are involved in the formation of pro-inflammatory complexes called inflammasomes.


Thus, in some embodiments, the additional adjuvant is a TLR ligand, a NOD ligand, an RLR ligand, a CLR ligand, and inflammasome inducer, a STING ligand, or a combination thereof. Such ligands are known in the art can obtained through commercial vendors such as InvivoGen.


As introduced above, the ligands and other adjuvants can be modified (e.g., through chemical conjugation, for example, maleimide thiol reaction, amine N-hydroxysuccinimide ester reaction, click chemistry, etc.) to include a lipid tail to facilitate incorporation of the adjuvant into the nanocage structure during self-assembly. Preferred lipids will include a 16:0 dipalmitoyl tail such as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], these, however, are non-limiting examples. For example, lipids of different lengths are also contemplated. In preferred embodiments, the lipid or lipids is/are unsaturated. Chemically functionalized lipids that that can be used for conjugation are known in the art and commercially available. See, for example, AVANTI® Polar Lipids, Inc. (e.g., “Headgroup Modified Lipids” and “Functionalized Lipids”).


The additional adjuvant can be an immunostimulatory oligonucleotide, preferable a lipidated immunostimulatory oligonucleotide. Exemplary lapidated immunostimulatory oligonucleotides and methods of making them are described in Liu, et al., Nature Letters, 507:519-22 (+11 pages of extended data) (2014)) (lipo-CpG) and U.S. Pat. No. 9,107,904, that contents of which are incorporated by reference herein in their entireties. In some embodiments, the immunostimulatory oligonucleotide portion of the adjuvant can serve as a ligand for PRRs. Therefore, the oligonucleotide can serve as a ligand for a Toll-like family signaling molecule, such as Toll-Like Receptor 9 (TLR9).


For example, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells in humans (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Therefore, the sequence of the oligonucleotide can include one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. The ‘p’ refers to the phosphodiester backbone of DNA, as discussed in more detail below, some oligonucleotides including CG can have a modified backbone, for example a phosphorothioate (PS) backbone.


In some embodiments, an immunostimulatory oligonucleotide can contain more than one CG dinucleotide, arranged either contiguously or separated by intervening nucleotide(s). The CpG motif(s) can be in the interior of the oligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9 with variations in the number and location of CG dinucleotide(s), as well as the precise base sequences flanking the CG dimers.


Typically, CG ODNs are classified based on their sequence, secondary structures, and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are Class A (Type D), Class B (Type K), Class C, Class P, and Class S (Vollmer, J & Krieg, A M, Advanced drug delivery reviews 61(3): 195-204 (2009), incorporated herein by reference). CG ODNs can stimulate the production of Type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also strong activators of natural killer (NK) cells through indirect cytokine signaling. Some classes are strong stimulators of human B cell and monocyte maturation (Weiner, G L, PNAS USA 94(20): 10833-7 (1997); Dalpke, A H, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).


Other PRR Toll-like receptors include TLR3, and TLR7 which may recognize double-stranded RNA, single-stranded and short double-stranded RNAs, respectively, and retinoic acid-inducible gene I (RIG-I)-like receptors, namely RIG-I and melanoma differentiation-associated gene 5 (MDAS), which are best known as RNA-sensing receptors in the cytosol. Therefore, in some embodiments, the oligonucleotide contains a functional ligand for TLR3, TLR7, or RIG-I-like receptors, or combinations thereof.


Examples of immunostimulatory oligonucleotides, and methods of making them are known in the art, see for example, Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein by reference.


In some embodiments, the oligonucleotide includes two or more immunostimulatory sequences.


Microbial cell-wall components such as Pam2CSK4, Pam3CSK4, and flagellin activate TLR2 and TLR5 receptors respectively and can also be used.


Any suitable ratios of the various particle components may be used. In one embodiment, comprising a lipid:additional adjuvant:sterol:saponin molar ratio of 2.5:1:10:10, or a variation thereof wherein the molar ratio of lipid, additional adjuvant, sterol, saponin or any combination thereof is increased or decreased by any value between about 0 and about 3. In a specific embodiment, the lipid is DPPC, the additional adjuvant is a natural or synthetic MPLA, the sterol is cholesterol, and the saponin is Quil A® in a molar ratio of 2.5:1:10:10. In another embodiment the Quil-A:chol:DPPC:MPLA are in a mass ratio of 10:2:1:1. See US20200085756 for exemplary methods for modifying the molar ratio or mass ratio of the particle components.


In another embodiment, the composition further comprises an antigen, either bound to the alum and/or the particle, or present in the composition unbound to the alum or the particle. In one embodiment, the antigen is bound to the alum and/or the particle. Any antigen may be used as appropriate for an intended use. In one embodiment, the antigen comprises an antigenic polypeptide. The antigen may be bound covalently or non-covalently. In one embodiment, the antigen is covalently bound to the alum and/or the particle. In another embodiment, the antigen or the antigenic polypeptide is covalently bound to the alum.


The antigen may be bound to the alum and/or particle via any suitable means. In some embodiments, the antigen is bound to the alum and/or particle via a linker comprising phosphoserine residues, as described in published U.S. patent application U.S. 20190358312, incorporated by reference herein in its entirety. In one embodiment, the antigen is bound to the alum via a linker comprising phosphoserine residues. As used herein, linkers comprising phosphoserine residues are referred herein as “phosphoserine linkers” (PS-linkers). In all embodiments, the linker may comprise any further residues suitable for linking a given antigen to the alum and/or particle. In some embodiment, the PS-linker comprises a polypeptide linker containing phosphoserine residues. In another embodiment the PS-linker comprises 1-12 consecutive PS residues followed by a short poly(ethylene glycol) spacer and N-terminal maleimide functional group. In another embodiment, the maleimide functional group at the N-terminal of the PS-linker is covalently via a thioether linkage to a thiol group on the antigen. In yet another embodiment, the multiple PS-linkers are conjugated to an antigen protein via azide functional groups and coupled to a DBCO-modified antigen. The linkers may be employed, for instance, to ensure that an antigen is positioned relative alum to ensure proper folding and formation of the antigen or to block or expose particular epitopes. In one embodiment, the antigen comprises at least one linker comprising 2-12 phosphoserine residues, wherein the antigen is covalently bound to the alum via the phosphoserine residues. In another embodiment, the antigen comprises at least one linker comprising 2-8 phosphoserine residues. In a further embodiment, the antigen polypeptide comprises at least one linker comprising 2-4 phosphoserine residues. The linker may be present at any suitable position on the antigen; in one embodiment, the at least one linker is present at the N-terminus or C-terminus of the antigenic polypeptide.


Any antigen may be used in the compositions of the disclosure as deemed suitable for an intended use. The compositions may comprise a single antigen, or may comprise a plurality (2, 3, 4, 5, or more) of different antigens.


E. Antigen

Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, or combinations thereof. The antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components or molecular components thereof.


Suitable antigens are known in the art and are available from commercial, government, and scientific sources. The antigens may be whole inactivated or attenuated organisms, or derived therefrom. These organisms may be infectious organisms, such as viruses, parasites and bacteria. These organisms may also be tumor cells, or derived therefrom. For example, the antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources. The antigens can be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. The antigens can be DNA encoding all or part of an antigenic protein. The DNA may be in the form of vector DNA such as plasmid DNA.


Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids. Exemplary antigens are provided below.


1. Viral Antigens


A viral antigen can be isolated from any virus including, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus and SARS-CoV-2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (for example, rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of Dengue protein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and Dengue D1NS3.


Viral antigens may be derived from a particular strain such as a papilloma virus, a herpes virus, e.g., herpes simplex 1 and 2; a hepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borne encephalitis viruses; parainfluenza, varicella-zoster, cytomegalovirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.


2. Bacterial Antigens


Bacterial antigens can originate from any bacteria including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.


3. Parasite Antigens


Parasite antigens can be obtained from parasites such as, but not limited to, an antigen derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoan antigens, Plasmodian antigens, such as all or part of a Circumsporozoite protein, a Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Merozoite surface protein.


4. Allergens and Environmental Antigens


The antigen can be an allergen or environmental antigen, such as, but not limited to, an antigen derived from naturally occurring allergens such as pollen allergens (tree-, herb, weed-, and grass pollen allergens), insect allergens (inhalant, saliva and venom allergens), animal hair and dandruff allergens, and food allergens. Important pollen allergens from trees, grasses and herbs originate from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including i.a. birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), Plane tree (Platanus), the order of Poales including e.g., grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including i.a. herbs of the genera Ambrosia, Artemisia, and Parietaria. Other allergen antigens that may be used include allergens from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blattella, Periplaneta, Chironomus and Ctenocephalides, those from mammals such as cat, dog and horse, birds, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Still other allergen antigens that may be used include inhalation allergens from fungi such as from the genera Alternaria and Cladosporium.


5. Cancer Antigens


A cancer antigen is an antigen that is typically expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in some instances it is expressed solely by cancer cells. The cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell. The cancer antigen can be MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated antigen (CRC)—0017-1A/GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD3-zeta chain, and CD20. The cancer antigen may be selected from the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad family of tumor antigens, 1 mp-1, PIA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, or c-erbB-2.


6. Tolerogenic Antigens


The antigen can be a tolerogenic antigen. Exemplary antigens are known in the art. See, for example, U.S. Published Application No. 2014/0356384. In some cases, the tolerogenic antigen is derived from a therapeutic agent protein to which tolerance is desired. Examples are protein drugs in their wild type, e.g., human factor VIII or factor IX, to which patients did not establish central tolerance because they were deficient in those proteins; or nonhuman protein drugs, used in a human. Other examples are protein drugs that are glycosylated in nonhuman forms due to production, or engineered protein drugs, e.g., having non-native sequences that can provoke an unwanted immune response. Examples of tolerogenic antigens that are engineered therapeutic proteins not naturally found in humans including human proteins with engineered mutations, e.g., mutations to improve pharmacological characteristics. Examples of tolerogenic antigens that have nonhuman glycosylation include proteins produced in yeast or insect cells.


Tolerogenic antigens can be from proteins that are administered to humans that are deficient in the protein. Deficient means that the patient receiving the protein does not naturally produce enough of the protein. Moreover, the proteins may be proteins for which a patient is genetically deficient. Such proteins include, for example, antithrombin-III, protein C, factor VIII, factor IX, growth hormone, somatotropin, insulin, pramlintide acetate, mecasermin (IGF-1), β-gluco cerebrosidase, alglucosidase-alpha, laronidase (α-L-iduronidase), idursuphase (iduronate-2-sulphatase), galsulphase, agalsidase-beta (α-galactosidase), α-1 proteinase inhibitor, and albumin.


The tolerogenic antigen can be from therapeutic antibodies and antibody-like molecules, including antibody fragments and fusion proteins with antibodies and antibody fragments. These include nonhuman (such as mouse) antibodies, chimeric antibodies, and humanized antibodies. Immune responses to even humanized antibodies have been observed in humans (Getts D R, Getts M T, McCarthy D P, Chastain E M L, & Miller S D (2010), mAbs, 2(6):682-694).


The tolerogenic antigen can be from proteins that are nonhuman. Examples of such proteins include adenosine deaminase, pancreatic lipase, pancreatic amylase, lactase, botulinum toxin type A, botulinum toxin type B, collagenase, hyaluronidase, papain, L-Asparaginase, rasburicase, lepirudin, streptokinase, antistreplase (anisoylated plasminogen streptokinase activator complex), antithymocyte globulin, crotalidae polyvalent immune Fab, digoxin immune serum Fab, L-arginase, and L-methionase.


Tolerogenic antigens include those from human allograft transplantation antigens. Examples of these antigens are the subunits of the various MHC class I and MHC class II haplotype proteins, and single-amino-acid polymorphisms on minor blood group antigens including RhCE, Kell, Kidd, Duffy and Ss.


The tolerogenic antigen can be a self-antigen against which a patient has developed an autoimmune response or may develop an autoimmune response. Examples are proinsulin (diabetes), collagens (rheumatoid arthritis), myelin basic protein (multiple sclerosis). For instance, Type 1 diabetes mellitus (T1D) is an autoimmune disease whereby T cells that recognize islet proteins have broken free of immune regulation and signal the immune system to destroy pancreatic tissue. Numerous protein antigens that are targets of such diabetogenic T cells have been discovered, including insulin, GAD65, chromogranin-A, among others. In the treatment or prevention of T1D, it would be useful to induce antigen-specific immune tolerance towards defined diabetogenic antigens to functionally inactivate or delete the diabetogenic T cell clones.


Tolerance and/or delay of onset or progression of autoimmune diseases may be achieved for various of the many proteins that are human autoimmune proteins, a term referring to various autoimmune diseases wherein the protein or proteins causing the disease are known or can be established by routine testing. In some embodiments, a patient is tested to identify an autoimmune protein and an antigen is created for use in a molecular fusion to create immunotolerance to the protein.


Embodiments can include an antigen, or choosing an antigen from or derived from, one or more of the following proteins. In type 1 diabetes mellitus, several main antigens have been identified: insulin, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD-65), GAD-67, insulinoma-associated protein 2 (IA-2), and insulinoma-associated protein 2.beta. (IA-213); other antigens include ICA69, ICA12 (SOX-13), carboxypeptidase H, Imogen 38, GLIMA 38, chromogranin-A, FISP-60, carboxypeptidase E, peripherin, glucose transporter 2, hepatocarcinoma-intestine-pancreas/pancreatic associated protein, S100p, glial fibrillary acidic protein, regenerating gene II, pancreatic duodenal homeobox 1, dystrophia myotonica kinase, islet-specific glucose-6-phosphatase catalytic subunit-related protein, and SST G-protein coupled receptors 1-5. In autoimmune diseases of the thyroid, including Hashimoto's thyroiditis and Graves' disease, main antigens include thyroglobulin (TG), thyroid peroxidase (TPO) and thyrotropin receptor (TSHR); other antigens include sodium iodine symporter (NIS) and megalin. In thyroid-associated ophthalmopathy and dermopathy, in addition to thyroid autoantigens including TSHR, an antigen is insulin-like growth factor 1 receptor. In hypoparathyroidism, a main antigen is calcium sensitive receptor. In Addison's disease, main antigens include 21-hydroxylase, 17α-hydroxylase, and P450 side chain cleavage enzyme (P450scc); other antigens include ACTH receptor, P450c21 and P450c17. In premature ovarian failure, main antigens include FSH receptor and .alpha.-enolase. In autoimmune hypophysitis, or pituitary autoimmune disease, main antigens include pituitary gland-specific protein factor (PGSF) 1a and 2; another antigen is type 2 iodothyronine deiodinase. In multiple sclerosis, main antigens include myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein. In rheumatoid arthritis, a main antigen is collagen II. In immunogastritis, a main antigen is H+, K+-ATPase. In pernicious angemis, a main antigen is intrinsic factor. In celiac disease, main antigens are tissue transglutaminase and gliadin. In vitiligo, a main antigen is tyrosinase, and tyrosinase related protein 1 and 2. In myasthenia gravis, a main antigen is acetylcholine receptor. In pemphigus vulgaris and variants, main antigens are desmoglein 3, 1 and 4; other antigens include pemphaxin, desmocollins, plakoglobin, perplakin, desmoplakins, and acetylcholine receptor. In bullous pemphigoid, main antigens include BP180 and BP230; other antigens include plectin and laminin 5. In dermatitis herpetiformis Duhring, main antigens include endomysium and tissue transglutaminase. In epidermolysis bullosa acquisita, a main antigen is collagen VII. In systemic sclerosis, main antigens include matrix metalloproteinase 1 and 3, the collagen-specific molecular chaperone heat-shock protein 47, fibrillin-1, and PDGF receptor; other antigens include Sc1-70, U1 RNP, Th/To, Ku, Jol, NAG-2, centromere proteins, topoisomerase I, nucleolar proteins, RNA polymerase I, II and III, PM-Slc, fibrillarin, and B23. In mixed connective tissue disease, a main antigen is U1 snRNP. In Sjogren's syndrome, the main antigens are nuclear antigens SS-A and SS-B; other antigens include fodrin, poly(ADP-ribose) polymerase and topoisomerase. In systemic lupus erythematosus, main antigens include nuclear proteins including SS-A, high mobility group box 1 (HMGB1), nucleosomes, histone proteins and double-stranded DNA. In Goodpasture's syndrome, main antigens include glomerular basement membrane proteins including collagen IV. In rheumatic heart disease, a main antigen is cardiac myosin. Other autoantigens revealed in autoimmune polyglandular syndrome type 1 include aromatic L-amino acid decarboxylase, histidine decarboxylase, cysteine sulfinic acid decarboxylase, tryptophan hydroxylase, tyrosine hydroxylase, phenylalanine hydroxylase, hepatic P450 cytochromes P4501A2 and 2A6, SOX-9, SOX-10, calcium-sensing receptor protein, and the type 1 interferons interferon alpha, beta and omega.


In some cases, the tolerogenic antigen is a foreign antigen against which a patient has developed an unwanted immune response. Examples are food antigens. Some embodiments include testing a patient to identify foreign antigen and creating a molecular fusion that comprises the antigen and treating the patient to develop immunotolerance to the antigen or food. Examples of such foods and/or antigens are provided. Examples are from peanut: conarachin (Ara h 1), allergen II (Ara h 2), arachis agglutinin, conglutin (Ara h 6); from apple: 31 kda major allergen/disease resistance protein homolog (Mal d 2), lipid transfer protein precursor (Mal d 3), major allergen Mal d 1.03D (Mal d 1); from milk: .alpha.-lactalbumin (ALA), lactotransferrin; from kiwi: actinidin (Act c 1, Act d 1), phytocystatin, thaumatin-like protein (Act d 2), kiwellin (Act d 5); from mustard: 2S albumin (Sin a 1), 11 S globulin (Sin a 2), lipid transfer protein (Sin a 3), profilin (Sin a 4); from celery: profilin (Api g 4), high molecular weight glycoprotein (Api g 5); from shrimp: Pen a 1 allergen (Pen a 1), allergen Pen m 2 (Pen in 2), tropomyosin fast isoform; from wheat and/or other cereals: high molecular weight glutenin, low molecular weight glutenin, alpha- and gamma-gliadin, hordein, secalin, avenin; from strawberry: major strawberry allergy Fra a 1-E (Fra a 1), from banana: profilin (Mus xp 1).


Many protein drugs that are used in human and veterinary medicine induce immune responses, which create risks for the patient and limits the efficacy of the drug. This can occur with human proteins that have been engineered, with human proteins used in patients with congenital deficiencies in production of that protein, and with nonhuman proteins. It would be advantageous to tolerize a recipient to these protein drugs prior to initial administration, and it would be advantageous to tolerize a recipient to these protein drugs after initial administration and development of immune response. In patients with autoimmunity, the self-antigen(s) to which autoimmunity is developed are known. In these cases, it would be advantageous to tolerize subjects at risk prior to development of autoimmunity, and it would be advantageous to tolerize subjects at the time of or after development of biomolecular indicators of incipient autoimmunity. For example, in Type 1 diabetes mellitus, immunological indicators of autoimmunity are present before broad destruction of beta cells in the pancreas and onset of clinical disease involved in glucose homeostasis. It would be advantageous to tolerize a subject after detection of these immunological indicators prior to onset of clinical disease.


7. Neoantigens and Personalized Medicine


In some embodiments the antigen is a neoantigen or a patient-specific antigen. Recent technological improvements have made it possible to identify the immune response to patient-specific neoantigens that arise as a consequence of tumor-specific mutations, and emerging data indicate that recognition of such neoantigens is a major factor in the activity of clinical immunotherapies (Schumacher and Schreidber, Science, 348(6230):69-74 (2015). Neoantigen load provides an avenue to selectively enhance T cell reactivity against this class of antigens.


Traditionally, cancer vaccines have targeted tumor-associated antigens (TAAs) which can be expressed not only on tumor cells but in the normal tissues (Ito, et al., Cancer Neoantigens: A Promising Source of Immunogens for Cancer Immunotherapy. J Clin Cell Immunol, 6:322 (2015) doi:10.4172/2155-9899.1000322). TAAs include cancer-testis antigens and differentiation antigens, and even though self-antigens have the benefit of being useful for diverse patients, expanded T cells with the high-affinity TCR (T-cell receptor) needed to overcome the central and peripheral tolerance of the host, which would impair anti-tumor T-cell activities and increase risks of autoimmune reactions.


Thus, in some embodiments, the antigen is recognized as “non-self” by the host immune system, and preferably can bypass central tolerance in the thymus. Examples include pathogen-associated antigens, mutated growth factor receptor, mutated K-ras, or idiotype-derived antigens. Somatic mutations in tumor genes, which usually accumulate tens to hundreds of fold during neoplastic transformation, could occur in protein-coding regions. Whether missense or frameshift, every mutation has the potential to generate tumor-specific antigens. These mutant antigens can be referred to as “cancer neoantigens” Ito, et al., Cancer Neoantigens: A Promising Source of Immunogens for Cancer Immunotherapy. J Clin Cell Immunol, 6:322 (2015) doi:10.4172/2155-9899.1000322. Neoantigen-based cancer vaccines have the potential to induce more robust and specific anti-tumor T-cell responses compared with conventional shared-antigen-targeted vaccines. Recent developments in genomics and bioinformatics, including massively parallel sequencing (MPS) and epitope prediction algorithms, have provided a major breakthrough in identifying and selecting neoantigens.


Methods of identifying, selecting, and validating neoantigens are known in the art. See, for example, Ito, et al., Cancer Neoantigens: A Promising Source of Immunogens for Cancer Immunotherapy. J Clin Cell Immunol, 6:322 (2015) doi:10.4172/2155-9899.1000322, which is specifically incorporated by reference herein in its entirety. For example, as discussed in Ito, et al., a non-limiting example of identifying a neoantigen can include screening, selection, and optionally validation of candidate immunogens. First, the whole genome/exome sequence profile is screened to identify tumor-specific somatic mutations (cancer neoantigens) by MPS of tumor and normal tissues, respectively. Second, computational algorithms are used for predicting the affinity of the mutation-derived peptides with the patient's own HLA and/or TCR. The mutation-derived peptides can serve as antigens for the compositions and methods disclosed herein. Third, synthetic mutated peptides and wild-type peptides can be used to validate the immunogenicity and specificity of the identified antigens by in vitro T-cell assay or in vivo immunization.


In all of these embodiments, any suitable molar ratio of alum:particle may be used in the compositions. In one embodiment, a molar ratio of alum:particle is between about 1:500 and about 500:1. Similarly, any suitable molar ratio of alum:antigen or antigenic polypeptide may be used; in one embodiment, the molar ratio is between about 1:500 and about 500:1.


In a further embodiment, the disclosure provides pharmaceutical compositions comprising the composition of any embodiment of the disclosure, and a pharmaceutically acceptable carrier. In this embodiment, the compositions are combined with a pharmaceutically acceptable carrier. Suitable acids which are capable of forming such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).


In some embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In some embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the immunogenic composition.


In some embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In some embodiments, an immunogenic composition can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in some embodiments, an immunogenic composition can be formulated as a lyophilizate using appropriate excipients such as sucrose.


The pharmaceutical compositions of the invention may be made up in any suitable formulation, preferably in formulations suitable for administration by parenteral delivery such as subcutaneous of intra-venous injection, inhalation, or oral delivery. Such pharmaceutical compositions can be used, for example, in the therapeutic methods disclosed herein.


The pharmaceutical compositions may contain any other components as deemed appropriate for a given use. In another embodiment, the disclosure provides vaccines comprising the composition of any embodiment of the disclosure in which an antigen is present. The compositions and vaccines may be used, for example in the methods of the disclosure.


In another aspect, the disclosure provides methods for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of (a) the composition of any embodiment herein in which an antigen is not bound to the alum and/or particle and (b) an antigen. In this embodiment, the composition and antigen are unlinked at the time of administration, and the antigen may be in the same or a separate pharmaceutical composition from the composition.


In another embodiment, the disclosure provides methods for generating an immune response, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition or vaccine of any embodiment herein in which the composition includes a bound antigen.


The “immune response” refers to responses that induce, increase, or perpetuate the activation or efficiency of innate or adaptive immunity. The immune response includes, but is not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.


In a further embodiment, the disclosure provides methods of treating a subject in need thereof comprising administering to the subject the composition or vaccine of any embodiment herein in an effective amount to induce an immune response against an antigen.


In some embodiments, the immunogenic compositions are administered as part of prophylactic vaccines or immunogenic compositions which confer resistance in a subject to subsequent exposure to infectious agents, or as part of therapeutic vaccines, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a viral antigen in a subject infected with a with an infectious agent or neoplasm. The desired outcome of a prophylactic or therapeutic immune response may vary according to the disease or condition to be treated, car according to principles well known in art. For example, an immune response against an infectious agent may completely prevent colonization and replication of an infectious agent, affecting “sterile immunity” and the absence of any disease symptoms. However, a vaccine against infectious agents may be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or reduces the transmission of an infectious agent. Similarly, immune responses against cancer, allergens or infectious agents may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease.


Methods for analyzing an antibody response in a subject are known to those of skill in the art. For example, in some embodiments an increase in an immune response is measured by ELISA assays to determine antigen-specific antibody titers. In some embodiments, the methods increasing broadly neutralizing antibodies in a subject. Methods for measuring neutralizing antibodies are known to those of ordinary skill in the art. In some embodiments, elicitation of neutralizing antibodies is measured in a neutralization assay. Methods for identifying and measuring neutralizing antibodies are known to those of skill in the art. Neutralizing antibodies are an indicator of the protective efficacy of a vaccine, but direct protection from a sub-lethal or lethal challenge of virus unequivocally demonstrates the efficacy of the vaccine. In an exemplary animal model system, a bacterial or virus challenge is conducted wherein the subjects are immunized, optionally more than once, and challenged after immune response to the vaccine has developed. Elicitation of neutralization may be quantified by measurement of morbidity or mortality on the challenged subjects.


In some embodiments, the administration of the composition or vaccine induces an improved B-memory cell response in immunized subjects. An improved B-memory cell response is intended to mean an increased frequency of peripheral blood B lymphocytes capable of differentiation into antibody-secreting plasma cells upon antigen encounter as measured by stimulation of in vitro differentiation. In some embodiments, the methods increase the number of antibody secreting B cells. In some embodiments, the antibody secreting B cells are bone marrow plasma cells, or germinal center B cells. In some embodiments, methods for measuring the number of antibody secreting B cells, includes, but are not limited to, an antigen-specific ELISPOT assay and flow cytometric studies of plasma cells, or germinal center B cells collected at various time points post-immunization.


In some embodiments, an immunogenic composition or vaccine, described herein, is useful for treating a disorder associated with abnormal apoptosis or a differentiative process (e.g., cellular proliferative disorders (e.g., hyperproliferative disorders) or cellular differentiative disorders, such as cancer). Non-limiting examples of cancers that are amenable to treatment with the methods of the present invention include but are not limited to carcinomas, sarcomas, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver. Accordingly, the compositions used herein, comprising an immunogenic composition or vaccine, can be administered to a patient who has cancer. The terms “cancer” or “neoplasm” are used to refer to malignancies of the various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, and the genitourinary tract, as well as to adenocarcinomas which are generally considered to include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The immunogenic composition can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macro globulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.


It will be appreciated by those skilled in the art that amounts for an immunogenic composition or vaccine that is sufficient to reduce tumor growth and size, or a therapeutically effective amount, will vary not only on the particular composition or vaccine selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist. The length of time during which the compound used in the instant method will be given varies on an individual basis. In some embodiments, the disclosure provides methods of reducing or decreasing the size of a tumor, or inhibiting a tumor growth in a subject in need thereof, comprising administering to the subject an immunogenic composition or vaccine described herein. In some embodiments, the disclosure provides methods for inducing an anti-tumor response in a subject with cancer, comprising administering to the subject an immunogenic composition or vaccine described herein


In some embodiments, an immunogenic composition or vaccine disclosed herein is useful for treating acute or chronic infectious diseases. Thus, in some embodiments an immunogenic composition or vaccine is administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), severe acute respiratory syndrome (e.g., SARS such as SARS-CoV-2), and common cold (e.g., human rhinovirus) viral infections. In some embodiments, pharmaceutical formulations including the immunogenic composition are administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts. In some embodiments, an immunogenic composition or vaccine is administered to treat systemic viral diseases, including, but not limited to, SARS, AIDS, influenza, the common cold, or encephalitis.


In some embodiments, the disclosure provides methods of reducing a viral infection in a subject in need thereof, comprising administering to the subject an immunogenic composition or vaccine described herein. In some embodiments, the disclosure provides methods for inducing an anti-viral response in a subject with cancer, comprising administering to the subject an immunogenic composition or vaccine described herein.


The “subject” may be any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.


In all embodiments, the antigen may be any antigen suitable for an intended use, including but not limited to those antigens disclosed herein. In one embodiment, the antigen is derived from tumor cells or a microbe; in such embodiments, the subject may have or be at risk of developing cancer or an infection associated with tumor cells or microbe. In all embodiments, administration of the compositions may be by any suitable route, including but not limited to subcutaneous, intramuscular, intradermal, or intravenous injection. In one embodiment, the alum is bound to the particle at the time of administering. In another embodiment, the antigen is bound to the alum and/or the particle at the time of administering. In a further embodiment, the antigen is bound to the alum at the time of administering.


Example 1
SUMMARY

There is a need for additional rapidly scalable, low-cost vaccines against SARS-CoV-2 to achieve global vaccination. Aluminum hydroxide (alum) adjuvant is the most widely available vaccine adjuvant but elicits modest humoral responses. We hypothesized that phosphate-mediated co-anchoring of the receptor binding domain (RBD) of SARS-CoV-2 immunogen together with molecular adjuvants on alum particles could potentiate humoral immunity by promoting extended vaccine kinetics and co-delivery of vaccine components to lymph nodes. Modification of RBD immunogens with phosphoserine (pSer) peptides enabled efficient alum binding and slowed antigen clearance in vivo, leading to striking increases in germinal center responses and neutralizing antibody titers in mice. Adding phosphate-containing CpG or saponin adjuvants to pSer-RBD:alum immunizations synergistically enhanced vaccine immunogenicity. Thus, phosphate-mediated co-anchoring of RBD and molecular adjuvants to alum is an effective strategy to enhance the efficacy of SARS-CoV-2 subunit vaccines.


INTRODUCTION

The coronavirus disease 2019 (COVID-19) pandemic was caused by the emergence of a novel beta-coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), leading to over 200 million confirmed cases and 4 million deaths worldwide. Viral entry into cells is mediated by the receptor binding domain (RBD) of the SARS-CoV-2 S protein, which binds the human angiotensin-converting enzyme 2 (hACE2) receptor (1, 2). Neutralizing antibody responses against the spike protein or the RBD have been shown to protect against SARS-CoV-2 infection in animal models (3-5) and are believed to be a correlate of protection against SARS-CoV-2 (6-9), making the RBD an attractive vaccine target for neutralizing responses (9-12).


Although safe and effective vaccines are already being deployed in many developed nations, there remains a significant need for strategies to facilitate global SARS-CoV-2 vaccine coverage. To this end, subunit vaccines are attractive for their ability to be produced at low cost, at scale and without the need for ultra-cold storage temperatures, but the global supply of adjuvants for accessible vaccines is unclear. The most common clinical vaccine adjuvant, alum, is well-suited to global vaccination campaigns due to its manufacturability and low cost, but alum has exhibited relatively poor immunogenicity with SARS-CoV-2 subunit vaccines to date (10, 11). Of equal importance to these practical issues is the ability of vaccines to promote neutralizing responses to SARS-CoV-2 variants that are now circulating globally (13-16). A number of preclinical studies have demonstrated that vaccines eliciting higher levels of neutralizing responses against the original Wuhan-Hu-1 virus tend to also elicit high neutralizing titers against these viral variants (17-21). Hence, approaches to enhance the immunogenicity of alum-adjuvanted subunit vaccines for SARS-CoV-2 may be important in the effort to achieve global vaccination coverage.


We recently described an approach to augment alum:protein subunit vaccines by site-specific introduction of phosphoserine (pSer) peptide tags onto protein immunogens (26). pSer tagging allows immunogens to bind to the surface of aluminum hydroxide via a ligand exchange reaction, providing tight binding that can be tuned by the valency of the pSer peptide tag sequence. Stable anchoring to alum was shown to prolong antigen delivery to lymph nodes via slow trafficking of alum particles, coincident with direct B cell triggering by antigen multivalently displayed on alum. These changes in the physical chemistry of vaccine delivery enhanced germinal center (GC) responses, serum antibody titers, and neutralizing antibody titers against human immunodeficiency virus (HIV) envelope (Env) immunogens (26).


Despite these promising data, alum remains an adjuvant that does not stimulate many of the innate immune recognition pathways that might be exploited to drive robust immune responses. We hypothesized that phosphate-mediated binding could be used to co-anchor SARS-CoV-2 and other antigens and complementary molecular adjuvants to alum particles to synergistically drive humoral immunity. To test this idea, we evaluated the potential of pSer-tagging to enhance the immunogenicity of alum:RBD subunit vaccines in mice. We assessed alum binding, antigen structural stability, and in vivo humoral immune responses for pSer-modified RBD proteins. Immunization with pSer-labeled RBD antigens was found to greatly enhance the immunogenicity of this antigen in combination with alum. To further amplify these responses, we combined pSer-tagged RBD with the Toll-like receptor (TLR)-9 ligand CpG or a saponin/phospholipid nanoparticle adjuvant (SMNP) that intrinsically contain phosphate residues (27), for co-adsorption to alum. We found that the persistence of these adjuvants in vivo could be significantly increased by complexing with pSer-RBD and alum, correlating with synergistic enhancements in vaccine immunogenicity. These findings indicate that phosphoserine modification is a promising way to enhance the efficacy of SARS-CoV-2 subunit vaccines, and that combining alum with molecular adjuvants capable of undergoing ligand-exchange-mediated binding can further substantially potentiate humoral immunity.


Results
Phosphoserine Peptide Modification Facilitates Stable Binding of SARS-CoV-2 RBD to Alum

We first tested whether coupling a pSer peptide tag to Wuhan-Hu-1 SARS-CoV-2 RBD could engender stable binding to alum without disrupting key epitopes on the antigen. RBD (amino acids 332-532 of SARS-CoV-2 S protein, table 1) modified with a histag for purification and containing an N- or C-terminal free cysteine was expressed in yeast, and then conjugated with a peptide tag containing a maleimide group linked to a 6-unit poly(ethylene glycol) spacer followed by four phosphoserine residues (FIG. 6). Measurement of the mean number of phosphates per protein using a malachite green assay revealed that the N-terminal-coupled pSer4-RBD (SEQ ID NO: 4-RBD) and C-terminal-modified RBD-pSer4 (RBD-SEQ ID NO: 4) gained the expected ˜4 phosphates per protein (FIG. 1A). Incubation of unmodified RBD with alum in tris-buffered saline led to adsorption of only ˜25% of the antigen (FIG. 1B “loading”), most of which desorbed following a 24-hour incubation of the alum in phosphate buffer containing 10% serum (FIG. 1B “10% serum”). By contrast, both pSer-RBDs exhibited high levels of alum binding in buffer, with the majority remaining bound after serum/phosphate exposure (FIG. 1B). To confirm that the alum-bound RBD conjugates were structurally intact relative to unmodified protein, we used a modified sandwich ELISA approach to probe the antigenicity of the constructs. Unmodified RBD was captured on plates coated with a histag-specific antibody, while pSer-conjugated RBD was captured on alum-coated plates (FIG. 1C). The immobilized RBD was then probed for binding to serial dilutions of recombinant hACE2 protein, the target receptor recognized by RBD, or monoclonal antibodies CR3022 (which recognizes a highly conserved epitope distal from the receptor binding site (28)), H4, or B38. As shown in FIGS. 1D-H, the pSer-modified RBDs had antigenicity profiles indistinguishable from unmodified RBD, and the proteins captured on alum retained recognition of both probes. Thus, pSer modification allowed substantially enhanced RBD binding to alum without disrupting its structure.









TABLE 1







Antigen sequences.








Protein
Sequence





RBD
ITNLCPFGEVFNATRFASVYAWNRKRISNCVADYS


C-terminal
VLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV


cysteine
IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAW



NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTE



IYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ



PYRVVVLSFELLHAPATVCGPKKSTNHHHHHHC



(SEQ ID NO: 1)





RBD
CITNLCPFGEVFNATRFASVYAWNRKRISNCVADY


N-terminal
SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF


cysteine
VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA



WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST



EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY



QPYRVVVLSFELLHAPATVCGPKKSTNHHHHHH



(SEQ ID NO: 2)





RBDJ
CITNLCPFGEVFNATRFASVYAWNRKRISNCVADY


N-terminal
SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF


cysteine
VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA



WNSNNLDSKVGGNYNYKYRLFRKSNLKPFERDIST



EIYQAGSTPCNGVEGFNCYWPLQSYGFQPTNGVGY



QPYRVVVLSFELLHAPATVCGPKKSTN (SEQ ID



NO: 3)









The amino acid sequences of RBD antigens used in these studies.


N-Terminal pSer Modification Enhances the Immunogenicity of RBD Antigens


We immunized BALB/c mice with pSer4-RBD (SEQ ID NO: 4-RBD), RBD-pSer4 (RBD-SEQ ID NO: 4), or unmodified RBD combined with alum and boosted at 6 weeks. Consistent with prior reports (10, 11), RBD:alum immunization elicited weak IgG responses, with none of the animals seroconverting by 3 weeks post-prime at this dose; post-boost, weak IgG titers were detected that steadily declined over time (FIG. 2A). Both pSer-modified immunogens exhibited stronger serum responses, and the N-terminally modified RBD was particularly effective, with titers 57-fold greater than the control group at the peak of response 2 weeks post-boost (FIG. 2A-B). Further, traditional alum:RBD immunization elicited no detectable neutralizing responses even after boosting, while 3 of 5 animals receiving pSer4-RBD (SEQ ID NO: 4-RBD):alum vaccination had pseudovirus (PSV) neutralizing titer ID50 (NT50)>103 post-boost (FIG. 2C-D). pSer4-RBD (SEQ ID NO: 4-RBD) also significantly augmented the number of antibody-secreting cells in the bone marrow at day 112 (FIG. 2E). Altogether, these data suggest that pSer conjugation to the N-terminus of RBD can substantially enhance humoral responses to alum:RBD immunization.


A Stabilized RBD Mutant Further Enhances the Immunogenicity of Alum:RBD Vaccines

We recently developed a novel RBD variant containing two point mutations (L452K, F490W; table 1) engineered to improve manufacturability and stability of the antigen. This variant (hereafter, RBDJ) was also more immunogenic than Wuhan-Hu-1 RBD (hereafter, wild-type RBD) in mice (29). Given the inconsistent neutralizing responses observed in mice immunized with pSer4-RBD (SEQ ID NO: 4-RBD), we thus tested whether RBDJ would benefit from pSer-tagging and assessed whether increasing the valency of the pSer tag could further enhance antibody responses. To this end, RBDJ N-terminally modified with a pSer4 or pSer8 (SEQ ID NOs: 4 or 5) tag was synthesized (FIG. 7A). Both pSer4-RBDJ and pSer8-RBDJ (SEQ ID NOs: 4-RBDJ & SEQ ID NO: 5-RBDJ) adsorbed efficiently to alum, and pSer8-RBDJ (SEQ ID NO: 5-RBDJ) showed slightly higher retention on alum over time on exposure to serum/phosphate buffer (FIG. 7B-C). As observed with the wild-type RBD, pSer-RBDJ protein bound to alum particles retained robust binding to hACE2, CR3022, H4, and B38 (FIG. 7D-E).


We previously found that pSer-modified HIV Env proteins trafficked to lymph nodes bound to alum particles, such that antigen-specific B cells directly internalized antigen-decorated alum particles (26). To gain insight into the behavior of pSer-tagged RBDJ and assess whether increased pSer valency impacted antigen availability kinetics in vivo, we fluorescently labeled the pSer-RBDJ proteins with an AlexaFluor™ 647 dye. Mice were immunized subcutaneously (s.c.) with these labeled vaccines near the tail base, and the kinetics of antigen clearance from the injection site over time were tracked by whole animal fluorescence imaging (FIG. 3A). Fluorescence from the RBD antigen steadily cleared from the immunization site, with the rate of decay ordered as RBDJ>pSer4-RBDJ (SEQ ID NO: 4-RBDJ)>pSer8-RBDJ (SEQ ID NO: 5-RBDJ) (FIG. 3B).


To determine whether these distinct vaccine kinetics impacted the immune response, we first quantified GC responses following alum:RBDJ immunization. Flow cytometry analysis of draining inguinal lymph nodes (dLNs) harvested at staggered time points post-injection revealed that phosphoserine-tagged RBDs elicited notably stronger GC responses than traditional alum:RBDJ immunization (FIG. 3C-F, FIG. 8A-C). The total GC response peaked at day 14, with pSer4-RBDJ (SEQ ID NO: 4-RBDJ) eliciting the strongest response (FIG. 3D and FIG. 8C). Even more striking was the impact on antigen-specific GC B cells: both pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and pSer8-RBDJ (SEQ ID NO: 5-RBDJ) primed a substantial population of RBD-specific GC B cells, while antigen-specific cells were very low in the alum:RBDJ control group across the entire time course (FIG. 3C, FIG. 3E-F, and FIG. 8C). In addition, pSer-conjugated RBDJ elicited ˜2-fold greater T follicular helper cell (TFH) responses than the unmodified immunogen (FIG. 3G). These enhanced GC and TFH responses correlated with greatly increased IgG antibody responses for mice immunized with pSer-RBDJ compared to RBDJ (FIG. 3I1), and these antibodies exhibited a significantly higher ability to block hACE2-RBD interactions (FIG. 9A). Intriguingly, total binding IgG ELISA titers and hACE2 binding inhibition trended to be higher with pSer4-RBDJ (SEQ ID NO: 4-RBDJ) vs. pSer8-RBDJ (SEQ ID NO: 5-RBDJ), but these differences did not reach statistical significance. Notably, the antigen drainage characterization (FIG. 3A-B) suggested a portion of pSer8-RBDJ (SEQ ID NO: 5-RBDJ) may be irreversibly trapped at the injection site. If we subtracted the plateau fluorescence signal from the total fluorescence of the pSer8-RBDJ (SEQ ID NO: 5-RBDJ) group over time, the resulting “bioavailable” pSer8-RBDJ (SEQ ID NO: 5-RBDJ) trajectory looks very similar to that of pSer4-RBDJ (SEQ ID NO: 4-RBDJ) (FIG. 9B). Further, longitudinal tracking of alum drainage from the injection site revealed that ˜58% of alum remains at the injection site 70 days post-immunization for pSer8-RBDJ (SEQ ID NO: 5-RBDJ) compared to ˜37% for pSer4-RBDJ (SEQ ID NO: 4-RBDJ) (FIG. 9C). We hypothesize that these altered kinetics for antigen and alum clearance observed with the longer pSer8-tagged (SEQ ID NO: 5-RBDJ) immunogen may reflect some level of inter-alum particle crosslinking mediated by the longer peptide tag, which inhibits disaggregation of alum particles and promotes their phagocytosis locally at the injection site, limiting delivery to the dLNs. Importantly, pSer4-RBDJ (SEQ ID NO: 4-RBD) elicited peak serum IgG titers ˜4-fold greater than the same dose of pSer4-RBD (SEQ ID NO: 4-RBD) with more consistent seroconversion (FIG. 2A and FIG. 3H). Altogether, these data indicate that pSer-RBDJ (SEQ ID NO: 4-RBDJ) vaccines elicit greatly enhanced GC and serum antibody responses compared to traditional immunization with admixed RBDJ and alum.


We also investigated the impact of alum dose and antigen density on humoral immune responses. Varying the amount of RBDJ added to a fixed amount of alum, we identified a range of antigen densities for which there was comparable pSer4-RBDJ (SEQ ID NO: 4-RBDJ) loading and retention on alum (FIG. 10A). Based on the reported surface area of Alhydrogel alum (30), antigen:alum mass ratios of 1:5, 1:10, and 1:20 correspond to an estimated average spacing between RBDs on alum particles of 9.6 nm, 13.5 nm, and 19.2 nm, respectively. To determine whether antigen density variation in this range affects the immune response, mice were immunized with a constant dose of pSer4-RBDJ (SEQ ID NO: 4-RBDJ) loaded on varying quantities of alum (50, 100, or 200 μg), and the serum antibody responses were tracked longitudinally. Interestingly, differences between these 3 groups were very modest and not statistically significant (FIG. 10B). Examining GC responses 14 days post-immunization, antigen-specific GC B cell frequencies showed a slight trend toward increased responses at lower antigen density/higher alum dose, but these differences again were not significant (FIG. 10C). These experiments are potentially confounded by the convolution of antigen density with alum amount, and thus we also devised a second experimental approach: immunizations were prepared by first loading antigen on alum at the specified ratios, and then supplementing in extra alum just prior to immunization to bring the alum dose to 200 μg for all mice. To confirm that pSer4-RBDJ (SEQ ID NO: 4-RBDJ) would not redistribute when additional alum was added, we imaged alum particles loaded with fluorophore-tagged pSer4-RBDJ (SEQ ID NO: 4-RBDJ) that were mixed with RBD-free alum tagged by a low density of pSer4-Alexa (SEQ ID NO: 4—Alexa) dye and incubated together for 2 days. As shown in FIG. 10D-E, no transfer of pSer4-RBDJ (SEQ ID NO: 4-RBDJ) (red) to the bare alum particles (cyan) was observed when the mixture was examined by microscopy. Therefore, using this approach we repeated immunizations varying the antigen density across the same RBD:alum mass ratios and assessed GC responses, serum IgG over time, and neutralizing antibody titers. Similar to the previous experiments, GC responses were not statistically different between the groups (FIG. 10F). There was a transient enhancement in humoral responses post-prime with increasing antigen density, but all groups responded similarly post-boost, and PSV NT50 were not different across the three pSer4-RBDJ (SEQ ID NO: 4-RBDJ) groups (FIG. 10G-H, FIG. 3I). The neutralizing responses elicited by pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum, however, were significantly higher than unmodified RBDJ and were notably more consistent than we observed with wild-type pSer4-RBD (SEQ ID NO: 4-RBD):alum, with all animals primed to produce high levels of neutralizing responses at a mean PSV NT50 of ˜5,270 two weeks post-boost (n.b., compare 1:10 antigen density in FIG. 3I with FIG. 2C). Thus, pSer4 (SEQ ID NO: 4) modification of RBDJ enhanced GC and neutralizing antibody responses, but these responses were not sensitive to the density of antigen loading on alum.


Conjugation of Antigen and Adjuvants to Alum Synergistically Amplifies Humoral Responses

Although pSer anchoring RBD to alum greatly enhanced its immunogenicity, alum remains an adjuvant with modest potency in large animals and humans. We hypothesized that combining alum with a molecular co-adjuvant employing the same ligand exchange reaction used to anchor RBD immunogen would synergistically enhance the immune response, by prolonging the exposure of dLNs to both antigen and inflammatory cues. We thus tested the behavior of two clinically relevant phosphate-containing co-adjuvants, CpG, a single-stranded DNA TLR9 agonist containing phosphorothioates in the oligonucleotide backbone, and SMNP (27), an ISCOMs-like ˜40 nm diameter nanoparticle formed by the self-assembly of phospholipids, saponin, and the TLR4 agonist monophosphoryl lipid A, which binds to alum via phosphate groups of the lipids and MPLA, in combination with pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and alum. Both CpG and SMNP demonstrated strong alum adsorption and retention on alum (3:10 and 1:20 mass ratios, respectively) in the presence of mouse serum, suggesting strong ligand exchange-mediated binding (FIG. 4A). We sequentially added pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and the two co-adjuvants to alum; importantly, we found that neither CpG nor SMNP displaced bound pSer4-RBDJ (SEQ ID NO: 4-RBDJ) (FIG. 4B). Whole animal fluorescence imaging of the injection site following immunization with labeled CpG or SMNP adsorbed to alum together with pSer4-RBD (SEQ ID NO: 4-RBD) revealed sustained drainage of the co-adjuvants compared to injection of these adjuvants in the absence of alum (FIG. 4C-D). Notably, addition of these co-adjuvants did not disrupt the sustained drainage of pSer4-RBDJ (SEQ ID NO: 4-RBDJ) when loaded on alum: when CpG or SMNP were adsorbed to alum with labeled pSer4-RBDJ (SEQ ID NO: 4-RBDJ), there was no significant difference in the kinetics of antigen clearance when compared to pSer4-RBDJ SEQ ID NO: 4-RBDJ) on alum alone (FIG. 11A).


In order to investigate the impact of these alum-bound co-adjuvants on humoral responses, we immunized mice with combinations of CpG or SMNP bound to alum with RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and tracked serum antibody responses over time. Notably, the addition of CpG to pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum or RBDJ:alum immunizations dramatically enhanced IgG antibody titers compared to pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum or soluble RBDJ plus CpG following the priming immunization (FIG. 4E, F). There were also trends of increased IgG antibody titers for alum-bound antigen and co-adjuvant SMNP (FIG. 4G). Examination of individual IgG isotypes showed that IgG1, IgG2a, and IgG2b titers were all substantially increased when pSer-RBDJ:alum was combined with each of the co-adjuvants (FIG. 4H), and the IgG2a/IgG1 and IgG2b/IgG1 ratios were increased with the addition of the co-adjuvants (FIG. 11B). The addition of CpG and SMNP to pSer:alum immunizations also elicited more functional antibody responses, as serum from immunized mice demonstrated stronger inhibition of hACE2-RBD binding both post-prime and post-boost (FIG. 4I). Notably, maximal hACE2 binding inhibition/neutralizing responses required that alum was combined with one of the co-adjuvants and that the RBD was pSer-modified. This finding was even more starkly illustrated by pseudovirus neutralizing antibody titers measured for animals immunized with pSer-RBDJ:alum±SMNP: immunization with pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum or RBDJ+SMNP elicited PSV NT50 titers ˜10-fold weaker than the pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum:SMNP combination (FIG. 4J, FIG. 11C). Notably, there was no statistically significant correlation between serum IgG ELISA binding titers and PSV NT50 at day 42 or 84 (FIG. 11D), suggesting that the serum IgG ELISA binding titers is not predictive of neutralizing titers for these groups.


To further investigate the basis of this enhanced neutralizing antibody response, we investigated the impact of CpG or SMNP co-adjuvants on the cellular localization of antigen. Mice were immunized with AlexaFluor™-labeled RBDJ, and the number of cells positive for antigen was assessed among B cells, monocytes, neutrophils, subcapsular sinus macrophages, medullary macrophages, and dendritic cells (FIG. 12A-B). B cells showed a significant increase in antigen uptake following pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum+SMNP immunization compared to RBDJ:alum and pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum, whereas there was a significant increase in monocyte uptake of antigen for pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum+CpG compared to RBDJ:alum and pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum. These differences in antigen distribution may contribute to the synergistic benefit of co-adjuvants with pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum. To elucidate the basis of the enhanced neutralization responses in mice immunized with pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum+SMNP despite comparable overall IgG titers with RBDJ+SMNP, we immunized mice with unmodified RBDJ or pSer4-RBDJ (SEQ ID NO: 4-RBDJ) and alum and/or SMNP and assessed the RBD-specific GC B cell responses in the dLNs at day 14 post-immunization. Notably, pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum+SMNP elicited significantly higher RBD-specific GC B cell responses compared to RBDJ+SMNP (FIG. 12C). These data suggest that stronger neutralizing responses observed in mice immunized with pSer4-RBDJ (SEQ ID NO: 4-RBDJ):alum+SMNP compared to RBDJ+SMNP are driven by more robust antigen-specific GC responses when alum anchoring and SMNP are combined. Hence, co-conjugation of molecular adjuvants and the immunogen with alum synergistically amplifies humoral immunity to RBD.


DISCUSSION

There is a need for additional safe and effective SARS-CoV-2 vaccines to facilitate global vaccine coverage. Given the emergence of novel SARS-CoV-2 variants, it is especially important that these vaccines elicit responses that retain activity against circulating variants of concern. Subunit vaccines are an attractive approach to achieve global coverage, as they can be rapidly scaled for manufacturing, and their distribution does not require ultra-cold storage temperatures. Here we describe an approach using alum, a low-cost adjuvant with widespread clinical use, that elicited potent humoral immune responses and neutralization in mice against SARS-CoV-2. By modifying the RBD antigen with a short peptide linker, the duration of antigen drainage from the injection site was substantially extended, leading to strong antigen-specific GC responses which lasted over a month post-immunization. Through the optimization of this immunization platform, testing the impact of N- versus C-terminal pSer conjugation, pSer valency, antigen density, and the addition of alum-binding co-adjuvants, the platform achieved continually higher and more consistent antibody and neutralization responses in mice (FIG. 13). Notably, the addition of CpG or SMNP co-adjuvants to pSer-RBD plus alum immunizations also promoted a more balanced Th1/Th2 bias to the antibody response.


The pSer modification approach employed here provides a simple and robust strategy to prolong antigen availability in a clinically translatable vaccine regimen. The alum-anchoring strategy used here has the additional capacity to help potentiate B cell responses by presenting many copies of antigen bound to a single alum particle, promoting BCR crosslinking and early signaling/B cell activation (26). However, in the case of RBD, varying antigen density did not impact any of the measures of the humoral responses assessed here, suggesting either that the RBD densities explored here did not cover a wide enough range to detect an effect on B cell triggering and/or that some release of pSer-RBD from alum particles occurs over time, thus diluting the “alum presentation” effect.


Studies applying repeated injections to achieve extended dosing in cancer vaccines have demonstrated the importance of sustained exposure to both antigen and inflammatory cues in peptide vaccines for optimal T cell responses (39), but the role of extended adjuvant exposure on humoral immunity is not well understood. To couple the kinetics of antigen and adjuvant delivery to lymph nodes, we tested here the use of two different molecular adjuvants, CpG and a nanoparticle-formulated saponin, each of which could undergo the same type of ligand exchange reaction with alum as employed in our pSer-modified immunogens. With each of these co-adjuvants, we observed sustained release from the injection site in the presence of alum. These altered vaccine kinetics correlated with enhanced antibody responses and neutralization that were much more than additive over the individual responses elicited by alum or the co-adjuvants in isolation, suggesting strong synergy induced by alum binding. We hypothesize that altered delivery kinetics achieved by ligand exchange binding to alum play an important role in the potency of this adjuvant combination. Altogether these findings suggest that the combination of pSer-mediated immunogen binding to alum with SMNP co-adjuvant delivery is an effective adjuvant combination.


As a platform, this technology promotes sustained antigen and co-adjuvant drainage from the injection site, inducing potent humoral immune responses against SARS-CoV-2 using alum, a low-cost adjuvant with widespread clinical use. In the context of more immunogenic antigens, this platform could also be beneficial to promote a dose-sparing strategy to increase vaccine availability. Our findings demonstrate that combinations of adjuvants enable new immunological mechanisms of action, providing vaccine formulations with activity greater than the individual components, and enhance the potency of subunit vaccine antigens.


Materials and Methods
Phosphoserine Peptide Synthesis

pSer peptide linkers were synthesized using solid phase synthesis on low-loading TentaGel™ Rink Amide resin (0.2 meq/g, Peptides International, catalog no. R28023) as described previously (26). Briefly, resin was deprotected with 20% piperidine (Sigma Aldrich, catalog no. 411027) in dimethylformamide (DMF, Sigma Aldrich, catalog no. 319937-4L), and peptide couplings were performed with 4 equivalents of Fmoc-Ser(PO(OBzl)OH)—OH (Millipore Sigma, catalog no. 8520690005) and 3.95 equivalents of hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU, Millipore Sigma catalog no. 148893-10-1) for 2 hours at 25° C. pSer residues were deprotected with 5% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, Sigma Aldrich, catalog no. 139009) in DMF. Double couplings were performed after the third residue. An Fmoc-protected 6-unit oligoethylene glycol linker (Peptides International, catalog no. DPG-5750) was then coupled to the peptide and subsequently deprotected and reacted with N-maleoyl-β-alanine (Sigma Aldrich, catalog no. 394815). Completion of each deprotection and coupling step was confirmed by a ninhydrin test (Sigma Aldrich, catalog no. 60017). pSer side chains were deprotected and the peptide was cleaved from the resin in 95% trifluoroacetic acid (Sigma Aldrich, catalog no. T6508), 2.5% H2O, and 2.5% triisopropylsilane (Sigma Aldrich, catalog no. 233781), for 2.5 hours at 25° C. The product was precipitated in 4° C. diethyl ether (Sigma Aldrich, catalog no. 673811) and dried under N2, then purified by HPLC on a C18 column (Agilent Zorbax™ 300SB-C18) using 0.1 M triethylammonium acetate buffer (Glen Research, catalog no. 60-4110-62) in an acetonitrile gradient. The peptide mass was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. For imaging experiments, the pSer4-AlexaFluor™ 488 (SEQ ID NO: 4—AlexaFluor™ 488) conjugate was synthesized as described for the pSer component of the linker, followed by deprotection and coupling to Fmoc-5-azido-pentanoic acid (Anaspec, catalog no. AS-65518-1). The peptide was deprotected with 20% piperidine in dimethylformamide prior to cleavage from the resin in 95% trifluoroacetic acid, 2.5% H2O, and 2.5% triisopropylsilane for 2.5 hours at 25° C. The product was then precipitated in 4° C. diethyl ether, and dried under N2, and purified by HPLC on a C18 column using 0.1 M triethylammonium acetate buffer in an acetonitrile gradient. The peptide mass was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This pSer4-azide (SEQ ID NO: 4-azide) linker was reacted with one equivalent of AlexaFluor™ 488-DBCO (Click Chemistry Tools, catalog no. 1278) overnight at 4° C. in a Cu-free click reaction in PBS (pH 7.2-7.4) and subsequently purified by HPLC on a C18 column using 0.1 M triethylammonium acetate buffer in an acetonitrile gradient.


Antigen Production and pSer Conjugation


RBD immunogens were expressed in yeast strains derived from Komagataella phaffii (NRRL Y-11430) as described previously (29). Protein was purified using the InSCyT purification module as described previously (58). Columns were equilibrated in buffer prior to each run. His-tagged RBDs were purified with a 1 ml HisTrap HP column (Cytiva Life Sciences, catalog no. 29051021) on an ÄKTA pure 25 L FPLC system (Cytiva Life Sciences, catalog no. 29018224). The column was equilibrated with a binding buffer composed of 25 mM imidazole, 25 mM sodium phosphate, 500 mM NaCl, pH 7.4. Protein-containing supernatant was applied to the column via a S9 sample pump (Cytiva Life Sciences, catalog no. 29027745) at a rate of 2 ml/min. After washing the column with binding buffer, the his-tagged RBD (amino acids 332-532 of SARS-CoV-2 Wuhan-Hu-1 S protein; GenBank: MN908947.3) was eluted with 500 mM imidazole, 25 mM sodium phosphate, 500 mM NaCl, pH 7.4. For non-histagged RBDs, protein-containing supernatant was adjusted to pH 4.5 using 100 mM citric acid and subsequently loaded into a pre-packed 5 ml CMM HyperCel™ column (Pall Corporation), re-equilibrated with 20 mM sodium citrate pH 5.0, washed with 20 mM sodium phosphate pH 5.8, and eluted with 20 mM sodium phosphate pH 8.0, 150 mM NaCl. Eluate from column 1 above 15 mAU was flowed through a 1 ml pre-packed HyperCel™ STAR AX™ column (Pall Corporation). Flow-through from column 2 above 15 mAU was collected.


Antigens expressed with a free terminal cysteine were reduced at 1 mg/ml with 2 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP, ThermoFisher, catalog no. 20490) and incubated at 25° C. for 10 minutes. TCEP was subsequently removed from reduced protein solutions using Amicon™ Ultra Centrifugal Filters (10 kDa MWCO, Millipore Sigma, catalog no. UFC501096) in tris-buffered saline (TBS, Sigma Aldrich, catalog no. T5912), and 1 mg/ml antigen was reacted with 2 molar equivalents of pSer-maleimide linkers for 16 hours at 4° C. in TBS (pH 7.2-7.4). Free pSer linker was subsequently removed using centrifugal filters in TBS, and pSer-antigen was buffer exchanged to PBS. The pSer4-cytochrome C (SEQ ID NO: 4-cytochrome C) used for antigenicity profiling of immunogens was prepared as described, using cytochrome C from Saccharomyces cerevisiae (Sigma Aldrich, catalog no. C2436). The number of pSer residues conjugated to the antigen was assessed using the Malachite Green Phosphoprotein Phosphate Estimation Assay Kit (Thermo Scientific, catalog no. 23270) against a standard curve of pSer-maleimide linker. Signal from pSer-antigen was compared to the background from an unconjugated antigen control. Fluorescently labeled protein used in imaging experiments were prepared by reacting 1 mg/ml antigen in 50 mM sodium bicarbonate buffer for 1 hour at 25° C. with 6 molar equivalents of AlexaFluor™ 647 NHS ester (Invitrogen, catalog no. A20006) for alum binding studies and whole-mouse imaging or AlexaFluor™ 555 NHS ester (Invitrogen, catalog no. A20009) for microscopy experiments. Labeled antigen was purified by centrifugal filtration.


SMNP Adjuvant Synthesis

Saponin-MPLA nanoparticles (SMNP) adjuvant was prepared as previously described (27). Briefly, solutions at 20 mg/ml were prepared of cholesterol (Avanti Polar Lipids, catalog no. 700000), DPPC (Avanti Polar Lipids, catalog no. 850355), and PHAD MPLA (Avanti Polar Lipids, catalog no. 699800P) in 20% MEGA-10 (Sigma, catalog no. D6277) detergent. Quil-A saponin (InvivoGen, catalog no. vac-quil) was dissolved in Milli-Q water at a final concentration of 100 mg/ml. These were mixed at a mass ratio of 10:2:1:1 (Quil-A:chol:DPPC:MPLA) and diluted in PBS to a final cholesterol concentration of 1 mg/ml. The solution was equilibrated overnight at 25° C. and then dialyzed against PBS using a 10 kDa MWCO cassette. The adjuvant was then sterile filtered, concentrated using Amicon™ Ultra Centrifugal Filters (50 kDa MWCO, Millipore Sigma, catalog no. UFC505096), and purified by FPLC using a Sephacryl S-500 HR size exclusion column. SMNP labeled with Cy7 was prepared as described incorporating 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(Cyanine 7) (Avanti Polar Lipids, catalog no. 810347) in place of 10 mol % of the MLPA.


Antigen and Adjuvant Alum Binding and Release

AlexaFluor™ 647-labeled antigen was loaded onto Alhydrogel (alum, InvivoGen, catalog no. vac-alu-250) in TBS at a 1:10 antigen:alum mass ratio, unless otherwise specified, for 30 minutes on a tube rotator at 25° C. To assess antigen binding to alum, samples were immediately centrifuged at 10,000×g for 10 minutes to pellet alum, and the fluorescence of the supernatant was measured against a standard curve of labeled antigen. To assess the release of antigen from alum, mouse serum was added to antigen-alum solutions post-loading to a final mouse serum concentration of 10 vol % and incubated at 37° C. for 24 hours, unless otherwise specified. Samples were subsequently centrifuged at 10,000 g for 10 minutes to pellet alum, and the fraction of protein bound to alum was measured by fluorescence using a Tecan Infinite M200 Pro plate reader. Experiments investigating CpG binding and release from alum were performed using FITC-labeled CpG 1826 (InvivoGen, catalog no. tlrl-1826f) with a 3:10 CpG:alum mass ratio. Experiments investigating SMNP binding and release from alum were performed using Cy7-labeled SMNP with a 1:20 SMNP:alum mass ratio.


Antigenicity Profiling of RBD Immunogens

Antigenicity profiling of antigens was completed by comparing antibody binding curves of pSer-conjugated RBD or RBDJ on alum against those of unmodified RBD or RBDJ. To capture alum on Nunc Maxisorp™ ELISA plates (Invitrogen, catalog no. 44-2404-21), plates were first coated with pSer4-conjugated cytochrome C (SEQ ID NO: 4-cytochrome C) at 2 μg/ml for 4 hours at 25° C. Alum was then added at 200 μg/ml and captured by pSer4-cytochrome C (SEQ ID NO: 4-cytochrome C) overnight at 4° C. To capture unmodified RBD, plates were coated with a rabbit anti-histag antibody (GenScript, catalog no. A00174-40) at 2 μg/ml overnight at 4° C. Plates were washed with 0.05% Tween-20 in PBS and incubated with 2 μg/ml protein in 2% BSA in PBS for 2 hours at 25° C. CR3022 monoclonal antibody (Abcam, catalog no. ab273073), hACE2-Fc chimera (InvivoGen, catalog no. fc-hace2), H4 (InvivoGen, catalog no. cov2rbdc1-mab1), or B38 (InvivoGen, catalog no. cov2rbdc2-mab1) was added at 5 μg/ml with 1:4 serial dilutions for 2 hours at 25° C. Plates were washed and antibody binding was detected with a goat anti-human HRP conjugated secondary antibody (BioRad, catalog no. 1721050) at 1:5000 dilution in PBS containing 2% BSA and then developed with 3,3′,5,5′-tetramethylbenzidine (ThermoFisher, catalog no. 34028), stopped with 2N sulfuric acid and immediately read (450 nm with 540 nm reference) on a BioTek Synergy2 plate reader.


Animals and Immunizations

Experiments and handling of mice were conducted under federal, state and local guidelines under an Institutional Animal Care and Use Committee (IACUC) approved protocol. Female 6-8-week-old BALB/c mice were purchased from the Jackson Laboratory (stock no. 000651). Immunizations were prepared by mixing 10 μg of antigen and 100 μg of alum in 100 μL sterile tris-buffered saline (TBS, Sigma Aldrich, catalog no. T5912) per mouse unless otherwise specified. Antigen was loaded onto alum for 30 minutes on a tube rotator prior to immunization. When CpG 1826 or SMNP was added into the immunization, antigen was first loaded onto alum for 30 minutes on a rotator, after which 30 μg of CpG 1826 or 5 μg of SMNP was added into the immunization and incubated with antigen-alum formulations for 30 minutes prior to immunization. This dose of SMNP corresponds to 5 μg of Quil-A and 0.5 μg MPLA. Experiments in which antigen density was altered but the total alum dose remained the same, antigen was loaded onto alum at the indicated antigen:alum mass ratio for 30 minutes, and supplemented alum added just prior to immunization to bring the total alum dose to 200 μg per mouse. Mice were immunized subcutaneously at the tail base with 50 μL on each side of the tail base and were subsequently boosted 6 weeks post-prime.


Antigen-Binding ELISA

Serum was collected from mice retro-orbitally using capillary tubes and stored at −20° C. until analysis. To determine serum IgG titers with RBD, Nunc Maxisorp™ plates (Invitrogen, catalog no. 44-2404-21) were coated with a rabbit anti-histag antibody (GenScript, catalog no. A00174-40) at 2 μg/ml for 4 hours at 25° C. in PBS and blocked with 2% BSA in PBS overnight at 4° C. Plates were washed with 0.05% Tween-20 PBS, and RBD was added at 2 μg/ml in 2% BSA in PBS for 2 hours. Serum dilutions (1:10 dilution followed by 1:50 dilution with 1:4 serial dilutions) were incubated in the plate for 2 hours. Plates are washed again, incubated with a goat anti-mouse IgG HRP-conjugated secondary (BioRad, catalog no. 1721011) at 1:5000 dilution, and then developed with 3,3′,5,5′-tetramethylbenzidine (ThermoFisher, catalog no. 34028), stopped with 2N sulfuric acid, and immediately read (450 nm with 540 nm reference) on a BioTek Synergy™ 2 plate reader. To determine serum IgG titers for mice immunized with RBDJ, protein was coated directly on Corning Costar High Binding 96-well plates (catalog no. 9018/3690) at 2 μg/ml in PBS overnight at 4° C. and blocked for 2 hours, and subsequently follow the protocol for RBD ELISAs. Isotype ELISAs followed the same protocol but used goat anti-mouse IgG1 HRP cross-adsorbed secondary antibody (Invitrogen, catalog no. A10551), goat anti-mouse IgG2a HRP cross-adsorbed secondary antibody (Invitrogen, catalog no. M32207), or goat anti-mouse IgG2b cross-adsorbed secondary antibody (Invitrogen, catalog no. M32407) at 1:2000 dilution.


ACE2 Competition ELISA

Surrogate virus neutralization ELISAs (GenScript, catalog no. L00847A) were performed following the manufacturer's protocol. Briefly, mouse serum samples were diluted at 1:10 with 1:3 serial dilutions and mixed 1:1 with RBD-HRP for 30 minutes at 37° C. Samples were then added to hACE2 coated plates and incubated for 15 minutes at 37° C. Plates were developed for 15 minutes with 3,3′,5,5′-tetramethylbenzidine, stopped with 1N sulfuric acid, and the absorbance at 450 nm was immediately read on a BioTek Synergy™ 2 plate reader. ID50 values were calculated using a nonlinear fit of individual dilution curves.


Pseudovirus Neutralization Analysis

To assess neutralization in mouse serum samples, SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated similar to an approach described previously (59, 60). Briefly, HEK293T cells were co-transfected with the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc™ (Addgene, catalog no. 17477), and spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT using lipofectamine 2000 (ThermoFisher, catalog no. 11668030). Pseudotype viruses were collected from culture supernatants 48 hours post-transfection and purified by centrifugation and 0.45 μm filtration. To assess the neutralization activity of the mouse serum samples, serum was inactivated at 56° C. for 30 minutes. HEK293T-hACE2 cells were seeded overnight in 96-well tissue culture plates at a density of 1.75×104 cells per well. Three-fold serial dilutions of heat inactivated serum samples were prepared and mixed with 50 μL of pseudovirus, followed by incubation at 37° C. for 1 hour before adding the mixture to HEK293T-hACE2 cells. After incubation for 48 hours, cells were lysed using Steady-Glo™ Luciferase Assay (Promega, catalog no. E2510) according to the manufacturer's instructions. SARS-CoV-2 pseudovirus neutralization titers were defined as the sample dilution at which a 50% reduction in relative light unit (RLU) was observed relative to the average virus control wells.


ELISPOT Analysis

Bone marrow ELISPOTs were performed in mice 16 weeks post-prime following the manufacturer protocol (MabTech, catalog no. 3825-2A) unless otherwise specified. Briefly, 96-well PVDF ELISPOT plates (Millipore Sigma, catalog no. MSIPS4510) were treated with 35% ethanol prior to coating with anti-mouse IgG at 15 μg/ml in sterile PBS overnight at 4° C. Cells were isolated from the femur and tibia of mice, ACK lysed, and 70 μm filtered in complete media (RPMI 1640 containing 10% FBS, 100 U/ml penicillin-streptomycin, and 1 mM sodium pyruvate). The next day, plates were blocked with complete media for at least 30 minutes prior to adding cells with three technical replicates per mouse. For total IgG and antigen-specific IgG, 100,000 and 500,000 cells were added per well, respectively, and incubated at 37° C. with 5% CO2 for 16 hours. Plates were then washed with PBS. Antigen-specific responses were determined by adding 1 μg/ml biotinylated RBD in PBS with 0.5% BSA to each well for 2 hours at 25° C. Total IgG responses were determined by adding 1 μg/ml anti-mouse IgG-biotin detection antibody in PBS with 0.5% BSA to each well for 2 hours at 25° C. Plates were washed again in PBS and incubated with 1:1000 streptavidin-alkaline phosphatase in PBS with 0.5% BSA for 1 hour at 25° C. After washing, plates were developed with BCIP/NBT substrate (MabTech, catalog no. 3650-10) and developed for 20 minutes, quenched with H2O, and dried prior to quantification on an ImmunoSpot CTL Analyzer.


Germinal Center and T Follicular Helper Responses

The inguinal lymph nodes were collected from immunized mice 14 days post-immunization unless otherwise specified. For germinal center analysis, cells were stained for viability (ThermoFisher Live/Dead Fixable Aqua, catalog no. L34957) and against CD3e (BV711, 145-2C11 clone; BioLegend, 100349), B220 (PE-Cy7, RA3-6B2 clone; BioLegend, catalog no. 103221), CD38 (FITC, 90 clone; BioLegend, catalog no. 102705), and GL7 (PerCP-Cy5.5, GL7 clone; BioLegend, catalog no. 144609), with antigen-specific staining completed using biotinylated RBD conjugated to streptavidin-BV421 (BioLegend, catalog no. 405226) and streptavidin-PE (BioLegend, catalog no. 405203). For T follicular helper analysis, cells were stained for viability (ThermoFisher LiveDead Fixable Aqua, catalog no. L34957) and against B220 (BV510, RA3-6B2 clone; BioLegend, catalog no. 103247), CD4 (FITC, GK1.5 clone; BioLegend, catalog no. 100405), CD44 (PE-Cy7, IM7 clone; BioLegend, catalog no. 103029), PD-1 (BV421, RMP1-30 clone; BD Biosciences, catalog no. 748268), and CXCR5 (PE, 2G8 clone; BD Biosciences, catalog no. 551960). Samples were analyzed by flow cytometry on a BD Celesta™ and analyzed on FlowJo™.


Cellular Uptake of Antigen

Mice were immunized with 10 μg of AlexaFluor™ 555 labeled antigen and 100 μg alum and 5 μg SMNP or 30 μg CpG, and the inguinal lymph nodes were collected 7 days post-immunization. Cells were stained for viability (ThermoFisher Live/Dead Fixable Near-IR, catalog no. L34975) and against CD3 (APC-Cy7, 17A2 clone; BioLegend, catalog no. 100221), NK1.1 (APC-Cy7, PK136 clone; BioLegend, catalog no. 108723), CD19 (PE-Cy7, 6D5 clone; BioLegend, catalog no. 115519), CD11b (BUV805, M1/70 clone; BD Biosciences, catalog no. 741934), CD11c (BUV496, HL3 clone; BD Biosciences, catalog no. 750483), Ly6C (BV650, HK1.4 clone; BioLegend, catalog no. 128049), Ly6G (BUV563, 1A8 clone; BD Biosciences, catalog no. 612921), F4/80 (BUV737, T45-2342 clone; BD Biosciences, catalog no. 749283), CD169 (BV421, 3D6.112 clone; BioLegend, catalog no. 142421), and MHC II (PE-Cy5, M5/114.15.2 clone; BioLegend, catalog no. 107611). Samples were analyzed by flow cytometry on a BD Symphony™ A3 and analyzed on FlowJo™.


Whole-Mouse Imaging of Vaccination Drainage

Mice were immunized subcutaneously at the tail base with fluorescently labeled antigen or adjuvant. Immunizations were prepared as described, using fluorescently labeled components as indicated. For studies including fluorescently labeled components, immunizations were prepared by loading antigen onto alum in sterile tris-buffered saline (TBS, Sigma Aldrich, catalog no. T5912) for 30 minutes on a tube rotator prior to adding co-adjuvants and incubating for 30 minutes on a tube rotator. Alum was labeled using 0.1 nmol of pSer4-AlexaFluor™ 488 (SEQ ID NO: 4—AlexaFluor™ 488). Imaging was completed using a PerkinElmer Xenogen Spectrum in vivo imaging system (IVIS), and the fluorescent signal at the injection site was quantified using LivingImage software. The radiant efficiency was tracked longitudinally to monitor drainage from the injection site.


Microscopy

Alum was incubated with AlexaFluor™ 555 labeled pSer4-RBDJ (SEQ ID NO: 4-RBDJ) or pSer4-AlexaFluor™ 488 (SEQ ID NO: 4—AlexaFluor™ 488) at 25° C. for 30 minutes in TBS. These solutions were mixed and incubated together for 2 days prior to imaging. Fluorescence images were acquired on an Applied Precision DeltaVision™ Microscope with a 100×/1.4 oil objective using the accompanying Softworx™ software. Image analysis was performed using Fiji (ImageJ™ version 2.1.0) by converting the images into a binary image, applying a Watershed transform, counting the number of particles (3D Objects Counter), and applying the Colocalization Threshold analysis to assess the number of particles for which there is colocalization of the two fluorescent signals. The number of alum particles with fluorescent colocalization was divided by the total number of alum particles detected in the image and reported as the fraction of particles with fluorescent colocalization.


Statistical Analysis

All data were plotted and all statistical analyses were performed using GraphPad Prism™ 8 software (La Jolla, Calif.). All graphs display mean values, and the error bars represent the standard deviation unless otherwise specified. No samples or animals were excluded from the analyses. Statistical comparison was performed using a one-way ANOVA followed by Tukey's post-hoc test for single timepoint data and two-way ANOVA followed by Tukey's post-hoc test for multi-timepoint longitudinal data. Statistical analysis of antibody titer was completed using log-transformed data. Data were considered statistically significant if the p-value was less than 0.05.


Example 2
SUMMARY

The potency of adjuvants is critical to the development of effective vaccines. Recent work from our lab has developed SMNP, an ISCOMs-like nanoparticle comprised of phospholipids, saponin, and the TLR4 agonist monophosphoryl lipid A which elicits strong humoral immune responses (Silva et al. Science Immunology 2021). Based on the presence of phosphates in the phospholipids of SMNP nanoparticles, we hypothesized that phosphate-mediated anchoring of SMNP on aluminum hydroxide (alum) particles could potentiate humoral immunity by promoting extended vaccine kinetics and co-delivery of vaccine components to lymph nodes. We found that the persistence of SMNP in vivo could be significantly increased by complexing with alum, correlating with synergistic enhancements in vaccine immunogenicity compared to SMNP or alum alone. Our results demonstrate that:

    • 1. SMNP demonstrates strong alum adsorption and retention on alum in the presence of mouse serum, suggesting strong ligand exchange-mediated binding (FIG. 4A).
    • 2. This alum binding behavior translated to sustained drainage of SMNP from the injection site in vivo. Whole animal fluorescence imaging of the injection site following immunization with labeled SMNP adsorbed to alum revealed sustained drainage of SMNP compared to injection in the absence of alum (FIG. 4D). This demonstrated that anchoring of SMNP to alum significantly prolonged the persistence of SMNP in vivo.
    • 3. The combination of alum and SMNP (alum:SMNP) synergistically enhances immune responses to immunization. To investigate the impact of the alum-bound co-adjuvant on humoral responses, we immunized mice with a SARS-CoV-2 antigen, receptor binding domain (RBD) of the S protein, with alum alone, SMNP alone, or alum:SMNP and tracked serum antibody responses over time. Given our lab's previous work using phosphoserine peptide linkers to enable ligand exchange interactions between antigens and alum (Moyer et al. Nature Medicine 2020), we also investigated the combination of alum binding antigen pSer-RBD with alum:SMNP. There were trends of increased IgG antibody titers for alum bound antigen and co-adjuvant SMNP (FIG. 4G). Examination of individual IgG isotypes showed that IgG1, IgG2a, and IgG2b titers were all substantially increased when pSer-RBD:alum was combined with SMNP (FIG. 4H). The addition of SMNP to pSer:alum immunizations also elicited more functional antibody responses, as serum from immunized mice demonstrated stronger inhibition of hACE2-RBD binding both post-prime and post-boost (FIG. 4I). Notably, maximal hACE2 binding inhibition/neutralizing titers required that alum was combined with SMNP and that the RBD was pSer-modified. This finding was even more starkly illustrated by pseudovirus neutralization titers measured for animals immunized with pSer-RBD:alum±SMNP: immunization with pSer-RBD:alum or RBD+SMNP elicited neutralizing titers ˜10 fold weaker than the pSer-RBD:alum:SMNP combination (FIG. 4J). Hence, co-conjugation of SMNP and the antigen with alum synergistically amplifies humoral immunity to RBD.
    • 4. The combination of alum and SMNP vaccine adjuvants also synergistically enhances humoral immune responses to a human immunodeficiency virus (HIV) envelope trimer antigen, MD39. To investigate the impact of SMNP when added to pSer-MD39+alum immunizations, we immunized mice with pSer-MD39+alum with varying doses of SMNP (0 μg, 5 μg, or 37.5 μg) and assessed serum antibody responses longitudinally. Notably, SMNP significantly enhanced responses post-prime and post-boost (FIG. 5). This supports the generalizability of this synergistic combination of alum and SMNP.


While alum alone is a weak adjuvant, the combination of SMNP and alum synergistically enhanced humoral immune responses. Given the wide availability of alum as an adjuvant, combining SMNP with alum is feasible and direct means to enhance immune responses to immunization. Phosphate-mediated co-anchoring of antigen and SMNP to alum is an effective strategy to enhance the efficacy of SARS-CoV-2 vaccines and subunit vaccines more broadly. This may enable the reduction in total vaccine dose required to elicit protective responses.


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Claims
  • 1. A composition, comprising: (a) alum; and(b) a non-liposome, non-micelle particle, wherein the particle comprises a lipid, a sterol, a saponin, and an optional additional non-alum adjuvant, wherein the particle is optionally bound to the alum.
  • 2.-3. (canceled)
  • 4. The composition of claim 1, wherein the lipid is a phospholipid, including but not limited to 2-Dipalmitoyl-snglycero-3-phosphocholine (DPPC).
  • 5. (canceled)
  • 6. The composition of claim 1, wherein the sterol comprises cholesterol or a derivative thereof.
  • 7. The composition of claim 1, wherein the saponin is a natural or synthetic saponin.
  • 8.-9. (canceled)
  • 10. The composition of claim 1, wherein the lipid is DPPC, the additional adjuvant is a natural or synthetic MPLA, the sterol is cholesterol, and the saponin is Quil A®.
  • 11. The composition of claim 1, wherein the additional adjuvant is present and comprises a TLR4 agonist, a pathogen-associated molecular pattern (PAMP), and/or a TLR ligand.
  • 12-18. (canceled)
  • 19. The composition of claim 1, comprising a lipid:additional adjuvant:sterol:saponin molar ratio of 2.5:1:10:10, or a variation thereof wherein the molar ratio of lipid, additional adjuvant, sterol, saponin or any combination thereof is increased or decreased by any value between about 0 and about 3.
  • 20.-21. (canceled)
  • 22. The composition of claim 1, wherein the alum comprises a salt of aluminum.
  • 23. The composition of claim 1, wherein the alum comprises aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or combinations thereof.
  • 24. (canceled)
  • 25. The composition of claim 1, wherein the alum and the particle are bound.
  • 26. The composition of claim 1, wherein the particle is covalently bound to the alum via phosphate residues in the particle.
  • 27. The composition of claim 1, further comprising an antigen or antigenic polypeptide bound to the alum and/or the particle.
  • 28. (canceled)
  • 29. The composition of claim 27, wherein the antigen or the antigenic polypeptide is covalently bound to the alum and/or the particle.
  • 30. (canceled)
  • 31. The composition of claim 27, wherein the antigen or the antigenic polypeptide comprises at least one linker comprising 2-12 phosphoserine residues, and wherein the antigen is covalently bound to the alum via the phosphoserine residues.
  • 32.-37. (canceled)
  • 38. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
  • 39. A vaccine comprising the composition of claim 27.
  • 40. A method for generating an immune response against an antigen, comprising administering to a subject an amount effective to generate an immune response in the subject of (a) the composition of claim 1; and(b) an antigen.
  • 41. (canceled)
  • 42. A method for generating an immune response, comprising administering to a subject an amount effective to generate an immune response in the subject of the composition of claim 27.
  • 43. A method of treating a subject in need thereof comprising administering to the subject the composition of claim 27 in an effective amount to induce an immune response against an antigen.
  • 44.-49. (canceled)
CROSS REFERENCE

This application claims priority to U.S. Provisional patent application Ser. No. 63/251,603 filed Oct. 2, 2021, incorporated by reference herein in its entirety.

FEDERAL FUNDING STATEMENT

This invention was made with government support under Grant Nos. AI144462 and 5-54176 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

Provisional Applications (1)
Number Date Country
63251603 Oct 2021 US