Patent Grant
11944679
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 1, 2023, is named 30435_0330USWO_SL.txt and is 51,949 bytes in size.
This invention relates to systems and methods for enhancing immune response and/or making pharmaceutical compositions.
Most viruses adapt rapidly to diverse selection pressures, posing a challenge for deploying safe and effective vaccines. Influenza viruses, for example, are characterized by large genetic diversity across subtypes and rapid antigenic drift and shift, which present problems for traditional vaccine strategies. Attenuation or inactivation of viruses tends to reduce the strength and breadth of immune responses, resulting in ineffective protection against antigenic alterations (1-3). Previous pandemics and recent influenza outbreaks highlight the need to develop safe vaccines that elicit effective immune responses and confer broad protection. Influenza A wild type WSN/1933(H1N1) strain is well known in the art and the complete sequence can be found in reference (51).
The type I interferon (IFN) system is the major component of innate immune responses (4-6). The IFN response provides the first line of defense against viral infections by inducing the expression of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral activities (7). The IFN response is also critical for dendritic cell maturation, development of B and T cells, and memory formation, bridging innate and adaptive immunity (8-12). Most viruses have evolved to efficiently suppress the production and function of IFN to allow replication in vivo. Removing the most well-characterized IFN modulator in influenza virus—namely, the NS1 protein—has shown promise in a vaccine candidate (delNS1) in phase ½ clinical trials (14, 15). Although studies have suggested that influenza proteins other than NS1 have IFN-modulating functions (16, 17), genome-wide identification and elimination of IFN-modulating functions without affecting viral replication fitness in vitro have remained challenging tasks.
Conventional vaccine and/or viral vectors used in immune therapy are typically built from the natural isolates or regenerated using existing knowledge. As discussed in detail below, we have discovered and developed a different approach for vaccine development, one that involves systematically identifying immune evasion functions of a pathogen genome used in the vaccine (e.g., a virus such as influenza), and then eliminating the immune evasion functions while maintaining or tuning the replication fitness of the virus genome. While influenza A is used as an illustrative working embodiment of the invention, this method is broadly applicable to vaccine development in a wide variety of pathogens.
Embodiments of our method are implemented using a systematic high-throughput mutagenesis system to reveal the potential IFN sensitive mutations across a viral genome (influenza A). This mutagenesis system can generate comprehensive information with amino acid or nucleotide resolution that enables construction of novel vectors with desired properties. Moreover, multiple screens can be performed to identify other mutations/features that enhance the properties of the products. In this context, by controlling a combination of mutations in the virus genome, a replication/immune response trade-off can be fine-tuned to achieve desirable efficiency and safety when the virus genome is administered as a vaccine or as a therapeutic agent. In an illustrative embodiment of the invention using the influenza virus, a carefully selected combination of nucleotide mutations generates an invention that is surprisingly capable of stimulating an elevated antibody response, while simultaneously being highly attenuated in vivo due to suppressed viral replication brought about by these mutations.
In the illustrative embodiments of the invention that are directed to the influenza A viral genome, combinations of nucleotide mutations are engineered using a genetic system. In this illustrative working embodiment, the invention provides a single-nucleotide resolution high-throughput genetics approach that simultaneously measures/profiles fitness effects of mutations at >50% of the nucleotide positions in the influenza A virus genome under different conditions. Applying this approach, we have systematically identified anti-interferon functional residues across the whole influenza A/WSN/33 genome, by measuring viral fitness in the presence or absence of interferon. Specifically, we identified and validated interferon sensitive mutations across the influenza A/WSN/33 genome and validated clusters of mutations at PB2 and M1 interacting with the interferon system. Interferon sensitive mutations were found on most segments, in addition to NS1. Clusters of mutations at PB2 and M1 can induce higher interferon production. By combining multiple mutations together (e.g., by combining multiple mutations on PB2, M1 and NS1), we generated a highly interferon sensitive strain as a live attenuated vaccine candidate, which we called a hyper-interferon-sensitive (HIS) virus strain (in one or more examples, also the DAI-1 (Deficient of Anti-Interferon) strain referred to in the priority U.S. Provisional Patent Application Ser. No. 62/471,269, filed on Mar. 14, 2017). The HIS strain can replicate efficiently in interferon-deficient cell lines (e.g., Vero cells) and mice (e.g., interferon-deficient mice such as IFNRA−/− mice). Despite the HIS being attenuated in normal cells and highly attenuated in wild type mice (BALBc), the HIS none-the-less generates surprisingly and unexpectedly robust antibody and T cell responses in mice that inhibit the challenge infection of wild type influenza virus.
Moreover, as the combination of multiple mutations can be located in different gene segments and are mostly neutral for viral replication without interferon selection, the risk of a revertant due to do novo mutation or gene assortment is greatly reduced.
The methods described herein are broadly applicable to the fabrication of genetically engineered pathogens useful as vaccines and/or therapeutic agents and/or pharmaceutical compositions. A therapeutic agent comprising a pathogen genetically engineered according to the method(s) described herein would be particularly useful for patients infected with a drug resistant virus.
Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.
Flow chart shows the experimental design and rationale of our vaccine development approach. Through the development of a quantitative high-throughput genomics system (18, 19, 59-61), we were able to measure the phenotypic effect of single nucleotide mutations in the viral genome under different selection conditions. We dissected the anti-IFN functions encoded by viral proteins from those that are essential for viral replication, and systematically identified IFN sensitive mutations on the viral genome. By combining multiple mutations, we generated a live attenuated vaccine candidate: Hyper-Interferon Sensitive (HIS) virus. This approach simultaneously satisfies the requirement for safety, efficacy and production of vaccine (14, 62).
(A) The induction of IFN-E expression is more significant for cells pre-treated with IFN. A549 cells were pretreated with 1000 U/ml IFN-D2 for 20 h or un-treated before infection. Cells were then infected with WT or indicated mutants at an MOI of 1. RNAs were extracted at 6 h post-infection. IFN-E gene expression was quantified by real-time PCR and calculated as fold of induction compared with WT infected cells (N=3). Error bars denote SD. *P<0.05, **P<0.01, ***P<0.001 (two-tailed t-test). (B&C). Induction of ISG54 expression is shown for WT and indicated mutants (N=3). A549 cells were infected at an MOI of 1. RNAs were extracted at 6 h post-infection. ISG54 gene expression was quantified by real-time PCR and shown as fold of induction normalized to mock infected cells (Ctl, B) or actual CT number (C) (69). Error bars denote SD. *P<0.05, **P<0.01, ***P<0.001 (two-tailed t-test compared with Ctl).
HA proteins is shown. 51 and 61 non-neutralized mutations were identified with more than one serum selection, in WT and HIS vaccinated groups respectively (N=5). ˜60% of these mutations are located in the head domain of the HA protein, while ˜40% of them are in the stem region. The percentage is similar between the two groups.
In the detailed description of the invention, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. A number of different publications are also referenced herein as indicated throughout the specification. A list of these different publications can be found below in the section entitled “REFERENCES”. All publications, patents, and patent applications cited herein (e.g. Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform” Journal of Virological Methods, Volume 193, Issue 2, November 2013, Pages 394-404) are hereby incorporated by reference in their entirety for all purposes.
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
Embodiments of the present invention describe a surprisingly different and radical way for developing live attenuated vaccines: controlling a combination of interferon sensitive mutations in the vaccine so as to fine-tune a replication/immune response tradeoff that achieves the best or desirable efficiency with sufficient safety. On one hand the interferon sensitivity attenuates the virus in vivo. On the other hand, the ability to induce higher interferon response induces a higher and more effective adaptive immune response.
Further aspects and embodiments of the invention are disclosed in the following examples.
The present disclosure describes the establishment of a high throughput genetic platform to examine the fitness effect of single mutations across the entire influenza genome.
To tackle the challenges described herein, we developed a quantitative high-throughput genomics system, which combines saturation mutagenesis and next-generation sequencing, to comprehensively identify IFN-modulating functions in the entire viral genome (18). This system has enabled us to quantitatively measure the replication capacity of a large number of mutants in parallel under specific conditions (18, 19). We performed comparative profiling of the entire influenza genome with and without IFN selection, which led to the identification of IFN-modulating functions on multiple viral segments. By combining eight IFN-sensitive mutations across the viral genome, we generated a hyper-interferon-sensitive (HIS) virus that is replication-competent in vitro but highly attenuated in IFN-competent hosts in vivo. The HIS virus showed desired properties as a safe and effective live attenuated influenza vaccine with robust humoral and cellular responses, and it provided broad protection against homologous and heterologous viral challenges in mice and ferrets.
Compared with previous methods, we significantly improved the quality and reproducibility, getting rid of the noise from multiple mutations. Moreover, to our knowledge, the genetic profiling system described herein is the first whole genome fitness profile for the influenza virus.
a. Fitness Profile of the Influenza a Viral Genome at Single-Nucleotide Resolution
An eight-plasmid reverse genetic system carrying the influenza A/WSN/33 (H1N1) virus genome was used for the construction of mutant plasmid libraries (20). The mutants were divided into 52 sublibraries, each of which contained single-nucleotide mutations in a small genome region of 240 base pairs that were generated by error-prone polymerase chain reaction (
b. Systematic Identification of IFN-Sensitive Mutations
The RF scores of most mutants are correlated in the presence and absence of exogenous IFN treatment; however, we observed a set of mutations that were nearly neutral in the absence of IFN but highly deleterious under IFN selection (
c. Combining Mutations Increases IFN Sensitivity and IFN Induction In Vitro
To maximize IFN sensitivity and IFN induction, we combined three IFN-inducing mutations on PB2 (N9D, Q75H, and T76A), three on M1 (N36Y, R72Q, and S225T), and two previously reported ones on NS1 (R38A and K41A) to create the HIS virus. The growth of HIS virus in IFN-competent A549 cells showed significant attenuation compared with that of WT virus (1.4-log decrease at 36 hours and 1.8-log decrease at 60 hours) but was fully restored in IFN-deficient Vero cells (
We further defined the phenotypes of HIS virus with a panel of human lung cells, including immortalized small airway epithelial cells, bronchial epithelial cells, primary alveolar epithelial cells, and primary alveolar macrophages (
d. HIS Virus is Highly Attenuated in IFN-Competent Mice and Ferrets
We next measured the replication and pathogenesis of HIS virus in mice and ferrets, the most commonly used animal models for influenza virus. BALB/c mice were intranasally inoculated with WT or HIS virus at different doses. Whereas the median lethal dose of WT virus was 5×105 TCID50 (50% tissue culture infective dose), and 1×103 TCID50 caused obvious body weight loss in all animals, neither weight loss nor indicative clinical symptoms were observed in HIS-infected mice given 1×107 TCID50, the highest dose that we have tested (
e. HIS Virus Induces Strong and Broad Adaptive Immune Responses
We then examined the ability of the HIS virus to induce humoral and cellular responses. Mouse sera and BAL samples were collected at day 28 after single-dose (1×104 TCID50) vaccination with WT, HIS, or WSN-CA virus. HIS virus induced robust antibody responses, as measured by ELISA (enzyme-linked immunosorbent assay) and hemagglutinin (HA) inhibition and neutralization antibody assays (
In addition to humoral responses, HIS virus elicited NP and PB1 antigen-specific CD8 T cell responses, similarly to WT virus and much more strongly than the WSN-CA, inactivated WT, and inactivated HIS viruses (
We analyzed the potential impact of immune responses on the viral genome at the population level. Our whole-genome fitness profiling provides a data set for examining the genetic flexibility of viral sequences. We calculated the fitness cost of mutations in the previously identified B and T cell epitopes. Mutations on several T cell epitopes, but not on antibody epitopes, were generally correlated with lower fitness scores (
f. HIS Virus Protects Against Homologous and Heterologous Viral Challenge
We examined whether HIS vaccination could offer protection against homologous and heterologous viral challenges. Immunized mice were challenged 28 days post-vaccination with 1×104 TCID50 of WT virus. Vaccination by HIS virus reduced viral replication by ˜3 log, with no sign of weight loss (
To test whether HIS vaccination provides protection against heterologous strains, we first challenged immunized mice with PR8 virus and examined viral titer at day 2 post-challenge. HIS vaccination reduced viral titer by ˜3 log compared with mock vaccination and significantly more than WSN-CA vaccination (
g. Discussion of Results for the Example
Although NS1 is the best studied IFN antagonist in influenza genome, the anti-IFN function has been recently appreciated in other proteins. It is reported that PB2 and PB1F2 could bind to MAVS and inhibit interferon production; ESIE motif in PB1 and PA can mediate type I interferon response, and NP of newly emerged H7N9 can counteract anti-viral ISG: MxA. Here, using a high-throughput genetic approach, we have identified multiple IFN sensitive mutations in the PB2, PA, PB1 and M1 proteins, suggesting the anti-IFN functions of corresponding WT proteins. As anti-IFN functions are essential for efficient viral replication in vivo, it is reasonable that the functions are distributed in different segments. Identified IFN sensitive mutations also work on different parts of IFN system. Mutations on PB2 and M1 can induce higher IFN production, indicating that they work at the upstream of IFN system. On the other hand, mutation on PB1 and PA cannot induce higher IFN production and response, and the sensitivity still remains in IFN deficient Vero cells, suggesting that they might be interacting with JAK/STAT pathway or a specific anti-viral ISG. Identification of these mutations open the path to dissect the underlying mechanisms.
Conventional approaches to develop vaccines render the virus avirulent but also reduce immunogenicity. We developed a quantitative high-throughput genomics approach to systematically identify and eliminate immune-modulating functions in the virus genome while maintaining replication fitness in vitro. This is a systems-based strategy to enhance viral immunogenicity while attenuating replication and pathogenesis. In this proof-of-principle study, we generated a HIS virus with a combination of eight IFN-sensitive mutations. These mutations also induced higher IFN production and response. We demonstrated that HIS virus is highly attenuated in vivo but is able to induce transient IFN responses, elicit robust and diverse humoral and cellular immunity, and provide protection against homologous and heterologous viral challenges in mice and ferrets.
Recent studies have suggested several strategies to design live attenuated vaccines (14, 15, 33-37). Our method is distinctive in the following aspects: (i) We systematically investigated the whole viral genome, and we eliminated immune-evasion functions at multiple loci to obtain a safe strain that has no detectable replication in vivo; (ii) we selected mutants that induce a higher IFN response, because a transient IFN response has been shown to be essential for adaptive immunity, including the strong and diverse T cell responses; (iii) HIS virus selectively induced a transient IFN response but no other tested inflammatory responses, which reduced potential pathogenesis or side effects for future clinical usage. We have also applied this approach to a DNA virus and generated an effective vaccine candidate.
In general, this unbiased and quantitative high-throughput genomics system can be widely applied to other pathogens to define the impact of genome-wide mutations under certain selection conditions. Similar profiling of a viral genome can be performed with other immune components, such as cytokines, natural killer cells, or T cells, in vitro and in vivo. Inactivating additional immune evasion functions in the virus will further increase the safety and immunogenicity of its derivatives for prevention or therapy.
II. Materials and Methods Used for the Examples in Section I (Influenza A Virus).
a. Viruses, Cells, Mice, and Ferrets
Influenza A/WSN/33 virus (WSN) and Influenza A/PR8/34 virus (PR8) were used to generate WT and mutant viruses. An eight-plasmid reverse genetics system was utilized to reconstitute WT virus (20,41). A live attenuated cold adapted WSN strain (WSN-CA) was generated by introducing 5 mutations in 3 polymerase genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). These 5 mutations were derived from A/Ann Arbor/6/60, which was identified in the backbone for vaccine FluMist (28, 42, 43). Influenza A/X-31 (H3N2) and A/Cal/04/09 (H1N1), A/Cal/07/09 (H1N1), in addition to WSN and PR8, were used for the challenge experiments in mice and ferrets.
293T cells were cultured in DMEM (Corning) with 10% FBS (Corning). Madin-Darby Canine Kidney (MDCK) cells were cultured in DMEM with 10% FBS, but changed to OPTI-MEM (Thermo Fisher) with 0.8 mg/ml TPCK trypsin for viral infection with PR8 backgrounds. A549 cells and THP1 cells were cultured in RPMI1640 (Corning) with 10% FBS. THP1 cells with knockout were generated by CRISPR-Cas9 using a lentivirus expressing a gRNA (targets STING or MAVS) and Cas9-T2A cassette (44). THP1 cells were transduced with the lentivirus and selected with 5 μg/ml puromycin (Life Technologies). Human small airway epithelial cells (HSAEC) were cultured in small airway epithelial cell basal media (SABM) with growth supplements (Lonza). Human bronchial epithelial cells (HBEC) were cultured in keratinocyte serum free media include bovine pituitary extract (BPE, 30 Pg/ml) and hrEGF supplements (0.2 ng/ml) (Invitrogen). Human alveolar macrophages were isolated from lungs of de-identified donors and cultured as previously described (45). For culture of human alveolar epithelial cells, the isolated ATII cells were plated in Dulbecoo modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) on rat tail collagen-coated tissue culture plate and after 2 days of adherence, media were switched to DMEM with 5% FBS. AECs were cultured for another 4 days before influenza virus infection. The Committee for Oversight of Research and Clinical Training Involving Decedents and the University of Pittsburgh Institutional Review Board approved the usage of these human tissues.
Female BALB/c mice (Jackson Laboratory) at the age of 6-8 weeks were used to determine safety, pathology, humoral immune responses and protection. Female C57BL/6J mice (Jackson Laboratory) of the same age were used for T cell studies. IFNAR−/− mice in C57BL/6J background were used for viral replication studies (46). Four-six month old female ferrets (body weight around 1 kg) were purchased from Wuxi Cay Ferret Farm (Jiangsu, China) to determine the safety and efficacy of vaccines.
b. Construction of Influenza Mutant Plasmid Library
Interferon sensitivity is defined using a high-throughput genetic system.
Libraries of influenza A/WSN/33 mutant viruses were generated using the eight-plasmid transfection system as previously described (18, 21-23, 47). The WSN strain was used to generate the mutant library as it provides flexibility for passaging large complex mutant libraries in a variety of cell lines. In brief, the whole length influenza genome was separated into 52 small fragments of 240 bp each. Random mutagenesis was performed with error-prone polymerase Mutazyme II (Stratagene) for each fragment separately. We have carefully controlled the mutation rate to maximize the proportion of point mutations. 0-3 mutations were expected to be generated for every template. For each small library, the mutated fragment was amplified and gel purified, BsaI or BsmbI (NEB) digested, ligated to the vector and transformed into MegaX DH10B T1R cells (Life Technologies). As each small library was expected to have 1,000 single mutations, 50,000 bacterial colonies were collected to cover the complexity at ˜50×. Plasmids from collected bacteria were midi-prepped as the input plasmid library. In each small library, single nucleotide mutation account for 30% of the entire population. Mutant libraries for PB1, PA and M segments were previously constructed (21, 22).
c. Reconstitution, Selection and Tittering of Virus Libraries
To reconstitute the mutant virus library, ˜30 million 293T cells were transfected with each plasmid library (one out of 52 small libraries) together with the other 7 plasmids encoding WT viral proteins (32 Pg of DNA in total) using Lipofectamine 2000 (Life Technologies). Media was changed at 24 h post transfection. Viruses were collected at 72 h post transfection. Viral titer (TCID50) was measured using limiting dilution with A549 cells by observing cell cytopathic effect (CPE). To passage viral libraries without IFN treatment, ˜10 million A549 cells were infected with individual mutant virus library at an MOI of 0.05. Cells were washed with PBS three times at 2 h post-infection. Viruses were collected at 24 h post-infection from supernatants, cleared of cell debris and stored at −80° C. in aliquots. For selection under exogenous type I interferon treatment, A549 cells were pre-treated with 1000 U/ml IFN-D2 (PBL Assay Science) for 20 h and then infected with viral libraries at an MOI of 0.05. Cells were washed with PBS three times at 2 h post-infection, and 1000 U/ml IFN-D2 was added back into culture media. Similarly, viruses were collected at 24 h post-infection from supernatants, clarified of debris and stored at −80° C. in aliquots.
d. Sequencing Mutant Libraries and Data Analysis
Sequencing libraries were prepared as previously described (21, 22). Briefly, viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen Sciences). DNaseI (Life Technologies) treatment was performed, followed by reverse transcription using Superscript III system (Life Technologies).
Real-time PCR was used to quantify viral copy number. At least 1×106 viral RNA copies were used to amplify the mutated fragment. The amplified fragment was then digested with BpuEI or BpmI and ligated with the sequencing adaptor, which had three-nucleotide multiplexing ID to distinguish among different samples.
Deep sequencing was performed with Illumina PE250. Raw sequencing reads were de-multiplexed using the three-nucleotide ID. Sequencing error was corrected by filtering out low quality reads (quality score <30) and un-matched base-pairs between forward and reverse reads. We achieved at least 20,000×sequencing coverage for each nucleotide position. Mutations were called by comparing sequencing reads with the wild-type sequence. Reads containing two or more mutations were excluded from the downstream analysis, which enables us to eliminate potential complications
RF scoremutant i=Relative Frequency of Mutant iinfection/Relative Frequency of Mutant iplasmid
Where Relative Frequency of Mutant i=Reads of Mutant i/Reads of wild type caused by multiple mutations. Relative fitness score (RF score) in each condition was calculated for individual mutations. To reduce sampling errors in the estimating relative fitness, only those mutations with frequency >0.05% in the input plasmid library were reported.
Where relative frequency of mutant i=reads of mutant i/reads of wild type.
All the data processing and analyses was performed with customized python scripts, which are deposited at https://github.com/YushenDu/Influenza-Vaccine
e. Construction of Viruses with Specific Mutation(s)
Viruses with specific mutation(s) were generated using a PCR-based site-directed mutagenesis strategy. To reconstitute mutant viruses on WSN background, 1.5 million 293T cells were transfected with 4 Pg plasmid DNA. Transfections were performed using Lipofectamine 2000 (Life Technologies). Viruses were collected at 72 h post-transfection. Mutant viruses were further amplified in A549 cells. Supernatants were collected, clarified of debris and stored at −80° C. in aliquots. Viral titers were measured by a TCID50 assay with A549 cells. Virion RNA was extracted, and reverse transcribed to cDNA. Coding sequences of all mutants were PCR amplified, and subjected to Sanger DNA sequencing for confirmation. To measure growth kinetics of mutant viruses, ˜1 million A549 cells were infected with each mutant virus at an MOI of 0.1 and supernatants were collected at indicated time. To generate mutants on PR8 background, 1.5 million 293T cells were transfected with 4 Pg DNA and co-cultured with MDCK cells. Media was changed to OPTI-MEM with 0.8 mg/ml TPCK Trypsin at 24 h post-transfection. Viruses were collected at 72 h post transfection. Viral titers were measured by TCID50 assay with MDCK cells. Mutant viruses were further amplified in MDCK cells, supernatants were collected, clarified of debris and stored at −80° C. in aliquots.
In one or more examples, using silent mutation as benchmark, we observed that on average, ˜60% of missense mutations are deleterious for influenza viral replication and only ˜2% are beneficial. Similarly to previous results, the high percentage of deleterious mutations suggested the general conserved nature of an RNA viral genome. However, different proteins in the influenza A genome also showed diverse levels of mutation tolerance. We quantified the average fitness cost as well as the percentage of lethal mutations of every viral protein. Polymerase subunits (PB2, PB1, PA and NP) are highly conserved while NS1 and M2 are relatively mutable.
f. Selection of Possible IFN Sensitive Mutations for Measuring IFN-Sensitivity Using Individually Constructed Mutant Viruses
Because anti-IFN function of NS1 protein has been extensively studied, we focused on proteins (PB2, PB1, PA, NP, M1, M2) for further validation. To select possible IFN sensitive mutations, we followed the following criteria: 1) RF scores >0.7 in A549 cells without IFN selection; 2) RF scores <0.3 under interferon selection; 3) Residues that give similar interferon sensitive phenotypes when mutated into different amino acids were preferred. Moreover, we mapped the potential interferon sensitive residues onto protein structures (PDB: 4WSB, 1RUZ, 2IQH, and 1EA3) (38-40, 48-50) and preferentially selected the ones clustered on the protein surface for validation. Up to 7 mutations per segment were selected.
To measure IFN-sensitivity using individually constructed mutant viruses, 1 million A549 cells were pre-treated with 1000 U/ml IFN-D2 (PBL Assay Science) for 20 h or left untreated. Then cells were infected with indicated virus at an MOI of 0.1. Cells were washed with PBS three times at 2 h post-infection, and 1000 U/ml IFN-D2 was added back into culture media. Supernatants were collected at 24 h post-infection and viral copy numbers were quantified by real-time PCR. Relative IFN-sensitivity for each mutant was calculated as the fold of viral copy number change with IFN selection, normalized to WT.
In one or more embodiments, well-known interferon sensitive mutations were captured by our interferon screen. For example, amino acids of RNA binding domain of NS1 protein, especially R37, R38, K41, R46 were shown to be highly interferon sensitive. This domain was reported to be important to sequester viral dsRNA from cytoplasmic RNA sensors and inhibit certain ISG functions. Our screen results correlate well with the reported key residues for this function.
g. Polymerase Activity Assay
Plasmid DNAs (100 ng of PB2, PB1, PA, and NP; 50 ng of virus-inducible luciferase reporter and 5 ng PGK-Renilla luciferase reporter) were co-transfected into 293T cells in 24-well plates (51). Cells were lysed at 24 h post-transfection and followed by the Dual-Luciferase Assay (Promega).
h. Immunofluorescence Assay
Intracellular localization of IRF3 protein was determined by immunofluorescence. Infected A549 cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton-X100, and blocked with 3% BSA and 10% FBS. Viral NP protein was detected with anti-NP monoclonal antibody (GeneTex). IRF3 was detected with anti-IRF3 rabbit polyclonal antibody (Cell signaling). Hoechst 33342 dye was used for staining DNA.
i. Cell Viability (CCK8) Assay
A549 cells were infected with WT, NS1 R38A/K41A or HIS virus at an MOI of 0.1 in 96-well plates. Mock infection were used as control. 72 h post-infection, CCK8 solution (VitaScientific) were added in each well and incubated for 4 h at 37° C. Signals were detected at OD 450 nm.
j. RNA-Seq Library Preparation and Sequencing
RNA-seq libraries were prepared using a modified method based on the ScriptSeq mRNA-Seq library preparation kit (Epicentre) (48). Multiplex sequencing was performed by 50 bp single-end read with Illumina HiSeq 2000 machine at UCLA Clinical Microarray Core. Raw reads were aligned to the human genome assembly (hg19) or the mouse genome assembly (mm10) using TopHat under default parameter (52, 53). Results were quantified by reads per million total reads (RPM). Differential expression analysis was performed with edgeR (54). Gene ontology enrichment analysis was performed through metascape (55). Genes related to certain cellular pathways were extracted from MsigDB (56,57).
k. Measuring Viral Replication in Mice
To measure viral replication, BALB/c mice were anesthetized with isoflurane (IsoFlo, Henry Schein) and intranasally inoculated with indicated virus in a volume of 30 Pl. Body weight loss was monitored daily for 14 days. To quantify viral growth in mouse lung tissues, mice were intranasally inoculated with indicated virus at a dose of 1×104 TCID50 and sacrificed at day 2 post-infection. DMEM was used as a control for infection. To quantify viral titer by TCID50, lung tissues were harvested, homogenized, and freeze-thawed three times to release the virus. To quantify the viral genome copy number, RNA was extracted from mouse lung tissues using Trizol (Thermo Fisher). Similarly, to quantify the viral growth kinetics and gene expression in mouse lung tissues, lung samples were collected at 2, 6, 24, 48 and 120 h post-infection. Viral copy number and gene expression was quantified by real-time PCR. All mice experiments were performed in accordance with the guidelines of the animal protocols approved by UCLA, CDC China, Trudeau Institute and University of Pittsburgh.
l. Measuring Pathogenesis in Mice
To determine the pathology and cytokine expression, BALB/c mice were intranasally inoculated with indicated virus at a dose of 1×104 TCID50. Bronchoalveolar lavage (BAL) and lung samples were collected at day 2 and day 9 post-infection. Albumin concentration in BAL was determined using mouse albumin ELISA Quantitation kit (Bethyl Alboratories) and cytokine response was analyzed by Lincoplex (BioRad). BAL cell cytospin slides were stained with a HEMA-3 stain kit (Fisher Scientific) for inflammatory cell differential counts. In addition, lung tissues were fixed with 10% neutral buffered formalin (EMD Millipore) and paraffin embedded for histology. Hematoxylin and Eosin (H&E) stained lung tissue slides were scored for their pathology with the following criteria (Score 1-5):
1=No Observable Pathology.
2=perivascular/peribronchus or lung parenchyma inflammatory infiltration <25% of the lobe section.
3=perivascular/peribronchus or lung parenchyma inflammatory infiltration 25%-50% of the lobe section.
4=perivascular/peribronchus or lung parenchyma inflammatory infiltration 50%-75% of the lobe section.
5=perivascular/peribronchus or lung parenchyma inflammatory infiltration >75% of the lobe section. Cytokines in BAL samples were measured by Bio-Plex Pro Mouse Cytokine 23-plex assay according to the product protocol (Bio-Rad)
m. Measuring Antibody and T Cell Responses in Mice
For measuring antibody responses, BALB/c mice were intranasally inoculated with the indicated virus at a dose of 1×104 TCID50. DMEM was used as a control. Mouse sera were collected at days 14, 21 and 28 post-infection. Sera and BAL samples were collected at day 28 post-infection. Sera were used for immunoglobulin G (IgG) antibody detection, hemagglutination inhibition and neutralization assays. BAL samples were used for immunoglobulin A (IgA) antibody detection.
To measure T cell response, C57BL/6J mice were intranasally inoculated with indicated virus at a dose of 1×104 TCID50. To examine primary T cell responses, lungs and spleens were harvested at day 10 post-infection. Fresh cells were used for tetramer staining and flow cytometry. For peptide stimulation of T cells, spleens were harvested at day 28 post-infection. To examine secondary T cell responses, lungs and spleens were harvested at 14 days post challenge of indicated virus at day 28 post-vaccination with HIS virus.
n. Protection from Challenging Infections in Mice
For protection studies, BALB/c mice were intranasally inoculated with the indicated virus at a dose of 1×104 or 1×106 TCID50. DMEM was used as control. Mice were then intranasally challenged with 1×104 TCID50 WT. Viral titers were quantified by both TCID50 and real-time PCR assays at day 2 post challenge.
To examine protection from homologous and heterologous viral challenges, C57BL/6J mice were intranasally vaccinated with HIS virus or PBS as a control. At day 28 post vaccination, A/PR8/34 (H1N1), A/Cal/04/09 (H1N1) and WSN (H1N1) were used for intranasal challenge at a dose of LD90 which was 6000 50% Egg Infective Dose (EID50), 8000 EID50 and 14,000 EID50 respectively. A/X-31(H3N2) was given at 45,000 EID50 (LD50) because it was difficult to reach LD90. Body weight loss was monitored twice daily for 14 days. The following clinical scores were used to quantify the clinical symptoms:
0=no visible signs of disease
1=slight ruffling of fur
2=ruffled fur, reduced mobility
3=ruffled fur, reduced mobility, rapid breathing
4=ruffled fur, minimal mobility, huddled appearance, rapid and/or labored breathing
5=found dead.
o. Enzyme-Linked Immunosorbent Assay (ELISA)
Viral protein specific IgG and IgA antibodies were detected using an enzyme-linked immunosorbent assay (ELISA). 96 well ELISA plates (Costar, Corning) were coated with 1 Pg/ml recombinant viral proteins (HA-WSN, HA-PR8, HA-HK68, HA-Viet04, NP, NA or M1) in bicarbonate/carbonate buffer (pH 9.5) at 4° C. overnight. Wells were washed with PBST between each step for 3-5 times. Wells were then blocked with 10% FBS in PBS for 1 h at room temperature. Serum or BAL samples were diluted in blocking buffer and added to wells for incubation at 4° C. overnight. HRP-conjugated anti-mouse IgG antibody (Cell Signaling) or HRP-conjugated anti-mouse IgA antibody (Thermo Fisher) was diluted in blocking buffer and added into wells for 1 h at room temperature. SIGMA FAST OPD (Sigma) was used to detect the signals at OD 450 nm.
Luminex multiplex assay was performed according to the manufacturer's protocol (Bio-Rad). IFN-D concentration in mouse BAL samples were determined using VeriKine Mouse Interferon Alpha ELISA kit (pbl Assay Science) according to the manufacturer protocol.
p. Hemagglutination Inhibition (HAI) Assay
Mouse sera were pre-treated at 56° C. for 30 min. 4 HA units of WT virus were incubated with 2-fold serially diluted sera at 37° C. for 1 h in V shaped 96-well plate. The starting concentration of sera was 1:4. Washed turkey red blood cells (Lampire) at a concentration of 0.5% were added into each well and incubated at room temperature for 30 min. The HA titer was then read as the highest dilution of serum that prevented hemagglutination.
q. Antibody Neutralization Assay
Mouse sera were pre-treated at 56° C. for 30 min. WT viruses were incubated with serial diluted serum at 37° C. for 1 h. Sera from mock-infected mice were used as control. Viral titer after incubation was measured by TCID50 assay using A549 cells.
r. Determining Mutations on HA Protein Non-Neutralized by Serum Antibodies
Dose response curve of mouse sera against WT viral replication was measured by an antibody neutralizing assay. Reconstituted mutant viral libraries on HA protein were incubated with individual mouse serum (5 mice from WT vaccinated group and 5 mice from HIS vaccinated group) at the concentration of IC80 at 37° C. for 1 h. Sera from unvaccinated mice were used as mock control. After incubation with a serum, mutant libraries were used to infect 30 million A549 cells, and washed with PBS twice at 2 h post-infection. Corresponding serum was added back into the culture media that matched IC80. Supernatants were collected at 48 h post-infection, viral RNA was extracted and reverse transcribed. The relative frequency of each mutant was determined by Illumina MiSeq PE250 as described before.
For each mouse serum selection condition, relative enrichment scores (RE scores) of HA point mutations were calculated by comparing the relative frequencies of the mutations with and without serum selection.
RF scoremutant i=Relative Frequency of Mutant iserum/Relative Frequency of Mutant iplasmid
Where
Relative Frequency of Mutant i=Reads of Mutant i/Reads of wild type.
Mutations with RE score >5 and with RF scores >0.05 were classified as non-neutralized mutations by the corresponding serum antibodies. Mutations that occurred in at least two mouse sera in the WT or HIS group were selected and compared.
s. Quantitation of Viral-Specific CD4 T Cells
Splenocytes were stimulated with WT WSN virus for 16 h at 37° C. with the presence of 1 Pg/ml brefeldin A. Cells were stained with CD3-efluorofore450, CD4-FITC and intracellularly stained with IFNJ-PE. Percentage of IFNJ positive CD3+CD4+ cells was quantified by flow cytometry.
t. Determining the Usage of T Cell Receptor Repertoire
The TCRE loci of influenza NP366-374 specific CD8 T cells from both primary and secondary responses were deep sequenced. For the primary response, mouse lung and spleen tissues were harvested at day 10 post-infection. For the secondary response, vaccinated mice were challenged with WT virus at day 28 post-infection, and tissues were harvested at day 10 post-challenge. To isolate NP-specific CD8 cells, mouse lung and spleen tissues were harvested and single cell suspensions were generated. T cell population was enriched using the EasySep mouse T cell isolation kit (Stemcell) and stained with CD3-efluorofore450, CD8a-FITC and NP366-374 tetramer conjugated with PE. NP positive CD8 T cells were sorted out by FACS. A total of 18 samples were deep sequenced (Adaptivebiotech), including 10 mice for the primary response (5 for WT vaccination and 5 for HIS vaccination), and 8 mice for the secondary response (4 for WT vaccination and 4 for HIS vaccination). VDJ recombination was analyzed for each sample by immunoSEQ Analyzer. Clonality was calculated for NP-specific T cells as the inverse of the normalized Shannon's entropy.
u. Determining Viral Replication and Protection Effects in Ferrets
Ferret experiments were performed in ABSL-2 laboratory in the animal facility at Center for Disease Control and Prevention, China. All animals were determined to be seronegative by HAI to circulating seasonal influenza viruses. To evaluate the replication of HIS and WT viruses, 18 ferrets were divided into 3 groups (6 ferrets each) and intranasally inoculated with 500 Pl of 1×106 TCID50 HIS or WSN. PBS was used as control. Body weight, body temperature and clinical symptoms were observed and recorded daily. At days 1, 3, 4, 7, 9 and 14 post-inoculation, nasal washes and rectal swabs were collected in 1.5 ml PBS. At day 3 post-inoculation, 3 ferrets in HIS or WSN groups and 1 ferret in PBS group were euthanized and tissue specimens (lungs, tracheas and turbinates) were collected.
To evaluate protection efficiency, 8 ferrets were inoculated with 1×106 TCID50 HIS. PBS was used as control. At day 35 post-inoculation, ferrets were intranasally challenged with either 1×107 TCID50 A/WSN/33 (H1N1) or 1×106 TCID50 A/California/07/09 (H1N1pdm) respectively. Body weight, body temperature and clinical symptoms were recorded daily. At days 1, 3, 4, 7 and 9 post challenge, nasal washes and rectal swabs were collected in 1.5 ml PBS. At day 3 post challenge, 2 ferrets in each group were euthanized. Tissue specimens (lungs, tracheas and turbinates) were collected and homogenized in
1 ml of PBS using stainless steel balls and a tissue lyser (TissueLyserII QIAGEN) operated at 25 Hz for 5 mins. Viral titers in supernatants of homogenized tissues, nasal washes and rectal swabs were examined using TCID50 on MDCK cells. Ferret experiments were approved by Animal Ethics Committee of National Institute for Viral Disease Control and Prevention, China.
v. Viral Titer Assay for Ferret Experiments
Viral titers (TCID50) of nasal washes, rectal swabs and tissue samples were detected in MDCK cells with virus culture medium containing 2 μg/ml TPCK-trypsin in 96-well flat-bottom cell culture plates. 50 μl of serial one-half log 10 dilutions for nasal washes and rectal swabs, or 10 fold dilutions for tissue samples were added to MDCK cells and incubated at 37° C. for 1 hour. 100 μl of virus culture medium was added to each well and incubated at 37° C. for 18 to 20 h. Cells were fixed by precooled 80% acetone for 10 mins, followed by ELISA using 1:4000 anti-influenza A NP monoclonal antibody pool (IRR) and 1:2000 goat anti-mouse IgG conjugated with HRP (KPL). Each sample was titrated in triplicate. TCID50 of each sample was calculated by the Reed Muench method.
w. Analyzing Fitness Cost of Mutations in T Cell and Antibody Epitope Regions
T cell epitopes, linear antibody epitopes and conformational antibody epitope sequences were extracted according to the Immune Epitope Database and Analysis Resource (IEDB) (58). We included the epitopes that with conservation >90% for WSN virus for analysis. For T cells epitopes, only human epitopes were considered. The RF scores for mutations within or outside of epitope regions were compared.
x. Mapping onto Protein Structures, Gene Expression, Viral Replication, Polymerase Activity,
We mapped the list of potential interferon sensitive residues onto protein structures. We reasoned that if a residue is interacting with interferon pathway, then it is more likely to be located on the exposable surface. Moreover, it is likely that multiple mutations around the same domain or same pocket showed a similar phenotype. For example, we observed two clusters of interferon sensitive mutations, located on the N terminus of PB2 protein and M1 protein, respectively. Thus, we preferably picked the residues that clustered on the protein surface for validation (
To further examine if each mutant can induce higher interferon production or is deficient in inhibiting interferon response, we infected A549 cells at MOI=1 with different mutants and examined gene expression at 6 h post infection by RT-qPCR. Compared with wild type, the three mutations on PB2 (N9D, Q75H, T76A) and three mutations on M1 (N36Y, R72Q, S225T) induced significantly higher IFN-β gene expression (
y. Safety, Efficiency, and In Vitro Growth
Safety and efficiency are two major considerations for vaccine development. Combinations of interferon sensitive mutations generate a virus that is more sensitive to interferons, and enables exploration of potential use as live attenuated vaccine candidate. On one hand, the interferon sensitive phenotype would make the virus highly attenuated in vivo, on the other hand, the higher induction of interferon system might induce stronger adaptive immune response. Moreover, as the combination of multiple mutations are located in different gene segments and are mostly neutral for viral replication without interferon selection, the risk of a revertant due to do novo mutation or gene assortment is greatly reduced.
Another important consideration for generating a live attenuated vaccine candidate is the capacity of viral growth in vitro. The strain should retain sufficient growth capacity in order for mass production. Thus, to generate a combination strain, we first picked those that can induce higher interferon production, then we tried to add additional interferon sensitive mutations while preserving the viral replication capacity in vitro. After testing a plurality of combinations, we selected one of the combinations, which we called HIS strain. It contains 3 mutations from PB2 (N9D, Q75H, T76A), three mutations on M1 (N36Y, R72Q, S225T), and two mutations on NS1 (R38A, K41A). HIS virus is highly sensitive to interferon treatment (
To examine if the IFN sensitive phenotype of HIS virus is independent of WSN background, we introduced the same 8 mutations into a PR8 background. As expected, HIS virus on the PR8 background also showed higher IFN sensitivity than WT PR8 or NS1 mutations.
We next investigated the replication and pathogenesis of HIS virus in a mouse model. 6-8 weeks Balb/c mice were intranasal inoculated with WT virus or HIS virus at a dose of 105 or 106 TCID50. Body weight loss was monitored daily for 10 days. 106 TCID50 WT virus resulted in significant weight loss of all animals, while same amount of HIS did not cause weight loss. 105 TCID50 WT virus caused very mild (˜5%) weight loss and quickly recovered. Thus, we consistently used 105 TCID50 as the vaccination dose for further studies.
The only FDA-approved LAIV is a recombined virus with internal (PB2, PB1, PA, NP, M and NS) segments derived from A/Ann Arbor/6/60 (H2N2) that is cold adapted, therefore temperature sensitive (TS) and attenuated. The temperature-sensitive phenotype comes form 5 mutations in 3 polymerase genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). Similar phenotypes can be achieved in different strains by introducing the same mutations, including A/WSN/33 strain that we used here. Thus, we constructed and used the temperature sensitive WSN (WSN-TS) strain as control. We further investigated the viral replication in lungs of vaccinated mice. Viral titers of mice lung were quantified by TCID50 and viral copy number at day 2 post vaccination (
To further examine global gene transcription change upon infection in vivo, we performed mRNA sequencing of lung samples at 6, 24, 48, 120 hours post infection of HIS and WT virus. Compared with mock infected controls, the IFN pathway was most significantly upregulated at 48 h post infection for wild type virus, correlated with viral replication. Although highly attenuated in replication, HIS virus also showed early but significant upregulation of certain IFN related genes (
z. Adaptive Immune Responses and Protection
As the HIS virus is highly attenuated in vivo, we then examined if it still can induce robust adaptive immune response for protection. We first evaluated the humeral response. Balb/c mice were intranasal infected with 105 TCID50 WT and HIS virus. At 14, 21 and 28 days post infection, serum samples were collected and HA-specific antibody were assessed through ELISA. We purified 4 types of HA protein from different strains of virus: WSN, PR8, HK68, and Viet04. WSN and PR8 belong to H1, HK68 is H3, and Veit04 is H5. According to the classification of HA proteins, H1 and H5 are class I, while H3 belongs to class II. Specific antibodies were detected for both WT and HIS vaccinated mice at all three time points against WSN, PR8 and Veit04 HA protein. The antibody titer kept increasing throughout the days post infection. HIS virus elicits a lower amount of specific antibodies compared with WT, majorly due to the attenuated replication. Notably, the antibody against HK58 HA was under the detection limit for both the WT and HIS virus using ELISA assay, suggesting there might be limited antibody that can recognize conserved epitope across class I and II HA. We also performed neutralization assay by hemagglutinin inhibition (HI) assays (
We then examined the ability of the HIS virus to induce T cell response. 6-8 week female C57/B6 mice were infected with 105 TCID50 of WT and HIS virus or mock infected with DMEM. A Single cell suspension was prepared from mice lung samples and T cell responses were examined by tetramer staining. HIS virus developed similar levels of NP-specific T cells compared with WT although the replication was three logs lower. Furthermore, we evaluate the memory T cell response 1 month post vaccination by peptide stimulation assay. Solenocytes were incubated with viral peptide of known epitopes overnight and the amount of IFN-g secreting CD8 T cells were quantified by intracellular cytokine staining. Again, similar levels of memory T cell response were observed between HIS and WT vaccinated mice. These results suggested HIS stimulate more robust T cell responses because of the knockout of immune evasion functions.
Finally, we examined if HIS virus can protect mice from challenges. 6-8 week female C57/B6 mice were intranasal vaccinated with 105 TCID50 of WT, NS1 R38A-K41A, HIS, WSN-TS virus or mock infected with DMEM. 4 mice were used per group. All mice were challenged with 105 TCID50 of WT virus 21 days post vaccination and viral titer in lung tissues were quantified at day 2 post challenge. By both RT-qPCR and TCID50 assay, we observed a ˜3 log titer drop in HIS vaccinated mice compare with mock vaccination. The decrease of viral titer is more significant than WSN-TS vaccinated group (
We then challenged the HIS vaccinated mice with three heterologous strains (PR8, ACal/04/09 and X31) at lethal dose. Strong protection was seen across the board in terms of survival rate, percentage of body weight loss, and clinic scores (
Although NS1 is the best studied IFN antagonist in influenza genome, the anti-IFN function has been recently appreciated in other proteins. It is reported that PB2 and PB1F2 could bind to MAVS and inhibit interferon production; ESIE motif in PB1 and PA can mediate type I interferon response, and NP of newly emerged H7N9 can counteract anti-viral ISG: MxA. Here, using a high-throughput genetic approach, we have identified multiple IFN sensitive mutations in the PB2, PA, PB1 and M1 proteins, suggesting the anti-IFN functions of corresponding WT proteins. As anti-IFN functions are essential for efficient viral replication in vivo, it is reasonable that the functions are distributed in different segments. Identified IFN sensitive mutations also work on different parts of IFN system. Mutations on PB2 and M1 can induce higher IFN production, indicating that they work at the upstream of IFN system. On the other hand, mutation on PB1 and PA cannot induce higher IFN production and response, and the sensitivity still remains in IFN deficient Vero cells, suggesting that they might be interacting with JAK/STAT pathway or a specific anti-viral ISG.
III. Process Steps
a. Identifying Interferon Sensitive Mutations Using a Genetic Platform
To systematically identify interferon sensitive mutations across the entire influenza genome, we performed the interferon selection using a high-throughput genetic platform.
The method comprises the following steps.
Block 2600 represents optionally constructing a mutant plasmid library.
In one example, a mutant plasmid library was constructed on the backbone of influenza A/WSN/1933 (H1N1) strain. In order to control mutant library size, the entire viral genome was divided into 240 bp small fragments, and error-prone polymerase was used to introduce random mutations into each small fragment so as to generate a mixed mutant population. In one or more examples, each resulting plasmid population is considered a mutant plasmid library, with ˜1000 different mutations, and total of 52 plasmid libraries are established to cover the whole genome.
Block 2602 represents co-transfecting a mutant DNA library with a plurality of plasmids encoding different fragments of a pathogen genome, so as to form a mutant pathogen library. In one or more examples, 30 million 293T cells are transfected with the DNA library together with seven other wild type plasmids to reconstitute the mutant virus library.
Block 2604 represents passaging the mutant pathogen library in host cells with and without interferons, so as to form a plurality of first infected segments obtained from the passaging with the host cells with the interferons and a plurality of second infected segments obtained from the passaging with the host cells without the interferons. In illustrative embodiments described herein, the mutant viral library was passaged in A549 cells under interferon selection at a concentration of 1000 U/ml, virus was collected 24 h post selection, and biological duplicates were also conducted. In one or more examples, 15 million A549 cells were used to passage the viral library with MOI 0.05 for 24 h. In one or more examples, biological duplications are conducted for both transfection and infection steps.
Block 2606 represents gene sequencing each of the infected segments so as to identify single nucleotide mutations or one or more nucleotide mutations.
Block 2608 represents calculating a relative fitness score (RF score) of each single nucleotide mutation, or each of one or more of the nucleotide mutations, in each of the infected segments, wherein the RF score is a ratio of a frequency of the single nucleotide mutation or one or more nucleotide mutations in the infected segment/infection library as compared with the mutant DNA library. For example, the relative fitness (RF) score of a mutant virus may be calculated as the ratio of the relative frequency in the selected virus library to that in the plasmid library. In one or more examples, ˜90% of nucleotide positions across the genome were covered, and ˜95% of single mutations were detected in the DNA library. In one or more embodiments, to further increase the measurement of viral fitness, filtering was performed to identify mutations that only occur <0.05% in the input library. In one or more embodiments and silent mutations form a normal distribution with RF score centered around 1. In one or more examples, there is a clear separation between silent mutations and lethal mutations, suggesting sufficient selection during passage.
Furthermore, in one or more embodiments, interferon sensitivity may be calculated as the difference of relative fitness with and without interferon selection, and correlation between biological duplicates may be observed.
Block 2610 represents selecting the mutations having an RF score less than 0.2 in the first infected segments and the RF score greater than 0.5 in the second infected segments, so as to form selected mutations. In one or more examples, the basic criteria for selection include RF scores of fitness >0.5; RF scores of fitness under interferon selection <0.2; and preferring residues that give similar interferon sensitive phenotypes when mutated into different amino acids.
Block 2612 represents forming the genome comprising the selected mutations. The genome composition of matter may be embodied in many ways, including, but not limited to, the following embodiments listed below.
Block 2614 represents optionally fabricating a vaccine, therapeutic agent, or other pharmaceutical composition comprising the composition of matter of Block 2612.
Thus,
Exemplary approaches described herein can be used to identify mutations that impact the production of INFs and the sensitivity to IFNs at single nucleotide or single amino acid resolution. In one or more embodiments, the high resolution enabled us to re-engineer the virus with precision that was not previously possible. For example, we are able to rationally change one amino acid at a defined location. Deletion mutation, on the other hand, as described in U.S. Pat. No. 9,387,240 (50) may impact other functions of the viral protein. However, as illustrated herein, we identified and utilized (e.g., 8) mutations that are different from the mutations described in the U.S. Pat. No. 9,387,240 (50). Each of the 8 mutations are very specific for INF induction and do not impact other functions. Moreover, the embodiments of the influenza A mutant described in the examples section herein are strongly suppressed in vivo and did not result in any death of vaccinated mice even at 1×10{circumflex over ( )}7 pfu. However, the U.S. Pat. No. 9,387,240 (50) describes 2 mice were dead after being vaccinated with NS1-99 at 5×10{circumflex over ( )}6 pfu.
b. Method of Fabricating a Composition of Matter and Generating an Immune Response
Block 2700 represents mutating the genome of the wild type influenza A virus so as to generate an influenza A viral genome having a plurality of mutations selected to (a) increase the influenza A virus sensitivity to type I interferon; and (b) induce an antibody and/or T cell response to the influenza A virus when used as an immunogen in vivo that is at least 5%, 10% 25% or 50% of the antibody and/or T cell response induced when a wild type influenza A viral genome lacking the plurality of mutations is used as an immunogen in vivo.
Block 2702 represents the end result, a composition of matter comprising an influenza A viral genome having a plurality of mutations selected to (a) increase the influenza A virus sensitivity to type I interferon; and (b) induce an antibody and/or T cell response to the influenza A virus as described in Block 2700. This genome composition of matter may be embodied in many ways including, but not limited to, the examples listed below.
The method illustrated in
With rapid genome replication, high mutation rate and genome assortment, influenza viruses constantly evolve and adapt to diverse selection pressures, including different host responses. The type I interferon system (IFN), one of the most critical components of innate immune responses, plays essential roles for limiting influenza replication. It is also the bridge between innate immune responses and adaptive immune responses. Determining which viral proteins, more specifically which domain or residues are involved in managing interferon responses, is crucial for better understanding the mechanisms of viral-host interactions and informative for vaccine design.
As these proteins are essential for viral replication, generating mutations is the most direct way to examine their potential anti-IFN function in the context of viral replication. Specifically, using the fact that type I IFN system is the key player of innate immune response, we engineered a virus to be IFN sensitive by mutating IFN evasion genes and so as to efficiently attenuate the virus in vivo. As the IFNs are critical for the development of T cell and B cell response, the IFN inducing strains used as a vaccine stimulated a more robust adaptive immune response (despite reduced replication of the virus). Moreover, the anti-IFN functions of the virus are usually on multi-loci: by selecting a combination of mutations that are introduced into the vaccine strain, the level of replication and the ability to induce protective immune response can be fine tuned in order to maximize the efficacy and limit the risk.
Finally, since NS1 is the major IFN counteracter among influenza genome, different versions of the truncated NS1 protein (deltaFlu) are also being used as vaccine candidates and show impressive CD4, CD8 T cell development and antibody production. However, one limitation is that by focusing on one protein, there is risk of a revertant by gene reassortment. Embodiments of the present invention, on the other hand, use a genome wide screen to systematically identify IFN sensitive mutations across the whole genome that are neutral in viral replication. By combining together multiple mutations located in different gene segments, the risk of a revertant due to do novo mutation or gene assortment is reduced while maintaining the efficiency of vaccination.
Thus, we disclose here a framework for developing a live attenuated vaccine: systematically identifying immune evasion functions on the virus genome, then eliminating the immune evasion functions while maintaining and/or tuning the replication fitness. This systematic method is a generally applicable way for developing live attenuated vaccines and/or therapeutic agents that stimulate antibody responses to viruses in general (including, but not limited to, influenza, Zika, and NiV viruses) as well as other types of pathogens. A therapeutic agent comprising a pathogen genetically engineered according to the method(s) described herein would be particularly useful for patients infected with drug resistant virus and such a patient population would be suitable for the first group of clinical trials. Moreover, the method(s) described herein are suitable for use in human and agricultural (e.g., poultry, pig) applications.
Further information on one or more embodiments of the present invention can be found in reference (47).
IV. Sequences for Proteins and Viruses Described Herein.
Note: This application references a number of different publications as indicated throughout the specification by reference numbers enclosed in brackets, e.g., (x). A list of these different publications ordered according to these reference numbers can be found below.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification.
This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.
This application claims the benefit under 35 U.S.C. Section 119(e) of co-pending and commonly-assigned U.S. Provisional Patent Application Ser. No. 62/471,269, filed on Mar. 14, 2017, by Yushen Du, Nicholas C. Wu, and Ren Sun, entitled “GENOME-WIDE IDENTIFICATION OF IMMUNE EVASION FUNCTIONS IN A VIRUS AT AMINO ACID RESOLUTION AND RATIONAL DEVELOPMENT OF IMMUNE THERAPIES-REDUCTION TO PRACTICE WITH ANTI-INTERFERON FUNCTIONS ON INFLUENZA VIRUS”, (2017-515-1); which application is incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/022465 | 3/14/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/170147 | 9/20/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5504005 | Bloom et al. | Apr 1996 | A |
| 6669943 | Palese et al. | Dec 2003 | B1 |
| 6699476 | Collins et al. | Mar 2004 | B1 |
| 9387240 | Palese et al. | Jul 2016 | B2 |
| 20050080034 | Standring et al. | Apr 2005 | A1 |
| 20050260601 | Whitt et al. | Nov 2005 | A1 |
| 20060140975 | Curtiss et al. | Jun 2006 | A1 |
| 20090010962 | Palese et al. | Jan 2009 | A1 |
| 20110070254 | Brown et al. | Mar 2011 | A1 |
| 20120122185 | Palese et al. | May 2012 | A1 |
| 20120331576 | Brass et al. | Dec 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| 102089423 | Jun 2011 | CN |
| 201736597 | Oct 2017 | TW |
| 201738241 | Nov 2017 | TW |
| 2015157189 | Oct 2015 | WO |
| 2015157189 | Oct 2015 | WO |
| 2018026547 | Feb 2018 | WO |
| Entry |
|---|
| Maamary et al., Attenuated Influenza Virus Construct with Enhanced Hemagglutinin Protein Expression, 2012, Journal of Virology, vol. 86, No. 10, pp. 5782-5790. |
| PCT International Search Report & Written Opinion dated Jul. 26, 2018, PCT/US18/22465. |
| Donelan et al. “A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces high levels of beta interferon and is attenuated in mice”, J Virol, Dec. 2003, vol. 77, No. 24, pp. 13257-13266. |
| Talon et al. “Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach”, Proc Nat Acad Sci. Apr. 11, 2000, vol. 97, No. 8, pp. 4309-4314. |
| Wu et al. “High-throughput identification of loss-of-function mutations for anti-interferon activity in the influenza A virus NS segment” J Virol. Sep. 1, 2014, vol. 88, No. 17, pp. 10157-10164. |
| Extended European Search Report dated Jan. 14, 2021 for European Patent Application No. 18767754.7. |
| Chinese Office Action dated Nov. 1, 2022 for Chinese Application No. 201880030897.9. |
| Donelan et al., “A Recombinant Influenza A Virus Expressing an RNA-Binding-Defective NSI Protein Induces High Levels of Beta Interferon and Is Attenuated in Mice”. Journal of Virology, Dec. 2003, p. 13257-13266. |
| Wu et al., “High-Throughput Identification of Loss-of-Function Mutations for Anti-Interferon Activity in the Influenza A Virus NS Segment”. Journal of Virology, Sep. 2014 vol. 88 No. 17, p. 10157-10164. |
| C. Mössler et al., “Phase I/II trial of a replication-deficient trivalent influenza virus vaccine lacking NS1”. Vaccine 31 , 6194-6200 (2013). |
| Du Y et al., “Genome-wide identification of interferon-sensitive mutations enables influenza vaccine design”. Science 359, 290-296 (2018). |
| Du Y et al., Supplementary Materials for “Genome-wide identification of interferon-sensitive mutations enables influenza vaccine design ”. Published Jan. 19, 2018, Science 359, 290 (2018) , www.sciencemag.org/content/359/6373/290/suppl/DC1. |
| H. Jin et al., Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60. Virology. 306, 18-24 (2003). |
| K. M. Graef et al., “The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon”. J. Virol. 84, 8433-8445 (2010). |
| UCLA Health: The flu vaccine could get a much-needed boost. Jan. 18, 2018, 5 pages, https://www.uclahealth.org/news/the-flu-vaccine-could-get-a-muchneeded-boost. |
| Vincent et al. “Live attenuated influenza vaccine provides superior protection from heterologous infection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease”. J Virol. Oct. 2012;86(19): 10597-605. doi: 10.1128/JVI.01439-12. Epub Jul. 18, 2012. |
| Blanchard et al., “Modified vaccinia virus Ankara undergoes limited replication human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine”. Journal of General Virology ( 1998), 79, 1159-1167. |
| Cidoncha et al., “An Unbiased Genetic Screen Reveals the Polygenic Nature of the Influenza Virus Anti-Interferon Response”. Journal of Virology p. 4632-4646, May 2014 vol. 88 No. 9. |
| Chinese Office Action dated Sep. 15, 2023 for Chinese Application No. 201880030897.9. |
| Number | Date | Country | |
|---|---|---|---|
| 20200129612 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62471269 | Mar 2017 | US |
