Universal t cell targeted influenza vaccine construct

Abstract

The present disclosure provides compositions and methods for generation of an anti-influenza immune response. In particular, multiple T cell epitopes have been identified and further screened for those for coverage of a wide variety of different influenza strains to ensure broad coverage approaching universality. Methods for vaccinating subjects with formulations of such peptides for the treatment or prevention of influenza infection also are described.

Description

DESCRIPTIONUNIVERSAL T CELL TARGETED INFLUENZA VACCINE CONSTRUCTPRIORITY CLAIM

[0001] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 716,369, filed November 5, 2024, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. R01AI130190 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML, created on October 22, 2025, is named USTLP0155WO.xml and is 177,092 bytes in size.BACKGROUND1. Field

[0004] The present disclosure relates generally to the fields of virology and immunology. More particularly, it concerns the identification of T-cell epitopes and the development of protein, DNA, adenovirus, and mRNA-based vaccines for the treatment and prevention of influenza.2. Description of Related Art

[0005] It is estimated that 250,000 - 500,000 deaths occur annually due to influenza (WHO, 2014). Each flu season, 5% to 10% of adults and 20% to 30% of children are infected with circulating flu strains. Influenza viruses undergo high mutation rates within the hemagglutinin (HA) and neuraminidase (NA) sequences, causing antigenic drift and partial escape from pre-existing flu immunity. The more dramatic and abrupt changes in viral composition led to pandemics and more severe disease because human populations have limited cross-protective immunity to the new reassortments (Belshe, 2005). The 1918 Spanish flu pandemic, resulting from a newly emerged avian-like influenza strain, infected 20%-40% of the world’s population and resulted in at least 50-100 million deaths(Taubenberger & Morens, 2006). Other pandemics occurred in 1957 (H2N2), 1968 (H3N2), and 2009 (H1N1), with the most recent 2009 strain infecting >60 million people in the U.S. alone (Shrestha et al. , 2011). Currently, avian H5 and H7 strains represent major public health threats that could lead to unprecedented morbidity and mortality. In 2013- 2014, new H7 avian influenza vims strains infected humans in China (Burke & Trock, 2018). These strains were highly virulent, killing roughly 1 / 3-1 / 2 of those infected. Fortunately, these H7 strains have not yet evolved for efficient human to human transmission (Fang et al., 2013). However, international public health officials agree there is significant risk to public health should H7 viruses evolve to become efficiently transmitted from person-to-person.

[0006] Licensed influenza vaccines currently focus on inducing neutralizing antibodies against seasonal viruses. A major limitation of this approach is the focus on strain-specific immunity that rarely induces optimal immunity against drifted strains that emerge from one flu season to the next. Even during a single flu season, viral drift can occur, which may make a newly generated seasonal influenza vaccine ineffective against the new strains (occurring most recently in 2014-2015 when most H3N2 isolates were antigenically different from the vaccine strain). A major consequence of targeting seasonal influenza strains is variable influenza vaccine effectiveness (VE), ranging from as low as 10% to 60% over the last decade (Treanor et al., 2012; Belongia et al., 2009; CDC, 2016). The predominant influenza vaccines used over the past >30 years are composed of inactivated purified components of split virions formulated to induce strain-specific anti- HA antibodies. Recombinant HA vaccines (rHA) and live attenuated influenza vaccines (LAIV; administered intranasally) have also been used extensively over the past several years. LAIV induces responses against internal viral proteins (unlikely to be effective targets of protective antibody responses), including T cells directed against conserved internal epitopes that can provide more broadly protective heterotypic immunity. LAIV has been shown to induce better protection in children (Belshe et al., 2007), potentially as a result of more broadly heterotypic T cell immunity (Belshe & Gruber, 2000).

[0007] T cells provide heterotypic protective immunity in both mice and humans (Schulman & Kilbourne, 1965; Liang et al., 1994; Benton et al., 2001; McMichael et al., 1983; Sonoguchi etal., 1985; Epstein, 2006). Infection induces heterotypic immunity that relies on both CD4+and CD8+T cells (Schulman & Kilbourne, 1965; Liang et al.,4901-0342-5651 , V. 11994). Furthermore, B cell knockout mice, unable to produce antibodies, develop protective heterotypic immunity after immunization by low dose respiratory infection (Benton et al., 2001). In addition, DNA vaccines expressing the internal influenza proteins nucleoprotein (NP) and matrix 1 and 2 (Ml / 2) have induced heterotypic T cell immunity (Mbawuike et al., 1994), including protective immunity directed against avian H5 strains (Ulmer et al., 1998; Epstein et al., 2002). Overall, these studies demonstrate that T cell-mediated heterotypic immunity can be protective against severe morbidity and death in mice.

[0008] Influenza-specific T cell-mediated heterotypic protective immunity also develops in humans. In 1983, McMichael demonstrated that human CD8+CTL can recognize diverse influenza strains (McMichael et al., 1983). It was more recently demonstrated that U.S. adults had both CD4+and CD8+T cells reactive with avian H5N1 viral sequences despite the absence of previous H5N1 exposure (Jameson et al., 1999). Also, children 6-35 months old develop T cells specific for highly conserved epitopes after LAIV vaccination (Hoft et al., 2011). Perhaps the best data supporting T cell-mediated heterotypic immunity in humans has been derived from epidemiological studies, documenting that persons previously infected with unrelated influenza strains are partially protected against pandemic strains without cross-neutralizing antibodies (Sonoguchi et al., 1985; Epstein, 2006).

[0009] The integration of the fields of bioinformatics and vaccinology has made possible the development of protective T cell -targeted multi -epitope vaccines. Immunoinformatic identification of conserved T cell epitopes in variola and vaccinia genomes (Moise et al., 2009) has been utilized to generate an epitope-based vaccine with demonstrated efficacy against poxviral lethal challenges in mice (Moise et al., 201 1). Furthermore, additional research has demonstrated that vaccines inducing responses against even a single T cell epitope can be sufficient to induce potent protection against virulent pathogen challenge (Moutaftsi et al. , 2006). T cell-targeted influenza and tularemia vaccines also have been shown to induce protective immunity against relevant pulmonary influenza and Francisella tularensis challenges, respectively (McMurry et al., 2007; Gregory et al., 2009; Moise et al., 2013). However, there remains a need for influenza vaccines that induce influenza- specific T cell-mediated heterotypic immunity in humans.4901-0342-5651 , V. 1SUMMARY

[0010] In accordance with the present disclosure, there is provided a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table A, a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table B or a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table C. The peptide / polypeptide may be ordered as in S EQ ID NO: 1. The peptide / polypeptide is fused to another amino acid sequence. The peptide / polypeptide may be lyophilized. The peptide / polypeptide may be disposed in a pharmaceutically acceptable buffer, diluent, or excipient, and / or in a virus-like particle, a nanoparticle of liposomal delivery vehicle.

[0011] Also provided is a nucleic acid encoding a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table A, a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table B or a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table C. The nucleic acid may be a DNA or an RNA. The nucleic acid may further comprise a promoter operably linked to the nucleic acid encoding the peptide. The nucleic acid may be an expression vector, such as a viral vector or a non- viral vector. The nucleic acid may be disposed in a lipid delivery vehicle or a viral or virus-like particle. The nucleic acid may be formulated in a pharmaceutically acceptable buffer, diluent, or excipient.

[0012] In another embodiment, there is provided a method of inducing an immune response in a subject comprising administering to a subject a peptide / polypeptide, a nucleic acid, or a formulation as described herein. Administration may comprise injection, such as subcutaneous or intramuscular injection. Administration may comprise inhalation, such as inhaling a nasal aerosol or mist. The method may further comprise administering an adjuvant to the patient, such as wherein the adjuvant is a squalene adjuvant, a cytokine adjuvant, a lipid adjuvant, or a TLR ligand. The peptide, nucleic acid, or composition may be administered at least a second time. The subject may be a human subject. The method may further comprise measuring a CD4+, a CD8+, and / or a yb T cell response in the subject following administration. The induced immune response may result in one or more of reduced weight loss, increased CD4+T cell activation, increased memory T cell activation, reduced viral load, and increased survival of an infected subject as compared to an infected subject not administered the peptide / polypeptide, nucleic acid or formulation.4901-0342-5651 , V. 1

[0013] As used herein, “essentially free,’’ in terms of a specified component, is used herein to mean that none of the specified components have been purposefully formulated into a composition and / or are present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0014] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0015] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0016] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0017] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0019] FIGS. 1A-C. Influenza-specific T cell responses induced by live attenuated influenza vaccination in children. CFSE-labeled PBMC samples from children vaccinated in a prime / boost fashion with TIV and / or TAIV were stimulated with media or live H3N2 for 7 days prior to analysis of proliferation by CFSE dilution and IFN- y production by intracellular cytokine staining. (FIG. 1 A) Representative CD8-gated FACS plots. (FIG. IB) Cumulative results from gated CD4 and CD8 T cells from n=1 -15 / group; *P<0.05 or **P<0.01 by Wilcoxon matched pairs comparing pre- and post- vaccination samples. Samples ordered left to right correspondence to top to bottom in key. (FIG. 1C) PBMC were stimulated in overnight IFN-y ELISPOT assays with pools of conserved influenza class I (peptide pool 1) or class II (peptide pool 2) predicted HLA binders. Samples ordered left to right correspondence to top to bottom in key. *P<0.05 by Mann- Whitney U test comparing the two groups; **P<0.05 by Wilcoxon matched pairs test comparing pre- and post-vaccination responses.

[0020] FIG. 2. Strategy to identify highly conserved influenza A CD4+and CD8+T cell epitopes relevant for human heterotypic immunity. Shown is an overview of the prediction strategy to identify highly conserved T cell epitopes. Note, that some putative peptides were not successfully synthesized, thus only 202 predicted MHC I supertype binders were evaluated in subsequent assays.

[0021] FIGS. 3A-B. Demonstration of HLA binding by newly identified conserved influenza A epitopes. RMA-S cells expressing HLA-A2 were incubated overnight with fluorescently labeled positive control peptide and serial dilutions of each test peptide. Shown in FIG. 3A are competitive binding assay results from 3 examples of newly identified conserved peptides (A2 66-68), and a predicted non-binding peptide (control peptide). The table presented in FIG. 3B shows the binding affinities for each of the conserved HLA-A2 predicted binders.{00901303} - 6 -4901-0342-5651 , V. 1

[0022] FIG. 4. Human T cell responses directed against highly conserved promiscuous class II influenza Immunogenic Consensus Sequences (ICS). PBMC from genetically diverse humans were expanded over the course of 8 days with a peptide pool comprised of 34 influenza ICS. On day 8, recovered cells were restimulated with individual peptides in overnight IFN-gamma ELISPOT assays. DMSO and the full peptide pool stimulations served as negative and positive controls, respectively. Shown are results of these assays from 2 different volunteers.

[0023] FIG. 5. Summary of T cell responses to predicted conserved influenza T cell epitopes. Shown are PBMC responses from 25 donors to highly conserved promiscuous class II influenza immunogenic consensus sequences (DR) and 13-16 donors for each MHC I supertype. Hit rate is defined as the number of PBMC sets expressing an MHC I supertype allele with a positive test divided by the number of PBMC sets tested. In summary, positive responses were detected in at least one donor to 161 of the 236 peptides tested.

[0024] FIG. 6. Epitope content of the 40 universally relevant long peptides used to create the Universal T cell Influenza Vaccine (UTIV) construct.

[0025] FIG. 7. Full length protein expression by vaccines encoding the UTIV ORF. HEK293 cells were cultured with or without pcDNA-UTIV (2 ug / ml mixed with Lipofcctaminc), Adenovirus expressing UTIV (MOI=1300), or VLPV-UTIV (MOI=20). Cells were collected 24 hours later, permeabilized, and stained with PE-labeled anti-HIS antibody, and analyzed by flow cytometry. Shown are overlay histograms compared with untreated control HEK cells.

[0026] FIGS. 8A-C. Immunogenicity of Adenovirus and saRNA vaccines engineered to express the UTIV open reading frame (ORF). HLA-A2 Tg mice were vaccinated twice, 25 days apart with negative control (NC) or UTIV ORF Ad5- or saRNA vaccines as shown. Adenovirus was delivered IN or SC, LNP-saRNA was administered i.m., and VLP-saRNA was given SC. Shown in FIG. 8A are conserved IAV peptidespecific splenic CD8+ T cell IFN-y ELISPOT results from 3 mice / group studied 3 days after the second vaccination. In FIG. 8B, frequencies of CD8+ / M158 tetramer+ cells in whole blood studied by flow cytometry 8 days post booster vaccination from 8-12 mice / group are shown. In FIG. 8C, PBMC collected 25 days after the final vaccination{00901303} - 7 -4901-0342-5651 , V. 1were studied in overnight IFN-y ELISPOT assays. The numbers of IAV peptide-specific T cells per million PBMC are shown (3-4 pools of 3-4 mice / pool). *** P<0.001 **** PcO.OOOl by ordinary 1 way ANOVA / Tukey’s multiple comparison tests.

[0027] FIG 9. Confirmation of CD4+ T cell immunity induced by UTIV vaccines in HLA DR1 and DR4 Tg mice. Groups of HLA-DR1 Tg mice (left) and HLA- DR4 Tg mice (right) mice devoid of murine class II expression were immunized twice, 2 weeks apart with Ad5- or VLPV- expressing UTIV (or controls). One month later, splenic CD4+ T cells were purified and stimulated with peptide -pulsed antigen presenting cells in overnight IFN-y ELISPOT assays. Shown are DMSO background subtracted mean (±SE) results from 3 mice / group.

[0028] FIGS. 10A-C. UTIV ORF vaccines provide protection against H1N1 and H3N2 challenges. HLA-A2 Tg mice were vaccinated twice, 25 days apart with negative control (NC) or UTIV ORF Ad5- or saRNA vaccines as shown. Adenovirus was delivered IN or SC, LNP-saRNA was administered i.m., and VLP-saRNA was given SC. Mice were challenged with H1N1 A / PR8 one month post-booster vaccination. FIG. 10A shows weight loss curves following a sublethal H1N1 challenge (day 0 weights used to calculate % weight loss). FIG. 10B shows survival curves from a second experiment involving a lethal challenge. FIG. 10C shows results from a similar experiment in which vaccinated mice were challenged with H3N2 A / Victoria / 3 / 75. Five days following challenge, mice were euthanized, and lungs were collected to measure viral burden. Lungs from mice vaccinated with adenovirus-UTIV or VLPV-UTIV contained significantly lower levels of infectious influenza compared to control vaccinated mice. FIG. 10A: * P<0.01 days 8-12 by 2-way ANOVA and Tukey’s multiple comparison tests as indicated compared to the control Ad group. FIG. 10B: ** P<0.04 by Mantel Cox and P<0.03 by Fisher exact tests compared to the VLP control. FIG. 10C: * P=0.0279 unpaired t test and P=0.01 by Mann- Whitney test.

[0029] FIG. 11. Peptides incorporated into the UTIV ORF induce memory T cells in genetically diverse healthy human donors. PBMC from 8 donors expressing diverse MHC I and II were stimulated with DMSO or UTIV peptide pool (UTIV-PP) in overnight IFN-y ELISPOT assays (FIG. HA). PBMC were cultured for 8 days in the presence of DMSO or UTIV-PP prior to restimulation in overnight IFN-y ELISPOT assays (FIG. 11B). * P<0.001 by 2 tailed MW test, ** P<0.01 by Wilcoxon matched pairs test.{00901303} - 8 -4901-0342-5651 , V. 1DETAILED DESCRIPTION

[0030] Influenza kills an estimated 250,000 to 500,000 people each year. Severe pandemics occur when new strains evolve that have not circulated in humans previously. Hemagglutinin, the current conventional vaccine target has a high viral mutation rate. Thus, new vaccines must be generated annually based on the prevalence of circulating strains. Reformulated influenza vaccines are effective only when well-matched with circulating viruses, and thus, efficacy varies substantially year-to-year. Influenza vaccines that induce greater cross-protective immunity are urgently needed.

[0031] Influenza vaccines have historically focused on inducing neutralizing antibodies protective against specific seasonally circulating viruses, but do not protect against drifted strains. In mice and humans, T cells provide heterotypic (more broadly protective) immunity. Mice lacking B cells develop heterotypic immunity (Benton et al. , 2001), and humans previously infected with unrelated influenza strains are partially protected against pandemic strains without cross-neutralizing antibodies (Sonoguchi et al., 1985). To date, influenza vaccines have not been optimized for generating influenzaspecific, T cell-mediated heterotypic immunity in humans, despite great potential for this strategy.

[0032] In this disclosure, the inventors applied the hypothesis that vaccines capable of inducing T cell responses directed against highly conserved internal viral proteins could provide broadly protective influenza immunity. They used cutting-edge immunoinformatic tools to identify T cell epitopes restricted by 9 alleles of HLA-DR and the six major class I supertypes, highly conserved among >73,000 diverse influenza A strains (including hemagglutinin types 1 / 2 / 3 / 5 / 7 / 9 / 10). The inventors validated these predictions, and then designed a multiepitope open reading frame (ORF) incorporating the validated MHC I and MHC II epitopes. This construct, named Universal T cell Influenza Vaccine (UTIV), can and has been incorporated into a variety of delivery platforms. The UTIV ORF vaccines were highly immunogenic in HLA transgenic mice and work is continuing to determine the broad protective efficacies of such vaccines.

[0033] These and other aspects of the disclosure are set out in detail below.{00901303} - 9 -4901-0342-5651 , V. 1I. Aspects of the Present Disclosure

[0034] In mice, T cells provide heterotypic protective immunity. Influenza infection of the lower respiratory tract induces heterotypic immunity dependent on both CD4 and CD8 T cells (Schulman & Kilbourne, 1965; Liang et al., 1994). B cell knockout mice, unable to produce antibodies, also develop protective heterotypic immunity after low dose respiratory infection (Benton et al., 2001). DNA vaccines expressing internal influenza proteins [nucleoprotein (NP), matrix protein 1 (Ml), and / or matrix protein 2 (M2)] can induce heterotypic immunity, including protection against avian H5 strains (Ulmer et al., 1998; Epstein et al., 2002). These studies demonstrate that T cell-mediated heterotypic immunity can protect mice against severe morbidity and death.

[0035] Influenza-specific T cell-mediated heterotypic protective immunity also develops in humans. In 1983, McMichael et al. demonstrated that human CD8+CTL can recognize diverse influenza strains and correlate with protection against human influenza challenge (McMichael et al., 1983). Jameson et al more recently demonstrated that U.S adults had both CD4+and CD8+T cells reactive with avian H5N1 viral sequences, despite the absence of previous H5N1 exposure (Jameson et al., 1999). Perhaps the best data supporting T cell-mediated heterotypic protective immunity in humans are provided by epidemiological observations that persons previously infected with unrelated influenza strains are partially protected against pandemic strains without cross-neutralizing antibodies (Sonoguchi et al., 1985; Epstein, 2006).

[0036] The inventors reported that children 6-35 months old develop T cells specific for highly conserved T cell epitopes after LAIV vaccination (Hoft et al., 2011). They enrolled 55 children over two flu seasons and randomized subjects to receive either two doses of TIV intramuscularly (i.m.), two doses of LAIV intranasally (i.n.), or one dose of each in both possible orders. PBMC were stimulated with a virulent H3N2 strain and with peptide pools predicted to contain “universally’’ relevant influenza CD4+and CD8+T cell epitopes (conserved among 1918 pandemic H1N1, seasonal H1N1 and H3N2, and avian H5N1 strains, and predicted to be presented by common HLA alleles) (Hoft et al., 2011). The inventors measured T cell responses in 7-day CFSE dilution, flow cytometric, and IFN-y ELLSPOT assays to assess expansion and effector cytokine production capacities. FIG. 1A presents dot plots from a representative LAIV recipient’s CFSE dilution assay after live H3N2 stimulation, gated on CD8+T cells. FIG. IB shows{00901303} - 10 -4901-0342-5651 , V. 1composite data for CD4+and CD8+T cell CFSE dilution results in all four vaccine groups (n=13-15 / group). LAIV uniquely induced influenza-specific CD4+and CD8+T cells in these children. Equally important, LAIV induced significant T cell IFN-y EEISPOT responses reactive with the universally relevant flu peptide pools the inventors designed (peptide pools 1 and 2 include epitopes predicted to induce universally cross-reactive influenza-specific CD8+and CD4+T cells, respectively; FIG. 1C). Heterotypic T cell responses could explain why LAIV has protected children significantly better than TIV. The inventors can also produce T cell-based vaccines that enhance conventional antibody responses in poorly responsive populations (e.g., young children and the elderly vaccinated with seasonal vaccines; and all ages vaccinated with pandemic vaccines), and directly induce protective T cell-based heterotypic immunity.IL Definitions

[0037] The phrases “isolated” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the disclosure preferably do not contain materials normally associated with the peptides in their in situ environment.

[0038] An “epitope,” also known as an antigenic determinant, is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cell receptors, or T cell receptors.

[0039] “Major histocompatibility complex” or “MHC” is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes (see Paul, 1993).

[0040] “Human leukocyte antigen” or “HLA” is a human class I or class II major histocompatibility complex (MHC) protein (see, e.g., Stites, 1994).

[0041] An “HLA supertype or family,” as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes. The terms HLA superfamily, HLA supertype{00901303} - 11 -4901-0342-5651 , V. 1family, HLA family, and HLA xx-like supertype molecules (where xx denotes a particular HLA type) are synonyms.

[0042] The term “motif’ refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

[0043] A “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Thus, a supermotif preferably is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.

[0044] “Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

[0045] A “protective immune response” refers to a T cell response to an antigen derived from an infectious agent, which prevents or at least partially arrests disease symptoms or infection. The immune response may also include an antibody response that has been facilitated by the stimulation of helper T cells.III. Influenza VirusA. General

[0046] The etiological cause of influenza, the Orthomyxoviridae family of viruses, was first discovered in pigs by Richard Shope in 1931. This discovery was shortly followed by the isolation of the virus from humans by a group headed by Patrick Laidlaw at the Medical Research Council of the United Kingdom in 1933. However, it was not until Wendell Stanley first crystallized tobacco mosaic virus in 1935 that the non-cellular nature of viruses was appreciated.

[0047] The first significant step towards preventing influenza was the development in 1944 of a killed-virus vaccine for influenza by Thomas Francis, Jr. This built on work by Australian Frank Macfarlane Burnet, who showed that the virus lost virulence when it was cultured in fertilized hen’s eggs. Application of this observation by{00901303} - 12 -4901-0342-5651 , V. 1Francis allowed his group of researchers at the University of Michigan to develop the first influenza vaccine, with support from the U.S. Army. The Army was deeply involved in this research due to its experience of influenza in World War I, when thousands of troops were killed by the vims in a matter of months.

[0048] Although there were scares in the State of New Jersey in 1976 (with the Swine Flu), worldwide in 1977 (with the Russian Flu), and in Hong Kong and other Asian countries in 1997 (with H5N1 avian influenza), there have been no major pandemics since the 1968 Hong Kong Flu. Immunity to previous pandemic influenza strains and vaccination may have limited the spread of the virus and may have helped prevent further pandemics.

[0049] The influenza virus is an RNA virus of the family Orthomyxoviridae, which comprises five genera: Influenza virus A, Influenza virus B, Influenza virus C, Isavirus and Thogotovirus. The Influenza virus A genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are:• H1N1, which caused Spanish flu in 1918 and has been identified as the serotype of the 2009 outbreak of swine flu originating from Mexico• H2N2, which caused Asian Flu in 1957• H3N2, which caused Hong Kong Flu in 1968• H5N1, a pandemic threat and agricultural concern (poultry, cattle)• H7N7, which has unusual zoonotic potential• H1N2, endemic in humans and pigs• H9N2• H7N2• H7N3{00901303} - 13 -4901-0342-5651 , V. 1H10N7

[0050] Influenza viruses bind to cells through sialic acid sugars on the surfaces of epithelial cells, typically in the nose, throat, and lungs of mammals and intestines of birds. The cell imports the virus by endocytosis. In the acidic endosome, part of the viral hemagglutinin protein fuses the viral envelope with the vacuole’s membrane, releasing the viral RNA (vRNA) molecules, accessory proteins, and RNA-dependent RNA polymerase into the cytoplasm. These proteins and vRNA form a complex that is transported into the cell nucleus, where the RNA-dependent RNA polymerase begins transcribing complementary positive-sense vRNA. The vRNA is either exported into the cytoplasm and translated, or remains in the nucleus. Newly synthesized viral proteins are either secreted through the Golgi apparatus onto the cell surface or transported back into the nucleus to bind vRNA and form new viral genome particles. Other viral proteins have multiple actions in the host cell, including degrading cellular mRNA and using the released nucleotides for vRNA synthesis and also inhibiting translation of host-cell mRNAs.

[0051] Negative-sense vRNAs that form the genomes of future viruses, RNA- dependent RNA polymerase, and other viral proteins are assembled into a virion. Hemagglutinin and neuraminidase molecules cluster into a bulge in the cell membrane. The vRNA and viral core proteins leave the nucleus and enter this membrane protrusion. The mature virus buds off from the cell in a sphere of host phospholipid membrane, acquiring hemagglutinin and neuraminidase with this membrane coat. As before, the viruses adhere to the cell through hemagglutinin; the mature viruses detach once their neuraminidase has cleaved sialic acid residues from the host cell. After the release of new influenza viruses, the host cell dies.

[0052] Because of the absence of RNA proofreading enzymes, the RNA- dependent RNA polymerase makes a single nucleotide insertion error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, the majority of newly generated influenza viruses are mutants, causing “antigenic drift.’’ The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one viral line has infected a single cell. The resulting rapid change in viral genetics produces antigenic shifts and allows the virus to infect new host species and quickly overcome protective immunity.{00901303}4901-0342-5651 , V. 1B. The 1918 “Spanish” Flu

[0053] The 1918 flu pandemic, commonly referred to as the Spanish Flu, was an influenza pandemic that spread to nearly every part of the world. It was caused by an unusually virulent and deadly Influenza A virus strain of subtype H1N1. Historical and epidemiological data are inadequate to identify the geographic origin of the virus. Most of its victims were healthy young adults, in contrast to most influenza outbreaks which predominantly affect juvenile, elderly, or otherwise weakened patients. The pandemic lasted from March 1918 to June 1920, spreading even to the Arctic and remote Pacific islands. It is estimated that anywhere from 20 to 100 million people were killed worldwide, or the approximate equivalent of one third of the population of Europe, more than double the number killed in World War I. This extraordinary toll resulted from the extremely high illness rate of up to 50% and the extreme severity of the symptoms, suspected to be caused by cytokine storms. The pandemic is estimated to have affected up to one billion people - half the world’s population at the time.

[0054] Scientists have used tissue samples from frozen victims to reproduce the virus for study. Among the conclusions of this research is that the virus kills via a cytokine storm, an overreaction of the body’s immune system, which explains its unusually severe nature and the concentrated age profile of its victims. The strong immune systems of young adults ravaged the body, whereas the weaker immune systems of children and middle-aged adults caused fewer deaths.

[0055] The global mortality rate from the 1918 / 1919 pandemic is not known, but is estimated at 2.5 to 5% of those who were infected died. Note this does not mean that 2.5-5% of the human population died; with 20% or more of the world population suffering from the disease to some extent, a case-fatality ratio this high would mean that about 0.5- 1 % (»50 million) of the whole population died. Influenza may have killed as many as 25 million in its first 25 weeks. Older estimates say it killed 40-50 million people while current estimates say 50 million to 100 million people worldwide were killed. This pandemic has been described as “the greatest medical holocaust in history’’ and may have killed more people than the Black Death.

[0056] As many as 17 million died in India, about 5% of India’s population at the time. In Japan, 23 million persons were affected, and 390,000 died. In the U.S., about 28% of the population suffered, and 500,000 to 675,000 died. In Britain as many as 250,000{00901303} - 15 -4901-0342-5651 , V. 1died; in France more than 400,000. In Canada approximately 50,000 died. Entire villages perished in Alaska and southern Africa. Estimates for the fatalities in the capital city, Addis Ababa, range from 5,000 to 10,000, with some experts opining that the number was even higher, while in British Somaliland one official there estimated that 7% of the native population died from influenza. In Australia an estimated 12,000 people died and in the Fiji Islands, 14% of the population died during only two weeks, and in Western Samoa 22%.

[0057] This huge death toll was caused by an extremely high infection rate of up to 50% and the extreme severity of the symptoms, suspected to be caused by cytokine storms. Indeed, symptoms in 1918 were so unusual that initially influenza was misdiagnosed as dengue, cholera, or typhoid. One observer wrote, “One of the most striking of the complications was hemorrhage from mucous membranes, especially from the nose, stomach, and intestine. Bleeding from the ears and petechial hemorrhages in the skin also occurred.” The majority of deaths were from bacterial pneumonia, a secondary infection caused by influenza, but the virus also killed people directly, causing massive hemorrhages and edema in the lung.

[0058] The unusually severe disease killed between 2 and 20% of those infected, as opposed to the more usual flu epidemic mortality rate of 0.1 %. Another unusual feature of this pandemic was that it mostly killed young adults, with 99% of pandemic influenza deaths occurring in people under 65, and more than half in young adults 20 to 40 years old. This is unusual since influenza is normally most deadly to the very young (under age 2) and the very old (over age 70) and may have been due to partial protection caused by exposure to a previous Russian flu pandemic of 1889. Another oddity was that this influenza outbreak was widespread in summer and fall (in the Northern Hemisphere). Typically, influenza is worse in the winter months.

[0059] People without symptoms could be stricken suddenly and within hours be too weak to walk; many died the next day. Symptoms included a blue tint to the face and coughing up blood caused by severe obstruction of the lungs. In some cases, the virus caused an uncontrollable hemorrhaging that filled the lungs, and patients drowned in their body fluids (pneumonia). In others, the flu caused frequent loss of bowel control and the victim would die from losing critical intestinal lining and blood loss.{00901303} - 16 -4901-0342-5651 , V. 1

[0060] In fast-progressing cases, mortality was primarily from pneumonia, by virus-induced consolidation. Slower-progressing cases featured secondary bacterial pneumonias, and there may have been neural involvement that led to mental disorders in a minority of cases. Some deaths resulted from malnourishment and even animal attacks in overwhelmed communities.

[0061] One theory is that the virus strain originated at Fort Riley, Kansas, by two genetic mechanisms - genetic drift and antigenic shift - in viruses in poultry and swine which the fort bred for food; the soldiers were then sent from Fort Riley to different places around the world, where they spread the disease. However, evidence from a recent reconstruction of the virus suggests that it jumped directly from birds to humans, without traveling through swine.

[0062] An effort to recreate the 1918 flu strain (a subtype of avian strain H1N1) was a collaboration among the Armed Forces Institute of Pathology, Southeast Poultry Research Laboratory and Mount Sinai School of Medicine in New York; the effort resulted in the announcement (on October 5, 2005) that the group had successfully determined the virus's genetic sequence, using historic tissue samples recovered by pathologist Johan Hultin from a female flu victim buried in the Alaskan permafrost and samples preserved from American soldiers.

[0063] Kobasa et al. (2007) reported that monkeys (Macaca fascicularis) infected with the recreated strain exhibited classic symptoms of the 1918 pandemic and died from a cytokine storm - an overreaction of the immune system. This may explain why the 1918 flu had its surprising effect on younger, healthier people, as a person with a stronger immune system would potentially have a stronger overreaction. In December 2008, research by Yoshihiro Kawaoka of University of Wisconsin linked the presence of three specific genes (termed PA, PB 1, and PB2) and a nucleoprotein derived from 1918 flu samples to the ability of the flu virus to invade the lungs and cause pneumonia. The combination triggered similar symptoms in animal testing.C. The 2009 “Swine” Flu

[0064] The 2009 swine flu outbreak is an epidemic that began in April 2009 with a new strain of influenza virus. The new strain is commonly called swine flu, but some parties object to the name and it has also been referred to as Mexican flu, swine-origin{00901303} - 17 -4901-0342-5651 , V. 1influenza, North American influenza, and 2009 H1N1 flu. On April 30, 2009, the World Health Organization called it influenza A(H1N1). The outbreak is believed to have started in March 2009. Local outbreaks of an influenza-like illness were first detected in three areas of Mexico, but the vims responsible was not clinically identified as a new strain until April 24, 2009. Following the identification, its presence was soon confirmed in various Mexican states and in Mexico City. Within days, isolated cases (and suspected cases) were identified elsewhere in Mexico, the U.S., and several other Northern Hemisphere countries.

[0065] By April 28, 2009, the new strain was confirmed to have spread to Spain, the United Kingdom, New Zealand, and Israel, and the virus was suspected in many other nations, with a total of over 3,000 candidate cases, prompting the World Health Organization (WHO) to change its pandemic alert phase to “Phase 5,” which denotes “widespread human infection.” Despite the scale of the alert, the WHO stated on April 29, 2009, that the majority of people infected with the vims have made a full recovery without need of medical attention or anti-viral dmgs. The common human H1N1 influenza vims affects millions of people every year according to the WHO, causing 250,000 and 500,000 deaths every year around the world. In industrialized countries, most of these deaths occur in those 65 or older.

[0066] In March and April 2009, over 3000 cases of suspected swine flu in humans were detected in Mexico and the southwestern United States. The disease was detected in several countries on multiple continents within weeks of its initial discovery. The strain appears to be unusually lethal in Mexico but not in other countries. There have also been cases reported in the states of San Luis Potosi, Hidalgo, Queretaro, and Mexico State. The Mexican fatalities are mainly young adults of 25 to 45, a common trait of pandemic flu.

[0067] The CDC has confirmed that U.S. cases contained genetic elements from four different flu vimses - North American swine influenza, North American avian influenza, human influenza, and swine influenza vims typically found in Asia and Europe - “an unusually mongrelized mix of genetic sequences.” Pigs have been shown to act as a potential “mixing vessel” in which reassortment can occur between flu vimses of several species. This new strain appears to be a result of the reassortment of two swine influenza vimses, which themselves are descended from previous reassortments in pigs. Influenza vimses readily undergo reassortment because their genome is split between eight pieces of{00901303} - 18 -4901-0342-5651 , V. 1RNA (see Orthomyxoviridae). The virus was resistant to amantadine and rimantadine, but susceptible to oseltamivir (Tamiflu®) and zanamivir (Relenza®).

[0068] Gene sequences for every viral gene were made available through the Global Initiative on Sharing Avian Influenza Data (GISAID). Preliminary genetic characterization found that the hemagglutinin (HA) gene was similar to that of swine flu viruses present in U.S. pigs since 1999, but the neuraminidase (NA) and matrix protein (M) genes resembled versions present in European swine flu isolates. The six genes from American swine flu are themselves mixtures of swine flu, bird flu, and human flu viruses. While viruses with this genetic makeup had not previously been found to be circulating in humans or pigs, there is no formal national surveillance system to determine what viruses are circulating in pigs in the U.S. The seasonal influenza strain Hl N1 vaccine is thought to be unlikely to provide protection.

[0069] The CDC has not fully explained why the U.S. cases were primarily mild disease while the Mexican cases had led to multiple deaths. However, research on previous pandemic strains has suggested that mortality can vary widely between different countries, with mortality being concentrated in the developing world. Differences in the viruses or co-infection are also being considered as possible causes. Of the fourteen initial samples from Mexico tested by the CDC, seven matched the American strain. The virus likely passes through several cycles of infection with no known linkages between patients in Texas and California, and that containment of the vims is “not very likely.’’D. Diagnosis

[0070] Symptoms of influenza can start quite suddenly one to two days after infection. Usually the first symptoms are chills or a chilly sensation, but fever is also common early in the infection, with body temperatures ranging from 38-39°C (approximately 100-103°F). Many people are so ill that they are confined to bed for several days, with aches and pains throughout their bodies, which are worse in their backs and legs. Symptoms of influenza may include:• Body aches, especially joints and throat• Extreme coldness and fever• Fatigue{00901303} - 19 -4901-0342-5651 , V. 1Headache• Irritated watering eyes• Reddened eyes, skin (especially face), mouth, throat and nose• Abdominal pain (in children with influenza B)

[0071] It can be difficult to distinguish between the common cold and influenza in the early stages of these infections, but a flu can be identified by a high fever with a sudden onset and extreme fatigue. Diarrhea is not normally a symptom of influenza in adults, although it has been seen in some human cases of the H5N1 “bird flu” and can be a symptom in children.

[0072] Since anti-viral drugs are effective in treating influenza if given early, it can be important to identify cases early. Of the symptoms listed above, the combinations of fever with cough, sore throat and / or nasal congestion can improve diagnostic accuracy. Two decision analysis studies suggest that during local outbreaks of influenza, the prevalence will be over 70%, and thus patients with any of these combinations of symptoms may be treated with neuraminidase inhibitors without testing. Even in the absence of a local outbreak, treatment may be justified in the elderly during the influenza season as long as the prevalence is over 15%.

[0073] The available laboratory tests for influenza continue to improve. The United States Centers for Disease Control and Prevention (CDC) maintains an up-to-date summary of available laboratory tests. According to the CDC, rapid diagnostic tests have a sensitivity of 70-75% and specificity of 90-95% when compared with viral culture. These tests may be especially useful during the influenza season (prevalence = 25%) but in the absence of a local outbreak, or peri-influenza season (prevalence = 10%).

[0074] Influenza’s effects are much more severe and last longer than those of the common cold. Most people will recover in about one to two weeks, but others will develop life-threatening complications (such as pneumonia). Influenza, however, can be deadly, especially for the weak, old, or chronically ill. The flu can worsen chronic health problems. People with emphysema, chronic bronchitis, or asthma may experience shortness of breath while they have the flu, and influenza may cause worsening of coronary heart disease or congestive heart failure. Smoking is another risk factor associated with more serious disease and increased mortality from influenza.{00901303} - 20 -4901-0342-5651 , V. 1

[0075] Common symptoms of the flu such as fever, headaches, and fatigue come from the huge amounts of proinflammatory cytokines and chemokines (such as interferon or tumor necrosis factor) produced from influenza-infected cells. In contrast to the rhinovirus that causes the common cold, influenza does cause tissue damage, so symptoms are not entirely due to the inflammatory response. This massive immune response can produce a life-threatening cytokine storm. This effect has been proposed to be the cause of the unusual lethality of both the H5N1 avian influenza, and the 1918 pandemic strain (see above).

[0076] In some cases, an autoimmune response to an influenza infection may contribute to the development of Guillain-Barre syndrome. However, as many other infections can increase the risk of this disease, influenza may only be an important cause during epidemics. This syndrome can also be a rare side-effect of influenza vaccines, with an incidence of about one case per million vaccinations.

[0077] People with the flu are advised to get plenty of rest, drink plenty of liquids, avoid using alcohol and tobacco and, if necessary, take medications such as paracetamol (acetaminophen) to relieve the fever and muscle aches associated with the flu. Children and teenagers with flu symptoms (particularly fever) should avoid taking aspirin during an influenza infection (especially influenza type B), because doing so can lead to Reye’s syndrome, a rare but potentially fatal disease of the liver. Since influenza is caused by a virus, antibiotics have no effect on the infection; unless prescribed for secondary infections such as bacterial pneumonia, they may lead to resistant bacteria. Anti-viral medication can be effective, but some strains of influenza can show resistance to the standard anti-viral drugs (see below).IV. Influenza Peptides / PolypeptidesA. Influenza Virus Structural Proteins

[0078] As discussed above, the three major genera of influenza virus are Influenza virus A, B, and C. Influenza virus A has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The{00901303} - 21 -4901-0342-5651 , V. 1influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses.

[0079] Influenza virus B has one species, influenza B vims. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animals known to be susceptible to influenza B infection are the seal and the ferret. This type of influenza mutates at a rate 2-3 times lower than type A and consequently is less genetically diverse, with only one influenza B serotype. As a result of this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at an early age. However, influenza B mutates enough that lasting immunity is not possible. This reduced rate of antigenic change, combined with its limited host range (inhibiting cross species antigenic shift), ensures that pandemics of influenza B do not occur.

[0080] Influenza vims C has one species, influenza C vims, which infects humans, dogs, and pigs, sometimes causing both severe illness and local epidemics. However, influenza C is less common than the other types and usually only causes mild disease in children.

[0081] Influenza viruses A, B, and C are very similar in overall structure. The vims particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous foims can occur. These filamentous forms are more common in influenza C, which can form cordlikc structures up to 500 micrometers long on the surfaces of infected cells. However, despite these varied shapes, the viral particles of all influenza viruses are similar in composition. These are made of a viral envelope containing two main types of glycoproteins, wrapped around a central core. The central core contains the viral RNA genome and other viral proteins that package and protect this RNA.

[0082] Unusually for a vims, its genome is not a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA, each piece of RNA contains either one or two genes. For example, the influenza A genome contains 11 genes on eight pieces of RNA, encoding for 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Ml, M2, NS 1, NS2(NEP), PA, FBI, PB1-F2, and PB2.

[0083] Hemagglutinin (HA) and neuraminidase (NA) are the two large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of{00901303} - 22 -4901-0342-5651 , V. 1the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins are targets for anti-viral drugs. Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA. These different types of HA and NA form the basis of the H and N distinctions in, for example, H5N1. There are 16 H and 9 N subtypes known, but only Hl, H2, and H3, and N1 and N2 are commonly found in humans.B. Peptide / Polypeptide Compositions

[0084] As used herein, an “amino acid” or “amino acid residue” refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimic known in the art, including modified or unusual amino acids. In certain embodiments, the natural residues of the peptide are sequential, without any non-amino acid interrupting the sequence of natural amino acid residues. In other embodiments, the sequence may comprise one or more non-natural amino acid moieties.

[0085] The peptides / polypeptides of the present disclosure can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), Houghten et al. (1985). In some embodiments, peptide synthesis is contemplated by using automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA). The peptides of the present disclosure may be isolated and extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle.

[0086] The term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. The term “polypeptide” generally refers to large peptide sequences.

[0087] An “immunogenic peptide” or “peptide epitope” is a peptide which comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a T cell response. Thus, immunogenic peptides of the disclosure are{00901303} - 23 -4901-0342-5651 , V. 1capable of binding to an appropriate HLA molecule and thereafter inducing a T cell response to the antigen from which the immunogenic peptide is derived.

[0088] As used herein, the term “biocompatible” refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions. In particular embodiments, biocompatible protein, polypeptide, or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens, and harmful immunogens.

[0089] An exemplary influenza poly-antigen sequence is shown below:MRELVRKTRFLPGMMMGMFNMLSTVLGVSILNLGQKNENPAHKSQLVWMACH SAAFEDLALASCMGLIYNRMGAVTTEAASVLVNTYQWIIRNWGPGPGAEIEDLIF LARSALILRGSVAHKSSLPKMEFEPFQSLVPKAIRGQRMFLAMITYITQGMEKLTI TYSSSMMWEINGPGPGMEVVFPNEVGARILRGDLNFVNRANQRLNPMHQLLRH FQKDAKVLFLNWKDEIRRIWRQANNGEDATLMSQSRTREILTKTTVDHMAIIKK GTMVMELIRMIKRGINDRNAQMALQLFIKDYRYTYLTDSQTATKSLLTEVETYV LSIVPSGPLKAEIHENRMVLASTDKRSYLIRALTLNTMTKDMDVNPTLLFLKYLE EHPSAGKKISPLMVAYMLGSGSGVDRFYRTCKLVGINMSKKKSYINRTGTFENSC LETMRTAYERMCNILKGKFQTAAQRAMMIGRFYIQMCTELKLSDYGPGPGMCS LMQGSTLQPTFSVQRNLPFERATVIAPIMFSNKMARLGKGYATYQRTRALVRTG MGTFGPVHFRNQVGPGPGSWIPKRNRSIRPVGISSMVEAMVLVSDGGPNLYNIRN LHIPEVSLKWELGSGSGILHLILWILGPGPGTFEFTSFFYRYGFVANFSMELPSFGV SGAAMGLRISSSFSFGGFTFKGPGPGSLRMKWMMAMKYPITADKQTYDWTLNR NQPAATALARPILSPLTKGILGFVFTLTVPSERGLQHHHHHH (SEQ ID NO: 1)

[0090] In addition, the following peptide components may be assembled into similar but distinct polyproteins for use as vaccine agents. The peptide / polypeptide may comprise all of the peptides in Table A, all of the peptides in Table B, or all of the peptides in Table C. The peptide or polyprotein may contain 50, 60, 64, 70, 80, 90, 100, 105, 110, 115, 120, 125 or all 127 peptides of Table A.{00901303} - 24 -4901-0342-5651 , V. 1Table A - Class I peptides inducing a positive response in human donor PBMCAl-1 LTEVETYVLS (SEQ ID NO: 2) Al-5 ALASCMGLIY (SEQ ID NO: 3) Al-6 LASCMGLIY (SEQ ID NO: 4) Al-7 GTFEFTSFF (SEQ ID NO: 5) Al-8 TFEFTSFFY (SEQ ID NO: 6) Al-9 GTFEFTSFFY (SEQ ID NO: 7) Al-11 LTDSQTATK (SEQ ID NO: 8) Al-17 VSDGGPNLY (SEQ ID NO: 9) Al-18 LVSDGGPNLY (SEQ ID NO: 10) Al-19 VSDGGPNLYN (SEQ ID NO: 11) Al-20 NAISTTFPY (SEQ ID NO: 12) Al-21 FLEESHPGIF (SEQ ID NO: 13) Al-22 NTETGAPQLN (SEQ ID NO: 14) A 1-24 ISSMVEAMV (SEQ ID NO: 15) Al-26 KISPLMVAY (SEQ ID NO: 16) Al-28 YLEEHPSAGK (SEQ ID NO: 17) Al-29 TMDTVNRTHQ (SEQ ID NO: 18) A 1-30 FSFGGFTFK (SEQ ID NO: 19) Al-35 CTELKLSDY (SEQ ID NO: 20) A2-37 LLTEVETYV (SEQ ID NO: 21) A2-38 SLLTEVETYV (SEQ ID NO: 22) A2-39 ALASCMGLI (SEQ ID NO: 23) A2-40 ILGFVFTLTV (SEQ ID NO: 24) A2-41 GMFNMLSTV (SEQ ID NO: 25) A2-42 GMFNMESTVE (SEQ ID NO: 26) A2-43 MGMFNMLSTV (SEQ ID NO: 27) A2-44 MMMGMFNML (SEQ ID NO: 28) A2-45 MMMGMFNMLS (SEQ ID NO: 29) A2-46 GMMMGMFNML (SEQ ID NO: 30) A2-47 GMMMGMFNM (SEQ ID NO: 31) A2-50 FVANFSMEL (SEQ ID NO: 32) A2-51 GILIILILWI (SEQ ID NO: 33) A2-52 GILHLILWIL (SEQ ID NO: 34) A2-53 ILHLILWIL (SEQ ID NO: 35) A2-54 FSMELPSFGV (SEQ ID NO: 36) A2-55 MLSTVLGVSI (SEQ ID NO: 37) A2-56 VLVNTYQW11 (SEQ ID NO: 38) A2-58 IMFSNKMARL (SEQ ID NO: 39) A2-59 VLTGNLQTL (SEQ ID NO: 40) A2-60 FLAMITYIT (SEQ ID NO: 41) A2-61 RMFLAMITYI (SEQ ID NO: 42) A2-62 QLVWMACHSA (SEQ ID NO: 43) A2-63 RMQFSSLTV (SEQ ID NO: 44) A2-64 GILGFVFTL (SEQ ID NO: 45) A2-65 NMLSTVLGV (SEQ ID NO: 46) A2-66 SLLEMCHST (SEQ ID NO: 47) A2-67 WMMAMKYPI (SEQ ID NO: 48) A2-68 MMAMKYPITA (SEQ ID NO: 49){00901303} - 25 -4901-0342-5651 , V. 1A3-70 ALTLNTMTK (SEQ ID NO: 50)A3-76 GTFEFTSFFY (SEQ ID NO: 51)A3-82 FSFGGFTFK (SEQ ID NO: 52)A3-83 SFSFGGFTFK (SEQ ID NO: 53)A3-84 AQMALQLFIK (SEQ ID NO: 54)A3-85 LILRGSVAHK (SEQ ID NO: 55)A3-86 ILRGSVAHK (SEQ ID NO: 56)A3-87 RMKWMMAMK (SEQ ID NO: 57)A3-90 QTYDWTLNR (SEQ ID NO: 58)A3-92 DVNPTLLFLK (SEQ ID NO: 59)A3-93 GVDRFYRTCK (SEQ ID NO: 60)A3-94 LVGINMSKK (SEQ ID NO: 61)A3-98 TVDHMAIIK (SEQ ID NO: 62)A3-99 TTVDHMAIIK (SEQ ID NO: 63)A3-100 TVDHMAIIKK (SEQ ID NO: 64)A3- 103 AMKYPITADK (SEQ ID NO: 65)A3-104 GTFGPVHFR (SEQ ID NO: 66)A24-105 MMMGMFNML (SEQ ID NO: 67)A24-106 MMMGMFNMLS (SEQ ID NO: 68)A24-107 GMMMGMFNML (SEQ ID NO: 69)A24-109 GMFNMLSTVL (SEQ ID NO: 70)A24-110 FYIQMCTEL (SEQ ID NO: 71)A24-111 FEFTSFFYRY (SEQ ID NO: 72)A24-112 LFIKDYRYTY (SEQ ID NO: 73)A24-113 RMCNILKGKF (SEQ ID NO: 74)A24-115 AYERMCNIL (SEQ ID NO: 75)A24-1 16 FYRYGFVANF (SEQ ID NO: 76)A24-118 MCSLMQGSTL (SEQ ID NO: 77)A24-120 SFSFGGFTF (SEQ ID NO: 78)A24-121 SSFSFGGFTF (SEQ ID NO: 79)A24-123 PYSHGTGTGY (SEQ ID NO: 80)A24-124 RLNPMHQLL (SEQ ID NO: 81)A24-125 SWIPKRNRSI (SEQ ID NO: 82)A24-127 TYQWIIRNW (SEQ ID NO: 83)A24-128 LMSQSRTREI (SEQ ID NO: 84)A24-131 MFLAMITYI (SEQ ID NO: 85)A24-132 RMFLAMITYI (SEQ ID NO: 86)A24-135 SYFFGDNAEE (SEQ ID NO: 87)A24-137 1FLARSAL1 (SEQ ID NO: 88)B7-149 RPILSPLTKG (SEQ ID NO: 89)B7-150 RPVGISSMV (SEQ ID NO: 90)B7-155 GPALSINEL (SEQ ID NO: 91)B7-156 LVRGNSPVF (SEQ ID NO: 92)B7-157 QPTFSVQRNL (SEQ ID NO: 93)B7-158 ATYQRTRAL (SEQ ID NO: 94)B7-159 FQTAAQRAMM (SEQ ID NO: 95)B7-162 VPSGPLKAEI (SEQ ID NO: 96)B7-163 SPLMVAYML (SEQ ID NO: 97)B7-164 SLRMKWMMAM (SEQ ID NO: 98)B7-165 GPVHFRNQV (SEQ ID NO: 99){00901303} - 26 -4901-0342-5651 , V. 1B7-168 IPEVCLKWEL (SEQ ID NO: 100) B7-170 FPNEVGARIL (SEQ ID NO: 101) B7-172 RTRALVRTGM (SEQ ID NO: 102) B7-173 RAAVSADPL (SEQ ID NO: 103) B44-175 FEFTSFFYRY (SEQ ID NO: 104) B44-176 TEVETYVLSI (SEQ ID NO: 105) B44-177 MEFEPFQSL (SEQ ID NO: 106) B44- 178 VES AVERGF (SEQ ID NO: 107)B44-179 VESAVLRGFL (SEQ ID NO: 108) B44- 181 MEVVFPNEVG (SEQ ID NO: 109)B44-182 TITGDNTKW (SEQ ID NO: 110) B44-I83 RELVRKTRFL (SEQ ID NO: 111) B44-184 RELVRKTRF (SEQ ID NO: 112) B44-185 MVSPLAVTWW (SEQ ID NO: 113) B44-186 MVSPLAVTW (SEQ ID NO: 114) B44-190 REILTKTTV (SEQ ID NO: 115) B44-191 NENPAHKSQL (SEQ ID NO: 116) B44-192 NENPAHKSQ (SEQ ID NO: 117) B44-193 SVLVNTYQW (SEQ ID NO: 118) B44- 194 HENRM VLAST (SEQ ID NO : 119)B44-195 AEIEDLIFL (SEQ ID NO: 120) B44-196 AEIEDLIFLA (SEQ ID NO: 121) B44-197 AEAIIVAMV (SEQ ID NO: 122) B44-200 MDVNPTLLF (SEQ ID NO: 123) B44-202 FENSCLETM (SEQ ID NO: 124) B44-204 KDAKVLFQNW (SEQ ID NO: 125) B44-205 EEMCHSTQI (SEQ ID NO: 126) B44-206 NENQNPRMF (SEQ ID NO: 127) B44-208 MEITTHFQR (SEQ ID NO: 128){00901303} - 27 -4901-0342-5651 , V. 1Table B - Immunogenic Consensus Sequences (ICS) inducing a positive response in human donor PBMCICS-1 PGMMMGMFNMLSTVLGVSI (SEQ ID NO: 129) ICS-2 FFYRYGFVANFSMELPSFGVSG (SEQ ID NO: 130) ICS-3 LTHMMIWHSNLNDATYQR (SEQ ID NO: 131) ICS-4 KGILGFVFTLTVPSERGLQ (SEQ ID NO: 132) ICS -5 DQSLI1AARN1VRRAS (SEQ ID NO: 133) ICS-6 RSALILRGSVAHKSSLP (SEQ ID NO: 134) ICS-7 ANVVRKMMTNSQDTE (SEQ ID NO: 135) ICS-8 SIGVTVIKNNMINNDLGPA (SEQ ID NO: 136) ICS-9 SNLFEKFFPSSSYRRPIG (SEQ ID NO: 137) ICS-10 PRMFLAMITYITRNQ (SEQ ID NO: 138) ICS - 11 DKRS YLIRALTLNTMTKD (SEQ ID NO : 139)ICS - 12 RGDLNFVNRANQRLNPMH (SEQ ID NO : 140)ICS-13 KSQLVWMACHSAAFEDL (SEQ ID NO: 141) ICS-14 IEDLIFLARSALILRGS (SEQ ID NO: 142) ICS-15 IAPIMFSNKMARLGKGY (SEQ ID NO: 143) ICS-16 QPTFSVQRNLPFERATV (SEQ ID NO: 144) ICS-17 TYDWTLNRNQPAATALA (SEQ ID NO: 145) ICS-18 GGPNLYNIRNLHIPEVSLK (SEQ ID NO: 146) ICS- 19 ALRMKWMMAMKYPITADR (SEQ ID NO: 147)ICS-20 KDEIRRIWRQANNGEDAT (SEQ ID NO: 148) ICS-21 IGRFYIQMCTELKLNDY (SEQ ID NO: 149) ICS-22 RTAYERMCNILKGKF (SEQ ID NO: 150) ICS-23 SDDFALIVNAPNHEGIQA (SEQ ID NO: 151) ICS-24 AAMGERISSSFSFGGFTF (SEQ ID NO: 152) ICS -25 GTMVMELIRMIKRGINDRN (SEQ ID NO : 153)ICS-26 LNPMHQLLRHFQKDAKVLFLNW (SEQ ID NO: 154) ICS-27 QGMEKLTITYSSSMMWEING (SEQ ID NO: 155) ICS-28 ASSMGLIYNRMGAVTTEAA (SEQ ID NO: 156) ICS-29 KHENRMVLASTTAKAMEQM (SEQ ID NO: 157) ICS-30 FGPVHFRNQVKIRRRVD (SEQ ID NO: 158) ICS-31 RMQFSSLTVNVRGSGLRI (SEQ ID NO: 159) ICS-32 KLHGINMSKKKSYINRTGT (SEQ ID NO: 160) ICS-33 RRRFVQNALNGNGDPN (SEQ ID NO: 161) ICS-34 KLEFEPFQSLVPKAIRGQ (SEQ ID NO: 162){00901303} - 28 -4901-0342-5651 , V. 1Table 3 - Long Peptides (LP)LP-01 SLLTEVETYVLSIVPSGPLKAEI (SEQ ID NO: 163)LP-02 RPILSPLTKGILGFVFTLTVPSERGLQ (SEQ ID NO: 164)LP-03 ALASCMGLIYNRMGAVTTEAA (SEQ ID NO: 165)LP-04 HENRMVLAST (SEQ ID NO: 166)LP-05 GILHLILWIL (SEQ ID NO: 167)LP-06 1GRFY1QMCTELKLSDY (SEQ ID NO: 168)LP-07 YLEEHPSAGK (SEQ ID NO: 169)LP-08 KDEIRRIWRQANNGEDAT (SEQ ID NO: 170)LP-09 ATYQRTRALVRTGM (SEQ ID NO: 171)LP-10 MCSLMQGSTL (SEQ ID NO: 172)LP-11 GTMVMELIRMIKRGINDRN (SEQ ID NO: 173)LP- 12 RTAYERMCNILKGKFQTAAQR AMM (SEQ ID NO : 174)LP-13 AEIEDLIFLARSALILRGSVAHKSSLP (SEQ ID NO: 175)LP-14 NENPAHKSQLVWMACHSAAFEDL (SEQ ID NO: 176)LP-15 QPTFSVQRNLPFERATV (SEQ ID NO: 177)LP-16 MDVNPTLLFLK (SEQ ID NO: 178)LP-17 FENSCLETM (SEQ ID NO: 179)LP-18 QTYDWTLNRNQPAATALA (SEQ ID NO: 180)LP-19 DKRSYLIRALTLNTMTKD (SEQ ID NO: 181)LP-20 RMFLAMITYIT (SEQ ID NO: 182)LP-21 IAPIMFSNKMARLGKGY (SEQ ID NO: 183)LP-22 PGMMMGMFNMLSTVLGVSILNLGQK (SEQ ID NO: 184)LP-23 GVDRFYRTCKLVGINMSKKKSYINRTGT (SEQ ID NO: 185)LP-24 GTFEFTSFFYRYGFVANFSMELPSFGVSG (SEQ ID NO: 186)LP-25 AQMALQLFIKDYRYTY (SEQ ID NO: 187)LP-26 LVSDGGPNLYNIRNLHIPEVSLKWEL (SEQ ID NO: 188)LP-27 SWIPKRNRSI (SEQ ID NO: 189)LP-28 RPVGISSMVEAMV (SEQ ID NO: 190)LP-29 LMSQSRTREILTKTTVDHMAIIKK (SEQ ID NO: 191)LP-30 SLRMKWMMAMKYPITADK (SEQ ID NO: 192)LP-31 GTFGPVHFRNQV (SEQ ID NO: 193)LP-32 MEVVFPNEVGARIL (SEQ ID NO: 194)LP-33 KISPLMVAYML (SEQ ID NO: 195)LP-34 RELVRKTRFL (SEQ ID NO: 196)LP-35 AAMGLRISSSFSFGGFTFK (SEQ ID NO: 197)LP-36 RGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFLNW (SEQ ID NO:198)LP-37 QGMEKLTITYSSSMMWEING (SEQ ID NO: 199)LP-38 SVLVNTYQWIIRNW (SEQ ID NO: 200)LP-39 KMEFEPFQSLVPKAIRGQ (SEQ ID NO : 201 )LP-40 LTDSQTATK (SEQ ID NO: 202)C. Variants

[0091] The present disclosure also contemplates modification of the disclosed peptides / polypeptides. Such peptide “variants” may include additional residues, such as{00901303} - 29 -4901-0342-5651 , V. 1additional N- or C-terminal amino acids, or altered / substituted / modified amino acids, and yet still comprise one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological activity.

[0092] The following is a discussion based upon changing the amino acids of a peptide to create a variant peptide. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0093] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartate (+3.0 + 1), glutamate (+3.0 + 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4), sulfur containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic, nonaromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 + 1), alanine (-0.5), and glycine (0); hydrophobic, aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (- 2.3).

[0094] It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred.

[0095] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their{00901303} - 30 -4901-0342-5651 , V. 1hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0096] A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second peptide or polypeptide. In particular, embodiments where multiple peptides of the present disclosure are linked in a “head-to- tail” fashion to create a polyptope molecule, i.e., an epitope multimer. The peptides may be linked to each directly though peptide bonds, or they may be separated by peptide “spacers,” or they may be attached using non-peptide or peptoid “linker,” which are well known in the art. In addition, inclusion of a cleavage site at or near the fusion junction or linker will facilitate removal or release of other peptide sequences. Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals, or transmembrane regions.D. Peptide Purification

[0097] In certain embodiments, the peptides / polypeptides of the present disclosure may be purified. The term “purified peptide” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

[0098] Generally, “purified” will refer to a peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

[0099] Protein / peptide purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide{00901303} - 31 -4901-0342-5651 , V. 1from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.

[0100] In purifying an HLA-restricted peptide of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. Although this preparation will be purified in an inactive form, the denatured material will still be capable of transducing cells. Once inside of the target cell or tissue, it is generally accepted that the polypeptide will regain full biological activity.

[0101] As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0102] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.{00901303} - 32 -4901-0342-5651 , V. 1

[0103] It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS / PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.V. Vaccine Protocols and Formulations

[0104] In an embodiment of the present disclosure, a method of treatment and prevention of influenza by the delivery of a peptide / polypeptide or peptide-encoding composition is contemplated. An effective amount of the vaccine composition, generally, is defined as that amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of the disease or condition or symptoms thereof. More rigorous definitions may apply, including elimination, eradication, or cure of disease.

[0105] Delivery modes include peptide / polypeptide delivery on or in vehicles, such as beads, liposomes, nanoparticles or vims-like particles. Alternatively, the delivery of nucleic acids (RNA or DNA) encoding the peptide / polypeptide, such as naked mRNA / DNA vaccines, viral delivery, non- viral expression constructs, which are optionally encapsulated in lipid delivery vehicles or nanoparticles.A. Administration

[0106] The peptides / polypeptides of the present disclosure may be used in vivo to produce anti -influenza vims immune response, and thus constitute therapeutic and prophylactic vaccines. Also, nucleic acids encoding peptides of the present disclosure may be used in vivo to produce anti-influenza vims immune response. Thus, peptides or nucleic acids can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Administration by the intradermal and intramuscular routes are specifically contemplated. The vaccine can also be administered by a topical route directly to the mucosa, for example by nasal drops or mist, inhalation, or by nebulizer.

[0107] The present disclosure also provides methods for inducing antigen- presenting cells using the peptides / polypeptides of this disclosure. The antigen-presenting cells can be induced by inducing dendritic cells from peripheral blood monocytes; and then contacting (stimulating) them with the peptides / polypeptides of this disclosure, in vitro or in vivo. Administering the peptides of this disclosure to subjects induces in the body of the{00901303} - 33 -4901-0342-5651 , V. 1subject antigen-presenting cells to which the peptides / polypeptides of this disclosure are immobilized. Alternatively, the peptides of this disclosure can be immobilized to the antigen-presenting cells to be administered to the subject as a vaccine.

[0108] Some variation in dosage and regimen will necessarily occur depending on the age and medical condition of the subject being treated, as well as the route chosen. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. In many instances, it will be desirable to have multiple administrations of the vaccine. Thus, the compositions of the disclosure may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Administrations will normally be at from one to twelve-week intervals, more usually from one to six- week intervals. Periodic re-administration will be desirable with recurrent exposure to the pathogen.

[0109] The administration may use various “unit doses.” Unit dose is defined as containing a predetermined quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.B. Measuring Immune Responses

[0110] One of ordinary skill in the art would know various assays to determine whether an immune response against peptides / polypeptides was generated. The phrase “immune response” includes both cellular and humoral immune responses. Various B lymphocyte and T lymphocyte assays are well known, such as ELISAs, cytotoxic T lymphocyte (CTL) assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and cytokine production assays. See Benjamini et al. (1991), hereby incorporated by reference.C. Injectable Formulations

[0111] One method for the delivery of a pharmaceutical according to the present disclosure is via injection. However, the pharmaceutical compositions disclosed herein may alternatively be administered intravenously, intradermally, intramuscularly, or even intraperitoneally as described in U.S. Patent 5,543,158; U.S. Patent 5,641,515 and U.S. Patent 5,399,363 (each specifically incorporated herein by reference in its entirety).{00901303} - 34 -4901-0342-5651 , V. 1

[0112] Injection may be by syringe or any other method used for injection of a solution, as long as the agent can pass through the particular gauge of needle required for injection. A novel needleless injection system has been described (U.S. Patent 5,846,233) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery.

[0113] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0114] The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 niL of isotonic NaCl solution and either added to 1000 mL of hypodermolysis fluid or injected at the proposed site of infusion, (see for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and{00901303} - 35 -4901-0342-5651 , V. 11570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0115] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0116] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

[0117] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions.{00901303} - 36 -4901-0342-5651 , V. 1

[0118] The phrase “pharmaceutically acceptable” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous injectable composition that contains a protein as an active ingredient is well understood in the art.D. Inhalable or Aerosol Formulations

[0119] A particular mode of administration contemplated by the inventor for the peptides of the present disclosure is via inhalation and / or administration to the nasal mucosa, i.e., intranasal administration. A variety of commercial vaccines (influenza, measles) are currently administered using a nasal mist formulation. The methods of the present disclosure can be carried out using a delivery similar to that used with the Flu- Mist® product, which employs the BD AccuSpray® System (Becton Dickinson). Also useful for this route are nebulizers, such as jet nebulizers and ultrasonic nebulizers.E. Additional Vaccine Components

[0120] In other embodiments of the disclosure, the antigenic composition may comprise an additional immunostimulatory agent. Immunostimulatory agents include but are not limited to additional antigens, immunomodulators, antigen presenting cells or adjuvants. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.1. Adjuvants

[0121] As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Adjuvants have been used experimentally to promote a generalized increase in immunity against unknown antigens (e.g., U.S. Patent 4,877,611). Immunization protocols have used adjuvants to stimulate responses for many years, and as such adjuvants are well known to one of ordinary skill in the art. Some adjuvants affect the way in which antigens are presented. For example, the immune response is increased when protein antigens are precipitated by alum. Emulsification of antigens also prolongs the duration of antigen presentation. Suitable molecule adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions.{00901303} - 37 -4901-0342-5651 , V. 1

[0122] Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis'), incomplete Freund’s adjuvants and aluminum hydroxide adjuvant. Other adjuvants that may also be used include IL-1, IL-2, IL-4, IL-7, IL- 12, interferon, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion also is contemplated. MHC antigens may even be used.

[0123] In one aspect, an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1 % solution in phosphate buffered saline. Alternatively, the antigen is made as an admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution. Adjuvant effect may also be made my aggregation of the antigen in the vaccine by heat treatment with temperatures ranging between about 70°C to about 101 °C for a 30 second to 2- minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin, mixture with bacterial cell(s) such as C. parvum, an endotoxin or a lipopolysaccharide component of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles, such as mannide mono-oleate (Aracel A), or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute, also may be employed.

[0124] Some adjuvants, for example certain organic molecules obtained from bacteria, act on the host rather than on the antigen. An example is muramyl dipeptide (N- acetylmuramyl-L alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. The effects of MDP, as with most adjuvants, are not fully understood. MDP stimulates macrophages but also appears to stimulate B cells directly. The effects of adjuvants, therefore, are not antigen specific. If they are administered together with a purified antigen, however, they can be used to selectively promote the response to the antigen.

[0125] In certain embodiments, hemocyanins and hemoerythrins may also be used in the disclosure. The use of hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be employed.{00901303} - 38 -4901-0342-5651 , V. 1

[0126] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al., 1989). The doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin el al., 1989). Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.

[0127] Another group of adjuvants are the muramyl dipeptide (MDP, N- acetylmuramyl-L alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of muramyl dipeptide, such as the amino acid derivative threonyl-MDP, and the fatty acid derivative MTPPE, are also contemplated.

[0128] U.S. Patent 4,950,645 describes a lipophilic disaccharide -tripeptide derivative of muramyl dipeptide which is described for use in artificial liposomes formed from phosphatidyl choline and phosphatidyl glycerol. It is the to be effective in activating human monocytes and destroying tumor cells, but is non-toxic in generally high doses. The compounds of U.S. Patent 4,950,645 and PCT Patent Application WO 91 / 16347, are contemplated for use with cellular carriers and other embodiments of the present disclosure.

[0129] BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell wall skeleton (CWS) may also be used as adjuvants, with or without trehalose dimycolatc. Trehalose dimycolatc may be used itself. Trehalose dimycolatc administration has been shown to correlate with augmented resistance to influenza virus infection in mice (Azuma et al., 1988). Trehalose dimycolate may be prepared as described in U.S. Patent 4,579,945. BCG is an important clinical tool because of its immunostimulatory properties. BCG acts to stimulate the reticulo-endothelial system, activates natural killer cells and increases proliferation of hematopoietic stem cells. Cell wall extracts of BCG have proven to have excellent immune adjuvant activity. Molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990). Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly 2 billion immunizations, BCG has a long record of safe use in man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and{00901303} - 39 -4901-0342-5651 , V. 1there is a worldwide distribution network with experience in BCG vaccination. An exemplary BCG vaccine is sold as TICE BCG (Organon Inc., West Orange, NJ).

[0130] Amphipathic and surface-active agents, e.g., saponin and derivatives such as QS21 (Cambridge Biotech), form yet another group of adjuvants for use with the immunogens of the present disclosure. Nonionic block copolymer surfactants (Rabinovich et al., 1994) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of the present disclosure.

[0131] Another group of adjuvants are the detoxified endotoxins, such as the refined detoxified endotoxin of U.S. Patent 4,866,034. These refined detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, detoxified endotoxins may be combined with other adjuvants to prepare multi-adjuvant-incorporated cells. For example, combination of detoxified endotoxins with trehalose dimycolate is particularly contemplated, as described in U.S. Patent 4,435,386. Combinations of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids is also contemplated (U.S. Patent 4,505,899), as is combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate, as described in U.S. Patents 4,436,727, 4,436,728 and 4,505,900. Combinations of just CWS and trehalose dimycolate, without detoxified endotoxins, is also envisioned to be useful, as described in U.S. Patent 4,520,019.

[0132] Those of skill in the art will know the different kinds of adjuvants that can be conjugated to cellular vaccines in accordance with this disclosure and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others. Certain adjuvants particularly contemplated for use are the teichoic acids from Gram-cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA), and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts may also be employed in connection with the disclosure (Takada et al., 1995).

[0133] Various adjuvants, even those that are not commonly used in humans, may still be employed in animals, where, for example, one desires to raise antibodies or to subsequently obtain activated T cells. The toxicity or other adverse effects that may result{00901303} - 40 -4901-0342-5651 , V. 1from either the adjuvant or the cells, e.g., as may occur using non-irradiated tumor cells, is irrelevant in such circumstances.

[0134] Adjuvants may be encoded by a nucleic acid (e.g., DNA or RNA). It is contemplated that such adjuvants may also be encoded in a nucleic acid (e.g., an expression vector) encoding the antigen, or in a separate vector or other construct. Nucleic acids encoding the adjuvants can be delivered directly, such as for example with lipids or liposomes.2. Biological Response Modifiers

[0135] In addition to adjuvants, it may be desirable to co-administcr biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines such as y-interferon, IL-2, or IL- 12 or genes encoding proteins involved in immune helper functions, such as B-7.3. Chemokines

[0136] Chemokines, nucleic acids that encode for chemokines, and / or cells that express such also may be used as vaccine components. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine coding sequence in combination with, for example, a cytokine coding sequence, to enhance the recruitment of other immune system components to the site of treatment. Such chemokines include, for example, R ANTES, MCAF, MIPl-a, MIP1-P, IP- 10 and combinations thereof. The skilled artisan will recognize that certain cytokines (e.g., IFN’s) are also known to have chemoattractant effects and could also be classified under the term chemokines.4. Immunogenic Carrier Proteins

[0137] The use of peptides / polypeptides for antibody generation or vaccination may require conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin, or bovine serum albumin. Means for conjugating a polypeptide or peptide to an immunogenic carrier protein are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-{00901303} - 41 -4901-0342-5651 , V. 1hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Other immunopotentiating compounds are also contemplated for use with the compositions of the disclosure such as polysaccharides, including chitosan, which is described in U.S. Patent 5,980,912, hereby incorporated by reference. Also, multiple (more than one) peptides may be crosslinked to one another (e.g., polymerized).F. Combination Treatments

[0138] In certain embodiments, it may prove useful to use the vaccines of the present disclosure in conjunction with an anti-viral therapy. Two well-known classes of anti-virals are neuraminidase inhibitors and M2 inhibitors (adamantane derivatives). Neuraminidase inhibitors are currently preferred for flu virus infections. The CDC recommended against using M2 inhibitors during the 2005-06 influenza season.

[0139] Anti-viral drugs such as oseltamivir (Tamiflu®) and zanamivir (Relenza®) are neuraminidase inhibitors that are designed to halt the spread of the vims in the body. These drugs are often effective against both influenza A and B and have been shown to be effective in combatting the recently emerged 2009 “swine” flu. The Cochrane Collaboration reviewed these drugs and concluded that they reduce symptoms and complications. Different strains of influenza viruses have differing degrees of resistance against these anti-virals, and it is impossible to predict what degree of resistance a future pandemic strain might have.

[0140] The anti-viral drags amantadine and rimantadine are designed to block a viral ion channel (M2 protein) and prevent the virus from infecting cells. These drags arc sometimes effective against influenza A if given early in the infection but are always ineffective against influenza B. Measured resistance to amantadine and rimantadine in American isolates of H3N2 has increased to 91% in 2005. In contrast to neuraminidase inhibitors, amantadine and rimantadine have not proven effect again the 2009 “swine” flu.VI. Examples

[0141] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light{00901303} - 42 -4901-0342-5651 , V. 1of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1

[0142] Scientific premise. As described above, conventional influenza vaccines are designed primarily to induce neutralizing antibody responses directed against specific HA present on circulating strains. These vaccines have not been developed to induce optimal T cell responses against more conserved influenza proteins or epitopes. It is well established that T cells directed against conserved influenza epitopes can provide heterotypic protective immunity. CD8+T cells are important for viral clearance, recognizing and destroying influenza-infected epithelial cells, and preventing viral propagation. CD4+T cells facilitate the development of both CD8+T cells and antibodysecreting B cells. Multi-epitope vaccines incorporating conserved influenza T cell epitopes protect against H1N1 and H3N2 challenges in HLA-A2 and HLA-DR1 transgenic mice (Eickhoff et al., 2019). These findings strongly supported the rationale of generating influenza vaccines against conserved influenza T cell epitopes. The inventors have employed state-of-the-art immunoinformatics to identify CD4+and CD8+T cell epitopes conserved in diverse influenza A strains to generate vaccines that provide long-term protective heterotypic immunity in genetically diverse human populations.

[0143] Experimental confirmation of innovative immunoinformatic tools identifying protective T cell epitopes. The inventors’ collaboration with EpiVax allows for novel immunoinformatic identification of T cell epitopes followed by experimental confirmation of the relevance for inclusion in protective vaccines. For example, shortly after the emergence of pandemic H1N1 (pH INI) virus in 2009, EpiVax computationally identified T cell epitopes highly conserved between the pandemic virus and the prepandemic seasonal vaccine strain, Brisbane H1N1, predicted to bind promiscuously to the most common HLA-DRB1 alleles (De Groot et al., 2009). These computational methods accurately determined broadly reactive CD4+T cell epitopes: cross-reactive pH INI - specific T helper cell responses were identified in the circulation of eight randomly chosen donors (Schanen et al., 2011). As donors were never exposed to pHINl (blood draws predated the outbreak), the results demonstrated pre-existence of cross-reactive pHlNl- specific T helper cells. This illustrates the power of immunoinformatics to define broadly{00901303} - 43 -4901-0342-5651 , V. 1reactive CD4+T cell epitopes and underscores the importance of cross-reactive immunity for influenza vaccine design.

[0144] Several groups have developed immunoinformatic algorithms for T cell epitope predictions. However, EpiVax has developed an innovative immunoinformatic toolkit that can facilitate T cell vaccine development, from epitope prediction to generation of optimally designed multi-epitope vaccines. Many algorithms can identify CD8 T cell epitopes restricted by the six major supertypes of HLA class I. The EpiVax toolkit component EpiAssembler, includes unique algorithms for identifying immunogenic consensus clusters of conserved CD4+T cell epitopes with promiscuous activity across nine major HLA-DR alleles expressed by >95% of humans. The resulting immunogens identified with this approach have higher epitope density and increased HEA binding diversity than epitopes identified with any other available algorithm. JanusMatrix, another unique component of the EpiVax toolkit, can be used to exclude peptides with homology to human self-peptides, and uniquely identifies TCR facing residues capable of inducing Treg cells or autoimmunity. VaxCAD™, a third unique component of the EpiVax toolkit can be used to optimally order a series of T cell epitopes in a single synthetic open reading frame, avoiding the generation of junctional neoepitopes. All three of these unique immunoinformatic tools will improve chances of success in the development of universal influenza T cell vaccines.

[0145] The use of innovative delivery formats for highly immunogenic T cell epitope-based vaccines. T cell-targeted vaccines have been generated that induce robust T cell immunity and protection against challenge in animal models (El Bissati et al., 2016; Kotturi et al., 2009; Moise et al., 2013c; Moise et al., 201 1 ; McMurry et al., 2007; Gregory etal., 2009). EpiVax algorithms have immunoinformatically identified conserved T cell epitopes in variola and vaccinia genomes (Moise et al., 2009) and generated epitopebased DNA and protein vaccines protective against lethal vaccinia challenge in HLA-DR3 Tg mice (Moise et al., 2011). These results indicate class II T cell epitopes alone can provide excellent protection in a HLA transgenic mouse model, and support other findings that a limited set of T cell epitopes may be sufficient to induce protective immune responses (Moutaftsi et al., 2006). EpiVax immunoinformatic tools have also been used to develop a tularemia T cell epitope-based vaccine that protects against pulmonary challenge (McMurry et al., 2007; Gregory et al., 2009) and a multi-epitope T cell-based vaccine for{00901303} - 44 -4901-0342-5651 , V. 1seasonal influenza (Moise et al., 2013c). These results clearly show that genome-derived, epitope-based vaccines can protect against virulent pathogens.

[0146] While a few T cell epitope vaccines have failed in clinical trials (Gorse et al., 2008; Wilson et al., 2008), there is increasing evidence that this approach can induce sufficient T cell responses in humans. For example, a recombinant protein expressing a limited number of CD4+and CD8+T cell influenza epitopes was shown to induce T cell responses, and enhance HA-specific antibody responses in human vaccinees (Atsmon et al., 2012; Atsmon et al., 2014). In addition, an adjuvanted vaccine formulation containing four short synthetic peptides from Ml, M2, and NP proteins which encompass both T and B cell epitopes (FLU-v) has proven to be immunogenic and offered protection against mild to moderate influenza disease in recent clinical trials (Pleguezuelos et al., 2020a; Pleguezuelos et al., 2020b; Pleguezuelos et al., 2015; Pleguezuelos et al., 2012). Further, several epitope-based cancer vaccines have proven immunogenic in humans (Lennerz et al., 2014; Asahara et al., 2013; Kenter et al., 2009; Bocchia et al., 2005; Ott et al., 2017; Sahin et al. , 2017). A major goal of this work is to improve vaccine delivery strategies for induction of potent T cell immunity in humans, generally applicable for vaccinology.

[0147] Previous investigator studies demonstrated success using a genome-to- vaccine approach for influenza (Eickhoff et al. , 2019). The inventors’ approach has focused on the most highly conserved proteins among diverse influenza A strains. Ml coats the inside of the virus and is involved in virus replication and assembly. M2 forms ion channels critical for viral uncoating and release of the segmented genome in infected cells. NP is a structural protein that encapsidates viral genetic material. All three are critical for the viral life cycle. The inventors’ previously used an immunoinformatic approach to develop T cell- targeted influenza vaccines. Epivax’s immunoinformatic tools were used to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). They found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class 11-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses.{00901303} - 45 -4901-0342-5651 , V. 1Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrated proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.

[0148] Generation of a Universal T cell Influenza Vaccine (UTIV). Here, the investigators utilized a more thorough approach to generate a universal T cell targeted influenza vaccine capable of inducing broadly protective immunity in diverse populations. An overview of this enhanced strategy is depicted in Fig. 2. Immunoinformatic tools developed at EpiVax were utilized to identify highly conserved influenza T cell epitopes from internal IAV proteins nucleoprotein (NP), matrix proteins 1 and 2 (M1 / M2), and viral polymerase subunits PB 1 and PB2. To accomplish this task, two datasets of these sequences were obtained. Dataset 1 contained protein sequences from 63 highly diverse influenza A strains which were used as an initial screen of epitope conservation. Dataset 2 was comprised of sequences from 73,989 diverse human, avian and swine IAV strains and was used to further determine the broad relevance of predicted CD4+ and CD8+ T cell epitopes. Highly conserved putative epitopes for the 6 class I supertypes (A*0101, A*0201, A*0301, A*2402, B*0702, and B*4403) and 9 common class II alleles (DRBl *0101 , DRBl *0301, DRBl*0401, DRBl*0701, DRBl*0801, DRBl*0901, DRBl*1101, DRBl*1301, and DRB 1*1501), each covering >95% of the world’s population, were first predicted based on dataset 1 using the EpiMatrix algorithm. EpiMatrix scores each individual 9- and 10- mer frame for predicted binding to the class I supertypes and common class II alleles above. The resulting Z-scores represent MHC binding potential, and scores >1.64 are considered as likely “hits”. Because class II molecules arc ‘open-ended’ Class II-cpitopc dense cores and overlapping frames can be combined into a single longer sequence with multiple binding frames predicted to bind multiple class II supertypes. Overlapping 9-mer frames predicted to bind diverse class II molecules were used to create immunogenic consensus sequences (ICS) using EpiAssembler. Typically, the core 9-mer is predicted to bind 4 or more different common class II supertype alleles, and the resulting ICS can range from 15- 30 amino acids and often contain over 20 individual hits. Class II clusters scores were determined based on the sum of the EpiMatrix hits in the ICS. High ranking putative class{00901303} - 46 -4901-0342-5651 , V. 1I epitopes and class II ICS were further evaluated for cross-conservation within human proteins and extended broad conservation in the dataset containing 73,989 IAV strains using the JanusMatrix algorithm. This tool examines relative homology of a given sequence with other sequences (eg - human proteome and diverse IAV strains) at both the MHC binding face (retained predicted MHC binding) and the TCR-facing face, thereby identifying similar sequences likely to trigger the same T cells. A weighted ranking of potential peptides identified 34 high value panDR ICS and 208 potential MHC I epitopes for further study. Putative peptides were synthesized to >80% purity (34 / 34 panDR ICS and 202 / 208 potential MHC I binders)

[0149] The investigators next validated these predictions using two methodologies. They performed MHC binding assays with a subset of the predicted epitopes (HLA-A2), and indeed all of the peptides were found to bind to HLA-A2 (Fig 3). The human relevance of these peptides were determined using human PBMC expansion ELISPOT assays. Here, PBMC from diverse individuals were stimulated with pools of peptides, and then after 1 week, T cell responses to individual peptides were measured using IFN-gamma ELISPOT assays. Shown in Fig. 4 are representative results from 2 individuals. Cumulative data obtained from 25 donors to highly conserved promiscuous class II influenza immunogenic consensus sequences (DR) and 13-16 donors for each MHC I supertype are shown in Fig. 5. In summary, positive responses were detected in at least one donor to 161 of the 236 peptides tested.

[0150] Construction of new vaccines expressing newly identified MHC I and MHC Il-restricted epitopes predicted to induce CD4+ and CD8+ T cells protective against all influenza A in >95% of the human population. A universal influenza vaccine construct was engineered based on results from human T cell expansion assays shown in Fig 5. Epitopes for inclusion were selected based on restriction by 9 common HLA-DR alleles and all 6 class I supertypes and human T cell responses shown in Fig 5. The combination of epitopes is predicted to induce responses in more than 95% of the world’s population. Scaled rankings were obtained based on the ranked hit rate, ranked magnitude, and ranked number of unique HLA alleles (of the hits). Next, overlapping peptides were assembled from epitopes with the highest scaled rankings. In total, 40 overlapping long peptides were generated; these contain 23 validated class II ICS and 106 validated class I epitopes. The epitope content (validated and predicted) within the{00901303} - 47 -4901-0342-5651 , V. 140 long peptides are shown in Fig 6. The 40 long peptides were engineered into a single open reading frame (741 amino acids) for creation of universal T cell targeted influenza vaccines. VaxCAD™ was utilized to minimize predicted class I and II junctional hits. Consensus aggregation-prone regions (APRs) were mapped based on predictions using 2 tools that had the best performance for prediction of APRs (Rawat PR, et al., 2021). The junctional APRs were manually rearranged. The resulting construct contains 5 predicted APRs, and only one is junctional. The remaining 4 are within individual long peptides. In addition, all predicted transmembrane helices were removed. The construct is likely to be insoluble; its overall predicted solubility is 27.65% based on an assessment using the Soluble Domain for Protein Expression (SoDoPE) tool. Next, a terminal 6x HIS tag and stop site was added. The sequence was then reverse engineered using codon harmonization tools optimized forexpression in humans. This construct (termed Universal T cell Influenza Vaccine, UTIV) was synthesized and subcloned into a pcDNA3 mammalian expression plasmid. The same UTIV ORF was subcloncd by collaborators into 1) adenoviral shuttle vectors for subsequent production of replication-deficient adenovirus vaccine constructs, and 2) an RNA replicon plasmid backbone for generation self-amplifying RNAs (saRNA). RNA replicon plasmid DNA was linearized by restriction digestion and then purified by phenol-chloroform extraction. Next, in vitro transcription was performed using Promega RiboMax large scale RNA production kits with rNTPs, T7 RNA polymerase, and m7G(5’)ppp(5’)G RNA capping. The saRNA encoding UTIV was encapsidated in vaccine-like particles (VLP) or lipid nanoparticles (LNP). FIG. 7 shows expression of UTIV by the various vaccine formats (plasmid, adenovirus, and VLP vectored saRNA) in treated IIEK293T cells (control treated cells are shown in red).

[0151] Next, comparative studies of Ad5 and saRNA vaccines were performed in HLA-A2 Tg mice, which express HLA-A2 but do not express murine class I molecules. Ad5-UTIV was delivered intranasally or subcutaneously, LNP-saRNA was delivered intramuscularly, and VLP vectored saRNA was delivered subcutaneously. Mice were vaccinated on days 0 and 25, and CD8+T cell responses were evaluated by EL1SPOT on day 28 after restimulation with mature dendritic cells pulsed with a pool of human MHC I conserved IAV peptides (FIG. 8A). Eight days post-booster vaccination, blood (9-12 mice / group) was stained with surface markers including CD45, CD3, CD4, and CD8, as well as with an HLA-A2 tetramer reagent to stain CD8+ T cells specific for an immunodominant conserved IAV matrix 1 peptide (FIG. 8B). To evaluate short term{00901303} - 48 -4901-0342-5651 , V. 1memory T cell responses, PBMC from 3-4 sets of pooled blood from 3-4 mice per set were stimulated with a pool of conserved human MHC I IAV peptides in overnight IFN-y ELISPOT assays (FIG. 8C). These immune studies (FIGS. 8A-C) demonstrated very potent and highly significant T cell responses elicited by Ad-UTIV s.c. and VLPV-UTIV s.c. vaccines. Similar results were observed in HLA-B7 transgenic mice with the VLP- UTIV vaccine, demonstrating that UTIV is capable of inducing CD8 T cell responses presented by a different class I supertype (not shown). To determine whether UTIV vaccines could induce CD4+ T cell immunity of human relevance, we vaccinated two strains of mice devoid of murine class II expression but instead expressing chimeric class II with binding domains from two different human class II supertypes (HLA DR1 and HLA DR4). FIG. 9 shows CD4+ T cell responses to UTIV peptides from vaccinated HLA-DR1 (left) and HLA-DR4 (right) transgenic mice (3 mice / group).

[0152] To determine whether the UTIV vaccines could provide protection against influenza infection and disease, groups of HLA-A2 mice (N=8-l l / group) were challenged i.n. with H1N1 (A / Puerto Rico / 8 / 1934). A second control group (Adenovirus expressing matched HA) was included in these studies as a positive control (N=4). Mice were weighed daily beginning the day of infection through 14 days post-infection. Shown in FIG. 10A are weight loss curves for groups of vaccinated mice after nonlethal challenge. Weight loss curves of the negative control vaccine group (Ad-NC) were very similar to groups vaccinated with Ad-UTIV (i.n.) and LNP-saRNA-UTIV. These mice lost -20% body weight, peaking at day 8-9 post challenge. The matched HA vaccine (Ad-HA) provided nearly absolute protection, as expected. Interestingly, the Ad-UTIV and VLP- saRNA-UTIV provided significant protection, with weight loss peaking 6 days post infection (PcO.Ol, days 8-12 post-infection compared to NC-Ad group). A lethal H1N1 challenge experiment was set up using the same vaccines described above (FIG. 10B). In this experiment, VLP-saRNA-UTIV provided significant protection against a highly lethal H1N1 challenge (P<0.04 by Mantel Cox and P<0.03 by Fisher exact tests compared to the VLP control). To determine if the UTIV construct could provide protection against another common seasonal influenza A subtype, groups of HLA-A2 mice were vaccinated with control or UTIV adenovirus or VLPV-saRNA vaccines. Mice were challenged with H3N2 (A / Victoria / 3 / 1975) and after 5 days, mice were euthanized and lungs were utilized to measure viral burdens. Lungs from mice vaccinated with adenovirus -UTIV or VLPV-{00901303} - 49 -4901-0342-5651 , V. 1UTIV contained significantly lower levels of infectious influenza compared to control vaccinated mice (FIG. 10C; P=0.01 by Mann- Whitney test).

[0153] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as defined by the appended claims.{00901303} - 50 -4901-0342-5651 , V. 1VII. References

[0154] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Asahara et al., J Transl Med. 11:291, 2013.Atsmon et al., J Clin Immunol. 32(3):595-603, 2012.Atsmon et al., Vaccine. 32(44):5816-23, 2014.Belongia et al., J. Infect. Dis., 199: 159, 2009.Belshe, N Engl J Med. 353(2 l):2209-l 1, 2005.Belshe et al., N. Engl. J. Med., 356(7):685-696, 2007.Belshe & Gruber, Pediatr. Infect. Dis. J., 19(5 Suppl):S66-71, 2000.Benton et al,, J. Immunol,, 166:7437, 2001.Bocchia et al., Lancet. 365(9460):657-62, 2005.Burke & frock, Emerg. Infect. Dis., 24(3):471-477, 2018.CDC, Seasonal Influenza Vaccine Effectiveness, 2005-2016, 2016 [cited April 25, 2016], De Groot et al.. Vaccine. 27(42):5740-7, 2009.Eickhoff et al. , Vaccine. 2019.El Bissati et al., JCI insight. l(15f.c85955, 2016.Epstein et al., Emerg. Infect. Dis., 8(8):796-801, 2002.Epstein, J. Infect. Dis., 193:49, 2006.Fang et al., Scientific Reports, 3:2722, 2013.Gregory et al., Vaccine, 27:5299, 2009.Gorse et al., Vaccine. 26(2):215-23, 2008.Hoft et al., J. Infect. Dis., 204(6):845-853, 2011.Jameson et al., J. Immunol., 162(12):7578-7583, 1999.Kenter et a / ., N Engl J Med. 361(19): 1838-47, 2009.Kotturi et al., PLoS Pathog. 5(12):cl000695, 2009.Lennerz et al., Cancer immunology, immunotherapy : CII.;63(4):381-94, 2014.Liang et al., J. Immunol., 152:1653, 1994.Mbawuikc et al,, Vaccine, 12(14):1340-1348, 1994.McMichael et al., N. Engl. J. Med., 309:13, 1983.{00901303} - 51 -4901-0342-5651 , V. 1McMurry et al., Vaccine. 25:3179, 2007.Moise et al., Vaccine, 27:6471, 2009.Moise et al.. Vaccine, 29:501, 201 1.Moise et al., Human Vaccines & Immunotherapeutics, 9:1577, 2013.Moise et al., Human Vaccines & Immunotherapeutics, 9:1598, 2013.Moise et al., Human Vaccines & Immunotherapeutics, 9:2060, 2013c.Moutaftsi et al., Nat. Biotechnol., 24:817, 2006.Ott et al., Nature. 547(7662):217-21 , 2017.Pleguezuelos et al., NPJ Vaccines. 5:22, 2020a.Pleguezuelos et al., Ann Intern Med. 172(7):453-62, 2020b.Pleguezuelos et al., Clin Vaccine Immunol. 22(7):828-35, 2015.Pleguezuelos et al., Vaccine. 30(31):4655-60, 2012.Sahin et al., Nature. 547(7662): 222-6, 2017.Schanen et al., Vaccine. 29(17):3299-309, 2011.Schulman & Kilbourne, J. Bacterial., 89:170, 1965.Shrestha et al. , Clin. Infect. Dis., 52(Suppl 1):S75, 2011.Sonoguchi et al., J. Infect. Dis., 151:81, 1985.Taubenberger & Morens, Emerg. Infect. Dis., 12:15, 2006.Treanor et al., Clin. Infect. Dis., 55:951, 2012.Ulmer et al., J. Virol., 72(7):5648-5653, 1998.Wilson et al., Clin Vaccine Immunol. 15(6):986-94, 2008.WHO, Fact sheet N°211 - Influenza (Seasonal). 2014.{00901303} - 52 -4901-0342-5651 , V. 1

Claims

WHAT IS CLAIMED IS:

1. A peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table A, a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table B or a peptide / polypeptide comprising a plurality of or all of the peptides set forth in Table C.

2. The peptide of claim 1, wherein the peptides are ordered as in SEQ ID NO: 1.

3. The peptide of claim 1, wherein the peptide / polypeptide is fused to another amino acid sequence.

4. The peptide of claim 1, wherein said peptide / polypeptide is lyophilized.

5. A pharmaceutical formulation comprising the peptide / polypeptide of any one of claims 1-4, formulated in a pharmaceutically acceptable buffer, diluent, or excipient.

6. A nucleic acid encoding the peptide of any one of claims 1-4.

7. The nucleic acid of claim 6, wherein the nucleic acid is a DNA.

8. The nucleic acid of claim 6, wherein the nucleic acid is an RNA.

9. The nucleic acid of claim 6 or claim 7, further comprising a promoter operably linked to the nucleic acid encoding the peptide.

10. The nucleic acid of any one of claims 5-9, wherein the nucleic acid an expression vector.

11. The nucleic acid of claim 10, wherein said expression vector is a viral vector.

12. The nucleic acid of claim 10, wherein said expression vector is a non- viral vector.

13. The nucleic acid of any one of claims 6-12, wherein nucleic acid is disposed in a lipid delivery vehicle.

14. The nucleic acid of any one of claim 6-12, wherein nucleic acid is disposed in a viral or vims-like particle.{00901303} - 53 -4901-0342-5651 , V.

115. A pharmaceutical formulation comprising the nucleic acid of any one of claims 6-14, formulated in a pharmaceutically acceptable buffer, diluent, or excipient.

16. A method of inducing an immune response in a subject comprising administering to a subject a peptide / polypeptide of any one of claims 1-4; a nucleic acid of any one of claims 6-14, or a formulation of claim 5 or claim 15.

17. The method of claim 16, wherein administration comprises injection.

18. The method of claim 17, wherein the injection comprises subcutaneous or intramuscular injection.

19. The method of claim 16, wherein administration comprises inhalation.

20. The method of claim 19, wherein inhalation comprises inhaling a nasal aerosol or mist.

21. The method of claim 16, further comprising administering an adjuvant to the patient.

22. The method of claim 12, wherein the adjuvant is a squalene adjuvant, a cytokine adjuvant, a lipid adjuvant, or a TLR ligand.

23. The method of claim 13, wherein the peptide, nucleic acid, or composition is administered at least a second time.

24. The method of claim 16, wherein the subject is a human subject.

25. The method of claim 16, further comprising measuring a CD4+, a CD8+, and / or a yS T cell response in the subject following administration.

26. The method of any one of claims 16-25, wherein the induced immune response results in one or more of reduced weight loss, increased CD4+T cell activation, increased memory T cell activation, reduced viral load, and increased survival of an infected subject as compared to an infected subject not administered the peptide / polypeptide, nucleic acid or formulation.{00901303} - 54 -4901-0342-5651 , V. 1

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