WO2023004307A1

WO2023004307A1 - T cell epitopes and related compositions useful in the prevention and treatment of respiratory syncytial virus infection

Info

Publication number: WO2023004307A1
Application number: PCT/US2022/073881
Authority: WO
Inventors: Anne De Groot; William Martin
Original assignee: Epivax, Inc.
Priority date: 2021-07-19
Filing date: 2022-07-19
Publication date: 2023-01-26

Abstract

The present disclosure generally relates to novel epitope-based compositions, including vaccines, against respiratory syncytial virus (RSV) infection and diseases caused by RSV. The disclosure relates to immunogenic polypeptides and the uses thereof, particularly in vaccine compositions. The disclosure also relates to nucleic acids, vectors, and cells which express the polypeptides and the uses thereof. The polypeptides more specifically comprise an agretope predicted to be a ligand of HLA class I and/or HLA class II MHC molecules, as well as an epitope that is predicted to be recognized by T-cells in the context of MHC class I and/or class II molecules. The compositions are particularly suited to produce vaccines, particularly for vaccinating against RSV infection and related diseases caused by RSV.

Description

T CELL EPITOPES AND RELATED COMPOSITIONS USEFUL IN THE PREVENTION AND TREATMENT OF RESPIRATORY SYNCYTIAL VIRUS INFECTION

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application 63/223,321, filed July 19, 2021, the content of which are hereby incorporated by reference in its entirety.

FIELD

The present disclosure generally relates to novel T-cell epitope-based compounds and compositions, including vaccines, effective against respiratory syncytial virus (RSV) infection and/or related diseases caused by RSV in a subject in need thereof. Such T-cell epitope compounds and compositions include immunogenic T-cell epitope polypeptides (including concatemeric polypeptides and chimeric or fusion polypeptides), as well as nucleic acids, plasmids, vectors (including expression vectors), and cells which express the polypeptides, pharmaceutical compositions, and vaccines. The present disclosure also generally relates to methods, assays, and kits for detecting a cell-mediated immune response, including a T cell response ( e.g ., CD8+ and/or CD4+ T cell response), against RSV.

SUMMARY

The present disclosure provides novel, therapeutic T cell epitope compounds and compositions (including one or more of e.g., peptides or polypeptides as disclosed herein, including polypeptides having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160 as disclosed herein; concatemeric peptides as disclosed herein, including concatemeric polypeptides comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 161-172 and 197-227 and variants and fragments thereof; chimeric or fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, expression cassettes, plasmids, expression vectors, recombinant viruses, or cells as disclosed herein; vaccine compositions or formulations as disclosed herein, and/or pharmaceutical compositions as disclosed herein), and use of the same, e.g., in methods of stimulating, inducing, and/or expanding an immune response, e.g., against RSV infection and related diseases caused by RSV, and methods of treating and/or preventing against RSV infection and related diseases caused by RSV in a subject.

In aspects, a T-cell epitope compound or composition of the present disclosure includes one or more peptides or polypeptides a disclosed herein. In aspects, the present disclosure is directed to a peptide or polypeptide having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160, or fragments or variants thereof. The phrase “consisting essentially of’ is intended to mean that a peptide or polypeptide according to the present disclosure, in addition to the sequence according to any of SEQ ID NOS: 1-160 or a fragment or variant thereof, contains additional amino acids or residues that may be present at either terminus of the peptide and/or on a side chain that are not necessarily forming part of the peptide or polypeptide that functions as an MHC ligand and provided they do not substantially impair the activity of the peptide to function as a T-cell epitope. The polypeptides of the present disclosure may be isolated, synthetic, and/or recombinant, and may comprise post-transcriptional modifications such as glycosylation, added chemical groups, etc. In aspects, the peptides or polypeptides can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation.

In aspects, the instant disclosure is directed to a peptide or polypeptide an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160. In aspects, the instant disclosure is directed to a peptide or polypeptide have a core amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments and variants thereof), and optionally having extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal of the core amino acid sequence, wherein the overall number of these flanking amino acids is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12, 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, wherein the flanking amino acids can be distributed in any ratio to the C-terminus and the N-terminus (for example, all flanking amino acids can be added to one terminus, or the amino acids can be added equally to both termini or in any other ratio). In aspects, the instant disclosure is directed to a peptide or polypeptide having a core sequence of SEQ ID NOS: 1- 160 (and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, wherein the overall number of these flanking amino acids is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12, 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, wherein the flanking amino acids can be distributed in any ratio to the C-terminus and the N-terminus (for example all flanking amino acids can be added to one terminus, or the amino acids can be added equally to both termini or in any other ratio), provided that the polypeptide with the flanking amino acids is still able to bind to a same HLA molecule (i.e., retain MHC binding propensity) as said polypeptide core sequence without said flanking amino acids. In aspects, said polypeptide with the flanking amino acids is still able to bind to a same HLA molecule (i.e., retain MHC binding propensity) and/or retain the same TCR specificity as said polypeptide core sequence without said flanking amino acids. In aspects, said polypeptide with the flanking amino acids is still able to bind to a same HLA molecule (i.e., retain MHC binding propensity) and/or retain the same TCR specificity, and/or retain anti-viral activity, including anti- RSV activity, as said polypeptide core sequence without said flanking amino acids. In aspects, said flanking amino acid sequences are those that also flank the peptides or polypeptides included therein in the naturally occurring protein from which the peptide or polypeptide is found. In aspects, said flanking amino acid sequences as described herein may serve as a MHC stabilizing region. In aspects, the use of a longer peptide may allow endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses. In aspects, the extension(s) may serve to improve the biochemical properties of the peptides or polypeptides (e.g., but not limited to, solubility or stability) or to improve the likelihood for efficient proteasomal processing of the peptide. In aspects, the polypeptides of the present disclosure may be islated, synthetic, and/or recombinant, and may comprise post-transcriptional modifications such as glycosylation, added chemical groups, etc. In aspects, the peptides or polypeptides can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation. In certain aspects, the peptides or polypeptides of the instant disclosure can be capped with an n-terminal acetyl and/or c-terminal amino group.

In aspects, the present disclosure is directed to a concatemeric polypeptide or peptide that comprises at one or more of the instantly-disclosed polypeptides or peptides (e.g., but not limited to, a peptide or polypeptide of SEQ ID NOS: 1-160, and optionally 1 to 12 additional amino acids distributed in any ratio on any terminus) linked, fused, or joined together (e.g., fused in- frame, chemically-linked, or otherwise bound) to an additional peptide or polypeptide. Such additional peptide or polypeptide may be one or more of the instantly instantly-disclosed polypeptides or peptides, or may be an additional peptide or polypeptide of interest. In aspects a concatemeric peptide is composed of 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more of the instantly-disclosed peptides or polypeptides. In other aspects, the concatemeric peptides or polypeptides include 1000 or more, 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less peptide epitopes. In yet other embodiments, a concatemeric peptide has 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75- 100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 ofthe instantly- disclosed peptides or polypeptides linked, fused, or joined together. Each peptide or polypeptide of the concatemeric polypeptide may optionally have one or more linkers, which may optionally be cleavage sensitive sites, adjacent to their N and/or C terminal end. In such a concatemeric peptide, two or more of the peptide epitopes may have a cleavage sensitive site between them. Alternatively two or more of the peptide epitopes may be connected directly to one another or through a linker that is not a cleavage sensitive site. In aspects, a concatemeric polypeptide of the present disclosure comprises, consists of, or consists essentially of one or more of SEQ ID NOS: 161-172 and 197-227 and/or fragments and variants thereof. In aspects, the instantly-disclosed concatermeric polypepide or peptide sequences do not correspond to a naturally occurring sequence, i.e., each of the one or more of the instantly-disclosed polypeptides or peptides are linked, fused, or joined together to an additional peptide or polypeptide in such a fashion such that the overall concatermic polypeptide does not correspond to a naturally occurring RSV sequence. In aspects, the concatemeric polypeptides of the present disclosure may be isolated, synthetic, and/or recombinant, and may comprise post-transcriptional modifications such as glycosylation, added chemical groups, etc. In aspects, the concatemeric polypeptides can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation. In certain aspects, the concatemeric polypeptides of the instant disclosure can be capped with an n-terminal acetyl and/or c-terminal amino group.

In aspects, one or more peptides or polypeptides or concatemeric polypeptides of the instant disclosure is joined to, linked to, and/or inserted into a heterologous polypeptide. In aspects, the one or more peptides or polypeptides or concatemeric polypeptides of the instant disclosure may be joined to, linked to, and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to, and/or inserted amino acid sequence, together with flanking amino acids of the heterologous polypeptide. In aspects, the present disclosure is directed to a chimeric or fusion polypeptide composition comprising one or more peptides, polypeptides, or concatemeric peptides of the present disclosure. In aspects, a chimeric or fusion polypeptide composition of the present disclosure comprises one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure joined to, linked to, and/or inserted into a heterologous polypeptide In aspects, the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure may be inserted into the heterologous polypeptide, may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide. In aspects of the above chimeric or fusion polypeptide compositions, the one or more peptide, polypeptides, or concatemeric peptides may be joined to, linked to, and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to, and/or inserted amino acid sequence, together with flanking amino acids of the heterologous polypeptide. In aspects, a chimeric or fusion polypeptide composition of the present disclosure comprises peptide, polypeptide, and/or concatemeric peptide having a sequence that is not naturally included in the heterologous polypeptide and/or is not located at its natural position in the heterologous polypeptide. For example, in aspects, the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure may be inserted into a RSV sequence in which the RSV sequence does not include the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure (e.g., the RSV sequence is mutated to not include the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure) or the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure is inserted into a RSV sequence but not at its natural position. In aspects, the one or more of peptide, polypeptide, and/or concatemeric peptide of the present disclosure can be joined, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into the heterologous polypeptide. In aspects of above-described chimeric or fusion polypeptide compositions, the chimeric or fusion polypeptides may be isolated, synthetic, or recombinant.

In aspects, the instant disclosure is directed to a nucleic acid (e.g., DNA or RNA, including mRNA) encoding one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein. For example, in aspects, the instant disclosure is directed to a nucleic acid encoding a peptide or polypeptide having an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160. Additionally, the instant disclosure is directed to a nucleic acid encoding a polypeptide having an amino acid sequence off SEQ ID NOS: 161-172 and 197-227. In aspects, the present disclosure is directed to a vector, such as an expression vector, comprising such a nucleic acid as described. In aspects, the present disclosure is directed to expression cassettes, plasmids, expression vectors, recombinant viruses, or cells comprising a nucleic acid as described herein. In aspects, the present disclosure is directed to a cell or vaccine comprising such a vector as described. In aspects, the present disclosure is directed to a cell comprising a vector of the present disclosure.

In aspects, the instant disclosure is directed to a pharmaceutical composition, the pharmaceutical composition comprising a T-cell epitope compound or composition of the instant disclosure (e.g., one or more of: polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, including nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, cells as disclosed herein) and a pharmaceutically acceptable carrier, excipient, and/or adjuvant. In aspects, the one or more nucleic acids encoding said peptides or polypeptides are DNA, RNA, or mRNA. In aspects of the above-described pharmaceutical compositions, the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000, peptides, polypeptides, and/or concatemeric peptides, as disclosed herein, including every value or range therebetween.

In aspects, the instant disclosure is directed to a vaccine comprising a T-cell epitope compound or composition of the instant disclosure (e.g., one or more of: polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, including nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, cells as disclosed herein; pharmaceutical compositions as disclosed herein; or vaccines as described herein) and, optionally, a carrier, excipient, and/or an adjuvant. The present disclosure also relates to methods of immunizing or inducing an immune response in a subject, said method comprising administering to said subject one more peptides, polypeptides, concatemeric peptides, chimeric or fusion polypeptides, nucleic acids, expression cassettes, plasmids, expression vectors, recombinant viruses, cells pharmaceutical compositions, or vaccines as described herein. In aspects, the subject is a human. In aspects, the present disclosure is directed to to methods of immunizing or inducing an immune response in a subject, comprising administering to said subject a T-cell epitope compound or composition of the instant disclosure (e.g., one or more of: polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, including nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, cells as disclosed herein; pharmaceutical compositions as disclosed herein; or vaccines as described herein). In aspects, the subject is a human. In aspects, the present disclosure is directed to a method of stimulating, inducing, and/or expanding an immune response to a RSV infection and/or related diseases caused by RSV, in a subject, comprising administering to said subject a T-cell epitope compound or composition of the instant disclosure.

The present disclosure also relates to methods of treating and/or preventing a RSV infection and/or related diseases caused by RSV, in a subject, such as a human, comprising administering to said subject a T-cell epitope compound or composition of the instant disclosure (e.g., one or more of: polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, including nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, cells as disclosed herein; pharmaceutical compositions as disclosed herein; or vaccines as described herein).

As should be understood, the T-cell epitope compounds or compositions of the instant disclosure as described herein may be used to induce an immune response and/or to vaccinate a subject. It is particularly useful to vaccinate against RSV infection and/or related diseases caused by RSV.

In aspects, the instant disclosure provide novel methods, assays, and kits for detecting an immune response, and in aspects a cell-mediated immune (“CMI”) response, including a T cell response (e.g., CD8+ and/or CD4+ T cell response) against RSV, as well as methods, assays, and kits for the diagnosis of a RSV infection, as well as diseases caused by RSV. The instantly-disclosed assays, methods, and kits use or include one or more T-cell epitope compounds and composiitons (including peptides or polypeptides) a disclosed herein.

In aspects, the present disclosure is directed to methods of measuring a CMI response against RSV infection in a subject by incubating a sample from the subject which comprises T-cells and/or other cells of the immune system with one or more peptides or polypeptides of the instant disclosure. In aspects, production of IFN-g or other cytokine or immune effector molecule(s) is then detected. The presence or level of immune effector is then indicative of the level of cell mediated responsiveness of the subject. In aspects, the sample is preferably whole blood collected in a suitable container comprising the antigen. Optionally, a simple sugar, such as dextrose, is added to the incubation mixture. Accordingly, one aspect of the present disclosure relates to a method for measuring a CMI response in a subject, said method comprising collecting a sample from said subject wherein said sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an antigen, incubating said sample with one or more peptides or polypeptides of the instant disclosure and then measuring the presence of or elevation in the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response against RSV. In aspects, the presence of or elevation in the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response against RSV.

In aspects, the present disclosure is directed to methods of assaying for RSV peptide- specific T-cells, the method comprising providing a fluid containing T-cells, adding one or more peptides or polypeptides of the instant disclosure to the fluid, incubating the fluid to cause cytokine release, and detecting the released cytokine. Preferably the method comprises providing the fluid containing T-cells in contact with a surface carrying an immobilized first antibody to the cytokine, adding the peptide or polypeptide to the fluid, incubating the resulting fluid mixture under conditions to cause any peptide or poleypeptide-specific T-cells that have been pre-sensitized in vivo to the peptide or polypeptide to secrete the cytokine, and detecting any secreted cytokine bound to the immobilized first antibody. In aspects, the cells are preferably peripheral blood mononuclear cells (PMBC). They may suitably be taken from a patient known to be suffering, or to have suffered, from RSV infection. In aspects, the cells used are fresh. In aspects, the assay is used to identify or quantitate peptide or polypeptide- specific T-cells e.g. CD8+ or CD4+ cells that have been activated or pre-sensitized in vivo to a particular peptide or polypeptide. In aspects, these are unrestimulated T-cells, i.e. cells capable of immediate effector function without the need to effect division/differentiation by in vitro culture. When a peptide or polypeptide in question is presented to such cells, the cells secrete various cytokines, of which any one may be selected for the purposes of this assay. In aspects, the cytokine selected is interferon-g (IFN g).

In aspects, the present disclosure provides a method of detecting an anti-RSV T cell response (which in aspects can included CD4+ and/or CD8+ T cell response) comprising contacting a population of T cells of an individual with a peptide or polypeptide of the instant disclosure, wherein one or more of said peptides or polypeptides may be substituted by an analogue which binds a T cell receptor that recognizes the peptide, and determining whether T cells of the T cell population recognize the peptide(s).

In aspects, the present disclosure provides a method of diagnosing a RSV infection in a host, or exposure of a host, to RSV comprising (i) contacting a population of T cells from the host with one or more peptides or analogues as disclosed here, and analogues thereof which can bind a T cell receptor which recognizes any of the said peptides; and (ii) determining whether the T cells of said T cell population recognize the peptide(s) and/or analogue(s).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a cartoon of the RSV virion as well as identifying the four main proteins used for identification of target antigens.

Fig. 2 shows an overview of the Class II analysis steps in identifying potential antigens within the four target RSV proteins.

Fig. 3 shows shows an overview of the Class I analysis steps in identifying potential antigens within the four target RSV proteins.

Fig. 4 shows an overview of the Class II binding assay protocol.

Fig. 5 shows the result summary for HLA Class II DRB 1*01:01.

Fig. 6 shows the result summary for HLA Class II DRB 1*03:01.

Fig. 7 shows the result summary for HLA Class II DRB 1*04:01.

Fig. 8 shows the result summary for HLA Class II DRB 1*07:01.

Fig. 9 shows the result summary for HLA Class II DRB 1*09:01.

Fig. 10 shows the result summary for HLA Class II DRB 1*11:01.

Fig. 11 shows the result summary for HLA Class II DRB 1*13:01.

Fig. 12 shows the result summary for HLA Class II DRB 1*15:01.

Fig. 13 shows a summary of the number of alleles bound by each peptide analyzed. All of the peptides bound to at least four alleles. Fig. 14 an overview of the Class I binding assay protocol.

Fig. 15 shows the results summary for HLA Class I A*01:01 binding.

Fig. 16 shows the results summary for HLA Class I A*02:01 binding.

Fig. 17 shows the results summary for HLA Class I A*03:01 binding.

Fig. 18 shows the results summary for HLA Class I A*24:02 binding.

Fig. 19 shows the results summary for HLA Class I B*07:02 binding.

Fig. 20 shows the results summary for HLA Class I B*44:03 binding.

Fig. 21 shows the Cluster scores and junctional scores for the Class II concatamers.

Fig. 22 shows an oiverview of the VaxCAD selection process for Class I peptides DETAILED DESCRIPTION OF THE INVENTION

The present disclosure generally relates to T-cell epitope-based compounds and compositions, including vaccines, for use against RSV infection and related diseases caused by RSV. The disclosure relates to immunogenic peptides, polypeptides, concatemeric peptides, and chimeric or fusion polypeptides and the uses thereof, particularly in pharmaceutical and vaccine compositions. The present disclosure also relates to nucleic acids, vectors (including expression vectors), and cells which express the peptides, polypeptides, concatemeric peptides, and chimeric or fusion polypeptides and the uses thereof. The peptides, polypeptides, concatemeric peptides, and chimeric or fusion polypeptides of the present disclosure more specifically comprise an agretope predicted to be a ligand of HLA class I and/or HLA class II MHC molecules, as well as an epitope that is predicted to be recognized by T-cells (including CD8+ and/or CD4+ T-cells) in the context of MHC class I and/or class II molecules. The instant disclosure is particularly suited to produce vaccines for humans, particularly for vaccinating against RSV infection and related diseases caused by RSV.

It is possible to exploit epitope-specific T-cells to induce an immune response against specific antigens. This discovery has implications for the design of therapeutic regimens and antigen-specific therapies against particular pathogens and infections. The instant disclosure and data relates to identified RSV T cell epitopes that are recognized in natural infection and stimulate pre-existing immunity to RSV. These epitopes are excellent candidates for T cell- directed vaccine development. A T cell targeting vaccine composed of conserved epitopes may provide rapid, effective and long-term immunity at sites of infection with production of tissue resident memory CD8⁺ T cells, as well as memory CD4⁺ T cells that support antibody responses. Influenza vaccination during the 2009 H1N1 pandemic demonstrated that memory CD4⁺ T cells are able to support naive B cell responses to a novel hemagglutinin. A T cell- directed RSV vaccine could generate robust CD4⁺ T cell memory that would provide early control of acute infection with a novel RS V virus in the absence of pre-existing cross-protective antibodies. Thus, administration of T-cell epitopes, including a T-cell epitope compound or composition of the present disclosure (including one or more of peptides or polypeptides having a sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160 as disclosed herein; concatemeric peptides as disclosed herein (including a concatemeric polypeptide of the present disclosure that comprises, consists of, or consists essentially of one or more of SEQ ID NOS: 161-172 and 197-227 and/or fragments and variants thereof); chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells which express such peptides, polypeptides, concatemeric peptides or chimeric of fusion polypeptide compositions as disclosed herein; vaccine compositions or formulations as disclosed herein; and/or pharmaceutical compositions or formulations as disclosed herein), optionally in conjunction with a drug (such as an antiviral drug), a protein, or a inactivated or live attenuated virus, can induce an immune response, e.g., against a pathogen, including RSV and related diseases caused by RSV. T-cell epitopes, including T-cell epitope compounds and compositions of the present disclosure, can be used to deliberately manipulate the immune system toward immunity.

For example, the T-cell epitope compounds and compositions of the present disclosure are useful in the selective engagement and activation of immunogenic T-cells. It is demonstrated herein that certain naturally occurring T-cells (in aspects, including CD4+ and CD8+ T-cells), can be engaged, activated, and/or applied to induce immunity or induce an immune response against pathogens such as RSV and related diseases caused by RSV. By using the T-cell epitope compounds and compositions of the present disclosure to selectively activate naturally occurring T-cells, it is herein shown that such T-cell epitope compounds and compositions can be used to stimulate, induce, and/or expand an immune response to a RSV and related diseases caused by RSV in a subject, and thus can be used in methods of treating and/or preventing RSV and related diseases caused by RSV in a subject.

DEFINITIONS

To further facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 25 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 25 may comprise 1 to 5, 1 to 10, 1 to 15, and 1 to 20 in one direction, or 25 to 20, 25 to 15, 25 to 10, and 25 to 5 in the other direction.

As used herein, the term “biological sample” refers to any sample of tissue, cells, or secretions from an organism.

As used herein, the term “medical condition” includes, but is not limited to, any condition and/or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders.

As used herein, the term “immune response” refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, malignant melanoma, invading pathogens (including a virus), cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. In aspects, an immune response includes a measurable cytotoxic T lymphocyte (CTL) response (e.g., against a virus expressing an immunogenic polypeptide) or a measurable B cell response, such as the production of antibodies, (e.g., against an immunogenic polypeptide). One of ordinary skill would know various assays to determine whether an immune response against a peptide, polypeptide, or related composition was generated, including use of the experiments and assays as disclosed in the Examples herein. Various B lymphocyte and T lymphocyte assays are well known, such as ELISAs, EliSpot assays, cytotoxic T lymphocyte CTL assays, such as chromium release assays, proliferation assays using peripheral blood lymphocytes (PBL), tetramer assays, and other cytokine production assays.

As used herein, the term "effective amount", “therapeutically effective amount”, or the like of a composition, including a T-cell epitope compound or composition of the present disclosure is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention of, or a decrease in, the symptoms and/or underlying causes associated with a disease that is being treated, such as RSV infection and related diseases caused by RSV, or an amount to measurably to inhibit inhibit virus (for example, RSV) replication or infectivity. The amount of a composition of the present disclosure administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The T-cell epitope compounds and compositions of the present invention can also be administered in combination with each other or with one or more additional therapeutic compounds.

As used herein, “anti-RSV activity”, “anti-RSV polypeptides”, “anti- RSV compounds and compositions”, and the like are intended to mean that the T-cell epitope compounds and compositions of the of the present diclsoure have anti-RSV activity and thus are capable of suppressing, controlling, and/or killing an invading RSV virus. For example, anti-RSV activity means that the instantly-disclosed therapeutic T-cell epitope comopounds and compositions are, in aspects: capable of stimulating, inducing, and/or expanding an immune response to RSV (e.g., a cellular (CD4+ and/or CD8+ T-cell response) or humoral immune response to RSV) and/or associated diseases in a subject; capable of stimulating, inducing, and/or expanding a RSV-specific IFNy response (e.g., by lymphocytes such as PMBC, or effector CD4+ and/or CD8+ T-cells), capable of inhibiting RSV viral replication or infectivity, and/or capable of inducing immunity against RSV. In aspects, a T-cell epitope compound or composition of the present disclosure having anti-RSV activity will reduce the disease symptoms resulting from RSV challenge by at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50% to about 90% or greater, including any value or range therebetween. Anti-RSV activity can be determined by various experiments and assays as known to those of skill in the art, including methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation, including use of experiments and assays as disclosed in the Examples herein.

As used herein, the term “T-cell epitope” means an MHC ligand or protein determinant, 7 to 30 amino acids in length, and capable of specific binding to human leukocyte antigen (HLA) molecules and interacting with specific T cell receptors (TCRs). As used herein, in the context of a T cell epitope that is known or determined (e.g. predicted) to engage a T cell, the terms “engage”, “engagement” or the like means that when bound to a MHC molecule (e.g. human leukocyte antigen (HLA) molecules), the T cell epitope is capable of interacting with the TCR of the T cell and activating the T cell. Generally, T-cell epitopes are linear and do not express specific three-dimensional characteristics. T-cell epitopes are not affected by the presence of denaturing solvents. The ability to interact with T-cell epitopes can be predicted by in silico methods (De Groot AS et ah, (1997), AIDS Res Hum Retroviruses, 13(7):539-41 ; Schafer JR et al., (1998), Vaccine, 16( 19): 1880-4; De Groot AS et al., (2001), Vaccine, 19(31):4385-95; De Groot AR et al, (2003), Vaccine, 21(27-30):4486-504, all of which are herein incorporated by reference in their entirety.

As used herein, the term “T-cell epitope cluster” refers to polypeptide that contains between about 4 to about 40 MHC binding motifs. In particular embodiments, the T-cell epitope cluster contains between about 5 to about 35 MHC binding motifs, between about 8 and about 30 MHC binding motifs; and between about 10 and 20 MHC binding motifs.

As used herein, the term “immune-stimulating T-cell epitope polypeptide” refers to a molecule capable of inducing an immune response, e.g., a humoral, T cell-based, or innate immune response.

As used herein, the term “regulatory T cell”, “Treg” or the like, means a subpopulation of T cells that suppress immune effector function, including the suppression or down regulation of CD4+ and/or CD8+ effector T cell (Teff) induction, proliferation, and/or cytokine production, through a variety of different mechanisms including cell-cell contact and suppressive cytokine production. In aspects, CD4+ Tregs are characterized by the presence of certain cell surface markers including but not limited to CD4, CD25, and FoxP3. In aspects, upon activation, CD4+ regulatory T cells secrete immune suppressive cytokines and chemokines including but not limited to IL-10 and/or TGF . CD4+ Tregs may also exert immune suppressive effects through direct killing of target cells, characterized by the expression upon activation of effector molecules including but not limited to granzyme B and perforin. In aspects, CD8+ Tregs are characterized by the presence of certain cell surface markers including but not limited to CD8, CD25, and, upon activation, FoxP3. In aspects, upon activation, regulatory CD8+ T cells secrete immune suppressive cytokines and chemokines including but not limited to IFNy, IL-10, and/or TGF . In aspects, CD8⁺ Tregs may also exert immune suppressive effects through direct killing of target cells, characterized by the expression upon activation of effector molecules including but not limited to granzyme B and/or perforin.

As used herein, the term “regulatory T cell epitope” (“Tregitope”) refers to a “T cell epitope” that causes a tolerogenic response (Weber CA et ah, (2009), Adv Drug Deliv, 61(11):965-76) and is capable of binding to MHC molecules and engaging (i.e. interacting with and activating) circulating naturally occurring Tregs (in aspects, including natural Tregs and/or adaptive Tregs). In aspects, upon activation, CD4+ regulatory T cells secrete immune suppressive cytokines and chemokines including but not limited to IL-10 and/or TGF . CD4+ Tregs may also exert immune suppressive effects through direct killing of target cells, characetized by the expression upon activation of effector molecules including but not limited to granzyme B and perforin leads to the expression of the immune suppressive cytokines including, but not limited to, IL-10 and TGF-b and TNF-a. In aspects, upon activation, regulatory CD8+ T cells secrete immune suppressive cytokines and chemokines including but not limited to IFNy, IL-10, and/or TGFfi. In aspects, CD8⁺ Tregs may also exert immune suppressive effects through direct killing of target cells, characetized by the expression upon activation of effector molecules including but not limited to granzyme B and/or perforin.

As used herein, the term “B-cell epitope” means a protein determinant capable of specific binding to an antibody. B-cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

The term “subject” as used herein refers to any living organism in which an immune response is elicited. The term subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

As used herein, the terms “the major histocompatibility complex (MHC)”, “MHC molecules”, “MHC proteins” or “HLA proteins” are to be understood as meaning, in particular, proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells. The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and/or foreign antigens and thus for regulating immunological processes. The major histocompatibility complex is classified into two gene groups coding for different proteins, namely molecules of MHC class I and molecules of MHC class II. The molecules of the two MHC classes are specialized for different antigen sources. The molecules of MHC class I present endogenously synthesized antigens, for example viral proteins and tumor antigens. The molecules of MHC class II present protein antigens originating from exogenous sources, for example bacterial products. The cellular biology and the expression patterns of the two MHC classes are adapted to these different roles. MHC molecules of class I consist of a heavy chain and a light chain and are capable of binding a peptide of about 8 to 11 amino acids, but usually 9 or 10 amino acids, if this peptide has suitable binding motifs, and presenting it to cytotoxic T-lymphocytes. The peptide bound by the MHC molecules of class I originates from an endogenous protein antigen. The heavy chain of the MHC molecules of class I is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is b-2-microglobulin. MHC molecules of class II consist of an a-chain and a b-chain and are capable of binding a peptide of about 12 to 25 amino acids if this peptide has suitable binding motifs, and presenting it to T-helper cells. The peptide bound by the MHC molecules of class II usually originates from an extracellular of exogenous protein antigen. The a-chain and the b-chain are in particular HLA- DR, HLA-DQ and HLA-DP monomers.

As used herein, the term “MHC complex” refers to a protein complex capable of binding with a specific repertoire of polypeptides known as HLA ligands and transporting said ligands to the cell surface.

As used herein, the term “MHC Ligand” means a polypeptide capable of binding to one or more specific MHC alleles. The term “HLA ligand” is interchangeable with the term “MHC Ligand”. Cells expressing MHC/Ligand complexes on their surface are referred to as “Antigen Presenting Cells” (APCs). Similarly, as used herein, the term “MHC binding peptide” relates to a peptide which binds to an MHC class I and/or an MHC class II molecule. In the case of MHC class I/peptide complexes, the binding peptides are typically 8-10 amino acids long although longer or shorter peptides may be effective. In the case of MHC class II/peptide complexes, the binding peptides are typically 10-25 amino acids long and are in particular 13- 18 amino acids long, whereas longer and shorter peptides may also be effective.

As used herein, the term “T Cell Receptor” or “TCR” refers to a protein complex expressed by T cells that is capable of engaging a specific repertoire of MHC/Ligand complexes as presented on the surface of cells, such as antigen presenting cells (APCs).

As used herein, the term “MHC Binding Motif’ refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele.

As used herein, the term “AAY cleavage motif’ refers to the short amino acid motif consisting of the sequence “alanine-alanine-tyrosine” capable of promoting proteasome- mediated cleavage of a peptide or protein, promoting the binding of the transporter associated with antigen processing to a peptide or protein, and/or increasing proteasome degradation at specific sites within a peptide or protein.

As used herein, the term “immune synapse” means the protein complex formed by the simultaneous engagement of a given T cell epitope to both a cell surface MHC complex and TCR.

The term "polypeptide" refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. As used herein, a polypeptide is said to be "isolated" or "purified" when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. A peptide or polypeptide (e.g., a polypeptide comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 or variants and fragments thereof, which in aspects may be isolated, synthetic, or recombinant) of the present disclosure, however, can be joined to, linked to, or inserted into another polypeptide (e.g., a heterologous polypeptide) with which it is not normally associated in a cell and still be "isolated" or "purified.” Additionally, one or more T- cell epitopes of the present disclosure can be joined to, linked to, or inserted into another polypeptide wherein said one or more T-cell epitopes of the present disclosure is not naturally included in the polypeptide and/or said one or more T-cell epitopes of the present disclosure is not located at its natural position in the polypeptide. When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.

As used herein, a “concatemeric” peptide or polypeptide refers to a series of at least two peptides or polypeptides linked together. Such linkages may form a string-of-beads design. In aspects, concatemeric polypeptides of the instant disclosure include concatemeric polypeptides comprising, consisting of, or consisting essentially of one or more of 161-172 and 197-227, and/or fragments and variants thereof (which in aspects may be isolated, synthetic, and/or recombinant). In aspects, earch of the peptides or polypeptides of concatermeric polypeptide may optionally be spaced by one or more linkers, and in further aspects neutral linkers. The term “linker” refers to a peptide added between two peptide domains such as epitopes or vaccine sequences to connect said peptide domains. In aspects, a linker sequence is used to reduce steric hindrance between each one or more identified peptides of the instant disclosure, is well translated, and supports or allows processing of the each one or more identified polypeptides of the instant disclosure. In aspects, the linker should have little or no immunogenic sequence elements. In aspects, each peptide or polypeptide of the concatemeric polypeptide may optionally have one or more linkers, which may optionally be cleavage sensitive sites, adjacent to their N and/or C terminal end. In such a concatemeric peptide, two or more of the peptides may have a cleavage sensitive site between them. Alternatively two or more of the peptides may be connected directly to one another or through a linker that is not a cleavage sensitive site.

As used herein, the term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

As used herein, the term “pharmaceutically acceptable excipient, carrier, or diluent” or the like refer to an excipient, carrier, or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

As used herein, the term “purpose built computer program” refers to a computer program designed to fulfill a specific purpose; typically to analyze a specific set of raw data and answer a specific scientific question.

As used herein, the term “z-score” indicates how many standard deviations an element is from the mean. A z-score can be calculated from the following formula: z = (X - m) / s ; where z is the z-score, X is the value of the element, m is the population mean, and s is the standard deviation.

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and/or.” As used herein, the term “and/or” and “one or more” includes any and all combinations of the associated listed items. For example, the term “one or more” with respect to the “one or more of SEQ ID NOS: 1-160 of the present disclosure” includes any and all combinations of SEQ ID NOS: 1-160. The term “or a combination thereof’ means a combination including at least one of the foregoing elements.

A “variant” peptide or polypeptide (including a variant T-cell epitope) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. In aspects, a variant peptide or polypeptide (including a variant T-cell epitope) can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these provided said variants retain MHC binding propensity and/or TCR specificity, and/or RSV activity.

The present disclosure also includes fragments of the peptide or polypeptides of the invention. The disclosure also encompasses fragments of the variants of the T-cell epitopes described herein, provided said fragments and/or variants at least in part retain MHC binding propensity and/or TCR specificity, and/or retain anti-RSV activity.

The present disclosure also provides chimeric or fusion polypeptides (which in aspects may be isolated, synthetic, or recombinant) wherein one or more of the instantly-disclosed peptides, polypeptides, or concatemeric peptides is a part thereof. In aspects, a chimeric or fusion polypeptide composition comprises one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure linked to a heterologous polypeptide. As previously stated, the term “heterologous polypeptide” is intended to mean that the one or more T-cell epitopes that are heterologous to, or not included naturally, in the heterologous polypeptide. In aspects, the one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure may be inserted into the heterologous polypeptide (e.g., through mutagenesis or other known means in the art), may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide. For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et ah, 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety).

In aspects, chimeric or fusion polypeptides comprise one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure operatively linked to a heterologous polypeptide. "Operatively linked" indicates that the polypeptide (e.g., the one or more T-cell epitope polypeptides of the present disclosure) and the heterologous protein are fused in-frame or chemically-linked or otherwise bound. In aspects, the instantly-disclosed chimeric or fusion polypeptides may be isolated, synthetic, or recombinant

An “isolated” peptide, polypeptide, concatemeric peptide (e.g., an isolated T-cell activating T-cell epitope or T-cell epitope polypeptide), or chimeric or fusion polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. In aspects, a peptide, polypeptide, or concatemeric peptide is produced by recombinant DNA or RNA techniques. For example, a nucleic acid molecule encoding the peptide, polypeptide, concatemeric peptide, or chimeric or fusion polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the peptide, polypeptide, concatemeric peptide, or chimeric or fusion polypeptide is expressed in the host cell. The peptide, polypeptide, concatemeric peptide, or chimeric or fusion polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.

For the purposes of the present disclosure, peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids; amino acid analogs; and mimetics. Further, in aspects, peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure can include retro-inverso peptides of the instantly disclosed peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure, provided said peptides, polypeptides, concatemeric peptides, or chimeric or fusion polypeptides of the instant disclosure at least in part retain MHC binding propensity and/or TCR specificity, and/or retain anti- RSV activity.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. Other features, objects, and advantages of the present disclosure will be apparent from the description and the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. POLYPEPTIDES, CONCATEMERIC POLYPEPTIDES, and CHIMERIC or FUSION POLYPEPTIDES

In aspects, the present disclosure provides a novel class of T-cell epitopes (which may be isolated, synthetic, or recombinant), which comprise a peptide or polypeptide chain derived from RSV proteins (e.g., encoded proteins from a RSV genome), including nucleoproteins (N), fusion (F) proteins, matrix (M) proteins, and non-structural 1 (NS) proteins, of RSV. T-cell epitopes of the present disclosure are highly conserved among known variants of their source proteins, and RSV “98-25147-X” subtype A (SEQ ID NO: 161)), RSV ATCC VR-26 subtype A, RSV LA2 106 subtype A, RSV DEU/107/2009 subtype A, RSV 9320 subtype B, and B/GZ/13-730 subtype B antigen sequences were obtained from GenBank at the National Center for Biotechnology Information. RSV epitopes were compared across sequences for T cell epitope mapping. RSV clinical isolate “98-25147-X”, subtype A was selected as the reference strain.

As further described in the Examples, T-cell epitopes of the present disclosure comprise at least one putative T cell epitope as identified by EpiMatrix™ analysis. EpiMatrix™ is a proprietary computer algorithm developed by EpiVax (Providence, Rhode Island), which is used to screen protein sequences for the presence of putative T cell epitopes. The algorithm uses matrices for prediction of 9- and 10-mer peptides binding to MHC molecules. Each matrix is based on position-specific coefficients related to amino acid binding affinities that are elucidated by a method similar to, but not identical to, the pocket profile method (Sturniolo, T. et al. , Nat. BiotechnoL, 17:555-561, 1999). Input sequences are, for example, parsed into overlapping 9-mer frames or 10-mer where each frame overlaps the last by 8 or 9 amino acids, respectively. Each of the resulting frames form the mutated peptide and the non-mutated peptide are then scored for predicted binding affinity with respect to MHC class I alleles (e.g., but not limited to, HLA-A and HLA-B alleles) and MHC class II alleles (e.g., but not limited to HLA-DRB1 alleles). Raw scores are normalized against the scores of a large sample of randomly generated peptides. The resulting “Z” scores are normally distributed and directly comparable across alleles. The resulting “Z” score is reported. In aspects, any 9-mer or 10- mer peptide with an allele-specific EpiMatrix™ Z-score in excess of 1.64, theoretically the top 5% of any given sample, is considered a putative T cell epitope.

As also further described in the Examples, peptides containing clusters of putative T cell epitopes are more likely to test positive in validating in vitro and in vivo assays. In aspects, the results of the initial EpiMatrix™ analysis are further screened for the presence of putative T cell epitope “clusters” using a second proprietary algorithm known as Clustimer™ algorithm. The Clustimer™ algorithm identifies sub-regions contained within any given amino acid sequence that contains a statistically unusually high number of putative T cell epitopes. Typical T-cell epitope “clusters” range from about 9 to roughly 30 amino acids in length and, considering their affinity to multiple alleles and across multiple 9-mer frames, can contain anywhere from about 4 to about 40 putative T cell epitopes. Each epitope cluster identified an aggregate EpiMatrix™ score is calculated by summing the scores of the putative T cell epitopes and subtracting a correcting factor based on the length of the candidate epitope cluster and the expected score of a randomly generated cluster of the same length. EpiMatrix™ cluster scores in excess of +10 are considered significant. In aspects, the T-cell epitopes of the instant disclosure contain several putative T-cell epitopes forming a pattern known as a T-cell epitope cluster.

Putative T-cell epitopes were also screened for cross-conservation with the human proteome using JanusMatrix, as further described in more detail in the Examples. The JanusMatrix system (EpiVax, Providence, Rhode Island) useful for screening peptide sequences for cross-conservation with a host proteome. JanusMatrix is an algorithm that predicts the potential for cross-reactivity between peptide clusters and the host genome or proteome, based on conservation of TCR- facing residues in their putative MHC ligands. The JanusMatrix algorithm first considers all the predicted epitopes contained within a given protein sequence and divides each predicted epitope into its constituent agretope and epitope. Each sequence is then screened against a database of host proteins. Peptides with a compatible MHC-facing agretope (i.e., the agretopes of both the input peptide and its host counterparty are predicted to bind the same MHC allele) and exactly the same TCR-facing epitope are returned. The JanusMatrix Homology Score suggests a bias towards immune tolerance. In the case of a therapeutic protein, cross-conservation between autologous human epitopes and epitopes in the therapeutic may increase the likelihood that such a candidate will be tolerated by the human immune system. In the case of a vaccine, cross-conservation between human epitopes and the antigenic epitopes may indicate that such a candidate utilizes immune camouflage, thereby evading the immune response and making for an ineffective vaccine. When the host is, for example, a human, the peptide clusters are screened against human genomes and proteomes, based on conservation of TCR-facing residues in their putative HLA ligands. The peptides are then scored using the JanusMatrix Homology Score. In aspects, peptides with a JanusMatrix Homology Score below 2.5 or below 3.0 (and even below 2.0 in certain apsects) indicate low tolerogenicity potential and may be useful for pharmaceutical formulations and vaccines for the treatment/prevention of RSV infection and related diseases caused by RSV, and in aspects may be included from the T cell epitope compositions and methods of the present disclosure. In aspects, peptides with a JanusMatrix Homology Score above 3.0 indicate high tolerogenicity potential and may not be useful for pharmaceutical formulations and vaccines for the treatment/prevention of RSV infection and related diseases caused by RSV, and in aspects may be excluded from the T cell epitope compositions and methods of the present disclosure.

In aspects, and as also described in further detail in the Examples, T-cell epitopes of the present disclosure are highly conserved among related RSV stains that infect humans including RSV “98-25147-X” subtype A, RSV ATCC VR-26 subtype A, RSV LA2 106 subtype A, RSV DEU/107/2009 subtype A, RSV 9320 subtype B, and B/GZ/13-730 subtype B. To identify potentially cross-reactive sequences, the TCR-face of select RSV epitopes was screened for homology with RSVs that infect humans, using the JanusMatrix algorithm.

In aspects, T-cell epitopes of the present disclosure bind to at least one and preferably two or more common HLA class I and/or class II alleles with at least a moderate affinity ( e.g ., in aspects, <1000 mM ICso, <500 mM ICso_, <400 mM ICso, <300 mM ICso, or <200 mM ICso in HLA binding assays based on soluble HLA molecules). In aspects, T-cell epitopes of the present disclosure are capable of being presented at the cell surface by cells in the context of at least one and, in other aspects, two or more alleles of the HLA. In this context, the epitope-HLA complex can be recognized by CD4+ and/or CD8+ T-cells having TCRs that are specific for the epitope-HLA complex and circulating in subjects. In aspects, the recognition of the epitope-HLA complex can cause the matching T-cell to be activated and to secrete activating cytokines (e.g., effector cytokines such as IKNg) and chemokines.

In aspects, a T-cell epitope compounds or compositions of the present disclosure includes one or more peptides or polypeptides a disclosed herein. In aspects, the present disclosure is directed to a peptide or polypeptide having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160, or fragments or variants thereof. The phrase “consisting essentially of’ is intended to mean that a peptide or polypeptide that contains additional amino acids or residues that may be present at either terminus of the peptide and/or on a side chain that are not necessarily forming part of the peptide or polypeptide that functions as an MHC ligand and provided they do not substantially impair the activity of the peptide to function as a T-cell epitope. In aspects, the peptides or polypeptides of the instant disclosure can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation. In aspects, the peptides or polypeptides of the instant disclosure can be capped with an n-terminal acetyl and/or c-terminal amino group. In aspects, peptides or polypeptides of the instant disclosure having SEQ ID NOS: 1-160 are capped with an n- terminal acetyl and a c-terminal amino group. Tables 1A and IB and 2A and 2B describes the MHC class II cluster address and gives the location of the MHC class II peptide within selected sequences that were provided for analysis. Table 1 A and Table 2A describes the core peptide (middle amino acids in bold, SEQ ID NO: in parentheses) which defines the actual cluster that was identified during the analysis. The stabilizing flanks (N-terminal and C-terminal, not bold) are included for use with the core sequence in cetain aspects, with the full sequence of the cluster and flanks being labeled in Table 1A and Table 2A by the SEQ ID NO: not listed in parentheses. Tables IB and 2B provide some Table 3 describes selected MHC class I peptides.

Table 1A:

Table IB:

Table 2A:

Table 2B:

Table 3:

In aspects, the instant disclosure is directed to a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160. In aspects, the instant disclosure is directed to a peptide or polypeptide have a core amino acid sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally having extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal of the core amino acid sequence, wherein the overall number of these flanking amino acids is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12, 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, wherein the flanking amino acids can be distributed in any ratio to the C-terminus and the N-terminus (for example all flanking amino acids can be added to one terminus, or the amino acids can be added equally to both termini or in any other ratio). In aspects, the instant disclosure is directed to a peptide or polypeptide have a core sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, wherein the overall number of these flanking amino acids is 1 to 12, 1 to 3, 2 to 4, 3 to 6, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12, 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, wherein the flanking amino acids can be distributed in any ratio to the C-terminus and the N-terminus (for example all flanking amino acids can be added to one terminus, or the amino acids can be added equally to both termini or in any other ratio), provided that the polypeptide with the flanking amino acids is still able to bind to the same HLA molecule (i.e., retain MHC binding propensity) as said polypeptide core sequence without said flanking amino acids. In aspects, said polypeptide with the flanking amino acids is still able to bind to the same HLA molecule (i.e., retain MHC binding propensity) and/or retain the same TCR specificity, and/or retain anti-RSV activity, as said polypeptide core sequence without said flanking amino acids. In aspects, the extension(s) may serve and be designed to improve the biochemical properties of the peptides or polypeptides (e.g., but not limited to, solubility or stability) or to improve the likelihood for efficient proteasomal processing of the peptide. In aspects, the polypeptides of the present disclosure may be islated, synthetic, and/or recombinant, and may comprise post-transcriptional modifications such as glycosylation, added chemical groups, etc. In aspects, said flanking amino acid sequences are those that also flank the peptides or polypeptides included therein in the naturally occurring protein (for example, as found in RSV clinical isolate “98-25147-X”, subtype A, RSV ATCC VR-26 subtype A, RSV LA2_106 subtype A, RSV DEU/107/2009 subtype A, RSV 9320 subtype B, or B/GZ/13-730 subtype B). In aspects, said flanking amino acid sequences are those that also flank the peptides or polypeptides included therein in the naturally occurring protein, for example, as described below:

• For a peptide or polypeptide have a core sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOS: 1, 2, 9, 10, 13, 14, 21-24, 31-34, 41, 42, 51, 52, 59, 60, 63, 64, 71,-74, 81-84, 91, or 92 (and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, the extensions of 1 to 12 amino acids are those found flanking the amino acid sequence of SEQ ID NOS: 1, 2, 9, 10, 13, 14, 21-24, 31-34, 41, 42, 51, 52, 59, 60, 63, 64, 71,- 74, 81-84, 91, or 92 in the amino acid sequence of a nucleocapsid (N) protein (e.g. any one of SEQ ID NO: 173-178) ofRSV.

• For a peptide or polypeptide have a core sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence ofSEQ ID NOS: 11, 12, 15-20, 25, 26, 35, 36, 43-48, 61, 62, 65-70, 75, 76, 85, 86, or 93-98 (and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, the extensions of 1 to 12 amino acids are those found flanking the amino acid sequence of SEQ ID NOS: 11, 12, 15-20, 25, 26, 35, 36, 43-48, 61, 62, 65-70, 75, 76, 85, 86, or 93-98 in the amino acid sequence of a fusion (F) protein (e.g., any one of SEQ ID NOS: 179-184) ofRSV.

• For a peptide or polypeptide have a core sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOS: 3-6, 29, 30, 37-40, 49, 50, 53-56, 79, 80, 87-90, 99, or 100(and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, the extensions of 1 to 12 amino acids are those found flanking the amino acid sequence ofSEQ ID NOS: 3-6, 29, 30, 37-40, 49, 50, 53-56, 79, 80, 87-90, 99, or 100 in the amino acid sequence of a matrix (M) protein (e.g., any one of SEQ ID NOS: 185-190) ofRSV.

• For a peptide or polypeptide have a core sequence comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence of SEQ ID NOS: 7, 8, 27, 28, 57, 58, 77, or 78 (and/or fragments and variants thereof), optionally with extensions of 1 to 12 amino acids on the C-terminal and/or the N-terminal, the extensions of 1 to 12 amino acids are those found flanking the amino acid sequence of SEQ ID NOS: 7, 8, 27, 28, 57, 58, 77, or 78 in the amino acid sequence of a non-structal 1 (NS1) protein (e.g., any one of SEQ ID NOS: 191- 196) ofRSV.

In aspects, said flanking amino acid sequences as described herein may serve as a MHC stabilizing region. The use of a longer peptide may allow endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses. In aspects, the peptides or polypeptides of the instant disclosure can be isolated, recombinant, and/or synthetic. In aspects, the peptides or polypeptides can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation. In aspects, the peptides or polypeptides of the instant disclosure can be capped with an n-terminal acetyl and/or c-terminal amino group.

In aspects, the instant disclosure is directed to one or more Class II polypeptides (“clusters”) of Table 1 and/or Table 2, (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of a polypeptide of Table 1 and/or Table 2.

In aspects, the instant disclosure is directed to one or more Class I polypeptides of Table 3, (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of a polypeptide of Table 3.

In aspects, the instant disclosure is directed to a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160 (and/or fragments thereof), wherein said polypeptide is still able to bind to a same HLA molecule (i.e., retain MHC binding propensity) and/or retain the same TCR specificity, and/or retain anti-RSV activity.

In aspects, the present disclosure is directed to a concatemeric polypeptide or peptide that comprises at one or more of the instantly-disclosed polypeptides or peptides (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160) linked, fused, or joined together (e.g., fused in- frame, chemically-linked, or otherwise bound) to an additional peptide or polypeptide. Such additional peptide or polypeptide may be one or more of the instantly instantly-disclosed polypeptides or peptides, or may be an additional peptide or polypeptide of interest. In aspects, a concatemeric peptide is composed of 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more of the instantly-disclosed peptides or polypeptides. In other aspects, the concatemeric peptides or polypeptides include 1000 or more, 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less peptide epitopes. In yet other embodiments, a concatemeric peptide has 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75- 100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 ofthe instantly- disclosed peptides or polypeptides linked, fused, or joined together. Each peptide or polypeptide of the concatemeric polypeptide may optionally have one or more linkers, which may optionally be cleavage sensitive sites, adjacent to their N and/or C terminal end. Such suitable linkers and cleavage sensitive sites, including AAY cleavage motifs or a poly GS linker which may be include on the N terminus of the C-terminal element, are known in the art. In such a concatemeric peptide, two or more of the peptide epitopes may have a linker, which may act as a cleavage sensitive site, between them. Alternatively two or more of the peptide epitopes may be connected directly to one another or through a linker that is not a cleavage sensitive site. In aspects, such linker is antigenically neutral, and the liker is preferably less than the length of a peptidyl backbone of 9 amino acids linearly arranged. In aspects, linker length is the length of a peptidyl backbone of between 2 and 8 amino acids, linearly arranged. In aspects, the spacer is unable to hydrogen bond in any spatially distinct manner to other distinct elements of the enhancing hybrid peptide.

In aspects, and with respect to antigenically neutral linker elements, various chemical groups may be incorporated as linkers instead of amino acids. Examples are described in U.S. Pat. No. 5,910,300, the contents of which are incorporated herein by reference. In apsects, a linker may be comprised of an aliphatic chain optimally interrupted by heteroatoms, for example a C2-C6 alkylene, or =N — (CH2)2-6 — N=. Alternatively, a spacer may be composed of alternating units, for example of hydrophobic, lipophilic, aliphatic and aryl-aliphatic sequences, optionally interrupted by heteroatoms such as O, N, or S. Such components of a spacer are preferably chosen from the following classes of compounds: sterols, alkyl alcohols, polyglycerides with varying alkyl functions, alkyl-phenols, alkyl-amines, amides, hydroxyphobic polyoxyalkylenes, and the like. Other examples are hydrophobic polyanhydrides, polyorthoesters, polyphosphazenes, polyhydroxy acids, polycaprolactones, polylactic, polyglycolic polyhydroxy-butyric acids. A linker may also contain repeating short aliphatic chains, such as polypropylene, isopropylene, butylene, isobutylene, pentamethlyene, and the like, separated by oxygen atoms.

Additional peptidyl sequences which can be used in as possible linkers are described in U.S. Pat. No. 5,856,456, the contents of which are incorporated herein by reference. In one embodiment, a linker has a chemical group incorporated within which is subject to cleavage. Without limitation, such a chemical group may be designed for cleavage catalyzed by a protease, by a chemical group, or by a catalytic monoclonal antibody. In the case of a protease- sensitive chemical group, tryptic targets (two amino acids with cationic side chains), chymotryptic targets (with a hydrophobic side chain), and cathepsin sensitivity (B, D or S) are favored. The term ‘tryptic target’ is used herein to describe sequences of amino acids which are recognized by trypsin and trypsin-like enzymes. The term ‘chymotryptic target’ is used herein to describe sequences of amino acids which are recognized by chymotrypsin and chymotrypsin-like enzymes. In addition, chemical targets of catalytic monoclonal antibodies, and other chemically cleaved groups are well known to persons skilled in the art of peptide synthesis, enzymatic catalysis, and organic chemistry in general, and can be designed into the hybrid structure and synthesized, using routine experimental methods.

In aspects, a concatemeric polypeptide of the instant disclosure is produced using the Epi Assembler System (EpiVax). The EpiAssembler system is useful for assembling overlapping epitopes to Immunogenic Consensus Sequences (ICS). EpiAssembler is an algorithm that optimizes the balance between pathogen and population coverage. EpiAssembler uses the information from the sequences produced by Conservatrix and EpiMatrix to form highly immunogenic consensus sequences. In aspects, the concatemeric peptides of the instant disclosure include those of Table 4, SEQ ID NOS: 161-172 and 197-227 (as well as nucleic acids (e.g., RNA mRNA, DNA, cDNA) encoding such concatemeric peptides). In aspects, the present disclosure provides a concatemeric polypeptide with at least 60%, 70%, 80%, 90%, or 95% homology to each of SEQ ID NOS: 161-172 and 197-227. In aspects, the present disclosure provides a concatemeric polypeptide having anti-RSV activity, said polyeptide having at least 60%, 70%, 80%, 90%, or 95% homology to each of SEQ ID NOS: 161-172 and 197-227. In aspects, the present disclosure provides concatemeric polypeptides with at least 60%, 70%, 80%, 90%, or 95% homology to those of SEQ ID NOS: 161-172 and 197-227. As previously described, anti-RSV activity means that the instantly- disclosed therapeutic T-cell epitope compounds and compositions are, in aspects: capable of stimulating, inducing, and/or expanding an immune response to RSV (e.g., a cellular (CD4+ and/or CD8+ T-cell response) or humoral immune response to RSV) and/or associated diseases in a subject; capable of stimulating, inducing, and/or expanding a RSV-specific IFNy response (e.g., by lymphocytes such as PMBC, or effector CD4+ and/or CD8+ T-cells), capable of inhibiting RSV viral replication or infectivity, and/or capable of inducing immunity against RSV. In aspects, a T-cell epitope compound or composition of the present disclosure having anti-RSV activity will reduce the disease symptoms resulting from RSV challenge by at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50% to about 90% or greater, including any value or range therebetween. Again, anti-RSV activity can be determined by various experiments and assays as known to those of skill in the art, including methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation, including use of experiments and assays as disclosed in the Examples herein.

Table 4: Class II Concatamers

SEQ ID

_N0 Sequence

161 LDKYKNAVTELQLLMQSTPAANNRKGAFKYIKPQSQFIVDLIDVFVHFGIAQSSTRGAS

162 NHKFTGLIGMLYAMSRLGREDTADLLIKELANVNILVKQISTPKGPSL

163 VSTYMLTNSELLSLINDMPITNRSGLTAVIRRANNVLKNETTNWKHTATRFAIK

164 RSGLTAVIRRANNVLKNETTNWKHTATRFAIKKEAGFYHILNNPKASLLSLTQFAH

165 KEAGFYHILNNPKASLLSLTQFAHTTEFKNAITNAKIIPYSALTEEFYQSTCSAVSKAY

166 VSTYMLTNSELLSLINDMPITNTTEFKNAITNAKIIPYSALTEEFYQSTCSAVSKAY

167 VLRWGVLAKSVKNIMLGHASVQAEMEGPSLRVMINSRSAVLAQMPSKFTISA

168 IPTYLRSISVRNKDLNNKDQLLSSSKYTIQRSTGDSIDTISQVNEKINQSLAFIRKSD

169 IKSALLSTNKAVVSLSNGVSVLTSKVYTDKLIQLTNALAKAVIHTIKLNGITF

170 DSGMIILCIAALVITKLAAGDRSALDQKKLMSNNVQIVRQQSYSIMSIIK

171 TGWYTSVITIELSTIKENKKTDVSSSVITSLGAIVSAY

172 IHTIKLNGIVFVHVITSSDISPNNYDVQKHINKLCGMLLITETVIEFQQKNNRLLEIT

227 ETVIEFQQKNNRLLEITKEAGFYHILNNPKASLLSLTQFAHKTDVSSSVITSLGAIVSAY

Table 5: Cl Concatamers

SEQ ID NO Sequence (w/ Spacers in bold)

197 TPNYDVQKHIAAYKSNFTTMPVAAYALRTGWYTSVAAYKFTGLIGML

198 KESIYYVTTAAYKEMKFEVLTLAAYTPKGPSLRVMAAYHFIDVFVHF

199 GLIDDNCEIAAYIPTYLRSISVAAYHTIKLNGIVAAYESIYYVTTNW

200 FPHFSSVVLGAAYLLKITCYTDKAAYFIDVFVHFGIAAYIYYVTTNWKH 201 MLRWGVLAKAAYFTGLIGMLYAAYNLFDNDEVALAAYALLKITCYT 202 VIRRANNVLKAAYMPVLQNGGYISQFIVDLGAYNIEIESRKSY

203 IKVRLQNLFAAYDEVALLKITCMEQVVEVYEYAAYKLHEGSTYT

204 VTDNKGAFKYAAYIVDLGAYLEKAAYKYIKPQSQFAAYCEIKFSKKL

205 VMLRWGVLAKAAYFPHFSSVVLAAYKYKNAVTELAAYTPKGPSLRV

206 PKDIANSFYAAYEEVLAYVVQLAAYMIKVRLQNLFAAYGSNSLSMIK

207 IQLTNALAKAAYYLEKESIYYVAAYILKDAGYHVAAYWEMMELTHC

208 SMIKVRLQNLAAYIWVPMFQSSMAAYITNAKIIPYAAYFIVDLGAYL

209 YLEKESIYYAAYKAVIHTIKLAAYVLTSKVLDLKAAYEVALLKITCY

210 VTDNKGAFKAAYGLIGMLYAMAAYKEMGEVAPEYAAYLSALRTGWY 211 SI YYVTTNWKAAYI VRQQSYSI AAYVVKSNFTTMAAYPFI FI DVFVFIF

212 EVALLKITCYAAYALRTGWYTSVAAYVTDNKGAFKAAYALLKITCYT

213 IQLTNALAKAAYGLIGMLYAMAAYKSNFTTMPVAAYLLKITCYTDK

214 FIDVFVHFGIAAYMLRWGVLAKAAYVMLRWGVLAKAAYSIYYVTTNWK

215 FTGLIGMLYAAYNLFDNDEVALAAYLSALRTGWYAAYNIEIESRKSY

216 PKDIANSFYAAYHTIKLNGIVVIRRANNVLKAAYYLEKESIYY

217 VTDN KGAFKYAAYI LKDAGYH VAAYITN AKI I PYAAYI VDLGAYLEK

218 M EQVVEVYEYAAYI VRQQSYSI AAYI KVRLQN LFAAYDEVALLKITC

219 TPKGPSLRVKYKNAVTELAAYTPKGPSLRVMAAYKYIKPQSQF

220 SMI KVRLQN LAAYI WVPM FQSSM AAYCEI KFSKKLAAYWEM M ELTHC 221 TPNYDVQKHIAAYMIKVRLQNLFAAYEEVLAYVVQLAAYKFTGLIGML 222 FPHFSSVVLGAAYFPHFSSVVLAAYKEMGEVAPEYAAYIPTYLRSISV

223 IYYVTTNWKHAAYKESIYYVTTAAYKEMKFEVLTLAAYSQFIVDLGAY

224 KAVIHTIKLAAYMPVLQNGGYIAAYGSNSLSMIKAAYHFIDVFVHF

225 PHFIDVFVHFAAYKLHEGSTYTAAYGLIDDNCEIAAYESIYYVTTNW

226 YLEKESIYYVAAYVVKSNFTTMAAYVLTSKVLDLKAAYFIVDLGAYL

In aspects, the concatemeric polypeptides of the instant disclosure can be isolated, recombinant, and/or synthetic. In aspects, the concatemeric peptides or polypeptides can be either in neutral (uncharged) or salt forms, and may be either free of or include modifications such as glycosylation, side chain oxidation, or phosphorylation. In aspects, the concatemeric peptides or polypeptides of the instant disclosure can be capped with an n-terminal acetyl and/or c-terminal amino group.

In aspects, one or more peptides or polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160; as well as the concatemeric polypeptides disclosed herein, including SEQ ID NOS: 161-172 and 197-227 of Tables 4 and 5) joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide. As previously described, with respect to the one or more T-cell epitopes of the instant disclosure, the term “heterologous polypeptide” is intended to mean that the one or more T-cell epitopes of the instant disclosure are heterologous to, or not included naturally, in the heterologous polypeptide. In aspects, a heterologous polypeptide may include, but are not limited to, e.g. monoclonal antibody, polyclonal antibody, mouse antibody, human antibody, humanized antibody, mono specific antibody, bispecific antibody, glycosylated antibody, Fc-modified antibody, or antibody-drug conjugates; an antibody of different class or subclass (e.g., IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD or IgE molecules) or antigen- specific antibody fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulfide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulfide-linked scFv, diabody)). In aspects, one or more of the instantly-disclosed polypeptides may be inserted into the heterologous polypeptide (e.g., through recombinant techniques, mutagenesis, or other known means in the art), may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide. In aspects, one or more of the instantly-disclosed polypeptides may be inserted into or replace amino acids in a Fc domain as disclosed in U.S. Patent No. 7,442,778, U.S. Patent No. 7,645,861, U.S. Patent No. 7,655,764, U.S. Patent No. 7,655,765, and/or U.S. Patent No. 7,750,128 (each of which are herein incorporated by reference in their entirety). For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et ah, 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety). In aspects, chimeric or fusion polypeptides comprise one or more of the instantly-disclosed polypeptides of the present disclosure operatively linked to a heterologous polypeptide. "Operatively linked" indicates that the one or more of the instantly-disclosed polypeptides and the heterologous protein are fused in-frame or chemically linked or otherwise bound. For example, in aspects, the one or more of the instantly-disclosed polypeptides may be covalently bound to one or more internal conjugation site(s) in an Fc domain as disclosed in U.S. Patent No. 8,008,453, U.S. Patent No. 9,114,175, and/or U.S. Patent No. 10,188,740 (each of which are herein incorporated by reference in their entirety). In aspects, the one or more peptides or polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160) may be joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted amino acid sequence, together with flanking amino acids of the heterologous polypeptide. In aspects, the present disclosure is directed to polypeptide (which, in aspects, may be an isolated, synthetic, or recombinant) having a sequence comprising one or more of SEQ ID NOS: 1-160 (and/or fragments or variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160), wherein said one or more of SEQ ID NOS: 1-160 is not naturally included in the polypeptide and/or said one or more of SEQ ID NOS: 1-160 is not located at its natural position in the polypeptide. For example, in aspects, the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure may be inserted into a RSV sequence in which the RSV sequence does not include the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure (e.g., the RSV sequence does not include, or is mutated to not include, the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure) or the one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure is inserted into a RSV sequence but not at its natural position. In aspects, the one or more peptides or polypeptides of the instant disclosure can be joined or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) to a small molecule (e.g., albumin or other known carriers and proteins), drug, or drag fragment, for example but not limited to, a drug or drug fragment that is binds with high affinity to defined HLAs.

As used herein, two polypeptides (or a region of the polypeptides) are substantially homologous or identical when the amino acid sequences are at least about 45-55%, typically at least about 70-75%, more typically at least about 80-85%, more typically greater than about 90%, and more typically greater than 95% or more homologous or identical. To determine the percent homology or identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide or nucleic acid molecule for optimal alignment with the other polypeptide or nucleic acid molecule). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position. As is known in the art, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Sequence homology for polypeptides is typically measured using sequence analysis software. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity". In aspects, the percent homology between the two sequences is a function of the number of identical positions shared by the sequences ( e.g percent homology equals the number of identical positions/total number of positions x 100).

In aspects, the present disclosure also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide of the instant disclosure (e.g., a polypeptide having a sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160; concatemeric peptides as disclosed herein, including the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227). Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, Met, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues His, Lys and Arg and replacements among the aromatic residues Trp, Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found (Bowie JU et al., (1990), Science, 247(4948):130610, which is herein incorporated by reference in its entirety).

In aspects, a variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Variant polypeptides can be fully functional (e.g., retain MHC binding propensity and/or TCR specificity, and/or retain anti-RSV activity) or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions; in this case, typically MHC contact residues provided MHC binding is preserved. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function (e.g., retain MHC binding propensity and/or TCR specificity, and/or retain anti-RSV activity). Alternatively, such substitutions can positively or negatively affect function to some degree. Non- functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region; in this case, typically TCR contact residues. In aspects, a variant and/or a homologous polypeptide retains the desired anti-RSV activity of the instant disclsoure (e.g.: capable of stimulating, inducing, and/or expanding an immune response to RSV (e.g., a cellular (CD4+ and/or CD8+ T-cell response) or humoral immune response to RSV) and/or associated diseases in a subject; capable of stimulating, inducing, and/or expanding a RSV-specific IFNy response (e.g., by lymphocytes such as PMBC, or effector CD4+ and/or CD8+ T-cells); and/or capable of inhibiting RSV viral replication or infectivity, and/or capable of inducing immunity against RSV). Alternatively, such substitutions can positively or negatively affect function to some degree. Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region; in this case, typically TCR contact residues. In aspects, funcational variants of a polypeptide having a sequence (or a core sequence) comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 as disclosed herein may contain one or more conservative substitutions, and in aspects one or more non conservative substitutions, at amino acid residues which are not believed to be essential for functioning (with amino acid residues considered being essential for functioning, including, e.g., retain MHC binding propensity and/or TCR specificity, and/or retain anti-RSV activity) of the instantly-disclosed polypeptides. For example, in aspects, a variant polypeptide having a sequence (or a core sequence) comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160, or fragments thereof as disclosed herein, or a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227 or a fragment thereof as disclosed herein, may contain one or more conservative substitutions (and in aspects, a nonconservative subsitution) in one or more HLA contact residues, provided HLA binding is preserved. MHC binding assays are well known in the art. In aspects, such assays may include the testing of binding affinity with respect to MHC class I and class II alleles in in vitro binding assays, with such binding assays as are known in the art. Examples include, e.g., the soluble binding assays as disclosed in U.S. 7,884,184 or PCT/US2020/020089, both of which are herein incorporated by reference in their entireties. Additionally, in aspects, a fully functional variant polypeptide having a sequence (or a core sequence) comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 as disclosed herein do not contain mutations at one or more critical residues or regions, such as TCR contact residues.

In aspects, the TCR-binding epitope (which can be referred to as TCR binding residues, TCR facing epitope, TCR facing residues, or TCR contacts) for a 9-mer identified epitope (which may be a 9-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class II molecule are at position 2, 3, 5, 7, and 8 of the identified epitope, while the MHC-binding agretope (which can be referred to as MHC contacts, MHC facing residues, MHC-binding residues, or MHC-binding face) for a 9-mer identified epitope (which may be a 9-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class II molecule are at position 1, 4, 6, and 9, both as counted from the amino terminal.

In aspects, the TCR binding epitope for a 9-mer identified epitope (which may be a 9- mer fragment of one or more of SEQ ID NOS: 1-160 or as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that binds to a MHC class I molecule are at position 4, 5, 6, 7, and 8 of the identified epitope, while the MHC binding agretope for a 9-mer identified epitope (which may be a 9-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class I molecule are at position 1, 2, 3, and 9, both as counted from the amino terminal.

In aspects, the TCR binding epitope for a 10-mer identified epitope that bind to a MHC class I molecule are at position 4, 5, 6, 7, 8, and 9 of the identified epitope (which may be a 10- mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 10-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227), while the MHC binding agretope for a 10-mer identified epitope (which may he a l 0-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 10-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class I molecule are at position 1, 2, 3, 9, and 10, both as counted from the amino terminal.

In aspects, the TCR-binding epitope for a 9-mer identified epitope (which may be a 9- mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class II molecule are at any combination of residues at positions 2, 3, 5, 7, and 8 (e.g., but not limited to, positions 3, 5, 7 and 8; positions 2, 5, 7, and 8; positions 2, 3, 5, and 7, etc.) of the identified epitope, while the MHC binding agretope for a 9-mer identified epitope (which may be a 9- mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) is the complementary face to the TCR facing residues, both as counted from the amino terminal. In aspects, the TCR binding epitope for 9-mer identified epitope (which may be a 9- mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class I molecule are at positions 4, 5, 6, 7, and 8; 1, 4, 5, 6, 7 and 8; or 1, 3, 4, 5, 6, 7, and 8 of the identified epitope, while the MHC binding agretope for a 9-mer identified epitope (which may be a 9-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 9-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) is the complementary face to the TCR facing residues, both as counted from the amino terminal.

In aspects, the TCR-binding epitope for a 10-mer identified epitope (which may be a 10-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 10-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) that bind to a MHC class I molecule are at any combination of residues at positions 1, 3, 4, 5, 6, 7, 8, and 9 of the identified epitope, while the MHC binding agretope for a 10-mer identified epitope (which may be a 10-mer fragment of one or more of SEQ ID NOS: 1-160 as disclosed herein or a 10-mer fragment of a concatemeric peptide of SEQ ID NOS: 161-172 and 197-227) is the complementary face to the TCR facing residues, both as counted from the amino terminal.

Based on the above, it should be understood that in apsects in which one or more 9- mers and/or 10-mer epitopes are contained within a longer polypeptide and are predicted to bind one or more Class I or Class II MHC molecules and are occurring in close proximity to each other in a naturally occurring sequence (e.g., wherein position 1 of each pair of binding 9-mers and/or 10-mers fall within, e.g., 3 amino acids of each other), such epitopes may be combined to form an epitope cluster. In a given cluster, any given amino acid may be, with respect to a given 9-mer epitope or 10-mer epitope, MHC facing and, with respect to another 9-mer epitope, TCR facing.

In aspects, the present disclosure also includes fragments of the instantly-disclosed polypeptides and concatemeric polypeptides. In aspects, the present disclosure also encompasses fragments of the variants of the instantly-disclosed polypeptides and concatemeric polypeptides as described herein. In aspects, as used herein, a fragment comprises at least about nine contiguous amino acids. In aspects, the present disclosure also encompasses fragments of the variants of the T-cell epitopes described herein. Useful fragments (and fragments of the variants of the polypeptides and concatemeric polypeptides described herein) include those that retain one or more of the biological activities, particularly: MHC binding propensity and/or TCR specificity, and/or anti-RSV activity. Biologically active fragments are, for example, about 9, 10, 11, 12, 1, 14, 15, 16, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids in length, including any value or range therebetween. Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Several fragments can be comprised within a single larger polypeptide. In aspects, a fragment designed for expression in a host can have heterologous pre- and pro polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment.

In aspects, the instantly disclosed polypeptides and concatemeric polypeptides of the present disclosure can include allelic or sequence variants (“mutants”) or analogs thereof, or can include chemical modifications ( e.g ., pegylation, glycosylation). In aspects, a mutant retains the same function, particularly MHC binding propensity and/or TCR specificity, and/or anti-RSV activity. In aspects, a mutant can provide for enhanced binding to MHC molecules. In aspects, a mutant can lead to enhanced binding to TCRs. In another instance, a mutant can lead to a decrease in binding to MHC molecules and/or TCRs. Also contemplated is a mutant that binds, but does not allow signaling via the TCR.

The manner of producing the polypeptides of the present disclosure will vary widely, depending upon the nature of the various elements comprising the molecule. For example, an isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. The synthetic procedures may be selected so as to be simple, provide for high yields, and allow for a highly purified stable product. For example, polypeptides of the instant disclosure can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques, such as recombinant techniques, mutagenesis, or other known means in the art. An isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis techniques. In aspects, a polypeptide of the instant disclosure is produced by recombinant DNA or RNA techniques. In aspects, a polypeptide of the instant disclosure can be produced by expression of a recombinant nucleic acid of the instant disclosure in an appropriate host cell. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression cassette or expression vector, the expression cassette or expression vector introduced into a host cell and the polypeptide expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternatively a polypeptide can be produced by a combination of ex vivo procedures, such as protease digestion and purification. Further, polypeptides of the instant disclosure can be produced using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety).

In aspects, the present disclosure also provides chimeric or fusion polypeptide compositions. In aspects, the present disclosure is directed to a chimeric or fusion polypeptide composition (which in aspects may be isolated, synthetic, or recombinant) comprising one or more peptides, polypeptides, or concatemeric peptides of the present disclosure (e.g., one or more peptides or polypeptides of the present disclosure have a sequence, e.g. but not limited to, comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160, and similarly may have a sequence, e.g. but not limited to, comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 161-172 and 197-227 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 161-172 and 197-227). In aspects, a chimeric or fusion polypeptide composition of the present disclosure comprises one or more peptides, polypeptides, and/or concatemeric peptides of the present disclosure joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide, such as an unrelated protein. As previously described, with respect to the one or more T-cell epitopes (e.g., peptide, polypeptides, or concatemeric peptides of the instant disclosure), the term “heterologous polypeptide” is intended to mean that the one or more T-cell epitopes of the instant disclosure are heterologous to, or not included naturally, in the heterologous polypeptide. In aspects, a heterologous polypeptide may include, but are not limited to, e.g. monoclonal antibody, polyclonal antibody, mouse antibody, human antibody, humanized antibody, mono specific antibody, bispecific antibody, glycosylated antibody, Fc-modified antibody, or antibody-drug conjugates; an antibody of different class or subclass (e.g., IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD or IgE molecules) or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulfide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulfide-linked scFv, diabody)). In aspects, one or more of the instantly-disclosed peptides, polypeptides, or concatemeric peptides may be inserted into the heterologous polypeptide (e.g., through recombinant techniques, mutagenesis, or other known means in the art), may be added to the C-terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide. In aspects, one or more of the instantly- disclosed polypeptides may be inserted into or replace amino acids in a Fc domain as disclosed in U.S. Patent No. 7,442,778, U.S. Patent No. 7,645,861, U.S. Patent No. 7,655,764, U.S. Patent No. 7,655,765, and/or U.S. Patent No. 7,750,128 (each of which are herein incorporated by reference in their entirety). For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety). In aspects, chimeric or fusion polypeptides comprise one or more of the instantly-disclosed peptides, polypeptides, or concatemeric peptides operatively linked to a heterologous polypeptide. "Operatively linked" indicates that the one or more of the instantly-disclosed peptides, polypeptides, or concatemeric peptides and the heterologous polypeptide are fused in- frame or chemically-linked or otherwise bound. For example, in aspects, the one or more of the instantly-disclosed polypeptides may be covalently bound to one or more internal conjugation site(s) in an Fc domain as disclosed in U.S. Patent No. 8,008,453, U.S. Patent No. 9,114,175, and/or U.S. Patent No. 10,188,740 (each of which are herein incorporated by reference in their entirety). In aspects, the one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure may be joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted into a heterologous polypeptide as a whole, although it may be made up from a joined to, linked to (e.g., fused in-frame, chemically-linked, or otherwise bound), and/or inserted amino acid sequence, together with flanking amino acids of the heterologous polypeptide. In aspects, a chimeric or fusion polypeptide composition comprises a peptide, polypeptide, or concatemeric peptide of the instant disclosure wherein said one or more of peptides, polypeptides, or concatemeric peptides is not naturally included in the heterologous polypeptide and/or said one or more of peptides, polypeptides, or concatemeric peptides is not located at its natural position in the heterologous polypeptide. In aspects, the one or more of peptide or polypeptides of the present disclosure can be joined, linked to (e.g., fused in- frame, chemically-linked, or otherwise bound), and/or inserted into the heterologous polypeptide. In aspects, chimeric or fusion polypeptide compositions comprise one or more of the instantly-disclosed T-cell epitopes (e.g., peptides, polypeptides, or concatemeric peptides of the instant disclosure) operatively linked to a heterologous polypeptide having an amino acid sequence not substantially homologous to the T-cell epitope. In aspects, the chimeric or fusion polypeptide does not affect function of the T-cell epitope per se. For example, the fusion polypeptide can be a GST-fusion polypeptide in which the T-cell epitope sequences are fused to the C-terminus of the GST sequences. Other types of fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Such fusion polypeptides, particularly poly-His fusions or affinity tag fusions, can facilitate the purification of recombinant polypeptide. In certain host cells (e.g. , mammalian host cells), expression and/or secretion of a polypeptide can be increased by using a heterologous signal sequence. Therefore, in aspects, the chimeric or fusion polypeptide contains a heterologous signal sequence at its N-terminus. In aspects of the above chimeric or fusion polypeptide compositions, the heterologous polypeptide or polypeptide comprises a biologically active molecule. In aspects, the biologically active molecule is selected from the group consisting of an immunogenic molecule, a T cell epitope, a viral protein, and a bacterial protein. In aspects, the one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure can be joined or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) to a small molecule, drug, or drug fragment. For example, the one or more peptides, polypeptides, or concatemeric peptides of the instant disclosure can be joined or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) to an unrelated peptide or protein, a small molecule (e.g., albumin or other known carriers and proteins), drug, or drag fragment, for example but not limited to, a drug or drug fragment that is binds with high affinity to defined HLAs. In aspects of the above-described chimeric or fusion polypeptide compositions, the chimeric or fusion polypeptide compositions can be recombinant, isolated, and/or synthetic.

A chimeric or fusion polypeptide composition can be produced by standard recombinant DNA or RNA techniques as are known in the art. For example, DNA or RNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, polymerase chain reaction (PCR) amplification of nucleic acid fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence(Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, (2^nd, 1992), FM Asubel et al. (eds), Green Publication Associates, New York, NY (Publ), ISBN: 9780471566355, which is herein incorporated by reference in its entirety). Further, one or more peptides, polypeptides or concatemeric of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160) can be inserted into a heterologous polypeptide or inserted into a non-naturally occurring position of a polypeptide through recombinant techniques, synthetic polymerization techniques, mutagenesis, or other standard techniques known in the art. For example, protein engineering by mutagenesis can be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety).

In aspects, the polypeptides, concatemeric polypeptides, and chimeric or fusion polypeptides can be purified to homogeneity or partially purified. It is understood, however, that preparations in which the T-cell epitope compounds and compositions are not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the composition, even in the presence of considerable amounts of other components. Thus, the present disclosure encompasses various degrees of purity. In one embodiment, the language "substantially free of cellular material" includes preparations of the polypeptides, concatemeric polypeptides, and chimeric or fusion polypeptides having less than about 30% (by dry weight) other proteins (e.g., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, less than about 5% other proteins, less than about 4% other proteins, less than about 3% other proteins, less than about 2% other proteins, less than about 1 % other proteins, or any value or range therebetween.

In aspects, when a polypeptide, concatemeric polypeptide, and chimeric or fusion polypeptide of the present disclosure is recombinantly produced, the composition can also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the the polypeptides, concatemeric polypeptides, and chimeric or fusion polypeptides preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of the the polypeptides, concatemeric polypeptides, and chimeric or fusion polypeptides in which it is separated from chemical precursors or other chemicals that are involved in the T-cell epitope’s synthesis. The language "substantially free of chemical precursors or other chemicals" can include, for example, preparations of the the polypeptides, concatemeric polypeptides, and chimeric or fusion polypeptides having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, less than about 5% chemical precursors or other chemicals, less than about 4% chemical precursors or other chemicals, less than about 3% chemical precursors or other chemicals, less than about 2% chemical precursors or other chemicals, or less than about 1% chemical precursors or other chemicals.

In aspects, the present disclosure also includes pharmaceutically acceptable salts of the T-cell epitope compounds and compositions (including one or more of e.g., peptides or polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric or fusion polypeptide compositions as disclosed herein (which in aspects may be isolated, synthetic, and/or recombinant). “Pharmaceutically acceptable salt” means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent peptide or polypeptide (e.g., peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as disclosed herein). As used herein, “pharmaceutically acceptable salt” refers to derivative of the instantly-disclosed polypeptides, concatemeric polypeptides, and/or chimeric or fusion polypeptides, wherein such compounds are modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc. NUCLEIC ACIDS

In aspects, the present disclosure also provides for nucleic acids (e.g., DNAs (including cDNA, RNAs (such as, but limited to mRNA), vectors, viruses, or hybrids thereof, all of which may be isolated, synthetic, or recombinant) that encode in whole or in part one or more one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides of the present disclosure as described herein. In aspects, the nucleic acid further comprises, or is contained within, an expression cassette, a plasmid, and expression vector, or recombinant virus, wherein optionally the nucleic acid, or the expression cassette, plasmid, expression vector, or recombinant virus is contained within a cell, optionally a human cell or a non-human cell, and optionally the cell is transformed with the nucleic acid, or the expression cassette, plasmid, expression vector, or recombinant virus. In aspects, cells are transduced, transfected, or otherwise engineered to contain within one or more of e.g., polypeptides of the present disclosure; isolated, synthetic, or recombinant nucleic acids, expression cassettes, plasmids, expression vectors, or recombinant viruses as disclosed herein; and/or isolated, synthetic, or recombinant chimeric or fusion polypeptide compositions as disclosed herein. In aspects, the cell can be a mammalian cell, bacterial cell, insect cell, or yeast cell. In aspects, the nucleic acid molecules of the present disclosure can be inserted into vectors and used, for example, as expression vectors or gene therapy vectors. Gene therapy vectors can be delivered to a subject by, e.g., intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (Chen SH et al, (1994), Proc Natl Acad Sci USA, 91(8):3054-7, which are herein incorporated by reference in their entirety). Similarly, the nucleic acid molecules of the present disclosure can be inserted into plasmids. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system. Such pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. In aspects of the above nucleic acids (e.g., DNAs, RNAs, vectors, viruses, or hybrids thereof) that encode in whole or in part at least one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein, the nucleic acids encode one or more peptides or polypeptides of the instant disclosure as described above (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160; as well as the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227). In aspects, the present disclosure is directed to a vector comprising a nucleic acid of the present disclosure encoding one or more polypeptides of the present disclosure or chimeric or fusion polypeptide composition of the present disclosure. In aspects, the present disclosure is directed to a cell comprising a vector of the present disclosure. In aspects, the cell can be a mammalian cell, bacterial cell, insect cell, or yeast cell.

The nucleic acid of the instant disclosure may be DNAs (including but not limited to cDNA) or RNAs (including but not limited to mRNA), single- or double-stranded. The nucleic acid is typically DNA or RNA (including mRNA). The nucleic acid may be produced by techniques well known in the art, such as synthesis, or cloning, or amplification of the sequence encoding the immunogenic polypeptide; synthesis, or cloning, or amplification of the sequence encoding the cell membrane addressing sequence; ligation of the sequences and their cloning/amplification in appropriate vectors and cells. The nucleic acids provided herein (whether RNAs, DNAs, vectors, viruses or hybrids thereof) that encode in whole or in part one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein can be isolated from a variety of sources, genetically engineered, amplified, synthetically produced, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems. In aspects nucleic acids provided herein are synthesized in vitro by well-known chemical synthesis techniques (as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066, all of which are herein incorporated by reference in their entirety). Further, techniques for the manipulation of nucleic acids provided herein, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature (see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993), all of which are herein incorporated by reference in their entirety).

A further object of the invention relates to a nucleic acid molecule encoding one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein. The nucleic acid may be used to produce the one or more peptides, polypeptides, concatemeric peptides, and/or chimeric or fusion polypeptides as described herein in vitro or in vivo, or to produce cells expressing the polypeptide on their surface, or to produce vaccines wherein the active agent is the nucleic acid or a vector containing the nucleic acid. The nucleic acid may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native or stabilized forms of polynucleotides as are known in the art.

As previously mentioned, the nucleic acid molecules according to the present disclosure may be provided in the form of a nucleic acid molecule per se such as naked nucleic acid molecules; a plasmid, a vector; virus or host cell, etc., either from prokaryotic or eukaryotic origin. Vectors include expression vectors that contain a nucleic acid molecule of the invention. An expression vector capable of expressing a polypeptide can be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the (e.g., cDNA, or RNA, including mRNA) is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA (e.g., cDNA, or RNA, including mRNA) may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into the host bacteria for cloning using standard techniques. The vectors of the present invention may, for example, comprise a transcriptional promoter, and/or a transcriptional terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator. One or more peptides or polypeptides of the present disclosure may be encoded by a single expression vector. Such nucleic acid molecules may act as vehicles for delivering peptides/polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA/RNA vaccines.

In aspects, the vector may be a viral vector comprising a nucleic acid as defined above. The viral vector may be derived from different types of viruses, such as, Swinepox, Fowlpox, Pseudorabies, Aujezky's virus, salmonella, vaccinia virus, BHV (Bovine Herpes Virus), HVT (Herpes Virus of Turkey), adenovirus, TGEV (Transmissible Gastroenteritidis Coronavirus), Erythrovirus, and SIV (Simian Immunodeficiency Virus). Other expression systems and vectors may be used as well, such as plasmids that replicate and/or integrate in yeast cells.

The instant disclosure also relates to a method for preparing a peptide, polypeptide, concatemeric peptide, and/or chimeric or fusion polypeptide of the instant disclosure, the method comprising culturing a host cell containing a nucleic acid or vector as defined above under conditions suitable for expression of the nucleic acid and recovering the polypeptide. As indicated above, the proteins and peptides may be purified according to techniques known per se in the art.

PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS

In aspects, the T-cell epitope compositions of the present disclosure (including one or more of e.g., polypeptides as disclosed herein; concatemeric peptides as disclosed herein; chimeric of fusion polypeptide compositions as disclosed herein; nucleic acids as disclosed herein, including nucleic acids encoding such peptides, polypeptides, concatemeric peptides, or chimeric of fusion polypeptide compositions as disclosed herein; expression cassettes, plasmids, expression vectors, recombinant viruses, or cells as disclosed herein, and vaccines as disclosed herein; hereafter referred to as “T-cell epitope compounds and compositions of the present disclosure”) may be comprised in a pharmaceutical composition or formulation. In aspects, the instantly-disclosed pharmaceutical compositions or formulations generally comprise a T-cell epitope composition of the present disclosure and a pharmaceutically- acceptable carrier and/or excipient. In aspects, a pharmaceutical composition or formulation comprises an adjuvant. In aspects, said pharmaceutical compositions are suitable for administration. Pharmaceutically-acceptable carriers and/or excipients are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the instantly-disclosed T-cell epitope compositions (see, e.g., Remington’s Pharmaceutical Sciences. (18^TH Ed, 1990), Mack Publishing Co., Easton, PA Publ)). In aspects, the pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Pharmaceutical compositions as disclosed herein are able for use in stimulating, inducing, and/or expanding an immune response to a RSV infection and/or related diseases caused by RSV, in a subject, and can be used in methods of treating and/or preventing RSV infection and/or related diseases caused by RSV, in a subject, such as a human.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,” and grammatical variations thereof, as they refer to compositions, carriers, excipients, and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” means, for example, an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. A person of ordinary skill in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular T-cell epitope compositions of the present disclosure.

In aspects, preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the T-cell epitope compounds and compositions of the present disclosure and as previously described above, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

In aspects, T-cell epitope compounds and compositions of the present disclosure are formulated to be compatible with its intended route of administration. The T-cell epitope compounds and compositions of the present disclosure can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; vaginally; intramuscular route or as inhalants. In aspects, T-cell epitope compounds and compositions of the present disclosure can be injected directly into a particular tissue where deposits have accumulated, e.g., intracranial injection. In other aspects, intramuscular injection or intravenous infusion may be used for administration of T-cell epitope compounds and compositions of the present disclosure. In some methods, T-cell epitope compounds and compositions of the present disclosure are administered as a sustained release composition or device, such as but not limited to a Medipad™ device. In aspects, T-cell epitope compounds and compositions of the present disclosure are administered intradermally, e.g., by using a commercial needle-free high- pressure device such as Pulse NeedleFree technology (Pulse 50TM Micro Dose Injection System, Pulse NeedleFree Systems; Lenexa, KS, USA). In aspects, said commercial needle- free high-pressure device (e.g., Pulse NeedleFree technology) confers one or more of the following benefits: non-invasive, reduces tissue trauma, reduces pain, requires a smaller opening in the dermal layer to deposit the composition in the subject (e.g., only requires a micro skin opening), instant dispersion of the composition, better absorption of the composition, greater dermal exposure to the composition, and/or reduced risk of sharps injury.

In aspects, T-cell epitope compounds and compositions of the present disclosure can optionally be administered in combination with other agents that are at least partly effective in treating various medical conditions as described herein.

In aspects, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include, but are not limited to, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Examples of excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene, dried skim milk, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

In aspects, pharmaceutical compositions or formulations suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition is sterile and should be fluid to the extent that easy syringeability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. In aspects formulations including a T-cell epitope compound or composition of the present disclosure may include aggregates, fragments, breakdown products and post-translational modifications, to the extent these impurities bind HLA and present the same TCR face to cognate T cells they are expected to function in a similar fashion to pure T-cell epitopes. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., 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. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.

In aspects, sterile injectable solutions (e.g., sterile solutions suitable for injectable and/or intradermal needle-free high-pressure device) can be prepared by incorporating the T- cell epitope compounds and compositions of the present disclosure 4- in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a 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, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Further, T-cell epitope compounds and compositions of the present disclosure can be administered in the form of a depot injection or implant preparation that can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

In aspects, oral compositions generally include an inert diluent or an edible carrier and can be enclosed in gelatin capsules or compressed into tablets. In aspects, for the purpose of oral therapeutic administration, the binding agent can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding compounds, and/or adjuvant materials can be included as part of the composition. In aspects, the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate or orange flavoring.

For administration by inhalation, T-cell epitope compounds and compositions of the present disclosure can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

In aspects, systemic administration of the T-cell epitope compounds and compositions of the present disclosure can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the T-cell epitope compounds and compositions may be formulated into ointments, salves, gels, or creams and applied either topically or through transdermal patch technology as generally known in the art.

In aspects, the T-cell epitope compounds and compositions of the present disclosure can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In aspects, the T-cell epitope compounds and compositions of the present are prepared with carriers that protect the T-cell epitope compounds and compositions against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically-acceptable carriers. These can be prepared according to methods known to those skilled in the art (U.S. Pat. No. 4,522,811, which is herein incorporated by reference in its entirety). In aspects, the T-cell epitope compounds and compositions of the present disclosure can be implanted within or linked to a biopolymer solid support that allows for the slow release of the T-cell epitope compounds and compositions to the desired site.

In aspects, it is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of binding agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the instant disclosure are dictated by and directly dependent on the unique characteristics of the binding agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such T-cell epitope compounds and compositions for the treatment of a subject.

In aspects of a pharmaceutical composition as described herein, the composition may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the instantly-disclosed peptides or polypetides (including concatemeric polypeptides) or nucleic acids encoding such peptides or polypeptides (including concatemeric polypeptides). For example, in aspects, a pharmaceutical composition can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 peptides or polypeptides (including up to 40 peptides or polypetides), including any value or range therebetween, comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160; concatemeric peptides as disclosed herein, including the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227 and nucleic acids (e.g., RNA mRNA, DNA, cDNA) encoding such peptides, polypeptides, or concatemeric peptides, and/or fragments and variants thereof, as described herein.

VACCINE COMPOSITIONS

The term “vaccine” as used herein includes an agent which may be used to cause, stimulate or amplify the immune system of animals (e.g., humans) against a pathogen. Vaccines of the invention are able to cause or stimulate or amplify an immune response against a RSV infection and/or related diseases caused by RSV.

The term “immunization” includes the process of delivering an immunogen to a subject. Immunization may, for example, enable a continuing high level of antibody and/or cellular response in which T-lymphocytes can kill or suppress the pathogen in the immunized animal, such as a human, which is directed against a pathogen or antigen to which the animal has been previously exposed.

Vaccines of the instant disclosure comprise an immunologically effective amount of a T cell epitope compound or composition of the instant disclosure as described above, and in aspects in a pharmaceutically acceptable vehicle and optionally with additional excipients and/or an adjuvant. As a result of the vaccination with a composition of the present disclosure, animals, and in aspects humans, become at least partially or completely immune to RSV infection and/or related diseases caused by RSV or resistant to developing moderate or severe RSV infection and/or related diseases caused by RSV. The instantly disclosed vaccines may be used to elicit a humoral and/or a cellular response, including CD4+ and CD8+ T effector cell responses. In aspects, an animal subject, such as a human, is protected to an extent to which one to all of the adverse physiological symptoms or effects of RSV infection and/or related diseases caused by RSV, are significantly reduced, ameliorated or totally prevented.

In practice, the exact amount required for an immunologically effective dose may vary from subject to subject depending on factors such as the age and general condition of the subject, the nature of the formulation and the mode of administration. An appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. For instance, methods are known in the art for determining or titrating suitable dosages of a vaccine to find minimal effective dosages based on the weight of the animal subject, including human subject, concentration of the vaccine and other typical factors. The dosage of the vaccines of the present disclosure will depend on the species, breed, age, size, vaccination history, and health status of the animal (e.g., swine/pig) to be vaccinated, as well as the route of administration, e.g., subcutaneous, intradermal, oral intramuscular or intravenous administration. The vaccines of the instant disclosure can be administered as single doses or in repeated doses. The vaccines of the instant disclosure can be administered alone, or can be administered simultaneously or sequentially administered with one or more further compositions, such as other porcine immunogenic or vaccine compositions. Where the compositions are administered at different times, the administrations may be separate from one another or overlapping in time. In aspects, the vaccine comprises a unitary dose of between 0.1-3000 pg, including any value or range therebetween of polypeptide and/or nucleic acid of the instant disclosure.

The dosage of the vaccine, concentration of components therein and timing of administering the vaccine, which elicit a suitable immune response, can be determined by methods such as by antibody titrations of sera, e.g., by ELISA and/or seroneutralization assay analysis and/or by vaccination challenge evaluation.

In aspects, the vaccine comprises a novel, therapeutic T cell epitope compounds or compositions as disclosed herein) in purified form, optionally in combination with any suitable excipient, carrier, adjuvant, and/or additional protein antigen.

In another aspect, the vaccine comprises a nucleic acid as defined above, optionally in combination with any suitable excipient, carrier, adjuvant, and/or additional protein antigen. In aspects, the vaccine comprises a viral vector containing a nucleic acid as defined above. In aspects, the vaccine comprises one or more plasmid vectors.

Vaccine constructs including a T-cell epitope compound or composition of the present disclosure upon administration to a subject may initiate a strong T-cell mediated immune response, but may not induce a humoral immune response. Therefore, aspects of a vaccine against RSV infection and/or related diseases caused by RSV, contains a combination of the putative T-cell epitopes together with either live attenuated virus (LAV, for example live attenuated RSV) or inactivated virus (for example inactivated RSV). This vaccine composition (including both the putative T-cell epitopes and an LAV or inactivated virus) upon administration to a subject may induce both cellular and humoral immune responses, thereby conferring comprehensive immunity against RSV infection and/or related diseases caused by RSV, in the animals, including humans.

Vaccines may comprise other ingredients, known per se by one of ordinary skill in the art, such as pharmaceutically acceptable carriers, excipients, diluents, adjuvants, freeze drying stabilizers, wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, and preservatives, depending on the route of administration.

Examples of pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, arachis oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as light liquid paraffin oil, or heavy liquid paraffin oil; squalene; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium salt, or hydroxypropyl methylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 -butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum tragacanth or gum acacia; and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the vaccine composition and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.

Examples of adjuvants include, but are not limited to, oil in water emulsions, aluminum hydroxide (alum), immunostimulating complexes, non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-a, IFN-b, IFN-g, etc.), saponins, monophosphoryl lipid A (MFA), muramyl dipeptides (MDP), MCA, and the like. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin(s) isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde. Further adjuvants may include, but are not limited to, poly- ICFC, 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSFIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRTX, Juvlmmune, FipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEE, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon. In aspects of the pharmaceutical compositions or vaccines as disclosed herein, the adjuvant comprises poly-ICLC. The TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand poly-ICLC are two of the most promising vaccine adjuvants currently in clinical development. In preclinical studies, poly-ICLC appears to be the most potent TLR adjuvant when compared to LPS and CpG. This appears due to its induction of pro-inflammatory cytokines and lack of stimulation of IL-10, as well as maintenance of high levels of co-stimulatory molecules in DCs. Poly-ICLC is a synthetically prepared double- stranded RNA consisting of polyl and polyC strands of average length of about 5000 nucleotides, which has been stabilized to thermal denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. The compound activates TLR3 and the RNA helicase-domain of MDA5, both members of the PAMP family, leading to DC and natural killer (NK) cell activation and mixed production of type I interferons, cytokines, and chemokines. Examples of freeze-drying stabilizer may be for example carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, and derivatives thereof.

Vaccines may additionally comprise at least one immunogen from at least one additional pathogen, e.g., a pig pathogen such as Actinobacillus pleuropneunomia Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Balantidium coli; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodyentheriae, B. pilosicoli, B. innocens, Brucella suis, preferably biovars 1, 2 and 3; Classical swine fever virus, Chlamydia and Chlamydophila spp., preferably C. pecorum and C. abortus; Clostridium spp., preferably Cl. difficile, Cl. perfringens types A, B and C, Cl. novyi, Cl. septicum, Cl. tetani Digestive and respiratory Coronavirus; Cryptosporidium parvum; Eimeria spp.; Eperythrozoonis suis currently named Mycoplasma haemosuis; Erysipelothrix rhusiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14; Hemagglutinating encephalomyelitis virus; lsospora suis; Japanese Encephalitis virus; Lawsonia intracellulars; Leptospira spp., preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagicae, Leptospira interrogans, Leptospira Pomona and Leptospira tarassovi; Mannheimia haemolytica; Mycobacterium spp., preferably M. avium, M. intracellular and M. bovis: Mycoplasma hyponeumoniae Parvovirus; Pasteurella multocida Porcine circovirus; Porcine cytomegolovirus; Porcine parovirus, Porcine reproductive and respiratory syndrome virus: Pseudorabies virus; Rotavirus; Sagiyama virus; Salmonella spp., preferably S. thyhimurium and S. choleraesuis; Staphylococcus spp., preferably S. hyicus; Streptococcus spp., preferably Strep suis; Swine cytomegalovirus; Swine herpes virus; Swine influenza virus; Swinepox virus; Toxoplasma gondii,· Vesicular stomatitis virus and virus of exanthema of swine; or other isolates and subtypes of porcine circovirus.

The vaccine compositions of the instant disclosure may be liquid formulations such as an aqueous solution, water-in-oil or oil-in-water emulsion, syrup, an elixir, a tincture, or a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents.

The route of administration can be percutaneous, via mucosal administration, or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccine compositions according to the present disclosure may be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. A vaccine of the present disclosure can conveniently be administered intranasally, transdermally (i.e., applied on or at the skin surface for systemic absorption), parenterally, ocularly, etc. The parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intradermal, and intraperitoneal routes and the like. In aspects, vaccines of the present disclosure are administered intradermally, e.g., by using a micro needle patch as is known in the art or by using a commercial needle-free high-pressure device such as Pulse NeedleFree technology (Pulse 50TM Micro Dose Injection System, Pulse NeedleFree Systems; Lenexa, KS, USA).

The present disclosure also relates to methods of immunizing or inducing an immune response in animals (e.g., humans) comprising administering to said animal a peptide, polypeptide, concatemeric peptide, chimeric or fusion polypeptide, nucleic acid, cell, vector, pharmaceutical, or vaccine as described above.

The present disclosure also relates to methods of treating and/or preventing RSV infection and/or related diseases caused by RSV in animals (e.g., humans) comprising administering to said animal a peptide, polypeptide, concatemeric peptide, chimeric or fusion polypeptide, nucleic acid, cell, vector, pharmaceutical, or vaccine as disclosed herein.

A vaccine of the present disclosure can conveniently be administered intranasally, transdermally (i.e., applied on or at the skin surface for systemic absorption), parenterally, ocularly, etc. The parenteral route of administration includes, but is not limited to, intramuscular, intravenous, and intraperitoneal routes and the like.

The dosage of the vaccines of the present disclosure will depend on the species, breed, age, size, vaccination history, and health status of the animal (e.g., human) to be vaccinated, as well as the route of administration, e.g., subcutaneous, intradermal, oral intramuscular or intravenous administration. The vaccines of the instant disclosure can be administered as single doses or in repeated doses. The vaccines of the instant disclosure can be administered alone, or can be administered simultaneously or sequentially administered with one or more further compositions, such as other porcine immunogenic or vaccine compositions. Where the compositions are administered at different times, the administrations may be separate from one another or overlapping in time.

In aspects, the present disclosure includes multiple rounds of administration of the instantly-disclosed vaccine compositions. For example, the vaccine can be boosted at one, two, three, and/or four week intervals.. Such are known in the art to improve or boost the immune system to improve protection against the pathogen. Additionally, the present disclosure may also include assessing a subject’s immune system to determine if further administrations of the instantly-disclosed vaccine compositions is warranted. In some aspects, multiple administrations may include the development of a prime boosting strategy of vaccination using the instantly-discloed vaccines (e.g.., polypeptide based or nucleic acid based as disclosed herein). Such may provide an opportunity to produce sequential immunogenic responses against RSV infection and/or related diseases caused by RSV. In some aspects, the vaccine can be boosted at 1, 2, 3, 4, 5, or 6 week intervals. In some aspects, the vaccine is boosted at 2 week intervals. In some apsects, the vaccine is boosted at 3 week intervals. In some aspects, peptide based vaccine and nucleice acid (e.g., RNA or DNA) vaccinations can be achieved in an alternative manner to provide a regimen of immunization with the same immunogen presented in different fashions to the subject’s immune system.

In one aspect, the vaccine compositions of the present disclosure are administered to a subject susceptible to or otherwise at risk for RSV infection and/or related diseases caused by RSV, to enhance the subject own immune response capabilities. The subject to which the vaccine is administered is, in one aspect, a human. The animal may be susceptible to infection by RSV infection (or a closely related virus) and/or related diseases caused by RSV.

In aspects of a vaccine as described herein, the vaccine may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the instantly-disclosed peptides or polypetides (including concatemeric polypeptides) or nucleic acids encoding such peptides or polypeptides (including concatemeric polypeptides). For example, in aspects, a vaccine can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 peptides or polypeptides (including up to 40 peptides or polypetides), including any value or range therebetween, comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence comprising, consisting of, or consisting essentially of one or more of SEQ ID NOS: 1-160 and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160; concatemeric peptides as disclosed herein, including the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227 and nucleic acids (e.g., RNA mRNA, DNA, cDNA) encoding such peptides, polypeptides, or concatemeric peptides, and/or fragments and variants thereof, as described herein. The present disclosure also provides a container comprising an immunologically effective amount of a polypeptide, nucleic acid or vaccine as described above. The present disclosure also provides vaccination kits comprising an optionally sterile container comprising an immunologically effective amount of the vaccine, means for administering the vaccine to animals, and optionally an instruction manual including information for the administration of the immunologically effective amount of the composition for treating and/or preventing RSV infection (or a closely related virus) and/or related diseases caused by RSV.

METHODS OF TREATMENT

Stimulating T-cells with T-cell epitope compounds and compositions of the present disclosure can stimulate, induce, and/or expand a corresponding naturally occurring immune response, e.g., stimulating, inducing, and/or expanding a corresponding naturally occurring immune response to a RSV infection and/or related diseases caused by RSV, including CD4+ and/or CD8+ T cell responses, and in aspects results in increased secretion of one or more cytokines and chemokines. In aspects, T-cells activated by the T-cell epitope compounds and compositions of the present disclosure stimulate cell-mediated immunity against RSV infection and/or related diseases caused by RSV, in a subject.

In aspects, T cells activated by the T-cell epitope compounds and compositions of the present disclosure stimulate cell-mediated immunity against RSV infection (or a closely related virus) and/or related diseases caused by RSV in a subject.

In aspects, the present disclosure is directed to a method of stimulating, inducing, and/or expanding an immune response, e.g., against RSV infection and/or related diseases caused by RSV, in a subject in need thereof by administering to the subject a therapeutically effect amount of a T-cell epitope composition (compound or composition of the present disclosure.

In aspects, the present disclosure is directed to a method of preventing, treating, or ameliorating a disease by RSV infection and/or related diseases caused by RSV, in a subject in need thereof by administering to the subject a therapeutically effect amount of a T-cell epitope compound or composition of the present disclosure.

Aspects

A 1st aspect is directed to a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160. A 2nd aspect is directed to a polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

A 3rd aspect is directed to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160.

A 4th aspect is directed to a polypeptide according to any one of apects 1-3, wherein said variant or fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160 retains MHC binding propensity and TCR specificity, and/or retains anti-RSV activity.

A 5^th aspect is directed to a polypeptide consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

A 6^th aspect is directed to a polypeptide consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

A 7^th aspect is directed to a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

An 8^th aspect is directed to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

A 9^th aspect is directed to a polypeptide according to aspect 8, wherein said fragment or variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227 retains anti-RSV activity.

A 10^th aspect is directed to a nucleic acid encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160. A 11^th aspect is directed to a nucleic acid encoding a polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

A 12^th aspect is directed to a nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

A 13^th aspect is directed to a nucleic acid encoding a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOS: 161-172 and 197-227 and/or fragments and variants thereof.

A 14^th aspect is directed to a nucleic acid consisting of a sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227 and fragments or variants thereof.

A 15^th aspect is directed to a nucleic acid consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

A 16^th aspect is directed to a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

A 17^th aspect is directed to a nucleic acid of any one of aspects 10-12, wherein said fragment or variant of the nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160 encodes a polypeptide that retains anti-RSV activity.

A 18^th aspect is directed to a nucleic acid of aspect 13, wherein said fragment or variant of the nucleic acid encoding a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOS: 161-172 and 197-227 encodes a polypeptide that retains anti-RSV activity.

A 19^th aspect is directed to a polypeptide or nucleic acid of any one of aspects 1-7, 10- 12, or 17, wherein said polypeptide is one or more Class II polypeptides (“clusters”) of Table 1 or Table 2, (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of a polypeptide of Table 1.

A 20^th aspect is directed to a polypeptide or nucleic acid of any one of aspects 8-9, or 13-16, or 18, wherein said polypeptide is one or more polypeptides comprising, consisting, or consisting essentially of an amino acid sequence of one or more of SEQ ID NOS: 1-172 and 197-227. A 21^st aspect is directed to a plasmid comprising a nucleic acid of any one of aspects

10-20.

A 22^nd aspect is directed to a vector comprising a nucleic acid according to any one of aspects 10-20.

A 23^rd aspect is directed to a pharmaceutical composition comprising a polypeptide according to any one of aspects 1-9 or 19-20 and a pharmaceutically-acceptable carrier and/or excipient.

A 24^th aspect is directed to a pharmaceutical composition comprising a nucleic acid according to any one of aspects 10-20 and a pharmaceutically-acceptable carrier and/or excipient.

A 25^th aspect is directed to a pharmaceutical composition comprising a plasmid according to aspect 21 and a pharmaceutically-acceptable carrier and/or excipient.

A 26^th aspect is directed to a pharmaceutical composition comprising a vector according to aspect 22 and a pharmaceutically-acceptable carrier and/or excipient.

A 27^th aspect is directed to a vaccine comprising a polypeptide according to any one of aspects 1-9 or 19-32 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

A 28^th aspect is directed to a vaccine comprising a nucleic acid according to any one of aspects 10-20 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

A 29^th aspect is directed to a vaccine comprising a plasmid according to aspect 21 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

A 30^th aspect is directed to a vaccine comprising a vector according to aspect 22 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

A 31^st aspect is directed to a method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of aspects 1-9 or 19-20.

A 32^nd aspect is directed to a method for inducing immunity against a RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of aspects 10-20.

A 33^rd aspect is directed to a method for inducing immunity against a RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to aspect 21. A 34^th aspect is directed to a method for inducing immunity RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to aspect 22.

A 35^th aspect is directed to a method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of aspects 23-26.

A 36^th aspect is directed to a method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of aspects 27-30.

A 37^th aspect is directed to a method according to any one of aspects 31-36, wherein the step of administration additionally includes administration of an RSV virus, wherein the virus is a live attenuated virus or inactivated virus.

A 38^th aspect is directed to a method for inducing an immune response against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more of a polypeptide according to any one of aspects 1-9 or 10-20.

A 39^th aspect is directed to a method for inducing an immune response against against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of aspects 10-20.

A 40^th aspect is directed to a method for inducing an immune response against against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to aspect 21.

A 41^st aspect is directed to a method for inducing an immune response against against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to aspect 22.

A 42^nd aspect is directed to a method for inducing an immune response against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of aspects 23-26.

A 43 ^rd aspect is directed to a method for inducing an immune response against against RSV infection and/or related diseases caused by RSV, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of aspects 27-30.

A 44^th aspect is directed to a method according to any one of aspects 50-55, wherein the step of administration additionally includes administration of a RSV virus, wherein the virus is a live attenuated virus or inactivated virus.

A 45^th aspect is directed to a chimeric or fusion polypeptide comprising a polypeptide of any one of aspects 1-9 or 10-20, wherein said polypeptide is joined, linked, or inserted into a heterologous polypeptide.

A 46^th aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more of a polypeptide according to any one of aspects 1-9 or 19-20.

A 47^th aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of aspects 10- 20.

A 48^th aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to aspect 21.

A 49^th aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection, the method comprising administering to the subject a therapeutically effective amount of a vector according to aspect 22.

A 50^th aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of aspects 23-26.

A 51 ^st aspect is directed to a method of preventing, treating, or ameliorating a disease by RSV infection, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of aspects 27-30. A 52^nd aspect is directed to a method according to any one of aspects 46-51, wherein the step of administration additionally includes administration of a RSV virus, wherein the virus is a live attenuated virus or inactivated virus.

A 53^rd aspect is directed to a polypeptide according to any one of aspects 1-9 or 19-20, wherein said polypeptide has one or more conservative substitutions compared to the polypeptide.

A 54^th aspect is directed to a polypeptide according to aspect 53, wherein said polypeptide retains MHC binding propensity and/or TCR specificity, and/or retains anti-RSV activity.

Further aspects and advantages of the instant disclosure are provided in the following section, which should be considered as illustrative only.

EXAMPLES

The examples that follow are not to be construed as limiting the scope of the invention in any manner. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.

EXAMPLE 1 : In-silico Identification of Potential Epitopes for HLA

T-cells specifically recognize epitopes presented by cells in the context of MHC (Major Histocompatibility Complex) Class I and II molecules. These T-cell epitopes can be represented as linear sequences comprising 7 to 30 contiguous amino acids that fit into the MHC Class I or II binding groove. A number of computer algorithms have been developed and used for detecting Class I and II epitopes within protein molecules of various origins (De Groot AS et al., (1997), AIDS Res Hum Retroviruses, 13 (7) :539-41; Schafer JR et al., (1998), Vaccine, 16(19): 1880-4; De Groot AS et al., (2001), Vaccine, 19(31):4385-95; De Groot AS et al., (2003), Vaccine, 21(27-30):4486-504). These “in silico ” predictions of T-cell epitopes have been successfully applied to the design of vaccines and the de-immunization of therapeutic proteins, i.e. antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics (Dimitrov DS, (2012), Methods Mol Biol, 899:1-26).

The Conservatrix system (EpiVax, Providence, Rhode Island) is an algorithm useful for identifying 9-mer polypeptide sequences from a larger set of data. The Conservatrix system parses input sequences into 9-mer sequences that are conserved amongst multiple inputted whole sequences, such as multiple strains of the same pathogen, for even the most mutable of potential vaccine targets. These 9-mer sequences may be searched for identically matched 9- mer sequences across data sets.

The EpiMatrix™ system (EpiVax, Providence, Rhode Island) is a set of predictive algorithms encoded into computer programs useful for predicting class I and class II HLA ligands and T cell epitopes. The EpiMatrix™ system uses matrices in order to model the interaction between specific amino acids and binding positions within the HLA molecule. In order to identify putative epitopes resident within any given input protein, the EpiMatrix™ System first parses the input protein into a set of overlapping n-mer frames (n=length of amino acids of epitope peptide being screen; e.g., n=9 or n=10) where each frame overlaps the last by n-1 amino acids. Each frame is then scored for predicted affinity to one or more common alleles of the HLA molecules. Briefly, for any given n-mer peptide specific amino acid codes (one for each of 20 naturally occurring amino acids) and relative binding positions (1 to n) are used to select coefficients from the predictive matrix. Individual coefficients are derived using a proprietary method similar to, but not identical to, the pocket profile method first developed by Sturniolo (Sturniolo T et al., 1999, Nat Biotechnol, 17(6):555-61). Individual coefficients are then summed to produce a raw score. EpiMatrix™ raw scores are then normalized with respect to a score distribution derived from a very large set of randomly generated peptide sequences. The resulting “Z” scores are normally distributed and directly comparable across alleles. It was determined that any peptide scoring above 1.64 on the EpiMatrix™ “Z” scale (approximately the top 5% of any given peptide set) has a significant chance of binding to the MHC molecule for which it was predicted. Peptides scoring above 2.32 on the scale (the top 1 %) are extremely likely to bind.

Peptides containing clusters of putative T cell epitopes are more likely to test positive in validating in vitro and in vivo assays. In aspects, the results of the initial EpiMatrix™ analysis is further screened for the presence of putative T cell epitope “clusters” using a second proprietary algorithm known as Clustimer™ algorithm. The Clustimer™ algorithm identifies sub-regions contained within any given amino acid sequence that contains a statistically unusually high number of putative T cell epitopes. Typical T-cell epitope “clusters” range from about 9 to roughly 30 amino acids in length and, considering their affinity to multiple alleles and across multiple 9-mer frames, can contain anywhere from about 4 to about 40 putative T cell epitopes. Each epitope cluster identified an aggregate EpiMatrix™ score is calculated by summing the scores of the putative T cell epitopes and subtracting a correcting factor based on the length of the candidate epitope cluster and the expected score of a randomly generated cluster of the same length. EpiMatrix™ cluster scores in excess of +10 are considered significant. In aspects, the T-cell epitopes of the instant disclosure contain several putative T-cell epitopes forming a pattern known as a T-cell epitope cluster.

The JanusMatrix system (EpiVax, Providence, Rhode Island) useful for screening peptide sequences for cross-conservation with a host proteome. JanusMatrix is an algorithm that predicts the potential for cross-reactivity between peptide clusters and the host genome or proteome, based on conservation of TCR- facing residues in their putative MHC ligands. The JanusMatrix algorithm first considers all the predicted epitopes contained within a given protein sequence and divides each predicted epitope into its constituent agretope and epitope. Each sequence is then screened against a database of host proteins. Peptides with a compatible MHC-facing agretope (i.e., the agretopes of both the input peptide and its host counterparty are predicted to bind the same MHC allele) and exactly the same TCR-facing epitope are returned. The JanusMatrix Homology Score suggests a bias towards immune tolerance. In the case of a therapeutic protein, cross-conservation between autologous human epitopes and epitopes in the therapeutic may increase the likelihood that such a candidate will be tolerated by the human immune system. In the case of a vaccine, cross-conservation between human epitopes and the antigenic epitopes may indicate that such a candidate utilizes immune camouflage, thereby evading the immune response and making for an ineffective vaccine. When the host is, for example, a human, the peptide clusters are screened against human genomes and proteomes, based on conservation of TCR-facing residues in their putative HLA ligands. The peptides are then scored using the JanusMatrix Homology Score. In aspects, peptides with a JanusMatrix Homology Score below 2.5 or below 3.0 indicate low tolerogenicity potential and may be useful for vaccines. In aspects, peptides with a JanusMatrix Homology Score above 3.0 indicate high tolerogenicity potential and may not be useful for vaccines, and in aspects may be excluded from the T cell epitope compositions of the present disclosure.

In aspects, the VaccineCAD system is useful for arranging potential epitopic vaccine candidates into a string to avoid creation of novel epitopes upon joining of the vaccine candidate sequences. Specifically, VaccineCAD designs potential vaccine candidates into a string-of-beads vaccine while minimizing any deleterious junctional epitopes that may appear in the joining process. VaccineCAD may use EpiMatrix to predict junctional epitopes. Particularly concatemeric peptides of interest developed using VaccineCad are those of SEQ ID NOS: 161-172 and 197-227. Similarly, particular nucleic acids of interest (including RNA, mRNA, DNA, etc.) are those encoding a peptide or polypeptide comprising, consisting of, or consisting essentially of one or more peptides or polypeptides having an amino acid sequence off SEQ ID NOS: 161-172 and 197-227. EXAMPLE 2: RSV Epitope Discovery and Vaccine Design Analysis was carried out on iVAX with the objectives of:

1) identification of top class I epitopes and class II clusters from Respiratory Syncytial Virus (RSV) strain 98-25147-X and confirmation that these are conserved with a set of RSV strains; and,

2) Subsequent vaccine design

Stage 1 - Selection of Top Scoring Epitopes The RSV strain selection identifed: strain: 98-25147-X, subtype A as a reference strain; and

Five circulating strains: ATCC VR-26 subtype A, LA2_106 subtype A, DEU/107/2009 subtype A, 9320 subtype B, B/GZ/13-730, subtype B.

Fig. 1 shows a cartoon overview of RSV and lists four antigens have been targeted for peptide selection: Nucleoprotein (N), Fusion protein (F). Matrix protein (M), and Non- structural 1 protein (NS1). SEQ ID NOs: 173-178 set forth the amino acid sequence of the nucleoprotein, SEQ ID NOs: 179-184 set forth the amino acid sequences for the Fusion protein, SEQ ID NOs: 185-190 set forth the amino acid sequences for the Matrix protein, and, SEQ ID NOs: 191-196 set forth the amino acvid sequences for NS1. Figs. 2 and 3 set forth an overview of the Class II and Class I analysis protocols. Figs. 4 and 14 show an overview of the Class II and Class I binding assays, respectively. The utilized amino acid sequences have the following GenBank Accession Nos.:ACY68428.1, ACY68430.1, ACY68432.1, ACY68435.1, AAX23994.1, AHX56976.1, AHA83760.1, AAR14266.1, AIY62428.1, AAX23991.1, AHX56973.1, AHA83757.1, AAR14263.1, AIY62425.1, AAX23989.1, AHX56971.1, AHA83755.1, AAR14261.1, AIY62423.1, AAX23987.1, AHX56969.1, AHA83753.1, AAR14259.1, AIY62421.1

The .pep files were uploaded on iVAX.

Class II analysis: Step 1 (Reference Sequence Analysis)

The antigen sequences for the reference strain were assessed for a standard Class II EpiMatrix Analysis. Fig. 2 provides an overview of the steps taken to identify the top 25 clusters, presented herein as SEQ ID Nos: 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, andlOO. Initially, all proteins were parsed into overlapping 9-mer frames and each frame was scored using a panel of nine Class II alleles. These alleles are: DRBU0101, DRBU0301, DRBU0401, DRBU0701, DRBU0801, DRBU0901,

DRB1*1101, DRB1*1301, and DRB1*1501, the Z scores and IC50 for which are shown in Figs. 5-12, respectively. Fig. 13 shows the number of alleles bound for each sequence analyzed. The protein summary and the full immunogenicity HTML report were generated

ClustiMer was then used to screen the results of the EpiMatrix Analysis and identify discrete regions containing unusually high densities of putative T cell epitopes. The significant EpiMatrix Scores contained within these regions were then aggregated to create an EpiMatrix Cluster Score. From this 46 clusters were identified with an EpiMatrix score above 10: The combined summary and full cluster HTML report were generated The epitope clusters identified by ClustiMer were screened against the immune epitope database (IEDB) at the La Jolla Institute for Allergy and Immunology for the presence of previously discovered T cell epitopes and MHC ligands. The analysis was performed with a threshold score of 80%.

Using the JanusMatrix algorithm, the putative T cell epitope clusters identified within the reference RSV strain antigens were then screened against the human proteome to look for potential cross-reactive signals. This analysis was carried out using the “Execute Class II JanusMatrix” link, and the “Choose Database to Search” parameter was selected to be “Human”.

Class analysis: Step 2 (RSV Conservation Analysis)

The epitope clusters from the reference strain identified by ClustiMer were then screened against the four antigen files (F, M, N, NS1) of the circulating RSV sequences to identify clusters conserved within this set of RSV strains. A threshold score of 80% was utilized to identify candidate sequences. Using the JanusMatrix algorithm, the putative T cell epitope clusters identified within the reference RSV strain antigens were screened against the set of RSV strains to identify epitopes that are cross-conserved at the TCR face with the Januse Matrix application.

Class analysis: Step 3 (Selection of Clusters)

Combined Cluster Results Table

The IEDB, RSV, and JanusMatrix Homology results were merged to create a combined cluster results table. The 46 clusters derived from the RSV reference strain were sorted in descending order based on the EpiMatrix els score. The table cells were coded to indicate the source antigen for each cluster: Fusion protein (F), Matrix protein (M), Nucleoprotein (N) and Non-structural 1 protein (NS1). Selection Criteria

Based on the EpiMatrix and JanusMatrix scores, three cluster tables were generated. These can be found in the spreadsheet titled “EpiVax Intravacc RSV Peptide- Selection_21Feb20”, in the tab titled “RSV TOP CLASS II”. This spreadsheet can be found at: Wepivax-srv02\Open COO ProjectsUntravacc - RSV\7 Data\03 EpiMatrix. The first table contains the top 25 selected cluster sequences. (F) = 9 clusters, (M) = 6 clusters, (N) = 8 clusters and (NS1) = 2 clusters. The following criteria have been applied for the top 25 cluster selection: High EpiMatrix Scores (>10).

Low JanusMatrix Scores (< 2).

Must include a cluster from each of the four targeted antigens.

Highly conserved and contains at least one EpiBar conserved across the 6 RSV strains (Cls 160 from the Matrix protein (19^th on the list) is an exception. The EpiBar is only conserved across 4 RSV subtype A strains).

No alarming negative IEDB Homology results.

Class analysis: Step 4 (peptide Synthesis checklist)

For each cluster, synthesis flags were added. These flags are as listed:

1) ^AQ in the first position; can be unstable or hard to process

2) includes cysteine; consider replacing with alanine or serine

3) ** No charge; can be difficult to synthesize and validate

4) † Includes methionine; consider replacing with leucine

5) } D followed by S, A, N, or G can induce dehydration of aspartic acid side chain; replacing

6) D with E would be a conservative alternative

7) i G or P in the last or second to last position can disrupt peptide structure; consider replacing with alanine

Based on the flags, a list of modified cluster sequences has been created and included in Table 2 herein.

Class analysis: Step 1 (Reference Sequence Analysis)

EpiMatrix Class I Analysis

The antigen sequences for the reference strain were submitted to a standard Class I EpiMatrix analysis. All proteins were parsed into overlapping 9-mer and 10-mer frames and each frame was scored using a panel of six Class I supertype alleles. These alleles are: A*0101, A*0201, A*0301, A*2402, B*0702, and B*4403, the results of which are shown is Figs. 15- 20, respectively..

The 9mer and lOmer epitopes identified by EpiMatrix were screened against the immune epitope database (IEDB) at the La Jolla Institute for Allergy and Immunology for the presence of previously discovered T cell epitopes and MHC ligands. A score threshold of 80% was required.

Next, using the JanusMatrix algorithm, the putative T cell epitopes identified within the reference RSV strain antigens were screened against the human proteome to look for potential cross-reactive signals.

Class analysis: Step 2 (RSV Conservation Analysis)

Conservatrix Analysis

The 9mer/10mer epitopes identified from the reference strain identified by EpiMatrix were screened against the four antigen files (F, M, N, NS1) of the circulating RSV sequences to identify epitopes conserved within this set of RSV strains.

Next, using the JanusMatrix algorithm, the putative T cell epitopes identified within the reference RSV strain antigens were screened against the set of RSV strains to identify epitopes that are cross-conserved at the TCR face.

Class analysis: Step 3 (Selection of Epitopes)

Combined Staircase Table

The IEDB, RSV and JanusMatrix Homology result tables were merged to create a combined 9mer/10mer staircase table for each antigen. 9mers/10mers identified from the RSV reference strain were sorted in a staircase order based on each frame’s z-score per class I supertype allele.

Selection Criteria

Based on the EpiMatrix, JanusMatrix and IEDB results, a total of 60 epitopes have been selected to move forward for synthesis and analysis, and are shown in Table 3 herein. For each Class I supertype allele, the top 10 epitopes have been selected: (F) = 1 epitope, (M) = 3 epitopes, (N) = 3 epitopes and (NS1) = 3 epitopes.

The following main criteria has been applied for the epitope selections: Top 1% hits (z-scores >2.32).

Low JanusMatrix Scores (< 2).

Conserved across the 6 RSV strains (100% or at the TCR face)

Low hydrophobicity

Several epitopes have been excluded for the following reasons:

The presence of a negative IEDB result that does not support the HLA predictions. Cysteine-containing epitopes (to avoid oxidation during peptide synthesis)

For some antigens and alleles, there were not enough top 1% hits that matched the criteria above, resulting in the inclusion of a few exceptions:

Six (6) top 1% epitopes are Cysteine-containing epitopes (only epitopes with one Cystine residue were selected). Epitopes 90 and 20 are also not conserved across the six (6) RSV strains

1) Epitope 21 EVALLKITCY (SEQ ID NO: 105)

2) Epitope 103 GLIDDNCEI (SEQ ID NO: 115)

3) Epitope 23 ALLKITCYT (SEQ ID NO: 116)

4) Epitope 24 LLKITCYTDK (SEQ ID NO: 125)

5) Epitope 109 CEIKFSKKL (SEQ ID NO: 155)

6) Epitope 90 WEMMELTHC (SEQ ID NO: 156)

7) Epitope 20 DEVALLKITC (SEQ ID NO: 157)

Seven (7) are in the top 5% hits, not the top 1%.

1) Epitope 47 HTIKLNGIV (SEQ ID NO: 107)

2) Epitope 15 NLFDNDEVAL (SEQ ID NO: 117)

3) Epitope 8 MIKVRLQNLF (SEQ ID NO: 135)

4) Epitope 9 IKVRLQNLF (SEQ ID NO: 136)

5) Epitope 7 SMIKVRLQNL (SEQ ID NO: 137)

6) Epitope 80 MPVLQNGGYI (SEQ ID NO: 146)

7) Epitope 43 KAVIHTIKL (SEQ ID NO: 147)

EXAMPLE 3: Administration of RSV Vaccine

Vaccine construct designs are developed, such as is demonstrated in the specification, and in aspects as specifically exemplified in Example 2. In aspects, this results in a concatemeric polypeptide vaccine or an “epistring” that consists of overlapping T-cell epitopes. As described throughout, such vaccines may be used for stimulating, inducing, and/or expanding an immune response against RSV infection and/or related diseases caused by RSV. In aspects, such vaccines initiate a strong T-cell mediated immune response and has potential of inducing a humoral immune response.

Therefore, a vaccine containing a combination of the epistring together with either live attenuated virus (LAV) or inactivated virus is administered in an immunization trial in an appropriate animal model , e.g., mice, rats, rabbits, hamsters, etc., or even humans, as are known in the art. Data from administration of this combination vaccine provides positive results on the safety and effectiveness of the vaccine. This vaccination approach is expected to induce both cellular and humoral immune responses, thereby stimulating, inducing, and/or expanding an immune response against RSV infection and/or related diseases caused by RSV, in humans.

EXAMPLE 4: Polypeptide Binding to MEIC

Methods for the Assessment Polypeptides as Disclosed Herein Binding to Soluble MHC.

Synthesis of peptides. The polypeptides of the present disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160) are produced by direct chemical synthesis or by recombinant methods (J Sambrook et ah, Molecular Cloning: A Laboratory Manual, (2ED, 1989), Cold Spring Harbor Laboratory Press, Cold Springs Harbor, NY (Publ)). Every peptide undergoes rigorous quality control characterization before release to determine purity, mass, and correct sequence. Peptides are assessed for purity by reversed phase high-pressure liquid chromatography (RP-HPLC). Peptides are > 90% pure, and each preparation will undergo Amino Acid Analysis to ensure that the equivalent molar amounts are used in assays for consistency and reproducibility between different lots of peptides, and will also allow for reliable comparison studies between peptide efficacy. Peptides are assessed for mass and correct sequence using tandem mass spectrometry and MS CheckT analysis. In certain aspects, the polypeptides as disclosed herein may be capped with an n-terminal acetyl and/or c-terminal amino group. HPLC, mass spectrometry and UV scan (ensuring purity, mass and spectrum, respectively) analysis of the selected polypeptides will indicate > 80% purity.

111 A Binding Assay. Binding activity is analyzed at EpiVax (Providence, Rhode Island) and is conducted for any polypeptides of the present disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160). The binding assay used (Steere AC et ah, (2006), J Exp Med, 2003(4):961-71) yields an indirect measure of peptide-MHC affinity. Soluble HLA molecules are loaded onto a 96-well plate with the unlabeled experimental polypeptides and labeled control peptide. Once the binding mixture reaches steady equilibrium (at 24 hours), the HLA-polypeptide complexes are captured on an ELISA plate coated with anti-human DR antibody and detected with a Europium-linked probe for the label (PerkinElmer, Waltham, MA). Time-resolved fluorescence measuring bound labeled control peptide is assessed by a SpectraMax® M5 unit (Spectramax, Radnor, PA). Binding of experimental polypeptides is expressed as the percent inhibition of the labeled control peptide (experimental fluorescence / control fluorescence multiplied by 100). The percent inhibition values for each experimental polypeptide (across a range of molar concentrations) is used to calculate the concentration at which it inhibits 50% of the labeled control polypeptide’s specific binding, i.e., the polypeptides’s IC50.

Select experimental polypeptides are solvated in DMSO. The diluted polypeptide is mixed with binding reagents in aqueous buffering solution, yielding a range of final concentrations from 100,000 nM down to 100 nM. The select polypeptides are assayed against a panel of eight common Class II HLA alleles: DRB 1*0101, DRB 1*0301, DRB 1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501. From the percent inhibition of labeled control peptide at each concentration, IC50 values are derived for each polypeptide/allele combination using linear regression analysis.

In this assay, the experimental polypeptides are considered to bind with very high affinity if they inhibit 50% of control peptide binding at a concentration of 100 nM or less, high affinity if they inhibit 50% of control peptide binding at a concentration between 100 nM and 1,000 nM, and moderate affinity if they inhibit 50% of control peptide binding at a concentration between 1,000 nM and 10,000 nM. Low affinity peptides inhibit 50% of control peptide binding at concentrations between 10,000 nM and 100,000 nM. Peptides that fail to inhibit at least 50% of control peptide binding at any concentration below 100,000 nM and do not show a dose response are considered non-binders (NB).

Peptide Characterization by Binding to HLA Class II Molecules

Soluble MHC binding assays are performed on any of the instantly disclosed polypeptides (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160). Soluble MHC binding assays are performed on selected polypeptides as disclosed herein according to the methods described previously. IC50 values (nM) will be derived from a six-point inhibition curve. EpiMatrix™ Predictions, calculated IC50 values, and results classifications are reported for each polypeptide and HLA allele. Binding curves are generated for certain polypeptides against the selected Class II HLA alleles, such as for the HLA DRB1 *0801 assay and the HLA DRB1 *1501 assay.

EXAMPLE 5: Peptide-Exposed APCs

Methods for Assessing the Phenotype of Peptide-Exposed APC

Surface expression of Class II HLA (HLA-DR) and CD86 by professional antigen presenting cells (APCs) is one way APCs modulate T cell response. In this assay, candidate polypeptides are tested for their ability to effect (e.g., upregulate) the expression of Class II HLA and the co-stimulatory molecule CD86 on the surface of professional APCs, specifically dendritic cells.

Polypeptides of the present disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160) are individually tested for effector potential using a proprietary APC phenotyping assay previously developed at EpiVax (EpiVax, Providence, Rhode Island). Previously harvested and frozen PBMC are thawed and suspended in chRPMI by conventional means. HLA typing is conducted on small, extracted samples of cellular material, provided by EpiVax, by Hartford Hospital (Hartford, Connecticut). On assay day 0, 0.5x106 cells are extracted, screened for the presence of surface marker CD1 lc (a marker specific to dendritic cells) and are analyzed for the presence of surface markers HLA-DR and CD86 by flow cytometry. The remaining cells are plated (4.0x106 cell per ml in chRPMI plus 800ul media) and are stimulated (50 pg/niL) with one of the selected peptides or positive and negative controls including buffer only (negative control), Tregitope 167 (negative control for effector activity) (21st Century Biochemicals, Marlboro, MA), Flu-HA 306-318 (positive control) (21 ST Century Biochemicals, Marlboro, MA) and Ova 323-339 (negative control) (21st Century Biochemicals, Marlboro, MA). Plated cells are incubated for seven days at 37°C. On assay day 7, incubated cells are screened by flow cytometry for the presence of surface marker CD1 lc. CD1 lc positive cells are then analyzed for the presence of surface markers HLA-DR and CD86. The experimental peptides are tested in samples drawn from five different human donors.

Leukocyte Reduction Filters are obtained from the Rhode Island Blood Center (Providence, RI) to filter white blood cells from whole blood obtained from healthy donors. After the whole blood is run through the filters, the filters are flushed in the opposite direction to push collected white blood cells out of the filter. The white blood cells are isolated using a conventional Ficoll™ separation gradient (GE Healthcare). The collected white blood cells are thereafter frozen for future use. When needed for use in an assay, the frozen white blood cells are thawed using conventional methods. For the GvHD studies discussed below, PBMCs are obtained (e.g., from HemaCare, VanNuys, CA).

Exposure to the instantly disclosed polypeptides as disclosed herein on the phenotypes of dendritic cells is measured by multiple means. First, for each experimental condition, dot- plots, contrasting surface expression of CD1 lc and HLA-DR, is produced. Dot-plots of cells exposed to all control and experimental peptides are overlaid onto dot-plots produced from control cells exposed to only the culture media. The overlay provides an effective method to visually observe shifts in HLA-DR distribution between polypeptide stimulated, and unstimulated CD1 lc-high cells (data not shown). Observed shifts in the distribution of HLA- DR are reported as a qualitative measure. Next, the change in intensity of HLA-DR expression for the CD 1 lc-high segment of each dot-plot is calculated. Percent change in intensity of HLA- DR expression equals Mean Florescence Index (MFI) of HLA-DR expression for peptide exposed cells minus MFI of HLA-DR expression for media exposed cells divided by MFI of HLA-DR expression for media exposed cells, times 100 (HLA-DRMFIpeptide - HLA- DRMFImedia / HLA-DRMFImedia * 100). Next, the percent change in the percentage of HLA-DR-low cells present among the CDl lc high population is calculated for each peptide relative to media control. Percent change in the percentage of HLA-DR-low cells is calculated, and equals the percent of HLA-DR-low for peptide exposed cells minus the percent of HLA- DR-low for media exposed cells divided by percent of HLA-DR-low for media exposed cells times 100 (HLA-DR-low%peptide - HLA-DR-low%media / HLA-DR-low%media * 100). In this assay, a negative change in observed HLA-DR MFI and a positive change in percentage of HLA-DR-low cells present in the CD 1 lc-high population indicates reduced expression of HLA and a shift to a regulatory APC phenotype. In this assay, a positive change in observed HLA-DR MFI and a negative change in percentage of HLA-DR-low cells present in the CD 1 lc-high population indicates increased expression of HLA and a shift to an effector APC phenotype. A similar process will be used to assess the impact of the instantly-disclosed polypeptides exposure on surface expression of CD86, which is a costimulatory molecule known to promote T cell activation. First, for each experimental condition, dot plots contrasting surface expression of CD1 lc and CD86 are produced. Dot plots of cells exposed to all control and experimental Tregitopes are overlaid onto dots plots produced from control cells exposed to only the culture media. The overlay provides an effective method to visually observe shifts in CD86 distribution between polypeptide stimulated and un-stimulated CD1 lc- high cells. Observed shifts in the distribution of CD86 are reported as a qualitative measure. Next, the change in intensity of CD86-high expression for the CDl lc-high segment of each dot plot is calculated. Percent change in intensity of CD86-high expression equals Mean Florescence Index (MFI) of CD86 expression for peptide exposed cells minus MFI of CD86- high expression for media exposed cells divided by MFI of CD86 expression for media exposed cells, times 100 (CD86-highMFIpeptide - CD86-highMFImedia / CD86-highMFImedia * 100). Next, the percent change in the percentage of CD86-low cells present among the CD1 lc high population is calculated. Percent change in the percentage of CD86-high cells equals the percent of CD86-high for peptide exposed cells minus the percent of CD86-high for media exposed cells divided by percent of CD86-high for media exposed cells, times 100 (CD86- low%Ipeptide - CD86-low%media / CD86-low%media * 100). In this assay, a negative change in observed CD86 MFI and a positive change in percentage of CD86-low cells present in the CD1 lc-high population indicates reduced expression of CD86 and a shift to a regulatory APC phenotype. In this assay, a positive change in observed CD86 MFI and a negative change in percentage of CD86-low cells present in the CDl lc-high population indicates increased expression of CD86 and a shift to an effector APC phenotype.

Characterization of Peptide Exposed APC

Dendritic cell phenotyping assays are performed on the polypeptides of the instant disclosure according to the methods described previously.

Dot plots representing the surface expression of CD 11 vs HLA-DR will be analyzed on assay day 7 across the five donors in the presence of various peptide stimulants. In aspects, it is expected that upward movement of the CD1 lc+/HLA-DR+ population will apparent in the samples treated with the effector polypeptides of the instant disclosure (e.g., select polypeptides of the instant disclosure (e.g., but not limited to, a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160), including the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227)), as compared to media control indicating an acquired effector phenotype.

Dot plots representing the surface expression of CD1 lc vs CD86 will be analyzed on assay day 7 across the five donors in the presence of various peptide stimulants. It is expected that an increase in CD86-hi cells present in the samples treated with effector polypeptides of the instant disclosure (e.g., a peptide or polypeptide comprising, consisting, or consisting essentially of an amino acid sequence of SEQ ID NOS: 1-160 (and/or fragments or variants thereof), and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160), including the concatemeric peptides of SEQ ID NOS: 161-172 and 197-227), as compared to media control, which indicates a shift to the acquired effector phenotype.

Claims

CLAIMS What is claimed is:

1. A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

2. A polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

3. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

4. A polypeptide according to any one of claims 1-3, wherein said variant or fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160 retains MHC binding propensity and TCR specificity, and/or retains anti-RSV activity.

5. A polypeptide consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

6. A polypeptide consisting essentially of an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

7. A polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% homology to any one of SEQ ID NOS: 1-160, and fragments thereof, wherein said polypeptide retains MHC binding propensity and the same TCR specificity, and/or retains anti-RSV activity.

8 A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

9. A polypeptide according to claim 8, wherein said fragment or variant of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 161- 172 and 197-227 retains anti-RSV activity.

10. A nucleic acid encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160.

11. A nucleic acid encoding a polypeptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C-terminus of the polypeptide of SEQ ID NOS: 1-160.

12. A nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160, and/or fragments and variants thereof, and optionally 1 to 12 additional amino acids distributed in any ratio on the N terminus and/or C- terminus of the polypeptide of SEQ ID NOS: 1-160.

13. A nucleic acid encoding a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOS: 161-172 and 197-227and/or fragments and variants thereof.

14. A nucleic acid encoding a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227and fragments or variants thereof.

15. A nucleic acid encoding a polypeptide consisting essentially of a sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

16. A nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 161-172 and 197-227, and fragments or variants thereof.

17. A nucleic acid of any one of claims 10-12, wherein said fragment or variant of the nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-160 encodes a polypeptide that retains anti-RSV activity.

18. A nucleic acid of any one of claims 13-16, wherein said fragment or variant of the nucleic acid encoding a polypeptide comprising an amino acid selected from the group consisting of SEQ ID NOS: 161-172 and 197-227 encodes a polypeptide that retains anti-RSV activity.

19. A plasmid comprising a nucleic acid of any one of claims 10-18.

20. A vector comprising a nucleic acid according to any one of claims 10-18.

21. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-9 and a pharmaceutically-acceptable carrier and/or excipient.

22. A pharmaceutical composition comprising a nucleic acid according to any one of claims 10-18 and a pharmaceutically-acceptable carrier and/or excipient.

23. A pharmaceutical composition comprising a plasmid according to claim 19 and a pharmaceutically-acceptable carrier and/or excipient.

24. A pharmaceutical composition comprising a vector according to claim 20 and a pharmaceutically-acceptable carrier and/or excipient.

25. A vaccine comprising a polypeptide according to any one of claims 1-9 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

26. A vaccine comprising a nucleic acid according to any one of claims 10-18 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

27. A vaccine comprising a plasmid according to claim 19 and a pharmaceutically- acceptable excipient, carrier, and/or adjuvant.

28. A vaccine comprising a vector according to claim 20 and a pharmaceutically-acceptable excipient, carrier, and/or adjuvant.

29. A method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of claims 1-9.

30. A method for inducing immunity against a RSV infection (and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 10-18.

31. A method for inducing immunity against a RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to claim 19.

32. A method for inducing immunity RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to claim 20.

33. A method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 21-24.

34. A method for inducing immunity against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of claims 25- 28.

35. A method according to any one of claims 29-34, wherein the step of administration additionally includes administration of an RSV virus, wherein the virus is a live attenuated virus or inactivated virus.

36. A method for inducing an immune response against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more of a polypeptide according to any one of claims 1-9.

37. A method for inducing an immune response against against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 10-18.

38. A method for inducing an immune response against against RSV infection (and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to claim 19.

39. A method for inducing an immune response against against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to claim

20.

40. A method for inducing an immune response against RSV infection and/or related diseases caused by RSV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 21-24.

41. A method for inducing an immune response against a RSV infection and/or related diseases caused by RSV, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of claims 25-28.

42. A method according to any one of claims 36-41, wherein the step of administration additionally includes administration of a RSV virus, wherein the virus is a live attenuated virus or inactivated virus.

43. A chimeric or fusion polypeptide comprising a polypeptide of any one of claims 1-9, wherein said polypeptide is joined, linked, or inserted into a heterologous polypeptide.

44. A method for measuring a CMI response against RSV in a subject, said method comprising; collecting a whole blood sample from said subject wherein said whole blood sample comprises cells of the immune system which are capable of producing immune effector molecules following stimulation by an antigen, incubating a mixture comprising the whole blood sample, at least one polypeptide according to any one of claims 1-9, optionally an amount of an isolated simple sugar effective to enhance the stimulation by the antigen, and optionally heparin, and measuring the presence of, or elevation in, the level of an immune effector molecule wherein the presence or level of said immune effector molecule is indicative of the capacity of said subject to mount a cell-mediated immune response.

45. The method of claim 44 wherein the subject is a human.

46. The method of claim 44 wherein the whole blood is collected in a tube comprising at least one polypeptide according to any one of claims 1-9.

47. The method of claim 44 wherein the whole blood is collected in a tube comprising heparin.

48. The method of claim 46 wherein the tube comprises heparin.

49. The method of claim 44 wherein the whole blood sample is incubated with the at least one polypeptide of any one of claims 1-9 for from about 5 to about 50 hours.

50. The method of claim 44 wherein the immune effector molecule is a cytokine.

51. The method of claim 50 wherein the cytokine is IFN-g.

52. The method of claim 50 wherein the cytokine is GM-CSF.

53. The method of claim 50 wherein the cytokine is an interleukin.

54. The method of claim 50 wherein the cytokine is a TNF-a.

55. The method of claim 44 or 45 wherein the subject is infected by RSV.

56. The method of claim 44 wherein the immune cells are selected from NK cells, T-cells, B- cells, dendritic cells, macrophages or monocytes.

57. The method of claim 56 wherein the immune cells are T-cells.

58. The method of claim 44 wherein the simple sugar is dextrose.

59. The method of claim 44 wherein the immune effectors are detected with antibodies specific for same.

60. The method of claim 59 wherein the immune effectors are detected using ELISA.

61. The method of claim 59 wherein the immune effectors are detected using ELISpot.

62. An assay for identifying RSV-specific immediate effector T cells in a subject, comprising: (a) providing a sample from said subject containing T cells; (b) exposing said T cells to an immunogenic amount of at least one polypeptide according to any one of claims 1-9; and (c) prior to the generation of new immediate effector T cells in the sample, determining whether said T cells are activated by said polypeptide by measuring secretion of a cytokine from said T cells; wherein activation of said T cells identifies the presence of RSV -specific immediate effector T cells that were present in the original sample, in said subject.

63. The method of claim 62, wherein said T cells are peripheral blood mononuclear cells.

64. The method of claim 62, wherein the activation of said T cells is determined by measuring secretion of interferon-g from said T cells.

65. The method of claim 60, wherein said subject is known to be suffering, or to have suffered from, infection with RSV.

66. The method of claim 65, wherein said infection is monitored.

67. An assay for identifying RSV-specific immediate effector T cells in a subject, comprising: (a) providing a sample from said subject containing T cells; (b) exposing said T cells to an immunogenic amount of at least one polypeptide according to any one of claims 1-9; (c) incubating said T cells for a period of time which is not sufficient to effect differentiation of quiescent T cells to immediate effector T cells; and (d) determining whether said T cells are activated by said polypeptide by measuring secretion of a cytokine from said T cells, wherein activation of said T cells identifies the presence of RSV-specific immediate effector T cells in said subject.

68. The method of claim 62 or 67, wherein said T cells are exposed to said polypeptide at around 37° C.

69. The method of claim 67, wherein said incubation time is from 4 hours to 24 hours.

70. The method of claim 67, wherein said incubation time is from 6 hours to 16 hours.

71. A method of detecting an anti-RSV CD8+ and/or CD4+ T cell response comprising contacting a population of CD8+ and/or CD4+ T cells of a human individual with one or more polypeptides according to any one of claims -19, wherein one or more polypeptides may be substituted by an analogue which binds a T cell receptor that recognizes the peptide, and determining whether CD8+ and/or CD4+ T cells of the CD8+ and/or CD4+ T cell population recognize the peptide(s).

72. A method according to claim 71 wherein a peptide panel is employed, wherein said panel includes one or more polypeptide according to any one of claims 1-9, wherein one or more peptides may be substituted by said analogue.

73. A method according to claim 71 wherein any analogue which is used is (i) at least 70% homologous, preferably at least 80% homologous, more preferably at least 90% homologous, to the entire polypeptide, and/or (ii) has one or more deletions at the N-terminus and/or C- terminus in comparison to the polypeptide, and/or (iii) has one or more conservative substitutions compared to the polypeptide.

74. A method according to claim 71 in which the recognition of the polypeptide(s) by the CD8+ and/or CD4+ T cells is determined by measuring secretion of a cytokine from the CD8+ and/or CD4+ T cells.

75. A method according to claim 74 in which IFN-g secretion from the T cells is measured.

76. A method according to claim 75 in which IFN-g secretion from the CD8+ and/or CD4+ T cells is determined by allowing secreted IFN-g to bind an immobilized antibody specific to the cytokine and then determining the presence of antibody/cytokine complex.

77. A method according to claim 71 in which the CD8+ and/or CD4+ T cells are freshly isolated ex vivo cells from peripheral blood.

78. A method according to claim 71 in which CD8+ and/or CD4+ T cells are pre-cultured in vitro with the peptide(s).

79. A method according to claim 71 wherein the population of CD8+ and/or CD4+ T cells is from an individual to whom an anti- RSV vaccine has been administered.

80. A method according to claim 71 which is carried out in vitro.

81. A method of diagnosing infection in a human host by, or exposure of a human host to a RSV, which method comprises the steps of: (i) contacting a population off cells from the host with one or more polypeptides according to any one of claims 1-9; and (ii) determining in vitro whether the T cells of said T cell population show a recognition response to said polypeptide.

82. The method of claim 81, wherein the T cells are freshly isolated.

83. The method of claim 81, wherein the T cells are isolated from blood.

84. The method of claim 81, wherein the T cell population comprises CD4+ and/or CD8+ T cells.

85. The method of claim 81, wherein the host is a healthy human host who has been exposed to RSV.

86 A kit comprising one or more polypeptides according to any one of claims 1-9, wherein said one or more polypeptides may be substituted by an analogue which binds a T cell receptor which recognizes the polypeptide, and optionally a means to detect recognition of the polypeptide(s) by CD8+ and/or CD4+ T cells.

87. A kit according to claim 86 which includes an antibody to IFN-g.

88. A kit according to claim 86 wherein said antibody is immobilized on a solid support and which optionally also includes a means to detect any antibody/IFN-g complex.

89. A kit according to claim 86 which includes the means to detect recognition of the peptide(s) by CD8+ and/or CD4+ T cells.

90. A kit according to claim 88 which includes the means to detect any antibody/IFN-g complex.

91. A method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more of a polypeptide according to any one of claims 1-9.

92. A method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 10-18.

93. A method of preventing, treating, or ameliorating a disease by RS V infectionin a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plasmid according to claim 19.

94. A method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector according to claim 20.

95. A method of preventing, treating, or ameliorating a disease by RSV infectionin a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 21-24.

96. A method of preventing, treating, or ameliorating a disease by RSV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition according to any one of claims 25- 28.

97. A method according to any one of claims 36-41, wherein the step of administration additionally includes administration of a RSV virus, wherein the virus is a live attenuated virus or inactivated virus.