Compositions and methods for eliciting viral immune responses

Abstract

Disclosed herein are compositions and methods for eliciting an immune response to an antigen. More particularly, disclosed herein are compositions suitable for eliciting immune responses to a plurality of viruses, including Epstein-Ban vims (EBV), cytomegalovirus (CMV), polyoma BK vims (BKV), polyoma JC vims (JCV), and adenovims (Adv), and methods of using such compositions for treatment and prophylaxis.

Description

TITLE OF THE INVENTION“COMPOSITIONSAND METHODS FOR ELICITING VIRAL IMMUNE RESPONSES”RELATED APPLICATIONS

[0001] This application claims priority to Australian Provisional Application No. 2024903316 entitled “Compositions And Methods For Eliciting Viral Immune Responses” filed 14 October 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The technology described herein relates to compositions and methods for eliciting an immune response to an antigen. More particularly, the technology described herein relates to compositions suitable for eliciting immune responses to a plurality of viruses, and methods of using such compositions for treatment and prophylaxis.BACKGROUND TO THE INVENTION

[0003] Opportunistic infections remain a significant cause of morbidity and mortality in immunocompromised patients, despite the ongoing development of new therapies1. Much of this disease burden is seen in solid organ transplant (SOT) and haematopoietic stem cell transplant (HSCT) recipients. Infectious complications of bacterial, fungal or viral origin occur in the majority of these transplant recipients who, due to underlying drug-induced immunosuppression, graft-versus-host disease or inborn errors of immunity (lEls), are unable to generate efficient adaptive cellular immune responses associated with pathogen control23. Current standard-of-care typically relies upon antimicrobial therapies, including antibiotics, antifungals and anti-viral therapies. Some of these therapies are provided pre-emptively or prophylactically, particularly in settings of latent viral infection, to prevent disease following reactivation. Although reduction in immunosuppression is also used as a clinical management strategy to control persistent infection, this may increase the risk of graft rejection following SOT or graft-versus-host disease following HSCT. Despite these effective measures, infections are still associated with 17% of all SOT recipient deaths, 24% of deaths following allogeneic HSCT and 15% of deaths following autologous HSCT45. The risk of infection-related morbidity and mortality is further increased in patients who receive a T cell-depleted graft in an allogeneic haploidentical HSCT6.

[0004] In Australia, Lindsay et al7reported that between 2013 and 2018, 409 allogeneic HSCT recipients, including 45 children, had infection-related mortality, equating to 34% of all-cause mortality during that period. In children, 61 % of the infection-related deaths were caused by a viral infection or virus-related cancer, whereas in adults, this rate was 25%. In SOT recipients, up to 20-30% of infection-related mortality is associated with a viral infection4. Prevalent among these viral causes of mortality are the reactivation of latent viruses normally associated with lifelong asymptomatic infection, including human cytomegalovirus (CMV)8, Epstein-Barr virus (EBV)9, BK polyomavirus (BKV) and John Cunningham virus (JCV)10 11, and infection with respiratory viruses including adenoviruses (AdV)12. Anti-viral drug therapies are either not available for these viruses or become ineffective due to drugresistance, particularly with CMV infections. Diseases caused by latent viral infections are also prevalent in patients with inborn errors of immunity (IEI) and in other actively immunosuppressed patients, such as those with severe autoimmune disease.SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a combination of peptides comprising five or more of the T cell epitopes listed in Tables 1 -4, wherein the five or more T cell epitopes comprise T cell epitopes from at least five different viruses.

[0006] In some embodiments, the five or more T cell epitopes comprise both HLA class I- restricted T cell epitopes and HLA class-ll restricted T cell epitopes.

[0007] In some embodiments, the combination includes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 or 135 of the T cell epitopes listed in Tables 1-4.

[0008] In some embodiments, the combination includes all of the T cell epitopes listed in Tables 1-4.

[0009] In some embodiments, the combination of peptides comprises a T cell epitope from Epstein Barr virus (EBV). In some embodiments, the T cell epitope(s) from EBV comprises at least one LMP2a epitope. In some embodiments, the T cell epitope(s) from EBV comprises at least one LMP1 epitope. In some embodiments, the T cell epitope(s) from EBV comprises an EBNA4 epitope. In some embodiments, the T cell epitope(s) from EBV comprises an EBNA3 epitope. In some embodiments, the T cell epitope(s) from EBV comprises an EBNA6 epitope. In some embodiments, the T cell epitope(s) from EBV comprises an EBNA2 epitope. In some embodiments, the T cell epitope(s) from EBV comprises an EBNA1 epitope.

[0010] In some preferred embodiments, the T cell epitopes from EBV comprise one or more EBNA1 epitopes, one or more EBNA2 epitopes, one or more EBNA6 epitopes, one or more EBNA4 epitopes, one or more EBNA3 epitopes, one or more LMP1 epitopes, and one or more LMP2a epitopes.

[0011] In some embodiments, the combination of peptides may comprise a T cell epitope from cytomegalovirus (CMV). In some embodiments, the T cell epitope(s) from CMV comprises a pp50 epitope. In some embodiments, the T cell epitope(s) from CMV comprises a pp65 epitope. In some embodiments, the T cell epitope(s) from CMV comprises an IE-1 epitope. In some embodiments, the T cell epitope from CMV comprises a gB epitope. In some embodiments, the T cell epitope from CMV comprises a gH epitope.

[0012] In some preferred embodiments, the T cell epitopes from CMV comprise one or more pp50 epitopes, one or more pp65 epitopes, one or more IE-1 epitopes, one or more gB epitopes, and one or more gH epitopes.

[0013] In some embodiments, the combination of peptides may comprise a T cell epitope from polyoma BK virus (BKV) or polyoma JC virus (JCV). In some embodiments, the T cell epitope(s) from BKV or JCV comprises a large T antigen (LTA) epitope. In some embodiments, the T cell epitope(s) from BKV or JCV comprises a VP1 epitope. In some embodiments, the T cell epitope(s) from BKV or JCV comprises a STA epitope. In some embodiments, the T cell epitope(s) from BKV or JCV comprises a VP2 epitope.

[0014] In some preferred embodiments, the combination of peptides may comprise T cell epitopes from BKV or JCV may comprise one or more VP1 epitopes, one or more LTA epitopes, one or more STA epitopes, and one or more VP2 epitopes.

[0015] In some of the same embodiments and some other embodiments, the combination of peptides may comprise a T cell epitope from adenovirus (ADV). In some embodiments, the T cell epitope(s) from ADV comprises a hexon protein epitope. In some embodiments, the T cell epitope(s) from ADV comprises a DNA polymerase epitope.

[0016] In some preferred embodiments, the T cell epitopes from ADV comprise at least one hexon epitope and a DNA polymerase epitope.

[0017] In some preferred embodiments, the combination of peptides includes T cell epitopes from EBV, CMV, BKV, JCV, and ADV.

[0018] In some embodiments, the T cell epitopes present as one or more polyepitope proteins.

[0019] In another aspect, the present invention provides pharmaceutical compositions comprising the combination of peptides as described above and / or elsewhere herein and a pharmaceutically acceptable carrier, excipient, or diluent.

[0020] In yet another aspect, the present invention provides methods of treating or preventing a viral infection in a subject comprising administering too the subject the pharmaceutical composition described above and / or elsewhere herein.

[0021] In some embodiments, the viral infection is an EBV, CMV, BKV, JCV, or ADV infection.

[0022] In still yet another aspect, the present invention provides methods of treating or preventing cancer in a subject comprising administering to the subject a pharmaceutical composition as described above and / or elsewhere herein.

[0023] In yet another aspect, the present invention provides methods of generating a population of antigen presenting cells (APCs) that collectively present epitopes from at least five viruses comprising contacting a sample comprising APCs with the combination of peptides described above and / or elsewhere herein.

[0024] In some embodiments of this type, the sample is a PBMC sample.

[0025] In some embodiments, the APCs comprise B cells. By way of an illustrative example, the APCs may comprise antigen-presenting T-cells, dendritic cells, and / or artificial antigen- presenting cells (e.g., aK562 cells).

[0026] In yet another aspect, the present invention provides a sample comprising the APCs generated according to the method described above and / or elsewhere herein.

[0027] In still yet another aspect, the present invention provides a method of generating multivirus-specific cytotoxic T cells (CTLs), the method comprising,(a) generating APCs that present epitopes from multiple viruses according to the method described above and / or elsewhere herein; and(b) incubating the APCs presenting multiple viruses of step (a) with CTLs, thereby generating multivirus-specific CTLs.

[0028] In another aspect, the present invention provides samples comprising the multivirusspecific CTLs generated according to the methods described above and / or elsewhere herein.

[0029] In still yet another aspect, the present invention provides a composition comprising the CTLs described above and / or elsewhere herein, and a pharmaceutically acceptable carrier.

[0030] In yet another aspect, the present invention provides methods of treating or preventing a viral infection in a subject comprising administering to the subject the composition described above and / or elsewhere herein.

[0031] In some embodiments of this type, the viral infection is an EBV, CMV, BKV, JCV, or ADV infection.

[0032] In some embodiments, the subject is immunocompromised.

[0033] In some embodiments, the CTLs in the composition are allogeneic to the subject.

[0034] In some of the same embodiments and some other embodiments, the CTLs in the composition are stored in a cell bank prior to administration to the subject.

[0035] In some embodiments, the CTLs in the composition are autologous to the subject.

[0036] In yet another aspect, the present invention provides a method of treating or preventing cancer in a subject comprising administering to the subject the composition described above and / or elsewhere herein.

[0037] In some embodiments, the CTLs in the composition are allogeneic to the subject. In some embodiments of this type, the CTLs in the composition are stored in a cell bank prior to administration to the subject.

[0038] In some alternative embodiments, the CTLs in the composition are autologous to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 provides schematic of the process used for the manufacture and administration of T cell therapy.

[0040] Figure 2 shows (A) graphical representative flow cytometry analysis showing multiple virus T cell specificities in a single T cell product. (B) Virus specificities of batches of T cell product within the multi-virus-specific T cell bank.

[0041] Figure 3 shows the frequency of CD4+T cells (A) and CD8+T cells (B) in each product specific for epitopes from different viruses. Data represents the frequency of viable IFN-y producing T cells subtract a no peptide control.

[0042] Figure 4 shows the correlation between IFN-y production by virus-specific CD8+T cells (A-C) and CD4+T cells (D-F) and TNF (A, D), IL-2 (B, E) and CD107a (C, F). Correlation analysis was performed using Pearson correlation coefficients.

[0043] Figure 5 shows a graphical representation of the participant allocation, follow-up, and analysis shows viral disease characteristics. (A) The viral causes of disease in the patient cohort. (B) The types of viral disease detected in patients.

[0044] Figure 6 shows graphical representation of SAS patients. (A) The number of SAS requests we received between 2008 and 2023. (B) The proportions of adult and paediatric patients in this cohort. (C) The geographic location of origin of the patients. (D) Underlying diseases in the patients. (E) The types of organ transplants within the patient cohort.

[0045] Figure 7 shows viral disease characteristics. (A) The viral causes of disease in the patient cohort. (B) The types of viral disease detected in patients.

[0046] Figure 8 provides a graphical representation of the clinical outcome following adoptive T cell therapy. (A) The proportion of patients who were treated with the supplied T cell therapy. (B) The proportion of patients who displayed clinical improvement. (C) Representative analysis of viraemia relative to the administration of T cells for a patient with CMV complications. (D) Representative analysis of viraemia relative to the administration of T cells for a patient.

[0047] Figure 9 shows a graphical representation of the comparison of T cell expansions with Multivirus pepmix and AdMVPpoly. Panels on the left-hand side shows expansion of both CD4+ and CD8+ T cells with new MVP pepmix., while AdMVPpoly which includes only CD8+ T cell epitopes only expand CD8+ T cells.DETAILED DESCRIPTION OF THE INVENTIONGeneral

[0048] In certain aspects, provided herein are compositions and methods related to the generation and use of multivirus-specific cytotoxic T cells (CTLs) for adoptive immunotherapy. In certain embodiments, provided herein are compositions and methods related to peptides, including combinations of peptides, that comprise T cell epitopes from different that are recognized by CTLs andthat are useful in the prevention and / or treatment of viral infections and / or cancer. In certain embodiments, provided herein are populations of antigen-presenting cells (APCs) that collectively present five or more T cell epitopes, from different viruses. In some embodiments, provided herein are populations of CTLs that collectively comprise T cell receptors (TCRs) that recognize five or more T cell epitopes, from different viruses.Definitions

[0049] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0050] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0051] As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein and / or a CTL provided herein.

[0052] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally- occurring amino acids; analogues, derivatives and congeners thereof; amino acid analogues having variant side chains; and all stereoisomers of any of any of the foregoing.

[0053] The term “antigen” as used herein refers to a structure of a macromolecule, typically protein (with or without polysaccharides) or made of proteic composition comprising one or more hapten(s) and comprising T cell epitopes. The term “antigenic protein” as used herein refers to a protein comprising one or more T cell epitopes.

[0054] The term “autologous” as used herein refers to a state of being derived from the same individual. In the context of the present disclosure, “autologous” APCs used for treatment of a subject are APCs originally obtained from the subject.

[0055] The term “binding” or “interacting” refers to an association, which may be a stable association, between two molecules, e.g., between a TCR and a peptide MHC, due to, for example, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions under physiological conditions. A TCR “recognizes” a T cell epitope that it is capable of binding to when the epitope is presented on an appropriate MHC.

[0056] The term “biological sample,” “tissue sample,” or simply “sample” each refers to a collection of cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritonealfluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.

[0057] The term “derivative” as used herein with reference to the peptides of the invention refers to molecules which contain at least the peptide active portion (i.e. , capable of eliciting cytolytic CD8+ or CD4+ T cell activity) and, in addition thereto comprises a complementary portion which can have different purposes such as stabilising the peptides or altering the pharmacokinetic or pharmacodynamic properties of the peptide.

[0058] A “diluent” includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, particularly for injectable solutions.

[0059] The term “epitope” means a protein determinant capable of specific binding to an antibody or TCR. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.

[0060] The term “MHC” refers to “major histocompatibility antigen”. In humans, the MHC genes are known as HLA (“human leukocyte antigen”) genes. Although there is no consistently followed convention, some literature uses HLA to refer to HLA protein molecules, and MHC to refer to the genes encoding the HLA proteins. As such the terms “MHC” and “HLA” are equivalents when used herein. The HLA system in man has its equivalent in the mouse (i.e., the H2 system). The most intensely- studied HLA genes are the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1 , HLA-DPB1 , HLA-DQA1 , HLAs DQB1 , HLA-DRA, and HLA-DRB1. In humans, the MHC is divided into three regions: Class I, II, and III. The A, B, and C genes belong to MHC class I, whereas the six D genes belong to class II. MHC class I molecules are made of a single polymorphic chain containing three domains (alpha 1 , 2, and 3), which associates with beta-2 microglobulin at the cell surface. Class II molecules are made of two polymorphic chains, each containing two chains (alpha 1 and 2, and beta 1 and 2). Class I MHC molecules are expressed on virtually all nucleated cells.

[0061] Peptide fragments presented in the context of class I MHC molecules are recognised by CD8+ T lymphocytes (cytolytic T lymphocytes or CTLs). CD8+ T lymphocytes frequently mature into cytolytic effectors which can lyse cells bearing the stimulating antigen. Class II MHC molecules are expressed primarily on activated lymphocytes and antigen-presenting cells. CD4+ T lymphocytes (helper T lymphocytes or Th) are activated with recognition of a unique peptide fragment presented by a class II MHC molecule, usually found on an antigen-presenting cell like a macrophage or dendritic cell. CD4+ T lymphocytes proliferate and secrete cytokines such as IL-2, IFN-y and IL-4 that support antibody-mediated and cell mediated responses.

[0062] Functional HLAs are characterised by a deep binding groove to which endogenous as well as foreign, potentially antigenic peptides bind. The groove is further characterised by a well-defined shape and physico-chemical properties. HLA class I binding sites are closed, in that the peptide termini are pinned down into the ends of the groove. They are also involved in a network of hydrogen bonds with conserved HLA residues. In view of these restraints, the length of bound peptides is limited to 8-10 residues. However, it has been demonstrated that peptides of up to 12 amino acid residues are also capable of binding HLA class I. Comparison of the structures of different HLA complexes confirmed a general mode of binding wherein peptides adopt a relatively linear, extended conformation, or can involve central residues to bulge out of the groove.

[0063] In contrast to HLA class I binding sites, class II binding sites are open at both ends. This allows peptides to extend from the actual region of binding, thereby “hanging out” at both ends. Class II HLAs can therefore bind peptide ligands of variable length, ranging from 9 to more than 25 amino acid residues. Similar to HLA class I, the affinity of a class II ligand is determined by a “constant” and a “variable” component. The constant part again results from a network of hydrogen bonds formed between conserved residues in the HLA class II groove and the main-chain of a bound peptide. However, this hydrogen bond pattern is not confined to the N- and C-terminal residues of the peptide but distributed over the whole chain. The latter is important because it restricts the conformation of complexed peptides to a strictly linear mode of binding. This is common for all class II allotypes.

[0064] The second component determining the binding affinity of a peptide is variable due to certain positions of polymorphism within class II binding sites. Different allotypes form different complementary pockets within the groove, thereby accounting for subtype-dependent selection of peptides, or specificity. Importantly, the constraints on the amino acid residues held within class II pockets are in general “softer” than for class I. There is much more cross reactivity of peptides among different HLA class II allotypes. The sequence of the + / - 9 amino acids of an MHC class II T cell epitope that fit in the groove of the MHC II molecule are usually numbered P1 to P9. Additional amino acids N- terminal of the epitope are numbered P-1 , P-2 and so on, amino acids C-terminal of the epitope are numbered P+1 , P+2 and so on.

[0065] The term “peptide” as used herein refers to a molecule comprising an amino acid sequence of between 2 and 200 amino acids, connected by peptide bonds, but which can comprise non-amino acid structures. Peptides according to the invention can contain any of the conventional 20 amino acids or modified versions thereof, or can contain non-naturally occurring amino acids incorporated by chemical peptide synthesis or by chemical or enzymatic modification.

[0066] As used herein, the phrase “pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit risk ratio.

[0067] As used herein, the phrase “pharmaceutically-acceptable carrier''1means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars (such as lactose, glucose and sucrose); starches (such as corn starch and potato starch); cellulose, and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients (such as cocoa butter and suppository waxes); oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil); glycols (such as propylene glycol); polyols (such as glycerin, sorbitol, mannitol and polyethylene glycol); esters (such as ethyl oleate and ethyl laurate); agar; buffering agents (such as magnesium hydroxide and aluminium hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0068] The terms “polynucleotide”, and “nucleic acid'1are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labelling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0069] As used herein, a therapeutic that “prevents” a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0070] The term “sequence identity” of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. In particular, the sequence identity is from 70% to 80%, from 81 % to 85%, from 86% to 90%, from 91 % to 95%, from 96% to 100%, or 100%.

[0071] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

[0072] The phrases “therapeutically-effective amount” and “effective amount” as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit / risk ratio applicable to any medical treatment.

[0073] “Treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment (e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening).

[0074] As used herein, a “vaccine” is a composition used for vaccination, e.g., for prophylaxis or therapy, that comprises one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a combination of two or more peptides; two or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides (e.g., a minigene that encodes a polyepitopic peptide). The “one or more peptides” can include any whole unit integer from 1 to 150 (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more) peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells.

[0075] The term “vector” refers to the means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.

[0076] The nomenclature used to describe peptides or proteins follows the conventional practice wherein the amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the residue located at the amino terminal end of the epitope, orthe peptide or protein of which it may be a part.

[0077] In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acid residues having D-forms is represented by a lower case single letter or a lower case three letter symbol. However, when three letter symbols or full names are used without capitals, they may refer to L amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or “G”. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.) In addition to these symbols, “B” in the single letterabbreviations used herein designates a-amino butyric acid. In some embodiments, a-amino butyric acid may be interchanged with cysteine.Combinations of Peptides

[0078] In certain aspects, provided herein are combinations of peptides that comprise five or more T cell epitopes (e.g., five or more of the T cell epitopes listed in Table 1), wherein the five or more T cell epitopes comprise T cell epitopes from at least five different viruses (e.g., Epstein Barr virus (EBV), cytomegalovirus (CMV), polyoma BK virus (BKV), polyoma JC virus, and / or adenovirus (ADV)). In some embodiments, the T cell epitopes are HLA class l-restricted T cell epitopes. In some of the same embodiments and some different embodiments, the T cell epitopes are HLA class Il-restricted T cell epitopes. For example, the combination of peptides may comprise T cell epitopes from EBV, CMV, BKV, JCV, and ADV. In some embodiments, the combination of peptides may comprise T cell epitopes from 6, 7, 8, 9, 10 or more different viruses.

[0079] In some embodiments, the combination of T cell epitopes is provided as a polyepitope protein (i.e., a single chain of amino acid residues comprising multiple T cell epitopes not linked in nature). In some embodiments, the T cell epitopes in the polyepitope protein are connected via an amino acid linker. In some embodiments, the T cell epitopes in the polyepitope protein are directly linked without intervening amino acids.

[0080] In some embodiments, combination of T cell epitopes comprise 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 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 38, at least 39, or at least 40 T cell epitopes. In some embodiments, the T cell epitopes comprise a T cell epitope from EBV (e.g., an LMP2A epitope, an LMP1 epitope, an EBNA1 epitope, an EBNA3 epitope, an EBNA4 epitope, and / or an EBNA6 epitope). In some embodiments, the T cell epitopes comprise a T cell epitope from CMV (e.g., a pp50 epitope, a pp65 epitope, an IE-1 epitope, a gb epitope, and / or a gH epitope). In some embodiments, the T cell epitopes comprise a T cell epitope from BKPyV (e.g. a large T antigen epitope, a small T antigen epitope, a VP1 epitope, and / or a VP2 epitope) In some embodiments, the T cell epitopes comprise a T cell epitope from JCPyV (e.g. a large T antigen epitope, a small T antigen epitope, a VP1 epitope, and / or a VP2 epitope) In some embodiments, the T cell epitopes comprise a T cell epitope from ADV (e.g., a hexon protein epitope).

[0081] In some embodiments, the combination of peptides comprise at least one T cell epitope selected from each of Tables 1-4. In some preferred embodiments, combination of peptides comprises all ofthe epitopes listed in Tables 1-4 (i.e., each of peptides of SEQ ID NOs: 1 -146). In some embodiments, the combination of peptides provided herein comprises at least 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37 or 38 of the T cell epitopes listed in Tables 1-4.TABLE 1LIST OF EXEMPLARY EBV T CELL EPITOPESTABLE 2LIST OF EXEMPLARY CMV T CELL EPITOPESTABLE 3LIST OF EXEMPLARY BKPYV / JCPYV T CELL EPITOPESTABLE 4LIST OF EXEMPLARY ADV T CELL EPITOPES

[0082] The combination of peptides may include any ratio of the each peptides .In some preferred embodiments, each of the peptides in the combination are present at an equimolar concentration. That is, each individual peptide epitope is present in the combination at an equal concentration. In some embodiments, each particular peptide is present at substantially the same numbers in the combination as another particular peptide.

[0083] In some alternative embodiments, the peptide epitopes are present in the combination at a concentration that corresponds to the immunogenicity of the peptide. In other words, the peptides that elicit a greater or stronger (or more dominant) immune response may be present in the combination at a lower concentration than those peptide epitopes that are determined to elicit a lower or weaker immune response.

[0084] In some embodiments, the combination of peptides consists of two or more epitopes from the five different viruses listed in Tables 1 to 4. In some embodiments, the combinations of peptides provided herein include no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids in addition to the epitopes listed in Tables 1 to 4 (i.e., sequences set forth SEQ ID NOs: 1-146).

[0085] In some embodiments, the sequence of the T cell epitopes comprise an epitope sequence provided herein except for one or more (e.g., 1 , 2, 3, 4 or 5) conservative sequence modifications. As used herein, the term “conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on an MHC. Such conservative modifications include amino acid substitutions, additions (e.g., additions of amino acids to the N or C terminus of the peptide) and deletions (e.g., deletions of amino acids from the N or C terminus of the peptide). Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues of the peptides described herein can be replaced with other amino acid residues from the same side chain family and the altered peptide can be tested for retention of TCR binding using methods known in the art. Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0086] Also provided herein are chimeric or fusion proteins (e.g., polyepitope proteins). As used herein, a “chimeric protein” or “fusion protein” comprises a peptide(s) provided herein (e.g., a peptide comprising an epitope listed in Tables 1-4) linked to a distinct peptide to which it is not linked in nature. For example, the distinct peptide can be fused to the N-terminus or C-terminus of the peptide either directly, through a peptide bond, or indirectly through a chemical linker. In some embodiments, the peptide is linked to peptides comprising other T cell epitopes. For example, in some embodiments of this type, the chimeric or fusion protein may comprise a plurality of epitopes selected from Tables 1 - 4. In some embodiments, the peptide provided herein is linked to peptides comprising epitopes from other viral and or infectious diseases. In some embodiments, the polyepitope provided herein is linked to a peptide encoding a cancer-associated epitope.

[0087] A chimeric or fusion peptide provided herein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different peptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety.

[0088] In some embodiments, the combination or peptides, or polyepitopes proteins, as described above an / or elsewhere herein, are part of a vaccine.

[0089] In some embodiments, provided herein are populations of cells that collectively contain the combination of peptides described herein. The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine and / or human cells. In some embodiments, the cells are a mammalian cell. In some embodiments, the cells may be HEK 293 cells. In some embodiments, the cells are antigen-presenting cells (APC) (including but not limited to antigen-presenting T cells, dendritic cells, B cells, or aK562 cells).

[0090] In the present methods, the combination of peptides can be administered to the population of cells, for example, as peptides without delivery vehicle, in combination with a delivery reagent.

[0091] In some embodiments, any delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. In some embodiments of the methods described herein, liposomes are used to deliver the combination of peptides to a cell or subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al., 1980, Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Patent Nos. 4,235,871 , 4,501 ,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.Nucleic Acid Compositions

[0092] In some embodiments the peptides and / or polypeptides of the disclosure described herein are encoded by a nucleic acid composition. Nucleic acids (for example, but not limited to RNA (mRNA, etc.), DNA (cDNA), vectors, viruses, or hybrids thereof (all of which may be isolated, synthetic or recombinant)).

[0093] In some embodiments, the nucleic acid further comprises or is contained within an expression cassette, plasmid and expression vector or recombinant virus, wherein optionally the nucleic acid or expression cassette, plasmid, The expression vector or recombinant virus is contained within a cell (optionally a human or non-human cell), optionally transfected with the nucleic acid or expression cassette, plasmid, expression vector or recombinant virus, be introduced. The cells can be mammalian, bacterial, insect, or yeast cells. The nucleic acid molecules of the disclosure can be inserted into vectors and used, for example, as expression vectors or gene therapy vectors. Gene therapy vectors can be administered, for example, by intravenous injection, topical administration (U.S. Pat. No. 5,328,470) or stereotactic injection (Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91 (8):3054-7). , which are incorporated herein by reference in their entireties).

[0094] Similarly, the nucleic acid molecules of the disclosure can be inserted into a plasmid. Pharmaceutical formulations of gene therapy vectors can include the gene therapy vector in an acceptable diluent or can consist of a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector can be produced directly from a recombinant cell, e.g., a retroviral vector, the pharmaceutical formulation can contain one or more cells that produce the gene delivery system.

[0095] A nucleic acid of the present disclosure can be RNA (including but not limited to mRNA) or DNA (including but not limited to cDNA), and can be single- or double-stranded. Nucleicacids are typically RNA (including mRNA) or DNA. Nucleic acids can be isolated using techniques well known in the art, such as synthesis, or cloning, or amplification of sequences encoding immunogenic polypeptides; synthesis, or cloning, or amplification of sequences encoding cell membrane address sequences; and by ligation of sequences and their cloning / amplification in cells.

[0096] Nucleic acids provided herein (RNA, DNA, vectors, viruses, or even hybrids thereof) can be isolated from a variety of sources, genetically amplified, synthetically produced , and / or recombinantly expressed / produced. Recombinant polypeptides produced from these (their) nucleic acids can be individually isolated or cloned and tested for the desired activity. Any recombinant expression system can be used, including in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.

[0097] In some embodiments, the nucleic acids provided herein are preferably synthesized in vitro by well-known chemical synthesis techniques (e.g., Adams (1983) J. Am. Chem. Soc. 105:661 ; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radio. Biol. Med.19:373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth. (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22: 1859). In addition, techniques for manipulation of nucleic acids provided herein, such as subcloning, labelled probes (e.g., random primer labelling with Klenow polymerase, nick translation, amplification), sequencing, hybridization, etc., are scientifically proven and well described in the literature (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. John Wiley & Sons, Inc., New York (1997); ed., New York (1997)).

[0098] Nucleic acid molecules according to the present disclosure, whether of prokaryotic or eukaryotic origin, may be provided in the form of nucleic acid molecules per se plasmids, vectors; viruses or host cells. Vectors include expression vectors containing the nucleic acid molecules of the invention. Expression vectors can be prepared that are capable of expressing a polypeptide. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the nucleic acid molecule (e.g., RNA, including mRNA) is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If desired, the nucleic acid molecule (e.g., RNA, including mRNA) may be ligated to appropriate transcriptional and translational control nucleotide sequences recognized by the desired host.

[0099] A vector of the invention can include, for example, a transcription promoter and / or a transcription terminator, wherein the promoter is operably linked to the nucleic acid molecule and the nucleic acid molecule is operably linked to the transcription terminator. One or more peptides or polypeptides of the disclosure may be encoded by a single expression vector. Such nucleic acid molecules can serve as vehicles for the in vivo delivery of peptides / polypeptides to subjects in need thereof, e.g., in the form of RNA vaccines.

[0100] In embodiments, the vector may be a viral vector comprising a nucleic acid as defined above. Viral vectors can contain viruses of different types, e.g., swinepox, fowlpox, pseudorabies, Aujeszky’s virus, salmonella, vaccinia virus, BHV (bovine herpes virus), HVT (turkishherpes virus), adenovirus, TGEV (infectious gastroenteritis coronavirus), erythrovirus, and SIV (simian immunodeficiency virus). Other expression systems and vectors can also be used, such as plasmids that replicate and / or deposit in yeast cells.

[0101] In some embodiments, the compositions disclosed herein may be formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent, and (ii) one or more nucleic acid molecules (preferably, an mRNA molecule), collectively encoding at least five of the peptide epitopes in Tables 1 -4. In such a nanoparticle composition, the lipid composition disclosed herein can encapsulate the nucleic acid molecules (preferably, an mRNA molecule), encoding at least five peptide epitopes.

[0102] Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0103] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and / or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

[0104] In some embodiments, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG- modified lipid, a phospholipid and a structural lipid.

[0105] As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.

[0106] In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable lipid. As used herein, the term “ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1 , or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, andimidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively-charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. Ionizable lipids can also be the compounds disclosed in International Publication Nos.: WO2017 / 075531 , WO2015 / 199952, WO2013 / 086354, or WO2013 / 116126, or selected from formulae CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each of which is hereby incorporated by reference in its entirety for this purpose.

[0107] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule. The terms “partial negative charge” and “partial positive charge” are given its ordinary meaning in the art. A “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

[0108] In some embodiments, the ionizable lipid is an ionizable amino lipid, sometimes referred to in the art as an “ionizable cationic lipid”. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure. In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group.

[0109] Vaccines of the present disclosure are typically formulated into lipid nanoparticles. In some embodiments, the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)-modified lipid.

[0110] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20- 40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable amino lipid.

[0111] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% noncationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipid.

[0112] In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25- 35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40- 55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.

[0113] In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, or 15% PEG-modified lipid.

[0114] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.Cells

[0115] In some aspects, provided herein are APCs that present on MHC two or more T cell epitopes (e.g., two or more of the T cell epitopes listed in Table 1), wherein the two or more T cell epitopes comprise T cell epitopes from at least five different viruses (e.g., Epstein Barr virus (EBV), cytomegalovirus (CMV), polyoma BK virus (BKV), polyoma JC virus (JCV) and / or adenovirus (ADV)). In some embodiments, the MHC is a class I MHC molecule. In some embodiments, the MHC is a class II MHC molecule. In some embodiments, the class I MHC has an a-chain polypeptide that is HLA-A, HLA-B, and HLA-C. In some embodiments, the class II MHC has an a-chain polypeptide that is HLA- DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA. In some embodiments, the class II MHC has a p- chain polypeptide that is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB. In some preferred embodiments, the population of APC comprise both class I MHC molecules and class II MHC molecules. In some embodiments, APCs present at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 T cell epitopes (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145 or all of the epitopes listed in Tables 1-4).

[0116] In some embodiments the APCs are B cells, antigen presenting T-cells, dendritic cells, or artificial antigen-presenting cells (e.g., aK562 cells). Dendritic cells for use in the process may be prepared by taking PBMCs from a patient sample and adhering them to plastic. Generally, the monocyte population adheres and all other cells can be washed off. The adherent population is then differentiated with IL-4 and GM-CSF to produce monocyte derived dendritic cells. These cells may be matured by the addition of IL-1 p, IL-6, PGE-1 and TNF (which upregulates the important co-stimulatory molecules on the surface of the dendritic cell) and are then contacted with the peptide combinations described herein.

[0117] In some embodiments, the APC is an artificial antigen-presenting cell, such as an aK562 cell. In some embodiments, the artificial antigen-presenting cells are engineered to express CD80, CD83, 4-1 BBL, and / or CD86. Exemplary artificial antigen-presenting cells, including aK562 cells, are described U.S. Patent Pub. No. 2003 / 0147869, which is hereby incorporated by reference.

[0118] In certain aspects, provided herein are methods of generating populations of APCs that present the five or more of the T cell epitopes described above and / or elsewhere herein comprising contacting an APC with the combination or peptides or polyepitope protein described herein. In someembodiments, the APCs are irradiated. In some preferred embodiments, the combination of peptides comprise, consist, or consist essentially of, each of the epitopes listed in Tables 1-4.

[0119] In some aspects, provided herein are methods of generating, activating and / or inducing proliferation of T cells (e.g., CTLs) that recognize two or more T cell epitopes from at least five different viruses. In some embodiments, the CTLs are incubated in culture with the population of APCs provided herein (e.g., the population of APCs that collectively present each of the T cell epitopes listed in Tables 1-4).

[0120] In some embodiments, the sample containing T cells are incubated two or more times with the population of APCs describe above and / or elsewhere herein. In some embodiments, the T cells are incubated with the APCs in the presence of at least one cytokine. In some embodiments, the cytokine is IL-4, IL-7, and / or IL-15. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in U.S. Patent Publication No. 2015 / 0017723, which is hereby incorporated by reference.

[0121] In some aspects, provided herein is a population of CTLs collectively comprising T cell receptors that recognize five or more T cell epitopes (e.g., five or more of the T cell epitopes listed in Tables 1-4), wherein the five or more T cell epitopes comprise T cell epitopes from at least five different viruses (e.g., Epstein Barr virus (EBV), cytomegalovirus (CMV), polyoma BK virus (BKV), polyoma JC virus (JCV), and / or adenovirus (ADV)). In some embodiments, the epitopes are HLA class l-restricted T cell epitopes. In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize a T cell epitope from EBV (e.g., an LMP2a epitope, an EBNA3A epitope, an EBNA3B epitope, an EBNA1 epitope, a BZLF1 epitope, and / or BMLF1 epitope). In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize a T cell epitope from CMV (e.g., a pp50 epitope, a pp65 epitope, an IE-1 epitope, and / or a ppl50 epitope). In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize a T cell epitope from BKV (e.g., a large T antigen epitope and / or a VPI epitope). In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize a T cell epitope from ADV (e.g., a hexon protein epitope, a DNA polymerase epitope, and / or DNA binding protein epitope). In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize T cell epitopes from at least three or four different viruses (e.g., Epstein Barr Virus (EBV), cytomegalovirus (CMV), polyoma BK virus (BKV), and adenovirus (ADV)). In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize T cell epitopes from any combination of the aforementioned viruses and / or from other viruses. In some embodiments, the population of CTLs collectively comprise T cell receptors that recognize at least , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 T cell epitopes T cell epitopes (e.g., at least , 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15, 120, 125, 130, 135, 140, 145 or all of the T cell epitopes of the epitopes listed in Tables 1-4).Pharmaceutical compositions

[0122] In some aspects, provided herein are compositions (for example, pharmaceutical compositions) comprising combinations of peptides as described above and / or elsewhere herein, ormultivirus-specific CTLs or APCs provided herein; and a pharmaceutically acceptable carrier, excipient or diluent. In some embodiments, such compositions are used in adoptive immunotherapy to boost multivirus-specific immunity in a subject by administering to the subject an effective amount of the composition. In some embodiments, the multivirus-specific CTLs and / or APCs are not autologous to the subject. In some embodiments, the T cells and / or APCs are autologous to the subject. In some embodiments, the T cells and / or APCs are stored in a cell bank before they are administered to the subject.

[0123] In some aspects, provided herein are compositions (e.g., a pharmaceutical composition), containing combination of peptides, and / or a polyepitope protein, and / or a CTL, and / or an APC provided herein. In some embodiments, the composition includes a combination of multiple (e.g., two or more) agents provided herein.

[0124] In some embodiments, the pharmaceutical compositions provided herein are vaccine compositions. In some embodiments, the pharmaceutical composition further comprises an adjuvant. As used herein, the term “adjuvant” broadly refers to an agent that affects an immunological or physiological response in a subject. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumour, help absorb an APC antigen, activate macrophages and lymphocytes, and / or support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent. Examples of adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, a-GalCer, aluminium phosphate, aluminium hydroxide (for example, Alhydrogel), calcium phosphate, p-Glucan peptide, CpG oligonucleotides (for example, CpG 1018), GPI-0100, monophophoryl lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D- isoglutamine, Pam3CSK4, quil A, and trehalose dimycolate.

[0125] In some embodiments, the combination of peptides are mixed with an adjuvant containing a stabilizing detergent, a micelle-forming agent, and / or an oil. In one embodiment, the adjuvant is MONTANIDE® ISA 51 VG, and may further include granulocyte macrophage colony stimulating factor (GM-CSF). Suitable stabilizing detergents, micelle-forming agents, and oils are detailed in U.S. Patent Nos. 5,585,103, 5,709,860, 5,270,202, and 5,695,770. A stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion. Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-1 ,2- ethanediyl; manufactured by ICI Americas, Wilmington, DE), TWEEN 40™, TWEEN 20™, TWEEN 60™, ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micellelike structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response. Examples of such agents include polymer surfactants described by BASF Wyandotte publications, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., 129: 1244, 1981 , PLURONIC™ L62LF, L101 , and L64, PEG1000, and TETRONIC™ 1501 , 150R1 , 701 , 901 , 1301 , and 130R1 . The chemical structures of such agents are well known in the art. In some embodiments, the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example between 0.5 and 10%, or in an amount between 1.25 and 5%.

[0126] The oil included in the composition is chosen to promote the retention of the antigen in oil-in- water emulsion (i.e., to provide a vehicle for the desired antigen) and may have a melting temperature of less than 65°C such that an emulsion is formed either at room temperature (about 20°C to 25°C), or once the temperature of the emulsion is brought down to room temperature.

[0127] Examples of such oils include squalene, squalane, EICOSANE™, tetratetracontane, glycerol, and peanut oil or other vegetable oils. In one specific, non-limiting example, the oil is provided in an amount between 1 and 10%, or between 2.5 and 5%. The oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse effects, such as granulomas, are evident upon use of the oil.

[0128] An adjuvant can be included in the composition. In one embodiment, the adjuvant is MONTANIDE® ISA 51 VG plus GM-CSF. In other embodiments, the adjuvant is a mixture of stabilizing detergents, micelle-forming agent, and oil available underthe name PROVAX® (Biogen Idee, San Diego, CA). An adjuvant can also be an immunostimulatory nucleic acid, such as a nucleic acid including a CpG motif.

[0129] In another embodiment, the combination of peptides composition includes one or more nucleic acids encoding one or more immunogenic combination of peptides. A therapeutically effective amount of the nucleic acid(s) encoding the combination of peptide(s) can be administered to a subject in order to generate an immune response. In one specific, non-limiting example, a therapeutically effective amount of the nucleic acid(s) is administered to a subject to treat a virus- associated concer, or any other tumour that expresses combination of the viral peptides. One approach to administration of nucleic acids to a subject is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequences encoding an combinations of peptide can be placed under the control of a promoter to increase expression of the molecule.

[0130] Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune- stimulating constructs, or ISCOMS™, negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and QUIL A™ (saponin). Protective immunity has been generated in a variety of experimental models of infection,including toxoplasmosis and Epstein-Barr virus-induced tumours, using ISCOMS™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 pg encapsulated in ISCOMS™ have been found to produce MHC class I mediated CTL responses(Takahashi et ah, Nature 344:873, 1990).

[0131] In another approach to using nucleic acids for immunization, immunogenic combinations of peptides can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus, herpes virus, retrovirus, or other viral vectors can be used to express the peptide, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover’ Nature 351 :456- 460, 1991).

[0132] In some embodiments, one or more nucleic acids encoding one or more immunogenic combinations of peptides are introduced directly into cells. For example, the nucleic acid(s) can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites, including tissues in proximity to metastases. Dosages for injection are usually around 0.5 g / kg to about 50 mg / kg, and typically are about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0133] The compositions (e.g., combination of peptides, a population of APCs loaded with combinations of peptides, or nucleic acids or vectors encoding the combination of peptides) can be administered for therapeutic treatments. In therapeutic applications, a therapeutically effective amount of the composition is administered to a subject suffering from a disease, such as prostate cancer, mesothelioma or breast cancer, or any other cancer that is associated with a viral infetion. Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the composition is administered in multiple doses, such as two, three, four, five, six, seven or eight doses. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject. Systemic or local administration can be utilized. For each dose, the composition can be administered using a single injection (a single site of injection) or can be administered using two or more injections (two or more sites of injection). In particular examples, the combination of peptide compositions are administered using two injections (such as one in each arm). In other cases, particularly if isolated combination of peptides are being administered (i.e. administered in the absence of APCs), one site per peptide may be required.

[0134] In some embodiments, any of the immunotherapies discussed above is augmented by administering a cytokine, such as IL-2, IL-3, IL-6, IL- 10, IL-12, IL-15, GM-CSF, or interferons, or a combination of two or more cytokines, such as 2, 3, 4, 5, 6, 7 or more cytokines.

[0135] Administration of the immunogenic combinations of peptide compositions disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments(such as surgical resection of a tumour). Any suitable anti-cancer agent can be administered in combination with the compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, cytotoxic chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. antiandrogens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy, antibodies that specifically target cancer cells, or antibodies to other immune modulating proteins such as CTLA-4, PD-1 , PD-L1 or TGF-p (transforming growth factor-beta).

[0136] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0137] Non-limiting examples of antimetabolites include folic acid analogues (such as methotrexate), pyrimidine analogues (such as 5-FU or cytarabine), and purine analogues, such as mercaptopurine or thioguanine.

[0138] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0139] Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

[0140] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone).

[0141] Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin®, Irinotecan (Camptosar, CPT- 11), Leustatin, Navelbine, Rituxan STI-571 , Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0142] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), IFN-y (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immuneglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, TNF (tumour necrosis factor; Genentech) and anti-CTLA-4 (ipilimumab, Bristol-Myers Squibb).

[0143] Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumour site to help eradicate the tumour or shrink it prior to surgical resection.

[0144] In particular embodiments, a subject having prostate cancer is administered a combination of peptides composition disclosed herein in combination with radiation therapy, brachytherapy, or cryotherapy. In other specific embodiments, a subject having prostate cancer, such as metastatic castration-resistant prostate cancer, is administered a combination of peptides composition disclosed herein in combination with chemotherapy.

[0145] In other embodiments, a subject with a virus-associated cancer, such as a CMV- associated cancer or an EBV-associated cancer, is administered a combination of peptide composition disclosed herein in combination with an agent that targets negative regulation of the immune system, such as anti-CTLA4, anti-PD-1 , anti-PD-L1 or anti-TGFp.Therapeutic Methods

[0146] In certain aspects, provided herein are methods of treating or preventing a viral infection (e.g., an EBV, CMV, BKV, JCV, or ADV infection) and / or a cancer in a subject comprising administering to the subject a pharmaceutical composition as described above and / or elsewhere herein.

[0147] In some embodiments, provided herein is a method of or preventing or treating a viral infection in a subject (e.g., an EBV, CMV, BKV, JCV, or ADV infection). In some embodiments, the subject to be treated is immunocompromised. For example, in some embodiments, the subject has a T cell deficiency. In some embodiments, the subject has leukemia, lymphoma or multiple myeloma. In some embodiments, the subject is infected with HIV and / or has AIDS. In some embodiments, the subject has undergone a tissue, organ and / or bone marrow transplant. In some embodiments, the subject is being administered immunosuppressive drugs. In some embodiments, the subject has undergone and / or is undergoing chemotherapy. In some embodiments, the subject has undergone and / or is undergoing radiation therapy.

[0148] In some embodiments, the subject has cancer. In some embodiments, the methods described herein may be used to treat any cancerous or pre-cancerous tumour. In some embodiments, the cancer expresses one or more of the T cell epitopes provided herein (e.g., the T cell epitopes listed in Tables 1-4). In some embodiments, the cancer includes a solid tumour. Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastro-intestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cellcarcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary Paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumour; malignant thecoma; malignant granulosa cell tumour; and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumour; malignant lipid cell tumour; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumour; mullerian mixed u; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant Brenner tumour; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing sarcoma; malignant odontogenic tumour; ameloblastic fibro-odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumour; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0149] In some embodiments, the subject is also administered an immune checkpoint inhibitor. Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1 , PD-L1 , PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 and VISTA. Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI- Al l 10, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.

[0150] In some embodiments, a composition provided herein (e.g., a vaccine composition provided herein) is administered prophylactically to prevent cancer and / or a viral infection. In some embodiments, the vaccine is administered to inhibit tumour cell expansion. The vaccine may be administered prior to or after the detection of cancer cells or virally infected cells in a patient. Inhibition of tumour cell expansion is understood to refer to preventing, stopping, slowing the growth, or killing of tumour cells. In some embodiments, after administration of a vaccine comprising nucleic acid vectors, recombinant adenoviruses, polyepitopes, CTLs or APCs described herein, a proinflammatory response is induced. The proinflammatory immune response comprises production of proinflammatory cytokines and / or chemokines, for example, interferon gamma (IFN-y) and / or interleukin 2 (IL-2). Proinflammatory cytokines and chemokines are well known in the art.EXPERIMENTALT cell Therapy Manufacturing and Characterisation

[0151] Twenty batches of allogenic multi-virus-specific T cells have been manufactured from a single blood collection (350-400 mL) from 20 healthy volunteers, producing an average of 1 15 vials (range 23-150 vials) of T cell therapy product (4 x 107T cells / vial) (Figure 4). The antigen specificity of each batch of T cells has been assessed using intracellular cytokine assays. Products were then selected for use based on HLA matching between patient and product, and the presence of the appropriate virus-specific T cells. Representative data from one of the multi-virus-specific T cell products with reactivity against AdV, BKV / JCV, CMV and EBV are shown in Figure 2A. Of the 20 T cell products, five showed reactivity against all four viruses, 11 against three viruses, while the remaining four products showed reactivity against one or two viruses (Figure 2B, Figure 3). Antigen-specific T cells in all products demonstrated a consistent functional profile, characterised by the correlative production of IFN-y with TNF, IL-2 and CD107a (Figure 4)

[0152] The initial approach undertaken to deliver T cells for compassionate use was through the generation of either autologous or allogeneic single-use T cell products. Between 2008 and 2019, 19 patients received T cells containing single-virus-specific CD8+T cells, CD4+T cells or both(Table 5). These T cell products were designed to provide CD4+and CD8+T cell specificity for multiple viruses (AdV, BKV / JCV, CMV and EBV) in a single batch (Table 6).TABLE 5SINGLE-USE T CELL THERAPIES GENERATED FOR COMPASSIONATE USETABLE 6BANKED ALLOGENEIC MULTIVIRUS-SPECIFIC T CELLSPatient Characteristics

[0153] From 2008 to the end of 2023, QIMR Berghofer Medical Research Institute has received 115 requests to provide T cells for compassionate use, with a significant increase in requests over the past 2 years (Figure 5 and 6A). These requests have come from clinical centres treating adult and paediatric patients (Figure 6B), predominantly in Queensland (QLD) and Victoria (VIC) (Figure 6C). More than half (54%) of these requests have been for the treatment of patients with underlying haematological malignancies (Figure 6D), with viral complications following allogeneic HSCT (Figure 5E). Of the HSCT recipients 15% had an underlying IEI that would have increased their susceptibility to opportunistic infections; the majority of these patients were paediatric. Paediatric patients with an IEI are often treated using T cell-depleted haploidentical HSCT, which renders them at increased susceptibility to post-transplant infectious complications. Requests have also been made for access to treatment for SOT recipients (n = 25) who are heavily immunosuppressed to prevent organ rejection and are unresponsive to standard-of-care treatment options, including anti-viral therapies and / or reduction of immunosuppression. The present inventors also provide T cell therapies for patients with EBV-associated head and neck cancers (n = 2) and autoimmune diseases (n = 6) that have been linked to EBV (Figure 4).

[0154] Most patients had complications associated with a single virus (Figure 7A), the most prevalent of which was CMV. However, several were experiencing post-transplant complications with multiple viruses. The most prevalent clinical indication associated with SAS requests was persistent viral reactivation (Figure 7B). Patients also presented with a variety of end-organ diseases in the lung, gut, liver, kidney, eyes or brain. BKV-specific T cell requests were for nephropathy (including nephritis) in renal transplant patients and haemorrhagic cystitis in HSCT patients, while JCV-specific T cell requests were for the treatment of progressive multifocal leukoencephalopathy (Figure 7B). The majority of EBV-associated lymphomas were post-transplant; however, 10 requests were received for non-transplant patients.Retrospective analysis of clinical outcomes following adoptive T cell therapy

[0155] From the 115 requests received, 78 patients had been treated with T cell therapy as of December 2023. The remaining patients did not receive therapy due to either rapid clinical deterioration / death (n=16), failure in manufacturing process of autologous T cell therapy (n = 2), access to T cell therapy from another source (n = 2) or clinical improvement prior to treatment (n = 8) (Figures 5 and 8A). We have previously reported on the clinical outcomes of some of these patients13 17. Response to T cell therapy was defined by resolution of persistent viraemia as assessed by monitoring of plasma viral DNA load, and improvement in clinical symptoms and signs from different disease manifestations assessed according to standard clinical practice. Of the 71 patients who had active disease at the time of T cell infusion, 46 (65%) showed clinical improvement, based on viral load reduction or disease improvement (Figure 7B). Representative analyses of viral loads pre and post T cell therapy from two of these patients with CMV viraemia and AdV viraemia are shown in Figures 8C and 8D respectively. Table 3 summarises retrospective clinical responses of adult and paediatric patients following adoptive T cell therapy stratified by viral infection and transplant status. It should be noted that many patients remained on standard-of-care treatment during their course of T cell therapy, e.g. ganciclovir or foscarnet to treat CMV. In addition, some patients showed evidence of a virological response to T cell therapy without an improvement in end-organ disease. Seven patients who were treated prophylactically due to a high risk of viral disease (due to either recurrent viral reactivation / disease, donor / recipient serostatus and underlying immunosuppression / immunodeficiency) also remained disease free following T cell therapy. We were unable to obtain clinical outcome data for one patient, while the remaining 23 patients who had late stage progressive disease and / or high levels of viral load showed no evidence of response to treatment. These results show that the compassionate use of virus-specific T cell therapy can provide therapeutic benefit, especially for patients with viral infections post-transplant, who have failed to respond to first- line therapy or are unable to tolerate standard therapies.TABLE 7SUMMARY OF CLINICAL RESPONSES TO ADOPTIVE T CELL THERAPYDiscussion

[0156] The increase in the number of solid organ and bone marrow transplants over the last three decades has had a significant impact on the lives of many people worldwide, who otherwise would have succumbed to their disease. Despite the remarkable success of transplanting tissue between genetically distinct individuals, the need to suppress or deplete the recipient’s immune system to prevent graft rejection leaves them highly susceptible to a variety of opportunistic infections1 3. While the development of anti-viral drugs, particularly against CMV, has had a dramatic impact on mortality associated with viral disease following transplantation, many patients still succumb to these opportunistic infections or develop toxicities from anti-viral drugs. There remains a lack of effective treatments for EBV, AdV and BKV / JCV. Virus-specific adoptive T cell immunotherapy has provided a last line of treatment for many patients without any remaining viable options. Although generally unproven in terms of effectiveness in randomised clinical studies, evidence over the last three decades has demonstrated real-world effectiveness of this approach18. Our own observations over the last 15 years provides additional support for the use of virus-specific T cell therapy to reduce the morbidity and mortality associated with viral infections in immunocompromised patients19.

[0157] The first evidence of virus-specific adoptive T cell therapy in transplant patients was provided in 1992 by Riddell and colleagues, who demonstrated that CMV-specific T cells could be generated from HSCT donors and administered to transplant recipients20. This was closely followed by work from Rooney and colleagues, who demonstrated the use of EBV-specific T cell therapy in HSCT recipients21. Due to the presence of genetic modification that allows long-term tracking, these EBV- specific T cells have since been shown to survive for more than two decades in the recipients2223. Sincethese early observations, multiple studies have reported the use of donor-derived virus-specific T cell therapy in more than 150 HSCT recipients12’24-26.

[0158] One limitation of the donor-derived approach is the need for the HSCT donor to have immunological memory to the virus in question. This approach is also not applicable to SOT patients when donor T cells are not available. We have shown that autologous T cell therapy can be generated from both SOT and HSCT recipients, demonstrating a good safety profile and clinical efficacy18. The present inventors used this autologous approach in several patients treated compassionately in the current report. However, this approach is not feasible for all patients, predominantly due to difficulties in manufacturing cells from heavily immunocompromised patients. To overcome this limitation, donors were selected to provide broad HLA coverage, allowing matching between the T cell donor and the patient with viral disease. Based on this approach, the allogeneic EBV-specific T cell therapy tabelecleucel (Ebvallo) was recently approved to treat EBV-associated PTLD in Europe2930. It is unclear if and when this will be available for patients in other regions of the world. This allogeneic approach has been extended to CMV and other virus-associated diseases in adult and paediatric settings, although most have not moved beyond early phase clinical studies31 33. We have also treated a number of patients included in the current report using allogeneic T cell therapy specific for a single virus.

[0159] Despite emerging data showing potential for allogeneic virus-specific T cell banks, it is challenging to generate a bank with broad HLA coverage that can target all known transplant- associated infectious complications, particularly those that are rare. To overcome this, Leen et al. pioneered the development of T cell therapy products containing multiple virus specificities34. Most patients in the current report were treated using T cells generated using a multi-virus-targeted approach. While EBV and / or CMV-specific T cells were typically dominant in these products, the majority also contained BKV / JCV- and AdV-specific CD4+T cells. Importantly, the present inventors didn’t see any obvious evidence of reduced efficacy against these viruses, indicating that the use of a multivirus- targeted approach does not impact the potency of virus-specific T cell therapy.

[0160] Based on this retrospective analysis, we can draw two key conclusions. First, adoptive T cell therapy (either autologous or allogeneic) was generally safe, with no reported serious adverse reactions. Importantly, there was no evidence of precipitation of graft rejection or graft-versus- host disease. Second, virus-specific T cell therapy seems to be more effective in patients who had less disease burden and were treated early rather than at a late stage of clinical symptoms, evidenced by 24 patients who had late-stage disease and did not respond to adoptive T cell therapy. Overall, the compassionate use of virus-specific T cell therapies provides an opportunity to deliver therapies to severely immunocompromised patients who otherwise have limited options.Comparison of new Multivirus Pepmix with Adenovirus Multivirus Polyepitope.

[0161] Comparative analysis was undertaken to compare HLA-coverage in the MVP pepmix with an Ad-MVPoly generated for a similar purpose. Table 8 demonstrates an increase HLA- coverage worldwide for the MVP pepmix compared to the Ad-MVPpoly. Figure 9A shows the frequency of virus-specific CD8+ T cells generated using the MVP pepmix peptide pools compared to the virus-specific CD8+ T cells generated using the AdMVPpoly in Figure 9B. Figure 9C shows the frequency of virus-specific CD4+ T cells generated using the MVP pepmix peptide pools. The AdMVPpoly does not CD4+ T cell epitopes.TABLE 8COMPARISON OF HLA COVERAGE OF ADMVPPOLY AND MVP PEPMIXMaterials & MethodsManufacture of Virus-Specific T cells

[0162] For autologous T cell therapy manufacture, peripheral blood mononuclear cells (PBMC) were isolated from patients then stimulated with clinical-grade custom HLA class I and class Il-restricted peptide epitopes from CMV, BKV, JCV or AdV. Autologous EBV-specific T cells were manufactured using the AdE1-LMpoly vector as previously described39. For allogeneic virus-specific T cell therapy manufacture, PBMC were isolated from healthy donor blood then cryopreserved prior to use. PBMC were thawed and stimulated with HLA class I and class Il-restricted peptide epitopes from CMV, BKV / JCV, EBV and AdV (Table 1-4: Figure 1). T cells were then cultured in RPMI 1640 medium supplemented with 5% human AB serum and recombinant interleukin 2 (IL-2; 200 lU / mL) for 14 to 17 days. T cell products were cryopreserved in Albumex 4 (CSL Behring) containing 10% dimethyl sulfoxide (WAK-Chemie Medical) (Figure 1). Antigen specificity and HLA restriction of T cell therapy batches were determined using intracellular cytokine assays as outlined below.Regulatory and Ethical Approvals

[0163] Compassionate access to autologous or allogeneic virus-specific T cell therapies was provided through the TGA SAS. Requests for access to T cell therapy were made to QIMR Berghofer Medical Research Institute by clinical centres across Australia. Each request was reviewed by a panel of three independent clinical experts and based on their advice regarding the appropriateness of T cell therapy for the case, the supply was approved by QIMR Berghofer. An SAS Category A notification form or SAS Category B application was also submitted to the TGA by the treating physician and informed consent was obtained from the patient or their parent / guardian. For each custom-manufactured batch of allogeneic T cell therapy from a donor selected by the requesting clinician, donor informed consent was also obtained. For SAS Category B applications, approval was obtained from the TGA prior to T cell manufacture and / or treatment. Access to T cell therapies was provided according to the TGA guidelines and did not require ethics committee approval.

[0164] To generate a bank of “off-the-shelf’ allogeneic virus-specific T cell therapies, healthy blood donors were recruited through the Australian Bone Marrow Donor Registry. Written informed consent was provided prior to participation. Approval for the cell therapy manufacturing study was obtained from the Australian Red Cross Lifeblood Human Research Ethics Committee (HREC) and the QIMR Berghofer Medical Research Institute Human Research Ethics Committee. Additional products were generated from blood donated by healthy adults participating in a clinical trial (ACTRN12620000141943). These products were added to the cell bank if excess material was available and the donor provided extended consent for use of their donated material to treat futurepatients. Ethics approval for the clinical trial was obtained through Children’s Health Queensland HREC Committee and the QIMR Berghofer Medical Research Institute HREC.Intracellular Cytokine Assays

[0165] Intracellular cytokine assays were used to define the specificity and HLA restriction of T cells generated for adoptive T cell therapy, in a two-step process. To define virus-specific reactivity, T cell therapy products were stimulated with pools of virus-specific peptides, then CD4+T cells and CD8+T cells were assessed as previously described19for the expression of interferon gamma (IFN-y), tumour necrosis factor (TNF), IL-2 and / or the mobilisation of CD107a, a surrogate for T cell-mediated cytotoxicity. Products containing virus-specific CD4+T cells and / or CD8+T cells were then assessed for reactivity to individual HLA-matched peptide epitopes using an IFN-y intracellular cytokine assay. Flow cytometric acquisition was performed using a BD LSR Fortessa cell analyser with FACSDiva software (BD Biosciences). T cell reactivity to a virus-specific peptide pool or HLA-restricted peptide epitope was considered positive if the response was greater than the mean plus three standard deviations of the response of T cells cultured in the absence of stimulus. Post-acquisition analysis was also performed using FlowJo software (FlowJo).Assessment of T cell Alloreactivity

[0166] Potential alloreactivity of T cells manufactured for allogeneic use was assessed using a panel of K562 cell lines, each transfected with a single HLA class I allele. Allogeneic T cell therapy products were incubated with each K562-HLA class I cell line at a responder-to-stimulator ratio of 10:1 and assessed for intracellular IFN-y production. Products were considered alloreactive against an HLA class I allele if >1 % of lymphocytes produced IFN-y following subtraction of the background response generated against untransfected K56240.Selection and Administration of Banked Allogeneic T cells

[0167] To select matching T cell products from the bank of allogeneic virus-specific T cells for compassionate use in patients, an in-house computer-based algorithm was used. This algorithm allowed identification of appropriate T cell products based on HLA matching (minimum of two HLA class I and / or class II alleles matched with the recipient), antigen specificity, strength of anti-viral reactivity and alloreactivity. Between four and twelve vials of T cells were provided to the site for each patient; in most cases, six vials were provided. For each infusion, one vial of T cell product was thawed and diluted to 20 mL in clinical-grade normal saline, then intravenously infused over 5-10 minutes. The dose provided to adult patients was 4 x 107cells / infusion, and the dose for paediatric patients was 2 x 107cells / m2body surface area. Infusions were generally administered fortnightly, or weekly at the discretion of the clinical team.Safety and Clinical Assessments

[0168] All patients were monitored for any evidence of adverse reactions for up to 4 hours following each infusion of T cell therapy. In addition, as part of standard clinical care, viral loadmeasurements, end-organ disease assessments and / or clinical symptoms were recorded. These records were used in the current retrospective analysis post T cell therapy, to assess clinical outcomes.REFERENCES1. Kotton, C. N., Huprikar, S. & Kumar, D. Transplant Infectious Diseases: A Review of the Scientific Registry of Transplant Recipients Published Data. Am J Transplant 17, 1439-1446, doi:10.1111 / ajt.14195 (2017).2 Timsit, J. F. et al. Diagnostic and therapeutic approach to infectious diseases in solid organ transplant recipients. Intensive Care Med 45, 573-591 , doi:10.1007 / S00134-019-05597-y (2019).3 van Delden, C. et al. Burden and Timeline of Infectious Diseases in the First Year After Solid Organ Transplantation in the Swiss Transplant Cohort Study. Clin Infect Dis 71 , e159-e169, doi:10.1093 / cid / ciz1113 (2020).4 Soborg, A. et al. Trends in underlying causes of death in solid organ transplant recipients between 2010 and 2020: Using the CLASS method for determining specific causes of death. PLoS One 17, e0263210, doi:10.1371 / journaL pone.0263210 (2022).5 Styczynski, J. et al. Death after hematopoietic stem cell transplantation: changes over calendar year time, infections and associated factors. Bone Marrow Transplant 55, 126-136, doi: 10.1038 / S41409-019-0624-z (2020).6 Esquirol, A. et al. Severe infections and infection-related mortality in a large series of haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. Bone Marrow Transplant 56, 2432-2444, doi:10.1038 / s41409-021-01328-4 (2021).7 Lindsay, J. et al. Infection-Related Mortality in Adults and Children Undergoing Allogeneic Hematopoietic Cell Transplantation: An Australian Registry Report. Transplant Cell Ther 27, 798 e791-798 e710, doi:10.1016 / j.jtct.2O21 .05.028 (2021).8 Stewart, A. G. & Kotton, C. N. What's New: Updates on Cytomegalovirus in Solid Organ Transplantation. Transplantation 108, 884-897, doi:10.1097 / TP.0000000000004855 (2024).9 Yamada, M., L'Huillier, A. G. & Green, M. A Focused Review of Epstein-Barr Virus Infections and PTLD in Pediatric Transplant Recipients: Guidance From the IPTA and ECIL Guidelines. J Pediatric Infect Dis Soc 13, S31-S38, doi: 10.1093 / jpids / piad097 (2024).10 Abudayyeh, A. et al. Poor immune reconstitution is associated with symptomatic BK polyomavirus viruria in allogeneic stem cell transplant recipients. Transpl Infect Dis 19, doi:10.1111 / tid.12632 (2017).11 Cortese, I., Reich, D. S. & Nath, A. Progressive multifocal leukoencephalopathy and the spectrum of JC virus-related disease. Nat Rev Neurol 17, 37-51 , doi:10.1038 / s41582-020- 00427-y (2021).12 Feghoul, L. et al. Adenovirus infection and disease in paediatric haematopoietic stem cell transplant patients: clues for antiviral pre-emptive treatment. Clin Microbiol Infect 21 , 701-709, doi:10.1016 / j.cmi.2015.03.011 (2015).18 Khanna, R. & Smith, C. Cellular immune therapy for viral infections in transplant patients. The Indian journal of medical research 138, 796-807 (2013).19 Smith, C. et al. Autologous Adoptive T-cell Therapy for Recurrent or Drug-resistant Cytomegalovirus Complications in Solid Organ Transplant Recipients: A Single-arm Open-label Phase I Clinical Trial. Clin Infect Dis 68, 632-640, doi:10.1093 / cid / ciy549 (2019).20 Riddell, S. R. et al. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T-cell clones. Science 257, 238-241 (1992).21 Rooney, C. M. et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr- virus-related lymphoproliferation. Lancet 345, 9-13, doi:S0140-6736(95)91150-2 [pii] (1995).22 Heslop, H. E. et al. Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat.Med. 2, 551-555 (1996).23 Rooney, C. M. et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein- Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92, 1549-1555 (1998).24 Savoldo, B. et al. Cellular immunity to Epstein-Barr virus in liver transplant recipients treated with rituximab for post-transplant lymphoproliferative disease. Am.J. Transplant 5, 566-572 (2005).Leen, A. M. et al. T-cell immunotherapy for adenoviral infections of stem-cell transplant recipients. Ann N Y Acad Sci 1062, 104-115, doi:1062 / 1 / 104 [pii] 10.1196 / annals.1358.013 (2005).Pakakasama, S. et al. Treatment of Epstein-Barr virus lymphoproliferative disease after hematopoietic stem-cell transplantation with hydroxyurea and cytotoxic T-cell lymphocytes. Transplantation 78, 755-757, doi:00007890-200409150-00022 [pii] (2004).Haque, T. et al. Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood 110, 1123- 1131 , doi:blood-2006-12-063008 [pii] 10.1182 / blood-2006-12-063008 (2007).Haque, T. et al. Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet 360, 436-442, doi:S0140-6736(02)09672-1 [pii] 10.1016 / S0140-6736(02)09672-1 (2002).Mahadeo, K. M. et al. Tabelecleucel for allogeneic haematopoietic stem-cell or solid organ transplant recipients with Epstein-Barr virus-positive post-transplant lymphoproliferative disease after failure of rituximab or rituximab and chemotherapy (ALLELE): a phase 3, multicentre, open-label trial. Lancet Oncol 25, 376-387, doi:10.1016 / S1470-2045(23)00649-6 (2024).Prockop, S. E. et al. Third-party cytomegalovirus-specific T cells improved survival in refractory cytomegalovirus viremia after hematopoietic transplant. J Clin Invest 133, doi:10.1172 / JCI165476 (2023).Withers, B. etal. Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program. Biol Blood Marrow Transplant 24, 2433-2442, doi:10.1016 / j.bbmt.2018.08.024 (2018).O'Reilly, R. J., Prockop, S. & Oved, J. H. Virus-specific T-cells from third party or transplant donors for treatment of EBV lymphoproliferative diseases arising post hematopoietic cell or solid organ transplantation. Front Immunol 14, 1290059, doi:10.3389 / fimmu.2023.1290059 (2023).Ma, C. et al. Adoptive transfer of CMV-specific TCR-T cells for the treatment of CMV infection after haploidentical hematopoietic stem cell transplantation. J Immunother Cancer 12, doi:10.1136 / jitc-2023-007735 (2024).Leen, A. M. et al. Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med 12, 1160-1166, doi:nm1475 [pii] 10.1038 / nm1475 (2006).Blyth, E. et al. BK virus-specific T cells for use in cellular therapy show specificity to multiple antigens and polyfunctional cytokine responses. Transplantation 92, 1077-1084, doi: 10.1097 / TP.0b013e31823328c0 (2011).Di Ciaccio, P. R. et al. Successful treatment of CMV, EBV, and adenovirus tissue infection following HLA-mismatched allogeneic stem cell transplant using infusion of third-party T cells from multiple donors in addition to antivirals, rituximab, and surgery. Transpl Infect Dis 23, e13528, doi:10.1111 / tid.13528 (2021).Jiang, W. et al. Third-party CMV- and EBV-specific T-cells for first viral reactivation after allogeneic stem cell transplant. Blood Adv 6, 4949-4966, doi: 10.1182 / bloodadvances.2022007103 (2022).Castellano-Gonzalez, G., Clancy, L. E. & Gottlieb, D. Prospects for adoptive T-cell therapy for invasive fungal disease. Current opinion in infectious diseases 30, 518-527, doi: 10.1097 / QCG.0000000000000403 (2017).Smith, C. & Khanna, R. Generation of cytotoxic T lymphocytes for immunotherapy of EBV- associated malignancies. Methods in Molecular Biology (Clifton, NJ) 651 , 49-59 (2010).Sinha, D. et al. 'Off-the-shelf allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies. J Immunother Cancer 9, doi:10.1136 / jitc- 2020-001608 (2021).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1 . A combination of peptides comprising five or more of the T cell epitopes listed in Tables 1-4, wherein the five or more T cell epitopes comprise T cell epitopes from at least five different viruses.

2. The combination of peptides of claim 1 , wherein the five or more T cell epitopes comprise both HLA class l-restricted T cell epitopes and HLA class-l I restricted T cell epitopes.

3. The combination of peptides of claim 1 or claim 2, wherein the combination incluudes at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15, 120, 125, 130 or 135 of the T cell epitopes listed in Tables 1-4.

4. The combination of peptides of any one of claims 1 to 3, wherein the combination includes all of the T cell epitopes listed in Tables 1-4.

5. The combination of peptides of any one of claims 1 to 4, wherein the vector encodes a T cell epitope from Epstein Barr virus (EBV).

6. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises at least one LMP2a epitope.

7. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises at least one LMP1 epitope.

8. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA4 epitope.

9. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA3 epitope.

10. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA6 epitope.11 . The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA2 epitope.

12. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA1 epitope.

13. The combination of peptides of claim 5, wherein the T cell epitope(s) from EBV comprises an EBNA1 epitope, EBNA2 epitope, an EBNA6 epitope, an EBNA4 epitope, an EBNA3 epitope, LMP1 epitope, and an LMP2a epitope.

14. The combination of peptides of any one of claims 1 to 13, wherein the vector encodes a T cell epitope from cytomegalovirus (CMV).21 . The combination of peptides of claim 14, wherein the T cell epitope(s) from CMV comprises a pp50 epitope.

22. The combination of peptides of claim 14, wherein the T cell epitope(s) from CMV comprises a pp65 epitope.

23. The combination of peptides of claim 14, wherein the T cell epitope(s) from CMV comprises an IE-1 epitope.24 The combination of peptides of claim 14, wherein the T cell epitope from CMV comprises a gB epitope.24 The combination of peptides of claim 14, wherein the T cell epitope from CMV comprises a gH epitope.

25. The combination of peptides of claim 14, wherein the T cell epitopes from CMV comprise a pp50 epitope, pp65 epitope, IE-1 epitope, gB epitope and a gH epitope.

25. The combination of peptides of any one of claims 1 to 25, wherein the vector encodes a T cell epitope from polyoma BK virus (BKV) or polyoma JC virus.

26. The combination of peptides of claim 25, wherein the T cell epitope(s) from BKV or JCV comprises a large T antigen (LT A) epitope.

27. The combination of peptides of claim 25, wherein the T cell epitope(s) from BKV or JCV comprises a VP1 epitope.

28. The combination of peptides of claim 25, wherein the T cell epitope(s) from BKV or JCV comprises a STA epitope.

29. The combination of peptides of claim 25, wherein the T cell epitope(s) from BKV or JCV comprises a VP2 epitope.

30. The combination of peptides of claim 25, wherein the T cell epitopes from BKV or JCV comprise a VP1 epitope, a LTA epitope, a STA epitope, and a VP2 epitope.31 . The combination of peptides of any one of claims 1 to 30, wherein the vector encodes a T cell epitope from adenovirus (ADV).

32. The combination of peptides of claim 31 , wherein the T cell epitope(s) from ADV comprises a hexon protein epitope.

33. The combination of peptides of claim 31 , wherein the T cell epitope(s) from ADV comprises a DNA polymerase epitope.

34. The combination of peptides of claim 31 , wherein the T cell epitopes from ADV comprise a hexon epitope and a DNA polymerase epitope.

35. The combination of peptides of any one of claims 1 to 34, wherein the combination of peptides includes T cell epitopes from EBV, CMV, BKV, JCV, and ADV.

36. The vector of any one of claims 1 to 35, wherein the T cell epitopes present as one or more polyepitope proteins.

37. A pharmaceutical composition comprising the combination of peptides of any one of claims 1 to 36 and a pharmaceutically acceptable carrier, exipient, or diluent.

38. A method of treating or preventing a viral infection in a subject comprising administering too the subject the pharmaceutical composition of claim 37.

39. The method of claim 38, wherein the viral infection is an EBV, CMV, BKV, JCV, or ADV infection.

40. A method of treating or preventing cancer in a subject comprising administering to the subject a pharmaceutical composition of claim 37.41 . A method of generating a population of antigen presenting cells (APCs) that present epitopes from at least five viruses comprising contacting a sample comprising APCs with the combination of peptides of any one of claims 1 to 35.

42. The method of claim 41 , wherein the sample is a PBMC sample.

43. The method of claims 41 or 42, wherein the APCs comprise B cells.

44. The method of any one of claims 41 to 43, wherein the APCs comprise antigen- presenting T-cells.

45. The method of any one of claims 41 to 44, wherein the APCs comprise dendritic cells.

46. The method of any one of claims 41 to 45, wherein the APC comprise artificial antigen-presenting cell.

47. The method of claim 46, wherein the artificial antigen-presenting cells are aK562 cells.

48. A sample comprising the APCs generated according to the method of any one of claims 41 to 47.

49. A method of generating multivirus-specific cytotoxic T cells (CTLs) comprising,(a) generating APCs that present epitopes from multiple viruses according to the method of any one of claims 41 to 47; and(b) incubating the APCs presenting multiple viruses of step (a) with CTLs, thereby generating multivirus-specific CTLs.

50. A sample comprising the multivirus-specific CTLs generated according to claim 49.

51. A composition comprising the CTLs of claim 50, and a pharmaceutically acceptable carrier.

52. A method of treating or preventing a viral infection in a subject comprising administering to the subject the composition of claim 51 .

53. The method of claim 52, wherein the viral infection is an EBV, CMV, BKV, JCV, or ADV infection.

54. The method of claim 52 or 53, wherein the subject is immunocompromised.

55. The method of any one of claims 52 to 54, wherein the CTLs in the composition are allogeneic to the subject.

56. The method of claim 55, wherein the CTLs in the composition are stored in a cell bank prior to administration to the subject.

57. The method of claims 52 or 53, wherein the CTLs in the composition are autologous to the subject.

58. A method of treating or preventing cancer in a subject comprising administering to the subject the composition of claim 51.

59. The method of claims 58, wherein the CTLs in the composition are allogeneic to the subject.

60. The method of claim 59, wherein the CTLs in the composition are stored in a cell bank prior to administration to the subject.61 . The method of claim 58, wherein the CTLs in the composition are autologous to the subject.

Images