Conjugated virus-like particles and uses thereof as anti-tumor immune redirectors

Abstract

Disclosed is a new class of conjugated virus-like particles (VLPs), These conjugated VLPs bind a wide variety of tumors and comprise epitopes recognized by a prior T cell immune response already existing in a host. These epitopes are derived from pathogens or previous vaccinations (such as early childhood vaccines). This provokes the body's pre-existing cytotoxic immunity obtained through previous infection or previous childhood vaccination to be redirected to the tumor cells for tire elimination of cancer, and form long-term anti-tumor immunity. The described conjugated VLPs are useful for tailoring a broad range of tumors towards a response from existing immunity circumventing the need to identify tumor antigens or generate tumor-specific immune responses, importantly, the compositions and methods described herein broadens opportunities for treatment for ail cancer types in subjects who previously had un-targetable cancers due to various technological and biological limitations of currently available immuno-therapeutic drugs.

Description

[0050] In various embodiments, disclosed are conjugated VLPs for treating cancer in a subject. In various embodiments, the compositions described herein comprise a capsid protein, wherein said capsid protein binds a cancer cell. In various embodiments the compositions described herein further comprise a fusion protein comprising at least: (1) a cleavage sequence, wherein the cleavage sequence is preferentially cleaved in the presence of a tumor; and (2) at least one recall protein, wherein said recall protein is a protein or fragment thereof having a sequence that is an epitope that is bound by an existing T cell in a patient. In various embodiments the cleavage sequence is bound to the capsid protein.

[0051] Capsid Proteins. In various embodiments, a VLP is provided. VLPs tire comprised of viral structural particles, i.e. capsid proteins that self-assemble into a particle that resembles a virus but lacks the viral genetic material of a virus. VLPs are excellent delivery molecules because they are non-infectious and can be re-engineered to specifically target or bind to tumor cells.

[0052] It has been reported that HPV capsids (VLP and pseudovirions (PsV)) have tumor tropism, and directly bind and infect tumor cells, including, e.g., ovarian and lung cancer cells. In various embodiments the conjugated VLPs described herein preferentially bind to tumor cells, e.g., they bind more to tumor cells than to non-tumor cells, and the presence of conjugated VLPs may result in a positive pro-inflammatory tumor microenvironment that attracts infiltrating CD8+ T cells. At the same time, the VLPs may stimulate a response by adaptive memory T cells that resulted from a previous vaccination or infection and that recognize the recall protein’s CD8+ T cell epitope. In various embodiments these strong responses are able to bypass immune tolerance, and render the tumor cells susceptible to this preexisting immunity thereby inhibiting the growth, progression and metastasis of the tumor.

[0053] Any VLP that preferentially binds to tumor cells relative to normal cells may be used with the present invention. In various embodiments, the VLP is an animal virusbased VLP. In various embodiments, the animal virus-based VLP is derived from HBVc or HPV. In various embodiments, the VLP is a bacteriophage-based VLP, such as MS2, Qp, or P22. In various embodiments, the VLP is a plant virus-based VLP such as cowpea chlorotic mottle virus (CCMV) or cowpea mosaic virus (CPMV). In various embodiments, the VLP is a currently commercialized prophylactic VLP-based vaccine, including GlaxoSmithKline's ENGERIX® (hepatitis B virus) and CERVARIX® (human papillomavirus), and Merck and Co., Inc.'s RECOMBIVAX HB® (hepatitis B virus) and GARDAS1L® (human papillomavirus). Other embodiments of the VLP include VLP-based vaccine candidates in clinical trials or preclinlcal evaluation, such as, influenza virus, parvovirus, and Norwalk virus. In various embodiments, the VLP is a hamster polyoma virus, Mucrobrachium Rosenbergii Nodavirus, HBsAg, HCV, retrovirus, or an HBc.

[0054] In one embodiment, the VLP is comprised of structural proteins derived from a papillomavirus. The papillomavirus virion contains 72 pentamers (capsomeres) of LI protein. The LI protein is capable of self-assembly into capsid-like structures that are morphologically indistinguishable from native virions when expressed in eukaryotic cells. The LI monomer contains 12 b-strands, 6 loops (BC, CD, DE, EF, FG, HI), and 5 helices (H1-H5). Most of the loops are highly exposed towards the outer surface of the capsid, attachment of a fusion protein to one of these loops, by, e.g., disulfide linkage, maleimide linkage, by “click” chemistry or by binding to a polyionic docking site, as described herein, in these areas will result in the recall protein being displayed on the outer surface of VLPs.

[0055] Papillomavirus VLPs. In various embodiments, a conjugated VLP is provided comprising a papilloma (PV) LI protein, or an PVL1 and PV L2 protein. The VLP in some embodiments comprises both papilloma LI and L2 proteins.

[0056] In an embodiment, the conjugated papillomavirus VLP comprises an LI capsid protein and a fusion protein. In other embodiments, the conjugated VLP comprises an LI capsid protein, an L2 capsid protein, and a fusion protein. The LI polypeptide can be full length LI protein or an 1.,1 polypeptide fragment. In specific embodiments, the full-length LI protein or LI polypeptide fragment is VLP assembly-competent; that is, the LI polypeptide will self-assemble to form capsomeres that tire competent for self-assembly into higher-order assemblies, thereby forming a VLP. In more specific embodiments, the VLPs comprise a fully assembled papillomavirus capsid, a structure of about 50 nm and composed of 72 capsomeres or 360 copies of LI protein.

[0057] The LI sequences are known for substantially all papillomavirus genotypes identified to date, and any of these LI sequences or fragments are contemplated as being employed in the present compositions. Examples of LI polypeptides include, without limitation, full-length LI polypeptides, e.g., HPV16 LI polypeptide, SEQ ID NO: , LI truncations that lack the native C-terminus, LI truncations that lack the native N-terminus, and LI truncations that lack an internal domain. The LI protein may be for example a modified LI protein, e.g., a modified HPV16 LI protein wherein the HPV16 L2 amino acids 17-36 (RG1 epitope) are inserted within the DE-surface loop of HPV16 LI. (See, Schellenbacher et al., 2013, J. Invest Dermatol; 133(12):2706-2713; Slupetzky et al., 2007, Vaccine. 25:2001-2010; Kondo et al., 2008, J. Med. Virol., 80:841-6; Schellenbacher et al., 2009,. / . Virol., 83:1008510095: and Caldeira et al., 2010, Vaccine, 28:4384-93).

[0058] The 1..2 polypeptide can be full-length L2 protein or an 1..2 polypeptide fragment. The L2 sequences are known for substantially all papillomavirus genotypes identified to date, and any of these L2 sequences or fragments can be employed in the present invention. Examples of L2 polypeptides include, without limitation, full-length L2 polypeptides, e.g., HPV16 L2 polypeptide, SEQ ID NO: , 1-2 truncations that lack the native C-terminus, L2 truncations that lack the native N-terminus, and L2 truncations that lack an internal domain.

[0059] The papillomavirus VLPs can be formed rising the LI and optionally L2 polypeptides from any animal papillomavirus, or derivatives or fragments thereof. Thus, any known (or hereafter identified) LI and optionally L2 sequences of human, bovine, equine, ovine, porcine, deer, canine, feline, rodent, rabbit, etc., papillomaviruses can be employed to prepare the VLPs or capsomeres of the present invention. (See de Villiers et al., Virology, 324:17-27, 2004, for a near complete listing of papillomavirus genotypes and their relatedness, incorporated herein by reference).

[0060] In various embodiments, the VLP is comprised of a papilloma virus (PV) LI protein, or a PV LI and PV L2 protein. Papillomaviruses tire small, double-stranded, circular DNA tumor viruses. The papilloma virion shells contain the LI major capsid protein and the L2 minor capsid proteins. Expression of the LI protein alone or in combination with the L2 protein in eukaryotic or prokaryotic expression systems is known to result in the assembly of capsomeres and VLPs. As used herein, the term “capsomere” is intended to mean a pentameric assembly of papillomavirus LI polypeptides, including full-length LI protein or fragments thereof. Native LI capsid proteins may self-assemble via intermolecular disulfide bonds to form pentamers (capsomeres).

[0061] The papilloma virus virion may contain 72 pentamers (capsomeres) of LI protein. (See, Trus et ah, Nat. Struct. Biol., 4:413-420,1997). The LI protein is capable of selfassembly into capsid-like structures that are morphologically indistinguishable from native virions when expressed in eukaryotic cells. (See, Buck et al., J. Virol., 5190-97, 2008; and Roy et al., Hum. Vaccin., 5-12, 2008, both incorporated herein by reference). The LI monomer contains about 12 strands, 6 loops (BC, CD, DE, EF, FG, HI), and 5 helices (H1-H5). Most of the loops are highly exposed towards the outer surface of the capsid, attachment of a fusion protein to one of these loops, by e.g., disulfide linkage, maleimide linkage, by "click" chemistry or by binding to a polyionic docking site, as described herein, in these areas will result in the fusion protein being displayed on the outer surface of VLPs.

[0062] In certain embodiments, the LI and optionally L2 polypeptides that are used to form the VLPs are from, a non-human papillomavirus or a. human papillomavirus genotype other than HPV-6, HPV-11, HPV-16, and HPV-18. For example, the LI and / or L2 proteins may be from HPV 1, 2, 3, 4, 5, 6, 8. 9, 15, 17, 23, 27, 31, 33, 35, 38, 39, 45, 51, 52,58, 66, 68, 70, 76, or 92.

[0063] In various embodiments, the conjugated VLP presented herein bind to one or more cancer cells. This is in part due to the VLP’s selectivity for proteins and / or molecules specific to tumor cells. In various embodiments, the VLP binds to heparin sulfate proteoglycan (HSPG), which is preferentially expressed on tumor cells. As used herein, “binding to a cancer cell” refers to the formation of non-covalent interactions between the capsid protein of the conjugated VLP and the tumor cell such that the conjugated VLP may come into close proximity to the tumor cell and the fusion protein may be cleaved from the VLP, and the recall protein may bind to the MHC receptor present on the tumor cell.

[0064] Animal Virus-based VLPs. In addition to the VLPs described elsewhere herein the VLP may be derived from a hepatitis B virus. The hepatitis B virus is comprised of an internal protein capsid and a lipid envelope containing other proteins. Two different VLPs can be produced from the virus, using either the core antigen that forms the internal capsid or the surface antigen that spontaneously combines with lipids to form nanoparticles (NP)s. The hepatitis B core (HBc) antigen is formed from 240 copies of a single protein. These proteins first form dimers, which then assemble with pentameric or pseudo-hexameric junctions in a T54 icosahedral geometry. The VLP has been produced using multiple technologies including Escherichia coli cytosolic accumulation and cell-free protein synthesis. The assembled VLPs are typically purified using size-exclusion chromatography or differential centrifugation. Individual coat proteins have been subsequently obtained by disassembling the VLPs with urea, which abows simultaneous cargo loading and VLP re-assembly.

[0065] Bacteriophage virus-based VLPs. In various embodiments, the VLP is derived from a bacteriophage-based VLP. The three bacteriophages, MS2, Qb, and Salmonella typhimurium P22, all infect enterobacteria, most notably E. coli. Although all three are composed of only a nucleic acid-filled viral capsid, P22 differs greatly from MS2 and Qb. MS2 and Qb fire composed of 90 homodimers and require a specific stem-loop hairpin secondary structure in their RNA genome to trigger VLP self-assembly by binding to the coat proteins. P22, on the other hand, is composed of up to 415 coat proteins, 100--300 scaffold proteins, and 12 portal proteins. However, the P22 VLP has been engineered to consist of 420 coat proteins and only the 100-300 scaffold proteins, which can subsequently be removed with guanidine hydrochloride, leaving only the coat proteins. Like the HBV VLP, these VLPs assemble with icosahedral geometry. All three can be produced in E. coli, but Qb can also be produced in yeast and both Qb and MS2 can be produced using cell-free protein synthesis. MS2 VLPs have been purified using size-exclusion chromatography, differential centrifugation, or immobilized metal affinity chromatography (for VLPs containing hexahistidine tags). Acids or urea can be used to disassemble the purified MS2 VLPs to obtain the dimers, which can then be reassembled after removal of the disassembly agent and the addition of the stem-loop RNA. Qb VLPs have been purified using size exclusion chromatography and the dimers can be obtained by disassembling the VLPs using acid, which can then be reassembled similar to MS2. P22 VLPs have been purified using size-exclusion chromatography or differential centrifugation and can also be disassembled using acid to obtain the coat proteins. Addition of scaffold proteins is required to reassemble the P22 VLP, but these can subsequently be removed. These bacteriophage-derived VLPs differ from HBc VLPs mainly in the assembly stimulus, using additional biomolecules (RNA or proteins) to initiate self-assembly instead of increasing the salt concentration.

[0066] Plant virus-based VLPs. In various embodiments, the VLP is a plant virusbased VLP. In various embodiments, the plant virus-based VLP is derived from the cowpea leaf: CCMV and CPMV. Neither virus has a lipid envelope. Both VLPs assemble with icosahedral geometry. The CCMV VLPs are formed from 90 homodimers and can be produced in E. coli or yeast. They have been purified using size exclusion chromatography or immobilized metal affinity chromatography, using coat proteins with hexahistidine extensions. 46,62 Dimers can be obtained by dialyzing the assembled VLPs against 0.5 M CaCh or by purifying hexahistidine tagged dimers directly. Combining the dimers with RNA in a 1:6 mass ratio and lowering the pH to 4--5 induces self-assembly. CPMV, on the other hand, is formed from 60 copies of the VP60 protein which must first be proteolyzed into the L and S coat proteins (60 copies of each). Unfortunately, the VLP cannot be produced using E. coli or yeast; insect cells or plants must be used. The VLPs have been purified using differential centrifugation, but the coat proteins cannot yet be obtained in usable quantities. The inability to produce the VLP in E. coll or obtain purified coat proteins adds another challenge for targeted drug delivery; however, CPMV has been actively evaluated for therapeutic use due to the ability to easily display ligands on its surface and load cargo through association with its genome. In other embodiments, tire plant virus-based VLP is a Tobacco Mosaic Virus (TMV).

[0067] Tumor Specificity of VLPs. In various embodiments, the VLP binds preferentially to tumor cells. The VLPs’ tumor preference may originate from several sources including the VLP’s charge (positive or negative), shape and size (different aspect ratio filaments and diameter spheres), shielding (self-proteins / peptides and polymers of various sizes and densities), and targeting (ligands for receptors or environmental factors displayed on different linkers at various densities).

[0068] In terms of charge, in various embodiments, the VLP contains a positive surface charge. Positive charged VLPs stay longer in circulation. Due to the abundant presence of proteoglycan in the cell membrane conferring a negative charge and collagen within the tumor interstitial space conferring a positive charge, positively charged particles are more likely to have enhanced binding to mammalian cells and are better able to avoid aggregation and penetrate tumor tissue. Some examples demonstrating these charge-based effects include poly arginine-decorated CPMV found to be taken up eight times more efficiently than native CPMV in a human cervical cancer.

[0069] With regards to shape, the shape and flexibility of the VLP will play an additional role in VLPs ability to diffuse throughout a tumor. E.g. A comparison between the diffusion profiles of a spherical and rod-shaped particle was performed with CPMV and TMV using a spheroid model, and it was shown that whereas CPMV (Sphere) experienced a steady diffusion profile, TMV (rod shape) exhibited a two-phase diffusion behavior that entailed an extremely rapid early loading phase, which could be attributed to its movement axially, acting like a needle. Some other advantageous properties that are conferred by elongated particles include better margination toward the vessel wall and stronger adherence due to greater surface area for interaction, which not only have implications for tumor homing but also for enhanced targeting of cardiovascular disease.

[0070] Besides passive tumor homing properties, natural interactions of viruses with certain cells can also be exploited. CPMV(cowpea mosaic virus) in particular exhibits unique specificity in interacting with surface vimentin, which is found on endothelial, cancer, and inflammatory cells. The native affinity of CPMV for surface vimentin allows for high-resolution imaging of microvasculature up to 500 pm in depth, which cannot be achieved through the use of other nanoparticles, as they tend to aggregate and block the vasculature. This interaction can be utilized for a range of applications, such as delivery to a panel of cancer cells including cervical, breast, and colon cancer cell lines, delineation of atherosclerotic lesions, and intravital imaging of tumor vasculature and angiogenesis. Another example of an existing endogenous association is CPV with transferrin receptor (TfR), an important receptor for iron transport into cells and highly upregulated by numerous cancer cell lines. Even after dye labeling, CPV retains its specificity for TfR and was shown to bind to receptors found on HeLa cervical cancer cells, HT-29 colon cancer cells, and MDA-MB-231 breast cancer cells.

[0071] In various embodiments, the VLP is capable of targeting a protein expressed preferentially on the tumor cell surface. Such proteins are typically overexpressed on the surface of tumor cells, but some if not all may also be found in the blood, i.e. serum Nonlimiting examples of such surface markers include: CEA (carcinoembryonic antigen), E-cadherin, EMA (epithelial membrane antigen; aka Ml C- h. vimentin, fibronectin, Her2 / neu (human epidermal growth factor receptor type 2, also called Erb b2), av£3 integrin, EpCAM (epithelial cell adhesion molecule), FR-a (folate receptor-alpha), PAR (urokinase-type plasminogen activator receptor ), and transferrin receptor (over expressed in tumor cells).

[0072] Peptides are often used to label cancerous cells based on recognition of their transmembrane proteins. The most commonly used peptide is arginylglycylaspartic acid (RGD), composed of L-arginine, glycine, and L-aspartic acid. RGD was first isolated from the cell-binding domain of fibronectin, a glycoprotein that binds to integrins, and is involved in cell-cell and cell-extracellular matrix (ECM) attachment and signaling by binding collagen, fibrin, and proteoglycans. RGD peptides have the highest affinity for a type of cell surface integrins, avP which are highly expressed in tumoral endothelial cells, but not in normal endothelial cells. In various embodiments such a peptide sequence is incorporated into the conjugated VLP.

[0073] Fusion Protein. In various embodiments the fusion protein comprises a cleavage sequence. The cleavage sequence can be any sequence capable of being preferentially cleaved by or near a tumor cell. The insertion of this cleavage sequence into the fusion protein, allows the protein to remain inactive until it enters the tumor micro-environment. By taking advantage of the elevated activities of particular proteases in cancer tissues, the recall protein is not released from, the VLP and able to actively coat MHC receptors until the recall protein enters the tumor microenvironment. Several proteases are known in the art to be active in the tumor microenvironment. For example, several metallo-, cysteine and serine proteases are known. From the standpoint of cancer therapy, an additional attraction is that because the proteases responsible for prodrug cleavage may come not just from cancer cells but also from the stromal components of tumors, release of tire active drug direction into the tumor microenvironment does not depend on a target expressed only by the cancer cells. Instead, it is the entire tumor ecosystem that represents the target.

[0074] Recall Protein. In various embodiments, the recall protein is an epitope, which is recognized by a T cell or T cell population, which already exists in a subject. In various embodiments, this existing T cell or T cell population exists because of a prior infection or vaccination. In various embodiments of the invention, the recall protein is an epitope that is capable of being, bound by a T cell. In various embodiments, the recall protein is an epitope capable of being bound by a T cell already present in a subject. In this context, “capable of being bound” means that a "epitope" is presented on the surface of a cell, where it is bound to MHC molecules directly. T cell epitopes presentable by MHC I can be bound by the T cell receptor of cytotoxic CD8 T lymphocytes (CDS T cells or CTLs). T cell epitopes presentable by MHC I are typically peptides of 9 to 12 amino acids in length. In various embodiments, a conjugated VLP is provided which allows release of a T cell response eliciting peptide, which is directly presentable via MHC class I. As the released recall protein does not require delivery to the antigen processing machinery in the cytosol, the T cell response eliciting peptide are presented on the surface of the target cell in a short amount of ti me. Hence, in one embodiment of the invention, in less than 8.5 hours after administration the target cell the T cell response eliciting peptide is presented on the surface of the target cell via an MHC class I molecule. In another embodiment of the invention, in less than 23.5 hours after introduction of the conjugated VLP to the target cell the T cell response eliciting peptide is presented on the surface of tlie target cell via an MHC class I molecule. In another embodiment of the invention, the conjugated VLP is capable of mediating T cell cytotoxicity against the target cell within less than 6 hours after administration of the conjugated VLP to the target cell.

[0075] In various embodiments, the fusion protein comprises at least two recall proteins. These recall proteins might be epitopes derived from different proteins, or they may be epitopes of the same protein. In various embodiments, the pathogen is a virus, a bacterium., a fungus, or a parasite.

[0076] In various embodiments, the preexisting T cells are specific to a vaccine epitope. In various embodiments the epitope is derived from, an early childhood vaccine. In various embodiments the preexisting immunity is the result of prior administration of a human vaccine.

[0077] Non-limiting examples of a virus includes , a vaccinia virus, a varicella zoster virus, a Herpes zoster virus, rubella, a hepatitis virus, e.g., hepatitis A virus or hepatitis B virus or hepatitis C viruse, influenza, e.g., type A or type B, a measles virus, a mumps virus, a polio virus, a variola (smallpox) virus, a rabies virus, a Dengue virus, an Ebola virus, a West Nile virus, a yellow fever virus, or a zika virus.

[0078] Non-limiting examples of a bacterium include, a Bordetella pertussis, chlamydia, trachomatis, Clostridium tetani, diphtheria, Hemophilus influenza, Meningococcus, pneumococcus, vibrio cholera, Mycobacterium tuberculosis, BCG, typhoid, E. coli, salmonella, Legionella pneumophila, rickettsia, Treponema pallidum, pallidum, Streptococcus group A or group B, Streptococcus pneumonia, Bacillus anthracis, Clostridium botulinum, or a Yersinia sp bacteria.

[0079] Non-limiting examples of a parasite include, Entamoeba histolytica, Toxoplasma gondii, a Trichinella sp., e.g., Trichinella spiralis, a Trichomonas sp., e.g., Trichomonas vaginalis, a Trypanosoma sp., e.g., Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, or a Trypanosoma cruzi, or a plasmodium, e.g., Plasmodium, falciparum, Plasmodium vivax, or Plasmodium malariae.

[0080] In various embodiments, the recall protein is selected from the list included in Table 1: Table 1: Recall Protein Epitopes Epitope Sequence SEQ ID NO Virus Type MHC aHGe Viral Protein SLPRSRTPI 4 Chicken Pox (VZV) A*02:01 IE62 SAPLPSNRV 5 Chicken Pox (VZV) A*02:01 IE62 GSAPLPSNRV 6 Chicken Pox (VZV) A*02:01 IE62 ALWALPHAA 1 Chicken Pox (VZV) A*02:01 IE62 SLSGLYVFV 8 Shingles vaccines A*02:01 Glycoprotein E YLGVYIWNM 9 Shingles vaccines A*02:01 Glycoprotein E KIHEAPFDL 10 Shingles vaccines A*02:01 Glycoprotein E LLCLVIFLI 11 Shingles vaccines A*02:01 Glycoprotein E DLLLEWLYV 12 Shingles vaccines A*02:01 Glycoprotein E SMYYAGLPV 13 Shingles vaccines A*02:01 Glycoprotein E ILHDGGTTL 14 Shingles vaccines A*02:01 Glycoprotein E WLYVPIDPT 15 Shingles vaccines A*02:01 Glycoprotein E VLMGFGIIT 16 Shingles vaccines A*02:01 Glycoprotein E CLVIFLICT 17 Shingles vaccines A*02:01 Glycoprotein E KEADQPWIV 18 Shingles vaccines A*02:01 Glycoprotein E VVSTVDHFV 19 Shingles vaccines A*02:01 Glycoprotein E FLICTAKRM 20 Shingles vaccines A*02:01 Glycoprotein E VLRTEKQYL 21 Shingles vaccines A*02:01 Glycoprotein E HMWNYHSHV 22 Shingles vaccines A*02:01 Glycoprotein E TVNKPVVGV 23 Shingles vaccines A*02:01 Glycoprotein E fvvyfnghv 24 Shingles vaccines A*02:01 Glycoprotein E W1VVNTSTL 25 Shingles vaccines A*02:01 Glycoprotein E VAYTVVSTV 26 Shingles vaccines A*02:01 Glycoprotein E FMYMSLLGV 27 measles A*02:01 m50 SLWGSLLML 28 measles A*02:01 C protein LLAVTFVMFL 29 measles A*02:01 H38 SMYRVFEVGV 30 measles A*02:01 H250-259 ILPGQDLQYV 31 measles A*02:01 H516-525 KLWCRHFCV 32 measles A*02:01 H576 KLWCRHFCVL 33 measles A*02:01 H576 RLSDNGYYTV 34 measles A*02:01 Ml 64 KLLRYYTEI 35 measles A*02:01 F205 KLWESPQEl 36 measles A*02:01 C 84 RLLDRLVRL 37 measles A*02:01 N50 KLMPNITLL 38 measles A*02:01 F57 TLLNNCTRV 39 measles A*02:01 F64 EMLTLATWV 40 HepB A*02:01 C64-72 FLPSDFFPSV 41 Hep B A*02:01 Core 18 FLPADFFPSV 42 Hep B A*02:01 Core 19 FLPSDFFPSI 43 Hep B A*02:01 Core 20 WLSLLVPF 44 Hep B A*02:01 ENV335 FLLTRILTI or FLLTRILTL 45 or 46 HepB A*02:01 ENV183 GLSPTVWLSV 47 HepB A*02:01 ENV348 LLDYQGMLPV 48 Hep B A*02:01 ENV260 LLCLIFLLV 49 Hep B A*02:01 ENV251 SIVSPFIPLL 50 Hep B A*02:01 ENV370 FLLTKILTI 51 Hep B A*02:01 ENV183 ILSPFLPLL 52 Hep B A*02:01 ENV371 FLLSLGIHL 53 Hep B A*02:01 POL 575 GLSRYVARL 54 Hep B A*02:01 POL 455 SLYADSPSV 55 HepB A*02:01 POL 816 YMDDVVLGA 56 Hep B A*02:01 POL 551 ALMPLYACI 57 Hep B A*02:01 POL 655 VLHKRTLGL 58 Hep B A*02:01 HBx 92 CLFKDWEEL 59 Hep B A*02:01 Hbxll5 STLPETTVVRR 60 Hep B A*03, AHI Core 141 EYLVSFGVW 61 Hep B A*31, A*68 core 117 FFPSIRDLL 62 HepB A*24 Core 23 SWLSLLVPF 63 HepB A*24 Env 334 KYTSFPWLL 64 Hep B A*24 Pol 756 HLSLRGLFV 65 Hep B A*02:01 HBx 52-60 CLFKDWEEL 66 Hep B A*02:01 HBx 115-123 LPSDFFPSV 67 Hep B B*51 Core 19 GILGFVFTL 68 Influenza HLA-A2 Ml ILGF V FTLTVPSERGLQRRRF 69 Influenza LIRHENRMVLASTTAKA 70 Influenza LQA YQKRMGV QMQR 71 Influenza YVYDHSGEAVK 72 WLSLLVPFV 73 (K)GH.£1FVFTL(T)(V) 74 KLSTRGVQIASNEN 75 RGLQRRRFVQNALNGNG 76 FMYSDFHFI 77 NLVPMVATV 3 VAIIEVDNEQPTTRAQKL 78 Any 9-mer sequence of GACV AIIEVDNEQPTTRAQKL.F AMWRITYKDTVQLRRKL 79 SVRDRLARL 80 LLDRVRFMGV 81 CLGGLLTMV 82 GLCTLVAML 83

[0081] In various embodiments the epitope is derived from an epitope of a human vaccine. In various embodiments the vaccine is an early childhood vaccine. Certain nonlimiting examples of suitable vaccines are listed in Table 2 below: Table 2: Approved Vaccines Containing Recall Protein Epitopes Type Commercial Name Form Source Responsible National Regulatory Authority Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Hemophilus influenzae type b Quinvaxem Liquid: ready to use Janssen Vaccines Corp. Ministry of Food and Drug Safety Diphtheria-Tetanus Adsorbed DT Vaccine Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Diphtheria- T etanu s ■ Pertussis (whole cell) DTP Vaccine Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Hepatitis B Hepatitis B Vaccine Recombinant Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Polio Vaccine - Oral (OPV) Trivalent Oral polio Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Polio Vaccine - Oral (OPV) Trivalent Oral polio Liquid: ready to use PT Bio Farma (Persero) National Agency of Drag and Food Control Indonesia Tetanus Toxoid TT vaccine Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Tetanus Toxoid TT vaccine Liquid: ready to use PT Bio Farma (Persero) National Agency of Drag and Food Control Indonesia Tetanus Toxoid TT vaccine Liquid: ready to use PT Bio Farma (Persero) National Agency of Drag and Food Control Indonesia Measles Measles vaccine Lyophilized Lyophilized active component to be reconstitute d with excipient diluent before use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Yellow Fever Yellow Fever Lyophilized Lyophilized active component to be reconstitute d with excipient Bio- Manguinho s / Fiocruz Agenda Nacional da Vigilancia Sanitaria diluent before use Yellow Fever Yellow Fever Lyophilized Lyophilized active component to be reconstitute d with excipient diluent before use Bio-Manguinho s / Fiocruz Agencia National da Vigilancia Sanitaria Yellow Fever Yellow Fever Lyophilized Lyophilized active component to be reconstitute d with excipient diluent before use Bio-Manguinho s / Fiocruz Agencia National da Vigilancia Sanitaria Hepatitis B Heberbiovac HB Liquid: ready to use Centro de Ingenieria Genetica y Biotecnolo gia Centro para el Control Estatal de la Calidad de los Medicamentos Hepatitis B Heberbiovac HB Liquid: ready to use Centro de Ingenieria Genetica y Biotecnolo gia Centro para el Control Estatal de la Calidad de los Medicamentos Rabies Rabipur Lyophilized active component to be reconstitute d with excipient diluent before use Chiron Behring V accines Private Ltd. Central Drags Standard Control Organization Rabies Rabipur Lyophilized active component to be GlaxoSmit hKHne Paul-Ehrlich- Institut reconstitute d with excipient diluent before use Vaccines GmbH Haemophilus influenzae type b Vaxeni HIB Liquid: ready to use Novartis Vaccines and Diagnostics S-r.f Agenzia Italiana del Farmaco Hepatitis B Engerix Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Hepatitis B Engerix Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Hepatitis B Engerix Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Trivalent Polio sabin Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Trivalent Polio sabin Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Measles, Mumps and Rubella Priorix Lyophilized active component to be reconstitute d with excipient diluent before use GlaxoSmit hKline Biologicals SA ’ Federal Agency for Medicines and Health Products Rotavirus Rotarix Liquid: ready to use GlaxoSmit hKline Federal Agency for Medicines and Health Products Biologicals SA ~ Polio Vaccine - Oral (OPV) Trivalent Polioviral vaccine Liquid: ready to use Haffkine Bio Pharmacal Corporatio n Ltd Central Drugs Standard Control Organization Yellow Fever Stabilized Yellow Fever Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Institut Pasteur de Dakar Ministere de la Sante publique Yellow Fever Stabilized Yellow Fever Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Institut Pasteur de Dakar Ministere de la Sante publique Yellow Fever Stabilized Yellow Fever Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Institut Pasteur de Dakar Ministere de la Sante publique BCG BCG Freeze Dried Glutamate vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Japan BCG Laboratory Chiba Local Government Hepatitis B EuvaxB Liquid: ready to use LG Chem Ltd Ministry of Food and Drug Safety Hepatitis B EuvaxB Liquid: ready to use LG Chem Ltd Ministry of Food and Drug Safety BCG BCG Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Bui Bio ■ National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency BCG BCG Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Bui Bio -National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Tetanus Toxoid Tetatox Liquid: ready to use Bui Bio -National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Tetanus Toxoid Tetatox Liquid: ready to use Bui Bio -National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Diphtheria-Tetanus Diftet Liquid: ready to use Bui Bio -National Center of Infectious and Parasitic Bulgarian Drug Agency Diseases Ltd. Diphtheria-Tetanus Diftet Liquid: ready to use Bui Bio ■ National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Diphtheria- T etanu s (reduced antigen content) Tetadif Liquid: ready to use Bui Bio -National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Diphtheria-Tetanus (reduced antigen content) Tetadif Liquid: ready to use Bui Bio -National Center of Infectious and Parasitic Diseases Ltd. Bulgarian Drug Agency Diphtheria- T etanu s ■ Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Easyfive-TT Liquid: ready to use P 3UHCC3. Biotec Ltd. Central Drags Standard Control Organization Diphtheria-Tetanus (reduced antigen content) IMOVAX dT adult Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine - Inactivated (IPV) IMOVAX POLIO Liquid: ready to use Sanofi Pasteur SA Agcncc nationale de securite du medicament et des produits de sante Polio Vaccine - Oral (OPV) Trivalent OPVERO Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine - Oral (OPV) Trivalent OPVERO Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine - Oral (OPV) Trivalent OPVERO Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Tetanus Toxoid TETAVAX Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Tetanus Toxoid TETAVAX Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Haemophilus influenzae type b Act-HIB Lyophilized active component to be reconstitute d with excipient diluent before use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Rabies VERORAB Lyophilized active component to be reconstitute d with excipient diluent before use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Y ellow Fever STAMARIL Lyophilized active component to be reconstitute d with excipient diluent before use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Meningococcal A+C POLYSACC HARIDE MENINGOC OCCAL A+C VACCINE Lyophilized active component to be reconstitute d with excipient diluent before use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine - Oral (OPV) Monovalent Type 1 ORAL MONOVAL ENT TYPE 1 POLIOMYE LITIS VACCINE Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante cholera: inactivated oral Dukoral Liquid: ready to use Valneva Sweden AB Medical Products Agency BCG BCG Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diph theria-T etanus Diphtheria and Tetanus Vaccine Adsorbed (Paediatric) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diphtheria-Tetanus Diphtheria and Tetanus V accine Adsorbed (Pediatric) Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diphtheria-Tetanus Diphtheria and Tetanus Vaccine Adsorbed (Pediatric) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-Tetanus (reduced antigen content) Diphtheria and Tetanus Vaccine Adsorbed for Adults and Adolescents Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diphtheria-Tetanus (reduced antigen content) Diphtheria and Tetanus Vaccine Adsorbed for Adults and Adolescents Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diph theria-T etanus (reduced antigen content) Diphtheria and Tetanus Vaccine Adsorbed for Adults and Adolescents Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-Tetanus-Pertussis (whole cell) DiphtheriaTetanusPertussis Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diph theria-Tetanus- Pertussis (whole cell) Diphtheria Tetanus Pertussis V accine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell) DiphtheriaTetanusPertussis Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B Diphtheria .Tetanus, Pertussis and Hepatitis B Vaccine Adsorbed Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B Diphtheria (Tetanus, Pertussis and Hepatitis B Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Vaccine Adsorbed Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B Diphtheria ,Tetanus, Pertussis and Hepatitis B Vaccine Adsorbed Liquid: ready to use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Hepatitis B Hepatitis B Vaccine (rDNA) (Adult) Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Hepatitis B Hepatitis B Vaccine (rDNA) (Adult) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Hepatitis B Hepatitis B Vaccine (rDNA) (Paediatric) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Hepatitis B Hepatitis B Vaccine (rDNA) (Paedriatic) Liquid: ready to use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Tetanus Toxoid Tetanus Toxoid Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Tetanus Toxoid Tetanus Toxoid V accine Adsorbed Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Tetanus Toxoid Tetanus Toxoid Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles and Rubella Measles and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization excipient diluent before use Measles and Rubella Measles and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles and Rubella Measles and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles and Rubella Measles and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles, Mumps and Rubella Measles, Mumps and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles, Mumps and Rubella Measles, Mumps and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization excipient diluent before use Measles, Mumps and Rubella Measles, Mumps and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles, Mumps and Rubella Measles, Mumps and Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles Measles Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Measles Measles Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Measles Measles Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization excipient diluent before use Measles Measles Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Rubella Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization Rubella Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Seram Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Rubella Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Rubella Rubella Vaccine, Live, Attenuated Lyophilized active component to be reconstitute d with Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization excipient diluent before use Tetanus Toxoid ShanTT Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drags Standard Control Organization Tetanus Toxoid ShanTT Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drags Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Shan-5 Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drugs Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Shan-5 Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drugs Standard Control Organization BCG BCG Vaccine SSI Lyophilized active component to be reconstitute d with excipient diluent before use AJ Vaccines A / S Danish Medicines Agency Rotavirus Rotateq Liquid: ready to use Merck Vaccines CBER / FDA Measles, Mumps and Rubella rHA M-M-R II Lyophilized active component to be reconstitute d with Merck Vaccines European Medicines Agency excipient diluent before use Rotavirus Rotarix Liquid: ready to use GlaxoSmit hKline Biologicals SA ' Federal Agency for Medicines and Health Products Yellow Fever Lyophilized active component to be reconstitute d with excipient diluent before use Federal State Budgetary Scientific Institution «Chumako v Federal Scientific Center for Reserch & Developme nt of Immune-And Biological Productsv, Russian Academy of Sciences Federal Service on Surveillance in Healthcare (ROSZDRAVNAD ZOR) of tire Russian Federation Yellow Fever Lyophilized active component to be reconstitute d with excipient diluent before use Federal State Budgetary Scientific Institution «Chumako v Federal Scientific Center for Reserch & Developme nt of Immune-And Biological Products», Russian Academy of Sciences Federal Service on Surveillance in Healthcare (ROSZDRAVNAD ZOR) of the Russian Federation Y ellow Fever Lyophilized active component to be reconstitute d with excipient diluent before use Federal State Budgetary Scientific Institution «Chumako v Federal Scientific Center for Reserch & Developme nt of Immune-And Biological Products», Russian Academy of Sciences Federal Service on Surveillance in Healthcare (ROSZDRAVNAD ZOR) of the Russian Federation Human Papillomavirus (Quadrivalent) Gardasil Liquid: ready to use Merck Vaccines European Medicines Agency Human Papillomavirus (Bivalent) Cervarix Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Human Papillomavirus (Bivalent) Cervarix Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Monovalent Type 1 Polio Sabin Mono T1 Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Monovalent Type 1 Polio Sabin Mono T1 Liquid: ready to use GlaxoSmit hKline Biologicals SA ‘ Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Polio Sabin One and Three Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Polio Sabin One and Three Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Haemophilus influenzae type b Haemophilus influenzae type b Conjugate Vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine - Oral (OPV) Monovalent Type 1 Monovalent type 1 Oral Poliomyelitis vaccine, IP (mOPVl) Liquid: ready to use Haffkine Bio Pharmaceut ical Corporatio n Ltd Central Drugs Standard Control Organization Polio Vaccine Oral (OPV) Monovalent Type 1 Monovalent Oral Poliomyelitis Vaccine Type 1 (mOPVl) Liquid: ready to use PT Bio Farma (Persero) National Agency of Drag and Food Control Indonesia Tetanus Toxoid None used on labelling for supply through UN agencies. Also marketed with labelled commercial name BEtt. Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Pneumococcal (conjugate) Synflorix Liquid: ready to use GlaxoSmit hKline Biologicals SA ' European Medicines Agency Diph theria-Tetanus- Pertussis (whole cell)- Hepatitis B-Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus Lyophilized active component to be reconstitute d with liauid Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization influenzae type b Conjugate Vaccine active component before use Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Bivalent Oral Poliomyelitis Vaccine Type 1&3 (bOPV 1 & 3) Liquid: ready to use PT Bio Farrna (Persero) National Agency of Drug and Food Control Indonesia Meningococcal A Conjugate 10 pg Meningococ cal A Conjugate MenAfriVac Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Haemophilus influenzae type b Quimi-Hib Liquid: ready to use Centro de Ingenieria Genetica y Biotecnolo gia Centro para el Control Estatal de la Calidad de los Medicamentos Pneumococcal (conjugate) Synflorix Liquid: ready to use GlaxoSmit hKline Biologicals SA European Medicines Agency Influenza, seasonal Fluvirin Liquid: ready to use Seqirus Vaccines Limited CBER / FDA Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Bivalent type 1&3 Oral Poliomyelitis vaccine, IP (bOPVl&3) Liquid: ready to use Haffkine Bio Pharmaceut ical Corporatio n Ltd. Central Drugs Standard Control Organization Influenza, seasonal Fluzone Liquid: ready to use Sanofi Pasteur-USA CBER / FDA Influenza, seasonal Fluzone Liquid: ready to use Sanofi Pasteur-USA CBER / FDA Diphtheria- T etanu s ■ Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus influenzae type b Conjugate Vaccine Lyophilized active component to be reconstitute d with liquid active component before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Influenza, seasonal GC FLU Multi inj. Liquid: ready to use Green Cross Corporatio n Ministry of Food and Drug Safety Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus influenzae type b Conjugate V accine Lyophilized active component to be reconstitute d with liquid active component before use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Influenza, pandemic H1N1 Panvax Liquid: ready to use Seqirus Limited Therapeutic Goods Administration Influenza, pandemic H1N1 Green Flu-S Liquid: ready to use Green Cross Corporatio n Ministry of Food and Drug Safety Influenza, pandemic H1N1 Influenza A (H1N1) 2009 monovalent vaccine Liquid: ready to use Medlmmun e CBER / FDA Influenza, pandemic HiM Celtura Liquid: ready to use Seqirus GmbH Paul-Ehrlich- Institut Influenza, pandemic HiXI ~ Focetria Liquid: ready to use Seqirus Vaccines Limited Influenza, pandemic H1NI Fluvirin-HINI Liquid: ready to use Seqirus Vaccines Limited CBER / FDA Influenza, pandemic HiM Panenza Liquid: ready to use Sanofi Pasteur SA Agcncc nationale de securite du medicament et des produits de sante Influenza, pandemic H1NI Influenza A (HINT) 2009 monovalent vaccine Liquid: ready to use Sanofi Pasteur-USA CBER / FDA Influenza, pandemic H1N1 Influenza A (H1N1) 2009 monovalent vaccine Liquid: ready to use Sanofi Pasteur-USA CBER / FDA Diphtheria-Tetanus-Pertussis (whole cell)-Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis and Haemophilus influenzae type b Conjugate Vaccine Lyophilized active component to be reconstitute d with liquid active component before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine -Inactivated (IPV) Poliorix Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Polio Vaccine -Inactivated (IPV) Poliorix Liquid: ready to use GlaxoSmit hKline Biologicals SA ' Federal Agency for Medicines and Health Products Pneumococcal (conjugate) Prevenar13 Liquid: ready to use Pfizer European Medicines Agency Diph theria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus influenzae type b Conjugate Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Vaccine Adsorbed Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus influenzae type b 0 onj u g at e Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-T etanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Diphtheria, Tetanus, Pertussis, Hepatitis B and Haemophilus influenzae type b Conjugate Vaccine Adsorbed Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine - Oral (OPV) Monovalent Type 3 Polio Sabin Mono Three (oral) Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Monovalent Type 3 Polio Sabin Mono Three (oral) Liquid: ready to use GlaxoSmit hKline Biologicals SA ’ Federal Agency for Medicines and Health Products Polio Vaccine - Inactivated (1PV) Poliomyelitis vaccine Liquid: ready to use Bilthoven Biologicals Medicines Evaluation Board (MEB) Polio V accine Inactivated (IPV) IPV Vaccine SSI Liquid: ready to use AJ V accines A / S Danish Medicines Agency Influenza, seasonal GC FLU inj Liquid: ready to use Green Cross Corporatio n Ministry of Food and Drug Safety Polio Vaccine - Oral (OPV) Monovalent Type 2 Polio Sabin Mono Two (oral) Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Polio Vaccine - Oral (OPV) Monovalent Type 2 Polio Sabin Mono Two (oral) Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Typhoid (Polysaccharide) Typhim-Vi Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Influenza, seasonal Vaxigrip Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 BIOPOLIO Bl / 3 Liquid: ready to use Bharat Biotech Intemation al Limited Central Drugs Standard Control Organization Diphtheria-Tetanus (reduced antigen content) none Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 none Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name ComBE Five (Reconstitute d). Lyophilized active component to be reconstitute d with liquid active component before use Biological E. Limited Central Drags Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name ComBE Five (Reconstitute d). Lyophilized active component to be reconstitute d with liquid active component before use Biological E. Limited Central Drags Standard Control Organization cholera: inactivated oral Shanchol Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drugs Standard Control Organization Measles, Mumps and Rubella Priorix Lyophilized active component to be reconstitute d with excipient diluent before use GlaxoSmit hKline Biologicals SA ’ Federal Agency for Medicines and Health Products Measles Measles vaccine Lyophilized active component to be reconstitute d with excipient diluent before use PT Bio Fatma (Persero) National Agency of Drug and Food Control Indonesia Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Poliomyelitis Vaccine (Oral), Bivalent types 1 and 3 Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Influenza, pandemic HIM NASOVAC Influenza Vaccine, Lyophilized active component Serum Institute of Central Drags Standard Control Organization Live Attenuated (Human) Freeze-Dried to be reconstitute d with excipient diluent before use India Pvt. Ltd. Influenza, pandemic H1N1 NASOVAC Influenza Vaccine, Live Attenuated (Human) Freeze-Dried Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Tetanus Toxoid None used on labelling for supply through UN agencies. Also marketed with labelled commercial name BEtt. Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Tetanus Toxoid None used on labelling for supply through UN agencies. Also marketed with labelled commercial name BEtt. Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Japanese Encephalitis Vaccine (Inactivated) 6ug JEEV® Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Hepatitis A (Human Diploid Cell), Inactivated (Adult) Havrix 1440 Adult Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Hepatitis A (Human Diploid Ceil), Inactivated (Paediatric) Havrix 720 Junior Liquid: ready to use GlaxoSmit hKline Biologicals SA ~ Federal Agency for Medicines and Health Products Diphtheria-T etanus- Pertussis (acellular) Boostrix Liquid: ready to use GlaxoSmit hKline Biologicals SA Federal Agency for Medicines and Health Products Meningococcal ACYW-135 (conjugate vaccine) Menveo Lyophilized active component to be reconstitute d with liquid active component before use GlaxoSmit hKline Vaccines S.r.l. European Medicines Agency Meningococcal ACYW-135 (conjugate vaccine) Menactra Liquid: ready to use Sanofi Pasteur- USA CBER / FDA Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Easyfive-TT Liquid: ready to use Panacea Biotec Ltd. Central Drugs Standard Control Organization Japanese Encephalitis Vaccine (live, attenuated) Japanese Encephalitis Vaccine Live (SA14-14-2) Lyophilized active component to be reconstitute d with excipient diluent before use Chengdu Institute of Biological Products Co.,Ltd National Medical Products Administration Japanese Encephalitis Vaccine (live, attenuated) Japanese Encephalitis Vaccine Live (SA14-14-2) Lyophilized active component to be reconstitute d with excipient diluent before use Chengdu Institute of Biological Products Co. .Ltd National Medical Products Administration Diphtheria-Tetanus- Pertussis (whole ceil ;■ Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name ComBE Five (Liquid). Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name ComBE Five (Liquid). Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Japanese Encephalitis Vaccine (live, attenuated) IMOJEV MD Lyophilized active component to be reconstitute d with excipient diluent before use GPO-MBP Co., Ltd. Thai Food and Drag Administration Diphtheria-T etanu s ■ Pertussis (whole cell) None used on labelling for supply through UN agencies. Also marketed with labelled commercial name TRIPVAC Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Diphtheria-Tetanus-Pertussis (whole cell) None used on labelling for supply through UN agencies. Also marketed with labelled commercial name TRIPVAC Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Diphtheria-T etanus (reduced antigen content) None used on labelling for supply through UN agencies. Also marketed with labelled commercial name BE Td Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Diph theria-Tetanus (reduced antigen content) None used on labelling for supply through UN agencies. Also marketed with labelled commercial name BE Td Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Poliomyelitis Vaccine (Oral), Bivalent types 1 and 3 Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine -Inactivated (IPV) Poliomyelitis vaccine multi dose, suspension for injection 2.5 mL Liquid: ready to use Bilthoven Biologicals Medicines Evaluation Board (MEB) Influenza, seasonal Nasovac-S Influenza Vaccine, Lyophilized active component Seram Institute of Central Drags Standard Control Organization Live, Attenuated (Human) to be reconstitute d with excipient diluent before use India Pvt. Ltd. Diphtheria-Tetanus- Pertussis (acellular)- Hepatitis B-Haemophilus influenzae type b-Polio (Inactivated) Hexaxim Liquid: ready to use Sanofi Pasteur SA European Medicines Agency Meningococcal A Conjugate 5 pg Meningococ cal A Conjugate 5 micrograms MenAfriV ac 5ug Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name Com BE Five (Liquid). Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b None used on labelling for supply through UN agencies. Also marketed with labelled commercial name ComBE Five (Liquid). Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Polio Vaccine - Inactivated (1PV) Poliomyelitis Vaccine (Inactivated) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drags Standard Control Organization Polio Vaccine - Oral (OPV) Trivalent BIOPOLIO Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization Polio Vaccine - Oral (OPV) Trivalent BIOPOLIO Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization Influenza, seasonal Influenza Vaccine (Split virion, inactivated) Liquid: ready to use Huai an Biological Bacterin Co., Ltd National Medical Products Administration Influenza, seasonal IL-YANG FLU Vaccine INJ. Liquid: ready to use IL-YANG PHARMA CEUTICA LCO., LTD. Ministry of Food and Drag Safety BCG BCG vaccine (Freeze Dried) -Intradermal Lyophilized active component to be reconstitute d with excipient diluent before use GreenSigna 1 Bio Pharma Limited Central Drugs Standard Control Organization Influenza, seasonal Quadrivalent Fluzone Quadrivalent Liquid: ready to use Sanofi Pasteur-USA CBER / FDA Influenza, seasonal Quadrivalent Fluzone Quadrivalent Liquid: ready to use Sanofi Pasteur- USA CBER / FDA Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Bivalent Oral Poliomyelitis Vaccine Type 1&3 (bOPV 1&3) Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia cholera: inactivated oral Euvichol Liquid: ready to use EuBiologic s Co., Ltd. Ministry of Food and Drug Safety Polio Vaccine - Oral (OPV) Monovalent Type 2 ORAL MONOVAL ENT TYPE 2 POLIOMYE LITIS VACCINE (mOPV2) Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Polio Vaccine Oral (OPV) Monovalent Type 3 ORAL MONOVAL ENT TYPE 3 POLIOMYE LITIS VACCINE Liquid: ready to use Sanofi Pasteur SA Agence nationale de securite du medicament et des produits de sante Meningococcal ACYW-135 (conjugate vaccine) Nimenrix Lyophilized active component to be reconstitute d with excipient diluent before use Pfizer European Medicines Agency Diphtheria- T etanu s ■ Pertussis (whole cell)-Hepatitis B-Haemophilus influenzae type b Eupenta Liquid: ready to use LG Chem Ltd Ministry of Food and Drug Safety Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Eupenta Liquid: ready to use LG Chem Ltd Ministry of Food and Drug Safety Human Papillomavirus (Ninevalent) Gardasil 9 Liquid: ready to use Merck Vaccines European Medicines Agency Influenza, seasonal Quadrivalent GCFLU Quadrivalent inj. Liquid: ready to use Green Cross Corporatio n Ministry of Food and Drug Safety Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Pentabio Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Diphtheria-Tetanus- Pertussis (whole cell)- Hepatitis B- Haemophilus influenzae type b Pentabio Liquid: ready to use PT Bio Farma (Persero) National Agency of Drug and Food Control Indonesia Hepatitis A (Human Diploid Cell), Inactivated (Adult) HEALIVE Liquid: ready to use Sinovac Biotech Co. Ltd National Medical Products Administration Varicella Varivax Lyophilized active component to be reconstitute d with excipient diluent before use Merck Vaccines CBER / FDA Rotavirus (live, attenuated) Rotavac Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization Diphtheria- T etanu s ■ Pertussis (acellular) Adacel Liquid: ready to use Sanofi Pasteur Limited Health Canada -Sante Canada Influenza, seasonal AGRIFLU Liquid: ready to use Seqirus Vaccines Limited Health Canada -Sante Canada Pneumococcal (conjugate) Prevenar 13 Multidose Vial Liquid: ready to use Pfizer European Medicines Agency Typhoid (Conjugate) Typbar-TVC Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Poliomyelitis Vaccine (live, oral attenuated, human Diploid Cell), type 1 and 3 Liquid: ready to use Beijing BioInstitute Biological Products Co.,Ltd National Medical Products Administration Japanese Encephalitis Vaccine (Inactivated) (3pg Pediatric) JEEV® Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization Rotavirus (live, attenuated) ROTASIIL Lyophilized active component to be reconstitute d with excipient diluent before use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine -Inactivated (IPV) Poliomyelitis Vaccine (Inactivated) Liquid: ready to use Serum Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Polio Vaccine -Inactivated (IPV) Poliomyelitis Vaccine (Inactivated) Liquid: ready to use Serum. Institute of India Pvt. Ltd. Central Drugs Standard Control Organization Influenza, seasonal Quadrivalent GCFLU Quadrivalent Multi inj. Liquid: ready to use Green Cross Corporatio n Ministry of Food and Drug Safety Influenza, seasonal Serinflu Liquid: ready to use Abbott Biologicals BV Medicines Evaluation Board {MLB; Polio Vaccine -Inactivated (IPV) ShanIPV Liquid: ready to use Shantha Biotechnics Private Limited (A Sanofi Company) Central Drugs Standard Control Organization Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 Bivalent OPV Type 1 and 3 Poliomyelitis Vaccine, Live (Oral) Liquid: ready to use Panacea Biotec Ltd. Central Drags Standard Control Organization cholera: inactivated oral Euvichol- Plus Liquid: ready to use EuBiologic s Co., Ltd. Ministry of Food and Drug Safety Polio Vaccine - Oral (OPV) Bivalent Types 1 and 3 BIOPOLIO Bl / 3 Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization BCG BCG Freeze Dried Glutamate vaccine Lyophilized active component to be reconstitute d with excipient diluent before use Japan BCG Laboratory Chiba Local Government Pneumococcal (conjugate) Synflorix Liquid: ready to use GlaxoSmit hKline Biologicals SA ’ European Medicines Agency Rotavirus (live, attenuated) Rotavac Liquid: ready to use Bharat Biotech Internation al Limited Central Drags Standard Control Organization Hepatitis A (Human Diploid Cell), Inactivated (Paediatric) HE ALIVE Liquid: ready to use Sinovac Biotech Co. Ltd National Medical Products Administration Typhoid (Conjugate) Typbar-TVC Liquid: ready to use Bharat Biotech Internation al Limited Central Drugs Standard Control Organization Rotavirus (live, attenuated) ROTASIIL Lyophilized active component to be reconstitute d with Seram Institute of India Pvt. Ltd. Central Drags Standard Control Organization excipient diluent before use Japanese Encephalitis Vaccine (Inactivated) 6ug JEEV® Liquid: ready to use Biological E. Limited Central Drags Standard Control Organization Japanese Encephalitis Vaccine (Inactivated) (3pg Pediatric) JEEV® Liquid: ready to use Biological E. Limited Central Drugs Standard Control Organization

[0082] In various embodiments, the recall protein epitope is released following proteolytic cleavage and the epitope binds to an MHC class I molecule. The MHC class I molecule may be from the HLA-A, B, and / or C families. The specific epitope that binds to the MHC class I molecule may be any of those recited in Table 1 or Table 2. The MHC class I molecule itself may be, one or more of the following non-limiting examples: HLA-A*02:01, HLA-A*03:01, HLA-A* 11:01, HLA-A*201, HLA-A*020101, HLA-A*0203, HLA-A*0206, HLA-A2, HLA-A2.1, or HLA-A*02.

[0083] In an aspect of the invention the recall protein is about 8 amino acid to about 50 amino acids in length, or about 8 amino acid to about 45 amino acids in length, or about 8 amino acid to about 40 amino acids in length, about 8 amino acid to about 35 amino acids in length, or about 8 amino acid to about 30 amino acids in length, about 8 amino acid to about 25 amino acids in length, about 8 amino acid to about 20 amino acids in length, or is about 8 amino acid to about 15 amino acids in length. In an aspect of the invention tire fusion protein is about 13 amino acid to about 50 amino acids in length, or about 13 amino acid to about 45 amino acids in length, or about 13 amino acid to about 40 amino acids in length, about 13 amino acid to about 35 amino acids in length, or about 13 amino acid to about 30 amino acids in length, about 13 amino acid to about 25 amino acids in length, about 13 amino acid to about 20 amino acids in length, or is about 13 amino acid to about 15 amino acids in length. In an aspect of the invention the CD8+ T cell epitope may be, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids in length.

[0084] Cleavage Sequence. In various embodiments, a cleavage sequence is provided that allows the recall protein to be released from the VLP so that the recall protein can bind to the MHC on the tumor cell surface. In various embodiments, the VLP must escape the endosome, disassemble, and release their therapeutic cargo to the cytosol in a functional form. In various embodiments the conjugated VLP and / or fusion protein of the conjugated VLP is susceptible to cleavage by a proteolytic enzy me within the tumor cell and tire position of the target cleavage sequence in the VLP or fusion protein is such that the cleave of the target site releases all or a portion of the recall protein comprising the CD8+ T cell epitope from the conjugated VLP, which complexes with an MHC class 1 molecule of the tumor cell. Sufficient amounts of conjugated VLP are readily determined by the skilled artisan and it will be appreciated that the amount will depend on, e.g., the characteristics of the subject, e.g., age, weight, gender, and / or medical condition of the subject, and the characteristics of the tumor, e.g., type, volume, and developmental status.

[0085] The cleavage sequence may be recognized by any protease present in a cell. At least 569 known proteases have been described (see, Lopez-Otin, et al., Nature Reviews Cancer, 7(10):800-808, 2007). All identified human proteolytic enzymes tire classified into five catalytic classes: metalloproteinases, serine, threonine, cysteine and aspartic proteases. A non-limiting list of potential proteases that could be targeted is demonstrated in Table 3, which is a table summarizing the most well studied proteases distributed into five broad classes (in order from greatest to least number): metalloproteinases, serine, cysteine, threonine, and aspartic proteases. Several of these proteases have been found to be over-expressed in cancer cells relative to health cells.

[0086] In various embodiments, the cleavage sequence is recognized by the protease furin, a matrix metalloproteinases (MMPs), e.g., MMP, 1, 2, 3, 7, 8, 9, 11, 13, 14, or 19, an ADAM (a disintegrin and metalloproteinase), e.g., ADAMS 8, 9, 10, 15, 17, or 28, a cathepsin, e.g., cathepsin D, G, H, or N. Elastase, proteinase-3, azurocidin, or ADAMTS-1. In various embodiments, the cleavage sequence is recognized by a furin protease. In various embodiments, the cleavage sequence comprises at least 4 amino acid residues, at least three of which are arginine residues. In various embodiments, the cleavage sequence comprises at least 4 amino acid residues, at least three of which are arginine residues and one of which is either a lysine residue or an arginine residue. In various embodiments, the cleavage sequence is R-X-R / K-R. In various embodiments, the cleavage sequence comprises additional residues. In various embodiments, tire cleavage sequence further comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 additional arginine residues. It is known that arginines are positively charged, and a longer chain of positive charged Arginine residues will bring the peptides closer to the surface of the VLP which is more negati vely charged. 'Table 3: Proteases and cancers associated with overexpressed proteases FamBy Pretense Location Cancer Ret Other Diseases Ref. General fotraceUidar, lysosomes Most Table in

[121] Cathepsin K Exkacelhilar, bone Breast

[178] Arthercaderoais, osteoporosis [179-182] Cysteine Extracellular Mid Breast, cervix, colon. Csthepsim Cathepsin B periceUular crdoractal, gastric, head and neck, liver, tang, melanoma, [31,38,81, 183-196] pstbclogicil cocdsticxs ovarian, pancreatic, prostate, thyroid Cathepsin L Breast, colorectal

[28] AD

[197] Aspartic Cathepsin E EtkdOMfiMt structures, ER, Lysosome Cervical, gastric, lung. pMSOCMHI sdeOOCflfCttkCOQMl [51-55] Cathepsins Cathepsin D Breast, colorectal, ovarian [47-49,198200] Atherosclerosis

[121] General IntraceUular, secreted Most Table in [15, 58] Kallikreins hKl Hypertension, wflanwimtinn

[24] W PSA(hK3) Prostate, ovarian [201-202] hK10 Colon, ovarian, pancreatic, head and neck [203-206] hKl 5 Ovarian, prostate [207-208] Seine uPA,uPAR Maribrane, Cervical, colorectal, gastric. [86,116, Proteases Pericellular prostate 209-210] CgSpaSQS IntraceUular Neurodegenerative disorders

[82] General Extracellular Most Table in PH] MMP-1,-8,-13 Breast [85,102-104, 211-212] Artherosclerosis, RA [213-214] MMPs Btcochicctasts, chronic MMP-2.-9 Breast, colorectal, tang, [91-94] [95- asthma, COTO, cystic fibrosis, HIV [87,113- malignunC gtinmaa ovarian 98] associated dementia, hypertension, stroke 117] MMP-14 Membrane Breast P12] ADAM Extracelhilar AD [105,107, 112] ’AbbreviaHaas: AD: Alzhasmer's disease; ADAM: a disntegrin and metalloproteinase domain protease; COPIA erratic obstractnre pulmonary disease; ER: endoplasmic reticulum; RA, rheumatoid ardmtis

[0087] In various embodiments, the fusion protein must be bound to the capsid proteins of the VLP. There are multiple methods of binding the fusion protein to the capsid protein. In various embodiments of the present invention the cleavage sequence is conjugated through a maleimide linkage or an amide linkage (discussed below). The fusion protein may be linked to any residue on the capsid protein of the VLP; however, disulfide linkages, maleimide linkages, and amide linkages tire formed by conjugating the recall protein to cysteine, lysine, or arginine residues. Surface Functionalization of the VLP

[0088] The VLPs described herein must be functionalized to deliver a recall protein, which must be presented on the surface of the conjugated VLP. In various embodiments, recall proteins may be conjugated to the VLP through cysteine residues on the capsid protein. Such cysteine molecule can be presented naturally, or by mutation on the surface of the VLP. In various embodiments, the VLP is subjected to reducing conditions sufficient to reduce the sulfhydryl groups of cysteine residues on the surface of the VLP while maintaining the capsid-like icosahedron structures of the VLP. Because of its free sulfhydryl group, cysteine will readily and spontaneously form disulfide bonds with other sulfhydryl-containing ligands under oxidative conditions. Alternatively, a series of compounds add on maleimide readily and irreversibly form thioester linkages with cysteine residues at a pH between 6.5 and 7.5.

[0089] In various embodiments, the fusion protein may also be conjugated to a lysine residue on the VLP. Lysine residues are easily modified because of its primary amine. Using reactions termed n-hydroxysuccinimide (NHS) ester reactions (because NHS is released as prat of the reaction), amide bonds are formed at surface-exposed lysine residues. The reaction occurs spontaneously between pH 7.2 and pH 9. This attachment chemistry has been used to display transferrin on MS2, which may allow the VLP to transcytose the blood-brain barrier that could open up a new library of therapies for neurological disorders.

[0090] In various embodiments, the fusion protein may also be conjugated to an aspartate- or glutamate residue. Unlike strategies involving cysteine and lysine, coupling to these residues requires multiple steps. First, the carboxylic acid must be activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Once activated, it will react with NHS to form an NHS ester. Now that the carboxylic acid side-chain has essentially become an NHS ester, a ligand with an exposed primary amine can be used to from a stable amide bond.

[0091] In various embodiments, the VLP comprises a region of negatively charged amino acids on a surface exposed area that is capable of binding to the fusion protein comprising a region of positively charged amino acids. In various embodiments, the region of negatively charged amino acids may be flanked, on one or on both sides, by one or more cysteine residues, referred to as polytinionic: cysteine or more specifically, polyglutamic acid:cysteine or polyaspartic acid:cysteine. In such cases, the conjugation of the VLP and the fusion protein would result from non-covalent binding between the complementary amino acid charges of the VLP and the fusion protein and a disulfide bond between the cysteines. In various embodiments, the cysteine(s) are one or more amino acids away from the region of charged amino acids such that any secondary / tertiary structure would bring the charged amino acid region in close proximity to the cysteine(s). In various embodiments of the invention the fusion protein comprises at least one recall protein and a polyionic:cysteine for attaching the fusion protein to the VLP comprising a complementary polyionic:cysteine sequence and an enzyme cleavage site positioned between the terminal cysteine and the CD8+ T cell epitope. In various embodiments, the fusion protein comprises, a terminal cysteine, at least one recall protein, and an enzyme cleavage sequence positioned between the terminal cysteine and the recall protein(s).

[0092] Negatively charged amino acids that can be used in producing the conjugated VLP include, e.g., glutamic acid and aspartic acid. These amino acids can be used singly, e.g., polyglutamic acid, or in combination. In a specific embodiment, the region comprises glutamic acid. The number of negatively charged amino acids can vary, and can include about 4 to about 20 amino acids, about 6 to about 18 amino acids, or about 8 to about 16 amino acids, and the like. In a specific embodiment, the region comprises about 8 negatively charged amino acids. In a more specific embodiment, the region comprises EEEEEEEEC (E8C) (SEQ ID NO: 84). In another embodiment, the region comprises CEEEEEEEEC (SEQ ID NO: 85). Methods for conjugating fusion proteins to a VLPs via disulfide bonding tire known. For instance, the presence of a polyargininecysteine moiety on the fusion protein allows docking of the peptide to the polyanionic site (EEEEEEEEC, E8C, SEQ ID NO: 84) present in the various loops of the LI particles. Covalent cross-linking between the two cy steine residues should render this association irreversible under oxidizing conditions. For the conjugation reactions, purified HPV particles are dialyzed in conjugation buffer (20 mM Tris / HCl pH = 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM CaCb) and then the peptide and the oxidizing reagents are added, allowing the reaction to proceed for 16 hrs at 4 °C. At the end of the incubation, the reaction mixtures are applied to a size-exclusion column (SEPHADEX® G-100, Pharmacia, New Jersey, US, volume 20 ml, flow' rate 1 ml / min, 10 mM Tris / HCl (pH = 7.4), 150 mM NaCl, 0.5 mM CaCh) to remove unconjugated peptide and exchange buffer. Conjugated particles that elute in the void volume are identified by the presence of the LI protein on SDS-PAGE. The conjugated particles are analyzed by electron microscopy. One of ordinary skill in the art can, through routine experimentation, create a VLP that includes a polyionic region in a surface exposed area, e.g., one or more loops, and that is VLP assembly competent.

[0093] In various embodiments, the fusion protein is genetically fused to the coat protein. In various embodiments, the fusion protein may be either covalently or non-covalently linked to the VLP. Rather than attaching the recall protein to the VLP via, e.g., binding of negatively and positively charged amino acids, or via maleimide based conjugation, a nucleic acid sequence encoding the fusion protein may be inserted into the nucleic acid encoding the capsid protein such that upon expression, a fusion protein is produced wherein the recall protein is inserted into a loop of the capsid protein and displayed on the surface of the VLP.

[0094] In various embodiments, non-natural amino acids may be used to conjugate the fusion protein to the VLP. Beyond the 20 natural amino acids, many non-natural amino acids have been used for site-specific protein conjugation reactions. For example, an azidohomoalanine (AHA) or a p-amino-phenylalanine (p AF) may be incorporated into the VLP coat protein for conjugation. These amino acids tire incorporated into proteins in two ways: global methionine replacement and amber stop codon suppression. Because AHA is very similar to methionine, AHA will be incorporated at each AUG codon if the methionine supply is rate limiting, this is termed global methionine replacement. Bacteria auxotrophic for methionine or cell-free protein synthesis can be used to limit-methionine availability. Amber stop codon suppression will incorporate pAF. Amber stop codon suppression uses nonnative synthetases and tRNAs that do not react with the natural amino acids to incorporate the nonnatural amino acid at the amber stop codon UAG. AHA, displaying an azide, will participate in in copper(l)-catalyzed azide-alkyne cycloaddition (“click” reaction) and form covalent triazole rings with alkyne-containing ligands.

[0095] In various embodiments, the conjugated VLP comprises, at least one-tenth of the viral coat proteins may display a recall protein. In various embodiments, at least onefifth of the viral coat proteins may display a recall protein. In various embodiments, about half of the viral coat proteins may display a recall protein. In various embodiments, about two-thirds of the viral coat proteins may display a recall peptide. In various embodiments, nearly all of the viral coat proteins may display a recall protein. Methods of Treatment

[0096] In various embodiments of the invention, a method for treating a cancer in a subject in need thereof by administering a conjugated VLP to patient in need thereof. The methods of this invention comprise administering the conjugated VLPs of this invention to a subject in need thereof in an amount sufficient to inhibit tumor growth, progression or metastasis. In various embodiments of the invention, the conjugated VLP is administered to a subject in need thereof in amount sufficient to stimulate cytokine production and / or cellular immunity, particularly innate immunity, including stimulating the cytotoxic activity of macrophages and natural killer cells. In various embodiments of this invention a subject in need thereof is a subject who has been previously treated for a tumor and is currently deemed cancer-free or disease free in accordance with medical standards.

[0097] Briefly, the mechanism, of action of the described conjugated VLPs is depicted in Figure 1. The VLPs specifically bind to a tumor cell. (Figure 1, left side, tumor cells). The recall protein epitope on the VLP is then proteolytically cleaved by furin, which is over-expressed in the tumor microenvironment. This in turn leads to release of the epitope from the conjugated VLP and the loading of the epitopes onto the tumor MHC class 1 molecules ("‘epitope coating”). (Figure 1, middle exploded view showing block MHI molecule loading with epitope from VLP). The epitope-coated tumor cell is then recognized as a pathogen-infected cell by one or more childhood vaccine T-cells and pre-existing CD8 T cells, yielding a triggered immune redirection response. (Figure 1, far right side of diagram). That is, this recognition event leads to the host’s pre-existing immune memory against pathogens and childhood vaccines activating against the tumor in the tumor microenvironment, attacking and destroying the tumors. Destruction of tumor cells can result in components of the preexisting immune response being exposed to cancer cell antigens. Thus, antigens released from the killed tumor cells will initiate an immune response to recruit additional tumor-specific CD8 T cells, or a “second wave” of T cells that then proceed to attack additional tumor cells in the area. This can result in elicitation of an endogenous immune response against the cancer cell antigens (commonly known as “epitope spreading”), and leads to anti-tumor immune memory.

[0098] Thus, the methods disclosed herein are methods of treating cancer in an individual by utilizing the individual's own pre-existing adaptive memory immune system to attack cancer cells. The methods described herein make use of the fact that individuals possess preexisting immune responses that were not originally elicited in response to a cancer, but that were elicited instead by routine vaccination scheduling or via microorganisms and pathogens present in the natural environment. Because the cancer cells would not normally express the microbial antigens that elicited the preexisting immune response, it would not be expected that such an immune response would attack a cancer. However, by way of the present methods, such preexisting immune responses can be recruited to attack, kill, and clear a cancer in a subject. This is achieved by introducing into or onto the surface of the cancer one or more antigens known to be recognized by the preexisting immune response in the subject, resulting in cells of the immune response attacking antigen-displaying cancer cells. Further, destruction of cancer cells can result in components of the preexisting immune response being exposed to additional cancer cell antigens. Thus, a general method of the invention can be practiced by recruiting a preexisting microbial or vaccine immune response in an individual to the site of a cancer, such that the preexisting immune response attacks the cancer. Thus, there are generally four steps involved in the method, including: 1) binding VLPs to the tumor cells, cleavage of the epitope from the VLP leading to MHC binding of the epitopes for display on the tumor cell surface, recognition of the loaded MHC by the subject’s pre-existing recalled immunity against the epitope, and triggering of a second wave and longer-term anti-tumoral immuni ty thereafter.

[0099] Data obtained from cell culture assays and animal studies may be used in formulating a range of dosages for use in humans. The dosages of such compositions lie preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the methods of the invention, the therapeutically effective dose may be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a halfmaximal inhibition of symptoms) as determined in cell culture. Such information may be used to accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0100] In many instances, it will be desirable to have multiple administrations of the VLP-containing composition, usually at most, at least, or not exceeding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more vaccinations including all ranges there between. The vaccinations will normally be at 1, 2, 3, 4, 5, 6, to 5, 6, 7, 8, 9, 10, 11, to 12 week / month / year intervals, including all values and ranges there between, more usually from three to five week intervals.

[0101] In various embodiments, a method is provided for stimulating the cytotoxic activity of macrophages and natural killer cells by administering to a subject in need thereof an effective amount of a conjugated VLP of this invention. The macrophages and natural killer cells may be those present in the tumor microenvironment. In an aspect of this invention, the conjugated VLPs are administered to the subject in an amount effective to stimulate the cytotoxic activity of macrophages and natural killer cells already present in the tumor microenvironment. In various embodiments of this invention, the conjugated VLPs are administered to the subject in an amount effective to attract macrophages and natural killer cells to the tumor microenvironment.

[0102] In various embodiments of the invention, the conjugated VLPs are administered to the subject in an amount effective to bind sufficient numbers of antibodies to the recall protein to attract and stimulate macrophages, neutrophils and natural killer cells.

[0103] In various embodiments of the invention, a method is provided for redirecting the cytotoxic activity of an existing memory CD8+ T cell to a tumor cell or tumor microenvironment by administering to a subject in need thereof an effective amount of the conjugated VLP of this invention. Preferably, the T cell epitope of the recall protein of the conjugated VLP is from a pathogen for which the subject has been actively vaccinated or from a pathogen that has previously infected the subject and the subject has memory CD8+ T cells that recognize the T cell epitope in complex with an MHC class I molecule on the tumor cells. In an aspect of this invention the effective amount of the conjugated VLP is an amount sufficient to attract the memory CD8+ T cell to the tumor microenvironment. In an aspect of this invention the effective amount of the conjugated VLP is an amount sufficient to stimulate the memory CD8+ T cell present in the tumor microenvironment.

[0104] In various embodiments, the tumor is a small lung cell cancer, hepatocellular carcinoma, liver cancer, hepatocellular carcinoma, melanoma, metastatic melanoma, adrenal cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, brain / CNS cancer, breast cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon / rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (gist), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, non-Hodgkin lymphoma, Hodgkin lymphoma, Burkitt's lymphoma, lymphoblastic lymphomas, mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal or nodal), mixed cell type diffuse aggressive lymphomas of adults, large cell type diffuse aggressive lymphomas of adults, large cell immunoblastic diffuse aggressive lymphomas of adults, small non-cleaved cell diffuse aggressive lymphomas of adults, or follicular lymphoma, head and neck cancer, endometrial or uterine carcinoma, non-small cell lung cancer, osteosarcoma, glioblastoma, or metastatic cancer. In a preferred embodiment, the cancer is a breast cancer, a cervical cancer, an ovarian cancer, a pancreatic cancer or melanoma,

[0105] The term "cancer" as used herein refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium., carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of toe small intestine, cancer of the endocrine system cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem, glioma, glio-blastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma and Ewing’s sarcoma, including refractory versions of any of toe above cancers, or a combination of one or more of the above cancers.

[0106] An aspect of the invention is a method for treating a cancer in a subject in need thereof by administering a conjugated VLP of this invention to the subject wherein the CD8+ epitope of the recall protein is of a failed therapeutic cancer vaccine against a Viral-induced cancer e.g. HPV cervical cancer, HPV+ oral cancer, EBV nasopharyngeal cancer (the “therapeutic vaccine”). The method comprises determining if toe subject has been actively vaccinated against toe vaccine but did not respond with an anti-tumor effect to the treatment. The patient is then administering to the subject an effective amount of a conjugated VLP of this invention wherein the CD8+ epitope of the recall protein is of the anti genic determinant in the vaccine previously administered to the subject that infected the subject.

[0107] In various embodiments, a method is provided for treating a cancer in a subject in need thereof by administering a conjugated VLP of this invention to the subject wherein the CD8+ epitope of toe recall protein is of a failed CAR-T cell therapy against a Viral-induced cancer e.g. HPV cervical cancer, HPV+ oral cancer, EBV nasopharyngeal cancer (the “CART”). The method comprises determining if the subject has been actively treated with the CART but did not respond with an anti-tumor effect to the treatment. The patient is then administering to the subject an effective amount of a conjugated VLP of this invention wherein the CD8+ epitope of the recall protein is of the antigenic determinant in the CAR-T previously administered to the subject that infected the subject.

[0108] In various embodiments, a method is provided for heating a cancer in a subject in need thereof by administering a conjugated VLP of this invention to the subject wherein the CD8+ epitope of the recall protein is of a failed Vaccine or CAR-T cell therapy against a cancer (the “CAR-T”). The method comprises determining if the subject has been actively heated with the CAR-T but did not respond with an anti-tumor effect to the treatment. The patient is then administered an effective amount of a conjugated VLP of this invention wherein the CD8+ epitope of the recall protein is of the antigenic determinant in the CAR-T previously administered to the subject that infected the subject.

[0109] VLPs have adjuvant properties. In some embodiments, the immunogenicity of the conjugated VLP compositions of this invention can be enhanced by the use of additional nonspecific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions such as alum..

[0110] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include 11.,-1, IL-2, IL-4, IL-7, IL-12, y-interferon, GM-C-SF, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MOP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL), or inactivated microbial agents. RIBI, which contains three components extracted from bacteria, MPL., trehalose dimycolate (TOM), and cell wall skeleton (CW-S) in a 2% squalene / Tween 80 emulsion. MHC antigens may even be used.

[0111] Various methods of achieving adjuvant affect for the conjugated VLP compositions includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1 % solution in phosphate buffered saline, admixture with synthetic polymers of sugars (CARBOPOL®) used as an about 0.25% solution, aggregation of a protein in the composition by heat treatment with temperatures ranging between about 70°C to about 101 °C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells, e.g., C. parvum, endotoxins or lipopolysaccharide components of Gramnegative bacteria; emulsion in physiologically acceptable oil vehicles, e.g., mannide monooleate (Aracel A™), or emulsion with a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a block substitute may also be employed to produce an adjuvant effect. A typical adjuvant is complete Freund's adjuvant (containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide.

[0112] For administration to humans, a variety of suitable adjuvants will be evident to a skilled worker. These include, e.g., Alum-MPL as adjuvant, or the comparable formulation, ASO4, which is used in the approved HPV vaccine CERVARIX®, AS03, AS02, MF59, montanide, saponin-based adjuvants such as GPI-0100, CpG-based adjuvants, or imiquimod. In embodiments of the invention, an adjuvant is physically coupled to the VLP, or encapsulated by the VLP, rather than simply mixed with them. In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA, US); or low-dose Cyclophosphamide (CYP; 300 mg / ml ) (Johnson / Mead, NJ, US) and cytokines such as y-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7. In embodiments of the invention, these genes are encapsulated by the VLP to facilitate their delivery into a subject.

[0113] The preparation of compositions that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art. Typically, such compositions are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the compositions may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. In specific embodiments, vaccines are formulated with a combination of substances.

[0114] The composi tions comprising the conjugated VLPs of the present invention are in biologically compatible form suitable for administration in vivo to subjects. The pharmaceutical composi tions further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the VLP is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be a carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose may be carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may be employed as liquid earners for injectable solutions. Suitable pharmaceutical excipients Include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried slim milk, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0115] The pharmaceutical compositions comprising the conjugated VLPs of the present invention can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. In a specific embodiment, a pharmaceutical composi tion comprises an effective amount of a conjugated VLP of the present invention together with a suitable amount of a pharmaceutically acceptable carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0116] The pharmaceutical compositions of the present invention may be administered by any particular route of administration including, but not limited to intravenous, intramuscular, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, oral, parenteral, subcutaneous, vaginal, rectal, buccal, sublingual, intranasal, iontophoretic means, or transdermal means. Most suitable routes are intravenous injection or oral administration. In particular embodiments, the compositions are administered at or near the target area, e.g., intratumoral injection.

[0117] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, intratumoral, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the indi vidual subject.

[0118] The conjugated VLP-containing compositions of this invention may be administered by inhalation. In certain embodiments a composition can be administered as an aerosol. As used herein the term "aerosol" or "aerosolized composition" refers to a suspension of solid or liquid particles in a gas. The terms may be used generally to refer to a composition that has been vaporized, nebulized, or otherwise converted from a solid or liquid form to an inhalable form including suspended solid or liquid drug particles. Such aerosols can be used to deliver a composition via the respiratory system. As used herein, "respiratory system" refers to the system of organs in the body responsible for the intake of oxygen and the expiration of carbon dioxide. The system generally includes all the air passages from, the nose to the pulmonary alveoli. In mammals it is generally considered to include the lungs, bronchi, bronchioles, trachea, nasal passages, and diaphragm. For purposes of the present disclosure, delivery of a composition to the respiratory system indicates that a drug is delivered to one or more of the air passages of the respiratory system, in particular to the lungs.

[0119] Additional formulations which are suitable for other modes of administration include suppositories (for anal or vaginal application) and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 % to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and toe like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain about 10% to about 95% of acti ve ingredient, preferably about 25% to about 70%.

[0120] The conjugated VLP compositions may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with toe free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium., ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0121] The pharmaceutical compositions of the present invention can also include an effective amount of an additional adjuvant. As noted herein, papillomavirus VLPs have adjuvant properties. Suitable additional adjuvants include, but are not limited to, Freund's complete or incomplete, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as Bacille Calmette-Guerin (BCG), Corynebacterium parvum, and non-toxic cholera toxin.

[0122] Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In all cases the pharmaceutical form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0123] The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, find vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0124] Sterile injectable solutions are prepared by incorporating the conjugated VLPs in the required amount in the appropriate solvent with various ingredients enumerated above, as required may be followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0125] Different aspects of the present invention involve administering an effective amount of a composition comprising die conjugated VLPs to a subject in need thereof. In some embodiments of the present invention, a conjugated VLP comprising a target peptide comprising a CD8+ T cell epitope is administered to the patient to treat a tumor or prevent the recurrence of such tumor. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Methods of Providing an Appropriate VLP

[0126] In various embodiments, a method for providing a conjugated VLP to a patient in need thereof is provided comprising: (1) measuring the preexisting immunity in a patient, and (2) selecting the appropriate conjugated VLP for administration of a patient in need. The appropriate conjugated VLP to administer to the patient will depend upon the patients T cell profile. The appropriate conjugated VLP will be one that is capable of eliciting a T cell response that is at least twice the baseline total of CD8+ cells. In various embodiments, the appropriate conjugated VLP will be one that is capable of eliciting a T cell response that is twice the baseline total of CD8+ or total CD8+ CD69+ T cells. The goal is to choose the appropriate conjugated VLP based on the patient’s vaccination history or prior exposure to a pathogen. Determining which conjugated VLP is appropriate can be achieved through: (1) patient interviews; (2) review of a patient’s medical records; and / or (3) assessing the patient’s T cell profile.

[0127] In various embodiments, more than one recall protein might be suitable for eliciting an immune response directed at a tumor. In various embodiments, a conjugated VLP carrying either recall protein will be appropriate. In various embodiments, more than one recall protein is expressed and bound to the VLP. In various embodiments, a single fusion protein will comprise more than one recall protein. In various embodiments, multiple fusion proteins comprising different recall proteins will be conjugated to the VLP. In various embodiments, the invention comprises a population of conjugated VLPs as described herein and a pharmaceutically acceptable excipient. In various embodiments, the conjugated VLPs administered to the subject are identical. In various embodiments, conjugated VLPs carrying different recall protein(s) are administered to a subject.

[0128] Selection Based on Prior Vaccination. In various embodiments of the invention, a method of selecting an appropriate conjugated VLP is provided in order to administer to a subject in need thereof. In various embodiments this involves ascertaining if the subject has been actively vaccinated against a given pathogen, e.g., a parasite, a bacterium, or virus, e.g., measles or polio, and then selecting and administering to the subject a conjugated VLP as disclosed herein wherein the CD8+ T cell epitope of tire recall protein is from the pathogen against which the subject has been immunized. In various embodiments, a subject’s vaccination history is obtained by reviewing the subject’s medical record. In various embodiments, a subject’s vaccination history is obtained by interviewing the subject.

[0129] Selection Based on Prior Infection. In various embodiments, the method of selecting an appropriate conjugated VLP for administration to a subject in need thereof involves ascertaining if a subject has been previously infected with a given pathogen, e.g., a parasite, a bacterium, or virus, e.g., measles or polio, and resolved the infection. In various embodiments, the subject is then administered a conjugated VLP comprising a recall protein which comprises said pathogen for which the subject has been previously infected.

[0130] One may ascertain if a subject has been infected with a particular pathogen by reviewing the subjects’ medical records or interviewing the subject. Non-limiting examples of CD8+ T cell epitopes that bind to particular MHC class I molecules fire set forth in Table 1. The method may also comprise determining which MHC class I determinant(s) the subject’s cells express and then administering a conjugated VLP of this invention wherein the CD8+ T cell epitope of the recall protein is a CD8+ T cell epitope of the antigenic component of the pathogen in the vaccine or of the pathogen that previously infected the subject that forms a complex with the subject’s MHC class I determinant(s).

[0131] Measuring T cell Responses. In various embodiments, a patient’s T cell profile is assessed in order to select an appropriate conjugated VLP using various techniques known in the art. This profile is then used to select the appropriate conjugated VLP to administer. Such techniques including measuring interferon-y levels, using flow7 cytometry to isolate Ag-specific CD8+ T cells, and / or cytotoxicity assays. To measure interferon-y (a marker of T cell activation), intracellular staining of isolated T cells. Alternatively, an enzyme-linked immunosorbent spot (ELISPOT) assay for interferon-y may be conducted. This technique allows for a high throughput assessment of a patient’s T cell profile. This method can potentially detect one in 100,000-300,000 cells. Briefly, a monoclonal antibody for a specific cytokine is pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate. CD8+ T cells are pipetted into the weiis along with dendritic cells and individual peptides and the microplate is placed into a humidified 37°C CO2 incubator for a period ranging from 24 to 48 h. During incubation, the immobilized antibody binds the cytokine secreted from the cells. After washing a detection antibody specific for the chosen analyte is added to the wells. Following the washes, enzyme conjugated to streptavidin is added and a substrate is added. A colored precipitate forms, according to the substrate utilized and appears as spot at the sites of cytokine secretion, with each individual spot representing a single producing cell.

[0132] In various embodiments, the invention described here in provides a method of determining the appropriate conjugated VLP to administer to a patient in need thereof, by assessing the patients T cell profile, comprising: (1) collecting PBMCs from patients / participants (pre-vaccination sample), (2) preparing the ELISPOT plates by coating with anti-IFN-y antibody (incubate overnight), (3) Incubating PBMCs with one of the pool of peptides of interest, ones expected to elicit a T cell response (incubate for 1-2 days), (4) washing the plates, adding a biotinylated secondary antibody (incubating for a few hours), (5) washing the plates, adding avidin conjugated horseradish peroxidase and incubating, (6) washing plates, adding aminoethyl carbazole (AEC) for a few minutes, (7) stopping the reaction (water), and (8) visualizing on an ELISPOT reader. The disclosed method can detect up to one in 100,000-300,000 cells. A two-fold increase in the frequency of antigen-specific T cells should be considered as a signal.

[0133] In various embodiments T cell proliferation can be measured by 3H (tritiated)-thymidine. Such methods are sensitive and can be used for high throughput assays. Such techniques may also include carboxyfluorescein succinimidyl ester (CFSE) and Ki64 intracellular staining.

[0134] Selecting Recall Proteins based on Tropism. It is known in the art that some viruses display a tropism for particular type of tissue. For example: viruses that display a tropism for brain tissue include without limitation, JC virus, measles, LCM virus, arbovirus and rabies; viruses that display a tropism for eye tissue include without limitation herpes simplex virus, adenovirus, and cytomegalovirus; viruses that display a tropism for nasal tissue include without limitation, rhinoviruses, parainfluenza viruses, and respiratory syncytial virus; viruses that display a tropism for oral tissue, e.g., oral mucosa, gingiva, salivary glands, pharynx, include without limitation, herpes simplex vims type I and type II, mumps virus, Epstein Bair virus, and cytomegalovirus; viruses that display a tropism for lung tissue include without limitation, influenza virus type A and type B, parainfluenza virus, respiratory syncytial virus, adenovirus, and SARS coronavirus; viruses that display a tropism for nerve tissue, e.g., the spinal cord, include without limitation poliovirus and HTLV-1; viruses that display a tropism for heart tissue, include without limitation, Coxsackie B viius; viruses that display a tropism for liver tissue, include without limitation, hepatitis viruses types A, B, and C; viruses that display a tropism for gastrointestinal tissue, e.g., stomach, and large and small intestine, include without limitation, adenovirus, rotaviius, norovirus, astrovirus, and coronavirus; viruses that display a tropism for pancreatic tissue, include without limitation, coxsackie B virus; viruses that display a tropism for skin tissue, include without limitation, varicella zoster vims, herpes simplex virus 6, smallpox virus, mohuscum contagiosum, papilloma viruses, parvovirus B19, rubella, measles and coxsackie A virus; and viruses that display a tropism for genital tissue, include without limitation, herpes simplex type 2, papillomaviruses, human immunodeficiency virus (HIV).

[0135] In various embodiments, a method for treating a cancer in a subject in need thereof is provided by administering a conjugated VLP of this invention to the subject wherein the recall protein is a CD8+ epitope of a pathogen that has a tropism for the tissue that is the source of the cancer (the “source tissue”). In various embodiments, the appropriate conjugated VLP is selected by first determining the source tissue of the tumor cell and then selecting a recall protein: (1) to which the patient already has existing CD8+ T cells, and (2) that has a tropism, for the source tissue of the tumor. The selected conjugated VLP(s) are then administered to the patient in need thereof.

[0136] In various embodiments, a method for treating a lung cancer comprising determining if a subject has been actively vaccinated against a pathogen that infects lung cells, e.g., an influenza virus, e.g., influenza virus type A or type B, then administering an effective amount of a conjugated VLP of this invention wherein the CD8+ T cell epitope of the recall protein is of the antigenic determinants of the pathogen contained in the vaccine and which T cell epitope forms a complex with an MHC molecule class I of the subject. In an aspect of a method of this invention for treating a lung cancer includes determining if a subject has been infected with pathogen that infects lung cells, e.g., an influenza virus, e.g., influenza virus type A or type B, then administering an effective amount of a conjugated VLP of this invention wherein the CD8+ T cell epitope of the recall protein is of that pathogen and which T cell epitope forms a complex with an MHC class I molecule of the subject.

[0137] An aspect of the invention is a method for treating an oral cancer, which are part of the group of cancers commonly referred to as head and neck cancers, by administering a conjugated VLP of this invention wherein the CD8+ epitope of the recall protein is of a pathogen that has a tropism for oral tissue, e.g., a mumps virus, Epstein Barr virus, cytomegalovirus, or a herpes simplex vims type I. The method comprises determining if a subject in need thereof has been actively vaccinated against, or infected with, e.g., a mumps virus, Epstein Barr virus, cytomegalovirus, or a herpes simplex virus type 1, and if the subject has been vaccinated or infected previously then administering to the subject a conjugated VLP of this invention wherein the CD8+ epitope of the recall protein is of a mumps virus or a measles virus or of the antigenic component of the vaccine the subject had received, or of the pathogen, i.e., mumps, measles, Epstein Bair virus, cytomegalovirus, or a herpes simplex virus type 1, that had previously infected the subject. Combination Therapy

[0138] In various embodiments, the conjugated VLP may be co-administered with other cancer therapeutics. Furthermore, in some embodiments, the conjugated VLPs described herein are administered in conjunction with other cancer treatment therapies, e.g., radiotherapy, chemotherapy, surgery, and / or immunotherapy. In some aspects of this invention, the conjugated VLPs described herein are administered in conjunction with checkpoint inhibitors. In various embodiments the chimer VLP may be administered in conjunction with an immune agonist. In various embodiments, the conjugated VLP may be administered in conjunction with treatment with a therapeutic vaccine. In various embodiments, the conjugated VLP may be administered in conjunction with treatment with a conjugated antigen receptor expressing T cell (CAR-T cell). In various embodiments, the conjugated VLP may be administered in conjunction with treatment with another immuno-oncology product. The conjugated VLPs of the present invention and other therapies or therapeutic agents can be administered simultaneously or sequentially by the same or different routes of administration. The determination of the identity and amount of therapeutic agent(s) for use in the methods of the present invention can be readily made by ordinarily skilled medical practitioners using standard techniques known in the art. EXAMPLES Example 1 Conjugated VLPs bind to mouse tumors in vivo

[0139] The ability of the described conjugated VLPs to bind to tumors is an important first step for the proposed mechanism of action (see Figure 1). Binding of VLP in vivo was assessed using a murine cervical cancer model, TC-1. Briefly, unconjugated VLPs are produced in Expi293F™ human cells (ThermoFisher Scientific, Waltham, MA, US) using the Expi293F™ Expression system. In this protocol, 360 x 106 cells in 120 mL of cultured media is prepared in a 250 mL shake flask. Following this, to express the HPV16 LI VLP, cells were transfected with the HPV16 LI gene expression vector (Cat #89910, Addgene, Cambridge, MA, US) via the manufacturer’s protocols (ThermoFisher Scientific, Wraltham, MA, US). Transfected cells were maintained for 72 hrs and were then harvested. HPV particles were released from Expi293F cells by detergent lysis and the lysate was incubated overnight at 37 °C with a mild detergent, e.g. Brij58 or Triton X-100. The VLP from the overnight incubated lysates were then solubilized by addition of sodium chloride. The lysate was then clarified by low-speed centrifugation. Capsids were separated from cell debris and detergent by high salt extraction followed by ultracentrifugation through an OPTIPREP™ (iodixanol) step gradient according to manufacturer's instructions (Sigma-Aldrich, St Louis, MI, US). Purity of the VLP preparation was determined by SDS- PAGE Coomassie stained gel (4-20% CRITERION™ gel) staining as per manufacturer’s protocol (Biorad Laboratories, Hercules, CA, US) and morphology of particles assessed by electron microscopy (EM). (See also, WO 2018 / 237115).

[0140] The HPV16 LI wild type protein sequence as reported elsewhere is as follows (SEQ ID NO: 86): Met Ser Leu Vai P r o V a 1 S e r Asn lie Tyr Tyr Pro Tyr Phe Pro Lys Vai Ser Gly Pro Asn Lys Phe Gin Arg Leu Vai Leu Gly Vai Asp Thr Glu Asn Arg Glu Cys lie Gly Cys Lys Pro Thr Asn Vai Lila Asn Thr Vai lie Trp Leu Pro Lys Vai Vai His Ala Gly lie Lys Lys Leu Gin Tyr Gly Phe Pro Trp Ala Cys Gly Tie Ser Ala Ser Ala Ser Met Asp Pro Tie Gly Vai Asn Pro Gin Asp Gly Ser Glu Ala Ser Thr Asp Thr Ser Arg Pro Asn Asn Arg Vai Phe Asp Thr Ser Vai Gly Vai Gly His Pro Tyr Ala Ala Tyr Lys Gin Glu His Trp Gly Asp Cys Aso Met Vai Thr Vai Tyr Glu Tyr Vai Leu Leu Ala Asn Lys Tie Arg Tie His Phe Tvr Asn Glu Vai Gly Leu Leu Asn Asn Ala Gly Thr Gin Leu Gly Lys Gly Pro Pro Leu Asp Thr Gly Leu Pro Pro Ala Arg Thr Vai Gly His Leu Vai Pro Leu Pro Asp Pro Asp Thr Arg Gly Gin Lys Leu Asp Vai Asp Asn Cys Leu Tie Ser Pro Cys Glu Leu Tie Phe Gly Ala Asp Phe Thr He Cys Thr Ser Glu Pro Tyr Gly Phe Vai Arg His Pro Asp Asp Leu Ser Asn Tyr Ala Gin lie Phe Asn Asn Gly He Thr Thr Arg Ser Glu Thr Thr Tyr Glu Glu Tyr Asp Thr Ala Asp Vai Glu Asp Trp Asn Asp Thr Tyr Arg Thr Pro Pro Ala Glu Vai Asn Leu Leu Gly Arg Lys Phe Thr Leu Gly Thr Thr Ala Lys Thr Leu Gin He Cys Lys Asp Ser Leu Leu Phe Asn Tyr lie Lys Phe Pro Thr Asn Lys Pro Cys Trp Gly Thr Asn Met Lys Asn Thr Leu Gin Phe Met Thr Tyr Phe Gly Leu Phe Vai Thr Pro Lys Glu Lys Glu Lys Phe Leu Leu Lys Arg Lys Arg Lys Lys Ala Asn Lys Tyr Pro Asp Phe Phe Tyr Arg Ala Gly Gly Ser Gly Pro Ser Gly Tyr Trp Leu Asn Gin Leu Ser Leu Cys Asn Phe Lys He Phe Gin He His Ser Gin Pro Pro Ser Gin Ala A s p P r o L e u Phe Ser Ala Gin Ala Gly Ala Thr Pro Arg Lys Leu Ser Glu Vai Tyr Tie Lys Leu Arg Arg I h r \7 a 1 a 1 y Ser Thr Ala Ser Met Vai Gin Arg Ala Phe Vai Thr Ala Ala He Glu Tyr Leu Leu Cys Lys Asn Ssr Pro Gly Gly He Ala Cys Lys Lys Tyr Asp Leu Asp Leu Lys Ala Thr Thr Ser Pro Leu Asp Met Vai Ser Glu Gin Miet Glu Asn Vai Asn Leu Ala Thr Ser Asp Gin Gly His Vai Vai Asp Ser Thr Ser Arg His Gly He Thr Leu Thr He Leu Thr Leu Glu Gin Lys His Thr Phe Trp Gin Phe Pro Lys Pro Lys Sgt Tnr Ssr

[0141] For functional assays, live C57BL / 6 mice (n=5, The Jackson Laboratory, Bar Harbor, MA, US) were subcutaneously injected with luciferase / green fluorescent protein (GFP)-expressing TC-1 tumor cells (2 x 105 cells). Mice were monitored for approximately 2 weeks to ensure consistency in tumor growth and also until palpable tumors were observed (about 7 mm in diameter). (See, FIG. 2A). Three mice were injected intravenously with 50 pg of VLP. Two mice served as untreated controls. After 16-24 hours post injection of the VLP, all 5 mice were sacrificed and the tumors were excised. Tumors were then digested with collagenase before being treated at a dilution of 1:100 at 4 °C in fluorescence activated cell sorting (FACs) staining buffer (phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA)), first with HPV16 anti-V5 anti-mouse antibody specific for VLP (Abeam, Burlingham., CA, US). After one hour, cells were then washed twice with FACs staining buffer and stained for another hour at a dilution of 1:100 at 4 °C with goat phycoerythrin (PE)-conjugated anti-mouse antibody specific for VLP (R&D systems, Minneapolis, MN, US). Cells were then washed twice again with FACs staining buffer before being analyzed by fluorescence activated cell sorting (FACS) cytometry: (1) first, gated for GFP to sort for only tumor cells; and (2) second, to assess the sorted tumor cells for the presence of the VLP (anti-Ll VLP antibody). (See FIG. 2B, right panel and left panel are data each generated from a different mice). FIG. 2B demonstrates that the conjugated VLPs were able to bind to the tumor, as indicated by rightward shift on the histogram plot of both the left and right panels. Example 2 Conjugated VLP bind to human tumors in vitro

[0142] To test for the ability of conjugated VLPs to bind to human tumors in vitro, five human tumor cells lines are to be assessed: OVCAR3, MDA-MB231, HCT116, PC-3, and SKOV3 (ATCC, Manassas, VA, US). The respective cell lines will be first seeded in one well within a 6 well plate. After 16 to 24 hours, cells will be dissociated using 0.05% trypsin. Cells will then be counted to achieve 1 x 106 cells, centrifuged, and the supernatant removed before being re-suspended in 100 pl. of FACS buffer (1% FBS in PBS). Next, 0.3 pg / ml of conjugated VLP will be added to the respective cultures in vitro and the cultures incubated for 24 hours under optimal growth conditions. To show that die binding is HSPG-specific, conjugated VLPs will be either pre-incubated with or without heparin (final concentration of 0.1 mg / ml) and incubated at 4°C (to prevent endocytosis) for 1 hr. After 1 hr of binding, cells will be washed twice with 1 mL of FACS buffer before being resuspended in 100 pL of FACS buffer again. The samples will then be incubated with PE-conjugated anti-mouse antibody specific for VLP at 4°C for 1 hour. After 1 hour, cells will be washed and re-suspended in FACS buffer for analysis by flow cytometry. Binding is then assessed either as a percent positive of total PE+ cells, or of the geometric mean fluorescence, as indicated by a rightward shift on the histogram plot, as in Example 1 and Figures 2A and 2B. Example 3 Conjugated VLP bind to human tumors in vivo

[0143] The described conjugated VLPs will be further tested to assess their ability to bind in vivo to human tumors. In a first experiment, mice will be injected with OVCAR3 tumor cells (3 x 10° cells), a human ovarian cancer cell line. At approximately two weeks after injection, the average tumor volume is expected to be approximately 10 mm3 in volume. Three weeks post injection, the tumor bearing mice will be administered a Cu2+ or Zn2+ radiolabeled VLPs. Biodistribution and tumor specificity of the VLP is to be assessed at 4, 12, 24, and 48 hours. The tumors are then removed for weighing, radioactive counting, viral micro distribution and cellular colocalization. In addition to tumors, various major organs such as the liver, spleen, kidneys will be assessed for VLPs presence using imaging methods as well as histology to assess tire efficacy of VLP homing to the tumor. In further experiments, mice are to be injected with other tumor cell lines such as HCT116, PC-3, or SKOV3 and the same experimental protocol followed. Example 4 VLP-induced T cell-mediated killing of tumor cells in vitro in a time-dependent manner

[0144] The ability of conjugated VLPs to deliver epitopes to MHC proteins on a tumor cell surface and subsequently redirect existing T cells to subject tumor cells leading to cytotoxic killing of tumor cells was tested in vitro. We chose to demonstrate proof-of-concept efficacy using pre-existing murine memory responses to the human papillomavirus HPV16 E7 antigen in a murine tumor model as this would be more clinically relevant. HPV16 E7is a true human viral antigen, as opposed to often used artificial antigens such as ovalbumin (OVA). It is known that several different E7 anti gen-specific CD8'H T-cells fire found in the peripheral blood of healthy human donors with different HLA-I (MHC-I) genes. Therefore, the use of E7 is relevant, provides proof-of-concept showing the redirection of pre-existing CD8+ T-cell responses to target a non-virally associated malignancy (tumor), and acts as a solid base from which to extend this system to other human viral antigens.

[0145] Conjugation of VLPs. To produce conjugated VLPs, recall proteins were synthesized to greater than 85% purity as polycationic 18-mer to 20-mer peptides comprising a maleimide molecule at the N-terminus, followed by a protease cleavage site and ending with a CD8+ recall epitope, i.e. N-terminal maleimide-RRRRRVKR-epitope (Genscript, New Jersey, US). Samples were sent as lyophilized materials and diluted to a concentration of 1 to 20 mM with dimethyl sulfoxide (DMSO). Conjugated VLP was prepared for conjugation via the following protocol. First, VLPs at a concentration of at least 1 mg / mL were dialyzed into conjugation reaction buffer (50 mM sodium phosphate, pH 6.5, 500 niM NaCl, 2 mM EDTA, 0.05% Tween 80) with buffer exchanged 3 times (3 + 1 h, 3 + 1 h, and overnight 16 + 3 h at 2 to 8 °C). The next day, VLPs were then treated with TCEP for 1 hour without shaking at room temperature at a TCEP:L1 (monomer) ratio of 10:1 molar ratio, where the concentration of LI monomer was 0.77 mg / mL. Subsequently, conjugation was performed by adding the 18- to 20-mer peptide at a molar ratio of peptide:Ll monomer of 5:1, bringing the final concentration of LI monomer to 0.58 mg / mL. The reaction was shaken at 200 rpm for 1 hour. Following conjugation, contents from the reaction were subjected to dialysis (PBS, pH 7.0 + 0.1, 500 mM NaCl, 0.05% Tween 80) using a dialysis cassette (MWCO = 1000 kDa) for about 3 + 1 hours in a cold room (2 to 8 °C). Next, the contents were subjected to density gradient ultracentrifugation (UC) OPTIPREP™ gradient (in DPBS, 0.8M NaCl) at 40,000 rpm, for 16 hours at 16 °C. Fractions (1-3) starting from the interface between 33 - 39% OPTIPREP™ were collected and then subjected to a final dialysis (MWCO = 1000 kDa) for 3 + 1,3+1, and 16 + 3 hours in a cold room. Following purification, samples were analyzed under gel electrophoresis / Coomassie staining to determine % conjugation via the shift in LI band beyond about 55 kDa.

[0146] Quantification of conjugated VLPs. Product concentrations, as well as % fusion protein conjugation, was determined by reducing SDS-PAGE (4 - 20% CRITERION™ TGX Stain-Free™ Precast Gels, 18 Well Comb, 30 pL, 1.0 mm, Bio-Rad, Hercules, CA, US). In addition, an unconjugated VLP control as well as five known BSA standards (Sigma Life Science, St. Louis, MO, US) of quantities between 5 pg to 2.5 pg will always be included as controls. Gels were run per manufacturer’s protocols and then subsequently subjected to Coomassie gel stain as per manufacturer’s protocols (Coomassie Brilliant Blue R-250 dye, BioRad, Hercules, CA, US). Amount and / or percent conjugation was determined via densitometry analysis utilizing BioRad software imaging analysis as per the manufacturer’s protocols.

[0147] Design of peptides. Peptides fire designed to include an N-terminal maleimide-RRRRRVKR-epitope, as described above. The peptides therefore contain a maleimide molecule at the N-terminus of the recall protein followed by a furin cleavage sequence: R X R / K R (SEQ ID NO: 89) upstream of the recall protein sequence. Exemplary furin cleavage sequence: ARG VAL LYS ARG (SEQ ID NO: 90).

[0148] For functional assays, murine Bl6 (melanoma / skin), and ID8 (ovarian) tumor cells overexpressing luciferase gene (B16-luc and ID8-luc) were grown in culture. Under normal circumstances, these two murine tumor cell lines B16 and ID8 will not be killed by murine HPV16 E7-specific CD8+ T cells since these cell lines do not express the HPV16 E7 antigen. Cells were grown in culture and seeded overnight. The next day, cells were then treated with either: (1) 3 different concentrations of HPV16 E7 peptide, RAHYNIVTF (SEQ ID NO: 1) (1 pg / ml, 100 pg / ml, 1 pg / mL), (2) HPV16 E7-conjugated VLP (2.5 pg / mL), (3) an unconjugated (control) VLP (2.5 pg / mL), or (4) were left untreated. The cells were then washed and co-cultured with murine CD8+ HPV16 E7-specific T cells at varying E:T ratios (Effector:Target ratio). The total final volume after co-culture was 200 pL. The cell viability was measured after a certain time of co-culturing by measuring the expression of luciferase, which is used as a surrogate marker for cell viability since these cells over-express luciferase. Reduced luciferase activity indicated more cell death suggesting greater immune redirection and hence greater cytotoxicity.

[0149] Responses to the HPV16 E7-conjugated VLP and HPV16 E7 peptide were timedependent and concentration-dependent in both cancer cell lines. After 5.5 hrs of co-culturing, both the E7 peptide only (1 pg / mL) and the conjugated VLP (2.5 pg / mL) demonstrated significant levels of T cell mediated cytotoxicity in the B16-luc. (See, FIG. 3A, B16-LUC, and FIG. 3C, ID8). Additional cytotoxicity was seen if co-culturing occurred for 8.5 hours, with cytotoxicity levels of approximately 50% in B16-LUC cells (FIG. 4B) and 38-58% in TD8-LUC cells (FIG. 4D) at E:T ratios of 10. Likewise, FIG. 4 shows similar results achieved over an even longer period of time, 22.5 hrs. Specifically, FIG. 4A and 4B show that cytotoxicity is improved between 8.5 hrs and 22.5 hrs, respectively, for E7-conjugated VLP incubated with B16-LUC cells and murine CD8+ HPV16 E7-specific T cells. Similarly, FIG. 4C and 4D show that cytotoxicity is improved between 8.5 hrs and 22.5 hrs, respectively, for E7-conjugated VLP incubated with ID8 cells and murine CD8+ HPV16 E7-specific T cells.

[0150] These results suggest that both an E7 peptide at higher concentrations (1 pg / mL) and an E7-conjugated conjugated VLP exhibit time dependent tumor cell killing. This further suggests that the E7-conjugated conjugated VLPs undergo furin cleavage and epitope coating of MHC receptors on the tumor cells in order to achieve tumor cell killing. Example 5 VLP-induced E7 T cell-mediated killing of tumor cells in a concentration-dependent manner

[0151] E7 T cell-mediated killing was also shown to be E7-conjugated VLP concentration dependent. Murine B16 (Melanoma / Skin) and ID8 (ovarian) tumor cells overexpressing luciferase gene (B16-luc and ID8-luc) were seeded. 24 hours later, the cells were then treated with: (1) an HPV16 E7 peptide RAHYNIVTF (SEQ ID NO: 1) (1 pg / ml, 1 ng / ml); (2) HPV16 E7 conjugated VLP (2.5 pg / ml, 0.025 pg / ml); (3) an unconjugated (control, 2.5 pg / ml) VLP; or (4) were left untreated. The cells were then washed and co-cultured with CD8+ HPV16 E7 specific T cells at varying E:T ratios (Effector:Target ratio). The total final volume after co-culture was 200 pL. The cell viability was measured after a certain time of coculturing by measuring the expression of luciferase, which is used as a surrogate marker for cell viability since these cells over-express luciferase. Reduced luciferase activity indicated greater immune redirection and hence greater cytotoxicity.

[0152] Responses to the HPV16 E7-conjugated VLP and HPV16 E7 peptide were concentration-dependent and in both cancer cell lines. Both the E7 peptide alone (1 pg / mL) and the E7 conjugated to VLP (2.5 pg / mL) demonstrated significant levels of T cell mediated cytotoxicity in both cell lines. (See, FIG. 3A and 3B, B16-LUC, and FIG. 3C and 3D, ID8). Figure 3 shows particularly that E7 peptide alone at 1 pg / mL exhibit little if any tumor cell killing, regardless of time point, whereas E7 peptide at 100 pg / mL and 1 pg / mL exhibited significant tumor cell cytotoxicity, regardless of time point. Similarly, concentration dependence of cytotoxicity was observed for E7-conjugated VLP in Figure 4. (See, FIG. 4A and 4B, B16-LUC, and FIG. 4C and 4D, ID8). In Figure 4, significant increases in cytotoxicity are observed between 0.025 pg / mL conjugated VLP vs. 2.5 pg / mL conjugated VLP for both cell lines regardless of time point (8.5 hr or 22.5 hr). Thus, Figures 3 and 4 show that cytotoxicity is improved as concentration increases for both E7 peptide and E7-conjugated VLP incubated with B16-LUC cells and murine CD8+ HPV16 E7-specific T cells. EXAMPLE 6

[0153] Batch consistency of VLP preparations. To ensure that the described process of creating conjugated VLPs that could perform immune redirection was consistent, several separately created batches of E7 peptide-conjugated VLP termed “Fl, F2, and F3” were examined. Murine B16 (Melanoma / Skin) and ID8 (ovarian) tumor cells overexpressing luciferase gene (B16-luc and ID8-luc) were seeded. 24 hours later the cells were incubated with: (1) an HPV16 E7 peptide RAHYNIVTF (SEQ ID NO: 1) (1 pg / ml); (2) HPV16 E7-conjugated VLP batch Fl, F2, or F3 (2.5 pg / ml); (3) an unconjugated (control, 2.5 pg / ml) VLP; or (4) were left untreated. All batches Fl - F3 were consistent and were able to activate E7 T cells, and showed killing of B16-luc and ID8-luc cells after 23 or 23.5 hr time points in two separate experiments respectively. (See, FIG. 5A and 5B, respectively, for B16-luc cells; FIG. 5C and 5D, respectively, for ID8-luc cells).

[0154] To further demonstrate batch consistency in another tumor cell line, MC38 murine colon tumor line, cells were grown in culture per manufacturer suggested conditions. Under normal circumstances, this cell line will not be killed by murine HPV16 E7-specific T cells since the MC38 cell line does not express the murine E7 antigen. MC38 cells were then treated with: (1) an E7 peptide RAHYNIVTF (SEQ ID NO: 1) (1 pg / ml,; (2) HPV16 E7-conjugated VLP, from batch Fl, F2, or F3 (2.5 pg / ml); (3) an unconjugated (control, 2.5 pg / ml) VLP; or (4) were left untreated. The cells were then washed and co-cultured with CD8+ HPV16 E7-specific T cells at varying E:T ratios (Effector:Target ratio). The total final volume after co-culture was 200 pL. The cell viability was measured after a certain time of co-culturing. In this example, because MC38 does not express luciferase, the cell viability was measured using an establish cell viability assay reagent: CELLTITER-GLO® (PROMEGA, WI, US). This assay provides a luciferase-expressing chemical probe that detects and binds to ATP, a marker of cell viability. Reduced luciferase activity hence indicates more MC38 cell death suggesting greater immune redirection and hence greater cytotoxicity. All batches F1-F3 were consistent in their functional activity and were able to activate E7 T cells, and showed killing of MC38 cells after 23 and 23.5 hr time points in two separate experiments respectively. (See, FIG. 6A and 6B, respectively). Example 7 In Vivo anti-tumor immune redirection in C57BL / 6 mice with E7-conjugated VLP

[0155] Efficacy of HPV16 E7-conjugated VLPs (conjugated-VLPs conjugated to murine MHC-restricted H-2Db HPV16 E7 peptide) was assessed in vivo. A total of 20 C57BL / 6 mice were injected intra-peritoneally (I.P.) with 1D8-Iuciferase cells (0.015 x 106 cells / mouse). On Day 6, mice were checked for tumor growth and establishment via luciferase luminescent imaging. (IVIS® Spectrum in vivo imaging system, Perkin Elmer, Waltham, MA, US). At day 5, mice are separated into treatment groups and then treated with: (1) buffer only, (2) nonconjugated VLP (100 pg), (3) murine CD8+ HPV16 E7 specific T cells plus E7 peptide conjugated-VLP (n=5, 100 pg), and (4) E7 peptide only (30 pg, n --- 5). After 1 hr all mice were injected with approximately 1 x 106 Day 4 post-stimulated CD8+ HPV16 E7 specific T cells (200 pL per mouse). On day 6 and day 7 treatments were administered a second time for each mouse, but no additional stimulated T cells were added. Mice are then monitored and imaged. Efficacy of treatment is monitored by luminescence imaging which acts as a surrogate for tumor growth and metastasis.

[0156] Results are provided in Figure 7 and show that E7 conjugated VLP therapy was able to redirect E7 specific T-cells to the ID8 murine tumor and control tumor formation even after 60 days post-treatment. (FIG. 7A and FIG. 7D). In contrast, controls such as nonconjugated VLP (FIG. 7C) or buffer only (FIG. 7B) failed to show any tumor protection or control. Although some protection was afforded by peptide only (FIG. 7E), it is important to recount that the overall molarity of peptides on E7 conjugated VLP versus peptide alone was less compared to peptide alone. Further, peptide provided incomplete protection, suggesting that the tumor specific targeting of the VLP is important compared to injection of exogenous peptide alone. Example 8 In Vivo anti-tumor immune redirection in B16.F10 mice with E7-conjugated VLP

[0157] Efficacy of HPV16 E7 VLPs was assessed in another murine tumor model (B16.F10, ATCC, Old Town Manassas, VA, USA). Unlike the ID8 ovarian murine model in which tumors are grown intraperitoneally, the BL16.F10 mouse tumor model is a subcutaneous tumor model. In this experiment, 20 mice were first vaccinated with HPV16 E7 peptide RAHYNIVTF (SEQ ID NO: 1). After 2 weeks, the mice were verified to have an immune response specific to the HPV 16 E7 peptide via HPV16 E7 tetramer (H-2Db / HPV16 E7 (RAHYNIVTF)(SEQ ID NO: 1) MHC Tetramer staining). Briefly, whole blood (2-3 drops) was collected from the tail vein from, the mice and checked for antigen specific CD8+ T cell responses by flow cytometry using E7 tetramer (H-2Db / HPV16 E7 (RAHYNIVTF)(SEQ ID NO: 1) MHC Tetramer, MBL International, Woburn MA, US). Mice were then injected subcutaneously with B16.F10-Iuciferase cells (0.5 x 10b cells / mouse) at the left flank of the thigh. Tumor growth volume was measured using Vernier calipers and volume was calculated using the formula (W2 x L) / 2, where W (width) is the shorter of the two orthogonal measurements and L (length) is the longer of the two. Mice were monitored every 2 days until palpable tumors (~7 mm in diameter) were observed. Subsequently, mice were divided into various treatment groups, as follows: (1) buffer only (n=5); (2) non-conjugated VLP (“Control” VLP) (n=5); (3) E7-conjugated VLP (n=5); and (4) peptide alone. All treatment groups received 5 pg indicated component per dose. A total of four treatments were administered to the mice, every other day. Mice were then monitored to assess tumor volume. Efficacy of treatment was monitored via tumor volume growth and survival in which the endpoint survival is when tumor growth volume reaches 1500 mm3. Results showed that a delayed effect in tumor growth was observed in control VLP, E7 conjugated VLP, and E7 peptide groups. (See, FIG. 8A and FIG. 8B). However, eventually all tumors reached the end point size of 1500 mm2 (FIG. 8A). All control mice reached their survival endpoint by day 17 (FIG. 8B). Treated mice survived up to day 19 to 22. One mouse from the E7 conjugated VLP-treated group had all tumors regressed, completely, and remained tumor free for over 80 days. (See, FIG. 8B).

[0158] To further test the importance of specific viral memory T-cells and whether their increased presence would lead to an improved therapeutic outcome, the mice were also separated into groups of “high” and “low” T-cell frequencies (see below for explanation), i.e. those mice that had a higher frequency of peripheral HPV16 E7 T cells in their PBMCs, vs. those that did not. Mice were first vaccinated with HPV16 E7 peptide RAHYNIVTF (SEQ ID NO: 1) to generate the anti-viral immune memory response. The vaccinated mice were left alone for approximately 6 weeks to establish immune memory to HPV16 E7. To check for immunogenicity, mice were separated into “high” and “low” T-cell frequencies. Briefly, whole blood (2-3 drops) was collected from the tail vein from the mice and checked for antigen specific CD8+ T cell responses by flow cytometry using E7 tetramer (H-2Db / HPV16 E7 (RAHYNIVTF) (SEQ ID NO: 1) MHC Tetramer, MBL International, Woburn MA, USA). The frequency of HPV16 E7 Tetramer-specific T cell response was between 1-50%. Mice that had a T cell frequency ranging between 1-14% were considered “low” and mice that had a T cell frequency ranging between “15%-50%” were considered “high.” All mice were then injected subcutaneously with B16.F10 melanoma tumor cells (0.5 x 106 cells / mouse) at the left flank of the thigh. Tumor growth volume was measured using Vemier calipers and volume was calculated using the formula (W2 x L) / 2, where W (width) is the shorter of the two orthogonal measurements, and L (length) is the longer of the two. Tumor volumes were allowed to reach 50 to 100 mm3 before starting treatments with E7 conjugated VLPs or control VLPs (nonconjugated VLP).

[0159] Results are depicted in Figure 8C and Figure 8D and show' that a delayed effect in tumor growth was observed in mice treated with E7 conjugated VLP but not in control groups treated with VLP alone. (See, FIG. 8C). However, eventually all tumors reached the end point of 1500 mnr by day 32 (FIG. 8D). All control mice reached their survival endpoint by day 17. (See, FIG. 8B). Treated mice survived up to day 24 for mice with “low” T-cell frequency, or up to day 32 for mice with “High” T-cell frequency. (FIG. 8D). In the absence of treatment, there was no difference in B16.F10 tumor growth between mice that had higher or lower frequency of E7 specific memory T-cells. (FIG. 8C). In contrast, tumor growth in the treatment groups seemed to suggest that the mice with higher E7 T-frequencies responded better to E7 conjugated VLP treatment, as the tumor growth was better controlled (FIG. 8D). However, further study of the data revealed no obvious trend when individual mice were scrutinized. This could be due to the “variable” tumor size as treatment was administered once all mice had tumors between 50 to 100 mm3. In general, it was observed that mice who had smaller tumors (closer to 50 mm3) responded better to the conjugated VLP treatment, but this did not necessarily correlate with a higher pre-existing T-cell PBMC frequency. This suggests that the immune redirection effect could be due to other potential E7-specific T cells, i.e. tissue resident memory T-cells. Nonetheless, these data suggest that immune redirection is possible even in a difficult B16.F10 mouse model that is known to be checkpoint refractory. Example 9 Detection of VLP-peptide loading onto MHC-I receptors on mouse tumor cells in vitro

[0160] An assay system, was developed to directly detect peptide-loading from conjugated-VLP onto MHC I receptors on tumor cells. This assay involves the use of OVA-conjugated VLPs (“OVA-VLPs) and an antibody (fluorophore-conjugated anti-OVA (SIINFEKL) / Kb (SEQ ID NO: 2) antibody, MBL International, Woburn, MA, US) that specifically recognizes the OVA (SnNFEKL) / Kb (SEQ ID NO: 2) complex, but not any other peptide / MHC-I complexes.

[0161] To this end, target cells (B16-LUC, ID8-LUC, and MC38) were plated at 0.02 x 106 cells / well on a 96-well plate. The next day, OVA-conjugated VLPs (2.5 pg / ml) were incubated with target tumor cells for 1 hr at 37 °C, followed by extensive washing to remove any unbound materials. Next, fluorophore-conjugated anti-OVA (SIINFEKL) / Kb (SEQ ID NO: 2) antibody was added directly to the cells at a dilution of 1:100 (total volume of 100 pL, i.e. 1 pL) for 30 minutes at 4 °C. Following this incubation, excess antibodies were washed off and the cells were analyzed for the presence of the OVA (SIINFEKL) / Kb (SEQ ID NO: 2) complex by FACS flow cytometry. As expected, the OVA (SIINFEKL) / Kb (SEQ ID NO: 2) complex was detected in three different mouse tumor cells after incubation with OVA-conjugated VLPs. (See, Figure 9).

[0162] FIG. 9 A shows FACS data resulting from incubation of ID8-LUC (top panel), B16-LUC (middle panel), and MC38 (lower panel) cells with OVA peptide alone or OVA-conjugated VLP. This indicates that OVA-conjugated VLPs bind to these tumor cells and their peptides were cleaved, followed by binding of the cleaved peptide to the tumor MHC-I molecules. Therefore, the design of the VLPs is verified to perform the intended functions, i.e. the epitope peptide is cleaved by furin and the peptide is bound by MHC I molecules on the tumor cell surface. These findings should be applicable to other viral peptides, other than OVA. It is also important to note that differential OVA (SIINFEKL) / Kb (SEQ ID NO: 2) complex was detected across the murine tumors and further studies suggest that loading might be dependent on availability of surface MHC Class 1 on tumor surfaces (not shown). FIG. 9B shows the same cells, ID8-LUC (top panel), B16-LUC (middle panel), and MC38 (lower panel), stained by binding of an H2-Db antibody (PE anti-mouse H-2D b antibody, anti-H-2Db, Biolegend, San Diego, CA, US) specific for MHC-I complex. Example 10 Redirection of human CD8+ T cell viral immunity against human immortalized tumor cell lines using VLP conjugated to HCMV pp65 antigen

[0163] The ability of the described conjugated VLP system to trigger immune redirection using other antigens or epitopes was further explored by investigating the ability of human cytomegalovirus (HCMV) pp65 antigen-conjugated VLP to redirect immunity. The conjugated VLPs were generated as described above. The rationale to choose HCMV was based on knowledge that HCMV is highly prevalent (infecting 50-90% of the human population) and mostly asymptomatic in healthy individuals. (See, Longmate et al., Immunogenetics, 52(3-4):165-73, 2001; Pardieck et al., FlOOORes, 7, 2018; and van den Berg et al., Med. Microbiol. Immunol, 208(3-4):365-373, 2019). Importantly, HCMV establishes a life-long persistent infection that requires long-lived cellular immunity to prevent disease. Hence, it is rational to hypothesize that a complex adaptive cell-mediated anti-viral immunity developed over many years to strongly control a viral infection in an aging person can be repurposed and harnessed to treat cancer.

[0164] In vitro cytotoxicity assays. Human target cells, HTC112, human colon caner, MCF7, human breast cancer or OVCAR 3, human ovarian cancer (ATCC, Manassas, VA, US) were seeded overnight at 0.01 to 0. 2 x 106 per well per 100 pL per 96 well plate. The next day (about 20 to 22 hrs later), each cell line was incubated for one hour at 37 °C under the following conditions: (1) CMV peptide at a final concentration of 1 ug I mL, (2) control VLP (no peptide) at a final concentration of 2.5 pg / mL, (3) CMV-conjugated VLP at a final concentration of 2.5 pg / mL, and CMV peptide at a final concentration of 14 pg / mL (equivalent to peptide conjugated to CMV-VLP at 2.5 pg / mL). After 1 hour, the cells were washed vigorously with 200 q L of media for three times to remove non-specific binding. Human patient donor CMV T cells (ASTARTE Biologies, Seattle, WA, US) were added at the E:T (effector celktarget cell) ratio of 10:1 and incubated in a tissue culture incubator for 24 hrs. The total final volume after co-culture was 200 pL. The cell viability was measured after a certain time of coculturing. In tills example, because the above human cells does not express luciferase, the cell viability was measured using an establish cell viability assay reagent: CELLTITER-GLO® (PROMEGA, WI, US). This assay provides a luciferase-expressing chemical probe that detects and binds to ATP, a marker of cell viability. Reduced luciferase activity hence indicates more human immortalized cell death suggesting greater immune redirection and hence greater cytotoxicity

[0165] The results are provided in Figure 10. CMV conjugated VLP was effective in killing human healthy donor CMV pp65-specific CD8+ T-cells (Astarte Biologies, Inc., Bothell, WA, US), immortalized HLA.A2 positive human colon cancer cells (HCT116, FIG. 10A), human ovarian cancer cells (OVCAR3, FIG. 10B) and human breast cancer cells (MCF7, FIG. 10C). In both the mouse and human systems, control VLPs, i.e., no peptide conjugation, did not induce killing of the target cells, nor did co-incubation of CD8+ T cells with tumor cells alone. In these series of in vitro studies using human cell lines, it was determined that 1 pg / mL of CMV peptide is equivalent to 1060 nM peptide, whereas the molar equivalent of CMV peptide conjugated to the surface of the VLP in 2.5 pg / mL of CMV-conjugated VLP was -0.024 nM. Thus, a comparable T-cell redirection killing of tumor cells was triggered by the conjugated VLP despite using almost 44,000 times less than the CMV peptide alone. Importantly, it was determined that this 0.024 nM molar equivalent concentration of CMV (pp65) peptide alone that is conjugated to VLP was 14 pg / mL. When cytotoxicity was tested at 14 pg / mL of the free peptide alone, little to no killing of tumor cells was observed in all three human target cells (Figure 10C). This highlights the potency of the conjugated VLP in enhancing peptide loading compared to exogenous free peptide loading-alone onto tumor cells to facilitate immune redirection. Example 11 VLP conjugation methods

[0166] Two conjugation approaches were tested as candidates for conjugation of the VLP to the fusion protein: (1) wild type HPV vaccine (WT-VLP); and (2) a conjugated HPV vaccine (RG1 VLP which has 2 additional cysteine residues per single LI monomer and therefore 720 extra cysteines on the RG1 VLP surface compared to WT VLP). For each test, the VLP was first reduced by reaction with tris(2-carboxyethyl)phosphine (TCEP), for 1 hour at room temperature. This reduction step was required to “release” free cysteines on the VLP surface for the next step - maleimide conjugation of the peptides to the VLP. Protocols followed for reduction and maleimide conjugation utilizing maleimide molecules directly synthesized upstream of the recall protein were as described above.

[0167] The RG1 VLP LI protein sequence with the additional RG1 peptide sequence insertion is as follows (SEQ ID NO: 87): MSLWLPSEAT LVPKVSGLQY PLLNKLDDTE CKPPIGEHWG PLDICTSICK GSTANLASSN NMSLCAAIST DWNFGLQPPP QFPLGRKFLL VYLPPVPVSK RVFRIHLPDP NASAYAQLYK K G S P C T N VA V YPDYIKMVSE YFPTPSGSMV SETTYKNTNF GGTEEDTYRF QAGEKAKPKF WSTDEYVAR NKFGFPDTSF TCKQAGTCPP NPGDCPPLEL PYGDS LFFYL TSDAQIFNKP KEYLRHGEEY VTSQAZACQK TLGKRKATPT TNIYYHAGTS YNPDTQRLW DIIPKVANAG INTVIQDGDM RREQMFVRHE Y W L Q R A Q G HN DLQFIFQLCK HTPPAPKEDP TSSTSTTAKR RLLAVGHPYF ACVGVEVGRG VDNRECISMD '7DTGFGAMDF FNRAGAVGEN NG1CWGNQLF ITLTADVMTY LKKYTFWEVN KKRKLSR P1KKPNNNKI QPLGVGISGH YKQTQLCLIG TTLQANKSEV VPDDLYIKGS VTWDTTRST IHSMNSTILE LKEKFSADLD

[0168] Little to no detectable conjugation occurs if there is no reduction step (see Figures 13, lane 3, and Figure 14, lane 5). The efficiency of conjugation is determined by performing Coomassie staining of the VLP on SDS-PAGE (CRITERION™ gels, 4-20% acrylamide, BioRad, Hercules, CA, GS) to determine relative amounts of conjugated and unconjugated VLP. (See, FIG. 11). If no conjugation occurs, there will only be a single band as seen in the TCEP:RG1 VLP 1:1 lane. In contrast, the multiple bands in the TCEP:RG1 ratios from 3:1-10:1 show conjugation occurring and therefore also validating the importance of the reduction step. It is also known that the VLPs are held by disulfide bonds and thus addition of TCEP might harm the VLP, however ratios of TCEP:RG1 of 5:1 and 3:1 show intact conjugated VLPs under TEM (transmission electron microscopy), thereby showing a TCEP amount suitable for RG1 conjugation without affecting particle integrity. (See, FIG. 12)

[0169] The relationship of how TCEP concentration determines the coupling efficiency of the fusion protein to the VLP was further investigated at additional ratios of reducing agent to VLP (up to 74:1). (See, FIG. 13). At the highest ratios of TCEP to RG1 (e.g. 74:1), up to 5 coupling sites were present on a single LI monomer. In contrast, WT-VLP, no matter what ratio of TCEP was tested, yielded only one coupling site per LI monomer. Example 12 Human Conjugated VLP trigger tumor cytotoxicity in vitro and in-vivo.

[0170] The ability to selectively load tumor cells with a common infectious viral antigenic peptide that can easily be recognized and be eliminated by a pre-existing immune recall response is to be evaluated both in vitro and in vivo using various murine models. As seen in Examples 1 through 11, using a murine MHC-restricted H-2Db HPV16 E7 peptide from the HPV (human papillomavirus) type 16 E7 antigen, a HPV16 E7-conjugated conjugated VLP (E7-VLP) was generated and shown to specifically bind to the tumor cells ID8 (ovarian), B16 (melanoma), and MC38 (colon). Cleavage of the E7 peptide from the E7-conjugated VLP leads to specific E7 peptide binding to MHC class I receptors on tumor cells thereby rendering those tumor cells susceptible to antigen-specific CTL-mediated killing, in this case murine CD8+ HPV16 E7 specific T cells. As seen in Example 10, using human cytomegalovirus (HCMV) pp65 antigen-conjugated VLPs, this result has also been extended to human healthy donor CMV pp65-specific CD8+ T-cells.

[0171] Since most human individuals have or might have pre-existing immunity against influenza, HPV16, CMV, EBV, and other childhood vaccines (MMR, chicken pox, HEPB), the same experimental methods may be adopted to conjugate the described VLPs to other immunogenic HLA-A2-restricted peptides. HLA-A2 restricted epitopes will be chosen for these experiments as these are the most prevalent human MHC class I allele in the U.S. Various VLPs were conjugated to an HLA-A2 restricted epitope with a furin cleavage site at its N2-terminus as described above. The specific epitopes are provided in Table 4. Table 4: VLPs Conjugated to an HLA-A2 restricted epitope comprising a furin cleavage site llilililiOiosiiiiiii Approved Human Vaccine where epitope can be detected (Source) Afluria-Trivalent or Flublok Quadrivalent if Epitope SEQ ID NO / / Influenza A aged 18-64, Fluzone High-dose OR Fluad if 65 and older, Flucelvax Quadrivalent for 4-18years old (and older actually) FMYSDFHFI GILGFVF1L 68 Hepatitis B Engerix (GSK) FLPSDFFPSV 41 Measles MMR-II, (Merck) KLWESPQEI 36 Chicken Pox VariVax (Merck) SLPRSRTPI 4 VariVax (Merck) SAPLPSNRV 5 Cytomegalovirus Natural Infection NLVPMVATV 3 Human Papilloma virus 16 E7 Natural Infection YMLDLQPET 88

[0172] Each construct was analyzed by SDS-PAGE as above. (See, FIG. 14). Figure 14A depicts conjugates of WT HPV VLP to the following peptides: MOV A2 (SIINFEKL (SEQ ID NO: 2), influenza VI, HepB VI, measles VI, and VZV VI. (fusion proteins are synthesized by Genescript, NJ, US, 80% purity as described above). Likewise, Figure 14B depicts conjugates of RG1 VLPs including M0VA2 (SIINFEKL), influenza VI, HepB VI, measles VI, and VZV VI.

[0173] These constructs will be tested in vitro for their ability to localize to tumor cells, bind to tumor cells, have their epitopes proteolytically cleaved, and the released epitope bound by the tumor MHCI receptors to trigger a T cell mediated immune response as in the examples above. Cell lines that tire positive for HLA-A2 will be used, as above. Such cell lines include, for example, human tumors such as MDA-MB231, HCT116, and PC-3. In addition, a subclone of Murine B16 (Melanoma / Skin), TC-1 (cervical), and ID8 (ovarian) tumor cell subclones were generated that overexpress a luciferase gene and over-express HLA-A2 (B16-AAD. TC1-AAD, and ID8-AAD, respectively). All of these cell lines can be grown in culture. Under normal circumstances, these cell lines will not be killed by the virus-specific CD8+T cells specific for the epitopes listed in the table above since these cell lines do not express or display the respective childhood vaccine or natural infection antigen on their cell surface. Cells will be then incubated with: (1) a specific virus antigen peptide from Table 4, above at dosages of 1 pg / ml, 100 pg / ml, and 1 pg / mL; (2) a specific virus antigen conjugated VLP (2.5 pg / mL); (3) an unconjugated (control) VLP; or (4) untreated (with buffer). The cells are then washed and co-cultured with the respective antigen specific T cells at varying E:T ratios (Effector:Target ratio). The cell viability is to be measured either by CELLTITER-GLO® assay for the human tumors or by measuring the expression of luciferase in the murine tumors. Reduced luciferase activity will indicate greater cytotoxicity, suggesting greater immune redirection.

[0174] To test this in vivo, naive transgenic HLA-A2 or HLA-AAD mice are to be vaccinated with childhood vaccines such as HepB, MMR, and chickenpox (see Table 2, above) to generate the pre-existing cytotoxic T lymphocyte (CTL) viral response. CTL responses are to be evaluated using the HLA-A2 specific tetramers to the respective viral peptides. Once sufficient CTL response is confirmed, the mice are then injected with luciferase expressing HLA-A2 positive tumor lines ID8-AAD (ovarian), B16-AAD (melanoma), or TC-1 AAD (cervical) tumor cells. Tumor growth is monitored daily. Once mice develop palpable tumors with average volume of 10 mm3, peptide-conjugated VLP are administered to the tumorbearing mice at 100 pg / mouse / week and continued for three weeks. The efficacy of anti-tumor immune redirection therapy as described herein using conjugated VLP is measured via tumor volume and survival against the untreated group. In a separate experiment, the same study will be conducted as the above except that the naive HLA-A2 or HLA-A AD mice will be directly vaccinated with the peptides instead of childhood vaccines. Example 13 Survival of mice treated with human tumors after exposure to conjugated-VLP

[0175] Immunodeficient mice are to be injected with human tumor cells that overexpress MHC class 1 HLA-A2. Examples include: PC-3, HCT112, and MDA-MB231. At the same time, the mice are injected with human Peripheral Blood Mononuclear Cells (PBMC)(106 cells) previously stimulated with a conjugated VLP that is conjugated to a fusion protein comprising one of the following epitopes (Table 5, below): Table 5: Recall Protein Epitopes from Approved Vaccines |||||||||iii|||||||l Approved Human Vaccine where epitope can he detected Epitope SEQ ID NO Influenza A Afluria-Trivalent or Flublok Quadrivalent if aged 18-64, Fluzone High-dose OR Fluad if 65 and older, Flucelvax Quadrivalent for 4-18years old (and older actually) FMYSDFHFI GILGFVFTL 77 68 Hepatitis B Energix (GSK) FLPSDFFPSV 41 Measles MMR-IL (Merck) KLWESPQEI 36 Chicken Pox VariVax (Merck) SLPRSRTPI 4 VariVax (Merck) SAPLPSNRV 5 Cytomegalovirus Natural Infection NLVPMVATV 3 Human Papilloma virus 16 E7 Natural Infection YMLDLQPET 88

[0176] The conjugated-VLP comprising a fusion protein containing a recall protein as described above is then injected into the mouse weekly for three weeks. A second injection of human PBMCs stimulated with the conjugated VLP will then be injected into the mouse at day 14. It is expected that tumor formation will be inhibited compared to controls that were not provided the conjugated VLPs. Example 14 Conjugated VLP immune redirection properties are dependent on furin cleavage

[0177] Tumor specificity is in part due to the increased presence of furin on tumor cells. In order to confirm the specificity of conjugated VLPs conjugated to a fusion protein containing a furin cleavage cite, two experiments will be conducted. In the first experiment, a conjugated VLP will be created without a furin cleavage site. A cytotoxic assay will then be conducted to demonstrate that without the furin cleavage site, the peptides cannot be loaded onto the tumor cell. Murine B16 (Melanoma / Skin) and ID8 (ovarian) tumor cells overexpressing luciferase gene (B16-luc and 1D8-Iuc) will be grown in culture. Cells will then be treated with: (1) an E7 peptide (1 pg / ml, 1 ng / ml); (2) HPV16 E7 (lacking the furin cleavage sequence) conjugated VLP (2.5 pg / ml, 0.025 pg / ml); or (3) an unconjugated (control, 2.5 pg / ml) VLP. The cells are then washed and co-cultured with CD8+ HPV16 E7 specific T cells at varying E:T ratios (Effector:Target ratio). The cell viability is then to be measured by detection of luciferase. Reduced luciferase activity indicates greater immune redirection and hence greater cytotoxicity.

[0178] In a second experiment, FD11, a furin deficient cell line will be engineered to over-express HLA-A2 MHC Class 1 molecules (FD11 / AAD). A cytotoxic assay will then be conducted using this cell line to further demonstrate that without furin, the peptides cannot be loaded onto the tumor cell. FD11 / AAD tumor cells overexpressing luciferase gene are to be grown in culture. Cells are then treated with: (1) an E7 peptide (1 pg / ml, 1 ng / ml); (2) HPV16 E7 conjugated VLP (2.5 pg / ml, 0.025 pg / ml); or (3) an unconjugated (control, 2.5 pg / ml) VLP. The cells are then washed and co-cultured with CD8+ HPV16 E7 specific T cells at varying E:T ratios (Effector:Target ratio). The cell viability is measured by measuring the activity / expression of luciferase. Reduced luciferase activity indicated greater immune redirection and hence greater cytotoxicity Example 15 Tumor Re-Challenge Experiments

[0179] The ability of mice previously cured of primary tumors by the described conjugated VLP to survive a tumor re-challenge is to be assessed. Mice (n = 20) that have survived treatment of a tumor with a conjugated VLP are to be re-challenged with same tumor (1 x 105 live cells) four weeks following disappearance of the last tumor. A group of naive mice (n = 20) that have not been exposed to either a tumor or a conjugated VLP are to be injected with the same tumor as a control. Three treated and three naive mice are then to be sacrificed pre-VLP therapy, post-VLP, and post re-challenge. Tumors and spleen are then prepared for TCRp high-throughput sequencing by ImmunoSEQ assay (Adaptive Biotechnologies Corp., Seattle, WA). Mice previously cured of primary tumors by conjugated VLPs are expected to exhibit resistance to secondary re-challenge, indicating that the conjugated VLP strategy is able to induce a protective system immune response against tumor recurrence. Different TCR clonal profiles of mice at various stages, such as pre-VLP therapy, post-VLP, and post re-challenge will be observed.

Claims

1. A virus-like particle (VLP), consisting of:at least one virus capsid protein, anda fusion protein comprising in order from amino terminus to carboxy terminus as a single peptide:a) one protease cleavage peptide sequence, attached directly tob) at least one recall protein comprising at least one epitope,wherein the at least one virus capsid protein is conjugated to one or more of the fusion protein through a disulfide bond, an ester bond, an amide bond, or a chemical linkage created by exposure of the at least one recall protein and at least one capsid protein simultaneously to a cross-linking agent under conditions amenable to formation of a chemical cross-link,wherein the fusion protein is 45 amino acids or less in length,wherein the one protease cleavage peptide sequence is recognized by a tumor microenvironment protease selected from one or more of: cathepsins, kallikreins, serine proteases, caspases, matrix metalloproteinases, disintegrin, and metalloproteinases (ADAMs), andwherein the at least one epitope is at least one human T cell epitope that is from 8 to17 amino acids in length.

2. A virus-like particle (VLP), comprising:at least one virus capsid protein, anda fusion protein consisting of, in order from amino terminus to carboxy terminus as a single peptide:i) one protease cleavage peptide sequence, attached toii) one recall protein comprising at least one epitope,wherein the fusion protein is 45 amino acids or less in length,2019414934   11 Sep 2026wherein the protease cleavage peptide sequence is recognized by a tumor microenvironment protease selected from one or more of: cathepsins, kallikreins, serine proteases, caspases, matrix metalloproteinases, disintegrin and metalloproteinases (ADAMs),wherein the at least one epitope is from 8 to 17 amino acids in length, andwherein the at least one capsid protein exhibits tropism for a specific type of tissue.

3. A virus-like particle (VLP), comprising in order from amino terminus to carboxy terminus as a single peptide:at least one virus capsid protein, anda fusion protein comprising:(A) at least one protease cleavage peptide sequence, attached to(B) at least one recall protein comprising at least one epitope,wherein the at least one protease cleavage peptide sequence is recognized by one or more tumor microenvironment protease selected from: cathepsins, kallikreins, serine proteases, caspases, matrix metalloproteinases, and disintegrin and metalloproteinases (ADAMs), andwherein the at least one capsid protein exhibits tropism for a specific type of tissue.

4. The VLP of any one of claims 1 to 3, wherein the at least one epitope:is a pathogen epitope,comprises a peptide sequence that complexes with an MHC molecule,has a sequence selected from one or more of SEQ ID NOS:1 to 83, and / oris from a childhood vaccine.

5. The VLP of any one of claims 1 to 4, wherein the capsid protein is from human papilloma virus, hepatitis B virus, bacteriophage MS2, bacteriophage QP,2019414934   11 Sep 2026bacteriophage P22, cowpea chlorotic mottle virus, cowpea mosaic virus, influenza virus, parvovirus, Norwalk virus, hamster polyoma virus, Macrobrachium rosenbergii nodavirus, hepatitis C virus, or a retrovirus.

6. The VLP of any one of claims 1 to 4, wherein the capsid protein is a papilloma virus L1 and / or L2 capsid protein, and wherein the L1 protein is from Bovine papilloma virus (BPV), Human papilloma virus (HPV), Rabbit papilloma virus (RPV), or Mouse papilloma virus (MPV).

7. The VLP of any one of claims 1 to 6, wherein the fusion protein comprises at least two recall proteins.

8. The VLP of any one of claims 1 to 7, wherein the at least one capsid protein exhibits tropism for a specific type of tissue, and wherein the tropism is a tropism for cells or tissues expressing heparin sulfate proteoglycan (HSPG).

9. The VLP of any one of claims 1 to 8, wherein the at least one epitope is:(I)     a viral epitope and is from vaccinia virus, varicella zoster virus, herpes zostervirus, rubella, hepatitis virus, influenza virus, measles virus, mumps virus, poliovirus, variola virus, rabies virus, dengue virus, Ebola virus, West Nile virus, yellow fever virus, zika virus, cytomegalovirus, or Epstein-Barr virus,(II) a bacterial epitope and is from Bordetella pertussis, Clostridium tetani, Chlamydia trachomatis, diphtheria, Hemophilus influenza, Meningococcus, Pneumococcus, Vibrio cholera, Mycobacterium tuberculosis, Bacillus Calmette-Guerin (BCG),2019414934   11 Sep 2026typhoid, Escherichia coli, Salmonella, Legionella pneumophila, Rickettsia, Treponema pallidum pallidum, Streptococcus, Bacillus anthracis, Clostridium botulinum, or Yersinia, or(III) a parasitic epitope and is from Entamoeba histolytica, Toxoplasma gondii, Trichinella, Trichomonas, Trypanosoma, or Plasmodium.

10. The VLP of any one of claims 1 to 9, wherein the fusion protein is conjugated to one capsid protein via a cysteine, lysine, or arginine residue.

11. A method of treating cancer, which comprises administering to a subject in need thereof a pharmaceutically effective amount of a virus-like particle (VLP),wherein:the VLP in the order from amino terminus to carboxy terminus as a single peptide comprises:at least one virus capsid protein, anda fusion protein comprising:at least one protease cleavage peptide sequence, attached toat least one recall protein comprising at least one epitope,wherein the at least one protease cleavage peptide sequence is recognized by one or more tumor microenvironment protease selected from: cathepsins, kallikreins, serine proteases, caspases, matrix metalloproteinases, and disintegrin and metalloproteinases (ADAMs), andwherein the fusion protein is 45 amino acids or less in length,wherein the at least one recall protein comprises at least one epitope that is from 8 to 17 amino acids in length,2019414934   11 Sep 2026wherein the at least one epitope is at least one human T cell epitope,wherein the cancer is selected from the group consisting of: lung, oral, cervical, epithelial, ovary, breast, colon, and prostate, andwherein the at least one virus capsid protein binds to cells of the cancer being treated.

12. The method of claim 11, further comprising detecting in the subject the presence of a preexisting immune response, wherein detecting in the subject the presence of a preexisting immune response comprises:obtaining a blood sample from the subject, andassaying the blood cells for the presence of one or more populations of T cells specifically recognizing one or more epitopes from one or more childhood vaccines.

13. The method of claim 12, wherein detecting in the subject the presence of a preexisting immune response further comprises determining whether the subject was ever immunized or vaccinated against a pathogen.

14. The method of any one of claims 11 to 13, wherein the epitope corresponds to an epitope present in a prior immunization or vaccination in the subject.

15. The method of any one of claims 11 to 14, wherein the epitope is one that is from a childhood vaccine and elicits an immune response in the subject, and wherein the method further comprises:obtaining from the subject a tumor tissue sample; and2019414934   11 Sep 2026identifying in the tumor tissue a peptide sequence of one or more major histocompatibility (MHC) molecules expressed by one or more tumor cells in the tumor tissue sample.

16. The method of claim 15, wherein the childhood vaccine is a shingles vaccine, a pneumococcal vaccine, a hepatitis vaccine, or a measles-mumps-rubella (MMR) vaccine.

17. The method of any one of claims 11 to 16, further comprising administering one or more additional therapies to the subject selected from: Chimeric Antigen Receptor T-cell (CAR-T) therapy, a vaccine, a check point inhibitor, an oncolytic virus, a neo-antigen vaccine, a neo-adjuvant, a chemotherapy, radiation, or surgery.

18. Use of a virus-like particle (VLP) in:(i) the treatment of cancer in a subject, and / or(ii) in the manufacture of a medicament for the treatment of cancer in a subject, wherein:the VLP in the order from amino terminus to carboxy terminus as a single peptide comprises:at least one virus capsid protein, anda fusion protein comprising:at least one protease cleavage peptide sequence,attached toat least one recall protein comprising at least one epitope,wherein the at least one protease cleavage peptide sequence is recognized by one or more tumor microenvironment protease selected from: cathepsins, kallikreins, serine2019414934   11 Sep 2026proteases, caspases, matrix metalloproteinases, and disintegrin and metalloproteinases (ADAMs), andwherein the fusion protein is 45 amino acids or less in length,wherein the at least one recall protein comprises at least one epitope that is from 8 to 17 amino acids in length,wherein the at least one epitope is at least one human T cell epitope,wherein the cancer is selected from the group consisting of: lung, oral, cervical, epithelial, ovary, breast, colon, and prostate, andwherein the at least one virus capsid protein binds to cells of the cancer being treated.

19. Use of a Virus Like Particle (VPL) according to any one of claims 1 to 10 in:(i) the treatment of cancer, and / or(ii) in the manufacture of a medicament for the treatment of cancer, wherein the at least one virus capsid protein binds to cells of the cancer being treated.

20. A method of treating cancer comprising administering to a subject in need thereof a pharmaceutically effective amount of a virus-like particle (VLP) according to any one of claims 1 to 10, wherein the at least one virus capsid protein binds to cells of the cancer being treated.

21. The use according to claim 19 or the method according to claim 20, wherein the Virus Like Particle (VPL) is a VPL as defined in claim 2 or claim 3.

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