T cell vaccine

Abstract

The present invention relates to antigen presentation to CD8+ T cells. Systems and methods are provided for efficient presentation of diverse peptide antigens and induction of stimulatory and regulatory CD8+ T cell responses.

Description

PATENT Docket No. Y9054-99007 T CELL VACCINE RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims priority to US provisional application Serial No. 63 / 650,634, filed May 22, 2024, incorporated by reference herein in its entirety.

[0002] The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. FIELD OF THE INVENTION

[0003] The present invention relates to antigen presentation to CD8+T cells. Systems and methods are provided for efficient presentation of diverse peptide antigens and induction of stimulatory and regulatory CD8+T cell responses. SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said .xml copy, created on May 22, 2025, is named Y9054-99007, and is 106,441 bytes in size. BACKGROUND OF THE INVENTION

[0005] Development of T cell responses relies on processing and MHC-mediated presentation of peptide antigens. Priming a robust T cell response requires existence of naïve antigen-specific CD8+T cells and effective antigenic peptide presentation by MHC-I molecules on the surface of antigen presenting cells.

[0006] MHC-I molecules bind peptides originating mainly from degradation of intracellular proteins. A portion of the peptides are transported to the endoplasmic reticulum via the transporter associated with antigen processing (TAP) complex, which consists of the TAP-1 and TAP-2 DM2\21145312.11PATENT Docket No. Y9054-99007 proteins, and is further regulated by different chaperones. Antigen transport is efficient for peptides of 8–14 residues. Once in the ER, peptides can be loaded on MHC-I molecules. These molecules form heterodimers with the β2m and the complex folds stably only upon binding to a peptide. Stable peptide–MHC-I complexes are then translocated to the cellular membrane, with the MHC-I peptide binding site in the extracellular milieu. The binding site of MHC-I molecules is highly specific both in terms of the amino acid sequence and peptide length. Only a small fraction of peptides available for loading in the ER can bind to a given MHC-I molecule.

[0007] Consequently, antigen selection for T cell vaccine use is also limited. Multiple factors affect surface levels of peptide-MHC complexes and efficiency of antigen presentation. These limitations include protein expression and abundance in the presenting cell, efficiency of proteasomal cleavage of an antigen that provides peptides of the appropriate length and sequence, efficiency of peptide loading onto MHC, and stability of the peptide-MHC complex on the cell surface. Furthermore, differences in antigen presentation may lead to certain antigenic peptides being immunodominant while others peptides from the same immunogen produce little or no response.

[0008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION

[0009] There is a need for new vaccines and systems of antigen presentation that are versatile and efficiently induce or regulate T cell immunity. In some aspects there are provided systems and methods for presentation of a target peptide to elicit CD8 T cell responses. The systems deliver the target peptide covalently attached to B2M or to B2M and an MHC alpha chain. In certain embodiments, the components are encoded by mRNA and delivered by lipid nanoparticles (LNPs) to antigen presenting cells (APCs) in a way that mimics presentation of native peptides. The systems promote delivery and presentation of the target peptide as a covalent complex with B2M and MHC and bypass the normal requirements of proteolytic processing, TAP mediated peptide translocation, and MHC loading which are inefficient and may be vulnerable to viral escape mechanisms. The covalent nature of the designs also overcomes limitations imposed by antigen processing and poor immunogenicity of certain peptides even though well presented.

[0010] Advantageously, when expressed as peptide—B2M—MHC the presenting MHC can be predefined. The single chain trimer (SCT) format also provides for the elicitation of non- DM2\21145312.12PATENT Docket No. Y9054-99007 classical MHC-Ib, e.g., HLA-E restricted CD8+responses. The target peptide is thus linked to B2M (peptide—B2M) and can form a complex with any selected class I MHC, loading the peptide on the MHC allele that has suitable affinity and presenting the peptide. The peptide—B2M linkage provides a product not restricted to a predefined MHC allele.

[0011] In an aspect, the invention provides improved HLA-mediated responses, including HLA-E restricted responses and can target viruses (e.g. HPV, CMV, EBV, HSV, VZV, and others) and cancers that evade T cell responses by downregulating class I MHC. In another aspect, the invention provides improved suppression of autoimmune activity.

[0012] The invention involves constructs (e.g., single chain trimer (SCT) constructs) that are independent of normal MHC-loading cellular machinery (e.g. TAP or peptide-loading complex chaperones. The constructs include some or all of the following features. Covalent linkage is demonstrated to improve stability of the peptide-MHC complexes. Covalent linkage increases surface expression of low abundance peptides and peptides with low presentation capacity. Stabilizing mutations within the constructs preserve T cell recognition. mRNA delivery provides preferential delivery to antigen-presenting cells (as opposed to DNA vaccines which generally target to muscle cells) the vaccines and can readily personalized. In certain embodiments, the lipid nanoparticle (LNP) delivery system is optimized to enhance cellular uptake and expression of the mRNA in specific target cells, thereby improving the immunogenic response. In certain embodiments, the method comprises codelivery of a costimulatory molecule.

[0013] Cytokines play a crucial role in shaping the magnitude, quality, and durability of CD8+ T cell responses, particularly in the context of memory formation. In certain embodiments, the LNP delivery system may be codelivered with specific cytokines for example to enhance the generation and persistence of memory CD8+ T cell populations. Useful cytokines include, without limitation, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, IL-33, IL-35, IL- 37, IFNy, TSLP, or TNFa.

[0014] In an aspect, there is provided a system for expressing an MHC-I single chain trimer (SCT) on a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising an antigen peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell; whereby the SCT is expressed and the antigen peptide presented. DM2\21145312.13PATENT Docket No. Y9054-99007

[0015] In some embodiments, the expressible mRNA encodes from 5’ to 3’, a signal sequence, the antigen peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

[0016] In some embodiments, the MHC-I alpha chain comprises a classical MHC I alpha chain, optionally an HLA-A, HLA-B, or HLA-C alpha chain.

[0017] In some embodiments, the MHC-I alpha chain comprises HLA-A*01:01, HLA-A*02:01, HLA-A*02:04, HLA-A*02:05, HLA-A*02:07, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-A*24:07, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*34:01, HLA-A*68:01, HLA-A*68:02, HLA-A*74:01, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*14:02, HLA-B*15:01, HLA-B*15:03, HLA-B*15:04, HLA-B*15:25, HLA-B*18:01, HLA-B*27:04, HLA-B*27:05, HLA-B*35:01, HLA-B*35:02, HLA-B*35:03, HLA-B*35:05, HLA-B*35:12, HLA-B*35:19, HLA-B*35:43, HLA-B*37:01, HLA-B*38:01, HLA-B*39:02, HLA-B*39:05, HLA-B*39:06, HLA-B*39:09, HLA-B*40:01, HLA-B*40:02, HLA-B*40:04, HLA-B*40:06, HLA-B*42:01, HLA-B*44:02, HLA-B*44:03, HLA-B*45:01, HLA-B*46:01, HLA-B*46:02, HLA-B*48:02, HLA-B*48:03, HLA-B*49:01, HLA-B*50:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01, HLA-B*55:01, HLA-B*56:01, HLA-B*56:02, HLA-B*57:01, HLA-B*57:03, HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*03:05, HLA-C*04:01, HLA-C*04:03, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*16:02, or HLA-C*17:01.

[0018] In some embodiments, the MHC-I alpha chain comprises H-2Db, or H-2Kb.

[0019] In some embodiments, the mRNA encoding the MHC-I SCT comprises SEQ ID NO:1.

[0020] In some embodiments, the MHC-I alpha chain comprises alanine at amino acid position 84, or the mRNA encoding the MHC-I SCT comprises SEQ ID NO:2.

[0021] In some embodiments, the MHC-I alpha chain comprises cysteine at amino acid position 84 and cysteine at amino acid position 139, or the mRNA encoding the MHC-I SCT comprises SEQ ID NO:3.

[0022] In some embodiments, the MHC-I alpha chain comprises cysteine at amino acid position 84 and the first linker comprises cysteine at amino acid position 2, or the mRNA encoding the MHC-I SCT comprises SEQ ID NO:4. DM2\21145312.14PATENT Docket No. Y9054-99007

[0023] In some embodiments, i) the MHC-I alpha chain comprises H-2Dband the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYCL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), or CSANNSHHYI (SEQ ID NO:14); or ii) the MHC-I alpha chain comprises H-2Kband the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), or YTVKYPNL (SEQ ID NO:13).

[0024] In some embodiments, the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, optionally an HLA-E, HLA-F, HLA-G, or HLA-H chain.

[0025] In some embodiments, the MHC-I alpha chain comprises HLA-E and the antigen peptide comprises VMPLSAPTL (SEQ ID NO:50), YLQPRTFLL (SEQ ID NO:51), VLWAHGFEL (SEQ ID NO:52), AMYTPHTVL (SEQ ID NO:53), or SLPINVIVF (SEQ ID NO:54)

[0026] In some embodiments, the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises SLQGRTLIL (SEQ ID NO:15), GMRFDKGYI (SEQ ID NO:27), RLPAKAPLL (SEQ ID NO:28), VMATRRNVL (SEQ ID NO:29), or VLRPGGHFL (SEQ ID NO:30).

[0027] In some embodiments, the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises ALFEGRNLV (SEQ ID NO:38), ALMDCIIFE (SEQ ID NO:39), ALMDLLMFS (SEQ ID NO:40), ALMDLLMFSTS (SEQ ID NO:41), AMMKSYVKS (SEQ ID NO:42), FLMFLQNLK (SEQ ID NO:43), LQMNANAYS (SEQ ID NO:44), SLFSRFRR (SEQ ID NO:45), SLMDKLRED (SEQ ID NO:46), SLMVSSFN (SEQ ID NO:47), TLMSIVSSL (SEQ ID NO:48), or VIQSVRRLY (SEQ ID NO:49).

[0028] In some embodiments, the system further comprises an expressible mRNA encoding a costimulatory molecule. In some embodiments, the system further comprises a T cell helper epitope. In some embodiments, the system further comprises an expressible mRNA encoding a cytokine.

[0029] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP), or a phospholipid bilayer, or a dried organic matrix, a carbon nanotube, an ionizable lipid nanoparticle; a cationic liposome; a lipoplex (lipid–mRNA complex); a polyplex (polymeric–mRNA complex); a dendrimer nanoparticle; a chitosan-based nanoparticle; a poly(β-amino ester) (PBAE) nanoparticle; a protamine–mRNA complex; a cell-penetrating peptide complex; a virus-like particle; an extracellular vesicle or exosome; a cationic nanoemulsion; a lipid–inorganic DM2\21145312.15PATENT Docket No. Y9054-99007 nanoparticle (LION); a squalene-based nanoemulsion; a hybrid nanoparticle; an inorganic nanoparticle (for example, a silica, gold, or iron oxide nanoparticle); a solid microparticle or depot matrix.

[0030] In an aspect, there is provided a vaccine formulation which comprises an expressible mRNA which encodes the MHC-I SCT comprising an antigen peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell.

[0031] In an aspect there is provided a method of inducing a CD8+ T cell mediated response against a disease or pathogen in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an antigen peptide from the disease or pathogen, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); wherein the MHC-I SCT is expressed and the antigen peptide presented to the CD8+T cell, whereby the CD8+T cell response is induced.

[0032] In some embodiments, the MHC-I alpha chain comprises a classical MHC-I alpha chain. In some embodiments, the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, optionally an HLA-E chain.

[0033] In some embodiments, the pathogen comprises Lymphocytic choriomeningitis virus (LCMV), and i) the MHC-I alpha chain comprises H-2Dband the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYCL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), or CSANNSHHYI (SEQ ID NO:14), or ii) the MHC-I alpha chain comprises H-2Kband the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), or YTVKYPNL (SEQ ID NO:13).

[0034] In some embodiments, the pathogen comprises influenza, the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises SLQGRTLIL (SEQ ID NO:15).

[0035] In an aspect, there is provided a method of inducing a CD8+ T cell response against a tumor antigen in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an antigen peptide of the tumor antigen operably linked to a β2-microglobulin (β2m) and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to antigen presenting cells,whereby the MHC-I SCT is expressed and an anti-tumor CD8+T cell response is induced.

[0036] In some embodiments the antigen peptide comprises a neoantigen peptide. DM2\21145312.16PATENT Docket No. Y9054-99007

[0037] In some embodiments, the composition comprises a second expressible mRNA which encodes a second MHC-I SCT comprising a second antigen peptide of the tumor antigen operably linked to a β2-microglobulin (β2m) and an MHC-I alpha chain; whereby the second MHC-I SCT is expressed and a second anti-tumor response CD8+T cell response is induced.

[0038] In some embodiments the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), CSANNSHHYI (SEQ ID NO:14), LCPGNKYEM (SEQ ID NO:18), VELCPGNKYEM (SEQ ID NO:19), AAVILRDAL (SEQ ID NO:20), AAVILRDALHM (SEQ ID NO:21), VILRDALHM (SEQ ID NO:22), or FPAAVILRDAL (SEQ ID NO:23), and the MHC-I alpha chain comprises H-2Dbor the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), YTVKYPNL (SEQ ID NO:13), SVYDFFVWL (SEQ ID NO:16), KYICNSSCM (SEQ ID NO:17), or AVILRDAL (SEQ ID NO:24), and the MHC-I alpha chain comprises H-2Kb.

[0039] In an aspect, there is provided a system for expressing an MHC-I single chain trimer (SCT) in a cell (APC), which comprises: an expressible mRNA which encodes the MHC-I SCT comprising an autoantigen peptide, a β2-microglobulin (β2m), and an HLA-E alpha chain; and a delivery vehicle for delivery of the expressible mRNA to the cell; whereby the SCT is expressed and the autoantigen peptide presented.

[0040] In some embodiments, the expressible mRNA encodes from 5’ to 3’: a signal sequence, the autoantigen peptide, a first linker, the β2m, a second linker, and the HLA-E alpha chain.

[0041] In some embodiments, the autoantigen peptide comprises FYAEATPML (SEQ ID NO:31), QMRPVSRAL (SEQ ID NO:89), QMRPVSRVL (SEQ ID NO:90), or GMKFDRGYI (SEQ ID NO:91).

[0042] In some embodiments, the system further comprises an expressible mRNA encoding a costimulatory molecule. In some embodiments, the system further comprises a T cell helper epitope. In some embodiments, the system further comprises an expressible mRNA encoding a cytokine.

[0043] In an aspect there is provided a vaccine formulation which comprises a system for expressing an MHC-I single chain trimer (SCT) in a cell (APC), which comprises: an expressible mRNA which encodes the MHC-I SCT comprising an autoantigen peptide, a β2-microglobulin (β2m), and an HLA-E alpha chain; and a delivery vehicle for delivery of the expressible mRNA to the cell. DM2\21145312.17PATENT Docket No. Y9054-99007

[0044] In an aspect, there is provided a method of inducing a CD8+ T cell regulatory response against a cell that is autoreactive in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an autoantigen peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehiccle for delivery of the expressible mRNA to an antigen presenting cell (APC) in the subject;wherein the MHC-I SCT is expressed and the autoantigen peptide is presented, whereby the CD8+T cell regulatory response is induced.

[0045] In an aspect, there is provided a method of reducing or inhibiting autoimmunity in a subject, which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an HLA-E alpha chain operably linked to a β2-microglobulin (β2m) and a peptide, wherein the SCT stimulates CD8+T cells to suppresses the autoimmunity when expressed by an antigen presenting cell of the subject, and a delivery vehicle for delivery of the expressible mRNA to cells of the subject; wherein the MHC SCT is expressed and the peptide presented; whereby the autoimmunity is suppressed.

[0046] In some embodiments, the peptide comprises FYAEATPML (SEQ ID NO:31), QMRPVSRAL (SEQ ID NO:89), QMRPVSRVL (SEQ ID NO:90), or GMKFDRGYI (SEQ ID NO:91).

[0047] In some embodiments, the autoimmunity comprises lupus or type 1 diabetes.

[0048] In an aspect, there is provided a system for expressing an MHC-I single chain trimer (SCT) in a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising a human immunodeficiency virus (HIV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell, whereby the SCT is expressed and the HIV peptide presented.

[0049] In some embodiments, expressible mRNA encodes from 5’ to 3’, a signal sequence, the HIV peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

[0050] In some embodiments the MHC-I alpha chain comprises a classical MHC-I alpha chain.

[0051] In some embodiments the MHC-I alpha chain comprises a non-classical MHC-I alpha chain or an HLA-E chain.

[0052] In some embodiments the MHC-I alpha chain comprises HLA-A*02:01 and the HIV peptide comprises an HLA-A*02:01-restricted peptide. DM2\21145312.18PATENT Docket No. Y9054-99007

[0053] In some embodiments the HIV peptide comprises YVDRFYKTL (SEQ ID NO:32), LTFGWCFKLV (SEQ ID NO:33), KLTPLCVTL (SEQ ID NO:34), PLTFGWCYKL (SEQ ID NO:35), LVGPTPVNI (SEQ ID NO:36), or VIYQYMDDL (SEQ ID NO:37).

[0054] In some embodiments the system encodes a second MHC-I SCT which comprises a second HIV peptide.

[0055] In some embodiments the system further comprises an expressible mRNA encoding a costimulatory molecule. In some embodiments the system further comprises a T cell helper epitope. In some embodiments the system further comprises an expressible mRNA encoding a cytokine.

[0056] In an aspect, there is provided a vaccine formulation which comprises an expressible mRNA which encodes the MHC-I SCT comprising a human immunodeficiency virus (HIV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell and a pharmaceutically acceptable carrier.

[0057] In an aspect, there is provided a method of inducing a CD8+ T cell mediated response against HIV in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human immunodeficiency virus (HIV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HIV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

[0058] In some embodiments, the MHC-I alpha chain comprises a classical MHC-I alpha chain. In some embodiments, the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, preferably an HLA-E chain.

[0059] In some embodiments, the MHC-I alpha chain comprises HLA-A*02:01 and the HIV peptide comprises an HLA-A*02:01-restricted peptide.

[0060] In some embodiments, the HIV peptide comprises comprises YVDRFYKTL (SEQ ID NO:32), LTFGWCFKLV (SEQ ID NO:33), KLTPLCVTL (SEQ ID NO:34), PLTFGWCYKL (SEQ ID NO:35), LVGPTPVNI (SEQ ID NO:36), or VIYQYMDDL (SEQ ID NO:37).

[0061] In an aspect, there is provided a system for expressing an MHC-I single chain trimer (SCT) in a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I DM2\21145312.19PATENT Docket No. Y9054-99007 alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell, whereby the SCT is expressed and the HPV peptide presented.

[0062] In some embodiments, the expressible mRNA encodes from 5’ to 3’: a signal sequence, the HPV peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

[0063] In some embodiments, the MHC-I alpha chain comprises a classical MHC-I alpha chain.

[0064] In some embodiments the MHC-I alpha chain comprises a non-classical MHC-I alpha chain or an HLA-E chain.

[0065] In some embodiments i) the MHC-I alpha chain comprises HLA-A2*02:01 and the antigen peptide comprises TLQDIVLHL (SEQ ID NO:92), or SLQDIEITC (SEQ ID NO:93), or GLYNLLIRC (SEQ ID NO:94), or SLQDIEITCV (SEQ ID NO:95); or ii) the MHC-I alpha chain comprises HLA-A*11.01 and the antigen peptide comprises ATLQDIVLH (SEQ ID NO:96), or iii) the MHC-I alpha chain comprises HLA-B*40.02 and the antigen peptide comprises QDIEITCVY (SEQ ID NO:97).

[0066] In some embodiments, the system further comprises an expressible mRNA encoding a costimulatory molecule.

[0067] In some embodiments, the system further comprises a T cell helper epitope.

[0068] In some embodiments, the system further comprises an expressible mRNA encoding a cytokine.

[0069] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP), or a phospholipid bilayer, an dried organic matrix, a carbon nanotube, an ionizable lipid nanoparticle; a cationic liposome; a lipoplex (lipid–mRNA complex); a polyplex (polymeric–mRNA complex); a dendrimer nanoparticle; a chitosan-based nanoparticle; a poly(β-amino ester) (PBAE) nanoparticle; a protamine–mRNA complex; a cell-penetrating peptide complex; a virus-like particle; an extracellular vesicle or exosome; a cationic nanoemulsion; a lipid–inorganic nanoparticle (LION); a squalene-based nanoemulsion; a hybrid nanoparticle; an inorganic nanoparticle (for example, a silica, gold, or iron oxide nanoparticle); a solid microparticle or depot matrix.

[0070] In an aspect, there is provided a vaccine formulation which comprises an expressible mRNA which encodes the MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2- DM2\21145312.110PATENT Docket No. Y9054-99007 microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell.

[0071] In an aspect, there is provided a method of inducing a CD8+ T cell mediated response against HPV in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HPV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

[0072] In an aspect, there is provided a method of treating or preventing cervical cancer or oropharyngeal cancer in a subject which comprises immunizing the subject with a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HPV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

[0073] In some embodiments, the MHC-I alpha chain comprises a classical MHC-I alpha chain.

[0074] In some embodiments, the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, preferably an HLA-E chain.

[0075] In some embodiments, i) the MHC-I alpha chain comprises HLA-A2*02:01 and the antigen peptide comprises TLQDIVLHL (SEQ ID NO:92), or SLQDIEITC (SEQ ID NO:93), or GLYNLLIRC (SEQ ID NO:94), or SLQDIEITCV (SEQ ID NO:95); or ii) the MHC-I alpha chain comprises HLA-A*11.01 and the antigen peptide comprises ATLQDIVLH (SEQ ID NO:96), or iii) the MHC-I alpha chain comprises HLA-B*40.02 and the antigen peptide comprises QDIEITCVY (SEQ ID NO:97).

[0076] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the DM2\21145312.111PATENT Docket No. Y9054-99007 product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0077] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” "comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes,” “included,” “including,” and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0078] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0080] Fig.1 is a cartoon to illustrate features of the invention.

[0081] Fig. 2 depicts several mRNA encoded single chain trimer (SCT) designs. SCTs are expressed as the peptide, beta-2-microglobulin (B2M), and MHC-I alpha chain covalently linked via two linkers. The MHC-I transmembrane (TM) domain is retained to enable native plasma membrane display of the SCT. A signal sequence N-terminal to the peptide targets SCTs to the secretory pathway and is cleaved prior to surface expression. Stabilizing mutations introduced in either the linker and / or the MHC-I are shown for each SCT construct.

[0082] Fig.3. Immunization with LCMV GP33 peptide SCT mRNA induces polyfunctional antigen-specific T cell responses in mice. Fig.3 depicts induction of LCMV GP33-specific T cell responses by SCT mRNA immunization. (A) Overview of experimental design. C57BL / 6 mice DM2\21145312.112PATENT Docket No. Y9054-99007 were immunized via the intramuscular (i.m.) route with 5 ug mRNA LNP on day 0 (1 dose, 1x) or 3 doses (3x) of 5 ug mRNA each on day 0, 2, and 4, or infected the intraperitoneal (i.p.) route with 2e5 focus forming units (ffu) of lymphocytic choriomeningitis virus (LCMV) Armstrong (n=3-5 per group). Peripheral blood or spleens were collected 10 days post immunization or infection (dpi). (B) Frequency of GP33 / H-2Db tetramer-specific cells among total peripheral CD8+ T cells at 10 dpi, as measured by flow cytometry. (C) Percentage of T cells with dual interferon gamma (IFNγ) and tumor necrosis factor alpha (TNF-α) production among splenic CD44hiCD8+T cells after GP33 peptide stimulation, as determined by intracellular cytokine staining (ICS). Mice were immunized with one dose of EGFP mRNA-LNP or a mixture of GP33 SCT mRNA-LNPs in one of the four variants (wY, noY, ds1, ds2), and one LCMV Armstrong-infected mouse was included as a control. Points represent individual animals, and bars and error bars represent mean and standard deviation, respectively (B-C).

[0083] Fig 4 depicts LCMV Clone13 viral challenge in GP33 SCT-vaccinated mice. (A) Overview of experimental design. C57BL / 6 mice immunized i.m. with 1 or 3 doses of 5 ug mRNA LNP were challenged with 2e6 ffu LCMV Clone 13 via the retro-orbital (r.o.) route 35 days after immunization (n=3-5 mice per group). Peripheral blood was collected 1 day prior to infection and 2 and 6 days after infection. (B) Frequency of GP33 / H-2Db tetramer-specific cells out of total peripheral CD8+ T cells, as determined by flow cytometry. Percentages for individual animals across longitudinal time points are shown as circles connected by lines. (C) Serum LCMV Clone13 viral titers, as determined by a focus forming assay, six days after viral challenge. Points represent individual animals, and bars and error ranges represent mean and standard deviation, respectively. The lower level of detection (LLOD) is shown as a dotted line. FFU / ml, focus forming units per ml.

[0084] Fig. 5 depicts vaccination with a cocktail of five LCMV T cell epitopes encoded by SCTs. (A) Overview of experimental design. C57BL / 6 mice were given an mRNA SCT cocktail comprising equimolar amounts of GP33 / H-2Db, GP276 / H-2Db, GP118 / H-2Kb, NP396 / H-2Db, and NP205 / H-2Kb SCTs via i.m. immunization. Immune responses were benchmarked against LCMV Armstrong infection (2e5 ffu i.p.), an mRNA cocktail encoding native full-length LCMV glycoprotein (GP) and nucleoprotein (NP), and an EGFP mRNA control. Mice were administered three injections on days 0, 2, and 4 with either high (5 ug), medium (1 ug), or low (0.1 ug) doses of total mRNA, e.g. 5 ug of the SCT mRNA cocktail corresponds to 1 ug of each LCMV SCT DM2\21145312.113PATENT Docket No. Y9054-99007 (n=5 mice per group). Peripheral blood was collected 10 days after the first immunization. (B). Frequency of IFNγ / TNF-α double-positive T cells among peripheral CD44hiCD8+T cells for each LCMV peptide, as determined by ICS. Peripheral blood from mice in each group was pooled prior to peptide stimulation. (C) Immunodominance patterns across LCMV epitopes are shown as the proportion of IFNγ / TNF-α double-positive T cells that were stimulated by each peptide for each immunization group.

[0085] Fig. 6 depicts induction of non-classical Qa-1b-restricted T cell responses by SCT vaccination. (A) Overview of experimental design. 3 ug mRNA flu SL9 / Qa-1b SCT constructs were injected i.m. on days 0, 2, and 4 in C57BL / 6 mice, and peripheral blood was collected 22 days post immunization for analysis (n=5 mice per group). (B) Frequency of T cells producing TNF-α and IFNγ among activated CD8+T cells after peptide stimulation with the flu SL9 peptide or an irrelevant Qa-1-restricted peptide Mycobacterium tuberculosis (Mtb) p68 (VLRPGGHEL), as assessed by ICS. Individual animals are shown as circles. Bar heights represent means, and error ranges represent standard deviation. Statistical comparisons were made by 2-way ANOVA with Dunnett’s multiple comparisons. **P < 0.01; ***P < 0.001.

[0086] Fig.7 depicts therapeutic neoantigen SCT vaccination in the B16F10 tumor model. (A) Experimental overview.6-week-old female C57BL / 6 mice were implanted s.c. on the right flank with 0.5 million B16F10 cells and injected with 3 ug of mRNA LNPs encoding a cocktail of neoantigen SCTs or control EGFP on days 3, 6, and 9 after tumor implantation. mRNA was injected into the right thigh intramuscularly or split across two footpads. Mice were treated intravenously (i.v.) with anti-PD-1 or isotype antibody (100 ug.) starting on day 8 and continuing every three days until day 20 (n=5 mice per group). Mice were euthanized prior to day 21 if tumors volumes exceeded pre-specified sizes. Remaining mice (n=3-5 per group) were sacrificed on day 21 and tumors excised for analysis. (B) Average tumor volumes for each treatment group over time, where error bars represent standard deviation. Tumors that were too small for measurement were assigned a tumor volume of 1 mm3. (C) Tumor volumes of individual mice 15, 18, and 20 days after tumor implantation. (D) Tumor weights after sacrifice on day 21. (E) Number of (left) total CD8+T cells and (right) Trp2 tetramer-positive CD44hiCD8+T cells, as enumerated by flow cytometry and calculated after normalization to tumor weight. (F) Percentage of IFNγ and TNF-α double-positive T cells among CD44hiCD8+TILs after peptide stimulation with (left) Trp2 and (right) mixture of M27, M33, and modified p53 peptides, as assessed by ICS. (C-F) Points DM2\21145312.114PATENT Docket No. Y9054-99007 represent individual animals and mean and standard deviation are shown by black lines and error bars, respectively.

[0087] Fig. 8 depicts in vitro stimulation and in vivo priming of HCMV-specific T cells by HLA A*02:01 SCT. (A) FACS plots of IFNγ and TNF-α cytokine production by CD8+T cells after direct in vitro stimulation of PBMCs isolated from a CMV-positive A*02:01 donor with mRNA LNPs encoding HCMV pp65 / A*02:01 SCT constructs, as measured by ICS. Stimulation with synthetic pp65 peptide and an irrelevant mRNA LNP (fLuc) were included as control. Gate percentages represent IFNγ / TNF-α double-positive cells among live CD8+CD3+T cells. (B) Frequency of all IFNγ-producing cells or IFNγ / TNF-α double-positive cells among CD8+T cells from two CMV-positive donor PBMCs after HCMV pp65 / A*02:01 SCT mRNA LNP stimulation, as measured by ICS. PBMCs were expanded ex vivo with HCMV pp65 for 8 days prior to LNP stimulation. Cytokine frequencies for each donor is shown across multiple stimulation conditions via connected lines. (C) Experimental overview. Transgenic HLA-A2.1 mice were immunized i.m. with 3 ug of an mRNA mixture comprising SCT constructs expressing pp65 / A*02:01 and US8 / A*02:01 on days 0, 2, and 4. Spleens were collected 10 days after the first immunization (n=3). (D) Percentage of (left) IFNγ-positive and (right) IFNγ / TNF-α-double-positive cells among activated splenic CD8+T cells after stimulation with HCMV US8 peptide, HCMV pp65 peptide, or DMSO, as assessed by ICS. Individual mice are shown as points connected by black lines. HCMV, human cytomegalovirus. fLuc, firefly luciferase.

[0088] Fig.9 depicts SCT-elicited T cells against all five LCMV epitopes are detectable 200 days after immunization. Fig. 9A depicts stimulation of CD8+T cells by full-length peptides compared to single-chain trimers comprising the peptides. Fig.9B depicts LCMV-specific T cells 10 days (D10 SCT, D10 FL) or 200 days (D200 SCT, D200 FL) following immunization.

[0089] Fig.10 depicts comparison of SCTs to mRNA-encoded full-length protein and peptide string vaccines. Immunodominance hierarchies were compared across a panel of 5 LCMV epitopes encoded within SCT mRNA, peptide string (PepStr) mRNA, full-length GP and NP mRNA, or infection by LCMV Armstrong and Clone13 strains. Overall SCT appears to elicit a more balanced CD8 T cell response with broad coverage of antigen specificities relative to the other mRNA vaccine constructs.Armstrong: LCMV Armstrong strain; Clone 13: LCMV Clone 13 is a persistent Armstrong variant. DM2\21145312.115PATENT Docket No. Y9054-99007

[0090] Fig. 11. Co-delivery of IL-2 skews T cells towards a short-lived, effector-like state. (A) Reduced GP92-specific cells by day 32. (B) Increased proportion of KLRG1hiIL-7Rlocells after IL-2 co-delivery immunization.

[0091] Fig. 12. IL-12 skews the antigen-specific response towards memory precursors. (A) Cytokine production by CD8+ T cells after LCMV peptide stimulation on Day 32. (B) Reduced proportion of KLRG1hiIL-7Rlocells after IL-12 co-delivery immunization.

[0092] Fig.13. Enhanced T cell responses by IL-4 and GM-CSF co-delivery at 32 days after immunization.

[0093] Fig.14. Screening of a panel of 10 CD40 helper, T cell co-stimulatory molecules and DC activation molecules for co-delivery with SCTs.

[0094] Fig.15. Co-delivery of CD4 helper molecules. The proportion of IFNγ+TNFα+double positive (left) and IFNγ+TNFα+IL-2+triple positive (right) CD8 T cells was determined following immunization with mRNA encoding a SCT (GP33 or GP276) and a CD4 helper molecule. The SCT and CD4 helper molecule were expressed from the same construct as separate proteins (IRES- li-PADRE) or a fusion (SCT-AAY-PADRE).

[0095] Fig.16. Costimulatory CD137L-CD40 chimera protein enhances polyfunctionality of T cell responses after SCT vaccination. The proportion of IFNγ+TNFα+double positive (left) and IFNγ+TNFα+IL-2+triple positive (right) CD8 T cells was determined following immunization with GP33 or GP276 SCT mRNA and co-delivery of CD137L-CD40 chimera protein.

[0096] Fig. 17. A chimeric CD70 (CD27 ligand)-CD40 molecule promotes development of cytotoxic CX3CR1+CD8+memory T cells. (A) The proportion of IFNγ+TNFα+double positive CD8+T cells or IL-2+CD8+T cells is depicted using SCT alone or SCT with CD70-CD40 chimera protein. (B) Left panel depicts CX3CR1 mean fluorescence intensity of tetramer+CD8 T cells using SCT alone or SCT with CD70-CD40 chimera protein. Right panel depicts proportion of effector memory T cells (CD127+CD62Llo) of tetramer+CD8 T cells.

[0097] Fig.18. Chimeric A*02:01 SCTs prime HIV antigen-specific T cells in humanized A02 mice. The proportion of IFNγ+TNFα+double positive CD8 T cells was determined for SCTs containing six different HIV A*02:01-restricted peptides. Nef 9205 (LTFGWCFKLV; SEQ ID NO:33); Gp120 121 (KLTPLCVTL; SEQ ID NO:34); Nef 9202 (PLTFGWCYKL; SEQ ID NO:35); protease 76 (LVGPTPVNI; SEQ ID NO:36); RT 179 (VIYQYMDDL; SEQ ID NO:37). DM2\21145312.116PATENT Docket No. Y9054-99007

[0098] Fig. 19. SCTs elicit T cells with polyfunctional cytokine profiles. Peptides: LCMV GP33 (KAVYNFATC; SEQ ID NO:9); LCMV GP276 (SGVENPGGYCL; SEQ ID NO:10); LCMV GP92 (CSANNSHHYI; SEQ ID NO:14); LCMV NP396 (FQPQNGQFI; SEQ ID NO:12); LCMV NP205 (YTVKYPNL; SEQ ID NO:13).

[0099] Fig. 20. Qa1-restricted T cell responses elicited by SCT immunization against exogenous and self-antigen-derived peptides. Peptides: Salmonella GroEL (GMRFDKGYI; SEQ ID NO:27); TB p44 (RLPAKAPLL; SEQ ID NO:28); TB p55 (VMATRRNVL; SEQ ID NO:29); TB p68 (VLRPGGHFL; SEQ ID NO:30); Flu PR8 SL9 (SLQGRTLIL; SEQ ID NO:15) 9mer epitope associated with influenza A virus (IAV) Puerto Rico / 8 / 1934 (PR8). The nonamer FL9 (FYAEATPML; SEQ ID NO:31) is a naturally processed peptide presented by Qa-1bmolecules.

[0100] Fig.21. (A) Classical MHC-I epitopes do not preclude induction of a nonclassical Qa- 1b-restricted T cell response. Mice were immunized with an SCT cocktail containing equimolar ratios of SL9 / Qa1, GP33 / Db, and GP276 / Db and the proportions of CD8+T cells that are IFNγ+(left) or IFNγ+TNFα+(right) after immunization were determined. (B) SL9 Qa-1b SCT elicits higher levels of antigen-specific T cells relative to mRNA encoding SL9 fused to the LCMV GP protein.

[0101] Fig. 22. Induction of T cell responses against novel LCMV Qa-1b-binding peptides discovered through the pMHC yeast display platform.

[0102] Fig. 23A-23B. Competition with Qa1- and Kb / Db-restricted peptides confirm that novel epitopes induced Qa-1b-restricted T cells. (A) Coexpression of IFNγ and CD44. (B) Percentage of IFNγ+of CD44hiCD8+T cells induced by FLK9 (left) or VIY9 (right).

[0103] Fig. 24. PBMCs from COVID convalescent donors exhibit enhanced responses to SARS-CoV-2 HLA-E SCTs. Peptides: P001 (VMPLSAPTL; SEQ ID NO:50); P003 (YLQPRTFLL; SEQ ID NO:51); P06 (VLWAHGFEL; SEQ ID NO:52); P013 (AMYTPHTVL; SEQ ID NO:53); P015 (SLPINVIVF; SEQ ID NO:54).

[0104] Fig.25. Immunization of self- protein peptides as a Qa-1b pepMHC SCT ameliorates disease in autoimmune mouse models. MRL-lpr mice were immunized with the FL9 / Qa-1b SCT and PBMCs were tested by peptide stimulation and ICS 10 days later.

[0105] Fig.26. Immunization in 7-week old mice prevents enlargement of lymph nodes in a spontaneous mouse lupus model. DM2\21145312.117PATENT Docket No. Y9054-99007

[0106] Fig. 27. Reduced incidence of type I diabetes in the spontaneous NOD model after Hsp60sp / Qa1 SCT immunization.

[0107] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION

[0108] Orchestration of immune responses generally depends on two subsets of T lymphocytes. CD8+T cells are cytotoxic and can eliminate virus-infected host cells. CD4+T cells generally provide signals that help the activation of immune cells, including antibody production by B lymphocytes and activation of CD8+T cells. CD8+T also have a regulatory role, including suppressing autoimmune CD4+T cells and reducing autoimmune disease severity.

[0109] Major histocompatibility complex (MHC) Class I: MHC class I molecules are heterodimers formed from two non-covalently associated proteins, the HLA heavy chain (also referred to as HLA α chain herein) and β2-microglobulin. The HLA heavy chain includes three distinct domains, αl, α2 and α3. The three-dimensional structure of the α l and α 2 domains forms the groove into which antigen fit for presentation to T-cells. The α 3 domain is an Ig-fold like domain that contains a transmembrane sequence that anchors the α chain into the cell membrane of the APC. MHC class I complexes, when associated with antigen (and in the presence of appropriate co-stimulatory signals) stimulate CD8 cytotoxic T-cells, which function to kill any cell which they specifically recognize.

[0110] Human leukocyte antigen (HLA): Proteins encoded by the MHC gene complex. HLAs from MHC Class I include classical HLA-A, HLA-B, and HLA-C genes and are highly variable, with up to hundreds of variant alleles at some loci. HLA loci are named with HLA, followed by the locus. Equivalent mouse HLA heavy chains are designated H-2K, H-2D, or H-2L heavy chain. HLAs from MHC Class I further include non-classical HLA-E, HLA-F, and HLA-G which exhibit little polymorphism. Mouse Qa-1a and Qa-1b are considered homologs of HLA-E. Mouse Qa-2 is considered the homolog of HLA-G.

[0111] Single chain trimer (SCT): A recombinant MHC Class I molecule including all components of the complex (HLA heavy chain, β2m, and peptide) as a single, linked molecule. The components may be joined with linkers. In some examples, SCT refers to a nucleic acid DM2\21145312.118PATENT Docket No. Y9054-99007 encoding an HLA heavy chain, β2m, peptide antigen, and one or more linkers. In some embodiments, an assembled nucleic acid encodes an SCT with protein subunits in the order (N- terminal to C-terminal): a secretion signal, a peptide (such as a peptide antigen or placeholder peptide), a first linker (LI), a β2m protein, a second linker (L2), and an HLA alpha chain (which may be referred to as an HLA heavy chain). Generally the HLA alpha chain includes a transmembrane (TM) domain. In some embodiments, the secretion signal is an HLA secretion signal. However, other secretion signals can be used. In certain examples set forth herein, the secretion signal of CD177 is employed.

[0112] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, CD4+T cells and CD8+T cells. A CD4+T cell is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+T cells carry the “cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8+T cell is a cytotoxic T lymphocyte (CTL). In another embodiment, a CD8+cell is a suppressor T cell.

[0113] Activated T cells can be detected by an increase in cell proliferation and / or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL- 18, IL-21, IL-27, IFNy, or TNFa). Activation of CD8+T cells can also be detected by an increase in cytolytic activity in response to an antigen. In certain embodiments, activated T cells can be detected by measuring the upregulation of surface activation markers.

[0114] In some embodiments, the HLA heavy chain described herein include one or more amino acid substitutions compared to a wild type HLA heavy chain. Amino acid substitutions may be selected to improve the properties or function of the SCT, such as increasing stability, peptide loading in the peptide binding groove, immunogenicity, and / or enabling dithiol linkage.

[0115] Exemplary amino acid substitutions include a leucine at an amino acid position corresponding to amino acid 74 (e.g., H74L or D74L), a cysteine or a leucine at an amino acid position corresponding to amino acid 84 (e.g., Y84C or Y84A or Y84L), a cysteine at an amino acid position corresponding to amino acid 139 (e.g., A139C), or any combination of two or more thereof.

[0116] Without being bound by theory, such mutations may be employed to modulate or enhance antigen presentation. For example, the Y84C, Y84A, and Y84L mutations are proposed DM2\21145312.119PATENT Docket No. Y9054-99007 to provide improved linker accommodation for peptides in the SCT peptide biding site. Other substitutions for Y84 would also be effective. The Y84C-A139C combination provides a disulfide linkage which allows normal PLC interaction and antigen presentation. The design is proposed to mimic the effects of bound high-affinity peptide and promote surface expression of the SCT. The Y84C-A139C combination may be referred to herein as “MHC-I (ds1)” Also described herein is a construct referred to as “MHC (ds2).” The construct includes Y84C combined with a cysteine substitution in the first linker is proposed to enhance binding of the peptide to HLA-I.

[0117] MHC Multimers

[0118] MHC multimers can be designed that comprise a complete set of functional components. In certain such embodiments, the N terminal to C terminal configuration (5’ to 3’ for an encoding nucleotide), including linkers, can be signal sequence-peptide-linker 1-β2m-linker 2- MHC-I alpha chain. In another embodiment, the elements can be configured to produce a trimer linked to or coordinately expressed with a costimulatory molecule. In another embodiment, the elements can be configured to produce a trimer linked to or coordinately expressed with a cytokine.

[0119] In some embodiments, an mRNA encoding an MHC-I alpha chain-containing trimer is operationally linked to an mRNA that encodes a costimulatory molecule or a cytokine. The components can be expressed separately from the same RNA for example by incorporating an IRES ribosome reentry site, or expressing the components as a single polypeptide with the peptide- joined through a self-cleaving 2A peptide (e.g., T2A, E2A, P2A or F2A).

[0120] The number of named HLA alleles identified according to the IPD-IMGT / HLA nomenclature is constantly increasing and observed diversity between human populations is shaped by geographic factors. Suitable classical human MHC-I alpha chains include, without limitation, HLA-A*01:01, HLA-A*02:01, HLA-A*02:04, HLA-A*02:05, HLA-A*02:07, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-A*24:07, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*34:01, HLA-A*68:01, HLA-A*68:02, HLA-A*74:01, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*14:02, HLA-B*15:01, HLA-B*15:03, HLA-B*15:04, HLA-B*15:25, HLA-B*18:01, HLA-B*27:04, HLA-B*27:05, HLA-B*35:01, HLA-B*35:02, HLA-B*35:03, HLA-B*35:05, HLA-B*35:12, HLA-B*35:19, HLA-B*35:43, HLA-B*37:01, HLA-B*38:01, HLA-B*39:02, HLA-B*39:05, HLA-B*39:06, HLA-B*39:09, HLA-B*40:01, HLA-B*40:02, HLA-B*40:04, HLA-B*40:06, HLA-B*42:01, HLA-B*44:02, DM2\21145312.120PATENT Docket No. Y9054-99007 HLA-B*44:03, HLA-B*45:01, HLA-B*46:01, HLA-B*46:02, HLA-B*48:02, HLA-B*48:03, HLA-B*49:01, HLA-B*50:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01, HLA-B*55:01, HLA-B*56:01, HLA-B*56:02, HLA-B*57:01, HLA-B*57:03, HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*03:05, HLA-C*04:01, HLA-C*04:03, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*16:02, or HLA-C*17:01. The enumerated list includes some of the most frequent HLA alleles around the world but is not limiting. (See, e.g., Sanchez-Mazas et al., The most frequent HLA alleles around the world: A fundamental synopsis. Best Pract Res Clin Haematol. 2024 Jun;37(2):101559).

[0121] Suitable MHC-I alpha chains further include non-classical MHC-I alpha chains. Unlike classical HLA genes, the non-classical HLA genes have a much smaller number of alleles. The most common HLA-E alleles are HLA-E*01:01 and HLA-E*01:03. Among HLA-F alleles, HLA- F*01:01 has the highest frequency, followed by HLA-F*01:03.

[0122] HLA-E is a highly conserved non-classical MHC class Ib molecule that exhibits limited polymorphism. Like classical MHC class Ia molecules, HLA-E associates with β2-microglobulin Unlike multiallelic MHC Ia genes, two HLA-E alleles represent most of the population. HLA-E typically binds peptides from signal sequences of classical MHC molecules HLA-A, HLA-B, HLA-C and HLA-G. The signal sequence 9-mer peptides, termed VL9, have similar sequences (VMAPRTL(L,I,V,F)F; SEQ ID NO:55). Upon assembly, the complex of HLA-E / β2m / VL9 is transported to the cell surface where it engages inhibitory NKG2A / CD94 receptors on NK cells, thus inhibiting NK-mediated killing of the presenting cell expressing the HLA-E complex. HLA- E can also present pathogenic peptides to the TCR of CD8+T cells. For example, HLA-E can present pathogen-derived peptides to the adaptive immune system, particularly to CD8+T cells. Unconventional HLA-E mediated CD8+T cell recognition has been observed for Mycobacterium tuberculosis (MTb), Salmonella typhi (S. typhi), cytomegalovirus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), and Hepatitis C Virus (HCV).

[0123] HLA-E can present self-peptides other than VL9. In certain viral infections and malignancies, expression of classical MHC class Ia proteins is inhibited, hence there are no HLA-derived peptides for HLA-E to present, and HLA-E complexes with a diverse set of self- peptides. There is herein provided a method of reducing or inhibiting autoimmune reactions and DM2\21145312.121PATENT Docket No. Y9054-99007 diseases. Without being bound by theory, it is understood that by stimulating appropriate populations of CD8+regulatory T cells, autoreactive T cells can be suppressed. Lampen, et al. identified over 500 peptides of different sequence and length (8- to 13-mer) presented by HLA-E in the absence of HLA class Ia expression. (Lampen et al., Alternative peptide repertoire of HLA-E reveals a binding motif that is strikingly similar to HLA-A2. Mol. Immunol.2013, 53, 126–131). Also, a small number of heat shock protein derived peptides have been identified that compete well for binding to HLA-E alleles and are similar if not identical between mouse and human. Expression of the HSPs, also known as chaperone proteins, while important for normal cellular function, is also implicated in various disease states. Without being bound by theory, a relatively small number of peptides may be useful for induction of regulatory responses that suppress autoreactive cells.

[0124] In certain embodiments, the MHC multimers comprise one or more peptide linkers, located for example in between the peptide, β2m, and HLA domains. The term “peptide linker” denotes a linear amino acid chain of natural and / or synthetic origin. The linker has the function to ensure that polypeptides conjugated to each other can perform their biological activity by allowing the polypeptides to fold correctly and to be presented properly. The peptide linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides. In some embodiments, the peptide linker has a length of from 2 to 50 amino acids. In some embodiments, the peptide linker is between 3 and 30 amino acids, between 5 to 25 amino acids, between 5 to 20 amino acids, or between 10 and 20 amino acids.

[0125] In some embodiments, the peptide linker is rich in glycine, glutamine, and / or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids. This small repetitive unit may be repeated for one to five times. At the amino- and / or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added. Other synthetic peptidic linkers are composed of a single amino acid, which is repeated between 10 to 20 times and may comprise at the amino- and / or carboxy-terminal end up to six additional arbitrary, naturally occurring amino acids. All peptidic linkers can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the linkers are themselves peptides, the polypeptide connected by the linker are connected to the linker via a peptide bond that is formed between two amino acids. DM2\21145312.122PATENT Docket No. Y9054-99007

[0126] Suitable peptide linkers are well known in the art, and are disclosed in, e.g., US2010 / 0210511 US2010 / 0179094, and US2012 / 0094909, which are herein incorporated by reference in its entirety. Other linkers are provided, for example, in U.S. Pat. Nos. 5,525,491; Alfthan et al., Protein Eng., 1995, 8:725-731; Shan et al., J. Immunol., 1999, 162:6589-6595; Newton et al., Biochemistry, 1996, 35:545-553; Megeed et al.; Biomacromolecules, 2006, 7:999- 1004; and Perisic et al., Structure, 1994, 12:1217-1226; each of which is incorporated by reference in its entirety.

[0127] In some embodiments, the polypeptide linker is synthetic. As used herein, the term “synthetic” with respect to a polypeptide linker includes peptides (or polypeptides) which comprise an amino acid sequence (which may or may not be naturally occurring) that is linked in a linear sequence of amino acids to a sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature. For example, the polypeptide linker may comprise non- naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or which comprise a first amino acid sequence (which may or may not be naturally occurring). Polypeptide linkers may be employed, for instance, to ensure that the binding portion (TCR or MHC), the multimerization domain and the Igg-Framework of each multimeric fusion polypeptide is juxtaposed to ensure proper folding and formation of a functional multimeric protein complex. Preferably, a polypeptide linker will be relatively non-immunogenic and not inhibit any non-covalent association among monomer subunits of a binding protein.

[0128] In some embodiments, the linker is a Gly-Ser polypeptide linker, i.e., a peptide that consists of glycine and serine residues. Non-limiting examples of such Gly-Ser linker include the following. One exemplary Gly-Ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n (SEQ ID NO:70), wherein n=1-6. In certain embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3. In certain embodiments, n=4. In certain embodiments, n=5. In certain embodiments, n=6. Another exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n (SEQ ID NO:71), wherein n=1-10. In certain embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3, i.e., Ser(Gly4Ser)3(SEQ ID NO:72). In certain embodiments, n=4, i.e., Ser(Gly4Ser)4 (SEQ ID NO:73). In certain embodiments, n=5. In certain embodiments, n=6. In certain embodiments, n=7. In certain embodiments, n=8. In certain embodiments, n=9. In certain embodiments, n=10. DM2\21145312.123PATENT Docket No. Y9054-99007

[0129] Other exemplary linkers include GS linkers (i.e., (GS)n) (SEQ IDNO:75), GGSG (SEQ ID NO:76) linkers (i.e., (GGSG)n) (SEQ ID NO:77), GSAT linkers (SEQ ID NO:78), SEG linkers, and GGS linkers (i.e., (GGSGGS)n) (SEQ ID NO:79), wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5), SSSGSSSGSAA (SEQ ID NO:80) linkers, G5linkers (GGGGG) (SEQ ID NO:81), (Gly4Ser)4(GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO:82); and (GS)2AG2SGSG3S linkers (GSGSAGGSGSGGGS) (SEQ ID NO:83).

[0130] In various embodiments, an MHC multimer expression construct comprises a linker at one or more of the following locations within the expression construct: between the MHC-binding peptide coding region and the MHC chain-encoding region; between the two MHC chain-encoding regions (e.g., between MHC Class I alpha chain and beta2-microglobulin chain coding regions or between the MHC Class II alpha chain and MHC Class II beta chain coding regions), between the MHC chain-encoding regions and the multimerization domain coding region, and / or between the multimerization domain coding region and a C-terminal tag-encoding region. In certain embodiments, the linker located between the MHC-binding peptide coding region and the MHC chain-encoding region comprises a cleavage site (e.g., a site cleavable by an enzyme, such as a protease). Suitable protease cleavage sites include those cleaved by proteases such as Factor Xa, thrombin, TEV, HRV3C, furin and the like.

[0131] In certain embodiments, an expression construct includes a signal sequence operatively linked at the N-terminal end of the coding region, such that the encoded fusion polypeptide is transcribed with a signal sequence to thereby facilitate secretion of the MHC multimer from the host cell (e.g., into the cell culture medium such that the MHC multimers can be recovered from the cellular supernatant). In certain embodiments, the signal sequence is a heterologous signal sequence (i.e., the signal sequence is not a native MHC signal sequence). In certain embodiments, the signal sequence is from an Ig supergroup member. In certain embodiments, the signal sequence is an immunoglobulin chain signal sequence.

[0132] Some exemplified constructs employ the signal sequence MSAVLLLALLGFILPLPGVQA (SEQ ID NO:56). In other non-limiting embodiments, suitable signal sequences include the signal sequence of an Ig Kappa chain V-III region CLL signal peptide, e.g., having the sequence MEAPAQLLFLLLLWLPDTTG (SEQ ID NO:57). Other suitable signal sequences include a human CD4 signal peptide, e.g., having the sequence MNRGVPFRHLLLVLQLALLPAAT (SEQ ID NO:58), a mouse Ig kappa chain V-III region DM2\21145312.124PATENT Docket No. Y9054-99007 signal peptide, e.g., having the sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO:59), a mouse H-2Kb signal peptide, e.g., having the sequence MVPCTLLLLLAAALAPTQTRA (SEQ ID NO:60), a human serum albumin signal peptide, e.g., having the sequence MKWVTFISLLFLFSSAYS (SEQ ID NO:61), a human IL-2 signal peptide, e.g., having the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:62), a human HLA-A*02:01 signal peptide, e.g., having the sequence MAVMAPRTLLLLLSGALALTQTWA (SEQ ID NO:63), and a human b2m signal peptide, e.g., having the sequence MSRSVALAVLALLSLSGLEA (SEQ ID NO:64).

[0133] The present invention encompasses nucleic acid sequences encoding any of the proteins (e.g., MHC multimer polypeptides) described herein. In one embodiment, the nucleic acid sequence is incorporated into a vector, such as a plasmid vector, a viral vector or a non-viral vector. The vector is selected to be suitable for use in the intended host cell (i.e., the vector incudes all necessary transcriptional regulatory elements to allow for expression of the encoded MHC multimer polypeptide in the host cell). Suitable vectors, including transcriptional regulatory elements for use in various host cells, including mammalian host cells, are well established in the art. In another embodiment, the nucleic acid sequence is incorporated in vector suitable for replicating the selected sequence. The replicated sequence provides a template for in vitro transcription to generate mRNA suitable for transfection and expression in a host cell. In another embodiment, a template for mRNA transcription is amplified from a suitable nucleic acid sequence prior to transcription. In yet another embodiment, a whole template nucleic acid is amplified from suitable segments by amplification followed by mRNA transcription from the template. Peptides and Antigens

[0134] As used herein, the term “peptide” in the context of a single chain trimer means a polypeptide capable of binding to one or more specific MHC alleles. Cells expressing MHC / peptide complexes on their surface are referred to as “Antigen Presenting Cells” (APCs). Similarly, as used herein, the term “MHC binding peptide” relates to a peptide which binds to an MHC class I and / or an MHC class II molecule. In the case of MHC class I / peptide complexes, the binding peptides are typically 8-10 amino acids long although longer or shorter peptides may be effective.

[0135] HLA / MHC binding DM2\21145312.125PATENT Docket No. Y9054-99007

[0136] In aspects of the invention, viral antigens, cancer antigens, and neoantigens are evaluated for immune presentation. Peptide antigens and neoantigens particularly may be individually evaluated for binding in vitro or to panels of mono-allelic cells. MHC binding can be determined by identifying peptides associated with MHC by liquid chromatography-tandem mass spectrometry (LC MS / MS) which allows identification of a large number of sequences that have undergone the entire peptide presentation pathway. (Creech et al., 2018, The Role of Mass Spectrometry and Proteogenomics in the Advancement of HLA Epitope Prediction. Proteomics 18(12):el700259). The LC MS / MS dataset published by Abelin et al. represents over 26,000 Class I peptides eluted across 16 HLA-A or HLA-B monoallelic cell lines. (Abelin et al., 2017, Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity 46(2):315-326). Such eluted peptides databases are useful to identify peptides that bind to MHC, to evaluate in silico predictions, to screen or rank peptides identified by in silico predictions, and to improve in silico prediction methods binding to HLA / MHC may be evaluated. In certain embodiment, a synthetic yeast display platform is employed that expresses SCTs with a proteolytically cleavable linker to identify peptides.

[0137] Several public in silico tools have been developed to predict MHC binding. NetMHCpan-2.0 is an in silico tool that generates quantitative predictions of the affinity of any peptide-MHC class I interaction. NetMHCpan-2.0 was trained on a large set of quantitative MHC binding data, covering human HLA-A and HLA-B. (Hoof I, Peters B, Sidney J, et al. NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics.2009;61: 1-13). NetMHCpan-3.0 captures differences in the length profile of binders to different MHC molecules leading to improved accuracy for ligand identification due to more uniform sampling of the MHC. (Nielsen M, Andreatta M. NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets. Genome Med.2016;8:33). NetMHCpan-4.0 is further iteration of the in silico tool and is trained on binding affinity and eluted ligand data. (Jurtz V, Paul S, Andreatta M, et al. NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. J. Immunol.2017;199:3360 LP-3368).

[0138] Peptide selection can include several considerations. In some embodiments, epitope selection of peptides may involve prediction software. In some embodiments, epitope selection of peptides may involve experimental data to evaluate binding and / or immunological activity. For DM2\21145312.126PATENT Docket No. Y9054-99007 example, Peng et al. used an activation assay to characterize presentation of HPV18 E7 / E6 epitopes. (See Peng et al., Identification of human MHC‑I HPV18 E6 / E7‑specific CD8 + T cell epitopes and generation of an HPV18 E6 / E7‑expressing adenosquamous carcinoma in HLA‑A2 transgenic mice. Journal of Biomedical Science, 2022, 29:80.

[0139] In some embodiments, it will be advantageous to identify peptides that have high sequence conservation and are predicted to be important for viral fitness, which may be referred to as “networked.” (See, e.g., Kaseke et al., HLA class-I-peptide stability mediates CD8+ T cell immunodominance hierarchies and facilitates HLA-associated immune control of HIV. Cell Rep. 2021;36(2):109378. HIV peptides exemplified herein were selected by such a process. The same or similar procedure may be used for epitope selection in other pathogens.

[0140] Exemplary peptides are provided in Table 2.

[0141] As used herein, the term “vaccine” refers to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, in particular a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a neoplasia (e.g., a cancer cell). A vaccine may be used for the prevention or treatment of a disease. Accordingly, vaccines are medicaments which include antigens and are used in humans or animals for generating specific defense and protective substance by vaccination. The term “personalized vaccine” or “personalized neoplasia vaccine” or the like term concerns a particular neoplasia patient and means that a neoplasia (e.g. cancer) vaccine is adapted to the needs or special circumstances of an individual patient.

[0142] Co-delivery of cytokines.

[0143] In an aspect of the invention, mRNAs encoding cytokines may be co-delivered with SCT trimers. In some embodiments, the cytokines may separated by a processing site such as a T2A site. In some embodiments, the cytokines may be separately encoded from the SCTs. In some embodiments, T cell antigen-specific phenotypes are skewed towards a short-lived, effector-like state. In a non-limiting example, IL-12 is co-delivered leading to an increased proportion of KLRG1hiIL7Rlocells after codelivery.

[0144] In some embodiments, T cell antigen-specific phenotypes are skewed towards memory precursors. In a non-limiting example, IL-2 is co-delivered leading to a reduced proportion of KLRG1hiIL7Rlocells after codelivery. In some embodiments, IL-15 is co-delivered promoting memory T cell activation and a reduced proportion of KLRG1hiIL7Rlocells. DM2\21145312.127PATENT Docket No. Y9054-99007

[0145] In some embodiments, T cell responses are enhanced by codelivery of IL-4 and / or GMCSF. Enhanced responses may be observed by increased proportions of CD8+T cells that are TNFα+IFNγ+.

[0146] Costimulatory molecules.

[0147] Costimulatory signals have been recognized as critical for optimal T cell responses and result from important interaction between receptors on the surface of T cells and their ligands on antigen presenting cells. To become fully activated, the initial activating signal from the TCR signal should be followed by an independent, co-stimulatory signal. Without this co-stimulatory signal, CD8+T cells become anergic and undergo apoptosis.

[0148] Herein provided are several costimulatory molecules, co-expressed as covalently attached to SCTs or expressed from the same RNA. Costimulatory molecules include, without limitation, CD40, CD70, CD137L, CD80, CD86 and fusions and combinations thereof.

[0149] CD4 helper molecules.

[0150] CD4 helper molecules comprise peptides designed to stimulate CD4+ T cells and may be coexpressed with SCTs. A common non-limiting example is PADRE (Pan HLA-DR Epitope) (AKFVAAWTLKAAA; SEQ ID NO:65), which binds to MHC-II receptors. In some embodiments, PADRE is presented by constructing an invariant (Ii) chain in which the class II- associated Ii peptide (CLIP) region is replaced with a CD4+T-helper epitope, PADRE (Ii- PADRE).

[0151] mRNAs

[0152] In some embodiments, an mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, and additionally comprises one or more of a 5' untranslated region, 3' untranslated region, 5' cap, and polyadenylation signal. In some embodiments, an mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, a 5' untranslated region, a 3' untranslated region, a 5' cap, and a polyadenylation signal. In some embodiments, an mRNA disclosed herein comprises modified ribonucleotides. In some embodiments, the mRNA comprises N1-methylpseudouridine or N1-ethylpseudouridine. In some embodiments, the 5' terminal cap is 7mG(5')ppp(5')NlmpNp. See, e.g., US20200261572, US20190351040, and US20190211065, each of which is incorporated herein by reference in its entirety.

[0153] Immunogenic Compositions DM2\21145312.128PATENT Docket No. Y9054-99007

[0154] In one aspect, provided herein are immunogenic compositions comprising an immunogenic polypeptide comprising a SCT described herein. In certain embodiments, an immunogenic composition described herein comprises at least one mRNA encoding a fusion polypeptide such as an SCT comprising an immunogenic peptide described herein. In some embodiments, the immunogenic composition comprises a cationic lipid nanoparticle (LNP) encapsulating mRNA having an open reading frame encoding at least one immunogenic peptide described herein, and a 5' terminal cap modified to increase mRNA translation efficiency. In some embodiments, the cationic lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. See, e.g., US20200261572, US20190351040, and US20190211065, each of which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the at least one mRNA encodes an SCT comprising a tumor specific neoantigen. In some embodiments, the at least one mRNA encoded SCT comprises a pathogen derived target peptide. In some embodiments, the pathogen derived target peptide is a viral, bacterial, or parasitic peptide. In some embodiments, the target peptides may be engineered.

[0156] In some embodiments, the target peptide comprises a viral peptide. In some embodiments, the viral peptide is selected from the group consisting of Betacoronavirus, Human Immunodeficiency Virus (e.g., HIV-Type 1 (HIV-1) and HIV-Type 2 (HIV-2), Chikungunya virus, Dengue virus, Ebola virus, Eastern Equine Encephalitis virus, Herpes Simplex virus, Human Cytomegalovirus, Human Papillomavirus. Human Metapneumovirus, Influenza virus, Hepatitis A, Hepatitis B, Japanese Encephalitis virus, Marburg virus, Measles, Parainfluenza virus, Respiratory Syncytial virus, Sindbis virus, Varicella Zoster virus, Venezuelan Equine Encephalitis virus, West Nile virus, Yellow Fever virus, Lassa virus, or Zika virus polypeptide and an immunogenic fragment thereof.

[0157] Betacoronavirus. In some embodiments, the BetaCoV is SARS-CoV2. In some embodiments, the BetaCoV is MERS-CoV. In some embodiments, the BetaCoV is SARS-CoV. The system of the invention is flexible and readily adaptable to seasonal CoVs and other evolving pathogens. In some embodiments, the at least one target peptide comprises a peptide of a Betacoronavirus structural protein. In some embodiments, the at least one target peptide comprises the spike protein (S) peptide, envelope protein (E) peptide, a nucleocapsid protein (N) peptide, or a membrane protein (M) peptide. In some embodiments, the at least one target antigen comprises a SI subunit peptide or a S2 subunit peptide of spike protein (S). In some embodiments, the at least DM2\21145312.129PATENT Docket No. Y9054-99007 one target peptide comprises a peptide of at least one accessory (e.g., protein 3, protein 4a, protein 4b, protein 5), at least one replicase peptide (e.g., protein la, protein lb), or a combination of at least one accessory peptide and at least one replicase peptide.

[0158] In some embodiments, the viral polypeptide is selected from the group consisting of MERS-CoV polypeptide, SARS-CoV polypeptide, SARS-CoV2 polypeptide and an immunogenic fragment thereof.

[0159] In some embodiments, the viral peptide comprises a SARS-CoV2 spike polypeptide (S), SARS-CoV2 envelope protein peptide (E), SARS-CoV2 nucleocapsid peptide (N), or a SARS- CoV2 membrane protein peptide (M).

[0160] Human Immunodeficiency Virus (e.g., HIV-Type 1 (HIV-1) and HIV-Type 2 (HIV-2). HIV is an example of a virus that interferes with antigen presentation by downregulating expression of classical MHC-I alleles. Advantageously, MHC-E presentation (which is not downregulated) can be employed. In some embodiments, the at least one target peptide comprises Gag, Pol, or other HIV gene. In some embodiments, the at least one target peptide comprises a networked epitope (i.e. including an amino acid position where mutation disproportionately impairs viral replication (see e.g., Gaiha et al., Structural topology defines protective CD8+ T cell epitopes in the HIV proteome, Science 2019 May 3; 364(6439): 480–484). Envelope glycoprotein (Env) peptides may be used but suffer from extreme diversity.

[0161] Chikungunya Virus. In some embodiments, the at least one target antigen comprises a CHIKV structural protein selected from an envelope protein (E) (e.g., El, E2, E3), a 6K protein, or a capsid (C) protein.

[0162] Dengue virus. In some embodiments, the at least one target peptide comprises a DENV capsid protein peptide, a DENV membrane protein peptide a DENV precursor-membrane protein peptide, a DENV precursor membrane (pry) and envelope (E) peptide (DENY prME), or a DENV non-structural peptide selected from NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. In some embodiments, the at least one target peptide comprises a polypeptide is from a DENV serotype selected from DENV-1, DENY-2, DENV-3, DENY-4, and DENY-5.

[0163] Ebola virus. In some embodiments, the at least one target peptide comprises an EBOV glycoprotein (GP) peptide, surface EBOV GP peptide, wild type EBOV pro-GP peptide, mature EBOV GP peptide, secreted wild type EBOV pro-GP peptide, secreted mature EBOV GP peptide, DM2\21145312.130PATENT Docket No. Y9054-99007 EBOV nucleoprotein (NP) peptide, RNA polymerase L peptide, and EBOV matrix protein peptide selected from VP35, VP40, VP24, or VP30.

[0164] Herpes Simpler virus. In some embodiments, the at least one target peptide comprises HSV (HSV-1 or HSV-2) glycoprotein B peptide, HSV (HSV-1 or HSV-2) glycoprotein C peptide, HSV (HSV-1 or HSV-2) glycoprotein D peptide, HSV (HSV-1 or HSV-2) glycoprotein E peptide, or HSV (HSV-1 or HSV-2) glycoprotein I peptide.

[0165] Human Cytomegalovirus. In some embodiments, the at least one target peptide comprises a HCMV gH, gL, gB, gO, gN, gM, UL83, UL123, UL128, UL130, or UL131A peptide.

[0166] Human Papillomavirus. In some embodiments, the at least one target peptide comprises an HPV El, E2, E4, E5, E6, E7, LI, or L2 peptide, e.g., obtained from HPV serotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 or 82.

[0167] Human Metapneumovirus, Parainfluenza virus and Respiratory Syncytial virus. In some embodiments, the at least one target peptide comprises a major surface glycoprotein G peptide. In some embodiments, the at least one peptide comprises a Fusion (F) glycoprotein (e.g., Fusion glycoprotein F0, Fl or F2) peptide. In some embodiments, the at least one peptide comprises a major surface glycoprotein G peptide and F glycoprotein peptide In some embodiments, the at least one peptide comprises a nucleoprotein (N) peptide, phosphoprotein (P) peptide, large polymerase protein (L) peptide, matrix protein (M) peptide, small hydrophobic protein (SH) peptide, nonstructural protein 1 (NS1) peptide, nonstructural protein 2 (NS2) peptide or an immunogenic peptide fragment thereof.

[0168] Influenza virus. In some embodiments, the at least one target peptide comprises an antigenic subdomain of HA, termed HA1, HA2, or a combination of HA1 and HA2 (or a combination of both, of any one of or a combination of any or all of H2, H3, H4, H5, H6, H7, H8, H9, H10, Hll, H12, H13, H14, H15, H16, H17, and / or H18). In some embodiments, the at least one target peptide comprises a neuraminidase (NA) peptide. In some embodiments, the at least one target peptide comprises nucleoprotein (NP) peptide, matrix protein 1 (Ml) peptide, matrix protein 2 (M2) peptide, nonstructural protein 1 (NS1) peptide or non-structural protein 2 (NS2) peptide.

[0169] Japanese Encephalitis virus. In some embodiments, the at least one target peptide comprises JEV E peptide, JEV Es, JEV prM, JEV capsidv, JEV NS1, or JEV prM and E peptide (prME). DM2\21145312.131PATENT Docket No. Y9054-99007

[0170] Marburg virus. In some embodiments, the at least one target peptide comprises a MARV glycoprotein (GP) peptide.

[0171] Measles. In some embodiments, the at least one target peptide comprises a hemagglutinin (HA) peptide. In some embodiments, the at least one target peptide comprises a Fusion (F) protein or an immunogenic fragment thereof. In some embodiments, the at least one peptide is from MeV strain D3 or B8, for example.

[0172] Varicella Zoster virus. In some embodiments, the at least one target peptide comprises a VZV glycoprotein peptide selected from VZV gE, gl, gB, gH, gK, gL, gC, gN, and gM.

[0173] West Nile virus, Eastern Equine Encephalitis virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. In some embodiments, the at least one target peptide comprises at least one Arbovirus peptide and / or at least one Alphavirus peptide.

[0174] Yellow Fever virus. In some embodiments, the at least one target peptide comprises a YFV peptide, a YFV capsid peptide, a YFV premembrane / membrane peptide, a YFV envelope peptide, a YFV non-structural protein 1 peptide, a YFV non-structural protein 2A peptide, a YFV non-structural protein 2B peptide, a YFV non-structural protein 3 peptide, a YFV non-structural protein 4A peptide, a YFV non-structural protein 4B peptide, or a YFV non-structural protein 5 peptide.

[0175] Zika virus antigens. In some embodiments, the at least one target peptide comprises a ZIKV polyprotein peptide, a ZIKV capsid protein peptide, a ZIKV premembrane / membrane protein peptide, a ZIKV envelope protein peptide, a ZIKV non-structural protein 1 peptide , a ZIKV non-structural protein 2A peptide, a ZIKV non-structural protein 213 peptide, a ZIKV non- structural protein 3 peptide, a ZIKV non-structural protein 4A peptide, a ZIKV non-structural protein 4B peptide, or a ZIKV non-structural protein 5 peptide.

[0176] In some embodiments, the target peptide comprises a bacterial peptide. A bacterial peptide is from an antigen encoded by a bacterial genome. In some embodiments, an immunogenic composition of described herein comprises a bacterial peptide antigen. Examples of bacterial peptide antigens include, but are not limited to, Chlamydia trachomatis antigen, Eyme Borrelia antigen and Streptococcal antigen. In some embodiments, the at least one target antigen comprises a major outer membrane peptide (MOMP or OmpA), e.g., from Chlamydia trachomatis serovar (serotype) H, F, E, D, I, G, J or K. In some embodiments, the at least one target peptide comprises DM2\21145312.132PATENT Docket No. Y9054-99007 a Borrelia OspA protein. In some embodiments, the target peptide comprises a tuberculosis peptide. In certain embodiments, it will be advantageous to employ HLA-E.

[0177] In some embodiments, the target peptide is a parasitic antigen peptide. A parasitic antigen is an antigen encoded by a parasitic genome. In some embodiments, an immunogenic composition described herein comprises a parasitic polypeptide antigen. Examples of parasitic antigen peptides include peptides from, without limitation, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, and Plasmodium knowlesi. In some embodiments, the at least one target peptide comprises a circumsporozoite (CS) protein or an immunogenic fragment thereof. In some embodiments, the at least one target peptide comprises a RTS hybrid protein. In some embodiments, the at least one target peptide comprises a merozoite surface protein-1 (MSP1), apical membrane antigen 1 (AMA1) peptide, or thrombospondin related adhesive protein (TRAP) peptide.

[0178] In some embodiments, the target peptide comprises a tumor specific neoantigen peptide. Any neoantigen known to a skilled artisan can be used in the context of an immunogenic composition described herein. Neoantigens are disclosed, for example, in US20160339090, US20170199961, US20190307868, US20190151428, and US20200279616, each of which is incorporated herein by reference in its entirety.

[0179] In some embodiments, the SCT comprises a target peptide suitable to stimulate an HLA-E restricted CD8+regulatory cell. Basic and preclinical studies have shown immunosuppressive activity of some heat shock proteins (HSPs). Regulation of autoreactive CD4 T cells is critical to maintenance of self-tolerance and prevention of autoimmune disease. Suppression of autoimmune responses by conserved epitopes of Hsp60 has been observed in murine models resembling rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA).

[0180] An aspect of the invention relates to stimulation of CD8+regulatory T cells. An aspect of the invention relates to treatment or suppression of autoimmune mediated diseases.

[0181] In some embodiments, the autoimmune disease comprises multiple sclerosis (MS). In some embodiments, the autoimmune disease comprises type 1 diabetes (T1D). In some embodiments, the autoimmune disease comprises a gastrointestinal disease including without limitation, celiac disease, Crohn’s disease, ulcerative colitis, or inflammatory bowel disease (IBD). In some embodiments, the autoimmune disease comprises a rheumatologic disease, such as, without limitation, rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). In some DM2\21145312.133PATENT Docket No. Y9054-99007 embodiments, the autoimmune disease comprises a hematologic or vascular disease, such as, without limitation autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP). In some embodiments, the autoimmune disease comprises a neuromuscular disease, for example, myasthenia gravis (MG). In some embodiments, the autoimmune disease comprises autoimmune- mediated rejection or graft-vs-host disease (GVHD). In some embodiments, the autoimmune disease comprises a chronic inflammatory or autoimmune skin condition, including without limitation, psoriasis, vitiligo, pemphigus and dermatomyositis.

[0182] In certain embodiments, the nucleic acids of the present invention may be delivered as a therapeutic mRNA.

[0183] The present invention contemplates expressing the herein disclosed immunogens as mRNAs, advantageously as mRNA vaccines. The disclosures of US Patent Nos. 9,675,668; 9,464,124; 9,447,164; 9,428,535; 9,334,328; 9,303,079; 9,301,993; 9,295,689; 9,283,287; 9,271,996; 9,255,129; 9,254,311; 9,233,141; 9,221,891; 9,220,792; 9,220,755; 9,216,205; 9,192,651; 9,186,372; 9,181,319; 9,149,506; 9,114,113; 9,107,886; 9,095,552; 9,089,604; 9,061,059; 9,050,297; 8,999,380; 8,980,864; 8,822,663; 8,754,062; 8,710,200; 8,680,069 and 8,664,194 and US Patent Publication Nos. 20220047518, 20200254086, 20200206362, 20180311336, 20180303929, 20170204152, 20160331828, 20160317647 and 20160194368 are herein incorporated by reference.

[0184] Exemplary aspects of the invention feature efficacious mRNA vaccines. Described herein are mRNA vaccines designed to achieve particular biologic effects. Exemplary vaccines of the invention feature mRNAs encoding a particular antigen of interest (or and mRNA or mRNAs encoding antigens of interest), optionally formulated with additional components designed to facilitate efficacious delivery of mRNAs in vivo. In exemplary aspects, the vaccines of the invention feature and mRNA or mRNAs encoding antigen(s) of interest, complexed with polymeric or lipid components, or in certain aspects, encapsulated in liposomes, or alternatively, in lipid nanoparticles (LNPs). Chemical modification of mRNAs can facilitate certain desirable properties of vaccines on the invention, for example, influencing the type of immune response to the vaccine. For example, appropriate chemical modification of mRNAs can reduce unwanted innate immune responses against mRNA components and / or can facilitate desirable levels of protein expression of the antigen or antigens of interest. Further description of such features of the invention is provided infra. DM2\21145312.134PATENT Docket No. Y9054-99007

[0185] In an embodiment, a novel vaccine platform is described, termed the RNA-lipid nanoparticle (RNA-LNP) vaccine. This platform involves the delivery of messenger RNA (mRNA) molecules encoding MHC-I single chain trimers (SCTs) using lipid nanoparticles as a carrier system. The mRNA-encoded SCTs comprise an antigen peptide, β2-microglobulin, and an MHC-I heavy chain, which can self-assemble into stable complexes capable of presenting the antigen to CD8+ T cells upon expression in antigen-presenting cells.

[0186] Provided herein are isolated nucleic acids (e.g., modified mRNAs encoding a peptide described herein) which may comprise a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid. For example, the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid. In certain embodiments, the nucleic acid may comprise RNA, DNA, TNA, GNA, or a hybrid thereof. In certain embodiments, the nucleic acid comprises messenger RNA (mRNA). In certain embodiments, the mRNA does not substantially induce an innate immune response of the cell into which the mRNA is introduced. In certain embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl- pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1- deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In certain embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- DM2\21145312.135PATENT Docket No. Y9054-99007 zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In other embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine. In yet other embodiments, the mRNA may comprise at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza- 8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0187] In some embodiments, the nucleic acids provided herein comprise a 5' untranslated region (UTR) and / or a 3'UTR, wherein each of the two different nucleoside modifications are independently present in the 5'UTR and / or 3'UTR. In some embodiments, nucleic acids are provided herein, wherein at least one of the two different nucleoside modifications are present in the translatable region. In some embodiments, nucleic acids provided herein are capable of binding to at least one polypeptide that prevents or reduces an innate immune response of a cell into which the nucleic acid is introduced.

[0188] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least one nucleoside modification, and (iii) at least one intronic nucleotide sequence capable of being excised from the nucleic acid.

[0189] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least two different nucleoside modifications, and (iii) a degradation domain. DM2\21145312.136PATENT Docket No. Y9054-99007

[0190] Further provided herein are non-enzymatically synthesized nucleic acids (e.g., modified mRNAs described herein) which may comprise at least one nucleoside modification, and which may comprise a translatable region encoding a peptide described herein. In certain embodiments, the non-enzymatically synthesized mRNA may comprise at least two different nucleoside modifications.

[0191] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise a noncoding region and at least one nucleoside modification that reduces an innate immune response of a cell into which the nucleic acid is introduced, wherein the nucleic acid sequesters one or more translational machinery components. In certain embodiments, the isolated nucleic acids which may comprise a noncoding region and at least one nucleoside modification described herein are provided in an amount effective to reduce protein expression in the cell. In certain embodiments, the translational machinery component is a ribosomal protein or a transfer RNA (tRNA). In certain embodiments, the nucleic acid may comprise a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

[0192] Further provided herein are isolated nucleic acids (e.g., modified mRNAs described herein) which may comprise (i) a first translatable region, (ii) at least one nucleoside modification, and (iii) an internal ribosome entry site (IRES). In certain embodiments, the IRES is obtained from a picornavirus, a pest virus, a polio virus, an encephalomyocarditis virus, a foot-and-mouth disease virus, a hepatitis C virus, a classical swine fever virus, a murine leukemia virus, a simian immune deficiency virus or a cricket paralysis virus. In certain embodiments, a translatable region encodes proteins or peptides joined through a self-cleaving 2A peptide (e.g., T2A, E2A, P2A or F2A). In certain embodiments, the isolated nucleic acid further may comprise a second translatable region. In certain embodiments, the isolated nucleic acid further may comprise a Kozak sequence. In some embodiments, the first translatable region encodes a peptide described herein. In some embodiments, the second translatable region encodes peptide described herein. In some embodiments, the first and the second translatable regions encode peptides described herein.

[0193] Provided herein are pharmaceutical compositions which may comprise: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; and (ii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl-cytidine, or a combination thereof, or ii) the mRNA does not comprise a substantial DM2\21145312.137PATENT Docket No. Y9054-99007 amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to a mammalian subject in need thereof. In some embodiments,

[0194] Further provided herein are pharmaceutical compositions which may comprise and / or consisting essentially of: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; (ii) a cell penetration agent; and (iii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl- cytidine or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to an animal (e.g., mammalian) subject in need thereof.

[0195] This invention provides nucleic acids, including RNAs such as mRNAs that contain one or more modified nucleosides (termed “modified nucleic acids”), which have useful properties including the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced. Because these modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity, these nucleic acids having these properties are termed “enhanced nucleic acids“ herein.

[0196] The term “nucleic acid,“ in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary nucleic acids for use in accordance with the present invention include, but are not limited to, one or more of DNA, RNA, hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc., described in detail herein.

[0197] Provided are modified nucleic acids containing a translatable region encoding a peptide described herein, and one, two, or more than two different nucleoside modifications. In some embodiments, the modified nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid. For example, the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, DM2\21145312.138PATENT Docket No. Y9054-99007 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid. Exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or a hybrid thereof. In preferred embodiments, the modified nucleic acid includes messenger RNAs (mRNAs). As described herein, the nucleic acids of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.

[0198] In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1- methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0199] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0200] In other embodiments, modified nucleosides include 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine. DM2\21145312.139PATENT Docket No. Y9054-99007

[0201] In certain embodiments it is desirable to intracellularly degrade a modified nucleic acid introduced into the cell, for example if precise timing of protein production is desired. Thus, the invention provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.

[0202] In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0203] Other components of nucleic acid are optional, and are beneficial in some embodiments. For example, a 5' untranslated region (UTR) and / or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence.

[0204] Further, nucleic acids encoding a peptide described herein, and containing an internal ribosome entry site (IRES) are provided herein. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic mRNA”). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

[0205] The therapeutic mRNAs may be prepared as described, for example, in US Patent Nos. 9,464,124; 9,447,164; 9,428,535; 9,334,328; 9,303,079; 9,301,993; 9,295,689; 9,283,287; 9,271,996; 9,255,129; 9,254,311; 9,233,141; 9,221,891; 9,220,792; 9,220,755; 9,216,205; 9,192,651; 9,186,372; 9,181,319; 9,149,506; 9,114,113; 9,107,886; 9,095,552; 9,089,604; DM2\21145312.140PATENT Docket No. Y9054-99007 9,061,059; 9,050,297; 8,999,380; 8,980,864; 8,822,663; 8,754,062; 8,710,200; 8,680,069 and 8,664,194 may be utilized for the present invention.

[0206] mRNA vaccine delivery

[0207] Direct injection of RNA without a carrier is not efficient as uptake is low and the RNA is rapidly degraded. Furthermore, it may be desirable to target mRNA vaccines to particular cells or subsets of cells.

[0208] Advantageously, the mRNA of the present invention may be formulated with a delivery vehicle to facilitate delivery and provide specificity. In some embodiments, the delivery vehicle for an mRNA vaccine comprises a lipid nanoparticle (LNP) formulation, such as a PEG lipid, which are useful in pharmaceutical compositions, cosmetic compositions, and drug delivery systems, e.g., for use in LNP formulations. The LNPs described in US Patent Publication Nos. 20220047518 and 20200254086 are exemplary for the delivery of an agent (e.g., therapeutic agent such as a nucleic acid) to a subject.

[0209] Lipid nanoparticles (LNPs) have been used successfully in clinical applications of mRNA therapeutics. In some embodiments, lipid nanoparticles (LNPs) are provided. In one embodiment, a lipid nanoparticle comprises lipids including an ionizable lipid (such as an ionizable cationic lipid), a structural lipid, a phospholipid, and mRNA. Each of the LNPs described herein may be used as a formulation for the mRNA described herein. In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol. In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of ionizable lipid: cholesterol:DSPC: PEG2000-DMG. DM2\21145312.141PATENT Docket No. Y9054-99007

[0210] However, while it is desirable to deliver vaccines to antigen-presenting cells, such as dendritic cells, LNP formulations tend to accumulate in the liver upon systemic administration. Advantageously there are several alternatives.

[0211] A phospholipid bilayer structure can be used to manufacture a delivery vehicle with inherent functions of cell membranes. In some embodiments, the delivery vehicle comprises a phospholipid bilayer structure. DCs express several receptors that could be targeted for vaccine delivery. Non-limiting examples include pathogen recognition receptors (PRR) and C-type lectin receptors (CLRs) that interact with molecules on the surface of pathogens and have a role in the capture and presentation of the antigen. DCs also express Toll-like receptors (from TLR1 to TLR10) which are involved in recognition of pathogen-associated molecular patterns (PAMPs) and Scavenger receptors like CD36, involved in the uptake of a variety of ligands, including lipids, apoptotic cells, and microbial components as well as several other receptors such as chemokines receptors, complement receptors and Fc receptors. Targeting of such receptors on DCs enhances antigen uptake, presentation, and immune response activation.

[0212] In some embodiments, mRNA vaccines are prepared in organic matrices. When these particles are prepared, for example, by a lyophilization process, dry powders may be produced which inhibit chemical degradation pathways and may optionally contain embedded antigens and adjuvants suitable for targeting DCs. (See Clausi, et al., Inhibition of aggregation of aluminum hydroxide adjuvant during freezing and drying. J. Pharm. Sci. (2008) 97: 2051–2061).

[0213] In some embodiments, mRNA vaccine formulations comprise carbon nanotubes. In a non-limiting example, Xu et al. employed a short carbon nanotube-based formulation to deliver an mRNA-based HIV vaccine. (See, Xu et al., Short Carbon Nanotube-Based Delivery of mRNA for HIV-1 Vaccines. Biomolecules. 2023 Jul 7;13(7):1088). The formulation was used to deliver or co-deliver a range of mRNAs encoding HIV-1 glycoproteins / peptides to antigen-presenting cells of the immune system via either intramuscular (IM) or intranasal (IN) routes.

[0214] In some embodiments, mRNA vaccines can be coated to control targeting and release. In a non-limiting example, vaccine particles can be coated by atomic layer deposition (ALD) to create nanometer-thick layered coatings providing control over location and timing of mRNA dosing. (See, e.g., Garcea et al., Single-administration, thermostable human papillomavirus vaccines prepared with atomic layer deposition technology. npj Vaccines (2020)5:45). DM2\21145312.142PATENT Docket No. Y9054-99007

[0215] Delivery vehicles include without limitation, an ionizable lipid nanoparticle (LNP); a cationic liposome; a lipoplex (lipid–mRNA complex); a polyplex (polymeric–mRNA complex); a dendrimer nanoparticle; a chitosan-based nanoparticle; a poly(β-amino ester) (PBAE) nanoparticle; a protamine–mRNA complex; a cell-penetrating peptide complex; a virus-like particle; an extracellular vesicle or exosome; a cationic nanoemulsion; a lipid–inorganic nanoparticle (LION); a squalene-based nanoemulsion; a hybrid nanoparticle; an inorganic nanoparticle (for example, a silica, gold, or iron oxide nanoparticle); a solid microparticle or depot matrix; and combinations thereof.

[0216] In other aspects the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 µg / kg and 400 µg / kg of the nucleic acid vaccine is administered to the subject. In some embodiments the dosage of the RNA polynucleotide is 1-5 µg, 5-10 µg, 10-15 µg, 15-20 µg, 10-25 µg, 20-25 µg, 20-50 µg, 30-50 µg, 40-50 µg, 40-60 µg, 60-80 µg, 60-100 µg, 50-100 µg, 80-120 µg, 40-120 µg, 40-150 µg, 50-150 µg, 50-200 µg, 80-200 µg, 100-200 µg, 120- 250 µg, 150-250 µg, 180-280 µg, 200-300 µg, 50-300 µg, 80-300 µg, 100-300 µg, 40-300 µg, 50- 350 µg, 100-350 µg, 200-350 µg, 300-350 µg, 320-400 µg, 40-380 µg, 40-100 µg, 100-400 µg, 200-400 µg, or 300-400 µg per dose. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal, intraperitoneal or intramuscular injection. Advantageously, the administration is an intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty-one.

[0217] In some embodiments, a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered DM2\21145312.143PATENT Docket No. Y9054-99007 to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.

[0218] Methods for the chemical conjugation of polypeptides, carbohydrates, and / or lipids are well known in the art (see, for example, Hermanson. Bioconjugate Techniques (Academic Press; 1992); Aslam and Dent, eds. Bioconjugation: Protein coupling Techniques for the Biomedical Sciences (MacMillan: 1998); and Wong Chemistry of Protein Conjugation and Cross-linking (CRC Press: 1991)). For instance, primary amino groups may be incorporated by reaction with ethylenediamine in the presence of sodium cyanoborohydride and sulfhydryls may be introduced by reaction of cysteamin dihydrochloride followed by reduction with a standard disulfide reducing agent. Heterobifunctional crosslinkers, such as, for example, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, which link the epsilon amino group on the D-lysine residues of copolymers of D- lysine and D-glutamate to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled, may be used as well. Chemical conjugation also includes anything covalently bonded directly via side chain bonds or via a linker or spacer group.

[0219] The nanoparticle formulations may be a carbohydrate nanoparticle which may comprise a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA). As a non- limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride- modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121; herein incorporated by reference in its entirety).

[0220] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin- MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg / kg dose to a 10 mg / kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining DM2\21145312.144PATENT Docket No. Y9054-99007 the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

[0221] The average diameter of the nanoparticle employed in the compositions of the invention can be at least one member selected from the group consisting of about 20 nanometers, about 25 nanometers, about 30 nanometers, about 40 nanometers, about 50 nanometers, about 75 nanometers, about 100 nanometers, about 125 nanometers, about 150 nanometers, about 175 nanometers and about 200 nanometers. In another embodiment, the average diameter of the particle is at least one member selected from the group consisting of between about 10 to about 200 nanometers, between about 0.5 to about 5 microns and between about 5 to about 10 microns. In another embodiment, the average diameter of the microparticle is selected from the group consisting of about 0.1 µm, about 0.2 µm, about 0.4 µm, about 0.5 µm, about 1 µm and about 2 µm.

[0222] Nanoparticles for use in the compositions of the invention can be made from lipids or other fatty acids (see, for example, U.S. Pat. Nos.5,709,879; 6,342,226; 6,090,406; Lian, et al., J. of Pharma. Sci.90:667-680 (2001) and van Slooten, et al., Pharm Res.17:42-48 (2000)) and non- lipid compositions (see, for example, Kreuter, J. Anat.189:503-505 (1996), the teachings of all of which are hereby incorporated by reference in their entirety). The compositions can be bilayer or multilamellar liposomes and phospholipid based. Polymerized nanoparticles, as described, for example, in U.S. Pat. No.7,285,289, the teachings of which are incorporated by reference in their entirety.

[0223] Metallic oxide nanoparticles for use in the compositions of the invention can be chemically substituted with at least one reactive moiety capable of forming a thioether bond employing conventionally techniques as described herein and in U.S. Pat. No. 6,086,881, the teachings of which are hereby incorporated by reference in their entirety. The antigen described herein can be coupled in a single step onto the metallic oxide particles by the formation of at least one thioether bond or it may be synthesized or assembled stepwise onto the metallic oxide particles after the initial thioether bond formation. The chemical derivatization reagents for the metallic oxide particles can include organosilane reagents that provide thioalkane functionality or other groups that may readily be converted into thiols or thiol-reactive moieties. Organosilane reagents which may be utilized for this purpose may be, but are not limited to, 3- DM2\21145312.145PATENT Docket No. Y9054-99007 mercaptopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-iodopropyltrimethoxysilane, 2-chloroethyltrichlorosilane, 3-glycidoxypropyltrimethoxysilane, vinyltrichlorosilane and 3- acryloxypropyltrimethoxysilane. Moieties that include one or more disulfide components may also be joined to the metallic oxide particle surface and thereby provide the corresponding reactive moiety able to enter into and form a thioether bond and juncture. Exemplary nanoparticles for use in the compositions of the invention include at least one member selected from the group consisting of poly (D,L-lactide-co-glycolide, also referred to as “poly(lactic-co-glycolic acid) and bisacyloxypropylcysteine.

[0224] Nanoparticles for use in the compositions of the invention can be made of inorganic material. Nanoparticles for use in the compositions of the invention can be made of a polymer material, such as at least one member selected from the group consisting of polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide- co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, a carbohydrate, carboxymethyl cellulose, hydroxyethyl cellulose, agar, gel, proteinaceous polymer, polypeptide, eukaryotic and prokaryotic cells, viruses, lipid, metal, resin, latex, rubber, silicone (e.g., polydimethyldiphenyl siloxane), glass, ceramic, charcoal, kaolinite and bentonite.

[0225] It is noted that these therapeutics may be a chemical compound, a composition which may comprise a polypeptide of the present invention and / or antibody elicited by such a chemical compound and / or portion thereof or a pharmaceutically acceptable salt or a composition which may comprise a polypeptide of the invention, and may be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, and vehicles, as well as other active ingredients.

[0226] The compounds or compositions may be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques.

[0227] It is noted that humans are treated generally longer than the mice or other experimental animals which treatment has a length proportional to the length of the disease process and drug DM2\21145312.146PATENT Docket No. Y9054-99007 effectiveness. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred. Thus, one may scale up from animal experiments, e.g., rats, mice, and the like, to humans, by techniques from this disclosure and documents cited herein and the knowledge in the art, without undue experimentation.

[0228] In a particularly advantageous embodiment, the mRNAs of the present invention are administered in combinations of a prime dose followed by one or more boost doses over time. mRNA doses of about 100 µg are advantageous, however, dosages of about 10 µg to about 1000 µg, about 20 µg to about 900 µg, about 30 µg to about 800 µg, about 40 µg to about 700 µg, about 50 µg to about 600 µg, about 60 µg to about 500 µg, about about 70 µg to about 400 µg, about 80 µg to about 300 µg, or about 900 µg to about 200 µg, are contemplated. Varying combinations are presented below as non-limiting examples.

[0229] The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient being treated. In some embodiments, the choice of MHC alleles in the mRNA may be personalized based on the HLA genotype of the individual patient, as determined by sequencing or serotyping.

[0230] When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0231] Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.

[0232] Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and DM2\21145312.147PATENT Docket No. Y9054-99007 the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.

[0233] Sterile injectable solutions may be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.

[0234] A pharmacological formulation of the present invention, e.g., which may comprise a therapeutic compound or polypeptide of the present invention, may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention may be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, iontophoretic, polymer matrices, liposomes, and microspheres.

[0235] A pharmacological formulation of the compound and composition which may comprise a polypeptide utilized in the present invention may be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques, which deliver the compound orally or intravenously and retain the biological activity, are preferred.

[0236] A pharmacological formulation of the compound and composition utilized in the present invention may be co-delivered with immunomodulatory molecules along with the MHC-I SCT-encoding mRNA. In certain embodiments, the immunomodulatory molecules are cytokines. In certain embodiments, the cytokines are IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, IL-33, IL-35, IL-37, IFNy, TSLP, or TNFa.

[0237] In one embodiment, a formulation of the present invention may be administered initially, and thereafter maintained by further administration. For instance, a formulation of the invention may be administered in one type of composition and thereafter further administered in a different or the same type of composition. For example, a formulation of the invention may be administered by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent DM2\21145312.148PATENT Docket No. Y9054-99007 upon the patient's condition, may be used. In the instance of a vaccine composition, the vaccine may be administered as a single dose, or the vaccine may incorporate set booster doses. For example, booster doses may comprise variants in order to provide protection against multiple clades of HIV. For example, one or more boost immunogens may be from HIV pseudo viruses (PSVs) or derivatives or mutations or a portion thereof.

[0238] The quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention and will vary from a few micrograms to a few milligrams for an average 70 kg patient, e.g., 5 micrograms to 5 milligrams such as 500 micrograms, or about 100 ng / kg of body weight to 100 mg / kg of body weight per administration and preferably will be from 10 pg / kg to 10 mg / kg per administration. Typically, however, the antigen is present in an amount on, the order of micrograms to milligrams, or, about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.

[0239] Of course, for any composition to be administered to an animal or human, including the components thereof, and for any particular method of administration, it is preferred to determine therefor: toxicity, such as by determining the lethal dose (LD) and LD50in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and / or RFFIT analysis. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation. For instance, dosages may be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Thus, the skilled artisan may readily determine the amount of compound and optional additives, vehicles, and / or carrier in compositions and to be administered in methods of the invention. Typically, an adjuvant or additive is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, DM2\21145312.149PATENT Docket No. Y9054-99007 this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation.

[0240] Examples of compositions which may comprise a therapeutic of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0241] Compositions of the invention, are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention may be in the “solid“ form of pills, tablets, capsules, caplets and the like, including “solid“ preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a metered dose or, a dose having a particular particle size.

[0242] Compositions of the invention may contain pharmaceutically acceptable flavors and / or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2500 to 5000 DM2\21145312.150PATENT Docket No. Y9054-99007 cps, since above that range they become more difficult to administer. However, above that range, the compositions may approach solid or gelatin forms, which are then easily administered as a swallowed pill for oral ingestion.

[0243] Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

[0244] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form).

[0245] Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and / or flavors may also be present. The compositions may be isotonic, i.e., it may have the same osmotic pressure as blood and lacrimal fluid.

[0246] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.

[0247] Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents. DM2\21145312.151PATENT Docket No. Y9054-99007

[0248] A pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.

[0249] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0250] It is generally envisaged that compounds and compositions of the invention will be administered by injection, as such compounds are to elicit anti-HIV antibodies, and the skilled artisan may, from this disclosure and the knowledge in the art, formulate compounds and compositions identified by herein methods for administration by injection and administer such compounds and compositions by injection.

[0251] The inventive compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally, the pH may be from about 3 to 7.5. Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0252] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art. DM2\21145312.152PATENT Docket No. Y9054-99007

[0253] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0254] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way. Examples Example 1

[0255] Materials and methods.

[0256] Overview of the single chain trimer (SCT) design.

[0257] The SCT construct includes the peptide, B2M, and Class I MHC alpha chain covalently connected through linkers. The SCT construct has the following generic design, where linkers are underlined: [peptide]-GGGASGGGGSGGGGS (SEQ ID NO:84)-[mouse or human B2M]- GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO:85)-[mouse or human MHC-I alpha chain] (Fig. 1). The SCT_wY construct encodes for the wildtype MHC-I alpha chain. Additionally, three SCT constructs containing stabilizing mutations were designed that encode the following mutations: for SCT_noY, Y84A in the MHC-I alpha chain; for SCT_ds1, Y84C and A139C in the MHC-I alpha chain; and for SCT_ds2, Y84C in the MHC-I alpha chain and mutation of the second glycine in the linker connecting peptide to B2M to cysteine (Fig.1). The same mutations were made for both mouse and human SCT constructs. All SCT constructs are expressed using the leader sequence MSAVLLLALLGFILPLPGVQA (SEQ ID NO:56), which targets the protein for surface expression and is cleaved prior to display of the protein on the cellular membrane.

[0258] The full protein sequences of LCMV GP33 / H-2Db SCT mRNA constructs used in Fig. 3 are listed below in Table 1. The LCMV GP33-41 peptide is bolded, the B2M is italicized, and the H-2Db MHC-I alpha chain is underlined. DM2\21145312.153PATENT Docket No. Y9054-99007 Table 1. Single Chain Trimers SEQ Sequence I L I L I L IDM2\21145312.154PATENT Docket No. Y9054-99007 ENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQSA GGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMA T Lthe exception of replacement of H-2Db alpha chain with the H-2Kb alpha chain: PHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEY WERETQKAKGNEQSFRVDLRTLLGYYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYA YDGSDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKN GNATLLRTDSPKAHVTHHSRPEDKVTLRCWALGFYPADITLTWQLNGEELIQDMELVET RPAGDGTFQKWASVVVPLGKEQYYTCHVYHQGLPEPLTLRWEPPPSTVSNMATVAVL VVLGAAIVTGAVVAFVMKMRRRNTGGKGGDYALAPGSQTSDLSLPDCKVMVHDPHSL A (SEQ ID NO:5).

[0260] Similarly, SCT constructs of Qa-1b-restricted peptides are expressed with the Qa-1b sequence: PHSLRYFTTAVSRPGLGEPRFIIVGYVDDTQFVRFDSDAENPRMEPRARWIEQEGPEYWE RETWKARDMGRNFRVNLRTLLGYYNQSNDESHTLQWMYGCDVGPDGRLLRGYCQEA YDGQDYISLNEDLRSWTANDIASQISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLG NETLQRSDPPKAHVTHHPRSEDEVTLRCWALGFYPADITLTWQLNGEELTQDMELVET RPAGDGTFQKWAAVVVPLGKEQYYTCHVYHEGLPEPLTLRWEPPPSTVSNMVIIAVLV VLGAVIILGAVVAFVMKRRRHIGVKGCYAHVLGSKSFQTSDWPQKA (SEQ ID NO:6).

[0261] Human HLA A*02:01 SCT constructs used in experiments in Fig.7 follow the same generic design as described previously except for the use of human B2M (amino acid sequence: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDW SFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM)(SEQ ID NO:7) and the HLA A*02:01 alpha chain (amino acid sequence): HSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWD GETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYA YDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE DM2\21145312.155PATENT Docket No. Y9054-99007 NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELV ETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGL VLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV (SEQ ID NO:8). Stabilizing mutations in the linker and MHC-I alpha chain were introduced at homologous amino acid positions.

[0262] Peptide sequences and MHC alleles used for each SCT vaccination experiment are listed below in Table 2. Table 2. Peptides presented by SCTs Ex eriment Pe tide Name Pe tide Se uence MHC Allele\ 53 .56PATENT Docket No. Y9054-99007 Fig.18 HIV Nef (9205-9234) LTFGWCFKLV A*02:01 33 Fig.18 HIV gp120 (121-129) KLTPLCVTL A*02:01 34DM2\21145312.157PATENT Docket No. Y9054-99007

[0263] As controls, mRNA LNPs were made that express EGFP (FPbase ID # R9NL8), firefly luciferase protein (Genbank ASL70198.1), full-length LCMV Armstrong glycoprotein (Uniprot # P09991), or LCMV Armstrong nucleoprotein (Uniprot # P09992).

[0264] Plasmid design for the generation of SCT mRNA.

[0265] DNA encoding the full amino acid sequence of the SCT is flanked on the 5’ by the T7 polymerase promoter and a UTR sequence; and on the 3’ by a UTR sequence, a 148 bp-polyA tail, and the linearization restriction enzyme BspQI cut site. All plasmids were synthesized by Genscript. As an example, the full mRNA sequence for the GP33 SCT_wY construct is shown below, where the T7 polymerase is in bold, the UTR regions are underlined, and the coding region is italicized. UAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAC CCCGGCGCCGCCACCAUGAGCGCCGUGCUGUUGCUGGCCCUGCUGGGCUUCAUCCUG CCCCUGCCCGGUGUGCAAGCCAGAGACCCAAUGCAUGUUAAUAACAUUUACAAGUGAAC CACUCGGGUCUCAGGUGGAGGUGCAUCUGGGGGUGGCGGUAGCGGAGGUGGUGGAUC CAUCCAGAAAACGCCCCAAAUCCAGGUGUACAGCCGACACCCUCCCGAAAACGGCAAAC CAAAUAUCCUGAACUGCUACGUGACCCAGUUCCAUCCGCCCCAUAUCGAGAUACAGAUG UUGAAGAAUGGCAAGAAGAUUCCCAAGGUUGAGAUGAGCGACAUGAGCUUCAGCAAGGA CUGGAGCUUCUACAUCCUGGCCCAUACCGAGUUCACUCCGACCGAGACAGAUACCUACG CCUGCCGGGUGAAGCACGCCAGCAUGGCCGAGCCCAAGACCGUGUACUGGGACAGGGA CAUGGGGGGAGGGGGCUCUGGUGGGGGGGGUUCCGGCGGAGGAGGAAGUGGAGGCG GUGGGAGCGGCCCUCACAGCCUCCGGUACUUCGUGACGGCCGUUUCCCGGCCUGGCC UGGGUGAACCCAGAUACAUGGAAGUGGGAUACGUGGAUGACACGGAAUUCGUCCGCUU CGAUAGCGACGCUGAAAACCCCAGGUACGAGCCACGCGCAAGGUGGAUGGAGCAGGAG GGUCCCGAGUACUGGGAACGCGAAACCCAGAAGGCAAAAGGCAACGAGCAGAGUUUCC GCGUGGACCUGCGCACCCUCUUGGGUUAUUACAACCAGAGCAAGGGUGGCUCACAUAC CAUCCAGGUCAUCAGCGGCUGUGAGGUGGGCUCAGACGGCCGCUUGCUGCGUGGCUA UCAGCAAUACGCCUACGACGGAAGCGAUUAUAUCGCGCUGAAUGAGGAUUUGAAAACGU GGACUGCUGCAGACAUGGCUGCUCUGAUUACCAAGCACAAGUGGGAGCAAGCCGGCGA GGCGGAGCGUCUCCGUGCUUAUCUGGAGGGUACAUGCGUGGAGUGGCUGCGCCGUUA CCUUAAGAACGGGAACGCGACUCUCCUGCGUACGGACUCACCCAAGGCUCACGUGACG CAUCACAGUCGCCCUGAAGACAAGGUUACACUGCGCUGCUGGGCGCUCGGCUUCUACC CUGCUGACAUUACACUGACGUGGCAGCUCAACGGCGAAGAGUUGAUCCAGGAUAUGGA ACUCGUGGAAACACGCCCCGCCGGUGACGGGACUUUCCAAAAAUGGGCCUCCGUCGUG DM2\21145312.158PATENT Docket No. Y9054-99007 GUCCCCCUGGGUAAGGAGCAGUAUUACACUUGUCAUGUGUAUCACCAGGGCCUCCCUG AACCCCUGACAUUGCGCUGGGAGCCACCCCCAAGCACUGUCUCCAAUAUGGCCACCGU UGCUGUGCUGGUGGUUCUGGGGGCCGCGAUCGUGACCGGGGCAGUAGUGGCUUUCGU GAUGAAGAUGCGUCGCCGGAACACUGGGGGCAAGGGCGGUGACUACGCCCUCGCUCCU GGCUCUCAGACUUCCGACCUUUCCCUGCCCGAUUGUAAGGUGAUGGUUCACGACCCCC AUUCUCUGGCUUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUC CCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA. (SEQ ID NO:66).

[0266] mRNA transcription and lipid nanoparticle (LNP) encapsulation.

[0267] Template Preparation. Circular plasmid DNA were linearized by restriction enzyme digestion, followed by incubation with 0.5% sodium dodecyl sulfate and Proteinase K (New England Biolabs #P8107S) at 56C for 30 minutes before extraction by phenol:chloroform:isoamyl acid (25:24:1, pH 8.0; Sigma-Aldrich #77617). After ethanol precipitation, template DNA was resuspended in water, quantified by Nanodrop, and stored at -20C or used immediately.

[0268] In Vitro Transcription (IVT). mRNA was in vitro transcribed from template DNA using the MEGAScript T7 Transcription Kit (Thermo #AM1334). Cotrascriptionally-capped mRNA was capped by including Anti-Reverse Cap Analog (ARCA, TriLink #N-7003) at a 4:1 ratio ARCA:GTP. Post-transcriptionally capped mRNA was capped using Faustovirus capping enzyme (New England Biolabs #M2081L) according to the manufacturer’s directions. All IVT reactions included 25 ng / µL – 50 ng / µL template, 7.5 mM ATP, 7.5 mM CTP, 7.5 mM GTP (or ARCA + GTP), and 7.5 mM 1N-methylpseudouridine (m1Ψ, TriLink #1081). mRNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs #T2050) and stored at -80C until use. mRNA concentration was determined by Ribogreen assay (Thermo #R11490) according to manufacturer’s directions and transcript length was determined by Agilent Tapestation.

[0269] Lipid Nanoparticle (LNP) Assembly. LNPs were prepared under sterile condition using a lipid mix comprising SM-102 (Cayman Chemical #33474), cholesterol (Cayman Chemical #9003100), 1,2-diasteroyl-sn-glycero-3-PC (1,2-DSPC, Cayman Chemical #15100), and 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG2000, Cayman Chemical #33945) at a 50:38.5:10:1.5 molar ratio (as is standard in the field). Prior to assembly, mRNA was brought to a concentration of 206 ng / µL in 50 – 100 mM sodium acetate, pH 5.2. Lipid mix and DM2\21145312.159PATENT Docket No. Y9054-99007 mRNA were microfluidically mixed using either a NanoAssemblr Ignite™ (3:1 mRNA:lipid mix (v:v) into 4 volumes DPBS, 12 mL / min) or NanoAssemblr Spark™ (2:1 mRNA:lipid mix (v:v) into 1 volume DPBS). Resulting LNP products were buffer exchanged at least 1000-fold into DPBS by spin filtration (Amicon® Ultra Centrifugal Filter, 10 kDa MWCO; Millipore #UFC801008). LNPs are stored at 4C for up to 14 days before use. For experiments where a cocktail of SCTs were used, mRNA transcripts were pooled before the IVT reaction, after IVT reaction, or after LNP assembly, as detailed for each experiment. Equal representation of transcripts were confirmed by sequencing of reverse transcribed cDNA.

[0270] LNP Characterization. Encapsulation efficiency and LNP cargo concentration were determined by modified Ribogreen assay. Briefly, a total mRNA concentration is determined by Ribogreen assay by adding a 1% Triton™-X100 (SigmaAldrich X100) to samples prior to quantification; unencapsulated mRNA is quantified by standard Ribogreen assay; and cargo concentration is determined to be the difference between the two values. Encapsulation efficiency (%) is calculated as 100% x (cargo concentration / total concentration), as previously described [https: / / doi.org / 10.1038 / nbt1122]. All LNPs used in reported experiments have encapsulation efficiency > 80%. Nanoparticle size and dispersity were measured by nanoparticle tracking analysis (Malvern NanoSight NS3000).

[0271] LCMV Virus. LCMV Armstrong and LCMV Clone 13 virus were passaged on baby hamster kidney cells as previously described (Welsh and Seedholm, 2008). Viral titers were quantified via focus forming assay on VeroE6 cells as previously described (Battegay et al., 1991). Example 2

[0272] Mouse immunization and LCMV challenge studies.

[0273] Experiments used 6-8-week-old sex-matched C57BL / 6 mice or transgenic HLA-A2 (C57BL / 6-Mcph1Tg(HLA-A2.1)1Enge / J) mice obtained from Jackson Laboratories. For LCMV Armstrong infection, mice were injected intraperitoneally with 2e5 focus forming units (ffu) virus in a 200 ul injection volume. For LCMV Clone13 challenge, mice were infected via r.o. infection with 2e6 ffu of virus in a 100 ul injection volume. mRNA LNPs were diluted in PBS and 50 ul of diluted LNP is administered via intramuscular immunization per mouse. For experiments including multiple LCMV SCT epitopes or proteins, IVT reactions were performed separately and DM2\21145312.160PATENT Docket No. Y9054-99007 mRNA was pooled prior to LNP encapsulation. HCMV A*02:01 SCTs for transgenic HLA-A2 mouse studies were pooled prior to IVT.

[0274] To characterize sera and peripheral T cell responses, blood was collected using heparinized capillary tubes via the r.o. route. Serum was collected following centrifugation of whole blood at 500 x g for 10 minutes. Sera were frozen at -80C or used immediately to determine serum LCMV viral titers using a focus forming assay described previously (Battegay et al., 1991). Red blood cells in the remaining cell pellet were lysed twice using ACK lysis buffer (Lonza, Basel, Switzerland) to isolate circulating lymphocytes for flow cytometric analysis.

[0275] Tetramer staining and intracellular cytokine stimulation (ICS).

[0276] For tetramer staining, splenocytes or PBMCs were distributed into round-bottom 96- well plates and incubated with fluorescently-conjugated tetramers (NIH Tetramer Core) diluted 1:200 in FACS buffer (1x PBS with 2% FBS, 2 mM EDTA) for 1 hr at room temperature (RT). Cells were then washed once with FACS buffer prior to incubating with other surface molecule antibodies, including unconjugated anti-mouse CD16, fluorescently conjugated antibodies targeting anti-mouse CD19, CD8, CD4, and CD44, and Ghost Violet 510 viability dye. Anti-CD16 Fc block was used after 1:50 dilution, surface antibodies are used after 1:200 dilution, and viability dye was used after 1:1000 dilution, for 30 min at 4C. Cells were washed twice with FACS buffer prior to analysis on the Cytek Aurora (Cytek Biosciences).

[0277] For peptide stimulation and ICS, splenocytes or PBMCs were added to round-bottom 96-well plates in complete T cell media 40 (10% FBS, 1x L-glutamine, 1x Pen / Strep, 1x Sodium Pyruvate, 10 mM HEPES, 1x β-mercaptoethanol), and stimulated with 2 mM peptide (custom synthesis at Genscript, >95% purity) or DMSO as a control. After 30 minutes of incubation with peptide, Brefeldin A, a protein transport inhibitor, was added at a final concentration of 3 ug / ml, and cells were incubated for 5-6 hours at 37C with 5% CO2. Following incubation, cells were washed out of T cell media and resuspended in FACS buffer for surface antigen staining as described above. Cells were then fixed with 4% PFA and permeabilized with BD Perm / Wash™ buffer (BD Biosciences). Intracellular cytokines were then stained with fluorescently conjugated anti-mouse TNF-α, IFNγ, and IL-2 antibodies diluted 1:200 in Perm / Wash buffer for 45 min at 4C. Cells were washed twice with FACS buffer prior to flow cytometric analysis.

[0278] Mice were immunized with one or three doses of LCMV GP33 peptide SCT mRNA vaccine doses as depicted in Fig. 3A and challenged with GP33 peptide after 10 days. Fig. 3B DM2\21145312.161PATENT Docket No. Y9054-99007 shows the percentage or CD8+T cells that were GP33 / H-2Dbtetramer specific cells among total peripheral CD8+T cells. Fig.3C demonstrates cytokine release after GP33 peptide stimulation.

[0279] Mice were immunized with one or three doses of LCMV GP33 peptide SCT mRNA vaccine doses and challenged with LCMV Clone 13 as depicted in Fig. 4A. Fig. 4B shows the frequency CD8+T cells that were GP33 / H-2Dbtetramer specific cells among total peripheral CD8+T cells. A single mRNA immunization reduced LCMV Clone 13 serum viral loads by nearly 1000- fold at 6 days post-infection (Fig.4C).

[0280] Mice were immunized with a cocktail of 5 LCMV peptide-MHC specificities (Fig.5A). mRNAs encoding peptide MHC SCTs produced cytokine responses against all 5 LCMV epitopes. By contrast, mRNA encoding native full length glycoprotein (GP) and nucleoprotein (NP) produced response against immunodominant epitopes. (Fig 5B). Fig. 5C shows immunodominance patterns as the proportion of IFNγ / TNF-α double-positive T cells that were stimulated for each immunization group. Example 3

[0281] Induction of T cells targeting an influenza epitope presented by Qa-1, the mouse homolog of HLA-E.

[0282] C57BL / 6 mice were immunized with 3 ug mRNA flu SL9 / Qa-1b SCT constructs, injected i.m. on days 0, 2, and 4 in, and peripheral blood was collected 22 days post immunization for analysis (n=5 mice per group) (Fig. 6A). Fig. 6B shows the frequency of T cells producing TNF-α and IFNγ among activated CD8+T cells after peptide stimulation with the flu SL9 peptide or an irrelevant Qa-1-restricted peptide. Example 4

[0283] Mouse tumor model.

[0284] 6-7 week-old female C57BL / 6 mice were subcutaneously implanted with 0.5 million B16F10 cells in the right flank. Mice were randomized by tumor size prior to the start of mRNA SCT vaccination and randomized again within each vaccination group by tumor size prior to initiation of PD-1 blockade. mRNA LNPs were diluted in 50 ul and immunized via intramuscular or footpad injection. The mRNA neoantigen SCT cocktail consists of the dominant Trp-2180-188 antigen, two CD8 T cell neoantigens (M27 and M33) each containing a single amino acid substitution reported by the Sahin group (Kreiter et al., 2015), and a modified p53 antigen reported DM2\21145312.162PATENT Docket No. Y9054-99007 by Mansour et al., 2007. The MHC restriction of M27 and M33 neoantigens were not determined and moreover, because neoantigens were reported as 27-mer peptides while MHC-I present shorter 8-12-mer peptides, the prediction algorithm NetMHCpan v4.1 was used to determine 8-12-mer peptides containing substitution mutations that most likely bind to C57BL / 6 alleles H-2Kb and H- 2Db. Predicted M27 and M33 variants were pooled prior to IVT, and LNPs encapsulated with Trp- 2, M27 pool, M33 pool, and modified p53 SCT mRNAs were mixed immediately before immunization. Starting 8 days after tumor implant, mice were treated with 100 ug anti-PD1 antibody (clone RMP1-14, Leinco Technologies Inc #P362) or IgG2a isotype control (Clone 1-1, Leinco Technologies Inc #I-1177) via i.v. injection every 3 days. (Fig.7A). Tumors were measured every 3 days starting 3 days after tumor implant. Mice were euthanized prior to the end of the study if tumor length and width exceeded 15 x 15 mm or if tumor growth extended into the abdominal cavity. Tumor volumes were calculated as (length x width x width) / 2.

[0285] Tumors were harvested 21 days after implant, weighed, and processed to isolate tumor infiltrating lymphocytes (TILs). Briefly, tumors were excised and weighed, and then filtered through a 100 uM cell strainer. Cells were subjected to ACK Lysis buffer for 5 min at room temperature, centrifuged, and resuspended in FACS buffer. To isolate TILs, cells were purified via a Percoll gradient and then further purified using magnetic bead-based CD45+ positive selection (Sepmate # 100-0350). TILs were stained with the Trp-2 tetramer (NIH Tetramer Core) and other surface antibodies. TILs were then fixed and permeabilized using the FoxP3 Fixation / Permeabilization kit (eBioscience, catalog# 00-5523-00), following manufacturer’s recommendations, and stained with antibodies targeting nuclear protein Ki-67 and Tcf-1 prior to flow cytometric analysis. Additionally, peptide stimulation and ICS was performed using either 2 mM Trp-2 peptide or a pool consisting of 2 mM each of M27, M33, and modified p53 (custom synthesis by Genscript, >95% purity). Average tumor volumes were substantially reduced in SCT- immunized groups (Fig. 7B). Fig. 7C demonstrates reduction of tumor volumes for individual SCT-immunized animals and Fig.7D shows reduced tumor weights upon sacrifice. Fig.7E shows elevated levels of CD8+ T cell infiltration into tumors in mRNA vaccinated mice. Fig.7F shows that the SCT vaccine results in polyfunctional tumor-infiltrating CD8+ T cells that recognize both dominant and subdominant epitopes. Example 5 DM2\21145312.163PATENT Docket No. Y9054-99007

[0286] Reactivation of circulating CMV-specific T cells from A*02:01 CMV-positive donors in vitro.

[0287] Peripheral blood mononuclear cells from HLA A*02:01 donors with known HCMV infection were obtained from Immunospot (Cellular Technology Limited). In vitro SCT mRNA LNP stimulation was performed using PBMCs either directly or following ex vivo culture and antigen-specific expansion. For direct in vitro stimulation, PBMCs were thawed and rested in complete T cell media overnight. The next day, PBMCs were incubated with 100 ng or 500 ng of LNPs for 3 hrs before adding 3 ug / ml brefeldin A, followed by 7 hrs of incubation. Cells were stained with antibodies targeting surface markers and intracellular cytokines as described previously. In parallel, PBMCs were expanded using the HCMV pp65495-503 peptide (JPT Peptide Technologies) ex vivo as described (Grant and Gras, 2022). Briefly, one-third of PBMCs were used as stimulators and loaded with peptide prior to incubation with the remaining two-third responder cells. Cells were cultured for 8 days with the addition of recombinant human IL-2 starting on day 4 and media changes as necessary. Expanded cells enriched for CMV pp65-specific T cells were used for LNP stimulation and ICS staining as described above. Fig.8A shows cytokine release by PBMCs from a CMV-positive donor after mRNA SCT stimulation. Fig.8B shows the proportion of CD+T cells that were positive for IFNγ or IFNγ and TNF-α. Fig.8C shows HCMV A*02:01 SCT mRNA primes antigen-specific T cells in transgenic mice expressing the human A*02:01 MHC-I allele. Example 6

[0288] SCT-elicited T cells against LCMV epitopes are detectable 200 day after immunization.

[0289] Mice immunized with either the SCT pool or full-length LCMV GP / NP proteins (as described in slide 14) were longitudinally tracked up to 200 days after vaccination. Importantly, we detect antigen-specific CD8 T cell memory elicited by the SCT at this time point across all five peptides that were included in the vaccination. Furthermore, immunodominance hierarchies across these peptides remained stable over time, with consistent representation across both immunodominant and subdominant antigens. DM2\21145312.164PATENT Docket No. Y9054-99007 Example 7

[0290] Benchmarking SCTs to mRNA-encoded full-length protein and peptide string vaccines.

[0291] C57BL / 6 mice were immunized with either mRNA SCT encoding peptides GP33, GP276, NP396, NP205, and GP118, an analogous mRNA-coded peptide string including the same peptides separated by AAY linkers, or full-length LCMV GP / NP proteins. Mice infected with 2e5 ffu LCMV Armstrong or 2e6 ffu LCMV Clone13 were included as controls. PBMCs taken 12 days after vaccination or infection were used to assess antigen-specific cytokine production by CD8 T cells. (Fig.10). SCTs elicited T cell responses with greater antigen breadth. Example 8

[0292] Tuning T cell phenotypes using cytokine co-delivery: IL-2 skews T cells towards a short-lived, effector-like state.

[0293] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP276 and GP92, with or without inclusion of recombinant mouse soluble IL-2. IL-2 is separated from SCTs via a P2A linker. Cytokine secretion was measured by peptide stimulation assays, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.11).

[0294] Co-delivery of SCTs encoding GP276 or GP92 (CSANNSHHYI; SEQ ID NO:14) with recombinant mouse single-chain IL-2 biases the T cell response towards short-lived CD8+ T cells, indicated by the higher frequency of antigen-specific CD8+ T cells at an early time point that wanes to levels below that of mice vaccinated with SCT without IL-2 by one month post- vaccination. Consistent with this observation, a higher fraction of LCMV-specific CD8+ T cells in the IL-2-codelivery group are KLRG1-high CD127-low, representative of an effector like phenotype. Example 9

[0295] IL-12 skews the antigen-specific response towards memory precursors.

[0296] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP276 and GP92, with or without inclusion of recombinant mouse soluble IL-12. IL-12 is separated from SCTs via a P2A linker. Cytokine secretion was measured by peptide stimulation assays, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.12). DM2\21145312.165PATENT Docket No. Y9054-99007

[0297] Co-delivery of SCTs encoding GP276 or GP92 with recombinant mouse single-chain IL-12 biases the T cell response towards memory precursors, indicated by a higher fraction of LRG1-low CD127-high CD8 T cells at day 11 post-vaccination. Example 10

[0298] Enhanced T cell responses by IL-4 and GMCSF codelivery at 32 days after immunization.

[0299] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP276 and GP92, with or without inclusion of recombinant mouse soluble GM-CSF and IL-4. GM-CSF and IL-4 are separated from SCTs via a T2A linker. Cytokine secretion was measured by peptide stimulation assays, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig. 13).

[0300] Co-delivery of dendritic cell maturation / activation cytokines GM-CSF and IL-4 induces higher levels of antigen-specific CD8 T cells at 32 days following vaccination. Taken together, these data indicate that co-delivery of both T cell directed (IL-2 and IL-12 in Fig.11) and DC-targeted (Fig. 12) cytokines can be used to skew the magnitude and phenotype of vaccine- induced T cells. Example 11

[0301] Co-delivery of a panel of 10 CD4 helper, T cell co-stimulatory molecules, and DC activation molecules.

[0302] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP33 and GP276, followed by an IRES sequence, and lastly, either CD4 helper or co-stimulatory molecules. Cytokine secretion was measured by peptide stimulation assays, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.14). The impact of co-delivery of either CD4 helper molecules or co-stimulatory markers were assessed using both GP33 and GP276 H-2Db SCTs. Helper and co-stimulatory proteins were expressed on a single mRNA separated from the SCT using the IRES sequence. Table 3 provides the IRES sequence and amino acid sequences of the costimulatory molecules. DM2\21145312.166PATENT Docket No. Y9054-99007 Table 3 IRES: A T C T T T D G Q P H G M P H G M

[0303] Chimeric CD137 / CD40. CD40L is a member of the TNF ligand family and forms a trimer, secreted by CD4+ T cells and recognized by CD40 on DCs. CD40 normally exists as a monomer but trimerizes upon binding CD40L, which causes the cytoplasmic domains to also DM2\21145312.167PATENT Docket No. Y9054-99007 trimerize, binding TRAF proteins resulting in signaling. Chimeric CD137 / CD40 combines the cytoplasmic domains of CD40 with the ECD of CD137L.

[0304] CD137 is another trimeric member of the TNF ligand family. When fused to the CD40 TM and CT, CD137 arranges in the same trimeric orientation as when CD40 is bound to CD40L and signals through TRAF.

[0305] Overall, co-delivery of these molecules can either increase or decrease the number of Gp33 / Gp276 tetramer-specific CD8 T cells induced by vaccination for fine tuning of the CD8 T cell response. Example 12

[0306] Increased cytokine secretion by mRNA encoding SCT followed by a CLIP-invariant chain construct.

[0307] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP33 and GP276, followed by an IRES sequence, and lastly, either CD4 helper or co-stimulatory molecules. Cytokine secretion was measured by ICS, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.15).

[0308] GP276 / H-2Db SCT co-delivered with CD4 helper peptide PADRE as a CLIP replacement in the invariant chain (Ii) induces more functional GP276-specific CD8 T cells that are IFNy+TNFa+ double-positive and IFNy+TNFa+IL-2+ triple-positive cytokine producers. Example 13

[0309] Costimulatory CD137L-CD40 chimera protein enhances polyfunctionality of T cell responses after SCT vaccination.

[0310] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP33 and GP276, followed by an IRES sequence, and lastly, either CD4 helper or co-stimulatory molecules. Cytokine secretion was measured by ICS, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.16).

[0311] GP33 / H-2Db and GP276 / H-2Db SCT co-delivered with a chimeric CD137L-CD40 (SEQ ID NO:86) molecule induces higher frequencies of IFNy+TNFa+ double-positive and IFNy+TNFa+IL-2+ triple-positive CD8+ T cells in response to peptide stimulation. DM2\21145312.168PATENT Docket No. Y9054-99007 Example 14

[0312] Chimeric CD70 (CD27 ligand)-CD40 molecule promotes the development of cytotoxic CX3CR1+ CD8+ memory T cells

[0313] C57Bl / 6 mice were immunized with a cocktail of SCTs containing GP33 and GP276, followed by an IRES sequence, and lastly, either CD4 helper or co-stimulatory molecules. Cytokine secretion was measured by ICS, and T cell phenotypes were quantified by surface marker staining and flow cytometry. (Fig.17).

[0314] GP33 / H-2Db SCT co-delivered with a chimeric CD70-CD40 (SEQ ID NO:88) protein induces higher frequencies of IFNy / TNFa+ and IL-2+ CD8+ T cells in response to peptide stimulation. GP33 and GP276 tetramer-specific CD8 T cells induced by CD70-C40 codelivered vaccination also exhibits a more effector-like phenotype, marked by elevated surface levels of the cytotoxicity marker CX3CR1 and higher proportion of effector memory (CD127-high CD62L- low) cells. Example 15

[0315] Chimeric A*02:01 SCTs prime HIV antigen-specific T cells in humanized A02 mice.

[0316] A*02:01 restricted peptides were chosen that have high sequence conservation across HIV strains and predicted to be important for viral fitness. A02-transgenic (C57BL / 6- Mcph1Tg(HLA-A2.1)1Enge / J) mice were immunized with mRNA A*02:01 SCT encoding HIV peptides following a triple prime schedule (5 ug per injection every other day from day 0-4) containing the following HIV-derived peptide sequences: HIV p24(164-172) YVDRFYKTL (SEQ ID NO:32); HIV Nef 9205-9234, LTFGWCFKLV (SEQ ID NO:33); HIV gp120 (121-129) KLTPLCVTL (SEQ ID NO:34); HIV Protease (76-84) LVGPTPVNI (SEQ ID NO:36); HIV Nef (9202-9231) PLTFGWCYKL (SEQ ID NO:35); HIV RT (179-187) VIYQYMDDL (SEQ ID NO:37). (Fig. 18). PBMCs from day 7 and splenocytes from day 30 were stimulated with individual HIV peptides and cytokine production was measured by ICS.

[0317] Fig.14 demonstrates priming of antigen specific T cells for all peptides employed. Example 16

[0318] SCTs elicit T cells with polyfunctional cytokine profiles.

[0319] Mice were immunized with a single injection of 1 ug mRNA encoding either a pool of H-2Db / Kb SCTs (GP33, GP276, NP396, NP205, and GP92) or an analogous mRNA-coded DM2\21145312.169PATENT Docket No. Y9054-99007 peptide string containing the same peptides separatetd by AAY linkers. Mice were challenged with 2e6 ffu LCMV Clone 13 36 days after vaccination, and serum titers were quantified by focus reduction assays. (Fig. 19). By day 7, SCT- and pepstring-vaccinated mice exhibited large reductions in serum viral titers or clearance in the sera. Example 17

[0320] Qa1-restricted T cell responses elicited by SCT immunization.

[0321] C57BL / 6 mice were immunized with an mRNA cocktail containing 5 exogenous antigen-derived peptides or the self FL9 peptide as peptide / Qa-1b SCTs, following a triple prime regimen as described in Fig.6. PBMCs were tested for antigen-specific cytokine secretion 12 days post-vaccination. Qa1-restricted T cell responses can be elicited by SCT immunization against a range of exogenous and self-antigen-derived peptides. (Fig.20).

[0322] C57BL / 6 mice were then immunized with a single dose of 3 ug mRNA cocktail containing equimolar ratios of SL9 / Qa1b, GP33 / H-2Db, and GP276 / H-2Db SCTs. PBMCs were assessed for cytokine secretion 16 days later. Fig.21 demonstrates that inclusion of classical MHC- I epitopes does not preclude the induction of a nonclassical Qa-1b-restricted T cell response. (Fig. 21). SL9 Qa-1b SCT elicited higher levels of antigen-specific T cells relative to mRNA encoding SL9 fused to the LCMV GP protein. (Fig.21). Example 18

[0323] Induction of T cell responses against novel LCMV Qa-1b-binding peptides discovered through the pMHC yeast display platform.

[0324] C57BL / 6 mice received three injections of mRNA LNPs containing equimolar ratios of either 1) ALV9, ALE9, ALS9, ALS11, AMS9, FLK9, or separately 2) LQA9, SLR8, SLD9, SLN8, TLL9, VIY9 formatted as Qa-1b SCTs. PBMCs were stimulated with individual peptides and cytokine secretion was measured by intracellular cytokine secretion assay (ICS) 14 days after the first immunization (Fig.22).

[0325] An exemplary Qa-1b SCT with the FLK9 peptide is as follows, which includes the leader sequence, FLK9 peptide (underlined), followed by a GS linker, B2M, GS linker, and the Qa-1b alpha chain containing disulfide mutations at Y84C and A139C: MSAVLLLALLGFILPLPGVQAFLMFLQNLKGGGASGGGGSGGGGSIQKTPQIQVYSRHP PENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTE DM2\21145312.170PATENT Docket No. Y9054-99007 TDTYACRVKHASMAEPKTVYWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFTTA VSRPGLGEPRFIIVGYVDDTQFVRFDSDAENPRMEPRARWIEQEGPEYWERETWKARD MGRNFRVNLRTLLGCYNQSNDESHTLQWMYGCDVGPDGRLLRGYCQEAYDGQDYISL NEDLRSWTANDICSQISKHKSEAVDEAHQQRAYLQGPCVEWLHRYLRLGNETLQRSDP PKAHVTHHPRSEDEVTLRCWALGFYPADITLTWQLNGEELTQDMELVETRPAGDGTFQ KWAAVVVPLGKEQYYTCHVYHEGLPEPLTLRWEPPPSTVSNMVIIAVLVVLGAVIILGA VVAFVMKRRRHIGVKGCYAHVLGSKSFQTSDWPQKA (SEQ ID NO:68). Example 19

[0326] PBMCs from COVID convalescent donors exhibit enhanced responses to SARS-CoV- 2 HLA-E SCTs.

[0327] To confirm that stimulated CD8+ T cells were Qa-1b-restricted and did not represent classical Ia-restrcted cells binding to novel LCMV peptides that are presented by classical H- 2Kb / Db alleles, a competitive peptide stimulation assay was used to confirm the successful induction antigen-specific Qa1-restricted CD8 T cells against two of the most immunogenic peptides FLK9 and VIY9.

[0328] Mice as in Example 18 were bled on day 18 post-vaccination and stimulated with either the FLK9 or VIY9 peptide in the presence of an excess of the Qa-1 competitive peptide Qdm, H-2Kb / Db competitive peptides (GP33, NP205, GP276), or DMSO negative control. Reduction in cytokine secretion in the presence of Qdm indicates the presence of FLK9- or VIY9-Qa-1- restricted CD8 T cells (Fig.23). Example 20

[0329] PBMCs from COVID convalescent donors exhibit enhanced responses to SARS-CoV- 2 HLA-E SCTs.

[0330] PBMCs from four COVID-19 convalescent donors were enriched for SARS-CoV-2 HLA-E-restricted T cells via co-culture with a mixture of the 5 peptides, supplemented with recombinant human IL-2 starting on day 5. P001 (VMPLSAPTL; SEQ ID NO:50); P003 (YLQPRTFLL; SEQ ID NO:51); P006 (VLWAHGFEL; SEQ ID NO:52); P013 (AMYTPHTVL; SEQ ID NO:53); P015 (SLPINVIVF; SEQ ID NO:54). An exemplary amino acid sequence of HLA-E SCT with the P001 peptide is shown, which includes the leader sequence, P001 peptide DM2\21145312.171PATENT Docket No. Y9054-99007 (underlined), followed by a linker, B2M, linker, and the HLA-E alpha chain containing disulfide mutations at Y84C and A139C: MSAVLLLALLGFILPLPGVQAVMPLSAPTLGGGASGGGGSGGGGSIQRTPKIQVYSRHP AENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE KDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSV SRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDT AQIFRVNLRTLRGCYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNE DLRSWTAVDTCAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPK THVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKW AAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVA AVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL. (SEQ ID NO:69)

[0331] An analogous mRNA-based peptide string encoding the same peptides (P001, P003, P006, P013, P015) was made as a direct comparator with the SCT, where each peptide was separated by an AAY cleavage linker.

[0332] After 8 days of culture, T cells were stimulated with 1 ug of mRNA LNPs encoding either an SCT pool of the 5 SARS2 HLA-E peptides, the analogous peptide string, or a negative luciferase mRNA control. IFNy secretion was measured among activated (CD137+) T cells by ICS after 8 hours of culture. (Fig.24).

[0333] These data show that the SCT format confers superior in vitro stimulation of IFNy production over the peptide string format across all four COVID-19 convalescent donors. Example 21

[0334] Immunization of self-protein peptides as a Qa-1b pepMHC SCT ameliorates disease in autoimmune mouse models.

[0335] MRL-lpr mice were immunized with the FL9 / Qa-1b SCT presenting the FL9 peptide (FYAEATPML; SEQ ID NO:31) derived from CYRI-b (gene Fam49b). PBMCs were tested by peptide stimulation and ICS 10 days later. In the spontaneous lupus erythematosus mouse model MRL / MpJ-Faslpr / J, the SCT induced FL9-specific CD8 T cells that produced IFNy and TNFa cytokines. (Fig.25). Example 22

[0336] Prevention of clinical symptoms in a spontaneous mouse lupus model. DM2\21145312.172PATENT Docket No. Y9054-99007

[0337] Mice were immunized as described in Example 21. Lymph node sizes were quantified by caliper measurements at week 15 (8 weeks after vaccination). MRL / MpJ-Faslpr / J I mice that received FL9 / Qa-1b SCT mRNA exhibited reduced lymph node sizes relative to mice that received the flu FL9 / Qa-1b SCT or PBS control, indicating that FL9-specific CD8 T cell responses had a protective effect against clinical symptoms of SLE. (Fig.26). Example 23

[0338] Reduced incidence of type I diabetes in the spontaneous NOD model after Hsp60sp / Qa1 SCT immunization.

[0339] NOD / ShiLtJ mice were used as a spontaneous T1D model. NOD / ShiLtJ mice were primed with either Hsp60sp / Qa-1b SCT or SL9 / Qa-1b SCT mRNA across 3 doses spread from week 5 through week 7 of age. Mice were again boosted at week 12 with 3 ug of the same SCT mRNA. Blood glucose was measured weekly and diabetes was diagnosed as having consecutive blood glucose levels >350 mg / dL in two measurements at least 3 days apart.

[0340] NOD mice immunized with Hsp60sp / Qa-1b SCT displayed delayed hyperglycemia and diabetes onset (as measured by blood glucose levels) compared with mice that received SCT encoding an irrelevant Qa1-restricted antigen SL9 derived from influenza virus. (Fig.27). * * *

[0341] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. DM2\21145312.173

Claims

PATENT Docket No. Y9054-99007 WHAT IS CLAIMED IS:

1. A system for expressing an MHC-I single chain trimer (SCT) in a cell (APC), which comprises: an expressible mRNA which encodes the MHC-I SCT comprising an autoantigen peptide, a β2-microglobulin (β2m), and an HLA-E alpha chain; and a delivery vehicle for delivery of the expressible mRNA to the cell; whereby the SCT is expressed and the autoantigen peptide presented.

2. The system of claim 1, wherein the expressible mRNA encodes from 5’ to 3’, a signal sequence, the autoantigen peptide, a first linker, the β2m, a second linker, and the HLA-E alpha chain.

3. The system of any one of claims 1 to 2, wherein the autoantigen peptide comprises FYAEATPML (SEQ ID NO:31), QMRPVSRAL (SEQ ID NO:89), QMRPVSRVL (SEQ ID NO:90), or GMKFDRGYI (SEQ ID NO:91).

4. The system of any one of claims 1 to 3, wherein the system further comprises an expressible mRNA encoding a costimulatory molecule.

5. The system of any one of claims 1 to 3, wherein the system further comprises a T cell helper epitope.

6. The system of any one of claims 1 to 4, wherein the system further comprises an expressible mRNA encoding a cytokine.

7. A vaccine formulation which comprises the system of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. Use of the system of any one of claims 1 to 6 to induce a CD8+T cell mediated regulatory response in a subject. DM2\21145312.174PATENT Docket No. Y9054-99007 9. A method of inducing a CD8+ T cell regulatory response against a cell that is autoreactive in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an autoantigen peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle for delivery of the expressible mRNA to an antigen presenting cell (APC) in the subject; wherein the MHC-I SCT is expressed and the autoantigen peptide is presented, whereby the CD8+T cell regulatory response is induced.

10. A method of reducing or inhibiting autoimmunity in a subject, which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an HLA-E alpha chain operably linked to a β2-microglobulin (β2m) and a peptide, wherein the SCT stimulates CD8+T cells to suppresses the autoimmunity when expressed by an antigen presenting cell of the subject, and; a delivery vehicle for delivery of the expressible mRNA to cells of the subject; wherein the MHC SCT is expressed and the peptide presented; whereby the autoimmunity is suppressed.

11. The method of claim 10, wherein the peptide comprises FYAEATPML (SEQ ID NO:31), QMRPVSRAL (SEQ ID NO:89), QMRPVSRVL (SEQ ID NO:90), or GMKFDRGYI (SEQ ID NO:91).

12. The method of any one of claims 10 or 11, wherein the autoimmunity comprises lupus or type 1 diabetes.

13. A system for expressing an MHC-I single chain trimer (SCT) on a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising an antigen peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell; whereby the SCT is expressed and the antigen peptide presented. DM2\21145312.175PATENT Docket No. Y9054-99007 14. The system of claim 13, wherein the expressible mRNA encodes from 5’ to 3’, a signal sequence, the antigen peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

15. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises a classical MHC I alpha chain, optionally an HLA-A, HLA-B, or HLA-C alpha chain.

16. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises HLA-A*01:01, HLA-A*02:01, HLA-A*02:04, HLA-A*02:05, HLA-A*02:07, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02, HLA-A*24:07, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*34:01, HLA-A*68:01, HLA-A*68:02, HLA-A*74:01, HLA-B*07:02, HLA-B*08:01, HLA-B*13:02, HLA-B*14:02, HLA-B*15:01, HLA-B*15:03, HLA-B*15:04, HLA-B*15:25, HLA-B*18:01, HLA-B*27:04, HLA-B*27:05, HLA-B*35:01, HLA-B*35:02, HLA-B*35:03, HLA-B*35:05, HLA-B*35:12, HLA-B*35:19, HLA-B*35:43, HLA-B*37:01, HLA-B*38:01, HLA-B*39:02, HLA-B*39:05, HLA-B*39:06, HLA-B*39:09, HLA-B*40:01, HLA-B*40:02, HLA-B*40:04, HLA-B*40:06, HLA-B*42:01, HLA-B*44:02, HLA-B*44:03, HLA-B*45:01, HLA-B*46:01, HLA-B*46:02, HLA-B*48:02, HLA-B*48:03, HLA-B*49:01, HLA-B*50:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01, HLA-B*55:01, HLA-B*56:01, HLA-B*56:02, HLA-B*57:01, HLA-B*57:03, HLA-B*58:01, HLA-C*01:02, HLA-C*02:02, HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*03:05, HLA-C*04:01, HLA-C*04:03, HLA-C*05:01, HLA-C*06:02, HLA-C*07:01, HLA-C*07:02, HLA-C*07:04, HLA-C*08:02, HLA-C*12:02, HLA-C*12:03, HLA-C*14:02, HLA-C*15:02, HLA-C*16:01, HLA-C*16:02, or HLA-C*17:

01.

17. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises H-2Db, or H-2Kb, or HLA-A*02:

01.

18. The system of any one of claims 13 or 14, wherein the mRNA encoding the MHC- I SCT comprises SEQ ID NO:

1. DM2\21145312.176PATENT Docket No. Y9054-99007 19. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises alanine at amino acid position 84, or wherein the mRNA encoding the MHC-I SCT comprises SEQ ID NO:

2.

20. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises cysteine at amino acid position 84 and cysteine at amino acid position 139, or wherein the mRNA encoding the MHC-I SCT comprises SEQ ID NO:

3.

21. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises cysteine at amino acid position 84 and the first linker comprises cysteine at amino acid position 2, or wherein the mRNA encoding the MHC-I SCT comprises SEQ ID NO:

4.

22. The system of any one of claims 13 or 14, wherein i) the MHC-I alpha chain comprises H-2Dband the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYCL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), or CSANNSHHYI (SEQ ID NO:14); or ii) the MHC-I alpha chain comprises H-2Kband the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), or YTVKYPNL (SEQ ID NO:13).

23. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, optionally an HLA-E, HLA-F, HLA-G, or HLA-H chain.

24. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises HLA-E and the antigen peptide comprises VMPLSAPTL (SEQ ID NO:50), YLQPRTFLL (SEQ ID NO:51), VLWAHGFEL (SEQ ID NO:52), AMYTPHTVL (SEQ ID NO:53), or SLPINVIVF (SEQ ID NO:54) 25. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises SLQGRTLIL (SEQ ID NO:15), GMRFDKGYI (SEQ ID NO:27), RLPAKAPLL (SEQ ID NO:28), VMATRRNVL (SEQ ID NO:29), or VLRPGGHFL (SEQ ID NO:30).

26. The system of any one of claims 13 or 14, wherein the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises ALFEGRNLV (SEQ ID NO:38), ALMDCIIFE (SEQ ID NO:39), ALMDLLMFS (SEQ ID NO:40), ALMDLLMFSTS (SEQ ID DM2\21145312.177PATENT Docket No. Y9054-99007 NO:41), AMMKSYVKS (SEQ ID NO:42), FLMFLQNLK (SEQ ID NO:43), LQMNANAYS (SEQ ID NO:44), SLFSRFRR (SEQ ID NO:45), SLMDKLRED (SEQ ID NO:46), SLMVSSFN (SEQ ID NO:47), TLMSIVSSL (SEQ ID NO:48), or VIQSVRRLY (SEQ ID NO:49).

27. The system of any one of claims 13 to 26, wherein the system further comprises an expressible mRNA encoding a costimulatory molecule.

28. The system of any one of claims 13 to 26, wherein the system further comprises a T cell helper epitope.

29. The system of any one of claims 13 to 26, wherein the system further comprises an expressible mRNA encoding a cytokine.

30. The system of any one of claims 13 to 29, wherein the delivery vehicle comprises a lipid nanoparticle (LNP), or a phospholipid bilayer, or a dried organic matrix, or a carbon nanotube.

31. A vaccine formulation which comprises the system of any one of claims 13 to 29 and a pharmaceutically acceptable carrier.

32. Use of the system of any one of claims 13 to 29 to induce a CD8+T cell mediated immune response in a subject.

33. A method of inducing a CD8+ T cell mediated response against a disease or pathogen in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an antigen peptide from the disease or pathogen, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); wherein the MHC-I SCT is expressed and the antigen peptide presented to the CD8+T cell, whereby the CD8+T cell response is induced.

34. The method of claim 33, wherein the MHC-I alpha chain comprises a classical MHC-I alpha chain. DM2\21145312.178PATENT Docket No. Y9054-99007 35. The method of claim 33, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, optionally an HLA-E chain.

36. The method of claim 33, wherein the pathogen comprises Lymphocytic choriomeningitis virus (LCMV), and i) the MHC-I alpha chain comprises H-2Dband the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYCL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), or CSANNSHHYI (SEQ ID NO:14), or ii) the MHC-I alpha chain comprises H-2Kband the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), or YTVKYPNL (SEQ ID NO:13).

37. The method of claim 33, wherein the pathogen comprises influenza, the MHC-I alpha chain comprises Qa-1b, and the antigen peptide comprises SLQGRTLIL (SEQ ID NO:15).

38. A method of inducing a CD8+ T cell response against a tumor antigen in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising an antigen peptide of the tumor antigen operably linked to a β2-microglobulin (β2m) and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to antigen presenting cells, whereby the MHC-I SCT is expressed and an anti-tumor CD8+T cell response is induced.

39. The method of claim 38, wherein the antigen peptide comprises a neoantigen peptide.

40. The method of claim 38, wherein the composition comprises a second expressible mRNA which encodes a second MHC-I SCT comprising a second antigen peptide of the tumor antigen operably linked to a β2-microglobulin (β2m) and an MHC-I alpha chain; whereby the second MHC-I SCT is expressed and a second anti-tumor response CD8+T cell response is induced.

41. The method of any one of claims 38 to 40, wherein the antigen peptide comprises KAVYNFATC (SEQ ID NO:9), SGVENPGGYL (SEQ ID NO:10), FQPQNGQFI (SEQ ID NO:12), CSANNSHHYI (SEQ ID NO:14), LCPGNKYEM (SEQ ID NO:18), VELCPGNKYEM (SEQ ID NO:19), AAVILRDAL (SEQ ID NO:20), AAVILRDALHM (SEQ ID NO:21), DM2\21145312.179PATENT Docket No. Y9054-99007 VILRDALHM (SEQ ID NO:22), or FPAAVILRDAL (SEQ ID NO:23), and the MHC-I alpha chain comprises H-2Dbor the antigen peptide comprises ISHNFCNL (SEQ ID NO:11), YTVKYPNL (SEQ ID NO:13), SVYDFFVWL (SEQ ID NO:16), KYICNSSCM (SEQ ID NO:17), or AVILRDAL (SEQ ID NO:24), and the MHC-I alpha chain comprises H-2Kb.

42. A system for expressing an MHC-I single chain trimer (SCT) in a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising a human immunodeficiency virus (HIV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell, whereby the SCT is expressed and the HIV peptide presented.

43. The system of claim 42, wherein the expressible mRNA encodes from 5’ to 3’, a signal sequence, the HIV peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

44. The system of any one of claims 42 or 43, wherein the MHC-I alpha chain comprises a classical MHC-I alpha chain.

45. The system of any one of claims 42 or 43, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain or an HLA-E chain.

46. The system of any one of claims 42 to 45, wherein the MHC-I alpha chain comprises HLA-A*02:01 and the HIV peptide comprises an HLA-A*02:01-restricted peptide.

47. The system of claim 46, wherein the HIV peptide comprises YVDRFYKTL (SEQ ID NO:32), LTFGWCFKLV (SEQ ID NO:33), KLTPLCVTL (SEQ ID NO:34), PLTFGWCYKL (SEQ ID NO:35), LVGPTPVNI (SEQ ID NO:36), or VIYQYMDDL (SEQ ID NO:37).

48. The system of any one of claims 42 to 47, wherein the system encodes a second MHC-I SCT which comprises a second HIV peptide. DM2\21145312.180PATENT Docket No. Y9054-99007 49. The system of any one of claims 42 to 48, wherein the system further comprises an expressible mRNA encoding a costimulatory molecule.

50. The system of any one of claims 42 to 48, wherein the system further comprises a T cell helper epitope.

51. The system of any one of claims 42 to 48, wherein the system further comprises an expressible mRNA encoding a cytokine.

52. A vaccine formulation which comprises the system of any one of claims 42 to 51, and a pharmaceutically acceptable carrier.

53. Use of the system of any one of claims 42 to 51 to induce a CD8+T cell mediated immune response in a subject.

54. A method of inducing a CD8+ T cell mediated response against HIV in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human immunodeficiency virus (HIV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HIV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

55. The method of claim 54, wherein the MHC-I alpha chain comprises a classical MHC-I alpha chain.

56. The method of claim 54, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, preferably an HLA-E chain.

57. The method of any one of claims 54 to 55, wherein the MHC-I alpha chain comprises HLA-A*02:01 and the HIV peptide comprises an HLA-A*02:01-restricted peptide.

58. The method of claim 57, wherein the HIV peptide comprises comprises YVDRFYKTL (SEQ ID NO:32), LTFGWCFKLV (SEQ ID NO:33), KLTPLCVTL (SEQ ID DM2\21145312.181PATENT Docket No. Y9054-99007 NO:34), PLTFGWCYKL (SEQ ID NO:35), LVGPTPVNI (SEQ ID NO:36), or VIYQYMDDL (SEQ ID NO:37).

59. A system for expressing an MHC-I single chain trimer (SCT) in a cell, which comprises: an expressible mRNA which encodes the MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to the cell, whereby the SCT is expressed and the HPV peptide presented.

60. The system of claim 59, wherein the expressible mRNA encodes from 5’ to 3’, a signal sequence, the HPV peptide, a first linker, the β2m, a second linker, and the MHC-I alpha chain.

61. The system of any one of claims 59 or 60, wherein the MHC-I alpha chain comprises a classical MHC-I alpha chain.

62. The system of any one of claims 59 or 60, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain or an HLA-E chain.

63. The system of any one of claims 59 or 60, wherein i) the MHC-I alpha chain comprises HLA-A2*02:01 and the antigen peptide comprises TLQDIVLHL (SEQ ID NO:92), or SLQDIEITC (SEQ ID NO:93), or GLYNLLIRC (SEQ ID NO:94), or SLQDIEITCV (SEQ ID NO:95); or ii) the MHC-I alpha chain comprises HLA-A*11.01 and the antigen peptide comprises ATLQDIVLH (SEQ ID NO:96), or iii) the MHC-I alpha chain comprises HLA-B*40.02 and the antigen peptide comprises QDIEITCVY (SEQ ID NO:97).

64. The system of any one of claims 59 to 63, wherein the system further comprises an expressible mRNA encoding a costimulatory molecule. DM2\21145312.182PATENT Docket No. Y9054-99007 65. The system of any one of claims 59 to 63, wherein the system further comprises a T cell helper epitope.

66. The system of any one of claims 59 to 63, wherein the system further comprises an expressible mRNA encoding a cytokine.

67. The system of any one of claims 59 to 66, wherein delivery vehicle comprises a lipid nanoparticle (LNP), or a phospholipid bilayer, an dried organic matrix, or a carbon nanotube.

68. A vaccine formulation which comprises the system of any one of claims 59 to 66.

69. Use of the system of any one of claims 59 to 66 to induce a CD8+T cell mediated immune response in a subject.

70. A method of inducing a CD8+ T cell mediated response against HPV in a subject which comprises administering to the subject a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HPV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

71. A method of treating or preventing cervical cancer or oropharyngeal cancer in a subject which comprises immunizing the subject with a composition which comprises: an expressible mRNA which encodes an MHC-I SCT comprising a human papilloma virus (HPV) peptide, a β2-microglobulin (β2m), and an MHC-I alpha chain; and a delivery vehicle that delivers the expressible mRNA to an antigen presenting cell (APC); whereby the MHC-I SCT is expressed, the HPV peptide is presented to the CD8+T cell, and the CD8+T cell response is induced.

72. The method of any one of claims 70 to 71, wherein the MHC-I alpha chain comprises a classical MHC-I alpha chain.

73. The method of any one of claims 70 to 71, wherein the MHC-I alpha chain comprises a non-classical MHC-I alpha chain, preferably an HLA-E chain. DM2\21145312.183PATENT Docket No. Y9054-99007 74. The method of any one of claims 70 to 71, wherein i) the MHC-I alpha chain comprises HLA-A2*02:01 and the antigen peptide comprises TLQDIVLHL (SEQ ID NO:92), or SLQDIEITC (SEQ ID NO:93), or GLYNLLIRC (SEQ ID NO:94), or SLQDIEITCV (SEQ ID NO:95); or ii) the MHC-I alpha chain comprises HLA-A*11.01 and the antigen peptide comprises ATLQDIVLH (SEQ ID NO:96), or iii) the MHC-I alpha chain comprises HLA-B*40.02 and the antigen peptide comprises QDIEITCVY (SEQ ID NO:97).DM2\21145312.184

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