Affinity targeted immunogens

Abstract

Provided herein are single chain trimer proteins engineered to have altered CD8 binding affinities. Because of these altered affinities, the recombinant proteins are particularly useful for eliciting immunogenic responses otherwise difficult to induce. Also provided are recombinant polypeptides encoding the single chain trimer proteins, cells and vaccines or other pharmaceutical compositions including the single chain trimer proteins, and treatment methods using the single chain trimer proteins, recombinant polypeptides, cells, and pharmaceutical compositions.

Description

PATENTAtorney Docket No. 070772-233510PC-1530971Client Ref. No. UC 2023-534-2AFFINITY TARGETED IMMUNOGENSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 724,185 filed November 22, 2024, the full disclosure of which is incorporated by reference in its entirety for all purposes.BACKGROUND

[0002] Current vaccines must meet pathogens on a pathologic playing field created by the adversaries themselves in the course of their own evolution. These confrontations are often losing battles because conventional adaptive immune responses are the normal context for such pathologies. This problem is illustrated by the clinical failure of T-cell vaccines against the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV). The most dramatic example of an effective T-cell response for HIV / SIV prophylaxis to date is that of major histocompatibility complex (MHC) class II and / or Mamu-E-restricted CD8+ T cells (i.e., unconventional responses) capable of clearing nascent SIV infection. That example aside, virtually all prophylactic T-cell vaccines against HIV, which generally elicit conventional T cell responses restricted by classical class la elements, have achieved limited success in primate models and either no success or even accelerated disease acquisition in humans.

[0003] On reflection, there are many likely reasons for failure of T-cell vaccines against HIV. Among the most important is the frustrating phenomenon of T-cell immunodominance, i.e., outgrowth of just a few epitope specificities at the expense of others that may have greater potential to control disease. Extreme concentration of the immune response lowers the bar for immune escape by the virus. More subtly, immunodominant T-cell responses restricted by common HLA alleles provide an evolutionary impetus to the virus, which over long periods selects for HIV sequences whose replication and transmission are not impeded by those responses. In fact, the evolution of HIV has been linked directly to viral adaptation to dominant epitopes presented by common HLA class la alleles (N. Frahm, et al., Nat. Immunol. 7, (2006): 173; Y. Kawashima et al., Nature 458, (2009): 641). Promising HLA class I-restricted epitopetargets for people with common class-I alleles therefore gradually vanish from circulating virus. In the case of HLA-A2, for example, there are targets such as SL9 that are thought to be associated with viral load decrements, but 70% of HLA-A2+ individuals already have this response — so even a very immunogenic vaccine targeting SL9 is expected to have at most a quantitative impact on virus replication. A similar example of virus evolution to evade T-cell responses restricted by the common HLA-A2 molecule has recently been reported for SARS- CoV-2 (G. Dolton et al., Cell 185, (2022): 2936). Therefore, for transformative impact society needs vaccines that can expand broadly targeted, “atypical” or “unconventional” memory T cells that viruses have rarely encountered.

[0004] A need therefore exists for germline targeting for T cells. Compared to B-cell immunogens, state-of-the-art T-cell immunogens are crude, consisting perhaps of fusion proteins that incorporate selected epitopes to which responses are thought most useful. Such immunogens do not begin to approach the elegance of germline-targeting B-cell immunogens that can selectively prime desired lineages from within the naive B-cell pool, with the goal of expanding adaptive immune receptors (e.g., antibodies) that would otherwise be rare. Currently available treatment methods could benefit greatly from immunogens that can target rare naive T cells of desired specificity, restriction, and / or affinity, and expand those cells into effectors that are otherwise rare. Such an approach could transform T-cell vaccines from crude interventions, which mostly rely upon simple exposure of the immune system to target epitopes and result in expansion of conventional responses, into sophisticated tools that can achieve highly specific immunologic goals. The present disclosure addresses this need and provides associated and other advantages.BRIEF SUMMARY

[0005] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. Covered embodiments of the disclosure are defined by the claims, not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0006] The present disclosure generally relates to single-chain trimer (SCT) proteins that are based on MHC-I, MHC-II, or MHC-E molecules and have been engineered for altered binding to CD8. As a result of this engineering, the provided single chain trimer proteins can elicit unique immune responses that are otherwise difficult to induce, tapping this underutilized immunologic resource.

[0007] In one aspect, the disclosure is to a single chain trimer that includes a target peptide, a P2-microglobulin, and an MHC class la a chain. The p2-microglobulin and / or MHC class la a chain is a variant. The single chain trimer has a CD8 binding affinity dissociation constant greater than 300 pM.

[0008] In another aspect, the disclosure is to a single chain trimer that includes a target peptide, a P2-microglobulin, and an MHC class lb a chain. The P2-microglobulin and / or MHC class lb a chain is a variant. The single chain trimer has a CD8 binding affinity dissociation constant less than 1000 pM.

[0009] In another aspect, the disclosure is to a single chain trimer that includes a target peptide, an a chain of a first MHC class II allele, and a P chain of a second MHC class II allele. The a and / or P chains are variants. The single chain trimer has a CD8 binding affinity dissociation constant less than 1000 pM.

[0010] In another aspect, the disclosure is to a single chain trimer that includes a target peptide, a P2-microglobulin, an MHC class lb a chain, and an anti-CD8 scFv fragment.

[0011] In another aspect, the disclosure is to a single chain trimer that includes a target peptide, an a chain of a first MHC class II allele, a P chain of a second MHC class II allele, and an anti-CD8 scFv fragment.

[0012] In another aspect, the disclosure is to a recombinant polynucleotide including a nucleic acid encoding a single chain trimer as disclosed herein.

[0013] In another aspect, the disclosure is to a system including a single chain trimer protein and an accessory CD8 binder protein. The single chain trimer protein includes a target peptide, an MHC a chain, and an MHC P chain. The accessory CD8 binder protein includes an anti- CD8 scFv fragment and a transmembrane domain.

[0014] In another aspect, the disclosure is to a system including a first recombinant polynucleotide and a second recombinant polynucleotide. The first polynucleotide includes afirst nucleic acid sequence encoding a single chain trimer protein. The single chain trimer protein includes a target peptide, an MHC a chain, and an MHC P chain. The second recombinant polynucleotide includes a second nucleic acid sequence encoding an accessory CD8 binder protein. The accessory CD8 binder protein includes an anti-CD8 scFv fragment and a transmembrane domain.

[0015] In another aspect, the disclosure is to a cell comprising a single chain trimer protein, recombinant polynucleotide, or system as disclosed herein.

[0016] In one aspect, the disclosure is to a pharmaceutical composition including a pharmaceutically acceptable excipient and a single chain trimer, recombinant polynucleotide, system, or cell as disclosed herein.

[0017] In another aspect, the disclosure is to a method for inducing an immune response or treating a health condition in a subject in need thereof. The method includes administering to the subject a single chain trimer, recombinant polynucleotide, system, cell, or pharmaceutical composition as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 presents an illustration of a complex of MHC-I and CD8, showing key interaction residues. The peptide binding groove is toward the top and the a3 domain and cell surface are below. Key interacting residues are seen through a transparent CD8 dimer (Protein Data Bank: 1AKJ).

[0019] FIG. 2 presents schematic illustrations of the ability of CD8inT immunogens in accordance with provided embodiments to restore CD8 binding to MHC-II- or MHC-E-based SCTs. These immunogens can recruit CD8-dependent naive cells at vaccination, which will develop into MHC-II- or MHC-E-restricted CD8+ memory and effector cells. Due to functional avidity maturation, memory T cells have a low threshold to activation. Thus, memory cells with alternative MHC restriction do not require the additional signals provided by RhCMV (middle) or CD8inT (right) to function.

[0020] FIG. 3 presents schematic illustrations of Class-I SCTs lacking CD8 binding or having reduced binding in accordance with provided embodiments.

[0021] FIG. 4 presents data showing that the MHC Class Ib-based SCT with enhanced CD8 engagement, Gag69-Mamu-E*02: 11 [Q 115E], is folded and achieves proper surface expressionon transfected cells. Cells transfected with conventional or CD8inT-plus constructs (Gag69- Mamu-E*02: l l or Gag69-Mamu-E*02: l l[Q115E]) were stained with antibodies specific for Mamu-E (clone 4D12, third column) or all class-I molecules (clone W6 / 32, fourth column). The figure demonstrates that both the conventional and CD8inT-plus SCTs are properly folded and reach the cell surface, where both are reactive with anti-Mamu-E and anti-class I

[0022] FIG. 5 presents data demonstrating stimulation of memory T cells by Class-I SCTs lacking CD8 binding or having reduced binding. The plots show interferon-gamma (IFN) and tumor necrosis factor alfa (TNF) production by effector-memory CD8+ T cells from a previously vaccinated macaque after incubation with stimulator cells expressing conventional CM9-Mamu-A*01 SCT, CD8inT-weak (CM9-Mamu-A*01[A245V] SCT), CD8inT-zero (CM9-Mamu-A*01[D227K / T228A] SCT), or CD8inT-plus (CM9-Mamu-A*O1[Q115E] SCT).

[0023] FIG. 6 presents additional data demonstrating stimulation of memory T cells by Class-I SCTs lacking CD8 binding or having reduced binding. The graph shows quantification of IFN production among effector-memory CD8+T cells in response to CD8inT immunogens.

[0024] FIG. 7 presents data demonstrating the proper folding, surface expression, and stimulatory capacity of conventional or CD8inT immunogens delivered as mRNA encapsulated into lipid nanoparticles. The CM9-Mamu-A*01[D227K / T228A] single-chain trimer (second row) carries two mutations that nearly eliminate CD8 binding. Shown are interferon-gamma (IFN) and tumor necrosis factor-alfa (TNF) production by CD8+T cells from an SIV-infected, Mamu-A*01+ macaque. The first row contains two negative controls (DMSO only or untransduced HEK 293 cells) and one positive control (CM9 peptide). The second row shows CD8+T-cell responses to HEK 293 stimulator cells transduced with mRNA / LNPs expressing either (i) conventional CM9-Mamu-A*01 SCT or (ii) CM9-CD8inT-zero SCT (CM9-Mamu-A*01 [D227K / T228A]).

[0025] FIG. 8 presents an experimental scheme for demonstrating antitumor effects of high- avidity and CD8-independent T cells elicited by CD8inT immunogens with reduced or absent CD8 engagement. Both prophylactic and therapeutic vaccination are tested in Groups A1-A4 and B1-B4, respectively. Groups A4 and B4 employ the heteroclitic peptide with improved TCR stimulatory capacity, SIIWFEKL.DETAILED DESCRIPTIONI. General

[0026] The present disclosure provides compositions and methods involving single chain MHC trimer (SCT) immunogens that are based on MHC-I (i.e., class la), MHC-II (i.e., class II), or MHC-E (i.e., class lb) molecules and have been engineered for altered binding to CD8. Because of these altered binding properties, the provided materials and approaches can be particularly useful for eliciting unique immune responses that are otherwise difficult to induce. For example, treatment methods such as immunization schemes can incorporate provided immunogens with “graded” CD8 affinity, thereby encouraging outgrowth of successive waves of responding T cells having different avidities for pMHC complexes. The advantages of these and other provided embodiments enable a vast and currently inaccessible part of human adaptive-immune potential. The disclosed compositions and methods thus improve capacities for immune surveillance through more potent and broadly reactive cytolytic T cells. Pathogens such as HIV, SARS-CoV-2, HCV, and herpesviruses therefore must contend with an entirely new class of immunologic effector cells. The disclosure enables design of new immunogens that can target rare naive T cells of desired specificity, restriction, and / or affinity and expand those cells into effectors that are otherwise rare.

[0027] Some provided class I single chain trimers lack CD8 binding (e.g., have an ablated CD8 binding site) or have limited CD8 binding and can therefore be used to select for higher- affinity T-cell receptors (TCRs) that are generally subdominant in natural infections, e.g., in HIV infection. Other provided single chain trimer proteins add CD8 binding capacity to MHC class-II or MHC-lb molecules, allowing the priming of MHC-II- or MHC-lb-restricted CD8+ naive T cells. In additional provided embodiments, exogenous CD8-binding molecules augment the critical interaction between professional antigen presenting cells and responding T cells, thus driving differentiation of cells with the desired alternate restriction. Many of the provided variant single chain trimer proteins have not been described before. These unique molecules include MHC-II molecules that are able to bind CD8 and single chain trimer proteins that incorporate (or are co-expressed with) exogenous CD8-binding molecules. Other variant single chain trimers have been created for in-vitro biochemical characterizations, but have not been used for immunizations because it was not previously predicted that such molecules would elicit uniquely beneficial T-cell responses.

[0028] Experimental evidence indicates that CD8-independent naive precursors are rare but that CD8-independent memory cells are fully functional once elicited. Additionally, memory T cells are defined in part by their high functional avidity - that is, by their sensitivity to low- abundance pMHC targets on cell surfaces. Thus, ~50X avidity maturation of memory T cells (M. Pihlgren, P.M. Dubois, M. Tomkowiak, T. Sjogren & J. Marvel, J. Exp. Med. 184, (1996): 2141; M.K. Slifka & J.L. Whitton, Nat. Immunol. 2, (2001): 711) permits design of vaccines that tap a vast, under-explored and under-utilized immune resource: cytotoxic T lymphocytes (CTLs) bearing TCRs that are CD8 dependent in the priming phase but fully CD8 independent in the memory phase. The memory cells produced by these vaccines include CTL that recognize either class I-restricted epitopes with very high avidity, class Il-restricted epitopes, or, for example, HLA / Mamu-E-restricted epitopes. When such unique naive T cells are preferentially primed by the provided immunogens and recruited into the memory compartment, the avidity maturation that defines T-cell memory makes them fully functional.

[0029] The disclosure provides a straightforward approach to priming naive CD8+ cytotoxic T lymphocytes with unusually high potential to control infectious diseases. The particular primed CD8+ T cells are special due to high affinity of their T-cell receptors for peptide-MHC complex (pMHC), or to their restriction by alternative class-II or class-Ib MHC elements, which are non-canonical restricting elements for these cells. In many cases, such memory T cells with non-canonical restriction have been experimentally shown to be “CD8 independent,” which makes intuitive sense, due either to high TCR:pMHC affinity or to absent / low CD8 binding by the alternative restricting elements. Their extraordinary potential for controlling disease has been repeatedly demonstrated in studies of the immune responses to infectious diseases (M.A. Alexander-Miller, G.R. Leggatt & J. A. Berzofsky, Proc. Natl. Acad. Sci. USA 93, (1996): 4102; A. Gallimore, T. Dumrese, H. Hengartner, R.M. Zinkemagel & H.G. Rammensee, J. Exp. Med. 187, (1998): 1647; R.H. Mealey, B. Zhang, S.R. Leib, M.H. Littke & T.C. McGuire, Virology 313, (2003): 537; I. Messaoudi, J. A. Guevara Patino, R. Dyall, J. LeMaoult & J. Nikolich-Zugich, Science 298, (2002): 1797; C. Sedlik et al., J. Virol. 74, (2000): 5769).

[0030] The provided vaccination strategy, referred to herein as “CD8inT” vaccination for CD8 independence targeting, involves in some embodiments non-traditional immunogen structures that incorporate the desired epitope target and restricting MHC element, and are additionally engineered to have an affinity for CD8 that allows selective recruitment of the desired TCRs from the naive pool. The provided vaccination approach further includes, in someembodiments, vaccine regimens that employ a series of different CD8inT boosts with graduated CD8 affinity to achieve recruitment of different clones in waves, and thereby achieve greater clonotypic breadth.

[0031] The advantageous materials and methods disclosed herein rely on a previously underappreciated awareness that the CD8 dependence of CD8+ T cells - i.e., the need for CD8 to engage a binding site on the peptide:MHC complex - is commonly restricted to cells in the naive state and can dissipate with the functional avidity maturation that accompanies differentiation into effector or memory cells. In fact, continued CD8 dependence in memory cells is associated with relatively lower TCR:pMHC affinity and poor cytolytic function. The CD8 dependence of most naive cells is indisputable based on their mechanisms of development and on the comparative rarity of MHC-II- or MHC-E-restricted T-cell responses to pathogens. That CD8 independence can commonly develop after appropriate priming, on the other hand, is clear from studies of immune responses to RhCMV / SIV vaccines, which elicit vast numbers of cells that do not require CD8:pMHC engagement for activation.II. Definitions

[0032] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0033] As used herein, the terms “single chain trimer” and “SCT” refer to a recombinant major histocompatibility complex (MHC) molecule that includes an MHC a chain segment (e.g., an MHC class I heavy chain segment or an MHC class II a chain segment), an MHC P chain segment (e.g., P2-microglobulin or an MHC class II P chain segment), and an antigen peptide or target peptide segment, together in a single molecule. In some configurations, a single chain trimer further includes one or more linkers connecting different segments of the single chain trimer molecule. In some configurations, a single chain trimer molecule further includes a leader peptide segment. A single chain trimer can also include additional segments, i.e., the single chain trimer can include three or more segments, four or more segments, or five or more segments, that can optionally be joined by linkers within the single chain trimer molecule.

[0034] In various embodiments of the present disclosure, the single-chain trimer (SCT) proteins and their encoding polynucleotides are organized and constructed in a defined linear order. Specifically, the polynucleotide constructs are arranged in the 5' to 3' direction to correspond to the N-terminus to C-terminus order of the expressed polypeptide, unless otherwise stated. In one exemplary embodiment, the SCT polynucleotide encodes, in sequential order: (i) a secretion signal peptide; (ii) a target peptide (antigenic sequence); (iii) a first linker domain (for example, a (G4S)n sequence where n is an integer); (iv) a p2-microglobulin domain (which may be wild-type or a variant); (v) a second linker domain (for example, a (G4S)nsequence, which may be the same as or different from the first linker); and (vi) an MHC a chain domain (such as an MHC class la, lb, or II a chain or, as appropriate, paired with a P chain). Each of these domains is encoded in-frame to produce a contiguous open reading frame, such that transcription and translation yields a contiguous polypeptide with the defined N- to C- terminal domain architecture. In some embodiments, a cysteine residue is engineered into the first linker and / or the MHC a chain (for example, a Y84C substitution) to facilitate a stabilizing disulfide bond between these regions.

[0035] In addition to the canonical linear arrangement of domains described above, the present disclosure contemplates alternative assembly strategies and permutations for the construction of the single-chain trimer (SCT) proteins and their encoding polynucleotides. In certain embodiments, the order, composition, or connection of domains may be varied without departing from the scope of the disclosure. For example, the length, sequence, or amino acid composition of linker domains connecting the target peptide, P2-microglobulin, and MHC a or P chains may be varied; such linkers may comprise any flexible peptide sequence, such as (G4S)n, where n is an integer from 1 to 10, or may be omitted entirely to allow direct fusion of domains.

[0036] In some embodiments, additional functional domains or segments may be inserted at various positions within the construct, such as anti-CD8 single-chain variable fragment (scFv) domains, transmembrane domains, or other accessory peptides, either between principal domains (e.g., between p2-microglobulin and the MHC a chain) or appended at the N- or C- terminus. The presence and position of such auxiliary domains may be specified for particular applications, and the linkers used to join them may vary in length and sequence.

[0037] The essential components of the provided SCT polypeptides are generally the target peptide, P2-microglobulin (or MHC class II P chain), and MHC a chain (or both a and P chainsfor class II SCTs), arranged in a manner that preserves the functional presentation of the peptide-MHC complex. Optional elements, such as secretion signal peptides, linker domains, engineered cysteine residues for disulfide trapping, anti-CD8 scFv fragments, and transmembrane domains, may be included as desired to enhance expression, stability, targeting, or immunogenicity.

[0038] The disclosure encompasses all such permutations, including but not limited to: (i) constructs with or without one or more linkers; (ii) constructs with alternative linker sequences or lengths; (iii) constructs in which auxiliary domains are inserted between any two principal domains or at the termini; (iv) constructs in which the order of MHC class II a and P chains or their subdomains is varied to facilitate proper folding and function; and (v) constructs with additional or fewer domains, provided that the essential function of the SCT is retained. Specific examples of alternative assemblies are provided throughout the detailed description and sequence listing, and all reasonable combinations and substitutions of domains, linkers, and auxiliary elements are contemplated.

[0039] As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to any polymer comprising two or more amino acid residues, regardless of length, sequence, structure, or biological origin, in which the amino acid residues are joined by covalent peptide bonds. The terms encompass full-length proteins, polypeptides, peptides, oligopeptides, and any other amino acid polymers, whether naturally occurring, recombinant, synthetic, semi synthetic, or chemically modified. As used herein, the terms include polymers containing only canonical amino acids, as well as those containing noncanonical amino acids, amino acid analogs, amino acid mimetics, artificial or unnatural amino acids, or any combination thereof, provided that the residues are linked by peptide or peptide-like bonds. The terms further encompass proteins or polypeptides that are post-translationally or chemically modified, including, but not limited to, glycosylated, phosphorylated, methylated, acetylated, ubiquitinated, PEGylated, lipidated, cyclized, or otherwise derivatized forms. The terms include molecules comprising a single contiguous amino acid chain, as well as multimeric proteins composed of two or more such chains associated by covalent or noncovalent interactions. Unless otherwise indicated, the terms are inclusive of any amino acid-based polymer of any size or sequence, whether derived from natural sources, produced by recombinant or synthetic means, or generated or modified by any method described or contemplated in this specification. Polypeptide sequences, when provided, are listed in the N-terminal to C-terminal direction, unless stated otherwise. Also, thesequential arrangement of domains in the polypeptide exactly reflects the order of their corresponding coding regions in the associated polynucleotide unless stated otherwise.

[0040] As used herein, the term “target peptide” refers to a peptide that can elicit T cells with a desired specificity. The target peptide can encode the exact sequence against which a T-cell response is desired, or the target peptide can be varied in ways that maintain the capacity to elicit T cells having the desired specificity. For example, the target peptide may be shorter or longer than the sequence against which a T-cell response is desired; or a target peptide may contain amino-acid changes (that is, it may be a “heteroclitic” variant) that impart desirable qualities while maintaining the possibility of eliciting T cells with a desired specificity (Capasso, Oncoimmunology 6, (2017): el319028)

[0041] As used herein, the term “variant” refers to any molecule, entity, or construct that is derived from, based on, or otherwise related to a reference molecule, and that exhibits one or more structural, sequence, or functional differences relative to the reference molecule. In the context of polypeptides or proteins, a “variant” encompasses any polypeptide or protein that shares substantial sequence or structural similarity with a reference polypeptide or protein, but that differs from the reference by virtue of one or more alterations in its amino acid sequence, such as substitutions, insertions, deletions, or modifications (including but not limited to post- translational modifications, incorporation of noncanonical amino acids, or chemical derivatization). Polypeptide or protein variants may have amino acid sequences that exhibit a degree of sequence identity to the reference sequence of at least 80%, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, as determined by methods known in the art. Variants may retain the biological activity or functional properties of the reference molecule, such as catalyzing the same chemical reaction or binding the same ligand, or may be specifically configured or selected to lack, alter, or enhance one or more biological activities or functional properties. The term “variant” further includes molecules generated by modification of the encoding polynucleotide sequence, such as by site-directed mutagenesis, random mutagenesis, recombination, synthetic gene construction, or other genetic engineering methods, as well as molecules produced by chemical synthesis or enzymatic modification. Unless otherwise indicated, “variant” as used herein is inclusive of any form — natural, synthetic, engineered, or otherwise — having one or more structural, sequence, or functional differences from a reference molecule, and is intended to encompass variants of polypeptides, proteins, nucleic acids, andother biomolecules described or utilized in the methods, systems, or compositions of this specification.

[0042] As used herein, the term “recombinant” when used with reference to a protein refers to a protein prepared via genetic engineering. A recombinant cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. A recombinant nucleic acid is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. A recombinant protein is made using recombinant techniques such as through the expression of a recombinant nucleic acid or DNA molecule as described above.

[0043] As used herein, the terms “nucleic acid” and “polynucleotide” refer to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine (Batzer et al., Nucleic Acid Res. 19, (1991): 5081; Ohtsuka et al., J. Biol. Chem. 260, (1985): 2605; and Rossolini et al., Mol. Cell. Probes 8, (1994): 91).

[0044] Non-limiting examples of polynucleotides or nucleic acids include DNA, RNA, coding or noncoding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA),ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro- RNA (miRNA), small nucleolar RNA(snoRNA), ribozymes, deoxynucleotides (dNTPs), or dideoxynucleotides (ddNTPs). Polynucleotides can also include complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification. Polynucleotides can also include DNA molecules produced synthetically or by amplification, genomic DNA (gDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, or primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Polynucleotide sequences, when provided, are listed in the 5' to 3' direction, unless stated otherwise.

[0045] Nucleic acids or polynucleotides can be double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive, for example, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands.

[0046] Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications include base modifications such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of S-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0047] Nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0048] In various embodiments, the polynucleotides encoding the single chain trimer proteins and their component domains as disclosed herein may be codon optimized forexpression in a selected host organism or cell type. As used herein, “codon optimized” refers to the alteration of the nucleotide sequence of a gene or coding region such that the codons used correspond to those most preferred or frequently utilized in the intended expression system (e.g., human, mouse, yeast, E. coli, plant), while maintaining the encoded amino acid sequence. Codon optimization may further include the removal or modification of cryptic splice sites, mRNA instability motifs, GC content adjustment, RNA secondary structure minimization, and / or the incorporation or removal of restriction sites.

[0049] The disclosure encompasses any nucleic acid sequence encoding the disclosed SCT polypeptides, including but not limited to: (i) wild-type or native coding sequences; (ii) codon- optimized sequences for any eukaryotic or prokaryotic expression system; (iii) chimeric or synthetic sequences comprising codons optimized for multiple hosts; and (iv) sequences further comprising regulatory elements or synthetic features facilitating transcription, translation, or stability. Where a specific amino acid sequence or SEQ ID NO is disclosed for a polypeptide, all nucleotide sequences encoding that polypeptide — including all possible codon substitutions, codon usage variations, and chemically modified nucleotides (e.g., pseudouridine, 5- methylcytidine, A7-methylpseudouridine) — are included within the scope of the disclosure and the claims, except where explicitly excluded.

[0050] Relatedly, the disclosure further encompasses chemically modified polynucleotides, including but not limited to messenger RNA (mRNA) comprising one or more modified nucleotides such as pseudouridine, A7-methylpseudouridine, 5-methylcytidine, 5- methyluridine, or other naturally occurring or synthetic nucleotide analogs, which may be incorporated to enhance stability, reduce immunogenicity, or improve translation efficiency. Codon-optimized mRNA sequences encoding the SCT constructs and their domains, as well as complementary DNA (cDNA), DNA templates for in vitro transcription, and all possible sequence variants encoding the same polypeptide, are expressly included within the scope of the invention.

[0051] As used herein, the terms “sequence identity,” “percent identity,” “percent alignment,” and the like refer to the degree of sequence similarity between two nucleic acid or amino acid sequences, expressed as a percentage, and determined by comparing the sequences after optimal alignment. Percent identity is calculated by aligning a candidate sequence and a reference sequence, and determining the proportion of identical residues (nucleotides or amino acids) at corresponding positions within the alignment, typically over the entire length of thereference sequence or over a defined region thereof. Optimal alignment of sequences for comparison may be performed by any method known in the art, including, without limitation, computerized sequence alignment algorithms such as the local homology algorithm of Smith and Waterman (Add. Appl. Math. 2:482, 1981), the global alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), or by implementations of these or similar algorithms in software tools such as BLAST (Basic Local Alignment Search Tool), BLAST 2.0, or other commercially available or publicly accessible sequence analysis platforms. Percent identity may also be calculated by manual alignment and visual inspection, provided the alignment is performed according to accepted principles in the art. When using alignment algorithms, default parameters may be employed unless otherwise specified, including gap penalties, scoring matrices, word size, and statistical thresholds as commonly utilized for nucleotide or protein sequence comparison. For example, BLASTN (for nucleotide sequences) commonly uses a word size of 28, expectation value of 10, and scoring parameters of match reward (M) of 1 and mismatch penalty (N) of -2, but alternative parameters may be used as appropriate for the specific comparison. Percent identity may be reported for any specified level, including, but not limited to, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to a given sequence. As used herein, “percent identity,” “sequence identity,” and “homology” may be used interchangeably, unless context clearly indicates otherwise. Unless otherwise indicated, percent identity is intended to encompass any method or algorithm for sequence comparison recognized in the art, and includes both local and global alignments, statistical analyses of similarity (such as smallest sum probability or expectation value), and any acceptable measure of the proportion of identical residues between aligned sequences.

[0052] Accordingly, in addition to the specific polypeptide and nucleic acid sequences disclosed herein (including all SEQ ID NOs), the present disclosure encompasses all polypeptides and nucleic acids having a specified degree of sequence identity to any such sequence. In particular, for each disclosed polypeptide sequence (including both wild-type and engineered sequences such as SCTs, scFv fusions, chimeric constructs, or other non- conventional SEQ ID NOs), the invention further encompasses: (a) any polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference SEQ ID NO and retaining the functional activity or binding property described for that reference sequence;(b) any nucleic acid (including DNA, RNA, cDNA, and chemically modified nucleic acids) encoding such a variant polypeptide, including all possible codon usages, codon-optimized sequences, degenerate codons, and complementary sequences; (c) all conservatively substituted, functionally equivalent, or otherwise modified variants of such polypeptides and nucleic acids, including those with insertions, deletions, or substitutions that do not materially alter the functional activity of the reference SEQ ID NO; (d) any chemically modified, chimeric, or synthetic variant of such polypeptides and nucleic acids, including but not limited to fusion proteins, scFv fusions, domain swaps, or other engineered modifications; (e) all sequences having at least the percent identity to the full-length reference sequence, or to a specified functional domain thereof (such as the target peptide, P2-microglobulin, linker, or MHC chain). Unless otherwise specified, all such variants are included within the scope of the present disclosure and any claims reciting SEQ ID NOs, polypeptide sequences, or nucleic acids encoding them.

[0053] As used herein, the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like. One of skill in the art will recognize that other pharmaceutically acceptable excipients and carriers are useful in the present disclosure.

[0054] As used herein, the term “vaccine” refers to a composition that provides active acquired immunity to one or more infectious or malignant diseases. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of a future infection by a pathogen), or therapeutic (e.g., to fight an infection or disease that has already occurred).

[0055] As used herein, the term “treatment” refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit imparts any relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested. A treatment can involve anyof ameliorating one or more symptoms of disease, preventing the manifestation of such symptoms before they occur; slowing down or completely preventing the progression of the disease (as may be evident by longer periods between reoccurrence episodes, slowing down or prevention of the deterioration of symptoms, etc.), enhancing the onset of a remission period, slowing down the irreversible damage caused in the progressive-chronic stage of the disease (both in the primary and secondary stages), delaying the onset of said progressive stage, or any combination thereof.

[0056] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as an independent, replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced. A vector can therefore refer to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.

[0057] As used herein, the term “cell” generally refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, abacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii. Chlamydomonas reinhardlii. Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, mollusk, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), etc. Sometimes a cell does not originate from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).

[0058] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, intradermal, or subcutaneous administration, intrathecaladministration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

[0059] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0060] As used herein, the term “therapeutically effective amount” refers to the quantity of a composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, or relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0061] As used herein, the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of’ is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of’ limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0062] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a pharmaceutically acceptable carrier” optionally includes a combination of two or more pharmaceutically acceptable carriers, and the like.

[0063] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0064] The terms “first” and “second” when used herein with reference to various elements and properties, are simply to more clearly distinguish the two elements or properties, when present, and are not intended to indicate order or to require that both elements or properties are present.III. Class la Single Chain Trimer Proteins Having Reduced CD8 Binding

[0065] In one aspect, the present disclosure provides various single chain trimer proteins that include a target peptide, a p2-microglobulin, and an MHC class la a chain. At least one of the P2-microglobulin and the MHC class la a chain is a variant polypeptide, e.g., a variant of a naturally occurring polypeptide, such that the P2-microglobulin and / or MHC class la a chains have one or more introduced mutations that result in altered CD8 binding affinity for the single chain trimer. The particular selection, composition, and arrangement of the target peptide segment, the P2-microglobulin segment, and the MHC class la a chain segment in the single chain trimer proteins provide the proteins with surprising characteristics useful for eliciting immune responses that are otherwise difficult to induce.

[0066] Inter-clonal competition forms part of the mechanisms of both immunodominance and T-cell avidity maturation (H. Turula, C.J. Smith, F. Grey, K.A. Zurbach & C.M. Snyder, Eur. J. Immunol. 43, (2013): 1252). Although higher-avidity CD8+ T cells have a selective advantage due partly to increased IL-15Ra expression and consequent cytokine acquisition, the resulting memory T-cell pool comprises cells with a range of TCR:pMHC affinities (S. Oh, L.P. Perera, D.S. Burke, T.A. Waldmann & J.A. Berzofsky, Proc. Natl. Acad. Sci. USA 101, (2004): 15154). Higher-avidity CTL can be identified within this pool by using pMHC tetramers that lack CD8 binding (D.A. Price et al., J. Exp. Med. 202, (2005): 1349; E.M. Choi et al., J. Immunol. 171, (2003): 5116; M. J. Pittet et al., J. Immunol. 171, (2003): 1844), because lower-avidity CTL generally require CD8 engagement for tetramer binding. The provided materials and methods are based in part on a recognition that lower-avidity CTLs that require CD8 binding as memory cells cannot be primed by class-I SCT immunogens that lack CD8 binding, and thus such cells would remain unprimed and not compete with higher-affinity clonotypes for IL- 15, antigen, or other trophic factors.

[0067] T-cell avidity is reflected in the sensitivity of a cell to cognate pMHC on the surface of target cells. High-avidity cells recognize antigen-presenting cells (APCs) bearing very low levels of peptide antigen, whereas low-avidity cells require much higher numbers of pMHC complexes to become activated. The avidity of a CD8+ T cell can greatly impact in vivo efficacy. In studies of vaccinia virus, adoptive transfer of high-avidity cells reduced titratable virus by -1000X but low-avidity cells had no effect (M.A. Alexander-Miller, G.R. Leggatt & J.A. Berzofsky, Proc. Natl. Acad. Sci. USA 93, (1996): 4102). Other examples of superior function ascribed to high-avidity cells include LCMV (A. Gallimore, T. Dumrese, H. Hengartner, R.M. Zinkernagel & H.G. Rammensee, J. Exp. Med. 187, (1998): 1647; C. Sedlik et al. J. Virol. 74, (2000): 5769), equine infectious anemia virus (R.H. Mealey, B. Zhang, S.R. Leib, M.H. Littke & T.C. McGuire, Virology 313, (2003): 537), and HSV (I. Messaoudi, J.A. Guevara Patino, R. Dyall, J. LeMaoult & J. Nikolich-Zugich, Science 298, (2002): 1797).

[0068] Cells with higher intrinsic avidity for pMHC depend less on the pMHC-CD8 interaction for stable binding of fluorescent tetramers. This avidity reflects not only the affinity of TCR for cognate pMHC-I ligand, but also factors such as the density, distribution, and mobility of TCR in the cell membrane. CD8+ T cells with high intrinsic avidity for antigen can therefore be identified using pMHC-I tetramers with mutations in the a3 domain that eliminate CD8 binding without affecting the TCR interaction (D. A. Price et al., J. Exp. Med. 202, (2005): 1349; E.M. Choi et al., J. Immunol. 171, (2003): 5116; M.J. Pittet et al., J. Immunol. 171, (2003): 1844).

[0069] MHC-I binds to CD8 using residues in the a3, a2, and P2-microglobulin domains. The key residues are known and conserved between humans and macaques, and the effects of various mutations are known and have been reported repeatedly (FIG. 1). The a3 domain makes the most extensive contacts with CD8, using a charged protruding loop. D227K / T228A mutations abolish the interaction and prevent CD8 binding (Kd > 10 mM) (S.L. Hutchinson et al., J. Biol. Chem. 278, (2003): 24285). A245V is outside the loop but causes a conformational change that reduces the affinity for CD8 from -130 to -500 pM (G.F. Gao et al., J. Biol. Chem. 275, (2000): 15232). These two mutations can therefore be used to abolish (D227K / T228A) or merely reduce (A245V) SCT affinity for MHC-Ia. The a2 domain makes a hydrogen bond contact to CD8 using QI 15. Mutation of this residue to glutamic acid (Q115E) shortens the hydrogen bond and improves binding affinity to -90 pM (L. Wooldridge et al., J. Biol. Chem. 280, (2005): 27491). Mutations of a2 residues 115, 122, or 128 to alanine interfere with CD8 interactions and with accessory functions (L. Shen et al., J. Exp. Med. 184, (1996): 1671; J.Sun et al., J. Exp. Med. 182, (1995): 1275). p2-microglobulin residue K58 participates in a hydrogen-bond network with CD8, and its mutation to glutamic acid lowers affinity of MHC- I more than 15-fold to > 2 mM.

[0070] In some embodiments, the MHC class la a chain and / or the p2-microglobulin of the provided single chain trimer include one or more amino acid substitutions. In some embodiments, the amino acid substitutions include one of 115A (a chain), 122A (a chain), 128 A (a chain), 245V (a chain), 227K (a chain), 228 A (a chain), 58E (P2-microglobulin), 58S (P2-microglobulin), 58R (P2-microglobulin), 58C (P2-microglobulin), 58 V (P2- microglobulin), 58D (P2-microglobulin), 58 Y (P2-microglobulin), 58W (P2-microglobulin), and 115E (a chain). In some embodiments, the amino acid substitutions consist of one of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include two of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions consist of two of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include three of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions consist of three of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include four of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions consist of four of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include five of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions consist of five of 115A, 122 A, 128 A, 245V, 227K, 228 A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include six of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions consist of six of 115A, 122A, 128A, 245V, 227K, 228A, 58E, 58S, and 115E. In some embodiments, the amino acid substitutions include 122A, 128A, 245V, 227K, 228A, 58E, and 115E. In some embodiments, the amino acid substitutions consist of 122A, 128A, 245V, 227K, 228A, 58E, and 115E. In some embodiments, the amino acid substitutions include 122A, 128A, 245V, 227K, 228A, 58S, and 115E. In some embodiments, the amino acid substitutions consist of 122A, 128A, 245V, 227K, 228A, 58S, and 115E. In some embodiments, the amino acid substitutions include 115A, 122A, 128A, 245V, 227K, 228A, and 58E. In some embodiments, the amino acid substitutions consist of 115A, 122A, 128A, 245V, 227K, 228A, and 58E. In some embodiments, the amino acid substitutions include 115A, 122A, 128A, 245V, 227K, 228A, and 58S. In someembodiments, the amino acid substitutions consist of 115A, 122A, 128A, 245V, 227K, 228A, and 58S.

[0071] In some embodiments, the amino acid substitutions include one of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of one of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and QI 15E. In some embodiments, the amino acid substitutions include two of QI 15A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of two of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and QI 15E. In some embodiments, the amino acid substitutions include three of QI 15 A, DI 22 A, El 28 A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of three of QI 15A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and QI 15E. In some embodiments, the amino acid substitutions include four of QI 15 A, DI 22 A, El 28 A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of four of QI 15A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions include five of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of five of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions include six of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and QI 15E. In some embodiments, the amino acid substitutions consist of six of Q115A, D122A, E128A, A245V, D227K, T228A, K58E, K58S, and Q115E. In some embodiments, the amino acid substitutions include D122A, E128A, A245V, D227K, T228A, K58E, and Q115E. In some embodiments, the amino acid substitutions consist of D122A, E128A, A245V, D227K, T228A, K58E, and Q115E. In some embodiments, the amino acid substitutions include D122A, E128A, A245V, D227K, T228A, K58S, and Q115E. In some embodiments, the amino acid substitutions consist of D122A, E128A, A245V, D227K, T228A, K58S, and Q115E. In some embodiments, the amino acid substitutions include Q115A, D122A, E128A, A245V, D227K, T228A, and K58E. In some embodiments, the amino acid substitutions consist of QI 15A, D122A, E128A, A245V, D227K, T228A, and K58E. In some embodiments, the amino acid substitutions include Q115A, D122A, E128A, A245V, D227K, T228A, and K58S. In some embodiments, the amino acid substitutions consist of Q115A, DI 22 A, El 28 A, A245V, D227K, T228A, and K58S. 1

[0072] The variant class la a chain and / or the variant P2-microglobulin of the provided single chain trimer are engineered to provide the single chain trimer with a reduced or eliminated binding affinity for CD8. The single chain trimer can have a binding affinity dissociation constant (Ka) that is, for example, between 300 pM and 1000 pM, e.g., between 300 pM and 720 pM, between 370 pM and 790 pM, between 440 pM and 860 pM, between 510 pM and 930 pM, or between 580 pM and 1000 pM. The single chain trimer can have a binding affinity dissociation constant that is, for example, between 1 mM and 10 mM, e.g., between 1 mM and 4 mM, between 1.3 mM and 5 mM, between 1.6 mM and 6.3 mM, between 2 mM and 7.9 mM, or between 2.5 mM and 10 mM. In terms of upper limits, the binding affinity dissociation constant of the single chain trimer can be, for example, less than 10 mM, e.g., less than 7.9 mM, less than 6.3 mM, less than 5 mM, less than 4 mM, less than 3.2 mM, less than 2.5 mM, less than 2 mM, less than 1.6 mM, less than 1.3 mM, less than 1000 pM, less than 930 pM, less than 860 pM, less than 790 pM, less than 720 pM, less than 650 pM, less than 580 pM, less than 510 pM, less than 440 pM, or less than 370 pM. In terms of lower limits, the binding affinity dissociation constant of the single chain trimer can be, for example, greater than 300 pM, e.g., greater than 370 pM, greater than 440 pM, greater than 510 pM, greater than 580 pM, greater than 650 pM, greater than 720 pM, greater than 790 pM, greater than 860 pM, greater than 930 pM, greater than 1000 pM, greater than 1.3 mM, greater than 1.6 mM, greater than 2 mM, greater than 2.5 mM, greater than 3.2 mM, greater than 4 mM, greater than 5 mM, greater than 6.3 mM, greater than 7.9 mM. In some embodiments, the binding affinity dissociation constant of the single chain trimer is greater than 10 mM. Lower dissociation constants, e.g., less than 300 pM, are also contemplated.

[0073] In some embodiments, the single chain trimer protein further includes a first linker connecting the target peptide and the P2-microglobulin in the single chain trimer. In some embodiments, the first linker includes a first cysteine amino acid, and the MHC class la a chain comprises a second cysteine amino acid bonded to the first cysteine amino acid, such that the linker forms a disulfide trap within the single chain trimer protein. In some embodiments, the second cysteine amino acid is the amino acid substitution Y84C. A disulfide bond can in some embodiments improve the immunogenicity of the provided single chain trimer protein. A disulfide bond between the subunit segments of the protein can increase the stability of the protein and prevent it from unfolding or dissociating under harsh conditions, such as high temperatures, low pH, or denaturing agents. The disulfide trap can also help the protein fold correctly and form the desired structure, stabilizing a particular conformation or orientation ofthe subunit segments. The presence of a disulfide bond can also facilitate purification of the single chain trimer, as the disulfide can be captured using a thiol-specific resin or antibody.

[0074] In some embodiments, the single-chain trimer protein further includes a second linker connecting the p2-microglobulin and the MHC class la a chain in the single chain trimer, which may comprise mutations introduced to the p2-microglobulin and / or MHC class la a chains. In some embodiments, at least one of the first linker and the second linker is a (G4S)nlinker, where n is an integer from 1 to 10. In some embodiments, the first linker and the second linker are each independently a (G4S)nlinker. In some embodiments, at least one of the first linker and the second linker is a (G4S)3 linker, i.e., GGGSGGGSGGGS. In some embodiments, the first linker and the second linker are each independently a (GiS)3 linker. In some embodiments, at least one of the first linker and the second linker is a (GiS)4 linker, i.e., GGGSGGGSGGGSGGGS. In some embodiments, the first linker and the second linker are each independently a (GiS)4 linker. In some embodiments, the first linker is a (G4S)3 linker and the second linker is a (GiS)4 linker.

[0075] In some embodiments, the variant MHC class la a chain of the provided single chain trimer is a variant HLA-A a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*01 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*02 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*03 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*09 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A* 10 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A* 11 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*24 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*28 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-A*29 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B *07 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B *08 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B* 12 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B* 13 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B* 14 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B* 18 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B *27 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B *44 a chain. In someembodiments, the variant MHC class la a chain is a variant HLA-B*58 a chain. In some embodiments, the variant MHC class la a chain is a variant HLA-B*62 a chain.IV. Class lb and II Single Chain Trimer Proteins Having High-Affinity CD8 Binding

[0076] In another aspect, the present disclosure provides various single chain trimer proteins that include a target peptide, p2-microglobulin, and an MHC class lb a chain. At least one of the p2-microglobulin and the MHC class la a chain is a variant polypeptide, e.g., a variant of a naturally occurring polypeptide, such that the P2-microglobulin and / or MHC class lb a chains have one or more introduced mutations that result in the trimer having altered CD8 binding affinity. The particular selection, composition, and arrangement of the target peptide segment, the P2-microglobulin segment, and the MHC class lb a chain segment in the single chain trimer proteins provide the proteins with surprising characteristics useful for eliciting immune responses that are otherwise difficult to induce.

[0077] In another aspect, the present disclosure provides various single chain trimer proteins that include a target peptide, an a chain of a first MHC class II allele, and a P chain of a second MHC class II allele. One or both of the a chain and the P chain are variant chains whose inclusion confers altered CD8 binding affinity on the single chain trimer. The particular selection, composition, and arrangement of the target peptide segment, the MHC class II a chain segment, and the MHC class II P chain segment in the single chain trimer proteins provide the proteins with surprising characteristics useful for eliciting immune responses that are otherwise difficult to induce.

[0078] Class II- and Ib-restricted CD8+ memory T cells can be elicited by certain pathogens that likely alter antigen presentation and costimulation pathways, for instance cytomegalovirus (FIG. 2, middle panel) (S.G. Hansen et al., Science 340, (2013): 1237874; S.G. Hansen et al., Science 351, (2016): 714). At least some class Il-restricted CD8+ T cells can also develop fortuitously after HIV infection (S. Ranasinghe et al., Immunity 45, (2016): 917). The expanded memory cells are CD8 independent because they can recognize cognate pMHC complex on the surface of K562 cells that do not express class I or any known CD8 binding partner (FIG. 2, lower right) (S.G. Hansen et al., Science 351, (2016): 714). It is thus certain that many naive CD8+ T cells bear receptors that bind peptide:MHC-II or peptide:MHC-E with sufficient affinity to become CD8-independent memory cells.

[0079] Importantly, the naive T cells capable of MHC-II or MHC-E restriction are themselves almost certainly not CD8 independent. That is, they are very unlikely to be primed by exposure to pMHC alone (FIG. 2, left panel). First, the cells are not primed and do not become memory cells under most conditions when pMHC-II or pMHC-E are produced. For example, it is clear that routine SIV infection produces pMHC-II and pMHC-E complexes (because infected PBMC can be recognized by RhCMV / SIV-elicited T cells with alternate restriction), but SIV infection alone produces very few or no T cells with alternative MHC restriction. Second, SCTs containing MHC-E-restricted “supertopes” have been created (these conventional MHC-E-based SCTs do not bind CD8) and macaques have been vaccinated using these constructs, under conditions that produce abundant responses to class la SCTs, and negligible or very low T-cell responses were observed even after repeated immunization. The naive T cells with potential alternative restriction are therefore rarely capable of development except under the special circumstances imposed by RhCMV infection or with the assistance of CD8 engagement provided by CD8inT immunogens (FIG. 2, middle and right panels).

[0080] Studies of SIV vaccines based on the fibroblast-adapted RhCMV strain, 68-1, demonstrated outgrowth of vaccine insert-specific CD8+ T cells that comprise more than 10% of the memory pool (S.G. Hansen et al., Nat. Med. 15, (2009): 293) and are exclusively MHC- II or MHC-E restricted (S.G. Hansen et al., Science 340, (2013): 1237874). Although a complete analysis of the clonotypic structure of this response has not been published, it is clear that the naive CD8+ T-cell pool contains sufficient TCRs with potential for pMHC-E or pMHC-II binding to generate a massive response. This work also generated a library of peptides from within SIV Gag that are known to be presented by MHC-E or MHC-II and for which the frequencies of responses in vaccinated macaques are known.

[0081] MHC-E molecules bind CD8 with extremely low affinity (Kd 1-2 mM (G.F. Gao et al., J. Biol. Chem. 275, (2000): 15232), > 10X weaker than for MHC-Ia). The overall conformation of HLA-E is very similar to that of HLA-A2, but the crucial 223-229 loop (see above) adopts a different conformation, most likely due to three amino-acid differences adjacent to the loop. It has been shown that mutation of these residues to those from MHC-Ia molecules confers on HLA-E a binding affinity for CD8 that is almost identical to the affinity of HLA-A2 for CD8 (G.F. Gao et al., J. Biol. Chem. 275, (2000): 15232). The converse is also true, i.e., placing the MHC-E residues into HLA-A2 degrades CD8 binding to have a Kd >1 mM. CD8inT-E immunogens (FIG. 2, upper right) are MHC-E-based SCTs that incorporate amino acids that favor CD8 binding so that the SCT can bind CD8 similarly to MHC-Ia.

[0082] The overall structure of HLA-DR1 is similar to that of HLA class I (J.H. Brown et al., Nature 364, (1993): 33). The ai and 012 domains of DR1 superimpose closely on the ai and P2-microglobulin chains of class I, and the Pi domain structure similarly follows that of HLA class-1012. The P2 domain of DR1 has a very similar structure to that of class-I as, but is rotated by ~15 degrees. However, the as domain of class-I molecules can change angle upon CD8 binding. Most of the well-conserved residues responsible for the very weak interaction with CD4 (Kd > 2 mM for human CD4 binding to human MHC-II) are located close to the analogous position of the CD8-binding loop in MHC-Ia.

[0083] These largely overlapping structures offer a number of opportunities for engineering CD8 binding into MHC-II based SCTs to create CD8inT-II immunogens. The overall approach is to replace residues, loops, or even larger sections of the MHC-II alpha and beta chains with analogous regions from MHC-Ia, which are known to be involved in CD8 binding (see above). Accordingly, in some embodiments, variants of alpha and / or beta chains in the provided single chain trimer proteins can include replacements of partial or entire loops or larger polypeptide regions, in addition to or as an alternative to point mutations (e.g., substitutions, insertions, or deletions).

[0084] The typical affinity for CD8 of MHC-II- or MHC-E-restricted TCRs found on CD8+ T cells is unknown. The precursors of MHC-II- or MHC-E-restricted T cells may therefore be characterized by a broad range of functional avidities for the CD8inT immunogens disclosed herein, and some clones may require high-affinity CD8 binding for successful priming (L. Wooldridge et al., Eur. J. Immunol. 37, (2007): 1323).

[0085] The CD8 molecule’s cytoplasmic tail is associated with the lymphocyte-specific tyrosine protein kinase p561ck, which initiates signal transduction by phosphorylating immunoreceptor tyrosine-based activation motifs (ITAMs) within the TCR complex. Most intuitively, CD8 assists or modulates the TCR:pMHC interaction by cooperatively binding with the TCR to the same pMHC target. CD8 plays a role in the distribution and organization of the TCR on the T cell surface, promoting its association with lipid rafts (A. Arcaro et al., J. Exp. Med. 194, (2001): 1485). Finally, CD8 may also have a role in cell-cell adhesion, helping to tether the T cell to its target (I.F. Luescher et al., Nature 373, (1995): 353). Thus, CD8 has disparate effects that can only partly be attributed to its extracellular interaction with pMHC. When memory T cells are found to be “CD8 independent,” the result means that the cells nolonger depend on this extracellular interaction, and not that CD8 is no longer involved in activation of the cell.

[0086] The cell-cell adhesion function of CD8 can contribute to signaling independently of the interaction of CD8 with the MHC molecules that are within the signaling complex, presenting the targeted peptide to specific TCR. In one published example, transduction of an HL A-DR1 -restricted signal to a CD8+ T cell was decreased when the interaction of CD8 with irrelevant HLA class I on the target cell was blocked (M.H. Heemskerk et al., Proc. Natl. Acad. Sci. USA 98, (2001): 6806). In this case the interaction of CD8 with MHC-I presenting irrelevant peptide was sufficient to facilitate signal transduction.1. Class lb Single Chain Trimer Proteins Having High-Affinity CD8 Binding

[0087] In some embodiments, the provided single chain trimer protein includes a target peptide, a p2-microglobulin, and an MHC class lb a chain. At least one of the p2-microglobulin and the MHC class la a chain is a variant polypeptide, e.g., a variant of a naturally occurring polypeptide, such that the p2-microglobulin and / or the MHC class lb a chain have one or more mutations that result in the trimer having altered CD8 binding affinity. In some embodiments, mutations in the MHC class lb a chain of the provided single chain trimer include one or more amino acid substitutions. In some embodiments, the amino acid substitutions include one of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of one of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include two of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of two of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include three of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of three of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include four of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of four of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include five of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of five of 115E,219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include six of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of six of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include seven of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of seven of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include eight of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of eight of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include nine of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of nine of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include ten of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of ten of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include eleven of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of eleven of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions consist of 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, and 229E. In some embodiments, the amino acid substitutions include one of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions consist of one of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions include two of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions consist of two of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions include three of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions consist of three of 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions include 115E, Q219R, G223D, and H224Q. In some embodiments, the amino acid substitutions consist of 115E, Q219R, G223D, and H224Q.

[0088] Mutations engineered into the variant class lb a chain or p2-microglobulin of the provided single chain trimer provide the single chain trimer with an increased binding affinity for CD8. The single chain trimer can have a binding affinity dissociation constant that is, for example, between 100 pM and 1000 pM, e.g., between 100 pM and 640 pM, between 190 pM and 730 pM, between 280 pM and 820 pM, between 370 pM and 910 pM, or between 460 pM and 1000 pM. In terms of upper limits, the binding affinity dissociation constant of the single chain trimer can be, for example, less than 1000 pM, e.g., less than 910 pM, less than 820 pM, less than 730 pM, less than 640 pM, less than 550 pM, less than 460 pM, less than 370 pM, less than 280 pM, or less than 190 pM. In terms of lower limits, the binding affinity dissociation constant of the single chain trimer can be, for example, greater than 100 pM, e.g., greater than 190 pM, greater than 280 pM, greater than 370 pM, greater than 460 pM, greater than 550 pM, greater than 640 pM, greater than 730 pM, greater than 820 pM, or greater than 910 pM. Higher dissociation constants, e.g., greater than 1000 pM, and lower dissociation constants, e.g., less than 100 pM, are also contemplated.

[0089] In some embodiments, the single chain trimer protein further includes a first linker connecting the target peptide and the p2-microglobulin in the single chain trimer. In some embodiments, the first linker includes a first cysteine amino acid, and the variant MHC class la a chain comprises a second cysteine amino acid bonded to the first cysteine amino acid, such that the linker forms a disulfide trap within the single chain trimer protein. In some embodiments, the second cysteine amino acid is the amino acid substitution Y84C. A disulfide bond can in some embodiments improve the immunogenicity of the provided single chain trimer protein. A disulfide bond between the subunit segments of the protein can increase the stability of the protein and prevent it from unfolding or dissociating under harsh conditions, such as high temperatures, low pH, or denaturing agents. The disulfide trap can also help the protein fold correctly and form the desired structure, stabilizing a particular conformation or orientation of the subunit segments. The presence of a disulfide bond can also facilitate purification of the single chain trimer, as the disulfide can be captured using a thiol-specific resin or antibody.

[0090] In some embodiments, the single-chain trimer protein further includes a second linker connecting the p2-microglobulin and the MHC class lb a chain in the single chain trimer. In some embodiments, at least one of the first linker and the second linker is a (G4S)nlinker, where n is an integer from 1 to 10. In some embodiments, the first linker and the second linker are each independently a (G4S)nlinker. In some embodiments, at least one of the first linker andthe second linker is a (G4S)3 linker, i.e., GGGSGGGSGGGS. In some embodiments, the first linker and the second linker are each independently a (GrS)3 linker. In some embodiments, at least one of the first linker and the second linker is a (GrS)4 linker, i.e., GGGSGGGSGGGSGGGS. In some embodiments, the first linker and the second linker are each independently a (GrS)4 linker. In some embodiments, the first linker is a (G4S)3 linker and the second linker is a (GrS)4 linker.

[0091] In some embodiments, the MHC class lb a chain of the provided single chain trimer is an HLA-E a chain.

[0092] In some embodiments, the provided single chain trimer protein also includes an anti- CD8 scFv fragment. The presence of the anti-CD8 scFv fragment in the single chain trimer protein can further increase the CD8 binding affinity of the single chain trimer protein. In some embodiments, the anti-CD8 scFv fragment is inserted within the class lb a chain segment of the single chain trimer protein. In some embodiments, the anti-CD8 scFv fragment is inserted between the p2-microglobulin segment of the single chain trimer protein and the class lb a chain segment of the single chain trimer protein, and is optionally connected to these two segments via linkers, each of which can independently be, for example, a (G4S)nlinker, e.g., a (G4S)3 linker or a (G4S)4 linker.2. Class II Single Chain Trimer Proteins Having High-Affinity CD8 Binding

[0093] In some embodiments, the provided single chain trimer protein includes a target peptide, an a chain of a first MHC class II allele, and a P chain of a second MHC class II allele, where one or both of the a chain and the P chain are independently a variant chain altering the CD8 binding affinity of the single chain trimer protein. In some embodiments, the MHC class II a chain of the provided single chain trimer protein includes one or more amino acid substitutions. In some embodiments, some, or all of the a chain amino acid substitutions are substitutions to introduce the amino acid located at a corresponding position in an analogous MHC class la allele, e.g., a naturally occurring MHC class la allele, or in an analogous P2- microglobulin allele. In some embodiments, the MHC class II P chain of the provided single chain trimer includes one or more amino acid substitutions. In some embodiments, some or all of the P chain amino acid substitutions are substitutions to introduce the amino acid located at a corresponding position in an analogous MHC class la allele, e.g., a naturally occurring MHC class la allele. In some embodiments, one or all of the P chain amino acid substitutions is the substitution introducing a glutamic-acid residue at the position structurally corresponding toposition 115 in an analogous MHC class la allele, e.g., a naturally occurring MHC class la allele. In some embodiments, the a chain of the single chain trimer protein and the P chain of the single chain trimer protein originate from different MHC class II alleles. In some embodiments, the a chain of the single chain trimer protein and the P chain of the single chain trimer protein originate from the same MHC class II allele.

[0094] In some embodiments, the MHC class II a chain or p chain of the provided single chain trimer protein is a variant class II a chain engineered to provide the single chain trimer with an increased binding affinity for CD8. The single chain trimer can have a binding affinity dissociation constant that is, for example, between 100 pM and 1000 pM, e.g., between 100 pM and 640 pM, between 190 pM and 730 pM, between 280 pM and 820 pM, between 370 pM and 910 pM, or between 460 pM and 1000 pM. In terms of upper limits, the binding affinity dissociation constant of the single chain trimer can be, for example, less than 1000 pM, e.g., less than 910 pM, less than 820 pM, less than 730 pM, less than 640 pM, less than 550 pM, less than 460 pM, less than 370 pM, less than 280 pM, or less than 190 pM. In terms of lower limits, the binding affinity dissociation constant of the single chain trimer can be, for example, greater than 100 pM, e.g., greater than 190 pM, greater than 280 pM, greater than 370 pM, greater than 460 pM, greater than 550 pM, greater than 640 pM, greater than 730 pM, greater than 820 pM, or greater than 910 pM. Higher dissociation constants, e.g., greater than 1000 pM, and lower dissociation constants, e.g., less than 100 pM, are also contemplated.

[0095] In some embodiments, the target peptide segment of the provided single chain trimer protein is connected directly or via a linker to the MHC class II a chain segment of the single chain trimer. In some embodiments, the target peptide segment of the provided single chain trimer protein is connected directly or via a linker to both the MHC class II a chain segment of the single chain trimer and the MHC class II P chain segment of the single chain trimer. In some embodiments, the sub-domains of the MHC class II a chain (al and a2 sub-domains) and the MHC class II P chain (pi and P2 sub-domains) are arranged within the single chain trimer protein to produce orderings chosen from among the set consisting of al - a2 - pi - P2, al - a2 - P2 - pi, al - P2 - a2 - pi, P2 - al - a2 - pi, P2 - al - pi - a2, al - P2 - pi - a2, al -Pl - P2 - a2, al - pi - a2 - P2, pi - al - a2 - P2, pi - al - P2 - a2, pi - P2 - al - a2, P2 -Pl - al - a2, P2 - pi - a2 - al, pi - P2 - a2 - al, pi - a2 - P2 - al, pi - a2 - al - P2, a2 -Pl - al - P2, a2 - pi - P2 - al, a2 - P2 - pi - al, P2 - a2 - pi - al, P2 - a2 - al - pi, a2 -P2 - al - pi, a2 - al - P2 - pi, and a2 - al - pi - P2. In some embodiments, the one or more optional linkers of the single chain peptide are each independently a (G4S)nlinker. In someembodiments, at least one of the one or more optional linkers is a (G4S)3 linker, i.e., GGGSGGGSGGGS. In some embodiments, the one or more optional linkers are each independently a (G4S)3 linker. In some embodiments, at least one of the one of the one or more optional linkers is a (G4S)4 linker, i.e., GGGSGGGSGGGSGGGS. In some embodiments, the one or more optional linkers are each independently a (G4S)4 linker. In some embodiments, at least one of the one of the one or more optional linkers is a (G4S)3 linker and at least one of the one of the one or more optional linkers is a (G4S)4 linker.

[0096] In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR1 allele, where the HLA-DR1 serotype / antigen comprises HLA-DRA with HLA-DRB 1*01 :01, HLA-DRB 1*01 :02, and HLA-DRB 1*01 :03. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR3 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR4 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR7 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR8 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR9 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR10 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR11 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR12 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR13 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR14 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR15 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR16 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DP allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DPA1 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DPB1 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ allele. In some embodiments,one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ2 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ4 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ5 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ6 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ7 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ8 allele. In some embodiments, one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DQ9 allele.

[0097] In some embodiments, the provided single chain trimer protein also includes an anti- CD8 scFv fragment. The presence of the anti-CD8 scFv fragment in the single chain trimer protein can further increase the CD8 binding affinity of the single chain trimer protein. In some embodiments, the anti-CD8 scFv fragment is inserted within the class II P chain segment of the single chain trimer protein. In some embodiments, the anti-CD8 scFv fragment is inserted between the class II P chain segment of the single chain trimer protein and the class II a chain segment of the single chain trimer protein, and is optionally connected to these two segments via linkers, each of which can independently be, for example, a (G4S)nlinker, e.g., a (G4S)3 linker or a (G4S)4 linker. In some embodiments, the anti-CD8 scFv fragment is inserted between sub-domains of the MHC class II a and / or p chains (al, a2, pi, and P2 sub-domains), and is optionally connected to these sub-domains via linkers, each of which can independently be, for example, a (G4S)nlinker, e.g., a (G4S)3 linker or a (G4S)4 linker.V. Systems With Single Chain Trimer Proteins and Accessory CD8 Binding Partners

[0098] In another aspect, the present disclosure provides various systems that include a class lb or II single chain trimer protein, and a separately expressed accessory CD8 binding protein. The presence of the accessory CD8 binding protein in the provided system allows for improved binding to CD8 beyond that achieved with only the class lb or class II single chain trimer protein. In some embodiments, the system includes a class lb single chain trimer protein as disclosed herein. In some embodiments, the system includes a class II single chain trimer protein as disclosed herein. In some embodiments, the accessory CD8 binding partner of the provided system includes an anti-CD8 scFv fragment. Typically, the accessory CD8 binding protein is a membrane protein that further includes a transmembrane domain. In someembodiments, the transmembrane domain is a receptor transmembrane domain. In some embodiments, the transmembrane domain is a platelet-derived growth factor receptor transmembrane domain. In some embodiments, the anti-CD8 scFv fragment of the accessory binding protein is connected to the transmembrane domain of the accessory binding protein via a linker. In some embodiments, the linker is a (G4S)n linker, e.g., a (G4S)3 linker or a (G4S)4 linker.VI. Polynucleotides and Cells

[0099] In another aspect, the present disclosure provides recombinant polynucleotides, e.g., isolated nucleic acids, encoding any of the single chain trimer proteins as described herein; vectors including such nucleic acids, and host cells into which the single chain trimer proteins, recombinant polynucleotides, or vectors are introduced.

[0100] In some embodiments, the provided recombinant polynucleotide is a vector that optionally includes, in addition to a nucleic acid encoding a disclosed single chain trimer protein, a promoter regulating expression of this nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. For certain applications, an inducible promoter can provide advantages relative to a constitutive promoter. For example, the constitutive expression of a provided single chain trimer protein in a cell can in certain circumstances have negative effects on the development of the cell . In such cases, the use of an inducible promoter can be beneficial.

[0101] A wide variety of cell types are suitable for use as the provided host cell. In some embodiments, the host cell is an immune cell, including any cell that is involved in an immune response. In some embodiments, the provided cell is an antigen-presenting cell (APC), for example a dendritic cell, a macrophage, or a B cell. Other cells suitable for including a single chain trimer protein, recombinant polynucleotide, or system as disclosed herein include, but are not limited to, epithelial cells, endothelial cells, mast cells, neurons, oligodendrocytes, Swann cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, hepatocytes, pancreatic beta cells, kidney cells, lung cells, skin cells, intestinal cells, or adipocytes.

[0102] In some embodiments, the host immune cell type includes granulocytes such as basophils, eosinophils, and neutrophils; mast cells; phagocytes such as monocytes which can develop into macrophages; antigen-presenting cells such as dendritic cells; and lymphocytes such as natural killer cells (NK cells), B cells, T cells, and innate lymphoid cells (ILC). In someembodiments, the host immune cell is an immune effector cell. An immune effector cell is an immune cell that can perform a specific function in response to a stimulus. In some embodiments, the host immune cell is a lymphocyte. In some embodiments, the lymphocyte is a NK cell.

[0103] In some embodiments, the host cell is a stem cell. The host cell can be, for example, an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), an adult stem cell, or a mesenchymal stem cell (MSC). In some embodiments, the host cell is a progenitor cell. The host cell can be, for example, a neural progenitor cell, a skeletal progenitor cell, a muscle progenitor cell, a fat progenitor cell, a heart progenitor cell, a chondrocyte, a fibroblast cell, or a pancreatic progenitor cell. The host cell can be, for example, a progenitor-differentiated cell, a stem cell-differentiated cell, an organoid, or an assembloid.

[0104] In another aspect, a population of host cells is provided. Each host cell of the population independently includes a single chain trimer protein as disclosed herein, a recombinant polynucleotide as disclosed herein, or a system as disclosed herein.VII. Pharmaceutical Compositions

[0105] In another aspect, the present disclosure provides pharmaceutical compositions including one or more pharmaceutically acceptable carriers, diluents, excipients, or buffers and one or more of the single chain trimers, recombinant polynucleotides, or cells provided herein. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a subject, for example, a human. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. The preparation of pharmaceutically acceptable carriers and excipients is described in, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Loyd V. Allen et al, editors, Pharmaceutical Press (2012).

[0106] In some embodiments, the composition also includes an additional active compound or other chemotherapeutic agent. In some embodiments, the pharmaceutical composition further includes one or more stabilizing compounds, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. In some embodiments, the pharmaceutical compositions also contain a pharmaceutically acceptable salt. Pharmaceutically acceptable salts can include, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like;and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in the provided pharmaceutical compositions. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization.

[0107] In some embodiments, the provided pharmaceutical composition is a vaccine. In some embodiments, the provided pharmaceutical composition includes recombinant DNA encoding a single chain trimer as disclosed herein. In some embodiments, the recombinant DNA of the pharmaceutical composition is a recombinant adenovirus vector. In some embodiments, the provided pharmaceutical composition includes recombinant mRNA. In some embodiments, the provided pharmaceutical composition further includes a lipid nanoparticle.VIII. Methods for Inducing an Immune Response or Treating a Health Condition

[0108] In another aspect, the present disclosure provides a method of inducing an immune response or treating a health condition or disorder. The method includes administering to a subject in need of such an immune response or treatment a therapeutically effective amount of a single chain trimer, polynucleotide, cell, or pharmaceutical composition disclosed herein, thereby treating the disorder. In some embodiments, the administration is given with a curative intent. In some embodiments, the administration is given with an aim to prolong the life of the subject. In some embodiments, the administration is given for the purpose of reducing symptoms associated with the disorder. In some embodiments, the treatment is given prophylactically to a subject.

[0109] The advantageous benefits of the provided materials and methods result at least in part from the population-wide rarity of the T cell responses that can be induced. For example, conventional vaccines elicit T-cell responses that are similar to those naturally generated in the course of HIV infection, differing primarily in the quantity or timing of the response. The vaccines disclosed herein will elicit unique T-cell responses, qualitatively different from those that the virus has evolved to confront. In prophylactic vaccination, the vaccines allow expansion of such unique T cells offering advantages for the prevention and / or mitigation of various infectious diseases. In the setting of therapeutic HIV vaccination, in contrast, there is some indication in the SIV model that alternative MHC restriction alone is insufficient to confer the capacity for post-treatment control (PTC) (A.A. Okoye et al., Nat. Med. 24, (2018): 1430). Nonetheless, class I-restricted CTL do contribute to PTC (L. Bailon et al., Top. Antivir. Med.29, (2021):48), so vaccines inducing outgrowth of an entirely new repertoire of high-affinity cells represent a significant contribution. Accordingly, the single chain trimers with altered CD8 binding disclosed herein provide notable advantages for post-treatment virus control.

[0110] In some embodiments, the provided method further includes, subsequent to administering a first single chain trimer, recombinant polynucleotide, cell, or pharmaceutical composition disclosed herein, administering a second single chain trimer, polynucleotide, cell, or pharmaceutical composition disclosed herein. In some embodiments, the first administration includes a single chain trimer having different CD8 binding affinity than that of a single chain trimer included in the second administration, thereby taking advantage of graded CD8 affinity to provide advantageous effects in inducing an immune response or treating a health condition or disorder. SCT-based immunogens that use graded CD8 affinity can be used to elicit waves of CTL having different avidities. For example, by using a range of immunogens developed according to provided embodiments, SCTs without CD8 binding can be administered first, to initially favor the highest-avidity clones, followed by SCTs with reduced CD8 binding to expand these high-avidity clones and recruit new clonotypes of intermediate avidity, followed finally by immunization with fully intact SCTs that will allow expansion of lower-avidity cells only after the higher-avidity cells are well established. In some embodiments, the first administration is of a recombinant polynucleotide, cell, or pharmaceutical composition including DNA encoding a provided single chain trimer protein. In some embodiments, the second administration is of a recombinant polynucleotide, cell, or pharmaceutical composition including mRNA encoding a provided single chain trimer protein. In other embodiments, the first administration can include mRNA encoding a provided single chain trimer protein, and the second administration can include DNA encoding a provided single chain trimer protein. In still further embodiments, both the first and second administrations can include DNA encoding a provided single chain trimer protein, or both can include mRNA encoding a provided single chain trimer protein.[OHl] In some embodiments, the provided method includes administering a first single chain trimer, polynucleotide, cell, or pharmaceutical composition including an MHC class la a chain having extremely low or no CD8 binding, e.g., having a CD8 binding affinity dissociation constant that is greater than 1 mM, e.g., greater than 1.3 mM, greater than 1.6 mM, greater than 2 mM, greater than 2.5 mM, greater than 3.2 mM, greater than 4 mM, greater than 5 mM, greater than 6.3 mM, greater than 7.9 mM, or greater than 10 mM. In some embodiments, the MHC class la a chain of the first administration includes at least one of the amino acidsubstitutions D227K and T228A. In some embodiments, the MHC class la a chain of the first administration includes the amino acid substitution D227K. In some embodiments, the MHC class la a chain of the first administration includes the amino acid substitution T228A. In some embodiments, the MHC class la a chain of the first administration includes the amino acid substitutions D227K and T228A.

[0112] In some embodiments, the provided method includes subsequently administering a second single chain trimer, polynucleotide, cell, or pharmaceutical composition including an MHC class la a chain having partially reduced CD8 binding, e.g., having a CD8 binding affinity dissociation constant that is between 300 pM and 1000 pM, e.g., between 300 pM and 720 pM, between 370 pM and 790 pM, between 440 pM and 860 pM, between 510 pM and 930 pM, or between 580 pM and 1000 pM. In some embodiments. In some embodiments, the MHC class la a chain of the second administration includes the amino acid substitution A245 V. In alternative embodiments, the MHC class la a chain of the second administration is a naturally occurring MHC class la a chain.IX. Exemplary Embodiments

[0113] The following embodiments are contemplated. All combinations of features and embodiments are contemplated. All amino acid positions in the following embodiments refer to positions in wild-type polypeptides (e.g., a chains or p chains) when expressed alone, i.e., not as part of a single chain trimer protein.

[0114] Embodiment 1 : A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a recombinant polynucleotide including a nucleic acid sequence encoding a single chain trimer protein, the single chain trimer protein comprising a target peptide, p2-microglobulin, and an MHC class la a chain, wherein: at least one of the P2- microglobulin and the MHC class la a chain is independently a variant of a naturally occurring polypeptide, and the single chain trimer protein has a CD8 binding affinity dissociation constant greater than 300 pM.

[0115] Embodiment 2: An embodiment of embodiment 1, wherein the pharmaceutical composition is a vaccine.

[0116] Embodiment 3: An embodiment of embodiment 1 or 2, wherein the recombinant polynucleotide comprises DNA.

[0117] Embodiment 4: An embodiment of embodiment 3, wherein the recombinant polynucleotide is a recombinant adenovirus vector.

[0118] Embodiment 5: An embodiment of embodiment 1 or 2, wherein the recombinant polynucleotide comprises mRNA.

[0119] Embodiment 6: An embodiment of any embodiment of embodiments 1-5, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0120] Embodiment 7: An embodiment of any embodiment of embodiments 1-6, wherein the MHC class la a chain is a variant MHC class la a chain comprising an amino acid substitution that is 115A, 122A, 128A, 245V, 227K, 228A, or 115E.

[0121] Embodiment 8: An embodiment of embodiment 7, wherein the variant MHC class la a chain comprises an amino acid substitution that is QI 15A, D122A, E128A, A245V, D227K, T228A, or Q115E.

[0122] Embodiment 9: An embodiment of any embodiment of embodiments 1-8, wherein the P2-microglobulin is a variant p2-microglobulin comprising an amino acid substitution that is 58E or 58S.

[0123] Embodiment 10: An embodiment of embodiment 9, wherein the variant P2- microglobulin comprises an amino acid substitution that is K48E or K58S.

[0124] Embodiment 11 : An embodiment of any embodiment of embodiments 1-10, wherein the single chain trimer protein further comprises a first linker connecting the target peptide and the P2-microglobulin.

[0125] Embodiment 12: An embodiment of embodiment 11, wherein the first linker comprises a first cysteine amino acid, and the MHC class la a chain comprises a second cysteine amino acid bonded to the first cysteine amino acid.

[0126] Embodiment 13: An embodiment of embodiment 12, wherein the second cysteine amino acid is the amino acid substitution Y84C.

[0127] Embodiment 14: An embodiment of embodiment 11, wherein the first linker has the amino acid sequence: GGGGSGGGGSGGGGS.

[0128] Embodiment 15: An embodiment of any embodiment of embodiments 1-14, wherein the single chain trimer protein further comprises a second linker connecting the P2- microglobulin and the MHC class la a chain.

[0129] Embodiment 16: An embodiment of embodiment 15, wherein the second linker has the amino acid sequence: GGGGSGGGGSGGGGSGGGGS.

[0130] Embodiment 17: An embodiment of any embodiment of embodiments 1-16, wherein the MHC class la a chain is an HLA-A*02 a chain or an HLA-B*27 a chain.

[0131] Embodiment 18: A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any embodiment of embodiments 1-17.

[0132] Embodiment 19: An embodiment of embodiment 18, wherein the pharmaceutical composition is a first pharmaceutical composition; the p2-microglobulin is a first P2- microglobulin; the MHC class la a chain is a first MHC class la a chain; the CD8 binding affinity dissociation constant is a first CD8 binding affinity dissociation constant; the method further comprises, subsequent to administering the first pharmaceutical composition, administering to the subject a second pharmaceutical composition comprising a second recombinant polynucleotide including a second nucleic acid sequence encoding a second single chain trimer protein; the second single chain trimer protein comprises an additional copy of the target peptide, a second p2-microglobulin, and a second MHC class la a chain; and the second single chain trimer protein has a second CD8 binding affinity dissociation constant less than the first CD8 binding affinity dissociation constant.

[0133] Embodiment 20: An embodiment of embodiment 19, wherein the first CD8 binding affinity dissociation constant is greater than 1,000 pM.

[0134] Embodiment 21 : An embodiment of embodiment 19 or 20, wherein the first MHC class la a chain comprises an amino acid substitution that is D227K and T228A.

[0135] Embodiment 22: An embodiment of any embodiment of embodiments 19-21, wherein the second MHC class la a chain comprises the amino acid substitution A245V.

[0136] Embodiment 23 : An embodiment of any embodiment of embodiments 19-21, wherein the second MHC class la a chain and the second p2-microglobulin are each independently naturally occurring polypeptides.

[0137] Embodiment 24: An embodiment of any embodiment of embodiments 19-23, wherein the recombinant polynucleotide of the first pharmaceutical composition comprises DNA, and the recombinant polynucleotide of the second pharmaceutical composition comprises mRNA.

[0138] Embodiment 25: An embodiment of any embodiment of embodiments 19-24, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0139] Embodiment 26: An embodiment of any embodiment of embodiments 19-25, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0140] Embodiment 27: A single chain trimer protein comprising a target peptide, a P2- microglobulin, and a MHC class lb a chain, wherein: at least one of the p2-microglobulin and the MHC class lb a chain is independently a variant of a naturally occurring polypeptide, and the single chain trimer protein has a CD8 binding affinity dissociation constant less than 1000 pM.

[0141] Embodiment 28: An embodiment of embodiment 27, wherein the MHC class lb a chain is a variant MHC class lb a chain comprising an amino acid substitution in the amino acid position 115, or the amino acid positions from 219 to 229.

[0142] Embodiment 29: An embodiment of embodiment 28, wherein the MHC class lb a chain comprises an amino acid substitution that is 115E, 219R, 220D, 221G, 222E, 223D, 224Q, 225T, 226Q, 227D, 228T, or 229E.

[0143] Embodiment 30: An embodiment of embodiment 29, wherein the MHC class lb a chain comprises an amino acid substitution that is Q219R, G223D, or H224Q.

[0144] Embodiment 31 : An embodiment of any embodiment of embodiments 27-30, wherein the variant MHC class lb a chain is a variant HLA-E a chain.

[0145] Embodiment 32: A recombinant polynucleotide comprising a nucleic acid sequence encoding the single chain trimer protein of any embodiment of embodiments 27-31.

[0146] Embodiment 33 : A cell comprising the single chain trimer protein of any embodiment of embodiments 27-31 or the recombinant polynucleotide of claim 34.

[0147] Embodiment 34: A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the single chain trimer protein of any embodiment of embodiments 27-31, the recombinant polynucleotide of embodiment 32, or the cell of embodiment 33.

[0148] Embodiment 35: An embodiment of embodiment 34, wherein the pharmaceutical composition is a vaccine.

[0149] Embodiment 36: An embodiment of embodiment 34 or 35, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 30, and the recombinant polynucleotide comprises DNA.

[0150] Embodiment 37: An embodiment of embodiment 35, wherein the recombinant polynucleotide is a recombinant adenovirus vector.

[0151] Embodiment 38: An embodiment of embodiment 34 or 35, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 32, and the recombinant polynucleotide comprises mRNA.

[0152] Embodiment 39: An embodiment of any embodiment of embodiments 34-38, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0153] Embodiment 40: A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the single chain trimer protein of any embodiment of embodiments 27-31, the recombinant polynucleotide of embodiment 32, the cell of embodiment 33, or the pharmaceutical composition of any embodiment of embodiments 34-39.

[0154] Embodiment 41 : An embodiment of embodiment 40, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0155] Embodiment 42: An embodiment of embodiment 40 or 41, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0156] Embodiment 43 : A single chain trimer protein comprising a target peptide, an a chain of a first MHC class II allele, and a P chain of a second MHC class II allele, wherein at least of the a chain and the P chain is independently a variant of a naturally occurring polypeptide, andthe single chain trimer protein has a CD8 binding affinity dissociation constant less than 1000 pM.

[0157] Embodiment 44: An embodiment of embodiment 43, wherein the a chain is a variant a chain comprising an amino acid substitution from an amino acid at a position of the first MHC class II allele to an amino acid at a corresponding position of a first naturally occurring MHC class la allele or of a p2-microglobulin.

[0158] Embodiment 45: An embodiment of embodiment 43 or 44, wherein the P chain is a variant P chain comprising an amino acid substitution from an amino acid at a position of the second MHC class II allele to an amino acid at a corresponding position of a second naturally occurring MHC class la allele.

[0159] Embodiment 46: An embodiment of embodiment 43, wherein the second MHC class II allele is the first MHC class II allele.

[0160] Embodiment 47: An embodiment of embodiment 43, wherein one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR allele.

[0161] Embodiment 48: A recombinant polynucleotide comprising a nucleic acid sequence encoding the single chain trimer protein of any embodiment of embodiments 43-47.

[0162] Embodiment 49: A cell comprising the single chain trimer protein of any embodiment of embodiments 43-47 or the recombinant polynucleotide of embodiment 48.

[0163] Embodiment 50: A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the single chain trimer protein of any embodiment of embodiments 43-47, the recombinant polynucleotide of embodiment 48, or the cell of embodiment 49.

[0164] Embodiment 51 : An embodiment of embodiment 50, wherein the pharmaceutical composition is a vaccine.

[0165] Embodiment 52: An embodiment of embodiment 50 or 51, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 48, and the recombinant polynucleotide comprises DNA.

[0166] Embodiment 53: An embodiment of embodiment 52, wherein the recombinant polynucleotide is a recombinant adenovirus vector.

[0167] Embodiment 54: An embodiment of embodiment 50 or 51, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 48, and the recombinant polynucleotide comprises mRNA.

[0168] Embodiment 55: An embodiment of any embodiment of embodiments 50-54, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0169] Embodiment 56: A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the single chain trimer protein of any embodiment of embodiments 43-47, the recombinant polynucleotide of embodiment 48, the cell of embodiment 49, or the pharmaceutical composition of any embodiment of embodiments 50-55.

[0170] Embodiment 57: An embodiment of embodiment 56, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0171] Embodiment 58: An embodiment of embodiment 56-57, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0172] Embodiment 59: A single chain trimer protein comprising a target peptide, P2- microglobulin, an MHC class lb a chain, and an anti-CD8 scFv fragment.

[0173] Embodiment 60: An embodiment of embodiment 59, wherein the MHC class lb a chain is a variant MHC class lb a chain comprising the anti-CD8 scFv fragment.

[0174] Embodiment 61 : An embodiment of embodiment 59, wherein the anti-CD8 scFv fragment is located between the p2-microglobulin and the MHC class lb a chain.

[0175] Embodiment 62: An embodiment of any embodiment of embodiments 59-61, wherein the MHC class lb a chain is an HLA-E a chain.

[0176] Embodiment 63: A recombinant polynucleotide comprising a nucleic acid sequence encoding the single chain trimer protein of any embodiment of embodiments 59-62.

[0177] Embodiment 64: A cell comprising the single chain trimer protein of any embodiments of embodiments 59-62 or the recombinant polynucleotide of embodiment 63.

[0178] Embodiment 65: A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the single chain trimer protein of any embodiment of embodiments 59-62, the recombinant polynucleotide of embodiment 63, or the cell of embodiment 64.

[0179] Embodiment 66: An embodiment of embodiment 65, wherein the pharmaceutical composition is a vaccine.

[0180] Embodiment 67: An embodiment of embodiment 65 or 66, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 63, and the recombinant polynucleotide comprises DNA.

[0181] Embodiment 68: An embodiment of embodiment 67, wherein the recombinant polynucleotide is a recombinant adenovirus vector.

[0182] Embodiment 69: An embodiment of embodiment 65 or 66, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 63, and the recombinant polynucleotide comprises mRNA.

[0183] Embodiment 70: An embodiment of any embodiment of embodiments 65-69, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0184] Embodiment 71 : A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the single chain trimer protein of any embodiment of embodiments 59-62, the recombinant polynucleotide of embodiment 63, the cell of embodiment 64, or the pharmaceutical composition of any embodiment of embodiments 65-69.

[0185] Embodiment 72: An embodiment of embodiment 71, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0186] Embodiment 73: An embodiment of embodiment 71 or 72, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0187] Embodiment 74: A single chain trimer protein comprising a target peptide, an a chain of a first MHC class II allele, a P chain of a second MHC class II allele, and an anti-CD8 scFv fragment.

[0188] Embodiment 75: The single chain trimer protein of embodiment 74, wherein the P chain is a variant P chain comprising the anti-CD8 scFv fragment.

[0189] Embodiment 76: An embodiment of embodiment 74, wherein the anti-CD8 scFv fragment is located between the P chain and the a chain.

[0190] Embodiment 77: An embodiment of any embodiment of embodiments 74-76, wherein the second MHC class II allele is the first MHC class II allele.

[0191] Embodiment 78: An embodiment of any embodiment of embodiments 74-76, wherein one or both of the first MHC class II allele and the second MHC class II allele is an HLA-DR allele.

[0192] Embodiment 79: A recombinant polynucleotide comprising a nucleic acid sequence encoding the single chain trimer protein of any embodiment of embodiments 74-78.

[0193] Embodiment 80: A cell comprising the single chain trimer protein of any embodiment of embodiments 74-78 or the recombinant polynucleotide of embodiment 79.

[0194] Embodiment 81 : A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the single chain trimer protein of any embodiment of embodiments 74-78, the recombinant polynucleotide of embodiment 79, or the cell of embodiment 80.

[0195] Embodiment 82: An embodiment of embodiment 81, wherein the pharmaceutical composition is a vaccine.

[0196] Embodiment 83: An embodiment of embodiment 81 or 82, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 79, and the recombinant polynucleotide comprises DNA.

[0197] Embodiment 84: An embodiment of embodiment 83, wherein the recombinant polynucleotide is a recombinant adenovirus vector.

[0198] Embodiment 85: An embodiment of embodiment 82 or 83, wherein the pharmaceutical composition comprises the recombinant polynucleotide of embodiment 79, and the recombinant polynucleotide comprises mRNA.

[0199] Embodiment 86: An embodiment of any embodiment of embodiments 81-85, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0200] Embodiment 87: A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the single chain trimer protein of any embodiment of embodiments 74-78, the recombinant polynucleotide of embodiment 79, the cell of embodiment 80, or the pharmaceutical composition of any embodiment of embodiments 81-86.

[0201] Embodiment 88: An embodiment of embodiment 87, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0202] Embodiment 89: An embodiment of embodiment 87 or 88, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0203] Embodiment 90: A system comprising: a single chain trimer protein comprising a target peptide, an MHC a chain, and an MHC P chain; and an accessory CD8 binder protein comprising an anti-CD8 scFv fragment and a transmembrane domain.

[0204] Embodiment 91 : An embodiment of embodiment 90, wherein the MHC a chain is an MHC class lb a chain, and the MHC P chain is P2-microglobulin.

[0205] Embodiment 92: An embodiment of embodiment 90, wherein the MHC a chain is an MHC class II a chain, and the MHC P chain is an MHC class II P chain.

[0206] Embodiment 93 : An embodiment of any embodiment of embodiments 90-92, wherein the single chain trimer protein further comprises a linker connecting the anti-CD8 scFv fragment and the transmembrane domain.

[0207] Embodiment 94: An embodiment of embodiment 93, wherein the linker has the amino acid sequence: GGGGSGGGGSGGGGS.

[0208] Embodiment 95: A system comprising: a first recombinant polynucleotide comprising a first nucleic acid sequence encoding a single chain trimer protein, wherein the single chain trimer protein comprises a target peptide, an MHC a chain, and an MHC P chain; and a second recombinant polynucleotide comprising a second nucleic acid sequence encoding an accessory CD8 binder protein comprising an anti-CD8 scFv fragment and a transmembrane domain.

[0209] Embodiment 96: An embodiment of embodiment 95, wherein the MHC a chain is an MHC class lb a chain, and the MHC P chain is P2-microglobulin.

[0210] Embodiment 97: An embodiment of embodiment 95, wherein the MHC a chain is an MHC class II a chain, and the MHC P chain is an MHC class II P chain.

[0211] Embodiment 98: An embodiment of any embodiment of embodiments 95-97, wherein the single chain trimer protein further comprises a linker connecting the anti-CD8 scFv fragment and the transmembrane domain.

[0212] Embodiment 99: An embodiment of embodiment 98, wherein the linker has the amino acid sequence: GGGGSGGGGSGGGGS.

[0213] Embodiment 100: A cell comprising the system of any embodiment of embodiments 90-99.

[0214] Embodiment 101 : A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the system of any embodiment of embodiments 90-99, or the cell of embodiment 100.

[0215] Embodiment 102: An embodiment of embodiment 101, wherein the pharmaceutical composition is a vaccine.

[0216] Embodiment 103: An embodiment of embodiment 101 or 102, wherein the pharmaceutical composition comprises the system of any embodiment of embodiments 95-99, and the first recombinant polynucleotide and the second recombinant polynucleotide each independently comprise DNA.

[0217] Embodiment 104: An embodiment of embodiment 103, wherein at least one of the first recombinant polynucleotide and the second recombinant polynucleotide is a recombinant adenovirus vector.

[0218] Embodiment 105: An embodiment of embodiment 101 or 102, wherein the pharmaceutical composition comprises the system of any embodiment of embodiments 95-99, and the first recombinant polynucleotide and the second recombinant polynucleotide each independently comprise mRNA.

[0219] Embodiment 106: An embodiment of any embodiment of embodiments 101-105, wherein the pharmaceutical composition further comprises a lipid nanoparticle.

[0220] Embodiment 107: A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the system of any embodiment of embodiments 90-99, the cell of embodiment 100, or the pharmaceutical composition of any embodiment of embodiments 101-106.

[0221] Embodiment 108: An embodiment of embodiment 107, wherein the method further comprises administering to the subject an additional pharmaceutical composition comprising the target peptide.

[0222] Embodiment 109: An embodiment of embodiment 107 or 108, wherein the method further comprises administering to the subject a further pharmaceutical composition comprising a heteroclitic variant of the target peptide.

[0223] Embodiment 110: An embodiment of embodiment 109, wherein the method further comprises administering to the subject the pharmaceutical composition of any embodiment of embodiments 34-39, 50-55, and 65-70.

[0224] Embodiment 111 : An embodiment of embodiment 110, wherein the administering of the pharmaceutical composition of any embodiment of embodiments 101-106 occurs subsequent to the administering of the pharmaceutical composition of any embodiment of embodiments 34-39, 50-55, and 65-70.EXAMPLES

[0225] The present disclosure will be better understood in view of the following non-limiting examples. The following examples are intended for illustrative purposes only and do not limit in any way the scope of the present disclosure.Example 1. T-cell repertoire and TCR properties after vaccination with Class- SCTs lacking CD8 binding or having reduced binding

[0226] Experiments are conducted by iterating on a platform of SIV-relevant SCTs that has been proven immunogenic in Mamu-B*08+ or -A*02+ macaques. New versions of the SCTs are created with altered CD8 binding, as confirmed by surface plasmon resonance (SPR). These SCTs are then engineered into Ad26 and mRNA / lipid nanoparticle (LNP) formats before administration to macaques. The TCR repertoire and its peptide avidity are followed during the vaccination regimen and afterward. Finally, vaccine regimens are tested that incorporateimmunogens with “graded” CD8 binding for the greatest potential to elicit and maintain a high frequency of high-avidity T cells having a broad TCR repertoire.

[0227] Mamu-B*08 is a classical class la allele associated with viral load control that has extraordinarily similar peptide-binding specificity to HLA-B27 (B.R. Mothe et al., Immunogenetics 65, (2013): 371). A successful SCT vaccine based onMamu-B*08 is therefore translatable to HLA-B27+ people. Since HLA-B27 and Mamu-B*08 are protective alleles, Mamu-A*02 is the second Mamu allele chosen for these analyses. Mamu-A*02 is a very common allele not associated with control, but capable of targeting epitopes in regions proven important to viral fitness. The most important of these is probably GY9, which overlaps with a vulnerable region including the human HLA-A2-restricted peptide, SL9 (P.J. Goulderet al., Nat. Med. 3, (1997): 212; A.K. Iversen et al., Nat. Immunol., (2006): 179; P.K. Kiepiela et al., Nat. Med. 13, (2007): 46). More important than these specific commonalities, however, is the idea that flexible mRNA vaccine technology makes it possible to apply these same ideas to develop effective vaccines for people inheriting many different HLA alleles, and to explore HL A supertype-based approaches to cover even larger proportions of human HLA genetics (J. Sidney, B. Peters, N. Frahm, C. Brander & A. Sette, BMC Immunol., (2008): 1). Overcoming HLA diversity is further facilitated by the observation that even single TCRs can target epitopes presented by several supertype alleles, thus making supertype-based applications feasible (S.C. Threlkeld et al., J. of Immunol. 159, (1997): 1648).

[0228] For each of the two Mamu MHC class la alleles, four SCTs are combined (Table 1) to create bespoke vaccines eliciting immune responses against epitopes covering sites with high importance for viral fitness. Efficacy of these highly targeted vaccines is compared to previous results obtained using full-length Gag / Env vaccines producing a preponderance of class I- restricted responses.Table 1. Mamu class la SCT vaccinesAllele or protein Epitope Evidence for fitness contribution Human relevanceMamu-A*02Gag GSENLKSLY low viral load, mutation, reversionGY9 overlaps SL9 (HLA-A2),Gag LSEGCTPY low viral load, mutation so success could be the basisPol FSIPLDEEF low viral load, mutation for an HLA-A2 vaccineNef YTYEAYVRY reversionMamu-B*08Gag YRRWIQLGL low viral load, mutation, fitness defectGag RQQNPIPVGNI low viral load, mutation Mamu-B*08 and HLA-B27Pol GRWPITHLH low viral load, mutation bind similar peptidesNef RRRLTARGLL reversion

[0229] Untrapped SCTs are created by fusion of the coding sequences for the signal sequence of P2-microglobulin, the target peptide, a 15-amino acid (G4S)3 linker, p2-microglobulin itself, a 20-amino acid (G4S)4 linker, and the MHC-I allele carrying a Y84A mutation. Disulfide- trapped SCTs are similar to untrapped SCTs except that the linker following the peptide includes a cysteine residue that can bond with a Y84C mutation in the MHC sequence. This mutation can improve immunogenicity. The sequence of a disulfide-trapped GY9-Mamu-A*02 SCT is given below as SEQ ID NO: 1.

[0230] A first series of experiments tests if class la SCTs with reduced CD8 binding or no binding, due to the MHC-I mutations mentioned above, can preferentially recruit CD8+ T cells bearing TCRs with high affinity for pMHC, and if memory cells derived from these primed cells have high functional avidity and efficacy against SIV. Additional experiments test if these immunogens can be used in sequence to subvert normal immunodominance patterns by expanding clonotypes of various TCR affinities in waves. All SCTs in Table 1 are prepared with either A245V (CD8inT-weak) or D227K / T228A (CD8inT-zero) mutations (FIG. 3). These versions of the GY9-Mamu-A*02 SCT, for example, are named GY9-Mamu- A*02[A245V] (CD8inT-weak version, SEQ ID NO: 2) and GY9-Mamu- A*02[D227K / T228A] (CD8inT-zero version, SEQ ID NO: 3).

[0231] Recent published data show that priming immunizations with mRNA leads to robust antibody responses but surprisingly poor T-cell responses, when compared to priming with DNA or adenovirus (Ad5 or Ad26) (A. Valentin et al., Front. Immunol. 13, (2022): 945706). mRNA vaccines, however, do provide a very potent boost for T-cell responses alreadyestablished by DNA or adenovirus. Accordingly, for immunogenicity experiments (Table 2), DNA priming is used, followed and mRNA / LNP boosting. For graded-vaccination experiments (Table 3), Ad26 priming is used, followed by two mRNA / LNP boosts.Table 2. Immunogenicity groups (DNA>mRNA; n=4 / group)""l A Mamu-A*02 SCTsIB Mamu-A*02 CD8inT-weak1C Mamu-A*02 CD8inT-zeroID Mamu-B*08 SCTsIE Mamu-B*08 CD8inT-weakIF Mamu-B*08 CD8inT-zeroTable 3. CD8-graded vaccines (Ad26>mRNA>mRNA; n=6)Mamu-A*02 SCTs x 32B zero > weak > SCTs2C SCTs > weak > zero2D Optimal CD8inT x 3

[0232] The immunogenicity experiments (Table 2) test the function of CD8inT immunogens using small numbers of macaques. The data from these immunizations show if CD8inT-weak or CD8inT-zero immunogens can elicit T cells of markedly higher affinity for pMHC and also of higher functional avidity. DNA priming and mRNA boosting were chosen to allow some animals to be re-used in subsequent vaccine experiments, because the nucleic-acid vaccines do not elicit anti-vector neutralizing antibodies.

[0233] The graded-vaccination experiments (Table 3) test if Mamu-A*02-based immunogens having different levels of CD8 engagement can be used sequentially to elicit different waves of responding clonotypes, thereby broadening the response at the clonotype level and perhaps strengthening it as well. The rationale is that indiscriminate recruitment of naive T cells (by an SCT with full CD8 binding) elicits a large number of clones that compete with each other, leading to loss of many high-avidity cells. One of these groups (2A-2D) generating the largest number of high-avidity T cells is chosen for SIV challenge in subsequent experiments. The Mamu-A*02-based immunogens were chosen for graded immunization andeventual challenge because Mamu-B*08 is a “controller allele” associated with lower viral loads, so macaques inheriting this allele can prove less informative in challenge studies.

[0234] Vaccine-induced T cell responses to the epitopes included are measured by ELISpot assays while their functionality and differentiation phenotypes are determined by flow cytometry. Both assays are performed in the absence or presence of anti-CD8 antibody to assess CD8 dependence. Functional avidity of vaccine-elicited responses is determined by ELISpot using serial 10-fold dilutions of the cognate epitope peptides, ranging generally from low pg / mL to pg / mL concentrations.

[0235] Circulating T cells specific for the vaccine epitopes are quantified using pMHC oligomers (e.g., dimers, tetramers, pentamers, or dextramers) with either an intact or ablated CD8 binding site. Intact tetramers measure the total response while CD8bs-ablated tetramers identify only higher-avidity cells (D.A. Price et al., J. Exp. Med. 202, (2005): 1349; E.M. Choi et al., J. Immunol. 171, (2003): 5116; M.J. Pittet et al., J. Immunol. 171, (2003): 1844). To sort cells for repertoire and transcriptomic analyses, 20M peripheral blood mononuclear cells (PBMC) are divided into two aliquots to be stained with either (i) surface markers including CD8 and intact tetramers, or (ii) surface markers including CD8 and CD8bs-ablated tetramers. Cells are then sorted into those that bind intact or ablated tetramers, of which the latter population contains higher-avidity cells (D.A. Price et al., J. Exp. Med. 202, (2005): 1349). The peripheral blood cells may also be stimulated with peptide first, before staining with surface markers including both CD8 and CD69. Cells are then sorted into those that express CD69 but do not bind tetramer (specific cells of lower avidity), those that bind intact tetramer (mixed medium and high avidity), and those that bind CDbs-ablated tetramers (higher intrinsic avidity). Thus, this sorting approach reflects graded CD8 dependence.

[0236] Combined TCR and transcriptomic analyses are conducted by 10X sequencing. When many antigen-specific cells are isolated or when 10X sequencing of -5000 cells are inadequate to the diversity of the response, TCRs from up to hundreds of thousands of cells are sequenced using Omniscope OS-T screens. Diversity indices, including Shannon entropy, are assessed for TCR repertoires during vaccination and in the memory phase. The number of public and private clonotypes are compared between immunogens and vaccine regimens. Similarity analyses of the identified TCR sequences are conducted to identify related features of high-affinity vs. total epitope-specific T-cell populations. For a select set of TCRs, stably transduced Jurkat T cellsare generated to determine the functional avidity by peptide titrations in the presence or absence of blocking CD8 antibodies.

[0237] Positive results are identified by determining the number of vaccine-elicited cells that stain with intact pMHC tetramers vs. those with an ablated CD8 binding site, the CD8 independence of the responding T cells in functional assays, the functional avidity of responding T cells, and the independently measured avidity of cloned TCRs expressed in Jurkats. Many of these outcomes are assessed at multiple time points because the effect of CD8inT immunization can vary greatly when used at prime or at boost. Because these immunogens act on naive cells, the differential effect can be greatest after priming.Example 2. MHC Class II and lb molecules that engage CD8

[0238] A series of MHC-E-based SCTs were created that are either unmodified or are modified to confer stronger CD8 binding, reflected, e.g., in a lower avidity constant. For example, Gag69-Mamu-E*02: l l[Q115E] harbors a mutation in an alpha? residue, Q 115, that shortens a hydrogen bond with CD8 and thereby provides greater affinity (Wooldridge, J. Biol. Chem. 280, (2005): 27491). Plasmids coding for either the control (conventional) Gag69- Mamu-E*02: l l SCT or Gag69-Mamu-E*02: l l[Q115E] were transfected into HEK 293 cells to test proper folding and surface expression. In each case, 2.5 pg of plasmid DNA were transfected into 6.25 * 106Expi 293 cells. The cells were then incubated for two days before collection for testing by flow cytometry. As shown in FIG. 4, results demonstrated that both the conventional SCT and CD8-inT SCTs were expressed on the cell surface and were reactive with the anti-Mamu-E antibody, 4D12 (FIG. 4, first and third rows). The conventional SCT was expressed on 44% of the cultured cell population while Gag69-Mamu-E*02: l l[Q115E] (Gag69-CD8inT-plus) was expressed on 53%. Both molecules were also shown to be reactive with the pan anti-MHC class I antibody, W6 / 32 (FIG. 4, second and fourth rows, last column). Both TCR and CD8 binding are then confirmed for each SCT individually (targeting four different peptides presented by Mamu-E). Macaques or other experimental animals are next immunized, and the resulting T-cell repertoire is examined.

[0239] Other SCTs were created that combined the HLA-E-restricted epitope from HIV Gag, RMYSPTSIL (RL9) (Yang, Sci. Immunol. 6, (2021): eabgl703), with a human HLA-E molecule (MHC Class lb) that engages CD8. The conventional SCT is designated RL9-HLA- E*01 :03 and presented below as SEQ ID NO: 4. The novel SCT with the ability to bind CD8 contains three mutations (Q219R, G223D, and H224Q) near the acidic loop responsible forCD8 binding. This CD8inT-plus immunogen is designated RL9-HLA- E*01:03[Q219R / G223D / H224Q] (SEQ ID NO: 5).

[0240] CD8inT-plus SCTs such as Gag69-Mamu-E*02: l l[Q115E] and RL9-HLA- E*01 :03[Q219R / G223D / H224Q] will elicit a larger population of epitope-reactive CD8+T cells because these immunogens provide higher avidity for the CD8 molecule. A series of MHC-II-based SCTs are also created that are modified to include key CD8-interacting residues from P2-microglobulin, the 012 domain of the MHC-I heavy chain, and / or the a. domain of the MHC-I heavy chain. Antigen presentation and CD8 binding by these SCTs are also confirmed before immunization of macaques and the resulting T-cell repertoire is also assessed.

[0241] The Mamu-E-based SCTs used in this study are shown in Table 4. The selected peptides, derived from SIV Gag, (i) elicit Mamu-E-restricted T-cell responses in most or all RhCMV / SIVgag recipients (S.G. Hansen et al., Science 340, (2013): 1237874), (ii) have been engineered into SCTs and shown to be expressed on the cell surface, and (iii) can also be presented on human HLA-E molecules, where they are targets for recognition by Mamu-E- restricted T cells from rhesus macaques that were vaccinated with RhCMV / SIVgag. CD8inT- E versions of these SCTs are prepared by introduction of QI 15E, Q219R, G223D, and H224Q. CD8 binding and the Kd for CD8inT-E immunogens are confirmed using SPR.Table 4. Mamu class II and lb SCTsAllele or protein Epitope NotesMamu-E*02:04Gag APLVPTGSEProven Mamu-E andGag GGNYPVQQIHLA-E binders prev.Gag RMYNPTNIL made into SCTsGag KPIKCWNCGMamu-DRB*03:01Gag MLKHWWAANELDR All bind multiple DRBGag GGNYVHLPLSPRTLN alleles and generateGag IPFAAAQQRGPRKP responses in 100% ofGag VTEDLLHLNSLFG RhCMV / SIV recip

[0242] In class II SCTs the sequentially fused segments are peptide, Pi, P2, ai, 012, and transmembrane domain. The Mamu-DR-based SCTs used in this study are shown in Table 4. One or more of these peptides, derived from SIVgag, were targeted by MHC-II-restricted T cells in 100% of macaques vaccinated with RhCMV / SIVgag (S.G. Hansen et al., Science 340,(2013): 1237874). Three of four were shown to be presented by more than one Mamu-DRB molecule in a set of only five tested, and the MHC-II-restricted CD8+ T cells elicited by these vaccines could respond to their specific peptide epitope in the context of the different allomorphs, including some not expressed by the vaccine recipient. Thus, the TCRs of these cells recognize the selected peptides either alone or with unvarying class II determinants on the MHC-II molecule. Similar conclusions have been drawn for pMHC-II recognition by CD4+ T cells (C. Corradin & A. Lanzavecchia, Int. Rev. Immunol. 7, (1991): 139). The fact that DR- restricted TCRs are so frequently able to respond to their cognate peptide in the context of multiple DR alleles implies a likelihood that these Mamu-DRB5* 03:01 will be able to elicit peptide-specific T cell responses both in macaques that express the allele and those that do not.

[0243] CD8inT-II immunogens are prepared by substitution of key CD8-binding residues and regions from MHC-Ia into the analogous position in Mamu-DRB5*03:01. For example, one combination includes insertion of K58(P2M), QI 15, A245, and the CD8-binding loop at their structurally analogous positions in the class II molecule. More extensive replacements are also assessed and inclusion of El 15 to enhance binding is tested. These possibilities are tested combinatorially and CD8 binding is assayed by SPR before choosing an optimal configuration for use in macaques.

[0244] Vaccination, T-cell responses, repertoire analysis, and avidity analyses are performed as described in Example 1. An immunogenicity experiment (Table 5) tests if the addition of CD8 binding to Mamu-E or Mamu-DR SCTs enhances the priming of naive T cells with appropriate receptors and leads to detection of more Mamu-E-restricted or Mamu-DR- restricted T cells. Because MHC-II- or MHC-E-restricted T-cell responses are virtually never elicited by DNA, mRNA, or adenovector immunization of primates, positive results are confirmed by the outgrowth of any substantial number of such cells.Table 5. Immunogenicity groups (DNA>mRNA; n=4 / group) "TG Mamu-E SCTs1H CD8inT-EII Mamu-DRB SCTs1J CD8inT-IIExample 3. Accessory CD8 binding partners

[0245] High-affinity CD8 binding is added to MHC-II / E SCT vaccines by either (i) insertion of anti-CD8 scFv fragment into the paralogue of the CD8-binding loop of MHC-II or Mamu- E, or (ii) co-expression of membrane-bound anti-CD8 scFv with the SCT vaccine sequence. Macaques are vaccinated with the resulting constructs and resulting T-cell response, TCR repertoire, and functional avidity are examined (Table 6 andTable 7).Table 6. Immunogenicity groups (n=4)IL CD8inT-E + anti-CD8-PDGFRtmIM CD8inT-II-scFvIN CD8inT-II + anti-CD8-PDGFRtmTable 7. CD8-graded vaccines (Ad26>mRNA>mRNA; n=6) 2E Optimal Mamu-E SCTs x 32F Mamu-E SCTs > CD8inT-E > CD8inT-E-scFv 2G CD8inT-E-scFv > CD8inT-E > Mamu-E SCTs 2H Optimal Mamu-II SCTs x 321 Mamu-E SCTs > CD8inT-E > CD8inT-E-scFv 2J CD8inT-E-scFv > CD8inT-E > Mamu-E SCTs

[0246] Graded vaccinations of different CD8 affinities are also tested as in Example 1, to elicit waves of MHC-II or MHC-E-restricted CTL having different TCR affinities for pMHC. SCTs without engineered CD8 binding are administered first, to favor the highest-affinity clones, followed by the administration of engineered SCTs developed in Example 2, followed finally by immunization with SCTs expressed with accessory CD8 binding partners (Table 7).

[0247] Anti-CD8 scFv fragments are derived from the rhesusized MT807R1 and human G10-1 antibodies. To create membrane-bound accessory CD8 binders these fragments were joined via (G4S)3 linker to the rhesus platelet-derived growth factor (PDGF) receptortransmembrane domain. One of these two membrane-based scFv’s is chosen for testing based on successful engineering of the same fragment into MHC-II or MHC-E SCTs.

[0248] MT807R1 and G10-1 scFv fragments are engineered into either (i) the a3 domain of Mamu-E SCTs or the P2 domain of Mamu-DRB SCTs described in Example 2, at a position that is analogous to the protruding CD8-binding loop in MHC-Ia, or (ii) between the P2- microglobulin and al domains of Mamu-E SCTs or between the P2 and al domains of Mamu- DR SCTs. The constructs (CD8inT-E-scFv or CD8inT-II-scFv) are first assessed for cellsurface expression and CD8 binding using fluorescent anti-HLA-DR antibodies or soluble CD8 protein, respectively. When expression and CD8 binding are demonstrated, then the constructs are expressed in RM3 cells and tested for ability to stimulate CD4+ or CD8+ memory T cells responsive to the encoded peptides, derived from previously vaccinated macaques.

[0249] Vaccination, T-cell responses, repertoire analysis, and avidity analysis are all performed as described above. Immunogenicity tests (Table 6) evaluate the value of high affinity CD8 engagement for eliciting MHC-E- or MHC-II-restricted responses, overcoming the rarity of TCRs that can engage pMHC-E or pMHC-II with high affinity in the absence of CD8 binding. CD8-graded vaccinations evaluate instead if a breadth of sequential CD8 engagement produces greater clonotypic breadth in the response. Graded immunizations use either scFv fusions or membrane-bound scFv’s, depending on immunogenicity results.Example 4. Efficacy of cytotoxic T cells with higher affinity or alternative MHC restriction for SIV prophylaxis or treatment

[0250] In prophylactic vaccination, it has been shown that MHC-E-restricted T-cell responses are associated with SIV replication “arrest” in -50% of cases (S.G. Hansen et al., Nature 473, (2011): 523; S.G. Hansen et al., Nature 502, (2013): 100; S.G. Hansen et al., Sci. Transl. Med. 11, (2019): eaaw2607), but it is uncertain if the vaccine-elicited, atypical T cells are sufficient for arrest. The vaccines disclosed herein allow expansion of such unique T cells in the absence of other immunomodulation due to RhCMV-vectored vaccination (N.R. Narayan et al., Gut Microbes 6, (2015): 28454; G.N. Mendez-Lagares et al., J. Clin. Invest. 131, (2021): el48542; C. Santos Rocha et al., J. Virol. 13, (2018): e00167-18) and thus allow clarification of how atypical T cells contribute to effective prophylaxis or therapy.

[0251] SIV “replication arrest” and eventual clearance is a unique mechanism of protection strongly associated with Mamu-E-restricted T cells. RhCMV strain 68-1-vectored SIVvaccines demonstrate a unique pattern of efficacy in which -50% of vaccine recipients show early, complete replication arrest followed by eventual complete clearance of the infection (S.G. Hansen et al., Nat. Med. 15, (2009): 293; S.G. Hansen et al., Nature 473, (2011): 523; S.G. Hansen et al., Nature 502, (2013): 100; S.G. Hansen et al., Sci. Transl. Med. 11, (2019): eaaw2607). This pattern has not been observed with any other vaccine modality, suggesting that it arises from unique characteristics of the RhCMV / SIV-elicited immune responses. To probe the character of the protective T cells, RhCMV / SIV variants were created that elicit different combinations of MHC-Ia-, MHC-II-, and / or MHC-E-restricted immune responses. Remarkably, vectors that manifested MHC-II- and / or MHC-Ia-restricted CD8+ T cell responses did not provide protection. Only a tropism-programmed “MHC-E-only” CD8+ T- cell vaccine was proven capable of achieving replication arrest equivalent to that produced by the unmodified RhCMV / SIV vaccine, suggesting that MHC-E-restricted T cells are uniquely capable of causing SIV replication arrest.

[0252] Macaques from each of the previous three Examples (Table 3 and Table 7) that achieve highly differentiated T-cell responses receive serial low-dose challenges with SIVmac251, as do a group of control macaques, with challenges stopped at the first detection of infection. The risk of infection, occurrence of replication arrest, and post-infection viremia are followed and compared between groups.

[0253] In a separate experiment, SIV-infected and antiretroviral therapy (ART)-treated macaques are therapeutically vaccinated using a regimen from Example 1 that is proven capable of eliciting a unique repertoire of high-affinity CTL clonotypes. ART is interrupted two months after the last vaccination and then posttreatment viremia is assessed. The results are compared to those obtained in a small number of contemporary controls and in group of 28 animals that were therapeutically vaccinated in previous studies.

[0254] To determine if high-avidity or alternatively restricted T cells can achieve replication arrest, i.e., complete clearance of nascent infection from the body (S.G. Hansen et al., Nature 502, (2013): 100), groups of animals with highly differentiated immune responses are selected from the graded-CD8 immunization groups. The groups chosen are those in which CD8inT strategies have had the greatest success in meeting their goals. One group for challenge is chosen from Table 3 (class-la restricted), one from 2E-2G of Table 7 (Mamu-E restricted), and one from 2H-2J of Table 7 (class-II restricted). At least three unvaccinated control animals are challenged contemporaneously. Historical control outcomes are from 50 macaques challengedequivalently, including a mixture of unvaccinated controls, RhCMV / SIV recipients, and Ad / SIV recipients.

[0255] The potential of high-avidity T cells to treat SIV infection is tested using an established chronic SIV infection-and-treatment model. A single vaccine regimen producing the most abundant high-avidity T cells (e.g., those binding tetramers that lack CD8 binding) is selected for therapeutic testing from the regimens evaluated in the groups of Table 3. Ten age- matched macaques (five males and five females) are infected intrarectally and treated with ART four weeks after infection. Eight of the ten are vaccinated using the selected vaccine regimen on weeks 24, 30, and 36 with Ad26 and mRNA, and ART is withdrawn at week 40. Outcomes in the eight treated animals are compared to those observed in the two contemporaneous controls and in 28 previously treated macaques, including a mixture of unvaccinated controls, RhCMV / SIV recipients, and Ad / SIV recipients.Example 5. Response of memory T cells to Class- SCTs lacking CD8 binding or having reduced binding

[0256] In some aspects, the provided materials and methods function via initially selective recruitment of naive T cells, which must later (after boosting vaccination or encounter with a pathogen) expand non-selectively. For example, Class-I SCTs lacking CD8 binding or having reduced binding are only able to stimulate naive cells bearing TCRs that have intrinsic avidity for peptide:MHC complex sufficient to make interaction with CD8 unnecessary. In the naive T-cell population, such high avidity is a rare quality. Stimulated naive T cells, however, develop into memory cells with intrinsically higher sensitivity to antigen via the process of avidity maturation. Due to this higher avidity / sensitivity, a substantial fraction of pre-existing memory cells should be capable of responding to Class-I SCTs lacking CD8 binding or having reduced binding.

[0257] Therefore, memory T cells were used to test the proper folding and stimulatory capacity of CD8inT immunogens that include peptide from a pathogen that is presented in the context of Class-I SCTs lacking CD8 binding or having reduced binding (FIG. 5). It was hypothesized that cells from a Mamu-A*01+ macaque previously sensitized by vaccination against SIV gag would harbor memory cells capable of responding to the Mamu-A*01- restricted CM9 peptide presented as a CD8inT immunogen (i.e., in the context of a Class-I SCT lacking CD8 binding or having reduced binding). To test this idea, HEK-293 cells were transfected with DNA coding for one of the following molecules: conventional CM9-Mamu-A*01 SCT (SEQ ID NO: 6), CM9-CD8inT-weak (CM9-Mamu-A*01[A245V] SCT; SEQ ID NO: 8), CM9-CD8inT-zero (CM9-Mamu-A*01[D227K / T228A] SCT; SEQ ID NO: 9), CM9- CD8inT-plus (CM9-Mamu-A*O1[Q115E] SCT; SEQ ID NO: 7), or Gag69-Mamu-E (negative control). Then, 400,000 transfected cells, which were acting as artificial antigen-presenting cells, were incubated with 600,000 PBMC from the Mamu-A*01+ macaque that had been presensitized to Gag. Interferon production by the CD8+memory T cells (CD3+CD8+CD95+CCR7 ) was measured to test the proper folding and stimulatory capacity of the CD8inT immunogens. Results showed robust stimulation by T cells by all tested constructs (FIG. 5), with only a minor decrement in interferon production in responses to cells producing CD8inT-zero (CM9-Mamu-A*01[D227K / T228A] SCT) or CD8inT-plus (CM9-Mamu- A*01[Q115E] SCT) (FIG. 6).Example 6. Expansion of high-avidity naive T cells by CD8inT immunogens delivered as mRNA / LNPs

[0258] To demonstrate the expansion of high-avidity T cells by Class-I SCTs lacking CD8 binding or having reduced binding, two groups of naive macaques were immunized using (A) CM9-Mamu-A*01 SCT or (B) CM9-CD8inT-zero (CM9-Mamu-A*01[D227K / T228A] SCT; n=3 each group). The immunogens were prepared as mRNA transcripts containing 1- methylpseudouridine and then packaged into lipid nanoparticles using a Precision NanoSystems Ignite instrument and GenVoy lipid. Proper expression of the SCT immunogens was tested by transducing human embryonic kidney (HEK) 293 cells with each LNP, waiting two days, and then incubating the transduced cells with PBMC from an SIV-infected, Mamu- A*01+ macaque that was known to have memory CD8+T-cell responses to the CM9 peptide. Despite use of these non-hematopoietic stimulator cells (i.e., embryonic kidney cells), the experiment demonstrated responsiveness to both CM9-Mamu-A*01 SCT and CM9-CD8inT- zero (FIG. 7) and thus delivery, proper expression, and immunologic function of both immunogens.

[0259] The six assigned macaques in Groups A and B were immunized with the SCT immunogens as mRNA / LNPs at the start of the experiment (week 0) to recruit responsive naive T cells. For Group B macaques, responding naive cells are only those with sufficiently high CD8-independent TCR affinity for the CM9:Mamu-A*01 complex. Both groups are boosted with full-length SIV Gag (also delivered as mRNA / LNP) at week 4 and with bacteriophage particles decorated with the CM9 peptide at week 8 (Van Rompay, J. Virol. 88, (2013): 2011). Group A received mRNA / LNPs encoding CM9-Mamu-A*01 SCT and Group B receivedmRNA / LNPs encoding CM9-CD8inT-zero. Blood is collected at least every two weeks through week 12. PBMCs purified from blood are then exposed to varying concentrations of the CM9 epitope to determine the avidity of the elicited CM9-responsive T cells. High-avidity T cells are responsive to low concentrations of peptide:MHC complex and thus retain responsiveness (as evidenced by cytokine production in the cytokine flow cytometry [CFC] assay) even when peptide is highly diluted. The CFC studies demonstrate that recipients of CM9-CD8inT-zero (Group B) retain more responsive T cells at low peptide concentrations than recipients of conventional SCT (Group A).

[0260] Two approaches are taken to demonstrate that the T cells responding to CM9- CD8inT-zero vaccination (Group B) can function without CD8 engagement, i.e., they are CD8 independent. First, the in vitro stimulation assays described above are conducted in the presence or absence of an anti-CD8 antibody that prevents engagement of class-I MHC molecules. Macaques immunized with CM9-CD8inT-zero have T cells with less dependence or no dependence on CD8, so T cells from these macaques can better respond to the CM9 epitope in the presence of the antibody. Second, the fraction of CD8-independent T cells is directly enumerated using tetramerized, soluble CM9-Mamu-A*01 complexes that are fluorescently labeled. Two complexes are prepared, one of which is a conventional CM9- Mamu-A*01 tetramer and the other of which contains Mamu-A*01 molecules that have been mutated to prevent CD8 engagement. Most T cells that are specific for CM9 in the context of Mamu-A*01 are bound by conventional CM9-Mamu-A*01 tetramers, while only CD8- independent clones can engage the CM9-Mamu-A*01 complexes containing mutations that disallow CD8 engagement. Macaques in Group B have a larger fraction of CD8-independent T cells and thus a larger fraction of CD8+T cells that bind to the latter tetramers vs. conventional tetramers.

[0261] Because these macaques had no prior exposure to Gag, this experiment demonstrates the ability of the CD8inT immunogen lacking CD8 binding — CM9-CD8inT-zero — to recruit naive T cells having higher avidity for the target epitope (CM9) and thus superior anti-SIV efficacy (Migueles, Science 382, (2023): 1270). These cells recruited in the priming phase by the CD8inT immunogen are then expanded in the boosting phase into a memory population that includes more high-avidity and CD8-independent T cells. These cells are more able to control SIV replication and their presence causes (i) greater resistance to SIV acquisition after low-dose challenge and (ii) better control over SIV replication after acquisition, reflected inlower peak viral loads, greater reduction in viremia from peak to plateau, and / or lower plateau viral loads.Example 7. Superior efficacy against cancer by T cells expanded by CD8inT comprising heteroclitic peptides

[0262] An additional experiment demonstrated that high-avidity cells elicited by Class-I SCTs lacking CD8 binding or having reduced binding (e.g., CD8inT-weak or CD8inT-zero) protect against B16-Ova tumor establishment and / or growth. As in the prior example, the immunogens are delivered as mRNAs packaged into lipid nanoparticles. Three different vaccines are assessed. The conventional SCT immunogen, SIINFEKL-H-2KbSCT (SEQ ID NO: 10), codes for a secretion signal, the SIINFEKL epitope, mouse P2-microglobulin, the H- 2KbMHC allele, and linker peptides (administered to Groups A2 and B2). SIINFEKL- CD8inT-zero (SEQ ID NO: 11) is the same except that the H-2Kbmolecule in this construct carries the D227K mutation that disrupts CD8 binding (Groups A3 and B3). SIIWFEKL- CD8inT-zero (SEQ ID NO: 12) is identical to SIINFEKL-CD8inT-zero except that the peptide target is a “heteroclitic” peptide containing a mutation (4W) that increases TCR stimulation (Capasso, Oncoimmunology 6(9): el319028, 2017). Such heteroclitic mutations, when used in association with CD8inT-weak or CD8inT-zero immunogens, compensate for reduced CD8 affinity of the MHC molecule with higher TCR affinity.

[0263] Mice are vaccinated either prophylactically (before tumor injection; groups A2-A4 in FIG. 8) or therapeutically (groups B2-B4). In prophylaxis groups (FIG. 8), the mice are first vaccinated and B16-Ova cells are later implanted. In therapy groups, the mice are implanted with tumor cells before vaccination. mRNA / LNP vaccines are produced by in-vitro translation using pseudouridine and CleanCap, followed by encapsulation into GenVoy particles on the Precision Nanosystems Ignite. They are characterized for mRNA content and size / polydispersity and function is confirmed by treatment of cultured cells before use. For each vaccination, 5 pg of mRNA are administered. Prophylactic mRNA vaccination (Groups A2-A4) is performed at days 0 and 28 of the experiment. Therapeutic vaccination (Groups B2- B4) is performed 6 days after tumor inoculation.

[0264] Adaptive immunity is fully assessed in groups A1-A4 six weeks post vaccination (n=6 sacrificed at this time point, before tumor inoculation) and in all groups Al -B4 after tumor inoculation. As in the prior example, the focus of the adaptive-immune assessment is on T-cell avidity and CD8 independence. In cytokine flow cytometry assays for magnitude and avidityof responses, splenocytes are stimulated with serial dilutions of the target Ova peptide, SIIWFEKL. Higher avidity of the vaccine-elicited cells is indicated by sensitivity to lower concentrations of peptide. CD8 independence, which is closely associated with high avidity, is assessed by performing the assays in the presence or absence of anti-CD8 antibody. The fraction of CD8-independent cells is also assessed by flow cytometric staining of splenoctyes with fluorescent SIINFEKL-H-2Kbtetramers, or with fluorescent SIINFEKL-H-2Kb[E227K] tetramers. A greater fraction of CD8-independent T cells elicited by the vaccines is reflected in better binding of the latter tetramers relative to the former — indicating tetramer engagement by TCRs despite CD8’s inability to bind H-2Kb[E227K],

[0265] Tumor inoculations is conducted with 3 x 105cells implanted subcutaneously. Tumor volumes are measured for 18 days (Groups A1-A4) or 34 days (Groups Bl -B4). At the time of sacrifice, the tumors are resected and tumor-infiltrating lymphocytes (TILs) harvested for analysis by CFC and tetramer staining as described above. The cells are simultaneously phenotyped for memory and exhaustion markers including PD-1 and TIM-3. Slides from the tumors are assessed for Ki-67 as a measure of growth rate; presence of macrophages, T cells, and NK cells; MHC class I and class II expression; and for angiogenesis.

[0266] Vaccination with CD8inT immunogens in groups A3, A4, B3, and B4 is associated with higher-avidity T-cell responses and better tumor control, or event regression. High avidity cells circulate and also migrate into the tumor and retain function. Improved tumor control is assessed primarily based on bulk growth rate, but is also evident in reduced Ki-67 expression or vascularization. Use of a heteroclitic peptide target in the CD8inT immunogens of groups A4 and B4 leads to recruitment of a larger absolute number of CD8-independent T cells and to optimal tumor control in these groups.INFORMAL SEQUENCE LISTING

[0267] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and a recombinant polynucleotide including a nucleic acid sequence encoding a single chain trimer protein, the single chain trimer protein comprising a target peptide, a P2- microglobulin, and an MHC class la a chain, wherein: at least one of the p2-microglobulin and the MHC class la a chain is independently a variant of a naturally occurring polypeptide, and the single chain trimer protein has a CD8 binding affinity dissociation constant greater than 300 pM.

2. The pharmaceutical composition of claim 1, wherein the MHC class la a chain is a variant MHC class la a chain comprising an amino acid substitution that is 115A, 122A, 128A, 245V, 227K, 228A, or 115E.

3. The pharmaceutical composition of claim 1 or 2, wherein the P2- microglobulin is a variant P2-microglobulin comprising an amino acid substitution that is 58E or 59S.

4. A method for inducing an immune response or treating a health condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1-3.

5. The method of claim 4, wherein the CD8 binding affinity dissociation constant is greater than 1000 pM.

6. The method of claim 4 or 5, wherein: the method further comprises, subsequent to administering the pharmaceutical composition, administering to the subject a second pharmaceutical composition comprising a second recombinant polynucleotide including a second nucleic acid sequence encoding a second single chain trimer protein; the second single chain trimer protein comprises an additional copy of the target peptide, a second p2-microglobulin, and a second MHC class la a chain; and the second single chain trimer protein has a second CD8 binding affinity dissociation constant less than the first CD8 binding affinity dissociation constant.

7. The method of claim 6, wherein the second MHC class la a chain and the second p2-microglobulin are each independently naturally occurring polypeptides.

8. The method of claim 6 or 7, wherein the recombinant polynucleotide of the pharmaceutical composition comprises DNA, and the recombinant polynucleotide of the second pharmaceutical composition comprises mRNA.

9. A single chain trimer protein comprising a target peptide, a P2- microglobulin, and an MHC class lb a chain, wherein: at least one of the p2-microglobulin and the MHC class lb a chain is independently a variant of a naturally occurring polypeptide, and the single chain trimer protein has a CD8 binding affinity dissociation constant less than 1000 pM.

10. The single chain trimer protein of claim 9, wherein the MHC class lb a chain is a variant MHC class lb a chain comprising an amino acid substitution in an amino acid at position 115, or at a position from 219 to 229.

11. A single chain trimer protein comprising a target peptide, an a chain of a first MHC class II allele, and a P chain of a second MHC class II allele, wherein: at least one of the a chain and the P chain is independently a variant of a naturally occurring polypeptide, and the single chain trimer protein has a CD8 binding affinity dissociation constant less than 1000 pM.

12. The single chain trimer protein of claim 11, wherein the a chain is a variant a chain comprising an amino acid substitution from an amino acid at a position of the first MHC class II allele to an amino acid at a corresponding position of a first naturally occurring MHC class la allele or of a P2-microglobulin.

13. The single chain trimer protein of claim 11 or 12, wherein the P chain is a variant P chain comprising an amino acid substitution from an amino acid at a position of the second MHC class II allele to an amino acid at a corresponding position of a second naturally occurring MHC class la allele.

14. A single chain trimer protein comprising a target peptide, a P2- microglobulin, an MHC class lb a chain, and an anti-CD8 scFv fragment.

15. The single chain trimer protein of claim 14, wherein the MHC class lb a chain is a variant MHC class lb a chain comprising the anti-CD8 scFv fragment.

16. The single chain trimer protein of claim 14, wherein the anti-CD8 scFv fragment is located between the p2-microglobulin and the MHC class lb a chain.

17. A single chain trimer protein comprising a target peptide, an a chain of a first MHC class II allele, a P chain of a second MHC class II allele, and an anti-CD8 scFv fragment.

18. The single chain trimer protein of claim 17, wherein the P chain is a variant P chain comprising the anti-CD8 scFv fragment.

19. The single chain trimer protein of claim 17, wherein the anti-CD8 scFv fragment is located between the P chain and the a chain.

20. A system comprising: a single chain trimer protein comprising a target peptide, an MHC a chain, and an MHC P chain; and an accessory CD8 binder protein comprising an anti-CD8 scFv fragment and a transmembrane domain.

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