Modulation of t cell responses by UL18 of human cytomegalovirus

Abstract

To provide methods of modulating T cell responses by UL18 of human cytomegalovirus, and also to provide methods of generating MHC-Ia, MHC-II, and / or MHC-E restricted CD8+ T cells.SOLUTION: There is provided a recombinant HCMV vector comprising a nucleic acid sequence encoding a heterologous antigen, wherein due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147, the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 889,310, filed August 20, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant Nos. AI059457 and AI128741 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the Sequence Listing in the electronically submitted ASCII text file submitted with this application (Name: 4153_013PC01_Seqlisting_ST25, Size: 11,029 bytes, and Creation Date: August 19, 2020) are hereby incorporated by reference in their entirety. [Background technology]

[0004] Rhesus cytomegalovirus (RhCMV) strain 68-1 has previously been shown to induce CD8+ T cells that recognize peptides presented by MHC-II and MHC-E, but not by conventional MHC-I. This effect was replicated in cynomolgus CMV (CyCMV), thus demonstrating that deletion of RhCMV and CyCMV homologs of HCMV UL128, UL130, UL146, and UL147 is necessary to induce MHC-E-restricted CD8+ T cells (WO2016 / 130693, WO2018 / 075591). Furthermore, these vectors induce MHC-II-restricted CD8+ T cells. However, inserting the target site of the endothelial cell-specific microRNA (miR) 126 into essential viral genes of these vectors abolishes the induction of MHC-II-restricted CD8+ T cells, resulting in an "MHC-E only" vector that exclusively induces MHC-E-restricted CD8+ T cells (WO2018 / 075591). In contrast, inserting the myeloid cell-specific miR142-3p into 68-1RhCMV prevents the induction of MHC-E-restricted CD8+ T cells, resulting in a vector that exclusively induces CD8+ T cells restricted by MHC-II (WO2017 / 087921). Similarly, deletion of the UL40 homolog Rh67 prevents the induction of MHC-E-restricted CD8+ T cells, resulting in an "MHC-II only" vector (WO2016 / 130693). Summary of the Invention

[0005] The present disclosure relates to a recombinant human CMV (HCMV) vector comprising a nucleic acid sequence encoding a heterologous antigen, wherein the recombinant HCMV vector does not express UL18.

[0006] In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the HCMV vector does not express UL128 and UL130.

[0007] The present disclosure also relates to a recombinant HCMV vector comprising a nucleic acid sequence encoding a heterologous antigen, wherein the HCMV vector does not express UL18, UL128, UL130, UL146, and UL147.

[0008] In some embodiments, the recombinant HCMV vector does not express the UL18 protein, the UL128 protein, the UL130 protein, the UL146 protein, and the UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.

[0009] In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof.

[0010] In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a microRNA expressed in endothelial cells. In some embodiments, the MRE expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, and miR-328.

[0011] In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a microRNA expressed in myeloid cells. In some embodiments, the MRE expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

[0012] In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host autoantigen. In some embodiments, the pathogen-specific antigen is selected from the group consisting of human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, and Mycobacterium tuberculosis.

[0013] In some embodiments, the pathogen-specific antigen is an MHC-E supertope. In some embodiments, the MHC-E supertope is an HIV epitope. In some embodiments, the MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), IVRMYSPVSILDIRQ (SEQ ID NO: 24), IVRMYSPVSILDIRQ (SEQ ID NO: 25), IVRMYSPVSILDIRQ (SEQ ID NO: 26), IVRMYSPVSILDIRQ (SEQ ID NO: 27), IVRMYSPVSILDIRQ (SEQ ID NO: 28), IVRMYSPVSILDIRQ (SEQ ID NO: 29), IVRMYSPVSILDIRQ (SEQ ID NO: 30), IVRMYSPVSILDIRQ (SEQ ID NO: 31), IVRMYSPVSILDIRQ (SEQ ID NO: 32), IVRMYSPVSILDIRQ (SEQ ID NO: 33), IVRMYSPVSILDIRQ (SEQ ID NO: 34), IVRMYSPVSILDIRQ (SEQ ID NO: 35), IVRMYSPVSILDIRQ (SEQ ID NO: 36), IVRMYSPVSILDIRQ (S 3), RMYSPVSIL (SEQ ID NO:24), QKQEPIDKELYPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0014] In some embodiments, the tumor antigen is associated with a cancer selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

[0015] In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor (TCR) or an antigen derived from the variable region of a B cell receptor.

[0016] The present disclosure also relates to a pharmaceutical composition comprising a recombinant HCMV vector and a pharmaceutically acceptable carrier.

[0017] The present disclosure also relates to an immunogenic composition comprising a recombinant HCMV vector and a pharmaceutically acceptable carrier.

[0018] The present disclosure also relates to a method of generating an immune response to at least one heterologous antigen in a subject, the method comprising administering to the subject a recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen.

[0019] The present disclosure also relates to the use of recombinant HCMV vectors in the manufacture of a medicament for use in generating an immune response in a subject.

[0020] The present disclosure also relates to recombinant HCMV for use in generating an immune response in a subject.

[0021] The present disclosure also relates to a method of treating or preventing cancer in a subject, comprising administering a recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response against at least one heterologous antigen.

[0022] The present disclosure also relates to the use of a recombinant HCMV vector in the manufacture of a medicament for use in treating or preventing cancer in a subject.

[0023] The present disclosure also relates to recombinant HCMV vectors for use in treating or preventing cancer in a subject.

[0024] The present disclosure also relates to a method of treating or preventing a pathogen infection in a subject, comprising administering to the subject a recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response against at least one heterologous antigen.

[0025] The present disclosure also relates to the use of a recombinant HCMV vector in the manufacture of a medicament for use in treating or preventing a pathogen infection in a subject.

[0026] The present disclosure also relates to recombinant HCMV vectors for use in treating or preventing a pathogen infection in a subject.

[0027] The present disclosure also relates to a method of treating an autoimmune disease or disorder in a subject, comprising administering to the subject a recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response against at least one heterologous antigen.

[0028] The present disclosure also relates to the use of a recombinant HCMV vector in the manufacture of a medicament for use in treating an autoimmune disease or disorder in a subject.

[0029] The present disclosure also relates to recombinant HCMV vectors for use in treating an autoimmune disease or disorder in a subject.

[0030] In some embodiments, at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or its orthologue, hi some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or its orthologue.

[0031] In some embodiments, at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or its orthologue, hi some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or its orthologue.

[0032] In some embodiments, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or its orthologue. In some embodiments, at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or its orthologue. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or its orthologue.

[0033] In some embodiments, a CD8+ TCR is identified from CD8+ T cells elicited by a recombinant HCMV vector, and the CD8+ TCR recognizes an MHC-II / heterologous antigen-derived peptide complex. In some embodiments, a CD8+ TCR is identified from CD8+ T cells elicited by an HCMV vector, and the CD8+ TCR recognizes an MHC-E / heterologous antigen-derived peptide complex. In some embodiments, a CD8+ TCR is identified from CD8+ T cells elicited by an HCMV vector, and the CD8+ TCR recognizes an MHC class Ia / heterologous antigen-derived peptide complex.

[0034] In some embodiments, the CD8+ TCR is identified by DNA or RNA sequencing.

[0035] In some embodiments, the CD8+ TCR recognizes an MHC-II supertope.

[0036] In some embodiments, the CD8+ TCR recognizes an MHC-E supertope. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), It is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of RMYSPVSIL (SEQ ID NO: 24), QKQEPIDKELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

[0037] The present disclosure also relates to a method for generating TCR-transgenic CD8+ T cells that recognize an MHC-E-peptide complex, the method comprising: (a) administering to a first subject a recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-E / heterologous antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, and / or UL147. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, or UL147 proteins, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a miRNA expressed in endothelial cells. In some embodiments, the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328. In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host autoantigen. In some embodiments, the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, or Mycobacterium tuberculosis.

[0038] The present disclosure also provides a method for generating TCR-transgenic CD8+ T cells that recognize an MHC-E-peptide complex, comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector of any one of claims 5-10, 12-13, or 16-17, and the first CD8+ TCR recognizes an MHC-E / heterologous antigen-derived peptide complex; and (b) isolating one or more CD8+ T cells from a second subject. and (c) transfecting one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises the CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR-transgenic CD8+ T cells that recognize an MHC-E peptide complex. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, and / or UL147. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, or UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an orthologue thereof.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a miRNA expressed in endothelial cells. In some embodiments, the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328. In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host autoantigen. In some embodiments, the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, or Mycobacterium tuberculosis.

[0039] In some embodiments, the first CD8+ T cell recognizes an MHC-E supertope. In some embodiments, the MHC-E supertope comprises a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), R It is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of MYSPVSIL (SEQ ID NO: 24), QKQEPIDKELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

[0040] In some embodiments, the second CD8+ T cells recognize an MHC-E supertope. In some embodiments, the MHC-E supertope comprises a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), R It is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of MYSPVSIL (SEQ ID NO: 24), QKQEPIDKELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

[0041] In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.

[0042] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0043] In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.

[0044] The present disclosure also provides a method for generating CD8+ T cells that recognize MHC-E peptide complexes, comprising: (a) administering to a non-human primate a recombinant rhesus CMV (RhCMV) or cynomolgus CMV (CyCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147, to express an HIV antigen in an amount effective to generate a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertope peptide; and (b) identifying a first CD8+ TCR from the set of CD8+ T cells, the first recognizing the MHC-E / supertope peptide complex. (c) isolating one or more CD8+ T cells from the second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more transfected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex.In some embodiments, the HIV epitope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23). , RMYSPVSIL (SEQ ID NO:24), QKQEPIDKELYPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).The present disclosure also provides a method for generating CD8+ T cells that recognize an MHC-E peptide complex, comprising: (a) identifying, from a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertope peptide, a first CD8+ TCR that recognizes an MHC-E / supertope peptide complex, wherein the set of CD8+ T cells is generated from a recombinant rhesus macaque (RhCMV) or cynomolgus macaque CMV (CyCCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147; and (b) identifying a first CD8+ TCR effective for generating the set of CD8+ T cells. (b) isolating one or more CD8+ T cells from the second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex.In some embodiments, the HIV epitope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23). , RMYSPVSIL (SEQ ID NO:24), QKQEPIDKELYPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0045] In some embodiments, the first subject is a non-human primate and the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR.

[0046] In some embodiments, administering the recombinant HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the recombinant HCMV vector to the first subject.

[0047] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

[0048] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent a pathogen infection. In some embodiments, the pathogen infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0049] In some embodiments, the transfected CD8+ T cells are administered to a second subject to induce an autoimmune response against host self-antigens.

[0050] The present disclosure also relates to a method for generating CD8+ T cells that recognize an MHC-II-peptide complex, the method comprising: (a) administering to a first subject a recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize an MHC-II / peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, where the first CD8+ TCR recognizes an MHC-II / heterologous antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, where the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-II+peptide complex. In some embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding a heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express the UL18 protein, the UL128 protein, the UL130 protein, the UL146 protein, and the UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, wherein the MRE comprises a target site for a miRNA expressed in myeloid cells. In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

[0051] The present disclosure also relates to a method for generating CD8+ T cells that recognize MHC-II-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-II / peptide complex derived from an MHC-II / heterologous antigen from a set of CD8+ T cells that recognize the MHC-II / peptide complex, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-II peptide complexes. In some embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding a heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express the UL18 protein, the UL128 protein, the UL130 protein, the UL146 protein, and the UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and deletions of all nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an ortholog thereof.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, wherein the MRE comprises a target site for a miRNA expressed in myeloid cells. In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

[0052] In some embodiments, the first CD8+ T cells recognize an MHC-II supertope. In some embodiments, the second CD8+ T cells recognize an MHC-II supertope.

[0053] In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.

[0054] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0055] In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.

[0056] In some embodiments, administering the HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the HCMV vector to the first subject.

[0057] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

[0058] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent a pathogen infection. In some embodiments, the pathogen infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0059] In some embodiments, the transfected CD8+ T cells are administered to a second subject to induce an autoimmune response against host self-antigens.

[0060] The present disclosure also relates to a method for generating CD8+ T cells that recognize an MHC-I-peptide complex, the method comprising: (a) administering to a first subject a recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize an MHC-I / peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, where the first CD8+ TCR recognizes an MHC-I / heterologous antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, where the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-I-peptide complex. In some embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding a heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express the UL18 protein, the UL128 protein, the UL130 protein, the UL146 protein, and the UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof.

[0061] The present disclosure also relates to a method for generating CD8+ T cells that recognize an MHC-I-peptide complex, the method comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-I / xenogenous antigen-derived peptide complex from a set of CD8+ T cells that recognize an MHC-I / xenogenous antigen-derived peptide complex, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector described in any one of claims 1 to 11; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-I-peptide complex.

[0062] In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.

[0063] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0064] In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.

[0065] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

[0066] In some embodiments, the transfected CD8+ T cells are administered to a second subject to treat or prevent a pathogen infection. In some embodiments, the pathogen infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0067] In some embodiments, the transfected CD8+ T cells are administered to a second subject to induce an autoimmune response against host self-antigens.

[0068] In some embodiments, the pathogen-specific antigen is selected from the group consisting of human immunodeficiency virus, simian immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, and Mycobacterium tuberculosis.

[0069] In some embodiments, the tumor antigen is associated with a cancer selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

[0070] In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor (TCR) or an antigen derived from the variable region of a B cell receptor.

[0071] The present disclosure also relates to a method of treating or preventing a pathogen infection in a subject, comprising administering CD8+ T cells to the subject.

[0072] The present disclosure also relates to the use of CD8+ T cells in the manufacture of a medicament for use in treating or preventing a pathogen infection in a subject.

[0073] The present disclosure also relates to CD8+ T cells for use in treating or preventing a pathogen infection in a subject.

[0074] The present disclosure also relates to a method of treating or preventing cancer in a subject, the method comprising administering CD8+ T cells to the subject.

[0075] The present disclosure also relates to the use of CD8+ T cells in the manufacture of a medicament for use in treating or preventing cancer in a subject.

[0076] The present disclosure also relates to CD8+ T cells for use in treating or preventing cancer in a subject.

[0077] The present disclosure also relates to a method of treating an autoimmune disease or disorder, comprising administering CD8+ T cells to a subject.

[0078] The present disclosure also relates to the use of CD8+ T cells in the manufacture of a medicament for use in treating an autoimmune disease or disorder.

[0079] The present disclosure also relates to CD8+ T cells for use in treating an autoimmune disease or disorder.

[0080] The present disclosure also relates to a method for inducing an autoimmune response against a host self-antigen, comprising administering CD8+ T cells to a subject.

[0081] The present disclosure also provides a nucleotide sequence having a length of 9 to 15 amino acids, including LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPV The present invention relates to a human immunodeficiency virus MHC-E supertope that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SIL (SEQ ID NO: 24), QKQEPIDKELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

[0082] In some embodiments, the recombinant HCMV vector comprises a nucleic acid encoding one or more human immunodeficiency virus antigens. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, or UL147 proteins, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a miRNA expressed in endothelial cells. In some embodiments, the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, wherein the MRE comprises a target site for an miRNA expressed in myeloid cells.In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125. [Brief explanation of the drawings]

[0083] [Figure 1] The mean frequencies of CD4+ or CD8+ T cells responding to SIV antigen-derived peptide pools are shown in the indicated cohorts. T cell frequencies were determined in peripheral blood mononuclear cells (PBMCs) at the indicated time points by intracellular cytokine staining (ICS) for IFNγ or TNFα in the presence of pools of overlapping (11A) 15-mer peptides representing SIV antigens. Cohort 1 was immunized with three "MHC-E only" 68-1RhCMV vectors containing mir126 recognition sites in the 3' untranslated regions of the essential genes Rh108 (UL79) and Rh156 (IE2) and expressing the 5' segments of the SIV antigens SIVgag, SIVretanef (a fusion of rev, tat, and nef), and SIVpol. Cohort 2 was immunized with three "MHC-II only" 68-1RhCMV vectors lacking Rh67 (UL40) and expressing the 5' segments of the SIV antigens SIVgag, SIVretanef, or SIVpol. Cohort 3 was immunized with three "MHC-II only" 68-1RhCMV vectors containing mir142 recognition sites in the 3' untranslated regions of the essential genes Rh108 (UL79) and Rh156 (IE2) and expressing the 5' segments of the SIV antigens SIVgag, SIVretanef, or SIVpol. Cohort 4 (control cohort) was immunized with three 68-1RhCMV vectors expressing the 5' segments of the SIV antigens SIVgag, SIVretanef, or SIVpol. [Figure 2]Figure 1 shows SIVgag-specific CD8+ T cell responses in PBMCs from three rhesus macaques (RM) in each cohort measured in the presence of individual peptides. Peptides resulting in specific CD8+ T cell responses are indicated by boxes, and box color indicates MHC restriction as determined by blocking with anti-pan MHC-I mAb W6 / 32, MHC-E blocking peptide VL9, and MHC-II blocking peptide CLIP. [Figure 3] Plasma viral loads (left panel) and SIVvif-specific CD8+ T cell responses (right panel) of RMs in cohorts 1, 2, and 3 after repeated dose-limited SIVmac239 challenge are shown. Animals that controlled SIV infection (RM controllers) are shown in white boxes, and non-controllers are shown in black boxes. One animal in cohort 2 initially controlled SIV infection but lost control when CD8+ T cells were deleted, consistent with this RM being a naturally occurring elite controller. [Figure 4] Immunoblots of SIV supertope fusion constructs are shown. Telomerized rhesus fibroblasts (TRF) were infected or uninfected, and lysates of the indicated RhCMV constructs and infected cells were separated by electrophoresis and immunoblotted. Fusion proteins containing the SIV supertope were visualized with an anti-HA antibody, and specific antibodies were used to detect the viral proteins IE1, Rh107, and Rh108. Protein bands observed in mock-infected or uninfected TRF lysates with the IE antibody are nonspecific. [Figure 5]Figure 5A shows the mean frequency of CD8+ T cells responding to SIV antigen-derived peptides in the PBMCs of cohort 5 animals (n = 8). Cohort 5 was immunized with the 68-1RhCMV vector, which contains mir126 recognition sites in the 3' untranslated regions of the essential genes Rh108 (UL79) and Rh156 (IE2) and expresses an MHC-E supertope fusion protein. T cell frequencies were determined in peripheral blood mononuclear cells (PBMCs) at the indicated time points by intracellular cytokine staining (ICS) for IFNγ or TNFα in the presence of pools of individual 15-mer peptides representing SIV supertopes. Figure 5B shows the frequency of CD8+ T cells responding to specific MHC-E-restricted supertopes in individual RMs. MHC-E-restricted supertopes (Gag69 and Gag120) and other MHC-E-restricted Gag epitopes are shown. [Figure 6] Figure 1 shows SIV plasma viral load (left panel) and SIVvif-specific T cell responses (right panel) following dose-limited SIVmac239 challenge of RMs in cohort 5. RM controllers are shown in white boxes, and non-controllers are shown in black boxes. SIVvif-specific responses indicate "taken" by SIV infection in controller animals. [Figure 7] Figure 7A shows the frequency of CD8+ T cells responding to SIV antigen peptide pools in RMs (n = 2) vaccinated with 68-1RhCMV expressing SIVgag, 68-1RhCMV expressing UL18 and SIVretanef, or 68-1RhCMV expressing UL18 and SIVpol. Figure 7B shows the frequency of CD8+ T cells responding to MHC-E-restricted supertopes in each RM. Figure 7C shows the frequency of CD8+ T cells responding to MHC-II-restricted supertopes in each RM. [Figure 8]Figure 1 shows SIVpol-specific CD8+ T cell responses in PBMCs obtained from three RMs vaccinated with 68-1RhCMV expressing UL18 and SIVpol. CD8+ T cell responses were measured in the presence of individual peptides. Peptides resulting in specific CD8+ T cell responses are indicated by boxes, and the color of the boxes indicates MHC restriction as determined by blocking with the anti-pan-MHC-I mAb W6 / 32, the MHC-E blocking peptide VL9, and the MHC-II blocking peptide CLIP. All peptide responses were blocked by W6 / 32 but not by the VL9 or CLIP peptides. Thus, CD8+ T cells are exclusively restricted by MHC-I. [Figure 9A] Dot plots showing the frequency of CD8+ T cells producing IFNγ or TNFα in response to SIVpol peptides from RMs vaccinated with 68-1RhCMV expressing UL18 and SIVpol are shown. [Figure 9B] Dot plots show the frequency of CD8+ T cells producing IFNγ or TNFα in response to SIVpol peptides from RMs vaccinated with 68-1RhCMV expressing the UL18D196S mutant and SIVpol. The frequency of CD8+ T cells responding to pools of overlapping 15-mer peptides, including SIVpol or the MHC-E-restricted supertope peptide SIVpol41 or the MHC-II-restricted supertope peptide SIVpol90, is shown. Intact UL18 prevents the induction of supertope responses, which is not observed with the UL18 D196S mutant. [Figure 10]Immunoblots of human MRC5 fibroblasts uninfected or infected with HCMV-TR3 (Caposio P. et al. 2019. Characterization of a live-attenuated HCMV-based vaccine platform. Sci Rep 9:19236) or an HCMV-TR3-based vector in which UL18 was replaced with HIV gag, HIV nef, and HIV pol fusion proteins are shown. Additionally, the UL18-deleted vector lacks UL128, UL130, UL146, and UL147, because previous studies have shown that these genes inhibit MHC-E-restricted CD8+ T cell responses (U.S. Patent No. 10,532,099). Additionally, the p24 fragment of HIV gag was added for control purposes. The top blot was probed with an antibody against the HIV gag protein. The bottom blot was probed with an antibody against the HCMV pp65 protein. [Figure 11] HIV gag-, nef-, and pol-specific CD8+ T cell responses in PBMCs obtained from RMs vaccinated with UL18-deleted vectors are shown (Figure 11, n=2). CD8+ T cell responses were measured 56 days post-vaccination using pools of overlapping peptides corresponding to each portion of the antigen. DETAILED DESCRIPTION OF THE INVENTION

[0084] I. Terminology Unless otherwise specified, terminology is used according to conventional usage.

[0085] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein or listed in the Application Data Sheet, including U.S. Patent Application No. 62 / 889,310, filed August 20, 2019, are incorporated by reference herein in their entirety for all purposes as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference.

[0086] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments of the disclosure, explanations of specific terms are provided below.

[0087] Unless the context otherwise requires, throughout the specification and claims, the word "comprise" and its variations, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, i.e., "including but not limited to." "Consisting of" means excluding trace elements and substantial method steps of other components disclosed herein. The term "consisting essentially of" limits the scope of a claim to specific materials or steps, or those that do not substantially affect the essential characteristics of the claimed invention. For example, a composition consisting essentially of components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, etc. Similarly, a protein consists essentially of a particular amino acid sequence if it contains additional amino acids that contribute up to 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alter the activity of the protein by 50% or less). Embodiments defined by each of the transition terms are within the scope of the present invention.

[0088] Antigen: As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that can induce an immune response in a subject. The term also refers to a protein that is immunologically active, in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response directed against the protein.

[0089] Antigen-specific T cells: CD8 that recognize specific antigens + or CD4 + Lymphocytes. Generally, antigen-specific T cells specifically bind to a particular antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0090] Administration: As used herein, the term "administration" means giving or administering an agent, such as a composition comprising an effective amount of a CMV vector containing an exogenous antigen, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, nasal, vaginal, and inhalation routes.

[0091] Effective amount: As used herein, the term "effective amount" refers to an amount of an agent sufficient to reduce or eliminate signs or symptoms of a condition or disease or to generate a desired response, such as inducing an immune response to an antigen, e.g., a CMV vector containing a heterologous antigen, or the amount of transfected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex, an MHC-II / heterologous antigen-derived peptide complex, or an MHC-I / heterologous antigen-derived peptide complex. In some examples, an "effective amount" treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases. An effective amount can be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with an infection or cancer, from developing.

[0092] Heterologous antigen: As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. The heterologous antigen may be a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, a host self-antigen, or any other antigen.

[0093] Hyperproliferative disease: A disease or disorder characterized by uncontrolled cell proliferation. Hyperproliferative diseases include, but are not limited to, malignant and non-malignant tumors.

[0094] Immune tolerance: As used herein, "immune tolerance" refers to a state of unresponsiveness of the immune system to substances capable of inducing an immune response. Self-tolerance to an individual's own antigens, e.g., tumor antigens, is achieved by both central and peripheral tolerance mechanisms.

[0095] Immunogenic peptide: A peptide that contains allele-specific motifs or other sequences, such as N-terminal repeats, such that the peptide binds to an MHC molecule and induces a cytotoxic T lymphocyte ("CTL") response or a B cell response (e.g., antibody production) against the antigen from which the immunogenic peptide is derived.

[0096] In some embodiments, immunogenic peptides are identified using sequence motifs or other methods known in the art, such as neural nets or polynomial determination. Typically, an algorithm is used to determine a peptide's "binding threshold" to select peptides that have a score that confers a high probability of binding with a particular affinity and are immunogenic. The algorithm is based on either the effect of a particular amino acid at a particular position on MHC binding, the effect of a particular amino acid at a particular position on antibody binding, or the effect of a particular substitution in a motif-containing peptide on binding. Within the context of immunogenic peptides, a "conserved residue" is a residue that appears at a particular position in a peptide with a frequency significantly higher than expected by random distribution. In some embodiments, a conserved residue is a residue that may provide a contact point for the MHC structure with the immunogenic peptide.

[0097] MicroRNA: As used herein, the term "microRNA" refers to a major class of biomolecules involved in the regulation of gene expression. For example, in the human heart, liver, or brain, miRNAs play a role in tissue specification or cell lineage determination. Furthermore, miRNAs affect various processes, including early development, cell proliferation, and cell death, as well as apoptosis and fat metabolism. The large number of miRNA genes, diverse expression patterns, and abundance of potential miRNA targets suggest that miRNAs may be an important source of genetic diversity.

[0098] Mature miRNAs are typically 8-25 nucleotide non-coding RNAs that regulate the expression of mRNAs containing sequences complementary to the miRNA. These small RNA molecules are known to control gene expression by regulating mRNA stability and / or translation. For example, miRNAs bind to the 3'UTR of target mRNAs and repress their translation. miRNAs can also bind to target mRNAs and mediate gene silencing via the RNAi pathway. miRNAs can also regulate gene expression by inducing chromatin condensation.

[0099] miRNAs silence the translation of one or more specific mRNA molecules by binding to miRNA recognition elements (MREs), defined as any sequence that interacts with miRNA by directly base-pairing with the miRNA somewhere on the miRNA transcript. MREs are often present in the 3' untranslated region (UTR) of an mRNA, but MREs can also be present in the coding sequence or 5' UTR. MREs are not necessarily perfectly complementary to the miRNA; they usually have only a few bases that are complementary to the miRNA, and often contain one or more mismatches within those complementary bases. MREs can be any sequence that can be sufficiently bound by the miRNA so that the translation of the gene to which the MRE is operably linked (such as a CMV gene that is essential for or enhances in vivo proliferation) is suppressed by a miRNA silencing mechanism such as RISC.

[0100] Mutation: As used herein, the term "mutation" refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus, or "wild-type" sequence. A mutant is any protein or nucleic acid sequence containing a mutation. Furthermore, a cell or organism having a mutation may be referred to as a mutant. Some types of coding sequence mutations include point mutations (individual nucleotide or amino acid differences), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences resulting in the loss of one or more nucleotides or amino acids, up to the deletion of the entire coding sequence of a gene), and frameshift mutations (differences resulting in a change in the amino acid sequence due to the deletion of a number of nucleotides that is not divisible by three). Mutations resulting in amino acid differences may also be referred to as amino acid substitution mutations. Amino acid substitution mutations can be described by the amino acid change relative to the wild-type at a specific position in the amino acid sequence.

[0101] Nucleotide sequence or nucleic acid sequence: The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence, including, but not limited to, messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid can be single-stranded or partially or completely double-stranded (duplex). A duplex nucleic acid can be homoduplex or heteroduplex.

[0102] Operably linked: As used herein, the term "operably linked" means that a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is positioned in such a manner that it affects the second nucleic acid sequence. Operably linked DNA sequences can be contiguous or operably spaced apart.

[0103] Promoter: As used herein, the term "promoter" can refer to any of a number of nucleic acid control sequences that direct transcription of a nucleic acid. Typically, eukaryotic promoters include a necessary nucleic acid sequence near the start site of transcription, such as in the case of a polymerase II type promoter, a TATA element, or any other specific DNA sequence recognized by one or more transcription factors. Expression by a promoter can be further regulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those skilled in the art. A nucleic acid containing a promoter operably linked to a nucleic acid sequence encoding a specific polypeptide can be referred to as an expression vector.

[0104] Recombinant: As used herein, the term "recombinant" with respect to a nucleic acid or polypeptide refers to one having a sequence that is not naturally occurring or that is created by the artificial combination of two or more separated sequence segments, such as a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid segments, e.g., genetic engineering techniques. A recombinant polypeptide can also refer to a polypeptide made using a recombinant nucleic acid, including a recombinant nucleic acid (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen) that is transcribed into a host organism that is not the natural source of the polypeptide.

[0105] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" is conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually contain an injectable fluid containing a pharmaceutically and physiologically acceptable fluid such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0106] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Polynucleotides consist of the four bases adenine, cytosine, guanine, and thymine / uracil (uracil is used in RNA). A coding sequence derived from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

[0107] Polypeptide: The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymers can be linear or branched, can comprise modified amino acids or amino acid analogs, and can be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as labeling or conjugation with a biologically active moiety.

[0108] Protein orthologs are typically characterized by having greater than 75% sequence identity across the full length of the amino acid sequence of a particular protein, using ALIGN with default parameters. Proteins with even greater similarity to the reference sequence, when assessed by this method, exhibit increasing percentages of sequence identity, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98%. Furthermore, sequence identity can be compared across the full length of a particular domain of the peptide of the present disclosure.

[0109] Sequence identity / similarity: As used herein, identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of the percentage identity, with the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of the percentage identity or similarity (taking into account conservative amino acid substitutions), with the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and also perform the same or similar functions to each other (e.g., proteins that perform the same function in different species or mutant forms of proteins that do not change the function or size of the protein) can be referred to as "homologs."

[0110] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv Appl Math 2, 482 (1981), Needleman & Wunsch, J Mol Biol 48, 443 (1970), Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988), Higgins & Sharp, Gene 73, 237-244 (1988), Higgins & Sharp, CABIOS 5, 151-153 (1989), Corpet et al, Nuc Acids Res 16, 10881-10890 (1988), Huang et al, Computer App Biosci 8, 155-165 (1992), and Pearson et al, Meth Mol Bio 24, 307-331 (1994). Additionally, Altschul et al., J Mol Biol 215, 403-410 (1990) provide a detailed discussion of sequence alignment methods and homology calculations.

[0111] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., (1990), supra) is available from several sources, including the National Center for Biotechnology Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI website.

[0112] BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. If the two compared sequences are homologous, the specified output file will present the homologous regions as aligned sequences. If the two compared sequences are not homologous, the specified output file will not present aligned sequences.

[0113] The number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in both sequences when aligned. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the specified sequence or the articulated length (such as 100 consecutive nucleotides or amino acid residues from the sequence shown in the specified sequence), and then multiplying the resulting value by 100. For example, a nucleic acid sequence with 1166 matches is 75.0 percent identical to a test sequence with 1154 nucleotides when aligned (1166÷1554*100=75.0). The percent sequence identity value is rounded down to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded down to 75.2. The length value is always an integer. In another example, a target sequence comprising a 20 nucleotide region that aligns with 20 consecutive nucleotides from a specified sequence contains a region having 75 percent sequence identity with the specified sequence (i.e., 15÷20×100=75), as follows:

[0114] For comparison of amino acid sequences greater than approximately 30 amino acids, the Blast2 sequence function is used, with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and per residue gap cost of 1). Homologs are typically characterized as having at least 70% sequence identity, counted over the full-length alignment with the amino acid sequence, using gapped blastp with databases such as NCBI Basic Blast2.0, the nr database, the swissprot database, and proprietary sequence databases. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, Comput Appl Biosci 10, 67-70 (1994)). Other programs use SEG. Additionally, manual alignments may be performed. Proteins with even greater similarity, when assessed by this method, exhibit increasing percentages of identity, such as at least approximately 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein.

[0115] When aligning short peptides (fewer than approximately 30 amino acids), alignments are performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap penalty of 9, extension gap penalty of 1). Proteins with even greater similarity to the reference sequence will exhibit increasing percentages of identity, such as at least approximately 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when assessed by this method. When comparing less than the entire sequence for sequence identity, homologs typically have at least 75% sequence identity over a short window of 10–20 amino acids, and may have at least 85%, 90%, 95%, or 98% sequence identity, depending on the identity to the reference sequence. Methods for determining sequence identity over such short windows are described on the NCBI website.

[0116] One indicator that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nevertheless, nucleic acid sequences that do not show a high degree of identity may encode the same or similar (conserved) amino acid sequences due to the degeneracy of the genetic code. This degeneracy can be used to alter the nucleic acid sequence to generate multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the nucleic acid encoding the protein.

[0117] Subject: As used herein, the term "subject" refers to living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.

[0118] Supertope: As used herein, the term "supertope" or "supertope peptide" refers to an epitope or peptide that is recognized by T cells in greater than about 90% of the human population, regardless of MHC haplotype, i.e., regardless of the presence or absence of a given MHC-I, MHC-II, or MHC-E allele.

[0119] Treatment: As used herein, the term "treatment" refers to an intervention that ameliorates the signs or symptoms of a disease or pathological condition. As used herein, the terms "treatment," "treat," and "treating" with respect to a disease, pathological condition, or symptom also refer to any observable beneficial effect of treatment. A beneficial effect may be evidenced, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease, a delay in the progression of a disease, a reduction in the number of disease recurrences, an improvement in the overall health or well-being of the subject, or other parameters well known in the art that are specific to a particular disease. A preventative treatment is a treatment administered to a subject who does not show signs of a disease or who shows only early signs, with the aim of reducing the risk of developing the condition. A therapeutic treatment is a treatment administered to a subject after the onset of signs and symptoms of a disease.

[0120] Vaccine: An immunogenic composition that can be administered to a mammal, such as a human, to confer immunity, such as active immunity, against a disease or other pathological condition. Vaccines can be used prophylactically or therapeutically. Thus, vaccines can be used to reduce the likelihood of developing a disease (such as a tumor or pathological infection), or to reduce the severity of symptoms of a disease or condition, or to limit the progression of a disease or condition (such as a tumor or pathological infection), or to limit the recurrence of a disease or condition (such as a tumor). In certain embodiments, the vaccine is a replication-deficient CMV that expresses a heterologous antigen, such as a tumor-associated antigen from a lung, prostate, ovarian, breast, colon, cervical, liver, kidney, bone tumor, or melanoma.

[0121] Vector: A nucleic acid molecule of a specific sequence can be incorporated into a vector, and then the vector is introduced into a host cell, thereby generating a transformed host cell.A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as a replication origin.A vector can also contain one or more selectable marker genes and other gene elements known in the art, including a promoter element that directs nucleic acid expression.The vector can be a viral vector, such as a CMV vector.Viral vectors can be constructed from attenuated viruses, including wild-type viruses or replication-defective viruses.

[0122] II. How HCMV UL18 regulates T cell responses Disclosed herein is a method for modulating T cell responses by UL18 of HCMV, comprising administering to a subject an effective amount of at least one recombinant HCMV vector comprising at least one heterologous antigen, wherein the HCMV vector does not express UL18.

[0123] In some embodiments, the method further comprises generating an immune response to at least one heterologous antigen, comprising administering to the subject an amount of an HCMV vector effective to induce a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating or preventing cancer in the subject, comprising administering to the subject an amount of an HCMV vector effective to induce a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating or preventing a pathogen infection in the subject, comprising administering to the subject an amount of an HCMV vector effective to induce a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating an autoimmune disease or disorder in the subject, comprising administering to the subject an amount of an HCMV vector effective to induce a CD8+ T cell response to the at least one heterologous antigen.

[0124] In some embodiments, the UL18-deficient HCMV vector also does not express the UL128, UL130, UL146, or UL147 protein due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147. Additionally, any of the UL18-deficient HCMV vectors may lack the US11 and / or UL82 protein due to the presence of a mutation in the nucleic acid sequence encoding US11 and / or UL82. The mutation may be any mutation that results in the lack of expression of an active protein. Such mutations may include point mutations, frameshift mutations, deletions of less than all of the protein-encoding sequence (truncating mutations), or deletions of all of the protein-encoding nucleic acid sequence, or any other mutation.

[0125] In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147, and expresses UL40 and US28.

[0126] In some embodiments, the HCMV vector comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE). In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147 (and optionally UL82) and expresses UL40 and US28, and the MRE comprises a target site for a microRNA expressed in endothelial cells. Examples of such miRNAs expressed in endothelial cells are miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, and miR-328. In some embodiments, the HCMV vector lacks UL18, and the MRE comprises a target site for a microRNA expressed in myeloid cells. Examples of such miRNAs expressed in myeloid cells are miR-142-ep, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, and miR-125.

[0127] The MRE can be any miRNA recognition element that silences expression in the presence of an miRNA expressed by endothelial cells. The MRE can be any miRNA recognition element that silences expression in the presence of an miRNA expressed by myeloid cells. Such an MRE can be the exact complement of the miRNA. Alternatively, other sequences can be used as the MRE for a given miRNA. For example, the MRE can be predicted from the sequence. In one example, miRNAs can be searched on the website microRNA.org (www.microrna.org). A list of mRNA targets for the miRNA is then listed. For each listed target on that page, the predicted MRE can be accessed by accessing "Alignment Details."

[0128] Those skilled in the art can select from the literature a reasonable, putative, or mutated MRE sequence that is predicted to induce silencing in the presence of miRNA expressed in myeloid cells, such as macrophages. Examples include the websites referenced above. Those skilled in the art can then obtain an expression construct in which a reporter gene (such as a fluorescent protein, enzyme, or other reporter gene) has its expression driven by a promoter, such as a constitutively active promoter or a cell-specific promoter. The MRE sequence can then be introduced into the expression construct. The expression construct can be transfected into appropriate cells, and the cells can be transfected with the miRNA of interest. The lack of expression of the reporter gene indicates that the MRE silences gene expression in the presence of the miRNA.

[0129] In some embodiments, the heterologous antigen may be a pathogen-specific antigen, a tumor antigen, a tumor-specific antigen, or a host autoantigen. In some embodiments, the host autoantigen is an antigen derived from the variable region of a T cell receptor (TCR) or a B cell receptor.

[0130] Pathogen-specific antigens can be derived from, for example, human immunodeficiency virus, simian immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, Clostridium tetani, and Mycobacterium tuberculosis.

[0131] Tumor antigens can be any protein that is relatively limited to tumor cells and induces an immune response. However, many tumor antigens are host (self) proteins, and therefore are typically not considered antigenic by the host immune system. Tumor antigens can also be aberrantly expressed by cancer cells. Tumor antigens can also be germline / testicular antigens expressed in cancer cells, lineage differentiation antigens that are not expressed in adult tissues, or antigens that are overexpressed in cancer cells.Tumor antigens include prostatic acid phosphatase (PAP); Wilms tumor suppressor protein (WT1); mesothelin (MSLN); Her-2 (HER2); human papillomavirus antigen E6 from HPV16 strains; human papillomavirus antigen E7 from HPV16 strains; human papillomavirus antigen E6 from HPV18 strains; human papillomavirus antigen E7 from HPV18 strains; fusion protein of human papillomavirus E6 and E7 from HPV16 and HPV18 strains; mucin 1 (MUC1); LMP2; epidermal growth factor receptor (EGFR); p53; New York esophagus 1 (NY-ESO-1); prostate-specific membrane antigen (PSMA); GD2, carcinoembryonic antigen (CEA); melanoma antigen a / melanoma antigen recognized by T cells 1 (MelanA / MART1); Ras; gp100, proteinase 3 (PR1), Bcr-abl; survivin; prostate-specific antigen (PSA); human telomerase reverse transcriptase (hTERT); EphA2; ML-IAP; alpha-fetoprotein (AFP); EpCAM; ERG; NA17; PAX3; ALK; androgen receptor (AR); cyclin B1; MYCN; RhoC; tyrosine-related protein 2 (TRP-2); GD3; fucosyl-GM1; PSCA; sLe(a); CYP1B1; PLCA1; GM3; BORIS; Tn; globoH (GloboH); Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETV6-AML); NY-BR-1; RGS5; squamous epithelial cell antigen rejection tumor or 3 (SART3); STn; carbonic anhydrase IX; PAX5; OY-TES1; sperm protein 17; LCK; HMWMAA; AKAP-4; SSX2; B7H3; legumain; These include, but are not limited to, Tie2; Page4; VEGFR2; MAD-CT-1; FAP; PDGFR; MAD-CT-2; Fos-related antigen 1; TAG-72; 9D7; EphA3; telomerase; SAP-1; BAGE family; CAGE family; GAGE ​​family; MAGE family; SAGE family; XAGE family; melanoma preferentially expressed antigen (PRAME); melanocortin 1 receptor (MC1R); beta-catenin; BRCA1 / 2; CDK4; chronic myeloid leukemia 66 (CML66); TGF-β.In certain embodiments, the host autoantigen includes prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.

[0132] In some embodiments, the tumor antigen is derived from a cancer, including acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, pediatric cerebellar or cerebral, basal cell carcinoma, bile duct cancer, extrahepatic, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma / malignant glioma, brain tumor, ependymoma, brain tumor, medulloblastoma, brain tumor, supratentorial primitive neuroectodermal tumor, brain tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's disease. Lymphoma; Carcinoid tumor, pediatric; Carcinoid tumor, gastrointestinal; Cancer of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, pediatric; Cerebral astrocytoma / malignant glioma, pediatric; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Colon cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing's tumor family; Extracranial germ cell tumor, pediatric; Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma ;Gallbladder cancer;Gastric (Stomach) cancer;Gastrointestinal carcinoid tumor;Gastrointestinal stromal tumor (GIST);Germ cell tumors: extracranial, extragonadal, or ovarian;Gestational trophoblastic tumor;Brain stem glioma;Glioma, childhood cerebral astrocytoma;Glioma, childhood visual pathway and hypothalamic;Gastric carcinoid;Hairy cell leukemia;Head and neck cancer;Carcinoma of the heart;Hepatocellular (liver) cancer;Hodgkin's lymphoma;Hypopharyngeal cancer;Hypothalamic and visual pathway glioma, childhood;Intraocular melanoma;Pancreatic islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney cancer (renal cell carcinoma); Laryngeal cancer;Leukemia;Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia);Leukemia, acute myeloid (also called acute myeloid leukemia);Leukemia, chronic lymphocytic leukemia (also called chronic lymphocytic leukemia);Leukemia, chronic myelogenous (also called chronic myeloid leukemia);Leukemia, hairy cell;Lip and oral cavity cancer;Liver cancer (primary);Lung cancer, non-small cell;Lung cancer, small cell;Lymphoma;Lymphoma, AIDS-related;Lymphoma, Burkitt;Lymphoma, cutaneous T-cell;Lymphoma, Hodgkin;Lymphoma, non-Hodgkin (old classification of all lymphomas except Hodgkin); Lymphoma, primary central nervous system; Marcus Whittle, fatal disease; Macroglobulinemia, Waldenstrom; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma, childhood; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma, adult malignant; Mesothelioma, childhood; Metastatic squamous cell carcinoma of the neck of unknown primary; Oral cancer; Multiple endocrine neoplasia syndrome, childhood; Multiple myeloma / plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplastic / myeloproliferative disorder; Myeloid leukemia, chronic; Myeloid leukemia, Adult acute; myeloid leukemia, pediatric acute; myeloma, multiple (cancer of the bone marrow); myeloproliferative disorders, chronic; nasal and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; non-Hodgkin's lymphoma; non-small cell lung cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer (superficial epithelial and stromal tumors); ovarian germ cell tumors; ovarian low malignant potential tumors; pancreatic cancer; pancreatic islet cell cancer; paranasal sinus and nasal cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal astrocytoma; pineal germinoma; pineoblastoma and supratentorial primitive neuroectodermal tumor, pediatric; pituitary gland Adenoma; Plasma cell neoplasm / multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell carcinoma; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sézary syndrome; Skin cancer (non-melanoma); Skin cancer (melanoma); Skin cancer, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (non-melanoma); Cervical squamous cell carcinoma of unknown primary, metastatic; Gastric cancer; Supratentorial primitive neuroectodermal carcinoma These include, but are not limited to, germinal tumors, pediatric; T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome); testicular cancer; throat cancer; thymoma, pediatric; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, pediatric; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, pregnancy; cancer of unknown primary site, adult; cancer of unknown primary site, pediatric; ureter and renal pelvis, transitional cell carcinoma; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; visual pathway and hypothalamic glioma, pediatric; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor (kidney cancer).

[0133] In some embodiments, the pathogen-specific antigen is an MHC-E supertope. In some embodiments, the MHC-E supertope is an HIV epitope. In some embodiments, the MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), Q KQEPIDKEL YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32) are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical. In some embodiments, one or more of the MHC-E supertopes are used to generate a fusion protein. The fusion protein may contain one or more of the MHC-E supertopes in any order.

[0134] In some embodiments, the HCMV vector is administered in an amount effective to induce a CD8+ T cell response against at least one heterologous antigen. In some embodiments, the CD8+ T cell response induced by the vector is characterized by having at least 10% of the CD8+ T cells directed against an epitope presented by MHC-E. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells are restricted by MHC-E. In some embodiments, the MHC-E-restricted CD8+ T cells recognized peptides carried by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against a supertope presented by MHC-E.

[0135] In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from CD8+ T cells induced from the HCMV vector.

[0136] The TCR can be identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes an MHC-E / foreign antigen-derived peptide complex. In some embodiments, the CD8+ TCR recognizes an MHC-E supertope. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), Q KQEPIDKEL YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32) are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0137] In some embodiments, the method further comprises using the supertope peptide to identify MHC-E-restricted CD8+ T cell receptors (TCRs) from CD8+ T cells induced by a non-human primate CMV, such as rhesus or cynomolgus macaque CMV (RhCMV or CyCMV), that lacks expression of orthologs of UL128, UL130, UL146, and UL147 (and optionally UL82) and expresses orthologs of UL40 and US28. The MHC-E-restricted CD8+ T cells are induced in rhesus macaques with RhCMV or in cynomolgus macaques with CyCMV.

[0138] In some embodiments, the CD8+ T cell response elicited by the HCMV vector is characterized by having at least 10% of the CD8+ T cells directed against an epitope presented by MHC-II. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells are restricted by MHC-II. In some embodiments, the MHC-II-restricted CD8+ T cells recognized peptides carried by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against a supertope presented by MHC-II.

[0139] In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from CD8+ T cells induced from the HCMV vector. The TCR may be identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes an MHC-II / heterologous antigen-derived peptide complex. In some embodiments, the CD8+ TCR recognizes an MHC-II supertope.

[0140] In some embodiments, the CD8+ T cell response elicited by a UL18-deficient HCMV vector that also lacks US11 is characterized by having at least 10% of the CD8+ T cells directed against epitopes presented by MHC-Ia. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or at least 95% of the CD8+ T cells are restricted by MHC-Ia.

[0141] In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from CD8+ T cells induced by the UL18 and US11 deleted HCMV vector. The TCR can be identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes an MHC-Ia / heterologous antigen-derived peptide complex.

[0142] Also disclosed herein are methods for generating CD8+ T cells that recognize MHC-E peptide complexes. The method includes administering to a first subject an HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-E / peptide complexes. The HCMV vector includes a first nucleic acid sequence encoding at least one heterologous antigen and does not express the UL18, UL128, UL130, UL146, and UL147 proteins. The vector may also lack the UL82 protein. In some embodiments, the HCMV vector expresses UL40 and US28. In some embodiments, the HCMV vector does not express the UL18, UL138, UL130, UL146, and UL147 proteins and includes nucleic acid sequences encoding UL40, US28, and a microRNA (miRNA) recognition element (MRE). In some embodiments, the MRE includes a target site for a microRNA expressed in endothelial cells. Examples of such miRNAs expressed in endothelial cells are miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, and miR-328.

[0143] The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor or a B cell receptor.

[0144] The method further includes identifying a first CD8+ T cell receptor from the set of CD8+ T cells, where the first CD8+ T cell receptor recognizes an MHC-E / heterologous antigen-derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector, where the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, where the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector can be isolated from the first subject or the second subject.

[0145] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from CD8+ T cells induced by an HCMV vector, wherein the CD8+ T cell receptor recognizes an MHC-E / heterologous antigen-derived peptide complex. In some embodiments, the method further comprises identifying an MHC-E-restricted CD8+ T cell receptor from CD8+ T cells induced by a non-human primate CMV, such as rhesus or cynomolgus macaque CMV (RhCMV or CyCMV), which lacks expression of orthologs of UL128, UL130, UL146, and UL147 and expresses orthologs of UL40 and US28. The MHC-E-restricted CD8+ T cells are induced in rhesus macaques using RhCMV or in cynomolgus macaques using CyCMV. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises identifying a CD8+ T cell receptor that recognizes an MHC-E supertope. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), Q KQEPIDKELYPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32) are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0146] Also disclosed herein is a method for generating TCR-transgenic CD8+ T cells that recognize an MHC-E-peptide complex, the method comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector, and the first CD8+ TCR recognizes an MHC-E / heterologous antigen-derived peptide complex; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR-transgenic CD8+ T cells that recognize an MHC-E-peptide complex.

[0147] (1) administering to a first subject an HCMV vector (deleting UL18, UL128, UL130, UL146, UL147, and in some embodiments, UL82, expressing UL40 and US28, and in some embodiments, expressing a nucleic acid sequence encoding a microRNA recognition element) in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex, wherein the recombinant HCMV vector comprises at least one heterologous antigen; and (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell recognizes the MHC-E / heterologous antigen-derived peptide. Also disclosed are TCR-transfected CD8+ T cells that recognize an MHC-E peptide complex prepared by a process comprising (1) identifying one or more CD8+ T cells that recognize or identify an MHC-E peptide complex from a first subject or a second subject; (2) isolating one or more CD8+ T cells from the first or second subject; and (3) transfecting an expression vector into one or more CD8+ T cells isolated from the first or second subject, thereby generating transfected T cells that recognize an MHC-E peptide complex, and the transfected CD8+ T cells generate an immune response to the MHC-E / heterologous antigen-derived peptide complex.

[0148] In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, the second CD8+ T cell receptor comprising the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more transected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or the second subject.

[0149] In some embodiments, the first and / or second CD8+ T cell receptors are identified by RNA or DNA sequencing. In some embodiments, the first and / or second CD8+ T cell receptors recognize an MHC-E supertope. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), Q KQEPIDKEL YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32) are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0150] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the first and / or second subject is a human or a non-human primate. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In some embodiments, the first subject is a non-human primate and the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

[0151] Also disclosed herein are methods for treating a disease, such as cancer, a pathogen infection, or an immune disease or disorder, comprising administering to a first or second subject transfected T cells that recognize an MHC-E peptide complex. Also disclosed herein are methods for inducing an immune response against a host self-antigen or a tissue-specific antigen, comprising administering to a first or second subject transfected T cells that recognize an MHC-E peptide complex.

[0152] Cancers include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, external genital cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma, and germ cell tumors.

[0153] Pathogen infections include, but are not limited to, human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0154] A method for generating CD8+ T cells that recognize MHC-E peptide complexes, comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-E / supertope peptide complex from a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertope peptide, wherein the set of CD8+ T cells is generated from a recombinant rhesus macaque (RhCMV) or cynomolgus macaque CMV (CyCCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147; and administering an HIV antigen in an amount effective to generate the set of CD8+ T cells. Also disclosed herein are methods comprising: (a) expressing a first CD8+ TCR from a first subject; (b) isolating one or more CD8+ T cells from the second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex.

[0155] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-II peptide complexes. The method includes administering to a first subject (or animal) an amount of a CMV vector effective to generate a set of CD8+ T cells that recognize MHC-II / peptide complexes. The CMV vector includes a first nucleic acid sequence encoding at least one heterologous antigen, and does not express the UL18 protein, UL128 protein, UL130 protein, UL146 protein, or UL147 protein, and in some embodiments, the UL82 protein.

[0156] In some embodiments, the UL18-deficient HCMV vector also comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE). In some embodiments, the MRE comprises a target site for a microRNA expressed in myeloid cells. Examples of such miRNAs expressed in myeloid cells are miR-142-ep, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, and miR-125.

[0157] The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor or a B cell receptor.

[0158] The method further includes identifying a first CD8+ T cell receptor from the set of CD8+ T cells, where the first CD8+ T cell receptor recognizes an MHC-II / heterologous antigen-derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector, where the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, where the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize an MHC-II / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector can be isolated from the first subject or the second subject.

[0159] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the HCMV vector-induced CD8+ T cells, wherein the CD8+ T cell receptor recognizes an MHC-II / heterologous antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises identifying a CD8+ T cell receptor that recognizes an MHC-II supertope.

[0160] The present invention relates to a method for generating CD8+ T cells that recognize MHC-II-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-II / heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize MHC-II / peptide complexes, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-II peptide complexes.

[0161] (1) administering to a first subject an amount of a UL18-deficient HCMV vector (deficient in UL128, UL130, UL146, or UL147 (or a combination thereof), and in some embodiments, UL82, and / or expressing a nucleic acid encoding a microRNA recognition element) that recognizes an MHC-II / peptide complex, wherein the recombinant CMV vector comprises at least one heterologous antigen; and (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell recognizes an MHC-II / heterologous antigen. Also disclosed are TCR-transfected CD8+ T cells that recognize MHC-II-peptide complexes prepared by a process comprising: (1) identifying a heterologous antigen-derived peptide complex; (2) isolating one or more CD8+ T cells from the first subject or the second subject; and (3) transfecting one or more CD8+ T cells isolated from the first or second subject with an expression vector, thereby generating transfected T cells that recognize MHC-II peptide complexes, and the transfected CD8+ T cells generate an immune response to the MHC-II / heterologous antigen-derived peptide complex.

[0162] In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, the second CD8+ T cell receptor comprising the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more truncated CD8+ T cells that recognize an MHC-II / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or the second subject.

[0163] In some embodiments, the first and / or second CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the first and / or second CD8+ T cell receptor recognizes an MHC-II supertope.

[0164] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the first and / or second subject is a human or a non-human primate. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In some embodiments, the first subject is a non-human primate and the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

[0165] Also disclosed herein are methods for treating a disease, such as cancer, a pathogen infection, or an immune disease or disorder, comprising administering to a first or second subject transfected T cells that recognize an MHC-II peptide complex. Also disclosed herein are methods for inducing an immune response against a host self-antigen or a tissue-specific antigen, comprising administering to a first or second subject transfected T cells that recognize an MHC-II peptide complex.

[0166] Cancers include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, external genital cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma, and germ cell tumors.

[0167] Pathogen infections include, but are not limited to, human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0168] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-Ia peptide complexes. The method includes administering to a first subject an UL18-deficient CMV vector that also lacks the US11 protein in an amount effective to generate a set of CD8+ T cells that recognize MHC-Ia / peptide complexes. The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and does not express the US11 protein and the UL18 protein. The vector may also lack the UL128 protein, the UL130 protein, or the UL146 protein, the UL147 protein, and / or the UL82 protein. The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable region of a T cell receptor or a B cell receptor.

[0169] The method further includes identifying a first CD8+ T cell receptor from the set of CD8+ T cells, where the first CD8+ T cell receptor recognizes an MHC-Ia / heterologous antigen-derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector, where the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, where the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize an MHC-Ia / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector can be isolated from the first subject or the second subject.

[0170] In some embodiments, the method further includes identifying a CD8+ T cell receptor from the CMV vector-induced CD8+ T cells, wherein the CD8+ T cell receptor recognizes an MHC-Ia / heterologous antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0171] Also disclosed is a method for generating CD8+ T cells that recognize an MHC-I-peptide complex, the method comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-I / xenogenous antigen-derived peptide complex from a set of CD8+ T cells that recognize an MHC-I / xenogenous antigen-derived peptide complex, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, and the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-I-peptide complex.

[0172] (1) administering to a first subject an amount of a CMV vector lacking US11 and UL18 (further, the vector may lack UL128, UL130, UL146, UL147, and / or UL82 and express UL40 and / or US28) effective to generate a set of CD8+ T cells that recognize an MHC-Ia / peptide complex, wherein the recombinant CMV vector comprises at least one heterologous antigen; and (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes an MHC-Ia / heterologous antigen-derived peptide. Also disclosed are transfected CD8+ T cells that recognize an MHC-Ia peptide complex prepared by a process that includes (1) identifying a CD8+ T cell that recognizes or identifies a peptide complex; (2) isolating one or more CD8+ T cells from the first subject or the second subject; and (3) transfecting one or more CD8+ T cells isolated from the first or second subject with an expression vector, thereby generating transfected T cells that recognize an MHC-Ia peptide complex, and wherein the transfected CD8+ T cells generate an immune response to the MHC-Ia / heterologous antigen-derived peptide complex.

[0173] In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, the second CD8+ T cell receptor comprising the CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more truncated CD8+ T cells that recognize an MHC-Ia / heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or the second subject.

[0174] In some embodiments, the first and / or second CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0175] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In some embodiments, the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

[0176] Also disclosed herein are methods for treating a disease, such as cancer, a pathogen infection, or an immune disease or disorder, comprising administering to a first or second subject transfected T cells that recognize an MHC-Ia peptide complex. Also disclosed herein are methods for inducing an immune response against a host self-antigen or a tissue-specific antigen, comprising administering to a first or second subject transfected T cells that recognize an MHC-Ia peptide complex.

[0177] Cancers include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, external genital cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma, and germ cell tumors.

[0178] Pathogen infections include, but are not limited to, human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

[0179] III. HIV Supertope Constructs A nucleotide sequence having a length of 9 to 15 amino acids, such as LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), Q KQEPIDKEL Also disclosed herein are human immunodeficiency virus antigens that are at least 90%, at least 95%, or 100% identical to the amino acid sequence of YPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

[0180] In some embodiments, the recombinant HCMV vector comprises a nucleic acid encoding one or more human immunodeficiency virus antigens. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, or UL147 proteins, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an orthologue thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a miRNA expressed in endothelial cells. In some embodiments, the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, wherein the MRE comprises a target site for an miRNA expressed in myeloid cells.In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

[0181] The CMV vectors disclosed herein can be used as immunogenic or vaccine compositions comprising a recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. Immunologic compositions comprising a recombinant CMV virus or vector (or its expression product) induce a local or systemic immune response. The response may be, but need not be, protective. Vaccine compositions induce a local or systemic protective or therapeutic response. Thus, the term "immunogenic composition" includes "vaccine composition" (as the former term may also refer to a protective composition).

[0182] The recombinant CMV vectors disclosed herein can be used in methods of inducing an immune response in a subject, comprising administering to the subject an immunogenic, immunological, or vaccine composition comprising the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.

[0183] The recombinant CMV vectors disclosed herein can be used in therapeutic compositions comprising a recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. The CMV vectors disclosed herein can be prepared by inserting DNA containing a sequence encoding a tumor antigen into an essential or non-essential region of the CMV genome. The method can further include deleting one or more regions from the CMV genome. The method can include in vivo recombination. Thus, the method can include transfecting CMV DNA into cells in a cell-compatible medium in the presence of donor DNA containing heterologous DNA flanked by DNA sequences homologous to a portion of the CMV genome, thereby introducing the heterologous DNA into the CMV genome, and optionally recovering the CMV modified by in vivo recombination. The method may include cleaving CMV DNA to obtain truncated CMV DNA, ligating heterologous DNA to the truncated CMV DNA to obtain a hybrid CMV-heterologous DNA, transfecting a cell with the hybrid CMV-heterologous DNA, and optionally then recovering CMV modified by the presence of the heterologous DNA. Because in vivo recombination is involved, the method also provides a plasmid containing donor DNA not naturally occurring in CMV that encodes a polypeptide foreign to CMV, the donor DNA being within a segment of CMV DNA that is otherwise co-linear with an essential or non-essential region of the CMV genome, such that DNA from an essential or non-essential region of CMV flanks the donor DNA. The heterologous DNA may be inserted into CMV in any orientation that results in stable integration of the DNA and, if desired, expression thereof, to generate a recombinant CMV.

[0184] The DNA encoding the heterologous antigen in the recombinant CMV vector may also contain a promoter. The promoter may be derived from any source, such as a herpesvirus, including endogenous cytomegalovirus (CMV) promoters, such as human CMV (HCMV), rhesus macaque CMV (RhCMV), mouse, or other CMV promoters. The promoter may also be a non-viral promoter, such as the EF1α promoter. The promoter may be a truncated transcriptionally active promoter containing a region transactivated by a viral transactivation protein and the minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter may consist of a minimal promoter and a DNA sequence corresponding to upstream regulatory sequences. The minimal promoter consists of a CAP site and an ATA box (the minimal sequence for the basal level of transcription, i.e., the unregulated level of transcription), and the "upstream regulatory sequences" consist of upstream elements and enhancer sequences. Furthermore, the term "truncated" indicates that the full-length promoter is not completely present, i.e., a portion of the full-length promoter has been removed. The truncated promoter can also be derived from a herpesvirus such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. Based on base pairs, the size can be up to 40%, or even up to 90%, reduced from the full-length promoter. The promoter can also be a modified non-viral promoter. For HCMV promoters, see U.S. Patent Nos. 5,168,062 and 5,385,839. For transfecting cells with plasmid DNA for expression, see Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561. For direct injection of plasmid DNA as a simple and effective vaccination method against various infectious diseases, see Science, 259:1745-49, 1993. Therefore, it is within the scope of this disclosure that vectors can be used by direct injection of vector DNA.

[0185] Also disclosed is an expression cassette that can be inserted into a recombinant virus or plasmid containing a truncated transcriptionally active promoter.The expression cassette can further comprise a functional truncated polyadenylation signal, such as a truncated but still functional SV40 polyadenylation signal.Considering that larger signals are naturally provided, it is quite surprising that the truncated polyadenylation signal is functional.The truncated polyadenylation signal addresses the problem of insert size limitations of recombinant viruses such as CMV.The expression cassette can also comprise heterologous DNA, and the DNA can be the heterologous DNA described herein, relative to the virus or system into which the heterologous DNA is inserted.

[0186] With regard to antigens for use in vaccines or immune compositions, see also Stedman's Medical Dictionary (24th ed., 1982, e.g., the definition of vaccine, for a list of antigens used in vaccine formulations). Any such antigens of interest or epitopes derived from those antigens may be used. With regard to tumor antigens, one skilled in the art can select tumor antigens and their encoding DNA without undue experimentation from knowledge of the amino acids and corresponding DNA sequences of peptides or polypeptides, as well as the properties of particular amino acids (e.g., size, charge, etc.) and the codon dictionary.

[0187] One method for determining the T epitope of an antigen involves epitope mapping. Overlapping peptides of tumor antigens are produced by oligopeptide synthesis. Then, each peptide is tested for its ability to induce T cell activation. This approach is particularly useful for mapping T cell epitopes, because T cells recognize short linear peptides complexed with MHC molecules.

[0188] Immune responses to tumor antigens are generally generated as follows: T cells recognize proteins only when they are cleaved into smaller peptides and presented in complexes called "major histocompatibility complexes (MHC)" located on the surface of other cells. There are two classes of MHC complexes, class I and class II, and each class consists of many different alleles. Different species and individual subjects have different types of MHC complex alleles. They are said to have different MHC species. One type of MHC class I molecule is called MHC-E (HLA-E in humans, Mamu-E in RM, and Qa-lb in mice). Unlike other MHC-I molecules, MHC-E is highly conserved within and between mammalian species.

[0189] It should be noted that the DNA containing the tumor antigen-encoding sequence may itself contain a promoter to drive expression in the CMV vector, or the DNA may be limited to the tumor antigen-encoding DNA. This construct may be operably linked to the promoter and positioned in an orientation relative to the endogenous CMV promoter so that it is expressed. Furthermore, multiple copies of the tumor antigen-encoding DNA, or the use of strong or early promoters or early and late promoters, or any combination thereof, may be used to amplify or increase expression. Thus, the tumor antigen-encoding DNA may be appropriately positioned relative to the CMV endogenous promoter, or the promoters may be repositioned so that they are inserted into a different location along with the tumor antigen-encoding DNA. Nucleic acids encoding two or more tumor antigens may be packaged in a CMV vector.

[0190] Pharmaceutical compositions and other compositions containing the CMV vectors of the present disclosure are also disclosed. Such pharmaceutical compositions and other compositions can be formulated for use in any administration procedure known in the art. Such pharmaceutical compositions can be administered via parenteral routes (intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous, or other). Administration can also be via mucosal routes such as oral, nasal, or genital.

[0191] Pharmaceutical compositions of the present disclosure can be prepared according to standard techniques well known to those skilled in the pharmaceutical arts. Such compositions can be administered at dosages and by techniques well known to those skilled in the medical arts, taking into account factors such as the breed or species, age, sex, weight, and condition of the particular patient, as well as the route of administration. The compositions can be administered alone or simultaneously or sequentially with other CMV vectors, or with other immunological, antigenic, or vaccine, or therapeutic compositions. Such other compositions can include purified native antigens or epitopes, or antigens or epitopes from expression by recombinant CMV or another vector system, and are administered taking into account the above factors.

[0192] Examples of compositions include liquid preparations, such as suspensions, syrups, or elixirs, for administration into an orifice, e.g., oral, nasal, anal, genital, e.g., vaginal, etc., as well as preparations, such as sterile suspensions or emulsions, for parenteral, subcutaneous, intraperitoneal, intradermal, intramuscular, or intravenous administration (e.g., injectable administration). In such compositions, the recombinant may be in a mixture with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, etc.

[0193] Antigen, immune, or vaccine compositions typically contain an adjuvant and a certain amount of CMV vector or expression product to elicit the desired response. For human use, alum (aluminum phosphate or aluminum hydroxide) is a typical adjuvant. Saponin and its purified component, QuilA, complete Freund's adjuvant, and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, e.g., those described in Goodman-Snitkoff et al., J Immunol. 147:410-415 (1991), encapsulation of proteins in proteoliposomes as described in Miller et al., J Exp. Med. 176:1739-1744 (1992), and encapsulation of proteins in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) can also be used.

[0194] The composition may be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or by orifice administration, e.g., sublingual (e.g., oral), intragastric, buccal, anal, vaginal, etc., administration. Again, the effective dosage and route of administration are determined by the nature of the composition, the nature of the expression product, the expression level if recombinant CMV is used directly, and known factors such as the species or species, age, sex, weight, condition, and characteristics of the host, as well as the LD50 and other known screening procedures that do not require undue experimentation. Doses of the expressed product may range from a few micrograms to hundreds of micrograms, e.g., 5 to 500 μg. The CMV vector may be administered in any suitable amount to achieve expression at these dosage levels. In a non-limiting example, the CMV vector may be administered in an amount of at least 102 pfu; thus, the CMV vector may be administered in at least this amount, or in a range of about 102 pfu to about 107 pfu. Other suitable carriers or diluents can be water or buffered saline, with or without preservatives. The CMV vector can be lyophilized or in solution for resuspension at the time of administration. "About" can mean within 1%, 5%, 10%, or 20% of a defined value.

[0195] It should be understood that the proteins of the present disclosure and the nucleic acids encoding them may differ from the exact sequences shown and described herein. Thus, the present disclosure contemplates deletions, additions, truncations, and substitutions to the sequences shown, as long as the sequences function according to the methods of the present disclosure. In this regard, substitutions are generally conservative in nature, i.e., substitutions that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, and histidine; (3) nonpolar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonably predictable that isolated substitutions of leucine with isoleucine or valine, or vice versa, of aspartate with glutamate, or vice versa, of threonine with serine, or similar conservative substitutions of amino acids with structurally related amino acids will not significantly affect biological activity. Thus, proteins having substantially the same amino acid sequence as the described proteins but with minor amino acid substitutions that do not substantially affect the immunogenicity of the proteins are within the scope of the present disclosure.

[0196] The nucleotide sequence of the present disclosure can be codon-optimized, for example, codons can be optimized for use in human cells.For example, any virus or bacterial sequence can be modified in this way.Many viruses, including HIV and other lentiviruses, use many rare codons, as described in Andre et al., J Virol.72:1497-1503,1998, and by modifying these codons to correspond to the commonly used codons in the desired target, the expression of tumor antigens can be enhanced.

[0197] Nucleotide sequences encoding functionally and / or antigenically equivalent variants and derivatives of CMV vectors and the glycoproteins contained therein are contemplated. These functionally equivalent variants, derivatives, and fragments exhibit the ability to retain antigenic activity. For example, DNA sequence changes that do not alter the encoded amino acid sequence, as well as changes resulting in conservative substitutions of amino acid residues, deletions or additions of one or several amino acids, and substitutions of amino acid residues with amino acid analogs, do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions include glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine / methionine, lysine / arginine, and phenylalanine / tyrosine / tryptophan. In some embodiments, variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to an antigen, epitope, immunogen, peptide, or polypeptide of interest.

[0198] Sequence identity or homology is determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence gap.In particular, sequence identity can be determined using any of many mathematical algorithms.A non-limiting example of the mathematical algorithm used to compare two sequences is the algorithm of Karlin & Altschul, Proc.Natl.Acad.Sci.USA 1990;87:2264-2268, modified as in Karlin & Altschul, Proc.Natl.Acad.Sci.USA 1993;90:5873-5877.

[0199] Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers & Miller, CABIOS 1988;4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing an align program for comparing amino acid sequences, a PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another algorithm useful for identifying regions of local sequence similarity and alignment is the FASTA algorithm described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988;85:2444-2448.

[0200] WU-BLAST (Washington University BLAST) version 2.0 software is advantageous for use with the present disclosure. A WU-BLAST version 2.0 executable program for some UNIX platforms can be downloaded. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266:460-480; Altschul et al., Journal of Molecular Biology 1990; 215:403-410; Gish & States, 1993; Nature Genetics 3:266-272; Karlin & Altschul, 1993; Proc. Natl. Acad. Sci. USA 90:5873-5877, all of which are incorporated herein by reference).

[0201] The various recombinant nucleotide sequences and antibodies and / or antigens of the present disclosure are produced using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See, for example, "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al. 1989).

[0202] Any vector that allows the virus of the present disclosure to be expressed can be used according to the present disclosure.In certain embodiments, the virus of the present disclosure can be used in vitro (for example, by using cell-free expression system) and / or in vitro grown cultured cells to produce the encoded heterologous antigen (for example, tumor virus antigen, HIV antigen, tumor antigen and antibody), which can be used for various applications such as the production of protein vaccines.For such applications, any vector that allows the virus to be expressed in vitro and / or cultured cells can be used.

[0203] For the tumor antigens of the present disclosure to be expressed, the protein-coding sequence of the tumor antigen must be "operably linked" to a regulatory sequence or nucleic acid control sequence that directs the transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be "operably linked" when they are covalently linked in such a manner that the expression, transcription, and / or translation of the coding sequence is under the influence or control of the nucleic acid control sequence. A "nucleic acid control sequence" can be any nucleic acid element, such as, but not limited to, a promoter, enhancer, IRES, intron, and other elements described herein, that directs the expression of a nucleic acid sequence or coding sequence operably linked to the nucleic acid element. The term "promoter" is used herein to refer to a group of transcriptional control modules that are clustered around the initiation site of RNA polymerase II and, when operably linked to a protein-coding sequence of the present disclosure, cause the expression of the encoded protein. The expression of the transgene of the present disclosure can be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when exposed to some specific external stimuli, such as, but not limited to, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals. The promoter can also be specific to a particular cell type, tissue, or organ. Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer can be used for the expression of the transgene of the present disclosure. For example, a suitable promoter and / or enhancer can be selected from the Eukaryotic Promoter Database (EPDB).

[0204] The vectors used in accordance with the present disclosure may include suitable gene regulatory regions, such as promoters or enhancers, so that the antigens of the present disclosure may be expressed.

[0205] The CMV vectors described herein may contain mutations that prevent host-to-host spread, thereby preventing the virus from infecting immunocompromised or other subjects that may suffer complications as a result of CMV infection. The CMV vectors described herein may also contain mutations that result in the presentation of immunodominant and non-immunodominant epitopes and atypical MHC restriction. However, the mutations in the CMV vectors described herein do not affect the vector's ability to reinfect subjects previously infected with CMV. Such CMV mutations are described, for example, in U.S. Patent Publication Nos. 2013-013676S, 2010-0142S23, 2014-014103S, and PCT Application Publication No. WO2014 / 13S209, all of which are incorporated herein by reference.

[0206] The CMV vectors of the present disclosure can be administered in vivo to generate immunogenic responses, including CD8+ immune responses, including immune responses characterized by a high proportion of CD8+ T cell responses restricted by MHC-E, MHC-II, or MHC-I (or their homologs or orthologs). For example, in some instances, it may be desirable to use the CMV vectors of the present disclosure for preclinical testing of immunogenic compositions and vaccines using RhCMV in laboratory animals such as rhesus monkeys. In other instances, it may be desirable to use the CMV vectors of the present disclosure for actual clinical use of immunogenic compositions using HCMV in human subjects, such as in clinical trials.

[0207] For such in vivo applications, the CMV vectors of the present disclosure are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions of the present disclosure are useful for stimulating an immune response against heterologous antigens, including tumor antigens, tumor virus antigens, or host self-antigens, and may be used as one or more components of a prophylactic or therapeutic vaccine against tumor antigens, tumor virus antigens, or host self-antigens for the prevention, amelioration, or treatment of cancer. The nucleic acids and vectors of the present disclosure are particularly useful for providing genetic vaccines, i.e., for delivering nucleic acids encoding the antigens of the present disclosure to a subject, such as a human, so that the antigen is then expressed in the subject and induces an immune response.

[0208] Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for a particular subject and immunogenic composition. Thus, the immunogen may be administered to a subject one or more times. Preferably, there is a set time interval between separate administrations of the immunogenic composition. This interval varies from subject to subject but typically ranges from 10 days to several weeks, and often is 2, 4, 6, or 8 weeks. In humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present disclosure, the intervals are longer, advantageously about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 weeks. The immunization regime typically involves one to six administrations of the immunogenic composition, but may be as few as one, two, or four. The method of inducing an immune response may also include administering an adjuvant with the immunogen. In some cases, the initial immunization protocol may be supplemented with booster immunizations annually, twice a year, or at other longer intervals (5-10 years). The methods of the present invention also include various prime-boost regimens. In these regimens, one or more priming immunizations are followed by one or more boosting immunizations. The actual immunogenic composition may be the same or different for each immunization, and the type of immunogenic composition (e.g., including a protein or expression vector), route, and formulation of the immunogen may also vary. For example, if an expression vector is used in the priming and boosting steps, it may be of the same or different types (e.g., DNA or bacterial or viral expression vectors). One useful prime-boost regimen provides two priming immunizations four weeks apart, followed by two boosting immunizations at weeks four and eight after the final priming immunization.It should also be readily apparent to one skilled in the art that several permutations and combinations may be encompassed to provide priming and boosting regimens using the DNA, bacterial, and viral expression vectors of the present disclosure. CMV vectors may be used repeatedly, expressing different antigens derived from different pathogens. [Example]

[0209] Example 1: Protection against SIV by induction of MHC-E-restricted CD8+ T cells Several studies have shown that strain 68-1-derived RhCMV vectors expressing SIV antigens can control and ultimately eliminate infection by the highly pathogenic SIVmac239 (Hansen 2019. A live-attenuated RhCMV / SIV vaccine shows long-term efficacy against heterologous SIV challenge. Science Translational Medicine 11:eaaw2607; Hansen 2013. Immune clearance of highly pathogenic SIV infection. Nature 502:100-4). This protection correlated with the ability of strain 68-1RhCMV to induce MHC-II and MHC-E restricted CD8+ T cells (Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science 351:714-20; Hansen. Cytomegalovirus Vectors Violate CD8+ T Cell Epitope Recognition Paradigms. Science 340:1237874-1237874). However, it was not known whether MHC-II and / or MHC-E restricted CD8+ T cells were required for this protection.

[0210] Thus, the ability to specifically program CD8 + T cells exclusively restricted by MHC-E or MHC-II allowed us to examine whether MHC-E or MHC-II-restricted CD8 + T cells are responsible for unique protection against SIVmac239.A cohort of four rhesus macaques (RM) was inoculated with different 68-1RhCMV strains as described below.

[0211] Cohort 1: Nine RMs were inoculated with three 68-1RhCMV "MHC-E only" vectors (one insert per vector) each containing three mir126 recognition sites in the 3' untranslated regions of the essential genes Rh108 (UL79) and Rh156 (IE2) and expressing the 5' segments of the SIV antigens SIVgag, SIVretanef (a fusion of rev, tat, and nef), and SIVpol, respectively.

[0212] Cohort 2: 15 RMs were inoculated with three 68-1RhCMV “MHC-II only” vectors lacking Rh67 (UL40) and expressing the 5′ segment of the SIV antigens SIVgag, SIVretanef (a fusion of rev, tat, and nef), and SIVpol (one insert per vector), respectively.

[0213] Cohort 3: Twelve RMs were inoculated with three 68-1RhCMV "MHC-II only" vectors (one insert per vector) each containing three mir142 recognition sites in the 3' untranslated regions of the essential genes Rh108 (UL79) and Rh156 (IE2) and expressing the 5' segments of the SIV antigens SIVgag, SIVretanef (a fusion of rev, tat, and nef), and SIVpol, respectively.

[0214] Cohort 4: (control cohort) 15 RMs were inoculated with three 68-1RhCMV vectors expressing the 5′ segment of the SIV antigens SIVgag, SIVretanef (a fusion of rev, tat, and nef), and SIVpol, respectively (one insert per vector).

[0215] The mean frequencies of CD4+ or CD8+ T cells responding to SIV antigen-derived peptide pools were quantified. T cell frequencies were determined in peripheral blood mononuclear cells (PBMCs) at the indicated time points by intracellular cytokine staining for IFNγ or TNFα in the presence of pools of overlapping (by 11A) 15-mer peptides representing SIV antigens. Each of the RMs generated robust CD4+ and CD8+ T cell responses to each of the SIV antigens (Figure 1).

[0216] Next, we analyzed the MHC restriction of SIVgag-specific CD8+ T cell responses. SIVgag-specific CD8+ T cell responses were measured in PBMCs obtained from three RMs in each cohort in the presence of individual peptides. MHC restriction was determined by blocking with the anti-pan-MHC-I mAb W6 / 32, the MHC-E blocking peptide VL9, and the MHC-II blocking peptide CLIP. All peptide responses in animals in cohort 1 were blocked by the VL9 peptide, while peptide responses in cohorts 2 and 3 were blocked by the CLIP peptide (Figure 2). Thus, CD8+ T cells in cohort 1 are exclusively restricted by MHC-E, and CD8+ T cells in cohorts 2 and 3 are exclusively restricted by MHC-II. CD8+ T cell responses in cohort 4 animals (not shown) were restricted by both MHC-II and MHC-E, as previously reported (Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science 351:714-20; Hansen 2013. Cytomegalovirus Vectors Violate CD8+ T Cell Epitope Recognition Paradigms. Science 340:1237874-1237874).

[0217] To determine whether MHC-E or MHC-II-restricted CD8+ T cells contribute to protection, cohorts 1, 2, and 3 were challenged with repeated intrarectal inoculations of dose-limited SIVmac239. RMs were challenged weekly until the first plasma viral load (pvl) or SIVvif response was detected (the onset of infection was defined as the previous challenge). Because the vaccine vector does not express SIVvif, the development of a de novo SIVvif response in the absence of a detectable SIV plasma viral load constitutes evidence of infection. Unlike non-controllers (black boxes), who, once infected, exhibit persistent viremia with a typical peak-and-plateau pattern, RMs were considered controllers if plasma viremia was not observed or became undetectable within 2 weeks of the first positive pvl and then remained below the threshold for at least 4 of the following 5 weeks (white boxes).

[0218] All animals in cohorts 2 and 3 developed systemic, progressive SIV viremia, suggesting that MHC-II-restricted CD8+ T cells failed to confer protection against SIVmac239 infection (Figure 3). In contrast, 6 / 9 (67%) of animals in cohort 1 vaccinated with the 68-1RhCMV / SIV / miR126 vector tightly controlled SIVmac239 infection. These data indicate that MHC-E-restricted CD8+ T cell responses conferred protection against virulent SIV.

[0219] It has previously been shown that strain 68-1-derived RhCMV vectors elicit CD8+ T cell responses exhibiting very high epitope density (i.e., the number of peptides recognized by T cells within a given antigen) (Hansen. 2013. Cytomegalovirus Vectors Violate CD8+ T Cell Epitope Recognition Paradigms. Science 340:1237874-1237874). It has further been shown that several of these MHC-E and MHC-II epitopes, so-called supertopes, are recognized in all animals (Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science 351:714-20). Supertopes have not been described in terms of "classical" epitopes presented by MHC-I molecules and therefore represent a unique feature of CMV-based vectors. To determine whether supertopes alone could explain the protection observed with the “MHC-E only” RhCMV vector described above, we generated artificial fusion proteins consisting of supertope sequences derived from individual SIV antigens ( Table 1 ; 15-mer and minimal supertope peptide sequences are underlined). [Table 1]

[0220] The sequence of the artificial fusion protein is as follows (the HA-epitope tag is underlined): MRRWRRRWQQLLALADRIYSFPDPTSSASNKPISNRTRHCQPEISMRRSRPSGGDLRQRLLRAEKLAYRKQNMDDIDEEDDDAQTSQWDDPWGEVLAWKFDYVRYPEEFGSKS GLSEEEVGGIGGFINTKEYKNVEIVLGKRNTPTFAIKKKDKNKWRMLIDFREWMGYELWPTKWKLQKIELPLGLQKCVRMYNPTNILDVKYMQLGKQQREKQRESREKPYKEV YPYDVPDYAD (SEQ ID NO: 12). Immunoblotting was performed to demonstrate expression of the SIV supertope fusion construct by probing with anti-HA antibodies (FIG. 4).

[0221] With the goal of focusing CD8+ T cell responses against a small set of MHC-E-restricted epitopes, we inserted an SIV MHC-E supertope fusion protein into 68-1RhCMV containing the mir126 targeting site. The resulting construct was inoculated into eight RMs (cohort 5). T cell frequencies were determined at the indicated time points in peripheral blood mononuclear cells (PBMCs) by intracellular cytokine staining for IFNγ or TNFα in the presence of pools of individual 15-mer peptides representing SIV supertopes (Figure 5). CD8+ T cells responded to SIV antigen-derived peptides (Figure 5A). CD8+ T cells responded to the MHC-E-restricted supertopes Gag69 and Gag120 but not to other MHC-E-restricted GAG epitopes commonly recognized by CD8+ T cells from RMs immunized with the 68-1RhCMV / gag vector expressing the entire SIVgag insert (Figure 5B). These results show that all animals elicited SIV-specific CD8+ T cell responses exclusively against the supertope.

[0222] To determine whether MHC-E supertope-restricted CD8+ T cells could recapitulate the protection observed with the "MHC-E-only" vector, cohort 5 was challenged by repeated intrarectal inoculations of low doses of SIVmac239 as described above. RMs were challenged weekly until the first plasma viral load (pvl) or SIVvif response was detected (the onset of infection was defined as the previous challenge). In contrast to non-controllers, who, once infected, exhibit persistent viremia with a typical peak-and-plateau pattern (black boxes), RMs were considered controllers if pvl became undetectable within 2 weeks of the first positive pvl and then remained below the threshold for at least 4 of the following 5 weeks (black boxes).

[0223] Importantly, 5 / 7 (71%) of the animals vaccinated with a single 68-1RhCMV / SIV / miR126 vector expressing the supertope fusion protein controlled infection with SIVmac239 (Figure 6). These data indicate that CD8+ T cells specific for the MHC-E supertope are involved in protection against highly pathogenic SIV.

[0224] To design HIV-based supertope antigens, we mapped HIV supertopes by inserting HIV antigens into 68-1RhCMV and inoculating RMs. Table 2 contains a list of the identified HIV supertopes. The optimal minimal peptide sequences are underlined. [Table 2]

[0225] Example 2: Expression of UL18 prevents the induction of MHC-E and MHC-II restricted CD8+ T cells. To determine the effect of UL18 on the ability of strain 68-1 RhCMV vector to elicit MHC-II and MHC-E restricted CD8+ T cell responses, two RhCMV constructs were generated:

[0226] Construct 1: 68-1RhCMV, which contains an expression cassette for the 5' fragment of SIVpol under the control of the EF1α promoter in the RhCMV gene Rh211 as the vector backbone. UL18 was inserted by replacing the gene Rh13.1, and is therefore expressed instead of Rh13.1. The inserted UL18 sequence corresponds to the UL18 of the HCMV TR isolate.

[0227] Construct 2: 68-1RhCMV, in which the gene Rh107 (a homologue of HCMV UL78) was replaced with a fusion protein of SIVrev, tat, and nef (SIVrtn) as the vector backbone. UL18 was inserted by replacing the gene Rh13.1.

[0228] 5x10 of construct 1 6 Plaque-forming units (PFU) of construct 2 were inoculated into three RhCMV-seropositive RMs on day 0, and the same amount of construct 2 was inoculated into two RhCMV-seropositive RMs on day 0. For control, RMs were inoculated with 68-1RhCMV expressing SIVgag under the control of the EF1α promoter.

[0229] On days 7, 14, and every other week thereafter, PBMCs were isolated from two RMs, and CD8+ T cell responses to SIV antigens elicited by constructs 1, 2, or control were measured by intracellular cytokine staining (ICS) for IFNγ and TNFα using overlapping 15mer peptide pools encompassing SIVpol, SIVrtn, or SIVgag, respectively. To specifically detect CD8+ T cells that recognize peptides in the context of MHC-E or MHC-II, it was advantageous for all animals to have a supertope within each SIV antigen (Hansen Science 2013, Hansen Science 2016). Therefore, each supertope peptide was tested individually by ICS in PBMCs from each RM.

[0230] Two animals from each group were analyzed for the frequency of CD8+ T cells responding to the SIV antigen peptide pool, representing the total antigen-specific response (Fig. 7A).The same two animals were also analyzed for the frequency of CD8+ T cells responding to MHC-E-restricted and MHC-II-restricted supertopes (Fig. 7B, 7C).

[0231] All animals generated CD8+ T cell responses to SIV antigens expressed by the RhCMV vector used for inoculation. However, supertope responses were observed only with 68-1RhCMV / SIVgag, and both vectors expressing UL18 failed to elicit T cells that recognized the supertope. These results indicate that UL18 prevents the induction of MHC-E- and MHC-II-restricted CD8+ T cells.

[0232] Next, MHC restriction mapping was performed to further determine whether MHC molecules were involved in eliciting SIVpol-specific responses in the three animals that received UL18-expressing 68-1RhCMV / SIVpol. SIVpol-specific CD8+ T cell responses were measured in PBMCs from three RMs vaccinated with construct 1 in the presence of individual peptides. CD8+ T cell responses to individual peptides within SIVpol were measured in the presence of specific reagents that block either MHC-I, MHC-II, or MHC-E presentation (MHC-I and MHC-E were blocked with antibody W6 / 32, MHC-II was blocked with an HLA-DR-specific antibody and CLIP peptide, and MHC-E was blocked with the VL9 peptide).

[0233] The results shown in Figure 8 reveal that stimulation of CD8+ T cells by each individual peptide was inhibited by the pan-MHC-I blocking antibody W6 / 32, but not by the MHC-E-specific peptide VL9 or the MHC-II-specific antibody and CLIP peptide. Thus, all CD8+ T cell epitopes are restricted by MHC-I. In contrast, CD8+ T cells from animals vaccinated with 68-1RhCMV-expressing SIV antigens recognize all peptides in the context of MHC-II or MHC-E (Hansen Science 2013, Hansen Science 2016).

[0234] These results indicate that UL18 reprogrammed CD8+ T cell responses, most likely by preventing the induction of MHC-II- and MHC-E-restricted CD8+ T cells. UL18 is known to associate with the host inhibitory receptor LIR-1 (Yang Z, Bjorkman PJ. 2008. Structure of UL18, a peptide-binding viral MHC mimic, bound to a host inhibitory receptor. Proc Natl Acad Sci USA 105:10095-100; Chapman TL, Heikeman AP, Bjorkman PJ. 1999. The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18. Immunity 11:603-13). Thus, a possible mechanism for this reprogramming is that UL18 prevents direct priming of CD8+ T cells by 68-1RhCMV (direct priming refers to T cells being primed by infected cells) by engaging inhibitory leukocyte inhibitory receptors (LIRs) on T cells. In the absence of direct priming, CD8+ T cells are indirectly induced by cross-priming, i.e., by uninfected cells (e.g., dendritic cells) that present antigens obtained from infected cells. Previously, UL18 has not been implicated in preventing T cell priming. These results are unexpected and unprecedented.

[0235] To determine whether interaction with the inhibitory receptor LIR1 contributes to UL18's ability to block the induction of MHC-II- and MHC-E-restricted CD8+ T cells, we mutated the UL18 coding region in construct 1 described above to replace the aspartate residue at amino acid position 196 in the α-3 domain with a serine (D196S). Previous structural studies have shown that this aspartate residue is involved in UL18 binding to LIR1 (Yang Z, Bjorkman PJ. 2008. Structure of UL18, a peptide-binding viral MHC mimic, bound to a host inhibitory receptor. Proc Natl Acad Sci USA 105:10095-100). Furthermore, this residue is conserved in all LIR1-binding HLA molecules but is absent in HLA-like molecules that do not bind LIR1. The D196S mutant of UL18 was inserted into 68-1RhCMV expressing SIVpol, and the resulting construct was inoculated into two RMs. On day 91, PBMCs were isolated, and CD8+ T cell responses to SIVpol were measured by ICS for IFNγ and TNFα using pools of overlapping 15-mer peptides encompassing SIVpol or the SIVpol MHC-E supertope peptide Pol41 (GFINTKEYKNVEIEV; SEQ ID NO: 33) or the MHC-II supertope Pol90 (LPQGWKGSPAIFQYT; SEQ ID NO: 34). In contrast to animals inoculated with 68-1RhCMV expressing intact UL18 (Figure 9A), T cell responses to both SIVpol supertopes were observed in animals inoculated with 68-1RhCMV expressing the D196S mutant of UL18 (Figure 9B). Thus, these results indicated that UL18 must engage the LIR1 receptor to prevent the induction of MHC-E and MHC-II restricted CD8 + T cells.

[0236] UL18 is thought to play a role in NK cell evasion (Prod'homme 2007. The human cytomegalovirus MHC class I homolog UL18 inhibits LIR-1+ but activates LIR-1- NK cells. J Immunol 178:4473-81). Because NK cell evasion may be important for vector function (Sturgill 2016. Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus. PLoS Pathog 12:e1005868), we hypothesized that deletion of UL18 from HCMV-based vectors would hinder their ability to elicit immune responses against heterologous antigens. To determine whether UL18-deleted HCMV vectors could elicit T cell responses against inserted antigens, we replaced UL18 with HIV antigens, thereby deleting UL18, and used the endogenous UL18 promoter to drive expression of the HIV gag / nef / pol fusion protein. Additionally, the genes UL128, UL130, UL146, and UL147 were also deleted from the UL18 deletion vector because these gene products have previously been shown to inhibit MHC-E and MHC-II-restricted CD8+ T cell responses (U.S. Patent No. 10,532,099). HCMV TR3 was used as the vector backbone (Caposio. 2019. Characterization of a live-attenuated HCMV-based vaccine platform. Scientific Reports 9:19236). Immunoblotting of human fibroblasts confirmed the expression of HIV fusion proteins in the resulting viral vector (HCMV TR3 ΔUL18 / HIV fusion ΔUL128-130 ΔUL146-147) (Figure 10).

[0237] UL18-deleted HCMV vectors were also inoculated into RMs, and immune responses to HIV antigens were determined in PBMCs by ICS 56 days after inoculation. As shown in Figure 11, the vectors induced CD8+ T cell responses to HIV gag, HIV nef, and HIV pol in RMs, as demonstrated by using pools of overlapping peptides containing each of these antigens. Therefore, we concluded that HCMV vectors lacking UL18 retain the ability to induce T cell responses to heterologous antigens.

Claims

1. A recombinant HCMV vector comprising a nucleic acid sequence encoding a heterologous antigen, wherein the recombinant HCMV vector does not express UL18.

2. The recombinant HCMV vector of claim 1 , wherein the recombinant HCMV vector does not express UL128.

3. The recombinant HCMV vector according to claim 1 or 2, wherein the recombinant HCMV vector does not express UL130.

4. The recombinant HCMV vector according to any one of claims 1 to 3, wherein the recombinant HCMV vector does not express UL128 and UL130.

5. The recombinant HCMV vector of claim 4, wherein the recombinant HCMV vector does not express UL146 and UL147.

6. 6. The recombinant HCMV vector according to any one of claims 1 to 5, wherein the recombinant HCMV vector does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147.

7. 7. The recombinant HCMV vector of claim 6, wherein the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.

8. The recombinant HCMV vector according to any one of claims 1 to 7, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof.

9. The recombinant HCMV vector according to any one of claims 1 to 8, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof.

10. The recombinant HCMV vector according to any one of claims 1 to 9, wherein the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof.

11. The recombinant HCMV vector according to any one of claims 1 to 10, wherein the recombinant HCMV vector does not express US11 or an orthologue thereof.

12. The recombinant HCMV vector of any one of claims 1 to 10, further comprising a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), wherein the MRE comprises a target site for a miRNA expressed in endothelial cells.

13. The recombinant HCMV vector of claim 12, wherein the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328.

14. The recombinant HCMV vector according to any one of claims 1 to 11, further comprising a nucleic acid sequence encoding an MRE, wherein the MRE comprises a target site for a miRNA expressed in myeloid cells.

15. The recombinant HCMV vector of claim 14, wherein the miRNA expressed in bone marrow cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

16. The recombinant HCMV vector according to any one of claims 1 to 15, wherein the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen.

17. 17. The recombinant HCMV vector of claim 16, wherein the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, or Mycobacterium tuberculosis.

18. The recombinant HCMV vector according to any one of claims 5 to 10 and 12 to 13, wherein the pathogen-specific antigen is an MHC-E supertope.

19. 19. The recombinant HCMV vector of claim 18, wherein the pathogen-specific antigen comprises an HIV epitope.

20. The HIV epitopes are LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), 20. The recombinant HCMV vector of claim 19, which is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to QKQEPIDKELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

21. The recombinant HCMV vector of claim 16, wherein the tumor antigen is associated with acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), or germ cell tumor.

22. 17. The recombinant HCMV vector of claim 16, wherein the host self-antigen is an antigen derived from the variable region of a T cell receptor (TCR) or an antigen derived from the variable region of a B cell receptor.

23. A pharmaceutical composition comprising the recombinant HCMV vector of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

24. An immunogenic composition comprising the recombinant HCMV vector of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

25. A method for generating an immune response to at least one heterologous antigen in a subject, comprising administering to the subject a recombinant HCMV vector described in any one of claims 1 to 22 in an amount effective to induce a CD8+ T cell response to the at least one heterologous antigen.

26. Use of a recombinant HCMV vector according to any one of claims 1 to 22 in the manufacture of a medicament for use in generating an immune response in a subject.

27. A recombinant HCMV vector according to any one of claims 1 to 22 for use in generating an immune response in a subject.

28. A method for treating or preventing cancer in a subject, comprising administering a recombinant HCMV vector of any one of claims 1 to 22 in an amount effective to induce a CD8+ T cell response against at least one heterologous antigen.

29. 23. Use of a recombinant HCMV vector according to any one of claims 1 to 22 in the manufacture of a medicament for use in treating or preventing cancer in a subject.

30. The recombinant HCMV vector of any one of claims 1 to 22 for use in treating or preventing cancer in a subject.

31. A method for treating or preventing a pathogen infection in a subject, comprising administering to the subject a recombinant HCMV vector described in any one of claims 1 to 22 in an amount effective to induce a CD8+ T cell response against at least one heterologous antigen.

32. 23. Use of a recombinant HCMV vector according to any one of claims 1 to 22 in the manufacture of a medicament for use in treating or preventing a pathogen infection in a subject.

33. 23. The recombinant HCMV vector of any one of claims 1 to 22 for use in treating or preventing a pathogen infection in a subject.

34. A method for treating an autoimmune disease or disorder in a subject, comprising administering to the subject a recombinant HCMV vector described in any one of claims 1 to 22 in an amount effective to induce a CD8+ T cell response against at least one heterologous antigen.

35. 23. Use of a recombinant HCMV vector according to claims 1 to 22 in the manufacture of a medicament for use in treating an autoimmune disease or disorder in a subject.

36. A recombinant HCMV vector according to any one of claims 1 to 22 for use in treating an autoimmune disease or disorder in a subject.

37. 37. The method, CMV vector for use, or use in manufacture of any one of claims 25 to 36, wherein at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof.

38. 38. The method, CMV vector for use, or use in manufacturing of claim 37, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or an orthologue thereof.

39. 37. The method, CMV vector for use, or use in manufacture of any one of claims 25 to 36, wherein at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof.

40. 40. The method, CMV vector for use, or use in manufacturing of claim 39, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-II or an orthologue thereof.

41. 37. The method, CMV vector for use, or use in manufacturing of any one of claims 25 to 36, wherein less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or an orthologue thereof.

42. 37. The method, CMV vector for use, or use in manufacture of any one of claims 25 to 36, wherein at least 10% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or an orthologue thereof.

43. 43. The method, CMV vector for use, or use in manufacture of claim 42, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or an orthologue thereof.

44. The method, CMV vector for use, or use in manufacturing of any one of claims 25 to 36, further comprising identifying a CD8+ TCR from the CD8+ T cells induced by the recombinant HCMV vector, wherein the CD8+ TCR recognizes an MHC-II / foreign antigen-derived peptide complex.

45. The method, CMV vector for use, or use in manufacturing of any one of claims 25 to 36, further comprising identifying a CD8+ TCR from the CD8+ T cells induced by the HCMV vector, wherein the CD8+ TCR recognizes an MHC-E / foreign antigen-derived peptide complex.

46. The method, CMV vector for use, or use in manufacturing of any one of claims 25 to 36, further comprising identifying a CD8+ TCR from the CD8+ T cells induced by the HCMV vector, wherein the CD8+ TCR recognizes an MHC class Ia / foreign antigen-derived peptide complex.

47. 47. The method, CMV vector for use, or use in manufacture of claims 44 to 46, wherein the CD8+ TCR is identified by DNA or RNA sequencing.

48. 45. The method, CMV vector for use, or use in manufacturing of claim 44, wherein the CD8+ TCR recognizes an MHC-II supertope.

49. 46. ​​The method, CMV vector for use, or use in manufacturing of claim 45, wherein the CD8+ TCR recognizes an MHC-E supertope.

50. 50. The method, CMV vector for use, or use in manufacture of claim 49, wherein the MHC-E supertope is a human immunodeficiency virus epitope.

51. The MHC-E supertope is LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), QKQEPID 51. The method of claim 50, the CMV vector for use, or the use in manufacturing, wherein the CMV vector is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of: KELYPLAS (SEQ ID NO: 25), KQEPIDKEL (SEQ ID NO: 26), SFSFPQITLWQRPLV (SEQ ID NO: 27), VRQYDQILIEICGKK (SEQ ID NO: 28), EPFRKQNPDIVIYQL (SEQ ID NO: 29), YVDGAANRETKLGKA (SEQ ID NO: 30), EEHEKYSNWRAMAS (SEQ ID NO: 31), or ILDLWVYHTQGYFPD (SEQ ID NO: 32).

52. 1. A method for generating CD8+ T cells that recognize MHC-E-peptide complexes, comprising: a. administering to a first subject a recombinant HCMV vector of any one of claims 5-10, 12-13, or 16-17 in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex; b. identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-E / foreign antigen-derived peptide complex; c. isolating one or more CD8+ T cells from the second subject; and d. transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex.

53. 1. A method for generating CD8+ T cells that recognize MHC-E-peptide complexes, comprising: a. identifying a first CD8+ TCR from a set of CD8+ T cells, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector of any one of claims 5-10, 12-13, or 16-17, and the first CD8+ TCR recognizes an MHC-E / foreign antigen-derived peptide complex; b. isolating one or more CD8+ T cells from a second subject; and c) transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR-transgenic CD8+ T cells that recognize an MHC-E peptide complex.

54. 54. The method of claim 52 or 53, wherein the first CD8+ T cells recognize an MHC-E supertope.

55. 55. The method of claim 54, wherein the MHC-E supertope comprises a human immunodeficiency virus epitope.

56. The MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), and RIVRMYSPVSIL (SEQ ID NO: 25).

56. The method of claim 54 or 55, wherein the amino acid sequence of the polypeptide of claim 54 is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any of the polypeptides of claim 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15

57. 57. The method of any one of claims 52 to 56, wherein the second CD8+ T cells recognize an MHC-E supertope.

58. 58. The method of claim 57, wherein the MHC-E supertope comprises a human immunodeficiency virus epitope.

59. The MHC-E supertope is selected from the group consisting of LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMYSPVSIL (SEQ ID NO: 24), and RIVRMYSPVSIL (SEQ ID NO: 25).

59. The method of claim 57 or 58, wherein the amino acid sequence of the polypeptide of claim 57 is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of any of the polypeptides of claim 57 or 58, including the polypeptides of claim 57, ...

60. 60. The method of any one of claims 52 to 59, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

61. 61. The method of any one of claims 52 to 60, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

62. 62. The method of any one of claims 52 to 61, wherein the first subject is a human.

63. 63. The method of any one of claims 52 to 62, wherein the second subject is a human.

64. 1. A method for generating CD8+ T cells that recognize an MHC-E peptide complex, comprising: a. Administering a recombinant rhesus CMV (RhCMV) or cynomolgus CMV (CyCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147 to a non-human primate to express an HIV antigen in an amount effective to generate a set of CD8+ T cells that recognize MHC-E in complex with the HIV supertope peptide of claims 18-20; b. identifying a first CD8+ TCR from the set of CD8+ T cells, the first recognizing an MHC-E / supertope peptide complex; c. isolating one or more CD8+ T cells from the second subject; and d. transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex.

65. 1. A method for generating CD8+ T cells that recognize an MHC-E peptide complex, comprising: a. Identifying a first CD8+ TCR that recognizes an MHC-E / supertope peptide complex from a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertope peptide according to claims 18-20, wherein the set of CD8+ T cells is generated from a recombinant rhesus monkey (RhCMV) or cynomolgus monkey CMV (CyCCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147; and expressing an HIV antigen in an amount effective to generate the set of CD8+ T cells. b. isolating one or more CD8+ T cells from a second subject; and c) transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex.

66. 66. The method of claim 64 or 65, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

67. 67. The method of any one of claims 64-66, wherein the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

68. 68. The method of any one of claims 64-67, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

69. 69. The method of any one of claims 64 to 68, wherein the second CD8+ TCR is a chimeric CD8+ TCR.

70. 70. The method of any one of claims 64 to 69, wherein administering the recombinant HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the recombinant HCMV vector to the first subject.

71. 71. The method of any one of claims 52-70, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

72. 72. The method of claim 71, wherein the cancer is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), or germ cell tumor.

73. 71. The method of any one of claims 52-70, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent a pathogen infection.

74. 74. The method of claim 73, wherein the pathogen infection is caused by human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, or Mycobacterium tuberculosis.

75. 71. The method of any one of claims 52-70, further comprising administering the transfected CD8+ T cells to the subject to induce an autoimmune response against the host self-antigen.

76. 1. A method for generating CD8+ T cells that recognize MHC-II-peptide complexes, comprising: a. administering to a first subject a recombinant HCMV vector of any one of claims 1 to 11, 14, or 15 in an amount effective to generate a set of CD8+ T cells that recognize an MHC-II / peptide complex; b. identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-II / foreign antigen-derived peptide complex; c. isolating one or more CD8+ T cells from the second subject; and d. transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-II peptide complex.

77. 1. A method for generating CD8+ T cells that recognize MHC-II-peptide complexes, comprising: a. Identifying a first CD8+ TCR that recognizes an MHC-II / peptide complex from a set of CD8+ T cells that recognize an MHC-II / peptide complex, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector of any one of claims 1 to 11, 14, or 15; b. isolating one or more CD8+ T cells from a second subject; and c) transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-II peptide complex.

78. 78. The method of claim 76 or 77, wherein the first CD8+ T cells recognize an MHC-II supertope.

79. 79. The method of any one of claims 76 to 78, wherein the second CD8+ T cells recognize an MHC-II supertope.

80. 80. The method of any one of claims 76 to 79, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

81. 81. The method of any one of claims 76 to 80, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

82. 82. The method of any one of claims 76 to 81, wherein the first subject is a human.

83. 83. The method of any one of claims 76 to 82, wherein the second subject is a human.

84. 84. The method of claims 76-83, wherein administering the HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the HCMV vector to the first subject.

85. 85. The method of any one of claims 76-84, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

86. 86. The method of claim 85, wherein the cancer is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

87. 85. The method of any one of claims 76-84, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent a pathogen infection.

88. 88. The method of claim 87, wherein the pathogen infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

89. 85. The method of any one of claims 76-84, further comprising administering the transfected CD8+ T cells to the subject to induce an autoimmune response against the host self-antigen.

90. 1. A method for generating CD8+ T cells that recognize MHC-I-peptide complexes, comprising: a. administering to a first subject a recombinant HCMV vector of any one of claims 1 to 11 in an amount effective to generate a set of CD8+ T cells that recognize an MHC-I / peptide complex; b. identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-I / foreign antigen-derived peptide complex; c. isolating one or more CD8+ T cells from the second subject; and d. transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-I peptide complex.

91. 1. A method for generating CD8+ T cells that recognize MHC-I-peptide complexes, comprising: a. Identifying a first CD8+ TCR that recognizes an MHC-I / xenoantigen-derived peptide complex from a set of CD8+ T cells that recognize an MHC-I / xenoantigen-derived peptide complex, wherein the set of CD8+ T cells is generated from a recombinant HCMV vector according to any one of claims 1 to 11; b. isolating one or more CD8+ T cells from a second subject; and c) transfecting the one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-I peptide complex.

92. 92. The method of any one of claims 90-91, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

93. 93. The method of any one of claims 90 to 92, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

94. 94. The method of any one of claims 90 to 93, wherein the first subject is a human.

95. 95. The method of any one of claims 90 to 94, wherein the second subject is a human.

96. 96. The method of any one of claims 90 to 95, wherein administering the HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the HCMV vector to the first subject.

97. 97. The method of any one of claims 90-96, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

98. 98. The method of claim 97, wherein the cancer is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), and germ cell tumors.

99. 97. The method of any one of claims 90-96, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent a pathogen infection.

100. 100. The method of claim 99, wherein the pathogen infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.

101. 97. The method of any one of claims 90-96, further comprising administering the transfected CD8+ T cells to the subject to induce an autoimmune response against the host self-antigen.

102. CD8+ T cells produced by the method of claims 25 to 101.

103. 103. The CD8+ T cell of claim 102, wherein the pathogen-specific antigen is human immunodeficiency virus, simian immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasite, or Mycobacterium tuberculosis.

104. The CD8+ T cell of claim 102, wherein the tumor antigen is associated with acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma (RCC), or germ cell tumors.

105. The CD8+ T cell of claim 102, wherein the host self-antigen is an antigen derived from the variable region of a T cell receptor (TCR) or an antigen derived from the variable region of a B cell receptor.

106. 104. A method of treating or preventing a pathogen infection in a subject, comprising administering to the subject the CD8+ T cells of claim 102 or 103.

107. 104. Use of the CD8+ T cell of claim 102 or 103 in the manufacture of a medicament for use in treating or preventing a pathogen infection in a subject.

108. 104. The CD8+ T cell of claim 102 or 103 for use in treating or preventing a pathogen infection in a subject.

109. 106. A method of treating or preventing cancer in a subject, comprising administering to the subject the CD8+ T cells of claim 102 or 104.

110. 106. Use of the CD8+ T cells of claim 102 or 104 in the manufacture of a medicament for use in treating or preventing cancer in a subject.

111. 105. The CD8+ T cell of claim 102 or 104 for use in treating or preventing cancer in a subject.

112. 106. A method for treating an autoimmune disease or disorder, comprising administering to a subject the CD8+ T cells of claim 102 or 105.

113. 106. Use of the CD8+ T cell of claim 102 or 105 in the manufacture of a medicament for use in the treatment of an autoimmune disease or disorder.

114. 106. The CD8+ T cell of claim 102 or 105 for use in treating an autoimmune disease or disorder.

115. 107. A method for inducing an autoimmune response against a host self-antigen, comprising administering to a subject the CD8+ T cells of claim 102 or 105.

116. 9-15 amino acids in length, and include LDAWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KKAQQAAADTGNSSQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQAISPRTLNAW (SEQ ID NO: 17), HQAISPRTL (SEQ ID NO: 18), NTMLNTVGGHQAAMQ (SEQ ID NO: 19), VGGHQAAMQ (SEQ ID NO: 20), STLQEQIGWMTNPP (SEQ ID NO: 21), STLQEQIGW (SEQ ID NO: 22), IVRMYSPVSILDIRQ (SEQ ID NO: 23), RMY A human immunodeficiency virus antigen that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of SPVSIL (SEQ ID NO:24), QKQEPIDKELYPLAS (SEQ ID NO:25), KQEPIDKEL (SEQ ID NO:26), SFSFPQITLWQRPLV (SEQ ID NO:27), VRQYDQILIEICGKK (SEQ ID NO:28), EPFRKQNPDIVIYQL (SEQ ID NO:29), YVDGAANRETKLGKA (SEQ ID NO:30), EEHEKYSNWRAMAS (SEQ ID NO:31), or ILDLWVYHTQGYFPD (SEQ ID NO:32).

117. 117. A recombinant HCMV vector comprising a nucleic acid encoding one or more of the human immunodeficiency virus antigens of claim 116.

118. 118. The recombinant HCMV vector of claim 117, wherein the recombinant HCMV vector does not express UL18.

119. 119. The recombinant HCMV vector of claim 117 or 118, wherein the recombinant HCMV vector does not express UL128.

120. The recombinant HCMV vector of any one of claims 117 to 119, wherein the recombinant HCMV vector does not express UL130.

121. The recombinant HCMV vector according to any one of claims 117 to 120, wherein the recombinant HCMV vector does not express UL128 and UL130.

122. 122. The recombinant HCMV vector of claim 121, wherein the recombinant HCMV vector does not express UL146 and UL147.

123. 123. The recombinant HCMV vector of any one of claims 117 to 122, wherein the recombinant HCMV vector does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147.

124. 124. The recombinant HCMV vector of claim 123, wherein the mutation in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.

125. The recombinant HCMV vector of any one of claims 117 to 124, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or an orthologue thereof.

126. The recombinant HCMV vector of any one of claims 117 to 125, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or an orthologue thereof.

127. The recombinant HCMV vector of any one of claims 117 to 126, wherein the recombinant HCMV vector does not express UL82 (pp71) or an orthologue thereof.

128. The recombinant HCMV vector of any one of claims 117 to 127, wherein the recombinant HCMV vector does not express US11 or an orthologue thereof.

129. The recombinant HCMV vector of any one of claims 117 to 128, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), the MRE comprising a target site for a miRNA expressed in endothelial cells.

130. The recombinant HCMV vector of claim 129, wherein the miRNA expressed in endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328.

131. The recombinant HCMV vector of any one of claims 117 to 130, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, and the MRE comprises a target site for a miRNA expressed in myeloid cells.

132. The recombinant HCMV vector of claim 131, wherein the miRNA expressed in bone marrow cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

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