Regulation of T cell responses by UL18 of human cytomegalovirus

Abstract

The present disclosure relates to methods for modulating T cell responses by UL18 of human cytomegalovirus. The present disclosure also relates to methods for generating MHC-Ia, MHC-II, and / or MHC-E restricted CD8+ T cells.

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 on August 20, 2019, which is hereby incorporated by reference in its entirety.

[0002] Description of Federally Sponsored Research or Development This invention was made with government support under grant numbers AI059457 and AI128741 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Reference to a Sequence Listing Submitted Electronically The content of the sequence listing in the ASCII text file submitted electronically together with this application (name 4153_013PC01_Seqlisting_ST25, size: 11,029 bytes, and creation date: August 19, 2020) is hereby incorporated by reference in its entirety.

Background Art

[0004] The RhCMV strain 68-1 has previously been shown to induce CD8+ T cells that recognize peptides presented by MHC-II and MHC-E, rather than conventional MHC-I. This effect has been reproduced in cynomolgus CMV (CyCMV), and thus it has been shown that deletion of the RhCMV and CyCMV homologs of HCMV UL128, UL130, UL146, and UL147 is required to enable the induction of MHC-E-restricted CD8+ T cells (WO2016 / 130693, WO2018 / 075591). Furthermore, these vectors induce MHC-II-restricted CD8+ T cells. However, by inserting the target site of endothelial cell-specific microRNA (miR) 126 into an essential viral gene of these vectors, induction of MHC-II-restricted CD8+ T cells is eliminated, and an “MHC-E only” vector that exclusively induces MHC-E-restricted CD8+ T cells is obtained (WO2018 / 075591). In contrast, by inserting myeloid cell-specific miR142-3p into 68-1 RhCMV, induction of MHC-E-restricted CD8+ T cells is blocked, and a vector that induces CD8+ T cells exclusively restricted by MHC-II is obtained (WO2017 / 087921). Similarly, deletion of Rh67, which is the UL40 homolog, blocks the induction of MHC-E-restricted CD8+ T cells, and a “MHC-II only vector” is obtained (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 UL18 is not expressed.

[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 and not expressing UL18, UL128, UL130, UL146, and UL147.

[0008] In some embodiments, the recombinant HCMV vector does not express the UL18 protein, UL128 protein, UL130 protein, UL146 protein, and 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 point mutations, frameshift mutations, truncation mutations, and deletions of all of the nucleic acid sequences encoding viral proteins.

[0009] 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 does not express UL82 (pp71) or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an ortholog thereof.

[0010] In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and the MRE comprises a target site for a microRNA expressed in endothelial cells. In some embodiments, the MREs expressed in endothelial cells are 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), and the MRE comprises target sites for microRNAs expressed in myeloid cells. In some embodiments, the MREs expressed in myeloid cells are miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, and miR-125.

[0012] In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen. 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 supertop. In some embodiments, the MHC-E supertop is an HIV epitope. In some embodiments, the MHC-E supertop is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to 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).

[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 syndromes, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin 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 against at least one heterologous antigen in a subject, the method comprising administering to the subject an effective amount of a recombinant HCMV vector to induce a CD8+ T cell response against the at least one heterologous antigen.

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

[0020] The present disclosure also relates to a 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, the method comprising administering an effective amount of a recombinant HCMV vector to induce 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 a recombinant HCMV vector 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, the method comprising administering to the subject a recombinant HCMV vector in an effective amount to induce 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 pathogen infections in a subject.

[0027] The present disclosure also relates to a method of treating an autoimmune disease or disorder in a subject, the method comprising administering to the subject an effective amount of a recombinant HCMV vector to induce 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 a recombinant HCMV vector 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 ortholog. 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%, or at least 95% of the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC-E or its ortholog.

[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 ortholog. In 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 ortholog.

[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 ortholog. 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 ortholog. 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 the CD8+ T cells induced by the recombinant HCMV vector are restricted by MHC class Ia or its ortholog.

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

[0036] In some embodiments, the CD8+ TCR recognizes an MHC-E supertop. In some embodiments, the MHC-E supertop is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertop has at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence 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), 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 of generating TCR transgenic CD8+ T cells that recognize an MHC-E-peptide complex, comprising: (a) administering to a first subject an effective amount of a recombinant HCMV vector 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 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 the 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 the UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequences 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 point mutations, frameshift mutations, truncation mutations, and all deletions of the nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or its ortholog.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and the MRE comprises a target site for an 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 self-antigen. 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 relates to a method of 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 the recombinant HCMV vector according to 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; (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 the CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more TCR transgenic CD8+ T cells that recognize the 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 the UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequences 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 point mutations, frameshift mutations, truncation mutations, and all deletions of the nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or its ortholog.In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and the MRE comprises target sites for miRNAs expressed in endothelial cells. In some embodiments, the miRNAs expressed in endothelial cells are 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 self-antigen. 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 has at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence 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), 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 cell recognizes an MHC-E supertop. In some embodiments, the MHC-E supertop comprises a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertop has at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with the amino acid sequence 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), 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 human. In some embodiments, the second subject is human.

[0044] The present disclosure also relates to a method of generating CD8+ T cells that recognize an MHC-E peptide complex, 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, and expressing an amount of an HIV antigen effective to generate a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertopical peptide; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first recognizes an MHC-E / supertopical 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 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 transfected CD8+ T cells that recognize an MHC-E / heterologous antigen-derived peptide complex.In some embodiments, the HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to 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 relates to a method of generating CD8+ T cells that recognize an MHC-E peptide complex, comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-E / supertop peptide complex from a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertop 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 expressing an effective amount of an HIV antigen 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 the CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize the MHC-E peptide complex.In some embodiments, the HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to 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, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR that includes the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR includes the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In some embodiments, the second CD8+ TCR includes 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 includes 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 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 parasite, 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 of generating CD8+ T cells that recognize an MHC-II-peptide complex, comprising: (a) administering to a first subject an effective amount of a recombinant HCMV vector 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 / 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 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 the 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, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and deletions of all of the nucleic acid sequences encoding the 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 does not express UL82 (pp71) or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding MRE, and MRE comprises a target site for 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 of generating CD8+ T cells that recognize an MHC-II-peptide complex, comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-II / peptide complex derived from a heterologous antigen 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; (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 the 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 orthologs, due to the presence of one or more mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and deletions of all of the nucleic acid sequences encoding the viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or its ortholog.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 does not express UL82 (pp71) or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding an MRE, wherein the MRE comprises target sites for miRNAs expressed in myeloid cells. In some embodiments, the miRNAs expressed in myeloid cells are 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 cell recognizes an MHC-II supertop. In some embodiments, the second CD8+ T cell recognizes an MHC-II supertop.

[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 human. In some embodiments, the second subject is human.

[0056] In some embodiments, administering the HCMV vector to the first subject includes 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 lymphocytic leukemia, non-Hodgkin 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 parasite, and Mycobacterium tuberculosis.

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

[0060] The present disclosure also relates to a method of generating CD8+ T cells that recognize an MHC-I-peptide complex, comprising: (a) administering to a first subject an effective amount of a recombinant HCMV vector 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 / 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 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 the 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, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and deletions 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 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 does not express UL82 (pp71) or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11 or an ortholog thereof.

[0061] The present disclosure also provides 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 / heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize MHC-I / heterologous antigen-derived peptide complexes, wherein the set of CD8+ T cells is generated from the recombinant HCMV vector according to any one of claims 1-11; (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 MHC-I-peptide complexes.

[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 human. In some embodiments, the second subject is 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 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 parasite, 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 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 for treating or preventing a pathogen infection in a subject, the method 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 for 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 for treating an autoimmune disease or disorder in a subject, the method comprising administering CD8+ T cells to the 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 in a subject.

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

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

[0081] The present disclosure also relates to a human immunodeficiency virus MHC-E supertop that is 9 to 15 amino acids in length and is at least 90%, at least 95%, or 100% identical to the amino acid sequence 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), 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 the UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, 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 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 all deletions 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 its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or its ortholog. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or its ortholog. In some embodiments, the recombinant HCMV vector does not express US11 or its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and 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, and the MRE comprises a target site for a miRNA expressed in myeloid cells.In some embodiments, the miRNAs expressed in bone marrow cells are miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.

Brief Description of the Drawings

[0083]

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Mode for Carrying Out the Invention

[0084] I. Terms Unless otherwise specified, technical terms are used according to their conventional usage.

[0085] All publications, patents, patent applications, Internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited in this specification or listed in an application data sheet that includes U.S. Patent Application No. 62 / 889,310, filed Aug. 20, 2019, are hereby incorporated by reference 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 the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. For ease of consideration of the various embodiments of the present disclosure, explanations of specific terms are provided below.

[0087] Except where the context requires otherwise, throughout this specification and the claims, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted in an open, inclusive sense, i.e., as "including, but not limited to". "Consisting of" means excluding trace elements of other components and substantial method steps disclosed herein. The term "consisting essentially of" limits the claim to the specified materials or steps, or those that do not substantially affect the basic characteristics of the invention recited in the claim. For example, a composition consisting essentially of the components defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc. Similarly, a protein consists essentially of a specific 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., change the activity of the protein by 50% or less). The embodiments defined by each of the transitional 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. This term also refers to a protein that is immunologically active in the sense that when administered to a subject (either directly or by administering a nucleotide sequence or vector encoding the protein), it can induce a humoral and / or cellular immune response directed against the protein.

[0089] Antigen-specific T cells: CD8 + or CD4 + lymphocytes. Generally, antigen-specific T cells specifically bind to a particular antigen presented by MHC molecules, but do not specifically bind to other antigens presented by the same MHC.

[0090] Administration: As used herein, the term "administration" means the giving or dispensing of a drug, such as a composition comprising a CMV vector containing an effective amount of an exogenous antigen, to a subject by any effective route. Exemplary administration routes 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" means an amount of a drug sufficient to produce a desired response, such as reducing or eliminating the signs or symptoms of a condition or disease or inducing an immune response to an antigen, e.g., the amount of 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 instances, an "effective amount" is one that treats (including preventing) one or more symptoms and / or underlying causes of any one of a disorder or disease. An effective amount can be a therapeutically effective amount, including an amount that prevents the development of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with an infectious disease or cancer.

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

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

[0094] Immune tolerance: As used herein, "immune tolerance" refers to a state of non-responsiveness of the immune system to a substance 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 other sequences such as an allele-specific motif or an N-terminal repeat 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, the immunogenic peptide is identified using a sequence motif or other methods known in the art such as neural networks or polynomial determination. Typically, an algorithm is used to determine a 「binding threshold」 for peptides to select peptides that have a high likelihood of binding with a specific 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 an immunogenic peptide, a 「conserved residue」 is a residue that appears at a particular position in a peptide with a significantly higher frequency than expected by random distribution. In some embodiments, the conserved residue is a residue at which the MHC structure may provide a contact point 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 roles in tissue specification or cell lineage determination, and further, miRNAs affect various processes including early development, cell proliferation, and cell death, as well as apoptosis and lipid metabolism. The abundance of numerous miRNA genes, diverse expression patterns, and potential miRNA targets suggests that miRNAs can 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 suppress translation. miRNAs can also bind to target mRNAs and mediate gene silencing via the RNAi pathway. miRNAs can also regulate gene expression by causing chromatin condensation.

[0099] miRNAs silence the translation of one or more specific mRNA molecules by binding to miRNA recognition elements (MREs), which are defined as any sequence that interacts with the miRNA by directly base - pairing with the miRNA somewhere on the miRNA transcript. MREs are often present in the 3’untranslated region (UTR) of mRNAs, but MREs can also be present in the coding sequence or 5’UTR. MREs are not necessarily perfect complements to the miRNA and usually have only a few bases that are complementary to the miRNA, often containing one or more mismatches within those complementary bases. An MRE can be any sequence that can be sufficiently bound by the miRNA such that the translation of the gene (such as a CMV gene that is essential for growth in vivo or enhances it) to which the MRE is operably linked is suppressed by an 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 sequence, a consensus sequence, or a "wild-type" sequence. A variant is any protein or nucleic acid sequence that contains a mutation. Further, a cell or organism having a mutation may be referred to as a variant. Some types of coding sequence mutations include point mutations (differences in individual nucleotides or amino acids), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences in which one or more nucleotides or amino acids are lost, including deletions of the entire coding sequence of a gene at most), and frameshift mutations (differences in which a deletion of a number of nucleotides not divisible by three results in a change in the amino acid sequence). A mutation that results in an amino acid difference may also be referred to as an amino acid substitution mutation. An amino acid substitution mutation 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), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acid can be single-stranded, or partially or completely double-stranded (duplex). A double-stranded nucleic acid can be a homoduplex or a heteroduplex.

[0102] Operably linked: When the term "operably linked" is used herein, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is arranged in such a manner as to affect the second nucleic acid sequence. An operably linked DNA sequence can be continuous or can act at a distance.

[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, a eukaryotic promoter includes the nucleic acid sequences necessary near the start site of transcription, such as in the case of a polymerase II type promoter, the 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 are well known to those of skill in the art. A nucleic acid comprising a promoter operably linked to a nucleic acid sequence encoding a particular 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 one having a sequence made by an artificial combination of two or more isolated sequence segments, such as a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques. A recombinant polypeptide can also refer to a polypeptide made using a recombinant nucleic acid that is transcribed in a host organism that is not the natural source of the polypeptide (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen).

[0105] Pharmaceutically acceptable carrier: As used herein, the “pharmaceutically acceptable carrier” used is conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995 describes compositions and formulations suitable for the pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as a vehicle. For solid compositions (such as in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions administered may include minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents such as, 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 are composed of the four bases adenine, cytosine, guanine, and thymine / uracil (uracil is used in RNA). Coding sequences derived from nucleic acids indicate the sequences of the proteins encoded by the nucleic acids.

[0107] Polypeptide: The terms "protein", "peptide", "polypeptide", and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer can be linear or branched, can include 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 naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as labeling or conjugation to a bioactive component.

[0108] Orthologs of a protein are typically characterized by having greater than 75% sequence identity as counted over the full-length alignment with the amino acid sequence of a particular protein, using ALIGN set to default parameters. Proteins having even greater similarity to the reference sequence will exhibit increased percentage identities, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity when evaluated by this method. Additionally, sequence identity can be compared over the full-length of specific domains of the peptides of the present disclosure.

[0109] Sequence Identity / Similarity: As used herein, identity / similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed with respect to the identity or similarity between the sequences. Sequence identity can be measured with respect to a percentage of identity, with higher percentages indicating more identical sequences. Sequence similarity can be measured with respect to a percentage of identity or similarity (taking into account conservative amino acid substitutions), with higher percentages indicating more similar sequences. Polypeptides or their protein domains 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 a protein that do not change the function or size of the protein) can be referred to as "homologs".

[0110] Methods for aligning arrays 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). Further, Altschul et al, J Mol Biol 215,403-410(1990) provides 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 related to the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found at the NCBI web site.

[0112] BLASTN is used for the comparison of nucleic acid sequences, and BLASTP is used for the comparison of amino acid sequences. If the two sequences being compared have homology, the specified output file presents those homologous regions as aligned sequences. If the two sequences being compared do not have homology, the specified output file does not present an aligned sequence.

[0113] When aligning, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is presented in both sequences. 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 concatenated length (such as 100 consecutive nucleotides or amino acid residues derived 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 the test sequence when aligned with a test sequence having 1554 nucleotides (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, as follows, a target sequence containing a 20-nucleotide region that aligns with 20 consecutive nucleotides from the specified sequence contains a region having 75 percent sequence identity with that specified sequence (i.e., 15÷20×100 = 75).

[0114] For comparison of amino acid sequences greater than about 30 amino acids, the Blast2 sequence function is used with the default BLOSUM62 matrix (gap existence cost 11 and per residue gap cost 1) set to default parameters. Homologs typically have at least 70% sequence identity counted over the full-length alignment with the amino acid sequence, using gapped blastp with databases such as the NCBI Basic Blast2.0, nr database, swissprot database, and the patent-acquired sequence database. 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 alignment can be performed. Proteins with even greater similarity show increased percentage identities such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when evaluated by this method.

[0115] When aligning short peptides (less than about 30 amino acids), the Blast2 sequence function is used to perform the alignment with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with even greater similarity to the reference sequence show increased percentage identities such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the protein when evaluated by this method. When comparing less than the full sequence for sequence identity, homologs typically have at least 75% sequence identity over a short window of 10 - 20 amino acids and can have at least 85%, 90%, 95%, or 98% sequence identity depending on the identity with the reference sequence. Methods for determining sequence identity in such short windows are described on the NCBI website.

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

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

[0118] Supertopes: 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. Beneficial effects can be demonstrated, for example, by delay in the onset of clinical symptoms of a disease in a subject prone to developing the disease, reduction in the severity of some or all of the disease clinical symptoms, delay in the progression of the disease, reduction in the number of recurrences of the disease, improvement in the overall health or well-being of the subject, or other parameters well known in the art specific to a particular disease. Preventive treatment is treatment administered to a subject who does not exhibit signs of, or exhibits only early signs of, a disease for the purpose of reducing the risk of developing the disease state. Therapeutic treatment is treatment administered to a subject after the signs and symptoms of a disease have manifested.

[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 infectious disease), or to reduce the severity of the symptoms of a disease or condition, or to limit the progression of a disease or condition (such as a tumor or infectious disease), 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 derived 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, which is then introduced into a host cell, thereby generating a transformed host cell. The vector may contain a nucleic acid sequence that enables its replication in the host cell, for example, an origin of replication. The vector may also contain one or more selectable marker genes and other gene elements known in the art, including promoter elements that direct nucleic acid expression. The vector can be a viral vector such as a CMV vector. The viral vector can be constructed from an attenuated virus, including a wild-type virus or a replication-deficient virus.

[0122] II. Method for regulating T cell response by UL18 of HCMV Disclosed herein is a method for regulating T cell response by UL18 of HCMV. The method includes 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 includes generating an immune response against at least one heterologous antigen, including administering to the subject an effective amount of the HCMV vector to induce a CD8+ T cell response against the at least one heterologous antigen. In some embodiments, the method further includes treating or preventing cancer in the subject, including administering to the subject an effective amount of the HCMV vector to induce a CD8+ T cell response against the at least one heterologous antigen. In some embodiments, the method further includes treating or preventing a pathogen infection in the subject, including administering to the subject an effective amount of the HCMV vector to induce a CD8+ T cell response against the at least one heterologous antigen. In some embodiments, the method further includes treating an autoimmune disease or disorder in the subject, including administering to the subject an effective amount of the HCMV vector to induce a CD8+ T cell response against the at least one heterologous antigen.

[0124] In some embodiments, the UL18 - deficient HCMV vector also does not express UL128, UL130, UL146, or UL147 due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147. Further, any of the UL18 - deficient HCMV vectors may lack US11 and / or UL82 proteins due to the presence of a mutation in the nucleic acid sequence encoding US11 and / or UL82. The mutation can be any mutation that results in the lack of expression of the active protein. Such mutations can include point mutations, frameshift mutations, deletions less than all of the protein - encoding sequence (truncation mutations), or deletions of all of the nucleic acid sequence encoding the protein, 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 contains 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), expresses UL40 and US28, and the MRE contains target sites for microRNAs 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 contains target sites for microRNAs 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] An MRE can be any miRNA recognition element that silences expression in the presence of miRNAs expressed by endothelial cells. An MRE can be any miRNA recognition element that silences expression in the presence of miRNAs expressed by myeloid cells. Such an MRE can be an exact complement of the miRNA. Alternatively, other sequences can be used as MREs for a given miRNA. For example, an MRE can be predicted from a sequence. In one example, a miRNA can be searched for at the website microRNA.org (www.microrna.org). A list of mRNA targets of the miRNA is then enumerated. For each of the listed targets on that page, one can access "Alignment Details" and access the putative MRE.

[0128] One of ordinary skill in the art can select a reasonable, putative, or mutated MRE sequence predicted to induce silencing in the presence of miRNAs expressed in myeloid cells such as macrophages from the literature. One example includes the above-referenced website. One of ordinary skill in the art can then obtain an expression construct in which a reporter gene (such as a fluorescent protein, an enzyme, or other reporter gene) has 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. Absence of expression of the reporter gene indicates that the MRE is silencing gene expression in the presence of the miRNA.

[0129] In some embodiments, the heterologous antigen can be a pathogen-specific antigen, a tumor antigen, a tumor-specific 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 (TCR) or from the variable region of 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 parasites, Clostridium tetani, and Mycobacterium tuberculosis.

[0131] A tumor antigen can be any protein that is relatively restricted to tumor cells and induces an immune response. However, many tumor antigens are host (self) proteins and are therefore typically not regarded as antigenic by the host immune system. Tumor antigens can also be abnormally expressed by cancer cells. Tumor antigens can also be germline / testis antigens expressed in cancer cells, cell lineage differentiation antigens not expressed in adult tissues, or antigens overexpressed in cancer cells.Tumor antigens include, but are not limited to, prostate acid phosphatase (PAP); Wilms tumor suppressor protein (WT1); mesothelin (MSLN); Her-2 (HER2); human papillomavirus antigen E6 of HPV16 strain; human papillomavirus antigen E7 of HPV16 strain; human papillomavirus antigen E6 of HPV18 strain; human papillomavirus antigen E7 of HPV18 strain; fusion protein of human papillomavirus E6 and E7 derived from HPV16 and HPV18 strains; mucin 1 (MUC1); LMP2; epidermal growth factor receptor (EGFR); p53; New York esophageal 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; globo H (GloboH); Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETV6-AML); NY-BR-1; RGS5; squamous epithelial antigen-rejected tumor or 3 (SART3); STn; carbonic anhydrase IX; PAX5; OY-TES1; sperm protein 17; LCK; HMWMAA; AKAP-4; SSX2; B7H3; legumain; 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; preferentially expressed antigen of melanoma (PRAME); melanocortin 1 receptor (MC1R); β-catenin; BRCA1 / 2; CDK4; chronic myeloid leukemia 66 (CML66); TGF-β.In certain embodiments, the host self-antigens include prostate acid phosphatase, Wilms' tumor suppressor protein, mesothelin, or Her-2.

[0132] In some embodiments, the tumor antigen is derived from cancer. Cancers include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical cancer; AIDS-related cancer; AIDS-related lymphoma; anal cancer; appendiceal cancer; astrocytoma, pediatric cerebellum or cerebrum; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem 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 lymphoma; carcinoid tumor, pediatric; carcinoid tumor, gastrointestinal; cancer of unknown primary origin; 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 sarcoma in the Ewing 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 tumor: extracranial, extragonadal, or ovarian; gestational trophoblastic tumor; glioma of the brainstem; glioma, pediatric cerebral astrocytoma; glioma, pediatric visual pathway and hypothalamus; gastric carcinoid; hairy cell leukemia; head and neck cancer; heart cancer; hepatocellular (liver) cancer; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma, pediatric; intraocular melanoma; islet cell cancer (endocrine pancreas); Kaposi 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 (also called chronic lymphocytic leukemia); leukemia, chronic myelogenous (myeloid) (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, Waldenström; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma, pediatric; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma, adult malignant; Mesothelioma, pediatric; Metastatic cervical squamous cell carcinoma of unknown primary; Oral cancer; Multiple endocrine neoplasia syndrome, childhood; Multiple myeloma / plasma cell neoplasms; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplasia / myeloproliferative disease; Myelogenous leukemia, chronic; Myelogenous leukemia, adult acute; Myelogenous leukemia, pediatric acute; Myeloma, multiple (cancer of the bone marrow); Myeloproliferative disease, chronic; Nasal and paranasal sinus cancer; Nasopharyngeal cancer; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (surface epithelial / stromal tumors); Ovarian germ cell tumors; Ovarian low-grade tumors; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germ cell tumors; Pinealoblastoma and supratentorial primitive neuroectodermal tumors, pediatric; Pituitary adenoma; Plasma cell neoplasms / 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, pediatric; Salivary gland cancer; Sarcoma, Ewing tumor family; 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); Metastatic cervical squamous cell carcinoma of unknown primary; Stomach cancer; Supratentorial primitive neuroectodermal tumors, pediatric; T-cell lymphoma, cutaneous (mycosis fungoides and Sézary syndrome); Testicular cancer; Throat cancer; Thymoma, pediatric; Thymoma and thymic cancer; Thyroid cancer; Thyroid cancer, pediatric; Transitional cell carcinoma of the renal pelvis and ureter; Gestational trophoblastic tumor; Cancer of unknown primary site, adult; Cancer of unknown primary site, pediatric; Transitional cell carcinoma of the ureter and renal pelvis; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, pediatric; Vulvar cancer; Waldenström macroglobulinemia; and Wilms tumor (kidney cancer) are included, but not limited to;

[0133] In some embodiments, the pathogen-specific antigen is an MHC-E supertop. In some embodiments, the MHC-E supertop is an HIV epitope. In some embodiments, the MHC-E supertop 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) and is 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 supertops are used to generate a fusion protein. The fusion protein may include one or more of the MHC-E supertops 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 epitopes 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 CD8+ T cells restricted by MHC-E recognize peptides carried by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against supertopes presented by MHC-E.

[0135] In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from the 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 / heterologous antigen-derived peptide complex. In some embodiments, the CD8+ TCR recognizes an MHC-E supertop. In some embodiments, the MHC-E supertop is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertop 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) and is 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.

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

[0138] In some embodiments, the CD8+ T cell response induced by the HCMV vector is characterized by having at least 10% of the CD8+ T cells directed against epitopes 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 CD8+ T cells restricted by MHC-II recognize peptides carried by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against supertopical epitopes presented by MHC-II.

[0139] In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from CD8+ T cells induced by the HCMV vector. The TCR can 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 supertopical epitope.

[0140] In some embodiments, the CD8+ T cell response induced by the UL18-deficient HCMV vector lacking 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 from a UL18- and US11-deficient 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 is a method for generating CD8+ T cells that recognize MHC-E peptide complexes. This method involves administering to a first subject an amount of an HCMV vector effective to generate a set of CD8+ T cells that recognize MHC-E / peptide complexes. The CMV vector contains a first nucleic acid sequence encoding at least one heterologous antigen and does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein. The vector may also lack UL82 protein. In some embodiments, the HCMV vector expresses UL40 and US28. In some embodiments, the HCMV vector does not express UL18, UL138, UL130, UL146, and UL147 proteins and contains a nucleic acid sequence encoding UL40, US28, and a microRNA (miRNA) recognition element (MRE). In some embodiments, the MRE contains 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] This method further comprises identifying a first CD8+ T cell receptor from a set of CD8+ T cells, wherein 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 comprise transfecting one or more CD8+ T cells with an expression vector, the 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, wherein the second CD8+ T cell receptor comprises CDR3α and CDR3β of the first CD8+ T cell receptor, and transfecting, 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 transfecting the expression vector can be isolated from a first subject or a 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 comprises further 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), that lacks the 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 monkeys using RhCMV or in cynomolgus monkeys using CyCMV. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes an MHC-E supertop. In some embodiments, the MHC-E supertop is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertop 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 KQEPIDKELIt is 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] 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 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 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 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, is also disclosed herein.

[0147] (1) Administering to a first subject an amount of a HCMV vector (deleted for 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) effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex, wherein the recombinant HCMV vector contains at least one heterologous antigen; (2) Identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cells recognize an MHC-E / heterologous antigen-derived peptide complex; (3) Isolating one or more CD8+ T cells from the first subject or a second subject; (4) 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 TCR-transfected CD8+ T cells prepared by the process, wherein the transfected CD8+ T cells generate an immune response against an MHC-E / heterologous antigen-derived peptide complex, are also disclosed.

[0148] In some embodiments, the method may further comprise transfecting an expression vector into one or more CD8+ T cells, the 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, wherein the second CD8+ T cell receptor comprises CDR3α and CDR3β of the first CD8+ T cell receptor, and transfecting, 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 transfecting the expression vector can 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 MHC-E supertopes. 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) and is 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.

[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 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, 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.

[0151] Also disclosed herein are methods of treating a disease such as cancer, a pathogen infection, or an immune disease or disorder, the method comprising administering to a first or second subject transfected T cells that recognize an MHC-E peptide complex. Also disclosed herein are methods of inducing an immune response against a host self-antigen or a tissue-specific antigen, the method 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 syndromes, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell cancer, 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 parasite, and Mycobacterium tuberculosis.

[0154] A method of generating CD8+ T cells that recognize an MHC-E peptide complex, comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-E / supertopep peptide complex from a set of CD8+ T cells that recognize MHC-E in complex with an HIV supertopep peptide, wherein the set of CD8+ T cells is generated from a recombinant rhesus CMV (RhCMV) or cynomolgus CMV (CyCCMV) vector lacking orthologs of UL128, UL130, UL146, and UL147, and expressing an effective amount of an HIV antigen 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 the CDR3α and CDR3β of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize an MHC-E peptide complex, is also disclosed herein.

[0155] A method of generating CD8+ T cells that recognize an MHC-II peptide complex is also disclosed herein. The method comprises administering to a first subject (or animal) an effective amount of a CMV vector effective to generate a set of CD8+ T cells that recognize an MHC-II / peptide complex. The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, or UL147 protein, and in some embodiments, 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 target sites for microRNAs 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 viral 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] This method further comprises identifying a first CD8+ T cell receptor from a set of CD8+ T cells, wherein 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 can further comprise transfecting one or more CD8+ T cells with an expression vector, the 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, wherein the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor, and transfecting, 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 transfecting the expression vector can be isolated from a first subject or a second subject.

[0159] 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-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 a CD8+ T cell receptor that recognizes an MHC-II supertop.

[0160] A method of generating CD8+ T cells that recognize an MHC-II-peptide complex, 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 an 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 the CDR3α and CDR3β of the first CD8+ TCR. Thereby generating one or more CD8+ T cells that recognize an MHC-II peptide complex.

[0161] (1) Administering to a first subject an amount of a UL18-deficient HCMV vector (UL128, UL130, UL146, or UL147 (or combinations thereof), and in some embodiments lacking UL82, and / or expressing a nucleic acid encoding a microRNA recognition element) effective to generate a set of CD8+ T cells that recognize MHC-II / peptide complexes, wherein the recombinant CMV vector contains at least one heterologous antigen; (2) Identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cells recognize MHC-II / heterologous antigen-derived peptide complexes; (3) Isolating one or more CD8+ T cells from the first subject or a second subject; and (4) Transfecting an expression vector into the one or more CD8+ T cells isolated from the first or second subject, thereby generating transfected T cells that recognize MHC-II peptide complexes, and the transfected CD8+ T cells generate an immune response against MHC-II / heterologous antigen-derived peptide complexes. Also disclosed are TCR-transfected CD8+ T cells that recognize MHC-II-peptide complexes prepared by a process that includes:

[0162] In some embodiments, the method may further include transfecting an expression vector into one or more CD8+ T cells, the expression vector including 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, wherein the second CD8+ T cell receptor includes CDR3α and CDR3β of the first CD8+ T cell receptor, and thereby generating one or more truncated CD8+ T cells that recognize MHC-II / heterologous antigen-derived peptide complexes. The one or more CD8+ T cells for transfecting 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 receptors are identified by RNA or DNA sequencing. In some embodiments, the first and / or second CD8+ T cell receptors recognize MHC-II supertopes.

[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 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 the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

[0165] Also disclosed herein are methods of treating a disease such as cancer, 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 of inducing an immune response against a host self-antigen or 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 syndromes, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell cancer, 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 of generating CD8+ T cells that recognize MHC-Ia peptide complexes. The method includes administering to a first subject an amount of a UL18-deficient CMV vector that is also deficient in US11 protein, which is effective to generate a set of CD8+ T cells that recognize MHC-Ia / peptide complexes. The CMV vector includes a first nucleic acid sequence encoding at least one heterologous antigen and does not express US11 protein and UL18 protein. The vector may also lack UL128 protein, UL130 protein, or UL146 protein, UL147 protein, and / or UL82 protein. The antigen can be any antigen including a pathogen-specific antigen, a tumor viral 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] This method further includes identifying a first CD8+ T cell receptor from a set of CD8+ T cells, the first CD8+ T cell receptor recognizing 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, the expression vector including 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 including the CDR3α and CDR3β of the first CD8+ T cell receptor, and transfecting, 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 transfecting the expression vector can be isolated from a first subject or a second subject.

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

[0171] A method for generating CD8+ T cells that recognize an MHC-I-peptide complex, comprising: (a) identifying a first CD8+ TCR that recognizes an MHC-I / heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize an MHC-I / heterologous 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 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 the CDR3α and CDR3β of the first CD8+ TCR. Also disclosed is a method for generating one or more CD8+ T cells that recognize an MHC-I peptide complex thereby.

[0172] (1) Administering to a first subject an amount of a CMV vector lacking US11 and UL18 effective to generate a set of CD8+ T cells that recognize an MHC-Ia / peptide complex (furthermore, the vector may lack UL128, UL130, UL146, UL147, and / or UL82 and express UL40 and / or US28), wherein the recombinant CMV vector comprises at least one heterologous antigen; (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 complex; (3) isolating one or more CD8+ T cells from the first subject or a second subject; and (4) transfecting the 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 the transfected CD8+ T cells generate an immune response against the MHC-Ia / heterologous antigen-derived peptide complex. Also disclosed are transfected CD8+ T cells that recognize an MHC-Ia peptide complex prepared by this process.

[0173] In some embodiments, the method may further comprise transfecting one or more CD8+ T cells with an expression vector, the 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, and transfecting, thereby generating one or more truncated CD8+ T cells that recognize MHC-Ia / heterologous antigen-derived peptide complexes. The one or more CD8+ T cells for transfecting the expression vector can be isolated from a first subject or a second subject.

[0174] In some embodiments, the first and / or second CD8+ T cell receptors are 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 is a method of treating a disease such as cancer, pathogen infection, or an immune disease or disorder, comprising administering to a first or second subject transfected T cells that recognize MHC-Ia peptide complexes. Also disclosed herein is a method of inducing an immune response against a host self-antigen or tissue-specific antigen, comprising administering to a first or second subject transfected T cells that recognize MHC-Ia peptide complexes.

[0177] Cancers include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar 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 Supertopes Constructs Amino acid sequences that are 9 to 15 amino acids in length and are at least 90%, at least 95%, or 100% identical to the amino acid sequences 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 also disclosed herein.

[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 the UL18 protein, the UL128 protein, the UL130 protein, the UL146 protein, and the UL147 protein, or their orthologs, due to the presence of one or more mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, cleavage mutations, and all deletions of the nucleic acid sequences encoding viral proteins. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 or its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 or its ortholog. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71) or its ortholog. In some embodiments, the recombinant HCMV vector does not express US11 or its ortholog. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and the MRE comprises a target site for an 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, and the MRE comprises a target site for an miRNA expressed in myeloid cells.In some embodiments, 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.

[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. Immunocompositions comprising a recombinant CMV virus or vector (or its expression product) induce a local or systemic immune response. The response may be protective, but need not be. Vaccine compositions induce a local or systemic protective or therapeutic response. Thus, the term "immunogenic composition" includes "vaccine composition" (since the former term may be a protective composition).

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

[0183] The recombinant CMV vectors disclosed herein can be used in a therapeutic composition 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 comprising a sequence encoding a tumor antigen into an essential or non-essential region of the CMV genome. The method can further comprise deleting one or more regions from the CMV genome. The method can comprise in vivo recombination. Thus, the method can comprise transfecting cells with CMV DNA in a cell-compatible medium in the presence of donor DNA comprising heterologous DNA flanked by DNA sequences homologous to portions of the CMV genome, thereby introducing the heterologous DNA into the genome of CMV, and optionally, then recovering CMV modified by in vivo recombination. The method can comprise cleaving CMV DNA to obtain cleaved CMV DNA, ligating heterologous DNA to the cleaved CMV DNA to obtain hybrid CMV-heterologous DNA, transfecting cells with the hybrid CMV-heterologous DNA, and optionally, then recovering CMV modified by the presence of the heterologous DNA. Because in vivo recombination is included, the method can also provide a plasmid comprising donor DNA that encodes a polypeptide that is foreign to CMV and that is not naturally present in CMV, wherein the donor DNA is within a segment of CMV DNA that is co-linear with an essential or non-essential region of the CMV genome such that DNA from the essential or non-essential region of CMV flanks the donor DNA. The heterologous DNA can be inserted into CMV to generate recombinant CMV in any orientation that results in stable integration of the DNA and, if desired, expression thereof.

[0184] DNA encoding a heterologous antigen in a recombinant CMV vector may also contain a promoter. The promoter can be derived from any source such as a herpes virus, including an endogenous cytomegalovirus (CMV) promoter, e.g., human CMV (HCMV), rhesus macaque CMV (RhCMV), mouse, or other CMV promoters. The promoter can also be a non-viral promoter such as the EF1α promoter. The promoter can be a truncated transcriptional active promoter, including a region transactivated by a transactivating protein provided by the virus and a minimal promoter region of the full-length promoter from which the truncated transcriptional active promoter is derived. The promoter can consist of the binding of a DNA sequence corresponding to a minimal promoter and upstream regulatory sequences. The minimal promoter consists of a CAP site and an ATA box (the minimal sequence for the basal level of transcription, which is the unregulated level of transcription), and the "upstream regulatory sequences" consist of upstream elements and enhancer sequences. Further, 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. Also, the truncated promoter can be derived from a herpes virus such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. Based on base pairs, there can be a size reduction of up to 40%, and further up to 90%, from the full-length promoter. The promoter can also be a modified non-viral promoter. For the HCMV promoter, see U.S. Pat. Nos. 5,168,062 and 5,385,839. For transfection of plasmid DNA for expression into cells, see Feigner et al. (1994), J Biol.Chem. 269, 2550-2561. Also, for direct injection of plasmid DNA as a simple and effective vaccination method against various infectious diseases, see Science, 259:1745-49, 1993. Thus, the use of the vector by direct injection of vector DNA is within the scope of the present disclosure.

[0185] Also disclosed are expression cassettes that can be inserted into recombinant viruses or plasmids containing a cleavage-type transcriptional activator promoter. The expression cassette may further comprise a functional cleavage-type polyadenylation signal, for example, the SV40 polyadenylation signal that has been cleaved but is still functional. Considering that a larger signal is naturally conferred, it is quite surprising that the cleavage-type polyadenylation signal is functional. The cleavage-type polyadenylation signal addresses the issue of the insertion size limit of recombinant viruses such as CMV. The expression cassette may also contain heterologous DNA, which may be the heterologous DNA described herein, in relation to the virus or system into which the heterologous DNA is inserted.

[0186] Regarding antigens for use in vaccines or immunological compositions, see also Stedman’s Medical Dictionary (24th edition, 1982), for example, the definition of vaccine (for a list of antigens used in vaccine formulations). Such antigens of interest or epitopes derived from those antigens may be used. Regarding tumor antigens, one of ordinary skill in the art can select tumor antigens and their encoding DNA from knowledge of the amino acids of the peptides or polypeptides and the corresponding DNA sequences, as well as the properties of specific amino acids (e.g., size, charge, etc.) and the codon dictionary, without undue experimentation.

[0187] One method for determining the T epitopes of an antigen involves epitope mapping. Overlapping peptides of the tumor antigen are generated by oligopeptide synthesis. The individual peptides are then tested for their 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] An immune response against a tumor antigen is generally generated as follows: T cells recognize a protein only when the protein is cleaved into smaller peptides and presented by a complex called the "major histocompatibility complex (MHC)" located on the surface of another cell. The MHC complex has two classes, 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 rhesus monkeys, Qa-lb in mice). Unlike other MHC-I molecules, MHC-E is highly conserved within and between mammalian species.

[0189] Note that the DNA containing the sequence encoding the tumor antigen may itself contain a promoter for driving expression in the CMV vector, or the DNA may be limited to the coding DNA of the tumor antigen. This construct can be operably linked to the promoter and arranged in the orientation with respect to the endogenous CMV promoter so as to be expressed thereby. Further, multiple copies of the DNA encoding the tumor antigen, or the use of a strong or early promoter or an early and late promoter, or any combination thereof, can be done to amplify or increase the expression. Thus, the DNA encoding the tumor antigen can be suitably arranged with respect to the CMV endogenous promoter, or those promoters can be repositioned to be inserted at another location together with the DNA encoding the tumor antigen. Nucleic acids encoding two or more tumor antigens can be packaged into the CMV vector.

[0190] Pharmaceutical compositions and other compositions containing the CMV vectors of the present disclosure are further 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 via parenteral routes (intradermal, intraperitoneal, intramuscular, subcutaneous, intravenous, or others). Administration can also be via mucosal routes such as oral, nasal, genital, etc.

[0191] The pharmaceutical compositions of the present disclosure can be prepared according to standard techniques well known to those skilled in the art in the pharmaceutical field. Such compositions can be administered at dosages and by techniques well known to those skilled in the medical field, taking into account factors such as the breed or species, age, gender, weight, and condition of a 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 immune, antigenic, or vaccine, or therapeutic compositions. Such other compositions can include purified natural 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 the compositions include liquid preparations for administration to an orifice, such as, for example, oral, nasal, anal, genital, such as vaginal, etc., for example, suspensions, syrups, or elixirs, as well as preparations for parenteral, subcutaneous, intraperitoneal, intradermal, intramuscular, or intravenous administration (for example, injectable administration), for example, sterile suspensions or emulsions. 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] An antigen, immune, or vaccine composition may typically include an adjuvant and an amount of a CMV vector or expression product to elicit a desired response. In human use, alum (aluminum phosphate or aluminum hydroxide) is a typical adjuvant. Saponin and its purified component Quil A, 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, lipid conjugates, for example, those described in Goodman-Snitkoff et al., J Immunol. 147:410-415 (1991), encapsulation of proteins within proteoliposomes as described in Miller et al., J Exp. Med. 176:1739-1744 (1992), and encapsulation of proteins within lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be used.

[0194] The composition can be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or administration at an orifice, such as sublingual (e.g., oral), intragastric, intraoral, intraanal, intravaginal, etc. mucosal administration. Again, the effective dosage and route of administration are determined by the nature of the composition, the nature of the expressed product, the expression level when recombinant CMV is used directly, as well as known factors such as the species or strain, age, sex, weight, condition, and nature of the host, and other known screening procedures that do not require excessive experimentation or LD50. The dosage of the expressed product can range from a few micrograms to several hundred micrograms, for example, in the range of 5 - 500 μg. The CMV vector can be administered in any suitable amount to achieve expression at these dosage levels. In non-limiting examples, the CMV vector can be administered in an amount of at least 102 pfu, and thus, the CMV vector can be administered at least this amount or in the 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 for resuspension at the time of administration or can be in solution. "About" can mean within 1%, 5%, 10%, or 20% of the 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. Accordingly, the present disclosure contemplates deletions, additions, truncations, and substitutions to the sequences shown, so 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 the following four families: (1) acidic - aspartate and glutamate, (2) basic - lysine, arginine, and histidine, (3) non - polar - 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 may be classified as aromatic amino acids. Isolated substitution of leucine by isoleucine or valine or vice versa, substitution of aspartate by glutamate or vice versa, substitution of threonine by serine or vice versa, or similar conservative substitutions of amino acids by structurally related amino acids are reasonably predictable not to have a major impact on biological activity. Accordingly, proteins having substantially the same amino acid sequence as the described proteins but having 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 sequences of the present disclosure can be codon - optimized. For example, the codons can be optimized for use in human cells. For example, any viral or bacterial sequences can be so modified. Many viruses, including HIV and other lentiviruses, use many rare codons and can have their codons modified to correspond to codons commonly used in the desired subject, as described in Andre et al., J Virol. 72:1497 - 1503, 1998, to achieve enhanced expression of tumor antigens.

[0197] Nucleotide sequences encoding CMV vectors and functionally and / or antigenically equivalent variants and derivatives of glycoproteins contained therein are contemplated. These functionally equivalent variants, derivatives, and fragments exhibit the ability to retain antigenic activity. For example, changes in the DNA sequence that do not alter the encoded amino acid sequence, as well as conservative substitutions of amino acid residues, deletions or additions of one or several amino acids, and changes that result in substitution of amino acid residues with amino acid analogs, are those that do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine / methionine, lysine / arginine, and phenylalanine / tyrosine / tryptophan. In some embodiments, the variant has 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 with the 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 gaps. In particular, sequence identity can be determined using any of a number of mathematical algorithms. Non-limiting examples of mathematical algorithms used for comparing two sequences are the algorithms of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990;87:2264-2268 as modified in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90:5873-5877.

[0199] Another example of a mathematical algorithm used for array comparison 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 array alignment software package. When using the ALIGN program to compare 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 according to the present disclosure. WU-BLAST version 2.0 executable programs for several 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 made using standard recombinant DNA and cloning techniques. Such techniques are well known to those skilled in the art. See, for example, ’’Molecular Cloning: A Laboratory Manual,’’ second edition (Sambrook et al. 1989).

[0202] Any vector that enables the expression of the virus of the present disclosure can be used in accordance with the present disclosure. In certain embodiments, the virus of the present disclosure can be used in vitro (e.g., using a cell-free expression system) and / or in cultured cells grown in vitro to produce encoded heterologous antigens (e.g., tumor viral antigens, HIV antigens, tumor antigens, and antibodies) for various applications such as the production of protein vaccines. For such applications, any vector that enables the expression of the virus in vitro and / or in cultured cells can be used.

[0203] For the tumor antigen of the present disclosure to be expressed, the protein coding sequence of the tumor antigen needs to be "operably linked" to regulatory sequences or nucleic acid control sequences that direct the transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are "operably linked" when they are covalently linked in such a way that the expression or transcription and / or translation of the coding sequence is placed under the influence or control of the nucleic acid control sequence. "Nucleic acid control sequence" is any nucleic acid element, such as, but not limited to, a promoter, enhancer, IRES, intron, and other elements described herein, that is operably bound to a nucleic acid sequence or directs the expression of a coding sequence operably linked thereto. The term "promoter" is used herein to refer to a group of transcriptional control modules that are clustered around the start site of RNA polymerase II and that cause the expression of the encoded protein when operably linked to the protein coding sequence of the present disclosure. 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 certain 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, suitable promoters and / or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).

[0204] Vectors used in accordance with the present disclosure can include suitable gene regulatory regions such as promoters or enhancers so that the antigen of the present disclosure can be expressed.

[0205] The CMV vectors described herein can include mutations that prevent spread from host to host, thereby rendering the virus unable to infect immunocompromised or other subjects who may develop complications as a result of CMV infection. The CMV vectors described herein can also include 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 ability of the vectors 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 disclosed CMV vectors can be administered in vivo for the purpose of generating an immunogenic response, including a CD8+ immune response, that includes an immune response characterized by a high proportion of CD8+ T cell responses restricted by, for example, MHC-E, MHC-II, or MHC-I (or homologs or orthologs thereof). For example, in some instances, it may be desirable to use the disclosed CMV vectors for preclinical testing of immunogenic compositions and vaccines using RhCMV in experimental animals such as rhesus monkeys. In other instances, it may be desirable to use the disclosed CMV vectors for the actual clinical use of immunogenic compositions using HCMV in human subjects such as in clinical trials.

[0207] For such in vivo uses, the CMV vectors of the present disclosure are administered as components 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 a heterologous antigen, including a tumor antigen, a tumor virus antigen, or a host self - antigen, and may be used as one or more components of a prophylactic or therapeutic vaccine against a tumor antigen, a tumor virus antigen, or a host self - antigen for the prevention, amelioration, or treatment of cancer. The nucleic acids and vectors of the present disclosure are particularly useful for providing a gene vaccine, i.e., a vaccine that delivers a nucleic acid encoding an antigen of the present disclosure to a subject such as a human, whereby the antigen is then expressed in the subject and an immune response is induced.

[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, an 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 the case of humans, the interval is typically 2 - 6 weeks. In particularly advantageous embodiments of the present disclosure, the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks, or 70 weeks. Immunization regimens typically have from 1 to 6 administrations of the immunogenic composition, but may be as few as 1, 2, or 4. Methods of inducing an immune response may also include administering an adjuvant along with the immunogen. In some cases, the initial immunization protocol may be supplemented by annual, biannual, or other long interval (5 - 10 years) booster immunizations. The methods of the present invention also include various prime - boost regimens. In these methods, one or more booster immunizations are performed after one or more priming immunizations. The actual immunogenic composition may be the same or different for each immunization, and the type, route, and formulation of the immunogen (including, for example, a protein or an expression vector) may also vary. For example, if an expression vector is used in the priming and boosting steps, it may be of either the same or different type (e.g., DNA or a bacterial or viral expression vector). One useful prime - boost regimen provides two priming immunizations 4 weeks apart, followed by two boosting immunizations at 4 weeks and 8 weeks after the last priming immunization.It should also be readily apparent to those skilled in the art that several substitutions and combinations are included to provide priming and boosting regimens using the DNA, bacterial, and viral expression vectors of the present disclosure. The CMV vector can be repeatedly used while expressing different antigens derived from different pathogens.

Examples

[0209] Example 1: Protection against SIV by induction of MHC-E-restricted CD8+ T cells In several studies, a strain 68-1-derived RhCMV vector expressing SIV antigens has been shown to control infection by highly pathogenic SIVmac239 and ultimately lead to clearance of the infection (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 68-1 RhCMV 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 testing whether MHC-E or MHC-II restricted CD8+ T cells are involved in unique protection against SIVmac239. As described below, four cohorts of rhesus macaques (RM) were inoculated with different 68-1RhCMV strains.

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

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

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

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

[0215] The mean frequency of generation of CD4+ or CD8+ T cells responding to SIV antigen-derived peptide pools was quantified. T cell frequency of generation was 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 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, the MHC restriction of the SIVgag-specific CD8+ T cell responses was analyzed. The SIVgag-specific CD8+ T cell responses in PBMC obtained from three RMs in each cohort were measured 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 the animals of cohort 1 were blocked by the VL9 peptide, and the peptide responses in cohorts 2 and 3 were blocked by the CLIP peptide (Figure 2). Thus, the CD8+ T cells in cohort 1 were exclusively restricted by MHC-E, and the CD8+ T cells in cohorts 2 and 3 were exclusively restricted by MHC-II. The CD8+ T cell responses in the animals of cohort 4 (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 are involved in protection, cohorts 1, 2, and 3 were challenged by repeated intrarectal inoculation of dose-limiting SIVmac239. RMs were challenged weekly until the first plasma virus load (pvl) or SIVvif response was detected (onset of infection was defined as the previous challenge). Since the vaccine vector does not express SIVvif, the occurrence of a new SIVvif response in the absence of detectable SIV plasma virus load is proof of infection. Unlike non-controllers (black boxes) that exhibit persistent viremia with a typical peak and plateau pattern once infected, an RM was considered a controller (white box) if plasma viremia was not observed within 2 weeks from the first positive pvl or became undetectable and then maintained below the threshold for at least 4 out of the next 5 weeks.

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

[0219] Previously, it has been shown that the RhCMV vector derived from strain 68-1 induces CD8+ T cell responses that exhibit a very high epitope density (= 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 some 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 are not described with respect to "classical" epitopes presented by MHC-I molecules and thus represent a unique feature of CMV-based vectors. To determine whether the supertopes alone can account for the protection observed with the above "MHC-E only" RhCMV vector, artificial fusion proteins consisting of supertope sequences derived from individual SIV antigens were generated (Table 1, 15-mer and minimal supertope peptide sequences are underlined).

Table 1

[0220] The sequence of the artificial fusion protein is as follows (HA-epitope tag is underlined): MRRWRRRWQQLLALADRIYSFPDPTSSASNKPISNRTRHCQPEISMRRSRPSGDLRQRLLRAEKLAYRKQNMDDIDEEDDDAQTSQWDDPWGEVLAWKFDYVRYPEEFGSKSGLSEEEVGGIGGFINTKEYKNVEIVLGKRNTPTFAIKKKDKNKWRMLIDFREWMGYELWPTKWKLQKIELPLGLQKCVRMYNPTNILDVKYMQLGKQQREKQRESREKPYKEV YPYDVPDYAD (SEQ ID NO: 12). Immunoblotting was performed to show the expression of the SIV supertop fusion construct by probing with anti-HA antibody (Figure 4).

[0221] Aiming to focus on the CD8+ T cell response to a small set of MHC-E restricted epitopes, the SIV MHC-E supertop fusion protein was inserted into 68-1RhCMV containing the mir126 targeting site. The resulting construct was inoculated into 8 RMs (cohort 5). Intracellular cytokine staining of IFNγ or TNFα in the presence of a pool of individual 15mer peptides representing the SIV supertop was used to determine the frequency of T cell generation at the indicated time points in peripheral blood mononuclear cells (PBMCs) (Figure 5). CD8+ T cells responded to SIV antigen-derived peptides (Figure 5A). CD8+ T cells responded to the MHC-E restricted supertops Gag69 and Gag120, but did not respond 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 SIV gag insert (Figure 5B). These results indicate that all animals induced an SIV-specific CD8+ T cell response exclusively to the supertop.

[0222] To determine whether MHC-E supertype-restricted CD8+ T cells can recapitulate the protection observed with the "MHC-E only" vector, cohort 5 was challenged by repeated low-dose rectal inoculation 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 that exhibit persistent viremia with a typical peak and plateau pattern once infected (black boxes), RMs were considered controllers (black boxes) if the pvl became undetectable within 2 weeks from the first positive pvl and then remained below the threshold for at least 4 of the subsequent 5 weeks.

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

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

Table 2

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

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

[0227] Construct 2: 68-1RhCMV, in which the gene Rh107 (a homolog 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] 5×10 6 Plaque-forming units (PFU) of Construct 1 were inoculated into three RhCMV-seropositive RMs, and the same amount of Construct 2 was inoculated into two RhCMV-seropositive RMs on day 0. For controls, RMs were inoculated with 68-1RhCMV expressing SIVgag under the control of the EF1α promoter.

[0229] On days 7, 14, and then every other week, PBMCs were isolated from two RMs, and the CD8+ T cell responses to SIV antigens induced by Construct 1, 2, or the control were measured by intracellular cytokine staining (ICS) for IFNγ and TNFα using overlapping 15-mer peptide pools containing 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 the supertopes within each SIV antigen (Hansen Science 2013, Hansen Science 2016). Therefore, each of the supertope peptides was individually tested by ICS in the PBMCs of each RM.

[0230] The frequency of generation of CD8+ T cells responsive to an SIV antigen peptide pool, which represents the total antigen-specific response in two animals per group, was analyzed (Figure 7A). For the same two animals, the frequency of generation of CD8+ T cells responsive to MHC-E-restricted supertopes and MHC-II-restricted supertopes was also analyzed (Figures 7B, 7C).

[0231] All animals generated a CD8+ T cell response to the SIV antigen expressed by the RhCMV vector used for inoculation. However, supertope responses were observed only for 68-1RhCMV / SIVgag, and neither of the two vectors expressing UL18 induced T cells that recognized the supertope. These results indicated that UL18 inhibits 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 the induction of SIVpol-specific responses in three animals that received UL18-expressing 68-1RhCMV / SIVpol. The SIVpol-specific CD8+ T cell response in PBMC obtained from three RMs inoculated with construct 1 was measured in the presence of individual peptides. The CD8+ T cell response to individual peptides within SIVpol was measured in the presence of specific reagents that block either MHC-I, MHC-II, or MHC-E presentation (MHC-I and MHC-E are blocked with antibody W6 / 32, MHC-II is blocked with HLA-DR-specific antibody and CLIP peptide, and MHC-E is blocked with VL9 peptide).

[0233] The results shown in Figure 8 revealed that the stimulation of CD8+ T cells by each individual peptide was inhibited by the pan-MHC-I inhibitory antibody W6 / 32, but not by the MHC-E specific peptide VL9 or 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 inoculated with 68-1 RhCMV-expressing SIV antigen recognize all peptides related to MHC-II or MHC-E (Hansen Science 2013, Hansen Science 2016).

[0234] These results most likely indicate that UL18 reprogrammed the CD8+ T cell response by preventing the induction of MHC-II- and MHC-E-restricted CD8+ T cells. UL18 is known to be involved 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 the direct priming of CD8+ T cells by 68-1RhCMV by engaging the inhibitory leukocyte receptor (LIR) on T cells (direct priming refers to T cells that are primed by infected cells). In the absence of direct priming, CD8+ T cells are induced indirectly by cross-priming, that is, by non-infected cells (e.g., dendritic cells) that present antigens obtained from infected cells. Until now, UL18 has not been implicated in preventing T cell priming. These results are unexpected and without precedent.

[0235] To determine whether the interaction with the inhibitory receptor LIR1 is involved in the ability of UL18 to prevent the induction of MHC-II and MHC-E restricted CD8+ T cells, the coding region of UL18 in construct 1 above was mutated to replace the aspartate at amino acid position 196 in the α-3 domain with serine (D196S). Previous structural studies have shown that this aspartate is involved in the binding of UL18 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 the CD8+ T cell response to SIVpol was measured by ICS for IFNγ and TNFα using pools of overlapping 15-mer peptides containing SIVpol or the SIVpol MHC-E supertopepptide Pol41 (GFINTKEYKNVEIEV; SEQ ID NO: 33) or the MHC-II supertop Pol90 (LPQGWKGSPAIFQYT; SEQ ID NO: 34). In contrast to animals inoculated with intact UL18-expressing 68-1RhCMV (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 needs to be involved in 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). Since NK cell evasion can be important for vector function (Sturgill 2016. Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus. PLoS Pathog 12:e1005868), deletion of UL18 from HCMV-based vectors was thought to impair their ability to induce an immune response against heterologous antigens. To determine whether UL18-deleted HCMV could induce a T cell response against an inserted antigen, UL18 was replaced with an HIV antigen, thereby deleting UL18 and driving the expression of an HIVgag / nef / pol fusion protein using the endogenous UL18 promoter. Furthermore, the genes UL128, UL130, UL146, and UL147 were also deleted from the UL18-deleted vector because these gene products had previously been shown to inhibit MHC-E and MHC-II restricted CD8+ T cell responses (U.S. Patent No. 10,532,099). As the vector backbone, HCMV TR3 was used (Caposio. 2019. Characterization of a live-attenuated HCMV-based vaccine platform. Scientific Reports 9:19236). The expression of the HIV fusion protein in the resulting viral vector (HCMV TR3 ΔUL18 / HIV fusion ΔUL128-130ΔUL146-147) was confirmed by immunoblot of human fibroblasts (Figure 10).

[0237] UL18 - deleted HCMV vectors were also inoculated into RMs, and the immune responses against HIV antigens were determined in PBMCs by ICS on day 56 post - inoculation. As shown in Figure 11, the vectors induced CD8+ T - cell responses against HIVgag, HIVnef, and HIVpol in RMs, as demonstrated by using pools of overlapping peptides containing each of these antigens. Thus, it was concluded that HCMV vectors lacking UL18 retained the ability to induce T - cell responses against heterologous antigens.

Claims

1. A recombinant human cytomegalovirus (HCMV) vector comprising a nucleic acid sequence encoding a heterologous antigen and retaining the ability to induce a T cell response against the heterologous antigen, (a) that does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, (b) wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40 protein Recombinant HCMV vector.

2. The recombinant HCMV vector according to claim 1, wherein the recombinant HCMV vector does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein due to the presence of one or more mutations in the nucleic acid sequences encoding UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein.

3. The recombinant HCMV vector according to claim 2, wherein the mutation in the nucleic acid sequences encoding UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein is selected from the group consisting of point mutations, frameshift mutations, cleavage mutations, and all deletions of the nucleic acid sequences encoding the viral proteins.

4. The recombinant HCMV vector according to any one of claims 1 to 3, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28 protein.

5. The recombinant HCMV vector according to any one of claims 1 to 4, wherein the recombinant HCMV vector does not express UL82 (pp71) protein.

6. The recombinant HCMV vector according to any one of claims 1 to 5, wherein the recombinant HCMV vector does not express US11 protein.

7. The recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) recognition element (MRE), and the MRE comprises a target site for a miRNA expressed in endothelial cells. The recombinant HCMV vector according to any one of claims 1 to 5.

8. The miRNA expressed in the endothelial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221 / 222, miR-378, miR-296, or miR-328. The recombinant HCMV vector according to claim 7.

9. 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 bone marrow cells. The recombinant HCMV vector according to any one of claims 1 to 8.

10. The miRNA expressed in the bone marrow cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125. The recombinant HCMV vector according to claim 9.

11. The heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen. The recombinant HCMV vector according to any one of claims 1 to 10.

12. The pathogen-specific antigen is derived from 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. The recombinant HCMV vector according to claim 11.

13. The pathogen-specific antigen is an MHC-E supertop. The recombinant HCMV vector according to any one of claims 1 to 5 and 7 to 8.

14. The recombinant HCMV vector according to claim 13, wherein the pathogen-specific antigen contains an HIV epitope.

15. The recombinant HCMV vector according to claim 14, wherein the HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LD AWEKIRLRPGGKK (SEQ ID NO: 13), DAWEKIRLR (SEQ ID NO: 14), KK AQQAAADTGNS SQ (SEQ ID NO: 15), KAQQAAADT (SEQ ID NO: 16), QMVHQ AISPRTLNAW (SEQ ID NO: 17), HQ AISPRTL (SEQ ID NO: 18), NTMLNT VGGHQ AAMQ (SEQ ID NO: 19), VGGHQ AAMQ (SEQ ID NO: 20), STLQ EQIGWMTNNPP (SEQ ID NO: 21), STLQ EQIGW (SEQ ID NO: 22), IVRMY SPVSIL DIRQ (SEQ ID NO: 23), RMY SPVSIL (SEQ ID NO: 24), QKQEP IDKELYPLAS (SEQ ID NO: 25), KQEP IDKEL (SEQ ID NO: 26), SFSF PQITLW QRPLV (SEQ ID NO: 27), VRQY DQILIEICGKK (SEQ ID NO: 28), EPFR KQNPDIVIYQL (SEQ ID NO: 29), YVDG AA NRETKL GKA (SEQ ID NO: 30), EEHE KYSNWRAMAS (SEQ ID NO: 31), or ILDLW VYHTQGYFPD (SEQ ID NO: 32).

16. The recombinant HCMV vector according to claim 11, wherein the tumor antigen is related to acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin 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.

17. The recombinant HCMV vector according to claim 11, 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.

18. A pharmaceutical composition comprising the recombinant HCMV vector according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. An immunogenic composition comprising the recombinant HCMV vector according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition for use in generating an immune response in a subject, comprising the recombinant HCMV vector according to any one of claims 1 to 17.

21. A pharmaceutical composition for use in the treatment or prevention of cancer in a subject, comprising the recombinant HCMV vector according to claim 11 or 16.

22. A pharmaceutical composition for use in the treatment or prevention of a pathogen infection in a subject, comprising the recombinant HCMV vector according to any one of claims 11 to 15.

23. A pharmaceutical composition for use in the treatment of an autoimmune disease or disorder in a subject, comprising the recombinant HCMV vector according to claim 11 or 17.

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