Tumor-associated antigen-specific t cell responses

Abstract

To provide a method for inducing an immune response against a tumor-associated antigen in a subject.SOLUTION: A method comprises administering to a subject a CMV vector encoding a tumor-associated antigen in an amount effective to induce a CD8+ T cell response against the tumor-associated antigen, where the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or respective orthologs thereof, and where the tumor-associated antigen comprises a specific amino acid sequence of 15 amino acid residues (MHC-E or MHC-II supertope peptide).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 878,511, filed July 25, 2019, and U.S. Provisional Patent Application No. 62 / 858,756, filed June 7, 2019, each of which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. R 44 CA 180177-03 awarded by the National Cancer Institute and Grant No. W81XWH-19-1-0358 awarded by the Department of Defense. The government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing in an ASCII text file submitted with this application (Name 4153_012PC02_SL_ST25; Size: 3,591 bytes; Created May 27, 2020) are hereby incorporated by reference in their entirety. [Background technology]

[0004] Cytomegalovirus (CMV)-based vaccines, as well as vaccines based on other herpesviruses, are expected to be promising near-term additions to our arsenal against infectious diseases and cancer. These herpesvirus-based vectors are unique not only in the high levels of T cell immunity they induce against their heterologously encoded pathogen (or cancer) target antigens, but also in the durability of that immunity and its "immediate effector" qualities.

[0005] Many tumor-associated antigens (TAAs) are autoantigens aberrantly expressed by cancer cells. A major challenge in eliciting T cell responses specific to TAAs is that "canonical" T cells, which strongly recognize peptides derived from these antigens in the context of MHC-I or MHC-II as autoantigens, are removed from the immune repertoire by negative selection. Therefore, effective cancer vaccines are expected to break immune tolerance by stimulating "non-canonical" T cells that escape negative selection by expressing low-affinity TCRs or by recognizing peptides that bind with low affinity to MHC. All currently available T cell-inducing vaccines, such as DNA, RNA, pox vector, adeno vector, or alphavirus systems, are designed to induce canonical T cells. Consequently, there remains a need in the art for therapeutic approaches that can break immune tolerance to tumor-associated antigens. Summary of the Invention

[0006] The present disclosure provides a method for generating an immune response to a tumor-associated antigen in a subject, comprising administering to the subject a CMV vector encoding the tumor-associated antigen in an amount effective to elicit a CD8+ T cell response against the tumor-associated antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHY ELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0007] The present disclosure also provides a method of treating cancer in a subject, comprising administering to the subject a CMV vector encoding a tumor-associated antigen in an amount effective to elicit a CD8+ T cell response against the tumor-associated antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen is selected from the group consisting of the amino acid sequences ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE ( SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0008] The present disclosure also relates to a CMV vector encoding a tumor-associated antigen for use in generating an immune response to the tumor-associated antigen in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNE SYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0009] The present disclosure also relates to a CMV vector encoding a tumor-associated antigen for use in treating cancer in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their respective orthologues, and the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0010] The present disclosure also relates to the use of a CMV vector encoding a tumor-associated antigen in the manufacture of a medicament for use in generating an immune response to the tumor-associated antigen in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen is selected from the group consisting of the amino acid sequences ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4). 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0011] The present disclosure also relates to the use of a CMV vector encoding a tumor-associated antigen in the manufacture of a medicament for the treatment of cancer, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their respective orthologues, and the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0012] The present disclosure also relates to a method of treating cancer caused by a tumor virus in a subject, comprising administering to the subject a CMV vector encoding a tumor virus antigen in an amount effective to induce a CD8+ T cell response against a tumor-associated antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues.

[0013] The present disclosure also relates to a method of treating cancer caused by an oncogenic virus in a subject, comprising administering to the subject a CMV vector encoding an oncogenic virus antigen in an amount effective to induce a CD8+ T cell response against the oncogenic virus antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues.

[0014] The present disclosure also relates to a CMV vector encoding a tumor virus antigen for use in treating cancer in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues.

[0015] The present disclosure also relates to the use of a CMV vector encoding a tumor virus antigen in the manufacture of a medicament for the treatment of cancer, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues.

[0016] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14). In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2). In some embodiments, the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3). In some embodiments, the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO:4). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO:5). In some embodiments, the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO:6). In some embodiments, the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO:7). In some embodiments, the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO:8). In some embodiments, the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO:9). In some embodiments, the tumor-associated antigen comprises the amino acid sequence IDESLI FYKKWELEA (SEQ ID NO:10). In some embodiments, the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO:11). In some embodiments, the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO:12). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13). In some embodiments, the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0017] In some embodiments, at least 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or its orthologue, or MHC-II or its orthologue. In another embodiment, 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 CMV vector are restricted by MHC-E or its orthologue. In another embodiment, less than 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC class 1a or its orthologue. In another embodiment, a portion of the MHC-E-restricted CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the vector.

[0018] In some embodiments, the specific MHC-E supertope comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 5. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 6. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 8. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 9. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 13. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 14.

[0019] In some embodiments, a subset of MHC-II-restricted CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the vector.

[0020] In some embodiments, the peptide comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-II epitope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 2. In another embodiment, the MHC-II epitope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 3. In another embodiment, the MHC-II epitope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 4. In another embodiment, the MHC-II epitope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:7.

[0021] The present disclosure also provides a method for generating CD8+ T cells that recognize MHC-E tumor-associated antigen peptide complexes, comprising the steps of: (a) administering to a first subject a recombinant CMV vector that does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / tumor-associated antigen-derived peptide complexes; and (b) activating a first CD8+ TCR that recognizes an MHC-E / tumor-associated antigen-derived peptide complex in said CD8+ T cells. (c) isolating a second set of one or more CD8+ T cells from a second subject; and (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-E / tumor-associated antigen peptide complexes.

[0022] The present disclosure also provides a method for generating CD8+ T cells that recognize MHC-E tumor-associated antigen peptide complexes, comprising the steps of: (a) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector that includes a nucleic acid that expresses a tumor-associated antigen, and does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue of each, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / tumor-associated antigen-derived peptide complexes; and (b) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector that includes a nucleic acid that expresses a tumor-associated antigen, and does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue of each, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / tumor-associated antigen-derived peptide complexes. (c) identifying a CD8+ TCR from the first set of CD8+ T cells; (c) isolating a second set of one or more CD8+ T cells from a second subject; and (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-E / tumor-associated antigen peptide complexes.

[0023] In some embodiments, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

[0024] In some embodiments, the tumor-associated antigen is associated with a cancer selected from the group consisting of prostate cancer, kidney cancer, mesothelioma, breast cancer, and cervical cancer. In other embodiments, the tumor-associated antigen is selected from the group consisting of prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, and Her-2, or orthologs thereof.

[0025] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14). In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2). In some embodiments, the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3). In some embodiments, the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO:4). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO:5). In some embodiments, the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO:6). In some embodiments, the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO:7). In some embodiments, the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO:8). In some embodiments, the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO:9). In some embodiments, the tumor-associated antigen comprises the amino acid sequence IDESLI FYKKWELEA (SEQ ID NO:10). In some embodiments, the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO:11). In some embodiments, the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO:12). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13). In some embodiments, the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0026] In some embodiments, the first CD8+ T cell recognizes a specific MHC-E supertope. In another embodiment, the specific MHC-E supertope comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 5. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 6. In some embodiments, the specific MHC-E supertope comprises a peptide derived from a Wilms tumor suppressor protein epitope. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 8. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 9. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 13. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:14.

[0027] In some embodiments, the second CD8+ T cells recognize a specific MHC-E supertope. In another embodiment, the specific MHC-E supertope comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 5. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 6. In another embodiment, the specific MHC-E supertope comprises a peptide derived from a Wilms tumor suppressor protein epitope. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 8. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 9. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 13. In another embodiment, the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:14.

[0028] In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.

[0029] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0030] In some embodiments, the first subject is a human or a non-human primate. In another embodiment, the first subject is a non-human primate and the second subject is a human, wherein 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 another embodiment, the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In another embodiment, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0031] In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In other embodiments, the second CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

[0032] In some embodiments, administering the CMV vector to the first subject comprises administering the CMV vector intravenously, intramuscularly, intraperitoneally, or orally to the first subject. In another embodiment, the transfected CD8+ T cells are administered to a second subject to treat or prevent cancer. In another embodiment, the cancer is prostate cancer, kidney cancer, mesothelioma, breast cancer, or cervical cancer.

[0033] The present disclosure also provides a method for generating CD8+ T cells that recognize MHC-II tumor peptide complexes, comprising the steps of: (a) administering to a first subject a recombinant CMV vector that includes a nucleic acid that expresses a tumor antigen, and that does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue of each, in an amount effective to generate a first set of CD8+ T cells that recognize an MHC-II / tumor antigen-derived peptide complex; and (b) activating a first CD8+ TCR that recognizes an MHC-II / tumor antigen-derived peptide complex in the CD8+ T cells. (c) isolating a second set of one or more CD8+ T cells from a second subject; and (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

[0034] The present disclosure also provides a method for generating CD8+ T cells that recognize MHC-II tumor antigen peptide complexes, comprising the steps of: (a) isolating a first set of CD8+ T cells from a first subject who has previously administered a recombinant CMV vector comprising a nucleic acid that expresses a tumor antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / tumor antigen-derived peptide complexes; and (b) isolating a first set of CD8+ T cells from a first subject who has previously administered a recombinant CMV vector comprising a nucleic acid that expresses a tumor antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / tumor antigen-derived peptide complexes. (c) identifying an 8+ TCR from the first set of CD8+ T cells; and (d) isolating a second set of one or more CD8+ T cells from a second subject. The method relates to a method for generating CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes, comprising: (a) identifying an 8+ TCR from the first set of CD8+ T cells; (b) isolating a second set of one or more CD8+ T cells from a second subject; and (c) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR, and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR.

[0035] In some embodiments, at least one recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

[0036] In some embodiments, at least one recombinant CMV vector does not express an active UL128 protein or its orthologue, does not express an active UL130 protein or its orthologue, does not express an active UL146 or its orthologue, does not express an active UL147 or its orthologue, or does not express an active US11 protein or its orthologue. In another embodiment, the mutation in the nucleic acid sequence encoding UL128, UL130, UL146, UL147, or US11 is selected from the group consisting of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequences encoding viral proteins.

[0037] In some embodiments, the tumor-associated antigen is associated with prostate cancer, kidney cancer, mesothelioma, breast cancer, or cervical cancer. In other embodiments, the tumor-associated antigen is prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2, or an ortholog thereof.

[0038] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14). In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2). In some embodiments, the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3). In some embodiments, the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO:4). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO:5). In some embodiments, the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO:6). In some embodiments, the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO:7). In some embodiments, the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO:8). In some embodiments, the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO:9). In some embodiments, the tumor-associated antigen comprises the amino acid sequence IDESLI FYKKWELEA (SEQ ID NO:10). In some embodiments, the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO:11). In some embodiments, the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO:12). In some embodiments, the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13). In some embodiments, the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0039] In some embodiments, the first CD8+ T cell recognizes an MHC-II supertope.

[0040] In some embodiments, the MHC-II supertope comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 2. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 3. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 4. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:7.

[0041] In some embodiments, the second CD8+ T cell recognizes an MHC-II supertope.

[0042] In some embodiments, the MHC-II supertope comprises a peptide derived from a prostatic acid phosphatase epitope. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 2. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 3. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO: 4. In another embodiment, the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:7.

[0043] In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.

[0044] In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0045] In some embodiments, the first subject is a human or a non-human primate. In other embodiments, the second subject is a human or a non-human primate.

[0046] In some embodiments, the first subject is a non-human primate and the second subject is a human, wherein 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 another embodiment, the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR. In another embodiment, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In another embodiment, the second CD8+ TCR is a chimeric CD8+ TCR. In another embodiment, the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

[0047] In some embodiments, administering the CMV vector to the first subject comprises administering the CMV vector intravenously, intramuscularly, intraperitoneally, or orally to the first subject. In another embodiment, the transfected CD8+ T cells are administered to a second subject to treat cancer. In another embodiment, the cancer is prostate cancer, kidney cancer, mesothelioma, breast cancer, or cervical cancer.

[0048] In some embodiments, CD8+ T cells are generated. In another embodiment, the CD8+ T cells are administered to a subject to treat or prevent cancer. In another embodiment, the CD8+ T cells are administered to a subject to induce an immune response against a host self-antigen. In some embodiments, the CD8+ T cells are used in the manufacture of a medicament for treating or preventing cancer. In some embodiments, the CD8+ T cells are administered to a subject to induce an immune response against a host self-antigen. In some embodiments, the CD8+ T cells are used to induce an immune response against a host self-antigen in a subject. In some aspects, the CD8+ T cells are used in the manufacture of a medicament for inducing an immune response against a host self-antigen.

[0049] The present disclosure also relates to an isolated MHC-E or MHC-II supertope peptide of about 8 to about 15 amino acids in length capable of being recognized by a CD8+ T cell receptor, wherein the supertope comprises a tumor-associated antigen.

[0050] In some embodiments, the peptide is ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 13).

[0051] The present disclosure also relates to a method for overcoming immune tolerance to a tumor-associated antigen in a subject in need thereof, the method comprising administering to the subject an effective amount of a cytomegalovirus (CMV) vector that expresses the tumor-associated antigen.

[0052] In some embodiments, the CMV vector is a human CMV vector or a rhesus CMV vector.

[0053] In some embodiments, the CMV vector does not express active UL128 or its orthologue, does not express active UL130 or its orthologue, does not express active UL146 or its orthologue, and does not express active UL147 or its orthologue. In another embodiment, the CMV vector does not express active UL128, active UL130, active UL146, or active UL147, or their orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL238, UL130, UL146, or UL147. In another embodiment, the mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147 is one or more of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of any nucleic acid sequence encoding a viral protein.

[0054] In some embodiments, the CMV vector is rhesus CMV strain 68-1.

[0055] In some embodiments, the CMV vector does not express active UL82 protein or its orthologue. In another embodiment, the CMV vector does not express active UL82 protein or its orthologue due to the presence of one or more mutations in the nucleic acid sequence encoding UL82. In another embodiment, the mutation in the nucleic acid sequence encoding UL82 is one or more of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of the nucleic acid sequence encoding UL82.

[0056] In some embodiments, the tumor-associated antigen is derived from prostate cancer, kidney cancer, mesothelioma, breast cancer, or cervical cancer. In other embodiments, the tumor-associated antigen is prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.

[0057] In some embodiments, an effective amount comprises an amount effective to induce a CD8+ T cell response against a tumor-associated antigen in a subject.

[0058] In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the CD8+ T cells are restricted by MHC-I or an ortholog thereof.

[0059] In some embodiments, the CD8+ TCR is identified from CD8+ T cells induced by a CMV vector, and the CD8+ TCR recognizes an MHC-I / tumor-associated antigen-derived peptide complex. In another embodiment, the CD8+ TCR is identified by DNA or RNA sequencing. In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0060] [Figure 1] This figure shows the mean T cell response frequencies elicited in six rhesus macaques (RMs) inoculated with RhCMV strain 68-1 (68-1 / PAP) expressing the cancer antigen PAP and RhCMV strain 68-1 (68-1 / SIVgag) expressing the SIV gag antigen. Three of the RMs were additionally co-vaccinated with RhCMV strain 68-1 (68-1 / WT1) expressing the cancer antigen WT1, and the other three were co-vaccinated with RhCMV strain 68-1 (68-1 / MSLN) expressing the cancer antigen MSLN. CD4+ and CD8+ T cell responses were measured in peripheral blood mononuclear cells (PBMCs) using overlapping peptide pools for each antigen by intracellular cytokine staining (ICS) at the indicated time points. The mean response frequencies are shown. [Figure 2]

[0023] Figure 1 shows the mean T cell response frequencies elicited in six female RMs inoculated with RhCMV strain 68-1 (68-1 / HER2), which expresses the cancer antigen HER2. CD4+ and CD8+ T cell responses were measured in peripheral blood mononuclear cells using overlapping peptide pools for HER2 by intracellular cytokine staining at each indicated time point. The mean response frequencies are shown. [Figure 3]

[0033] Figure 1 shows the mean T cell response frequencies elicited in eight female RMs inoculated with either RhCMV strain 68-1 (68-1 / HPV) (solid line) or RhCMV strain 68-1.2 (68-1.2 / HPV) (dashed line), which express a fusion protein of the E6 and E7 proteins of HPV16 and HPV18. CD4+ and CD8+ T cell responses were measured in PBMCs by ICS using overlapping peptide pools for HPV antigens at each indicated time point. Individual response frequencies are shown. [Figure 4] Figure 3 shows MHC-E-dependent recognition of HPV antigens by CD8+ T cells from RMs immunized with 68-1 / HPV. Four female RMs were inoculated with 68-1 expressing a fusion protein of the E6 and E7 proteins of HPV16 and HPV18 (see Figure 3). T cell responses were measured by ICS for TNFα and IFNγ. CD8+ T cells responding with both TNFα and IFNγ production appear in the upper right quadrant. VMAPRTLLL (SEQ ID NO: 1) (VL9) is an MHC-E ligand peptide. [Figure 5] Figure 1 shows MHC-E-dependent recognition of PAP by CD8+ T cells from RMs immunized with 68-1 / PAP. CD8+ T cells were isolated and co-incubated with K562 cells expressing MHC-E and either MHC-E and PAP or MHC-E and HPV fusion proteins. T cell responses were measured by ICS for TNFα and IFNγ. CD8+ T cells responding with both TNFα and IFNγ production appear in the upper right quadrant. VMAPRTLLL (SEQ ID NO: 1) (VL9) is an MHC-E ligand peptide. [Figure 6]Figure 1 shows MHC-E-dependent recognition of PAP and WT1 by CD8+ T cells from RMs immunized with 68-1 / PAP and 68-1 / WT1. Six male RMs were co-inoculated with 68-1 / PAP and 68-1 / WT1. CD8+ T cells were isolated and co-incubated with K562 cells expressing MHC-E and either MHC-E and PAP or MHC-E and WT1. T cell responses were measured for TNFα and IFNγ by ICS. [Figure 7] Figure 1 shows CD4+ and CD8+ T cell responses in PBMCs were measured by ICS using overlapping peptide pools for TNFα and IFNγ at the indicated time points. The frequency of PAP-specific T cells among memory T cells is shown. [Figure 8] Figure 1 shows MHC restriction analysis of PAP-specific CD8+ T cells. CD8+ T cell responses to individual peptides are shown as boxes along the PAP sequence. Peptide responses blocked by the MHC-I-specific antibody W6 / 32 indicate MHC-I restriction, peptide responses blocked by the MHC-II-specific peptide CLIP indicate MHC-II restriction, and peptide responses blocked by the MHC-E-specific peptide indicate MHC-E restriction. Peptides not yet tested in the presence of blocking reagents are also shown. Gray boxes: pending restriction; white boxes: conventional MHC-Ia restriction; dotted boxes: non-conventional MHC-E restriction; dashed boxes: non-conventional MHC-II restriction; black boxes: indeterminate blocking. [Figure 9] Figure 1 shows peptide mapping of PAP-, WT1-, and MSLN-specific CD8+ T cells. CD8+ T cell responses to individual peptides are shown as boxes along the sequence. Peptides have not yet been tested in the presence of blocking reagent. "Supertope" peptides that produced responses in all 68-1 / PAP animals are boxed: gray dashed boxes: MHC-II or MHC-E supertopes; black boxes: MHC-II supertopes (based on results in Figure 8); black dashed boxes: MHC-E supertopes (based on results in Figure 8). [Figure 10] Figure 1 shows MHC restriction analysis of WT1-specific CD8+ T cells. CD8+ T cell responses to individual peptides are shown as boxes along the WT1 sequence. Peptide responses blocked by the MHC-I-specific antibody W6 / 32, the MHC-II-specific peptide CLIP, and an MHC-E-specific peptide were blocked, as well as peptides not yet tested in the presence of blocking reagents. The following "supertope" peptides induced responses in all 68-1 / WT1-immunized animals: #3, #13, #14, and #58. Results from three animals suggest that all of these supertopes are MHC-E restricted. Gray boxes: reserved restriction; black boxes: conventional MHC-IA restriction; white boxes: non-conventional MHC-IE restriction; dashed boxes: non-conventional MHC-II restriction; dotted boxes: indeterminate blocking. DETAILED DESCRIPTION OF THE INVENTION

[0061] I. Terminology Unless otherwise specified, technical terms are used according to conventional usage.

[0062] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent 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 as such.

[0063] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0064] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open and inclusive sense, i.e., "including, but not limited to." "Consisting of" shall mean excluding more than trace elements of other components and substantial method steps disclosed herein. The term "consisting essentially of" limits the claim to specific materials or steps, or those that do not materially affect the essential characteristics of the claimed invention. For example, a composition consisting essentially of the elements specified herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, and the like. Similarly, a protein consists essentially of a particular amino acid sequence if it includes additional amino acids that contribute up to 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alter the activity of the protein by 50% or less). Embodiments defined by each transition phrase are included within the scope of the invention.

[0065] About: As used herein, the term "about" can mean within 1%, 5%, 10%, or 20% of a stated value.

[0066] Antigen: As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that can induce an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response against the protein.

[0067] Antigen-specific T cells: CD8 that recognize specific antigens + Lymphocytes or CD4 + Lymphocytes. Generally, antigen-specific T cells specifically bind to a particular antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0068] Administration: As used herein, the term "administration" means providing or giving an agent, such as a composition comprising an effective amount of a CMV vector containing an exogenous antigen, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0069] Effective amount: As used herein, the term "effective amount" refers to an amount of an agent, such as a CMV vector containing a heterologous antigen or transfected CD8+ T cells that recognize an MHC-E / xenogenous antigen-derived peptide complex, an MHC-II / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex, that is sufficient to reduce or eliminate the signs or symptoms of a condition or disease, or to produce a desired response, such as inducing an immune response to the antigen. In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of a disorder or disease. An effective amount can be a therapeutically effective amount, including an amount that prevents the onset 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.

[0070] Heterologous antigen: As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. The heterologous antigen may be a pathogen-specific antigen, a tumor virus antigen, a tumor-associated antigen, a host self-antigen, or any other antigen.

[0071] Hyperproliferative disease: A disease or disorder characterized by uncontrolled proliferation of cells. Hyperproliferative diseases include, but are not limited to, malignant and non-malignant tumors.

[0072] Immune tolerance: As used herein, "immune tolerance" refers to a state of unresponsiveness of the immune system to substances that have the potential to induce an immune response. Self-tolerance to an individual's own antigens, such as tumor-associated antigens, is achieved by both central and peripheral tolerance mechanisms.

[0073] Epitope: As used herein, an "epitope" is one that contains an allele-specific motif or other sequence, such as an N-terminal repeat, such that a peptide containing said motif 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 epitope is derived.

[0074] In some embodiments, epitopes are identified using sequence motifs or other methods, such as neural nets or polynomial determination, known in the art. Typically, an algorithm is used to determine the "binding threshold" of a peptide and select peptides with scores that result in a high probability of binding with a certain affinity and are immunogenic. The algorithm is based on either the effect of a specific amino acid at a specific position on MHC binding, the effect of a specific amino acid at a specific position on antibody binding, or the effect of a specific substituent on binding in a motif-containing peptide. In the context of an epitope, a "conserved residue" is a residue that appears significantly more frequently than would be expected by random distribution at a specific position in a peptide. In some embodiments, a conserved residue is a residue that may provide a contact point for the MHC structure with the epitope.

[0075] Mutation: As used herein, the term "mutation" refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus, or "wild-type" sequence. A mutant is any protein or nucleic acid sequence containing a mutation. Furthermore, a cell or organism having a mutation may also be referred to as a mutant. Some types of coding sequence mutations include point mutations (individual nucleotide or amino acid differences), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences in which one or more nucleotides or amino acids are missing, including deletions of the entire coding sequence of a gene or less), and frameshift mutations (differences in which deletion of a number of nucleotides not divisible by three results in a change in the amino acid sequence). Mutations that result in amino acid differences may also be referred to as amino acid substitution mutations. Amino acid substitution mutations can be described by the amino acid change at a specific position in the amino acid sequence compared to the wild-type.

[0076] Nucleotide sequence or nucleic acid sequence: The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence, including, but not limited to, messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid can be single-stranded or partially or completely double-stranded (duplex). A duplex nucleic acid can be a homoduplex or a heteroduplex.

[0077] Operably linked: As used herein, the term "operably linked" means that a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned so that it affects the second nucleic acid sequence. Operably linked DNA sequences may be contiguous, or they may be operably spaced apart.

[0078] Promoter: As used herein, the term "promoter" can refer to any of several nucleic acid control sequences that direct transcription of a nucleic acid. Typically, eukaryotic promoters contain necessary nucleic acid sequences near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element or some other specific DNA sequence recognized by one or more transcription factors. Expression by a promoter can be further regulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those skilled in the art. A nucleic acid containing a promoter operably linked to a nucleic acid sequence encoding a specific polypeptide can be called an expression vector.

[0079] Recombinant: As used herein, the term "recombinant" when referring to a nucleic acid or polypeptide refers to one having a sequence that is not naturally occurring or that is made by the artificial combination of two or more otherwise separated segments of sequence, such as a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques. Recombinant polypeptide can also refer to a polypeptide made using recombinant nucleic acid, including a recombinant nucleic acid (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen) that has been introduced into a host organism that is not the natural source of the polypeptide.

[0080] Operably linked: As used herein, the term "operably linked" means that a coding sequence and a nucleic acid regulatory sequence or promoter are covalently linked in such a way that the expression or transcription and / or translation of the coding sequence is under the influence or control of the nucleic acid regulatory sequence. A "nucleic acid regulatory sequence" can be any nucleic acid element, including, but not limited to, a promoter, enhancer, IRES, intron, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence operably linked thereto. In order to express the disclosed tumor-associated antigens, the protein-coding sequence of the tumor-associated antigen should be "operably linked" to a regulatory sequence or nucleic acid regulatory sequence that directs the transcription and translation of the protein.

[0081] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" is conventional. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1995, 19th Edition, by E.W. Martin, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will vary depending on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as excipients. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0082] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Polynucleotides are composed of four bases: adenine, cytosine, guanine, and thymine / uracil (uracil is used in RNA). A coding sequence from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

[0083] Polypeptide: The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymers can be linear or branched, can comprise modified amino acids or amino acid analogs, and can be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as labeling or conjugation with a biologically active moiety.

[0084] Protein orthologs are typically characterized by having greater than 75% sequence identity across a full-length alignment with the amino acid sequence of a particular protein using ALIGN set to default parameters. Proteins with even greater similarity to the reference sequence will exhibit increasing 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, as assessed by this method. Additionally, sequence identity can be compared across the entire length of a particular domain of the disclosed peptide.

[0085] Promoter: As used herein, the term "promoter" refers to a group of transcriptional control modules that are centered around the initiation site of RNA polymerase II and that, when operably linked to a protein-coding sequence of the present disclosure, induce expression of the encoded protein. 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 upon exposure to some specific external stimulus, such as, but not limited to, an antibiotic such as tetracycline, a hormone such as ecdysone, or a heavy metal. 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 expression of the transgene of the present disclosure. For example, a suitable promoter and / or enhancer can be selected from the Eukaryotic Promoter Database (EPDB).

[0086] Sequence identity / similarity: As used herein, identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity, with the higher the percentage, the more identical the sequences. Sequence similarity is measured in terms of percentage identity or similarity (taking into account conservative amino acid substitutions), with the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and function identically or similarly to each other (e.g., proteins that perform the same function in different species or mutant forms of the protein that do not change the function or magnitude of the protein) can be referred to as "homologs."

[0087] Sequence identity or homology is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence gaps.In particular, sequence identity can be determined using any of several mathematical algorithms.A non-limiting example of the mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc.Natl.Acad.Sci.USA 1990;87:2264-2268, modified as in Karlin and Altschul, Proc.Natl.Acad.Sci.USA 1993;90:5873-5877.

[0088] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv Appl Math 2, 482 (1981); Needleman and Wunsch, J Mol Biol 48, 443 (1970); Pearson and Lipman, Proc Natl Acad Sci USA 85, 2444 (1988); Higgins and Sharp, Gene 73, 237-244 (1988); Higgins and 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). Furthermore, Altschul et al., J Mol Biol 215, 403-410 (1990) present a detailed discussion of sequence alignment methods and homology calculations.

[0089] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., (1990) supra) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Maryland 20894) and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Further information can be found on the NCBI website.

[0090] BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, the specified output file will present those homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences.

[0091] Once the alignment is complete, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches by the length or concatenated length of the sequence shown in the identified sequence (e.g., 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a nucleic acid sequence with 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0% identical to the test sequence (1166 ÷ 1554 × 100 = 75.0). The percent sequence identity value is rounded to the nearest whole number. 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 up to 75.2. Length values ​​are always integers. In another example, a target sequence containing a 20-nucleotide region that matches 20 consecutive nucleotides from a sequence identified as follows contains a region that shares 75% sequence identity with the identified sequence (i.e., 15÷20×100=75).

[0092] For comparison of amino acid sequences longer than approximately 30 amino acids, the Blast2 alignment function is used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, per-residue gap cost of 1). Homologs are typically characterized as having at least 70% sequence identity counted over a full-length alignment with the amino acid sequence using NCBI Basic Blast 2.0, gapped blastp, and databases such as the nr database, swissprot database, and patent sequence databases. Queries searched with the blastn program were filtered using DUST (Hancock and Armstrong, Comput Appl Biosci 10, 67-70 (1994)). Other programs use SEG. Additionally, manual alignments may be performed. Proteins with even higher similarity, as assessed by this method, will exhibit increasing percentage identities, such as at least approximately 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the protein.

[0093] When aligning short peptides (fewer than approximately 30 amino acids), alignments are performed using the Blast2 sequence function, employing the PAM30 matrix set to default parameters (9 penalties for open gaps, 1 for extension gaps). Proteins with even higher similarity to the reference sequence, as assessed by this method, will exhibit increasing percentage identities, such as at least approximately 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the protein. When comparing less than the entire sequence for sequence identity, homologs will typically have at least 75% sequence identity over a short window of 10–20 amino acids and may have at least 85%, 90%, 95%, or 98% sequence identity, depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described on the NCBI website.

[0094] One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nevertheless, nucleic acid sequences that do not show a high degree of identity may encode identical or similar (conserved) amino acid sequences due to the degeneracy of the genetic code. Alterations in nucleic acid sequences can be made using this degeneracy to generate multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the nucleic acid encoding the protein.

[0095] Subject: As used herein, the term "subject" refers to a living, multi-cellular, vertebrate organism, a category that includes both human and non-human mammals. The term "subject" includes all animals, including non-human primates and humans, although "animal" includes all vertebrate species except humans, and "vertebrate" includes all vertebrates, including animals (as "animal" is used herein) and humans. And, of course, a subset of "animal" is "mammal," which, for purposes of this specification, includes all mammals except humans.

[0096] Supertope: As used herein, the term "supertope" or "supertope peptide" refers to an epitope or peptide that is recognized by T cells in greater than about 90% of the human population, regardless of MHC haplotype, i.e., in the presence or absence of a given MHC-I, MHC-II, or MHC-E allele.

[0097] Tumor-associated antigen: As used herein, the term "tumor-associated antigen" refers to a self-antigen that is aberrantly expressed by cancer cells. TAAs include (i) germline / testis antigens expressed by cancer cells, (ii) lineage differentiation antigens not expressed in adult tissues, or (iii) antigens overexpressed by cancer cells. Tumor-associated antigens can be any protein that is relatively restricted to tumor cells and induces an immune response. However, many tumor-associated antigens are host (self) proteins and are therefore typically not recognized as antigenic by the host immune system. Tumor-associated antigens can also be aberrantly expressed by cancer cells. Tumor-associated antigens can also be germline / testis antigens expressed by cancer cells, lineage differentiation antigens not expressed in adult tissues, or antigens overexpressed by cancer cells.

[0098] Oncovirus: As used herein, the term "oncovirus," "cancer virus," or "oncovirus" refers to a virus that induces the development of cancer in some cases (e.g., after chronic infection, in individuals with compromised immune systems, etc.).

[0099] Treatment: As used herein, the term "treatment" refers to an intervention that improves the signs or symptoms of a disease or pathological condition. As used herein, the terms "treatment," "treat," and "treating" in relation to a disease, pathological condition, or symptom also refer to any observable beneficial effect of treatment. A beneficial effect can be evidenced, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease, a delay in the progression of a disease, a reduction in the number of recurrences of a disease, an improvement in the overall health or well-being of a subject, or other parameters known in the art specific to a particular disease. A preventive treatment is a treatment administered to a subject who does not show signs of a disease or who only shows early signs, with the aim of reducing the risk of developing a pathology. A therapeutic treatment is a treatment administered to a subject after signs and symptoms of a disease have occurred.

[0100] 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, a vaccine can be used to reduce the likelihood of developing a disease (such as a tumor or pathological infection), or to reduce the severity of symptoms of a disease or condition, to limit the progression of a disease or condition (such as a tumor or pathological infection), or to limit the recurrence of a disease or condition (such as a tumor). In certain embodiments, the vaccine is a replication-deficient CMV that expresses a heterologous antigen, for example, a tumor-associated antigen derived from a lung, prostate, ovarian, breast, colon, cervical, liver, kidney, bone, or melanoma tumor.

[0101] Vector: A nucleic acid molecule of a specific sequence can be incorporated into a vector, and then the vector can be introduced into a host cell, thereby producing a transformed host cell. A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also contain other genetic elements known in the art, including one or more selectable marker genes and promoter elements that direct nucleic acid expression. A vector can be a viral vector, such as a CMV vector. A viral vector can be constructed from wild-type or attenuated viruses, including replication-deficient viruses.

[0102] Any vector that allows the expression of the virus of the present disclosure can be used in accordance with the present disclosure.In certain embodiments, the disclosed virus can be used in vitro (for example, using a cell-free expression system) and / or in vitro grown cultured cells to produce the encoded heterologous antigen (e.g., tumor virus antigen, HIV antigen, tumor-associated antigen, and antibody), and then used for various applications, such as the production of proteinaceous vaccines.For the aforementioned applications, any vector that allows the expression of the virus in vitro and / or in cultured cells can be used.The vector used in accordance with the present disclosure can include an appropriate gene regulatory region, such as a promoter or enhancer, so that the antigen of the present disclosure can be expressed.

[0103] II. Methods of Treating and Preventing Cancer Disclosed herein are methods for treating or preventing cancer. The methods include administering to a subject an effective amount of at least one recombinant CMV vector comprising at least one tumor-associated antigen or tumor virus antigen. In some embodiments, the methods also include administering T cells comprising an MHC-E-restricted T cell receptor.

[0104] Animal studies have demonstrated that CMV vaccines are unique in that they a) induce and maintain high frequencies of extralymphoid T cell responses (so-called effector memory T cells), b) superinfect CMV-positive hosts, and c) remain immunogenic even when host-to-host spread is insufficient. Furthermore, experiments in animal models have shown that vaccine vectors derived from animal CMV induce protective immune responses against infectious diseases and cancer (U.S. Patent Application Publication Nos. 20080199493, 20100142823, 20130136768, and 20140141038). Of particular note is the finding that a rhesus CMV (RhCMV)-vectored simian immunodeficiency virus (SIV) vaccine was not only able to prevent AIDS in non-human primates but was also able to ultimately cure these animals of SIV (Hansen SG et al., Nature 502, 100-104 (2013)).

[0105] A major challenge in eliciting tumor-associated antigen (TAA)-specific T cells is that "canonical" T cells, which strongly recognize peptides derived from these antigens in the context of MHC-I or MHC-II as self-antigens, are eliminated from the immune repertoire by negative selection. Therefore, cancer vaccines must break immune tolerance by stimulating "non-canonical" T cells that escape negative selection by expressing low-affinity TCRs or by recognizing peptides that bind with low affinity to MHC. All currently available T cell-inducing vaccines, such as DNA, RNA, pox vector, adeno vector, or alphavirus systems, are designed to elicit canonical T cells. As a result, these vectors are difficult to break immune tolerance. Furthermore, anti-vector immunity precludes the repeated use of the same vector to enhance immunity, resulting in complex heterologous prime / boost vaccine regimens or the need for combination with tolerance-breaking checkpoint inhibitors. For example, PROSTVAC is a poxvirus-based vaccine against PSA-expressing prostate cancer. The immunization regimen required one immunization with PSA expressed by vaccinia virus, followed by six booster immunizations with fowlpox virus encoding PSA. Despite this effort, PROSTVAC was only able to elicit CD8+ T cells, accounting for approximately 0.03% of total CD8+ T cells. As a result, phase III clinical trials were discontinued due to futility.

[0106] In some embodiments, the methods provide treatment for cancer associated with a tumor-associated antigen. In some embodiments, the treatment results from breaking tolerance in the subject, such that an immune response is mounted against the TAA.

[0107] In some embodiments, the cancer is caused by a pathogen. In some embodiments, the pathogen is an oncogenic virus, and the antigen is a protein derived from the oncogenic virus. Oncogenic viruses include, but are not limited to, human T-lymphotropic virus, hepatitis B virus, hepatitis C virus, human papillomavirus (HPV), human polyomavirus, Kaposi's sarcoma-associated herpesvirus, Merkel cell polyomavirus, and Epstein-Barr virus. In some embodiments, the oncogenic virus antigen is E6 and E7 from HPV strain 16 or E6 and E7 from HPV strain 18. In some embodiments, the oncogenic virus antigen is a fusion of E6 and E7 from HPV. The oncogenic virus antigen can be a protein derived from any part of the oncogenic virus. For example, in some embodiments, the oncogenic virus antigen can be derived from a core, envelope, surface, or polymerase protein.

[0108] Tumor-associated antigens include, but are not limited to, prostatic 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 from HPV16 and HPV18; mucin 1 (MUC1); LMP2; epidermal growth factor receptor (EGFR) (EGF) GFR); 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-linked protein 2 (TRP-2); GD3; fucosyl-GM1; PSCA; sLe(a); CYP1B1; PLCA1; GM3; BORIS; Tn; GloboH; Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETV6-AML); NY-BR-1; RGS5; squamous cell tumor rejection antigen or 3 (SART3); STn; carbonic anhydrase IX; PAX5; OY-TES1; sperm protein 17; LCK; HMWMAA; AKAP-4; SSX2; B7H3; leguminous These include: 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 in melanoma (PRAME); melanocortin 1 receptor (MC1R); β-catenin; BRCA1 / 2; CDK4; chronic myeloid leukemia 66 (CML66); and TGF-β.In certain embodiments, the host autoantigen includes prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.

[0109] In some embodiments, the method relates to the prevention or treatment of cancer, including, but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, pediatric cerebellar or cerebral, basal cell carcinoma, bile duct cancer, extrahepatic, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma / malignant glioma, brain tumor, ependymoma, brain tumor, medulloblastoma, brain tumor, supratentorial primitive neuroectodermal tumor, brain tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Brain astrocytoma / malignant glioma, childhood; 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 family of tumors; Extracranial germ cell tumor, childhood; Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye Cancer, retinoblastoma;gallbladder cancer;gastric (stomach) cancer;gastrointestinal carcinoid tumor;gastrointestinal stromal tumor (GIST);germ cell tumors: extracranial, extragonadal or ovarian;gestational trophoblastic tumor;glioma of the brain stem;glioma, childhood brain astrocytoma;glioma, childhood visual pathway and hypothalamus;gastric carcinoid;hairy cell leukemia;head and neck cancer;cardiac cancer;hepatocellular (liver) cancer;Hodgkin's lymphoma;hypopharyngeal cancer;hypothalamic and visual pathway glioma, childhood;intraocular melanoma;islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;kidney cancer (renal cell carcinoma);laryngeal cancer;leukemia;leukemia , acute lymphoblastic (also called acute lymphocytic leukemia); leukemia, acute myeloid (also called acute myeloid leukemia); leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); leukemia, chronic 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's lymphoma);Lymphoma, primary central nervous system; Marcus Whittle, fatal disease; Waldenström macroglobulinemia; Malignant fibrous histiocytoma / osteosarcoma of bone; Medulloblastoma, childhood; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma, adult malignant tumor; Mesothelioma, childhood; Metastatic squamous cell neck cancer of unknown primary; Oral cancer; Multiple endocrine neoplasia syndrome, childhood; Multiple myeloma / plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplastic / myeloproliferative disorder; Myeloid leukemia, chronic; Myeloid leukemia, adult acute; Myeloid leukemia, childhood acute; Myeloma, multiple (Bone and bone marrow cancer); Myeloproliferative disorders, chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin's lymphoma; Non-small cell lung cancer; Oral cavity cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (surface epithelial-stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic islet cell cancer; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germ cell tumor; Pineoblastoma and supratentorial primitive neuroectodermal tumor, childhood; Pituitary adenoma; Plasma cell neoplasm / multiple Primary myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell carcinoma; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family tumor; Sarcoma, Kaposi's; Sarcoma, soft tissue; Sarcoma, uterine; Sézary syndrome; Skin cancer (non-melanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (non-melanoma); Occult primary metastatic squamous cell neck cancer; Gastric cancer; Supratentorial primitive neuroectodermal tumor , childhood; T-cell lymphoma, skin (mycosis fungoides and Sézary syndrome); testicular cancer; throat cancer; thymoma, childhood; thymoma and thymic carcinoma; thyroid cancer; thyroid cancer, childhood; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, gestational; carcinoma of unknown primary site, adult; cancer of unknown primary site, childhood; ureter and renal pelvis, transitional cell carcinoma; urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; optic pathway and hypothalamic glioma, childhood; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor (kidney cancer).

[0110] In some embodiments, the method relates to treating or preventing tumor virus-positive cancer. In some embodiments, the method relates to generating an immune response against a tumor-associated antigen in a subject.

[0111] In some embodiments, the methods of the present disclosure provide for the administration of a CMV vector that does not express active UL128, UL130, UL146, and UL147 proteins due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, and UL147, or their homologs or orthologues (homologous genes in CMV that infect other species). In some other embodiments, the vector does not express active UL128, UL130, UL146, UL147, and US11 proteins due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, UL147, and US11, or their homologs or orthologues (homologous genes in CMV that infect other species). The mutation can be any mutation that results in the lack of expression of active UL128, UL130, UL146, UL147, or US11 proteins. Such mutations may include point mutations, frameshift mutations, deletions of less than the entire protein-encoding sequence (truncating mutations), or deletions of the entire protein-encoding nucleic acid sequence, or any other mutations. Exemplary vectors are described in U.S. Patent Nos. 9,783,823 and 9,862,972, and U.S. Patent Application Publication No. 2018 / 0298404, which are incorporated herein by reference.

[0112] In a further example, the CMV vector does not express, or the vector does not express, active UL128, UL130, UL146, and US11 proteins due to the presence of a nucleic acid sequence in the vector comprising an antisense or RNAi sequence (siRNA or miRNA) that inhibits expression of the UL128, UL130, UL146, UL147, or US11 proteins. Mutations and / or antisense and / or RNAi, in any combination, can be used to generate CMV vectors lacking active UL128, UL130, UL146, UL147, or US11.

[0113] In some embodiments, the CD8+ T cell response elicited by the vector is characterized by at least 10% of the CD8+ T cells being directed against an epitope presented by MHC-E. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or at least 95% of the CD8+ T cells are restricted by MHC-E. In some embodiments, the MHC-E-restricted CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against a supertope presented by MHC-E. In some embodiments, the CD8+ T cell response elicited by the vector is characterized by at least 10% of the CD8+ T cells being directed against an epitope presented by MHC-II. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or at least 95% of the CD8+ T cells are restricted by MHC-II. In some embodiments, the MHC-II-restricted CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against a supertope presented by MHC-II.

[0114] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from CD8+ T cells induced by either a human UL128-130 and UL146-147 deleted HCMV vector, a rhesus macaque RhCMV strain 68-1 vector, or a cynomolgus macaque UL128-130 and UL146-147 deleted CyCMV vector. 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 supertope or an MHC-II supertope. In some embodiments, the MHC-E supertope comprises a peptide derived from PAP, WT1, MSLN, HER2, HPV E6 and E7 from strain 16, HPV E6 and E7 from strain 18, or an HPV E6 / E7 fusion protein.

[0115] In some embodiments, the MHC-E or MHC-II supertope peptide has 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence corresponding to PAP: (ARAASLSLGFLFLLF) (SEQ ID NO:2); (KELKFVTLVFRHGDR) (SEQ ID NO:3); (QLTQLGMEQHYELGE) (SEQ ID NO:4); (LNESYKHEQVYIRST) (SEQ ID NO:5); (NHMKRATQMPSYKKL) (SEQ ID NO:6); or (MVLLFIHIRRGPCWQ) (SEQ ID NO:7). In some embodiments, the MHC-E or MHC-II supertope peptide has 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% identity to the amino acid sequence corresponding to WT1 (VPEPASQHTLRSGPG) (SEQ ID NO: 8) (SAERLQGRRSRGASG) (SEQ ID NO: 9); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14). In some embodiments, the MHC-E or MHC-II supertope peptide has 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence (IDESLIFYKKWELEA) (SEQ ID NO: 10); (PFTYEQLDVLKHKLD) (SEQ ID NO: 11); or (FMKLRTDAVLPLTVA) (SEQ ID NO: 12) corresponding to MSLN.

[0116] In some embodiments, the method further includes identifying a CD8+ T cell receptor from CD8+ T cells induced by either a human UL128-130 and UL146-147 deleted HCMV vector, a rhesus macaque RhCMV strain 68-1 vector, or a cynomolgus macaque UL128-130 and UL146-147 deleted CyCMV 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.

[0117] In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes a specific MHC-II supertope, hi some embodiments, the specific MHC-II supertope comprises a peptide derived from PAP, WT1, MSLN, HER2, HPV E6 and E7 from strain 16, HPV E6 and E7 from strain 18, or an HPV E6 / E7 fusion protein.

[0118] In some embodiments, the recombinant CMV vector expresses active UL128 and active UL130, and inactive US11. In some embodiments, the CD8+ T cell response elicited by this vector is characterized in that at least 10% of the CD8+ T cells are directed against epitopes presented by MHC-I. 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-I.

[0119] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-I / foreign antigen-derived peptide complex. In some embodiments, the T cell receptor is derived from a human or monkey T cell. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0120] In some embodiments, the recombinant CMV vector is administered to prevent or treat cancer. In some embodiments, the cancer is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, renal 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, or germ cell tumor.

[0121] In some embodiments, the recombinant CMV vector is administered to prevent or treat tumor virus-positive cancer. In some embodiments, the tumor virus-positive cancer is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma, or germ cell tumor.

[0122] The term oncovirus includes, but is not limited to, human T-lymphotropic virus, herpes simplex virus, hepatitis B virus, hepatitis C virus, human papillomavirus (HPV), human polyomavirus, Kaposi's sarcoma-associated herpesvirus, Merkel cell polyomavirus, and Epstein-Barr virus. In some embodiments, the oncovirus antigen is E6 and E7 from HPV strain 16 or E6 and E7 from HPV strain 18. In some embodiments, the oncovirus antigen is a fusion of E6 and E7 from HPV. The oncovirus antigen can be a protein derived from any part of the oncovirus. For example, in some embodiments, the oncovirus antigen can be derived from the core, envelope, surface, or polymerase protein.

[0123] Also disclosed herein is a method for generating an immune response to at least one tumor-associated antigen in a subject. The method comprises administering to the subject an effective amount of a recombinant CMV vector comprising at least one tumor-associated antigen. In some embodiments, the CMV vector is characterized by having a nucleic acid sequence that does not express an active UL128 protein, an active UL130 protein, or an active US11 protein.

[0124] In some embodiments, the vector does not express active UL128, UL130, or US11 protein due to a mutation in the nucleic acid sequence encoding UL128, UL130, or US11, or a homolog thereof, or an ortholog thereof (a homologous gene of CMV that infects other species). 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 of less than the entire protein-encoding sequence (truncating mutations), or deletions of the entire protein-encoding nucleic acid sequence, or any other mutation.

[0125] In some further examples, the vector does not express active UL128, UL130, or US11 protein due to the presence of a nucleic acid sequence in the vector that includes an antisense or RNAi sequence (siRNA or miRNA) that inhibits expression of the UL128, UL130, or US11 protein. Mutations and / or antisense and / or RNAi, in any combination, can be used to generate CMV vectors that lack active UL128, UL130, or US11.

[0126] In some embodiments, the method further includes identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-I heterologous antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0127] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-E-peptide complexes. The method includes administering to a first subject (or animal) a CMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-E / peptide complexes. The CMV vector includes a first nucleic acid sequence encoding at least one heterologous antigen, and does not express an active UL128 protein or its orthologue, an active UL130 protein or its orthologue, an active UL146 protein or its orthologue, or an active UL147 protein or its orthologue. The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor-associated 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.

[0128] In some embodiments, the tumor-associated antigen has 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence corresponding to PAP (ARAASLSLGFLFLLF) (SEQ ID NO: 2); (KELKFVTLVFRHGDR) (SEQ ID NO: 3); (QLTQLGMEQHYELGE) (SEQ ID NO: 4); (LNESYKHEQVYIRST) (SEQ ID NO: 5); (NHMKRATQMPSYKKL) (SEQ ID NO: 6); or (MVLLFIHIRRGPCWQ) (SEQ ID NO: 7). In some embodiments, the tumor-associated antigen has 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% identity to the amino acid sequence corresponding to WT1 (VPEPASQHTLRSGPG) (SEQ ID NO: 8) (SAERLQGRRSRGASG) (SEQ ID NO: 9); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14). In some embodiments, the tumor-associated antigen has 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence corresponding to MSLN (IDESLIFYKKWELEA) (SEQ ID NO: 10); (PFTYEQLDVLKHKLD) (SEQ ID NO: 11); or (FMKLRTDAVLPLTVA) (SEQ ID NO: 12).

[0129] The method further includes administering to the first subject a recombinant CMV vector containing a nucleic acid expressing a tumor-associated antigen in an amount effective to generate a first set of CD8+ T cells that recognize an MHC-E / peptide complex, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof. In some embodiments, the method can further include identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-E / tumor-associated antigen-derived peptide complex. In some embodiments, the method can further include isolating a second set of one or more CD8+ T cells from the second subject. In some embodiments, the method may include transfecting a second set of 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 CD8+ T cells that recognize an MHC-E / tumor-associated antigen peptide complex tumor-associated antigen.

[0130] The method includes isolating a first set of CD8+ T cells from a first subject, the first subject having been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor-associated antigen in an amount effective to generate the first set of CD8+ T cells that recognize MHC-E / peptide complexes, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof. In some embodiments, the method further includes identifying a first CD8+ TCR from the first set of CD8+ T cells, the first CD8+ TCR recognizing an MHC-E / tumor-associated antigen-derived peptide complex. In some embodiments, the method further includes isolating a second set of one or more CD8+ T cells from a second subject. In some embodiments, the method comprises transfecting a second set of 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 CD8+ T cells that recognize MHC-E / tumor-associated antigen peptide complexes. One or more CD8+ T cells for transfection with the expression vector can be isolated from the first subject or the second subject.

[0131] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0132] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-E / 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-E supertope. In some embodiments, the MHC-E supertope comprises a peptide derived from one or more of PAP, WT1, MSLN, HER2, and E6 or E7 of HPV16 or HPV18.

[0133] The method also includes the steps of: (a) administering to a first subject a recombinant CMV vector comprising a nucleic acid expressing a tumor-associated antigen in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / peptide complexes, wherein the recombinant CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof; (b) identifying from the first set of CD8+ T cells a first CD8+ TCR that recognizes the MHC-E / tumor-associated antigen-derived peptide complex; and (c) identifying from a second subject one or more CD8+ T cells that recognize the MHC-E / tumor-associated antigen-derived peptide complex. (d) isolating a second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, to generate CD8+ T cells that recognize an MHC-E / tumor-associated antigen peptide complex tumor-associated antigen.

[0134] The method also includes the steps of: (a) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor-associated antigen in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / peptide complexes, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes the MHC-E / tumor-associated antigen-derived peptide complex; and (c) identifying a second pair of CD8+ TCRs from the first set of CD8+ T cells that recognize the MHC-E / tumor-associated antigen-derived peptide complex. Disclosed are transfected CD8+ T cells that recognize MHC-E peptide complexes, prepared by a method comprising: (a) isolating a second set of one or more CD8+ T cells from an elephant; and (b) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, to generate CD8+ T cells that recognize MHC-E / tumor-associated antigen peptide complexes.

[0135] The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen, and does not express an active UL128 protein or its orthologue, an active UL130 protein or its orthologue, an active UL146 protein or its orthologue, or an active UL147 protein or its orthologue. The expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ T cell receptor, wherein the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor. The heterologous antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, or a host self-antigen.

[0136] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0137] In some embodiments, the first CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0138] Also disclosed herein are methods for treating diseases such as cancer, pathogenic infections, or immune diseases or disorders, comprising administering transfected T cells that recognize MHC-E peptide complexes to a first or second subject. Also disclosed herein are methods for inducing an immune response against a host self-antigen or a tissue-specific antigen, comprising administering transfected T cells that recognize MHC-E-peptide complexes to a first or second subject. Also disclosed herein are uses of CD8+ T cells in the manufacture of a medicament for treating or preventing cancer. Also disclosed herein are uses of CD8+ T cells in the manufacture of a medicament for inducing an immune response against a host self-antigen in a subject.

[0139] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-II-peptide complexes. The method includes administering to a first subject a recombinant CMV vector containing a nucleic acid expressing a tumor antigen in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / peptide complexes, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, or active UL147 protein, or their orthologs. In some embodiments, the method can further include identifying a first CD8+ TCR from the first set of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-II / tumor antigen-derived peptide complex. In some embodiments, the method can further include isolating a second set of one or more CD8+ T cells from the second subject. In some embodiments, the method may include transfecting a second set of 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 CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

[0140] In some embodiments, the method includes isolating a first set of CD8+ T cells from a first subject, where the first subject has been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor antigen in an amount effective to generate the first set of CD8+ T cells that recognize an MHC-II / peptide complex, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof. In some embodiments, the method further includes identifying a first CD8+ TCR from the first set of CD8+ T cells, where the first CD8+ TCR recognizes an MHC-II / tumor antigen-derived peptide complex. In some embodiments, the method further includes isolating a second set of one or more CD8+ T cells from a second subject. In some embodiments, the method comprises transfecting a second set of 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 CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

[0141] The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen, and does not express an active UL128 protein or its orthologue, an active UL130 protein or its orthologue, an active UL146 protein or its orthologue, or an active UL147 protein or its orthologue. The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor-associated antigen, a tissue-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 or a B cell receptor. In some embodiments, the host self-antigen can be a tumor-associated antigen (TAA) that is aberrantly expressed by cancer cells. TAAs include, but are not limited to, i) germline / testis antigens expressed in cancer cells, ii) lineage differentiation antigens not expressed in adult tissues, or iii) antigens overexpressed in cancer cells. In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0142] In some embodiments, the method further includes identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-II / foreign antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0143] In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes a specific MHC-II supertope, hi some embodiments, the specific MHC-II supertope comprises a peptide derived from PAP, WT1, MSLN, HER2, HPV E6 or E7 from strain 16, HPV E6 and E7 from strain 18, or an HPV E6 / E7 fusion protein.

[0144] The method also includes the steps of: (a) administering to a first subject a recombinant CMV vector comprising a nucleic acid expressing a tumor antigen, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / peptide complexes, and that does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof; (b) identifying from the first set of CD8+ T cells a first CD8+ TCR that recognizes an MHC-II / tumor antigen-derived peptide complex; and (c) identifying one or more CD8+ T cells from a second subject. and (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

[0145] The method also includes the steps of: (a) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof, in an amount effective to generate a first set of CD8+ T cells that recognize an MHC-II / peptide complex; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-II / tumor antigen-derived peptide complex; and (c) isolating a first set of CD8+ T cells from a second subject who has previously been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or an orthologue thereof, in an amount effective to generate a first set of CD8+ T cells that recognizes an MHC-II / tumor antigen-derived peptide complex. and (d) isolating a second set of one or more CD8+ T cells from the first CD8+ TCR; and (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

[0146] In some embodiments, the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLI FYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

[0147] The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen, but does not express an active UL128 protein or its orthologue, an active UL130 protein or its orthologue, an active UL146 protein or its orthologue, or an active UL147 protein or its orthologue. The expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ T cell receptor, wherein the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor. The heterologous antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tissue-specific antigen, or a host self-antigen. In some embodiments, the first CD8+ T cell receptor is identified by RNA or DNA sequencing. Also disclosed herein are methods for treating a disease, such as cancer, a pathogenic infection, or an immune disease or disorder, comprising administering to a first or second subject transfected T cells that recognize an MHC-II peptide complex. Also disclosed herein are methods for inducing an immune response against a host self-antigen or a tissue-specific antigen, comprising administering to a first or second subject transfected T cells that recognize an MHC-II-peptide complex.

[0148] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-I-peptide complexes. The method includes administering to a first subject (or animal) a CMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-I / peptide complexes. The CMV vector includes a first nucleic acid sequence encoding at least one heterologous antigen and expresses an active UL128 protein or its orthologue, an active UL130 protein or its orthologue, an active UL146 protein or its orthologue, and an active UL147 protein or its orthologue. The antigen can be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor-associated antigen, a tissue-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 or a B cell receptor.

[0149] In some embodiments, the method further includes identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-I / foreign antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing.

[0150] In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes a specific MHC-I epitope, in some embodiments, the specific MHC-I epitope comprises one or more of PAP, WT1, MSLN, HER2, HPV E6 or E7 from strain 16, HPV E6 or E7 from strain 18, and an HPV E6 / E7 fusion protein.

[0151] In some embodiments, the method may further include administering one or more transfected T cells to a first or second subject to treat a disease, such as cancer, a pathogenic infection, or an immune disease or disorder. In some embodiments, the method may further include administering one or more transfected T cells to a first or second subject to induce an immune response against a tumor-associated antigen.

[0152] The CMV vectors disclosed herein can be used as immunogenic, immunological, or vaccine compositions containing a recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. Immunological compositions containing a recombinant CMV virus or vector (or its expression product) induce a local or systemic immunological response. The response may be, but need not be, protective. Immunogenic compositions containing a recombinant CMV virus or vector (or its expression product) similarly induce a local or systemic immunological response that may be, but need not be, protective. Vaccine compositions induce a local or systemic protective response. Thus, the terms "immunological composition" and "immunogenic composition" include "vaccine composition" (as both terms can be protective compositions).

[0153] The recombinant CMV vectors disclosed herein can be human cytomegalovirus vectors, rhesus cytomegalovirus vectors, or cynomolgus vectors.

[0154] The recombinant CMV vectors disclosed herein can be used in methods of inducing an immunological response in a subject, comprising administering to the subject an immunogenic, immunological, or vaccine composition comprising the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.

[0155] The recombinant CMV vectors disclosed herein can be used in therapeutic compositions containing the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. The CMV vectors disclosed herein can be prepared by inserting DNA containing a sequence encoding a tumor-associated antigen into an essential or non-essential region of the CMV genome. This method can further include deleting one or more regions from the CMV genome. This method can include in vivo recombination. Thus, this method can include transfecting CMV DNA into cells in a cell-compatible medium in the presence of donor DNA containing heterologous DNA flanked by DNA sequences homologous to a portion of the CMV genome, thereby introducing the heterologous DNA into the CMV genome, and then optionally recovering the modified CMV by in vivo recombination. This method can also include cleaving CMV DNA to obtain the cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMV DNA to obtain a hybrid CMV-heterologous DNA, transfecting cells with the hybrid CMV-heterologous DNA, and then optionally recovering the CMV modified by the presence of the heterologous DNA. Because in vivo recombination is involved, this method also results in a plasmid containing donor DNA not naturally occurring in CMV that encodes a polypeptide foreign to CMV, where the donor DNA is within a segment of CMV DNA that is otherwise colinear with an essential or nonessential region of the CMV genome, such that DNA from an essential or nonessential region of CMV flanks the donor DNA. Heterologous DNA can be inserted into CMV to generate recombinant CMV in any orientation that results in stable integration and expression of that DNA.

[0156] The DNA encoding the heterologous antigen in the recombinant CMV vector can also contain a promoter. The promoter can be derived from any source, such as a herpesvirus, including an endogenous cytomegalovirus (CMV) promoter, such as human CMV (HCMV), rhesus CMV (RhCMV), mouse, or other CMV promoter. The promoter can also be a non-viral promoter, such as the EF1α promoter. The promoter can be a truncated transcriptionally active promoter, which contains a region transactivated by a viral transactivation protein and a minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter can be composed of an assembly of DNA sequences corresponding to a minimal promoter and upstream regulatory sequences. A minimal promoter is composed of a CAP site plus an ATA box (a minimal sequence for the basal level of transcription; a non-regulated level of transcription), and the "upstream regulatory sequence" is composed of multiple upstream elements and multiple enhancer sequences. Furthermore, the term "truncated" indicates that the full-length promoter is not present in its entirety, i.e., a portion of the full-length promoter has been removed. Truncated promoters can also be derived from herpesviruses such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. Based on base pairs, the size can be reduced by up to 40%, or even up to 90%, from the full-length promoter. The promoter can also be a modified non-viral promoter. For HCMV promoters, see U.S. Patent Nos. 5,168,062 and 5,385,839. Transfecting cells with plasmid DNA to achieve cellular expression is described in Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561. For direct injection of plasmid DNA as a simple and effective method for vaccination against various infectious diseases, see Science, 259:1745-49, 1993. Therefore, it is within the scope of this disclosure that vectors can be used by direct injection of vector DNA.

[0157] Also disclosed is an expression cassette that can be inserted into a recombinant virus or plasmid containing a truncated transcriptionally active promoter.The expression cassette can further contain a functional truncated polyadenylation signal, such as a truncated but functional SV40 polyadenylation signal.Considering that larger signals are provided in nature, it is quite surprising that the truncated polyadenylation signal is functional.The truncated polyadenylation signal addresses the insert size limitation problem of recombinant viruses such as CMV.The expression cassette can also contain heterologous DNA related to the virus or system into which it is inserted, and the DNA can be the heterologous DNA described herein.

[0158] For antigens for use in vaccines or immunological compositions, see also Stedman's Medical Dictionary (24th ed., 1982), e.g., the definition of vaccine (for a list of antigens used in vaccine preparations). Such antigens or epitopes of interest from these antigens can be used. With regard to tumor-associated antigens, those skilled in the art should be able to select tumor-associated antigens and their encoding DNA without undue experimentation from knowledge of the amino acids and corresponding DNA sequences of peptides or polypeptides, as well as from the properties of specific amino acids (e.g., size, charge, etc.) and codon dictionaries.

[0159] One method for determining T-cell epitopes of an antigen involves epitope mapping. Overlapping peptides of tumor-associated antigens are generated by oligopeptide synthesis. Individual peptides are then tested for their ability to induce T-cell activation. This approach has been particularly useful for mapping T-cell epitopes, as T cells recognize short, linear peptides complexed with MHC molecules.

[0160] The immune response to tumor-associated antigens generally occurs as follows: T cells recognize proteins only when they are cleaved into smaller peptides and presented in complexes called "major histocompatibility complexes (MHC)" located on the surface of other cells. There are two classes of MHC complexes, class I and class II, and each class is composed of many different alleles. Different species and individual subjects have different types of MHC complex alleles, and they are said to have different MHC types. One type of MHC class I molecule is called MHC-E (HLA-E in humans, Mamu-E in RM, and Qa-lb in mice). Unlike other MHC-I molecules, MHC-E is highly conserved within and between mammalian species.

[0161] It is known that the DNA containing the sequence encoding the tumor-associated antigen may itself contain a promoter for driving expression in the CMV vector, or the DNA may be limited to the DNA encoding the tumor-associated antigen. This construct may be oriented relative to the endogenous CMV promoter so that it is operably linked to the promoter and expressed thereby. Furthermore, multiple copies of the DNA encoding the tumor-associated antigen, or the use of strong or early promoters, or early and late promoters, or any combination thereof, may be used to amplify or increase expression. Thus, the DNA encoding the tumor-associated antigen may be appropriately positioned relative to the CMV endogenous promoter, or the promoters may be translocated so that they are inserted into a different position along with the DNA encoding the tumor-associated antigen. Nucleic acids encoding two or more tumor-associated antigens can be packaged into a CMV vector.

[0162] Pharmaceutical and other compositions containing the disclosed CMV vectors are further disclosed. The aforementioned pharmaceutical and other compositions can be formulated for use in any administration procedure known in the art. The aforementioned pharmaceutical compositions can be administered via parenteral routes (intradermal, intramuscular, subcutaneous, intravenous, etc.). Administration can also be via mucosal routes, such as oral, nasal, genital, etc.

[0163] The disclosed pharmaceutical compositions can be prepared according to standard techniques well known to those skilled in the art. The compositions can be administered in dosages and by techniques well known to those skilled in the art, taking into account factors such as the race or species, age, sex, weight, and condition of the particular patient, as well as the route of administration. The compositions can be administered alone, or co-administered or sequentially with other CMV vectors, or other immunological, antigenic, vaccine, or therapeutic compositions. The other compositions can include purified native antigens or epitopes or antigens or epitopes expressed by recombinant CMV vectors or other vector systems, and are administered taking into account the above factors.

[0164] Examples of compositions include liquid preparations such as suspensions, syrups, or elixirs for administration via orifices, for example, oral, nasal, anal, or genital (e.g., vaginal) routes, and preparations for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable liquid administration), such as sterile suspensions or emulsions. In the above-mentioned compositions, the recombinant may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, or glucose.

[0165] Antigenic, immunological, or vaccine compositions typically contain an adjuvant and a certain amount of CMV vector or expression product to elicit the desired response. For human applications, alum (aluminum phosphate or aluminum hydroxide) is a typical adjuvant. Saponin and its purified components, Quil A, Freund's complete adjuvant, and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. Chemically defined preparations, such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described in Goodman-Snitkoff et al., J Immunol. 147:410-415 (1991), encapsulation of proteins within proteoliposomes, such as those described in Miller et al., J Exp. Med. 176:1739-1744 (1992), and encapsulation of proteins in lipid vesicles, such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH), can also be used.

[0166] The composition may be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or mucosal administration, including orifice administration, e.g., perilingual (e.g., oral), intragastric, buccal, anal, vaginal, etc. Similarly, the effective dosage and route of administration are determined by the nature of the composition, the nature of the expression product, the expression level if recombinant CMV is used directly, and known factors such as the species or race of the host, age, sex, weight, condition, and nature, as well as LD50 and other screening procedures that are known and do not require undue experimentation. The dosage of the expression product can range from a few micrograms to hundreds of micrograms, e.g., 5 μg to 500 μg. The CMV vector can be administered in any appropriate amount to achieve expression at these dosage levels. In a non-limiting example, the CMV vector is administered in an amount of at least 10 μg. 2 pfu, i.e., the CMV vector may be administered in at least this amount, or about 10 2 pfu~about 10 7The CMV vector may be administered in an amount ranging from 1000 to 10000 pfu. Other suitable carriers or diluents include water or buffered saline, with or without preservatives. The CMV vector may be lyophilized or in a dissolved state for resuspension at the time of administration.

[0167] It goes without saying that the proteins of the present disclosure and the nucleic acids encoding them may differ from the exact sequences specifically 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 single family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, and histidine; (3) nonpolar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonably expected that a single substitution of leucine with isoleucine or valine, or vice versa, a single substitution of aspartate with glutamate, or vice versa, a single substitution of threonine with serine, or vice versa, or similar conservative substitution of amino acids with structurally related amino acids, will not significantly affect biological activity.Therefore, proteins that have substantially the same amino acid sequence as the described proteins but have minor amino acid substitutions that do not substantially affect the immunogenicity of the proteins are included within the scope of the present disclosure.

[0168] The nucleotide sequence of the present disclosure may be codon-optimized, for example, the codons may be optimized for use in human cells.For example, any virus or bacterial sequence can be modified in this way.Many viruses, including HIV and other lentiviruses, use many rare codons, and by modifying these codons to correspond to the codons commonly used in the desired target, the expression of tumor-associated antigens can be enhanced, as described in Andre et al., J Virol.72:1497-1503, 1998.

[0169] Nucleotide sequences encoding functionally and / or antigenically equivalent variants and derivatives of the CMV vector and the glycoproteins contained therein are contemplated. These functionally equivalent variants, derivatives, and fragments exhibit the ability to retain antigenic activity. For example, DNA sequence changes that do not alter the encoded amino acid sequence, as well as changes resulting in conservative substitutions of amino acid residues, deletions or additions of one or a few amino acids, and substitutions of amino acid residues with amino acid analogs, do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions include glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine / methionine, lysine / arginine, and phenylalanine / tyrosine / tryptophan. In some embodiments, a 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 to an antigen, epitope, immunogen, peptide, or polypeptide of interest.

[0170] The various recombinant nucleotide sequences and antibodies and / or antigens of the present disclosure are produced using standard recombinant DNA and cloning techniques. Such techniques are well known to those skilled in the art. See, for example, "Molecular Cloning: A Laboratory Manual," 2nd Edition (Sambrook et al., 1989).

[0171] The CMV vectors described herein may contain mutations that can prevent host-to-host transmission, thereby preventing the virus from infecting immunocompromised subjects or other subjects who may face complications as a result of CMV infection. The CMV vectors described herein may also contain mutations that result in the presentation of immunodominant and non-immunodominant epitopes and non-canonical MHC restriction. However, the mutations in the CMV vectors described herein do not affect the vector's ability to reinfect subjects previously infected with CMV. The aforementioned CMV mutations are described, for example, in U.S. Patent Application Publication Nos. 2013-013676S, 2010-0142S23, 2014-014103S, and PCT Application Publication No. WO2014 / 13S209, all of which are incorporated herein by reference.

[0172] The disclosed CMV vectors can be administered in vivo to generate immunogenic responses, including CD8+ immune responses, including immune responses characterized by a high percentage of CD8+ T cell responses restricted by 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 in laboratory animals, such as rhesus monkeys, to conduct preclinical testing of immunogenic compositions and vaccines using RhCMV. In other instances, it will be desirable to use the disclosed CMV vectors in human subjects, such as in clinical trials and for actual clinical use of immunogenic compositions using HCMV.

[0173] For such in vivo applications, the disclosed CMV vectors are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions of the present disclosure are useful for stimulating an immune response against heterologous antigens, including tumor-associated antigens, tumor virus antigens, or host self-antigens, and can be used as one or more components of prophylactic or therapeutic vaccines against tumor-associated antigens, tumor virus antigens, or host self-antigens for the prevention, amelioration, or treatment of cancer. The nucleic acids and vectors of the present disclosure are particularly useful for providing genetic vaccines, i.e., vaccines for delivering nucleic acids encoding the disclosed antigens to subjects, such as humans, so that the antigens are expressed in the subject and thereby elicit an immune response.

[0174] Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for a particular subject and immunogenic composition. Accordingly, the immunogen may be administered to the subject once or multiple times. Preferably, a set time interval is allowed between individual administrations of the immunogenic composition. This interval varies from subject to subject, but typically ranges from 10 days to several weeks, often 2, 4, 6, or 8 weeks. For humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present disclosure, the intervals are longer, preferably about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 weeks. The immunization regimen typically includes one to six administrations of the immunogenic composition, but may include as few as one, two, or four administrations. The method of inducing an immune response may also include administering an adjuvant with the immunogen. In some cases, the initial immunization protocol may be supplemented with booster immunizations annually, twice a year, or at longer intervals (5-10 years). The present method also includes various prime-boost regimens. These methods involve one or more priming immunizations followed by one or more boosting immunizations. The actual immunogenic composition can be the same or different for each immunization, and the type of immunogenic composition (e.g., including a protein or expression vector), route, and formulation of the immunogen can also vary. For example, if an expression vector is used for the priming and boosting steps, it can be of the same type or different types (e.g., DNA or bacterial or viral expression vectors). One useful prime-boost regimen involves two priming immunizations spaced four weeks apart, followed by two boosting immunizations four and eight weeks after the final priming immunization.It will also be apparent to those skilled in the art that there are several permutations and combinations encompassed using the DNA, bacterial, and viral expression vectors of the present disclosure to achieve priming and boosting regimens. CMV vectors can be used repeatedly, expressing different antigens derived from different pathogens. [Example]

[0175] Example 1: CMV vaccines can overcome immune tolerance Vaccine vectors based on the rhesus cytomegalovirus (RhCMV) strain 68-1 induce CD8+ T cell responses that recognize SIV, TB, or malaria peptides in the context of MHC-E and MHC-II instead of the classical MHC-Ia molecule (Hansen et al., 2019; Cytomegalovirus vectors expressing Plasmodium knowlesi antigens induce immune responses that delay parasitemia upon sporozoite challenge, PLoS One 14:e 0210252; Hansen et al., Science, 2013; Hansen et al., Science, 2016).

[0176] Several factors make HLA-E a particularly attractive target for cancer immunotherapy, including: (a) It has been reported that cancer cells upregulate HLA-E in some cancers (Kamiya, 2019, J Clin Invest). Because HLA-E is a ligand for the inhibitory NKG2A receptor, this may be the result of cancer cell selection for NK cell evasion. (b) Normal tissues (except endothelial cells and some immune cells) express HLA-E at low levels. (c) Cancer cells are often selected to express low levels of classical HLA molecules, likely as a means of escaping T cell control. (d) Unlike classical HLA molecules, which are highly polymorphic, HLA-E is highly conserved. Therefore, HLA-E-restricted TCRs can be universally applied to transgenic T cells.

[0177] CMV-based vectors can be used in two ways as cancer immunotherapy: directly as a cancer vaccine in humans or indirectly as a vehicle to elicit MHC-E-restricted TCRs in non-human primates. However, both approaches require demonstration that (a) CMV can elicit MHC-E-restricted CD8+ T cells against cancer antigens and (b) cancer cells can present cancer antigens. Because cancer antigens are often self-antigens, they are subject to immune tolerance. Breaking immune tolerance is difficult for most vector systems, as exemplified by the fact that pox vectors (e.g., PROSTVAC) have failed in clinical trials and likely require combination with checkpoint inhibitor antibodies to elicit effective prostate antigen-specific immune responses (https: / / www.onclive.com / web-exclusives / prostvac-misses-phase-iii-goal-in-prostate-cancer). Furthermore, HLA-E is thought to be a highly selective receptor for a single peptide, VL9 (itself derived from the signal peptide of a polymorphic HLA molecule), and it is not known how other peptides are commonly loaded onto HLA-E.

[0178] To evaluate the ability of CMV to induce MHC-E-restricted CD8+ T cell responses in the context of cancer antigens, we constructed 68.1 strain vectors expressing cancer antigens and administered them to rhesus macaques (RM). RhCMV strain 68-1 lacks the RhCMV homologs of UL128, UL130, UL146, and UL147. Vectors were designed to express one of the following inserts: 1) rhesus PAP, 2) human WT1 Ag, 3) human MSLN, 4) human HER2, and 5) HPV 16 / 18 E6+E7.

[0179] Six male RMs were inoculated with RhCMV strain 68-1 (68-1 / PAP), which expresses the cancer antigen prostatic acid phosphatase (PAP, rhesus macaque). As a control, RMs were also inoculated with SIV gag antigen (68-1 / SIVgag). Three of the six RMs were additionally inoculated with RhCMV strain 68-1 (68-1 / WT1), which expresses the cancer antigen Wilms tumor suppressor protein (WT1, human), and the other three RMs were additionally inoculated with RhCMV strain 68-1 (68-1 / MSLN), which expresses the cancer antigen mesothelin (MSLN, human). Each RM received a booster immunization on day 140, and all monkeys received all antigens at this time. CD4+ and CD8+ T cell responses were measured in peripheral blood mononuclear cells (PBMCs) using overlapping peptide pools for each antigen by intracellular cytokine staining (ICS) at the indicated time points. Infected RMs were able to induce high-frequency T cell responses against each antigen (Figure 1). These results are highly significant because they demonstrate that a CMV-based vaccine can overcome immune tolerance and induce T cell responses against self-antigens and viral oncogenes. Importantly, no negative side effects were observed, despite the fact that the T cells were autoreactive.

[0180] Next, six female RMs were inoculated with RhCMV strain 68-1 (68-1 / HER2), which expresses the human cancer antigen HER2. CD4+ and CD8+ T cell responses were measured in PBMCs using overlapping peptide pools for HER2 by ICS at the indicated time points. As shown in Figure 2, infected RMs were able to induce high-frequency T cell responses against HER2 (Figure 2).

[0181] Four female RMs were inoculated with RhCMV strain 68-1 (68-1 / HPV) and four with RhCMV strain 68-1.2 (68-1.2 / HPV), which expresses a fusion protein of the E6 and E7 proteins of HPV16 and HPV18. RhCMV strain 68-1.2 was "repaired" for UL128-130 as described in Lilja AE and Shenk T, Proc Natl Acad Sci USA 105, 19950-19955 (2008). The vectors were engineered to express one of these proteins, and CD4+ and CD8+ T cell responses were measured in PBMCs by ICS using overlapping peptide pools for HPV antigens at each time point shown in Figure 3. As shown in Figure 3, all inoculated RMs were able to elicit high-frequency T cell responses to the E6 and E7 oncogenes of HPV16 and HPV18. The 68-1 strain induced CD8+ T cells restricted by MHC-II and MHC-E, whereas the 68-1.2 strain induced CD8+ T cells restricted by MHC-I.

[0182] Example 2: Cancer cells can present cancer antigens via HLA-E To further determine whether the T cells generated above can recognize cancer cells expressing these antigens, we measured T cell responses from CD8+ T cells incubated with K562 (human chronic myeloid leukemia) cells expressing MHC-E. Because K562 cells do not express other MHC molecules, peptide presentation to T cells is mediated by MHC-E.

[0183] Four female RMs were inoculated with 68-1 (68-1 / HPV), which expresses a fusion protein of the E6 and E7 proteins of HPV16 and HPV18. CD8+ T cells were isolated and co-incubated with K562 cells expressing either MHC-E or the same fusion protein of MHC-E and HPV. T cell responses were measured by intracellular cytokine staining for TNFα and IFNγ (Figure 4). CD8+ T cells responding with both TNFα and IFNγ production appear in the upper right quadrant. MHC-E-expressing K562 cells transfected with the HPV fusion protein (K562-E) were recognized by CD8+ T cells from two of the four RMs immunized with 68-1 / HPV. Peptide presentation by MHC-E was further demonstrated by adding the peptide VMAPRTLLL(VL9) (SEQ ID NO: 1), a high-affinity ligand for MHC-E. Addition of VL9 inhibited CD8+ T cell responses, demonstrating that RM-elicited TCRs can recognize human MHC-E-presented peptides.

[0184] Next, we determined whether the CMV vector could generate T cells capable of recognizing cancer cells expressing host self-antigens, such as rhesus macaque PAP. Three male RMs were inoculated with 68-1 / PAP. CD8+ T cells were isolated and co-incubated with K562 cells expressing MHC-E and either MHC-E and PAP or MHC-E and HPV E6-E7 fusion proteins. T cell responses were measured by intracellular cytokine staining (ICS) for TNFα and IFNγ (Figure 5). CD8+ T cells responding by producing both TNFα and IFNγ appear in the upper right quadrant. PAP-specific MHC-E-restricted CD8+ T cell responses were blocked by the MHC-E ligand peptide VMAPRTLLL(VL9) (SEQ ID NO: 1). PAP-transfected K562-E, but not HPV fusion protein-transfected K562-E cells, were recognized by CD8+ T cells isolated from 68-1 / PAP-immunized RMs, demonstrating specific recognition. Addition of VL9 inhibited PAP-specific CD8+ T cell responses, demonstrating that recognition was mediated by MHC-E.

[0185] In the second experiment, six male RMs were co-inoculated with 68-1 / PAP and 68-1 / WT1. CD8+ T cells were isolated and co-incubated with K562 cells expressing MHC-E and K562 cells expressing MHC-E and PAP (rhesus macaque) or MHC-E and WT1 (human). T cell responses were measured by intracellular cytokine staining for TNFα and IFNγ (Figure 6). CD8+ T cells responding by both TNFα and IFNγ production appear in the upper right quadrant. K562-E transfected with PAP or WT1 was recognized by CD8+ T cells from six RMs immunized with 68-1 / PAP and 68-1 / WT1.

[0186] In these experiments, cancer cells expressing both MHC-E and TAAs were recognized by MHC-E-restricted CD8+ T cells.

[0187] The results show that CMV-based cancer vaccines overcome some of the challenges faced by immune tolerance, i.e. (i) anti-vector immunity does not affect the ability of CMV vectors to elicit T cell responses against inserted antigens (Hansen et al., 2010. Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus. Science 328:102-106.), and (ii) CMV vectors induce CD8+ T cells against TAAs at frequencies similar to those elicited against foreign antigens. Therefore, CMV-based vaccines are highly effective at breaking immune tolerance. Breaking immune tolerance can occur in two ways: (a) by eliciting MHC-I-restricted CD8+ T cells directed against non-canonical MHC-I-restricted epitopes (e.g., 68-1.2 / PAP) or (b) by eliciting MHC-E and MHC-II-restricted CD8+ T cells directed against non-conventional epitopes (e.g., 68-1-based vectors). This point is further illustrated by the observation that CMV-based vectors lacking US11 failed to elicit CD8+ T cells. In contrast, US11 deletion results in vectors that elicit MHC-I-restricted CD8+ T cells that recognize canonical, or immunodominant, epitopes against non-self antigens (Hansen Science 2010, Hansen PlosONE 2019). The most likely explanation for the inability of US11-deleted vectors to induce MHC-I-restricted CD8+ T cells to self-antigens is that these T cells are eliminated in the thymus (=central tolerance). Central tolerance likely explains why other vaccine and vector systems are inferior to CMV in inducing CD8+ T cells to cancer antigens, due to their inability to induce CD8+ T cells to non-canonical or subdominant MHC-I-restricted epitopes and to induce CD8+ T cells to MHC-II or MHC-E-restricted epitopes. Targeting MHC-E is likely to be particularly effective, as MHC-E is often upregulated on cancer cells, whereas MHC-I is downregulated. Currently, no other vector systems are capable of inducing MHC-E-restricted CD8+ T cells to cancer antigens.

[0188] Example 3: Identification of MHC-II and MHC-E supertopes Chimeric antigen receptor (CAR)-expressing transgenic T cells (CAR-T cells) have revolutionized the treatment of several cancers, particularly leukemia. In most cases, CARs contain antibody-derived binding domains that recognize cancer cell surface proteins (e.g., CD20 for B-cell lymphoma). However, to avoid rejection of the transgenic T cells, new CAR-T cells are generated for every patient, making this treatment very expensive. Commercially available CAR-T cells that can be used in every patient are under development, but none have been approved for clinical use to date.

[0189] Because CAR-T cells can eliminate all cells expressing a given antigen (e.g., all B cells express CD20), they can have the side effect of rendering patients immunosuppressed or susceptible to immune disease complications. Engineered TCR-T cells, i.e., T cells transgenic for a T cell receptor (TCR) that recognizes tumor-specific peptides in the context of MHC, offer another therapeutic approach that can reduce these side effects. However, any given TCR will only recognize specific MHC / peptide complexes. Because MHC is highly polymorphic, the use of engineered TCRs is limited to individuals with precise MHC alleles, a severe limitation of engineered TCR-T cells. In contrast, because MHC-E is non-polymorphic within the human population, MHC-E-restricted TCRs are "universal" (i.e., they can be used by any human). Indeed, because MHC-E is conserved between non-human primates and humans, RM-induced TCRs recognize human MHC-E and HLA-E / presented peptides. Therefore, MHC-E-restricted, TAA-specific TCRs generated in RM can be used to generate universal, pre-manufactured human TCR T cells. A key step in identifying such T cells is the identification of MHC-E-restricted supertopes. The identified supertope peptides can then be used to identify TCRs in activated T cells.

[0190] Deletion of the US11 homolog Rh189 has been shown to elicit "canonical" MHC-I-restricted CD8+ T cell responses (i.e., high-affinity peptide binding to CD8+ T cells expressing MHC-I and T cell receptors with high affinity for the peptide / MHC-I complex results in a peptide that is immunodominant in the context of a conventional vaccine). Taking advantage of this property, we prepared three distinct constructs encoding rhesus PAP, one of which does not encode active Rh189: 68-1.2 / PAP (predicted to elicit MHC-I-restricted CD8+ T cell responses), 68-1 / PAP (predicted to elicit MHC-II- and MHC-E-restricted CD8+ T cell responses), and 68-1 / PAPΔRh189 (predicted to elicit MHC-II- and MHC-E-restricted C and canonical MHC-I-restricted CD8+ T cell responses).

[0191] Eight male RMs were inoculated with either 68-1.2 / PAP, 68-1 / PAP, or 68-1 / PAPΔRh189(US11). CD4+ and CD8+ T cell responses in PBMCs were measured by ICS using overlapping peptide pools for TNFα and IFNγ at the indicated time points (Figure 7). The frequency of PAP-specific T cells among memory T cells is shown in Figure 7, indicating that immunized RMs elicited high-frequency T cell responses.

[0192] Next, we performed a restriction analysis of PAP-specific CD8+ T cells (Figure 8). CD8+ T cell responses to individual peptides are shown as boxes aligned with the PAP sequence. Peptide responses blocked by the MHC-I-specific antibody W6 / 32 (but not by the peptide VL9) are shown as open boxes. Peptide responses blocked by the MHC-II-specific peptide CLIP are shown as dashed boxes. Peptide responses blocked by MHC-E-specific peptides are shown as dotted boxes. As expected, CD8+ T cells from 68-1.2 / PAP-immunized animals were exclusively restricted by MHC-I, whereas those from 68-1 / PAP-immunized animals were restricted by either MHC-II or MHC-E. Interestingly, deletion of Rh189 / US11 did not result in further induction of "canonical" MHC-I-restricted responses, as observed for viral antigens (Hansen, Science, 2013). However, this observation is consistent with PAP undergoing "central" immune tolerance, i.e., CD8+ T cells expressing high-affinity TCRs are eliminated by negative selection in the thymus. This result also suggests that the MHC-I-restricted CD8+ T cells induced by 68-1.2 / PAP are "non-canonical," i.e., CD8+ T cells that recognize subdominant epitopes.

[0193] To identify MHC-E and MHC-II restricted supertopes, we performed restriction analyses on all six animals immunized with 68-1 RhCMV / PAP, 68-1 RhCMV / WT1 (see Example 1), and 68-1 RhCMV / MSNL (see Example 1) (Figures 9 and 10, which show the results for 68-1 RhCMV / WT1 after testing in the presence of a blocking reagent). In Figures 9 and 10, CD8+ T cell responses to individual peptides are shown as boxes along the TAA sequence, with the supertope peptides indicated in parentheses. The color of the box indicates whether the supertope is MHC-E restricted, MHC-II restricted, or whether the restriction has yet to be determined. The supertope peptides and their sequences are listed in Table 1. [Table 1]

[0194] MHC-E restricted supertope peptides can be used to identify MHC-E restricted TCRs, and MHC-II restricted supertope peptides can be used to identify MHC-II restricted TCRs.

Claims

1. 1. A method of generating an immune response to a tumor-associated antigen in a subject, comprising administering to the subject a CMV vector encoding the tumor-associated antigen in an amount effective to elicit a CD8+ T cell response against the tumor-associated antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYE LGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

2. 1. A method of treating cancer in a subject, comprising administering to the subject a CMV vector encoding a tumor-associated antigen in an amount effective to elicit a CD8+ T cell response against the tumor-associated antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or their respective orthologues, and the tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4). Sequence number 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

3. 1. A CMV vector encoding a tumor-associated antigen for use in generating an immune response against said tumor-associated antigen in a subject, wherein said CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their orthologues, and wherein said tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

4. 1. A CMV vector encoding a tumor-associated antigen for use in treating cancer in a subject, the CMV vector not expressing an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues, wherein the tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); A CMV vector comprising: HMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

5. 1. Use of a CMV vector encoding a tumor-associated antigen in the manufacture of a medicament for use in generating an immune response against said tumor-associated antigen in a subject, wherein said CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their respective orthologues, and said tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); Uses including LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

6. 1. Use of a CMV vector encoding a tumor-associated antigen in the manufacture of a medicament for the treatment of cancer, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their orthologues, and the tumor-associated antigen is selected from the group consisting of the amino acid sequences ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRS T (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

7. A method for treating cancer caused by a tumor virus in a subject, comprising administering to the subject a CMV vector encoding a tumor virus antigen in an amount effective to induce a CD8+ T cell response against the tumor virus antigen, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their respective orthologues.

8. A CMV vector encoding a tumor virus antigen for use in treating cancer in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their respective orthologues.

9. Use of a CMV vector encoding a tumor virus antigen in the manufacture of a pharmaceutical for the treatment of cancer, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their respective orthologues.

10. The tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SAERLQGRR 7. The method, CMV vector for use, or use in production of any one of claims 1 to 6, comprising SRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

11. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2).

12. The method, the CMV vector for use, or the use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3).

13. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO: 4).

14. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO: 5).

15. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO: 6).

16. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO: 7).

17. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO: 8).

18. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO: 9).

19. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence IDESLIFYKKWELEA (SEQ ID NO: 10).

20. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO: 11).

21. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO: 12).

22. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13).

23. 7. The method, CMV vector for use, or use in production according to any one of claims 1 to 6, wherein the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

24. 24. The method, CMV vector for use, or use in manufacturing of any one of claims 1 to 23, wherein at least 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or an orthologue thereof, or MHC-II or an orthologue thereof.

25. 25. The method, CMV vector for use, or use in manufacturing of claim 24, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or an orthologue thereof.

26. 26. The method, CMV vector for use, or use in manufacture of any one of claims 1 to 25, wherein less than 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC-class 1a or an orthologue thereof.

27. 27. The method, CMV vector for use, or use in manufacture of any one of claims 1 to 26, wherein a portion of the CD8+ T cells restricted by MHC-E recognize an epitope shared by at least 90% of other subjects immunized with the vector.

28. 28. The method, CMV vector for use, or use in manufacture of any one of claims 1 to 6 and 10 to 27, wherein the epitope recognized by the CD8+ T cells comprises a peptide derived from prostatic acid phosphatase.

29. 28. The method, CMV vector for use, or use in manufacture of any one of claims 1 to 6 and 10 to 27, wherein the epitope recognized by the CD8+ T cells comprises a peptide derived from Wilms tumor suppressor protein.

30. 29. The method, CMV vector for use, or use in manufacturing of claim 28, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

5.

31. 29. The method, CMV vector for use, or use in manufacturing of claim 28, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

6.

32. 30. The method, CMV vector for use, or use in manufacturing of claim 29, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

8.

33. 30. The method, CMV vector for use, or use in manufacturing of claim 29, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

9.

34. 30. The method, CMV vector for use, or use in manufacturing of claim 29, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

13.

35. 30. The method, CMV vector for use, or use in manufacturing of claim 29, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

14.

36. 25. The method, CMV vector for use, or use in manufacture of claim 24, wherein a portion of the CD8+ T cells restricted by MHC-II recognize an epitope shared by at least 90% of other subjects immunized with the vector.

37. 37. The method, CMV vector for use, or use in manufacture of claim 36, wherein the epitope comprises a peptide derived from prostatic acid phosphatase.

38. 38. The method, CMV vector for use, or use in manufacturing of claim 37, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

2.

39. 38. The method, CMV vector for use, or use in manufacturing of claim 37, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

3.

40. 38. The method, CMV vector for use, or use in manufacturing of claim 37, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

4.

41. 38. The method, CMV vector for use, or use in manufacturing of claim 37, wherein the epitope recognized by the CD8+ T cells has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

7.

42. 1. A method for generating CD8+ T cells that recognize MHC-E tumor-associated antigen-peptide complexes, comprising: (a) administering to a first subject a recombinant CMV vector comprising a nucleic acid expressing a tumor-associated antigen, and not expressing an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / peptide complexes; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-E / tumor-associated antigen-derived peptide complex; (c) isolating a second set of one or more CD8+ T cells from a second subject; (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize an MHC-E / tumor-associated antigen peptide complex tumor-associated antigen.

43. 1. A method for generating CD8+ T cells that recognize MHC-E tumor-associated antigen-peptide complexes, comprising: (a) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor-associated antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their respective orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-E / peptide complexes; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-E / tumor-associated antigen-derived peptide complex; (c) isolating a second set of one or more CD8+ T cells from a second subject; (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-E / tumor-associated antigen peptide complexes.

44. The method according to any one of claims 42 to 43, wherein the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

45. The method according to any one of claims 42 to 44, wherein the tumor-associated antigen is associated with prostate cancer, kidney cancer, mesothelioma, breast cancer and cervical cancer.

46. The method according to any one of claims 42 to 45, wherein the tumor-associated antigen is prostatic acid phosphatase, Wilms' tumor suppressor protein, mesothelin and Her-2, or an orthologue thereof.

47. The tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGP 47. The method of claim 46, comprising: G (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

48. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2).

49. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3).

50. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO: 4).

51. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO: 5).

52. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO: 6).

53. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO: 7).

54. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO: 8).

55. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO: 9).

56. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence IDESLIFYKKWELEA (SEQ ID NO: 10).

57. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO: 11).

58. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO: 12).

59. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13).

60. 48. The method, CMV vector for use, or use in manufacture of any one of claims 47, wherein the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

61. 61. The method of any one of claims 42 to 60, wherein the first CD8+ T cells recognize a specific MHC-E supertope.

62. 62. The method of claim 61, wherein the specific MHC-E supertope comprises a peptide derived from a prostatic acid phosphatase epitope.

63. 62. The method of claim 61, wherein the specific MHC-E supertope comprises a peptide derived from a Wilms' tumor suppressor protein epitope.

64. 63. The method of any one of claims 42 to 62, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

5.

65. 63. The method of any one of claims 42 to 62, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

6.

66. 64. The method of any one of claims 42 to 63, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

8.

67. 64. The method of any one of claims 42 to 63, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

9.

68. 64. The method of any one of claims 42 to 63, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

13.

69. 64. The method of any one of claims 42 to 63, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

14.

70. 70. The method of any one of claims 42 to 69, wherein the second CD8+ T cells recognize a specific MHC-E supertope.

71. 71. The method of claim 70, wherein the specific MHC-E supertope comprises a peptide derived from a prostatic acid phosphatase epitope.

72. 71. The method of claim 70, wherein the specific MHC-E supertope comprises a peptide derived from Wilms tumor suppressor protein.

73. 72. The method of claim 71, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

5.

74. 72. The method of claim 71, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

6.

75. 73. The method of claim 72, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

8.

76. 73. The method of claim 72, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

9.

77. 73. The method of claim 72, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

13.

78. 73. The method of claim 72, wherein the MHC-E supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

14.

79. 79. The method of any one of claims 42 to 78, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

80. 80. The method of any one of claims 42 to 79, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

81. 81. The method of any one of claims 42 to 80, wherein the first subject and / or the second subject is a human or a non-human primate.

82. 82. The method of any one of claims 42 to 81, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

83. 82. The method of any one of claims 42 to 81, wherein the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

84. 84. The method of any one of claims 42 to 83, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

85. 85. The method of any one of claims 42 to 84, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

86. The method of any one of claims 42 to 85, wherein the second CD8+ TCR is a chimeric CD8+ TCR.

87. 87. The method of any one of claims 42 to 86, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

88. 88. The method of any one of claims 42 to 87, wherein the step of administering the CMV vector to the first subject comprises administering the CMV vector to the first subject intravenously, intramuscularly, intraperitoneally, or orally.

89. 89. The method of any one of claims 42-88, further comprising administering the transfected CD8+ T cells to the second subject to treat or prevent cancer.

90. 55. The method of claim 54, wherein the cancer is prostate cancer, kidney cancer, mesothelioma, breast cancer, and cervical cancer.

91. 1. A method for CD8+ T cells that recognize an MHC-II tumor peptide complex, comprising: (a) administering to a first subject a recombinant CMV vector comprising a nucleic acid that expresses a tumor antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their respective orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / peptide complexes; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-II / tumor antigen-derived peptide complex; (c) isolating a second set of one or more CD8+ T cells from a second subject; (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

92. 1. A method for generating CD8+ T cells that recognize MHC-II tumor antigen peptide complexes, comprising: (a) isolating a first set of CD8+ T cells from a first subject who has previously been administered a recombinant CMV vector comprising a nucleic acid expressing a tumor antigen and not expressing an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein, or their respective orthologues, in an amount effective to generate a first set of CD8+ T cells that recognize MHC-II / peptide complexes; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-II / tumor antigen-derived peptide complex; (c) isolating a second set of one or more CD8+ T cells from a second subject; (d) transfecting the second set of one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-II / tumor antigen peptide complexes.

93. The method according to any one of claims 91 to 92, wherein the at least one recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

94. The method of claim 91 or 93, wherein the at least one recombinant CMV vector does not express an active UL128 protein or its orthologue, does not express an active UL130 protein or its orthologue, does not express an active UL146 or its orthologue, does not express an active UL147 or its orthologue, or does not express an active US11 protein or its orthologue.

95. 95. The method of any one of claims 91 to 94, wherein the mutation in the nucleic acid sequence encoding UL128, UL130, UL146, UL147, or US11 is one or more of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.

96. 96. The method of any one of claims 91 to 95, wherein the tumor-associated antigen is associated with prostate cancer, kidney cancer, mesothelioma, breast cancer, and cervical cancer.

97. 97. The method of claim 96, wherein the tumor-associated antigen is prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, and Her-2, or an orthologue thereof.

98. The tumor-associated antigen has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGP G (SEQ ID NO: 8); SAERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); or HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

99. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2).

100. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence KELKFVTLVFRHGDR (SEQ ID NO: 3).

101. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence QLTQLGMEQHYELGE (SEQ ID NO: 4).

102. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence LNESYKHEQVYIRST (SEQ ID NO: 5).

103. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence NHMKRATQMPSYKKL (SEQ ID NO: 6).

104. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence MVLLFIHIRRGPCWQ (SEQ ID NO: 7).

105. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence VPEPASQHTLRSGPG (SEQ ID NO: 8).

106. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence SAERLQGRRSRGASG (SEQ ID NO: 9).

107. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence IDESLIFYKKWELEA (SEQ ID NO: 10).

108. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence PFTYEQLDVLKHKLD (SEQ ID NO: 11).

109. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence FMKLRTDAVLPLTVA (SEQ ID NO: 12).

110. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence LQGRRSRGASGSEPQ (SEQ ID NO: 13).

111. 99. The method, CMV vector for use, or use in manufacture of any one of claims 98, wherein the tumor-associated antigen comprises the amino acid sequence HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

112. 112. The method of any one of claims 91 to 111, wherein the first CD8+ T cells recognize an MHC-II supertope.

113. 63. The method of claim 62, wherein the MHC-II supertope comprises a peptide derived from a prostatic acid phosphatase epitope.

114. 114. The method of any one of claims 113, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

2.

115. 114. The method of any one of claims 113, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

3.

116. 114. The method of any one of claims 113, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

4.

117. 114. The method of any one of claims 113, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

7.

118. The method of any one of claims 91 to 117, wherein the second CD8+ T cells recognize an MHC-II supertope.

119. 119. The method of claim 118, wherein the MHC-II supertope comprises a peptide derived from a prostatic acid phosphatase epitope.

120. 119. The method of any one of claims 118, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

2.

121. 119. The method of any one of claims 118, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

3.

122. 119. The method of any one of claims 118, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

4.

123. 119. The method of any one of claims 118, wherein the MHC-II supertope has 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% identity to the amino acid sequence corresponding to SEQ ID NO:

7.

124. 124. The method of any one of claims 91 to 123, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

125. 125. The method of any one of claims 91 to 124, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

126. 126. The method of any one of claims 91 to 125, wherein the first subject and / or the second subject is a human or a non-human primate.

127. 127. The method of any one of claims 91 to 126, wherein the second subject is a human or non-human primate.

128. 128. The method of any one of claims 91 to 127, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR.

129. 128. The method of any one of claims 91 to 127, wherein the second CD8+ TCR comprises the non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

130. 128. The method of any one of claims 91 to 127, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

131. 131. The method of any one of claims 91 to 130, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

132. The method of any one of claims 91 to 131, wherein the second CD8+ TCR is a chimeric CD8+ TCR.

133. 133. The method of any one of claims 91 to 132, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

134. 134. The method of any one of claims 91 to 133, wherein the step of administering the CMV vector to the first subject comprises administering the CMV vector to the first subject intravenously, intramuscularly, intraperitoneally, or orally.

135. 135. The method of any one of claims 91-134, further comprising administering the transfected CD8+ T cells to the second subject to treat cancer.

136. 136. The method of claim 135, wherein the cancer is prostate cancer, kidney cancer, mesothelioma, breast cancer, and cervical cancer.

137. CD8+ T cells produced by the method of any one of claims 42 to 136.

138. 138. A method of treating or preventing cancer in a subject in need thereof, comprising administering to said subject the CD8+ T cells of claim 137.

139. The CD8+ T cell of claim 137 for use in treating or preventing cancer in a subject.

140. 138. Use of the CD8+ T cells of claim 137 in the manufacture of a medicament for treating or preventing cancer.

141. 138. A method of inducing an immune response against a host self-antigen, comprising administering to a subject the CD8+ T cells of claim 137.

142. The CD8+ T cell of claim 137 for use in inducing an immune response against a host self-antigen in a subject.

143. Use of the CD8+ T cells of claim 137 in the manufacture of a medicament for inducing an immune response against a host self-antigen.

144. An isolated MHC-E or MHC-II supertope peptide of about 8 to about 15 amino acids in length capable of being recognized by a CD8+ T cell receptor, wherein the supertope comprises a tumor-associated antigen.

145. The peptide has the amino acid sequence ARAASLSLGFLFLLF (SEQ ID NO: 2); KELKFVTLVFRHGDR (SEQ ID NO: 3); QLTQLGMEQHYELGE (SEQ ID NO: 4); LNESYKHEQVYIRST (SEQ ID NO: 5); NHMKRATQMPSYKKL (SEQ ID NO: 6); MVLLFIHIRRGPCWQ (SEQ ID NO: 7); VPEPASQHTLRSGPG (SEQ ID NO: 8); SA The supertope peptide of claim 144 is selected from the group consisting of ERLQGRRSRGASG (SEQ ID NO: 9); IDESLIFYKKWELEA (SEQ ID NO: 10); PFTYEQLDVLKHKLD (SEQ ID NO: 11); FMKLRTDAVLPLTVA (SEQ ID NO: 12); LQGRRSRGASGSEPQ (SEQ ID NO: 13); and HEDPMGQQGSLGEQQ (SEQ ID NO: 14).

146. A method for overcoming immune tolerance to a tumor-associated antigen in a subject in need thereof, comprising administering to the subject an effective amount of a cytomegalovirus (CMV) vector expressing the tumor-associated antigen.

147. The method of claim 146, wherein the CMV vector is a human CMV vector or a rhesus CMV vector.

148. The method of claim 146, wherein the CMV vector does not express active UL128 or its orthologue, does not express active UL130 or its orthologue, does not express active UL146 or its orthologue, and does not express active UL147 or its orthologue.

149. The method of claim 146, wherein the CMV vector does not express active UL128, active UL130, active UL146, or active UL147, or their respective orthologues, due to the presence of one or more mutations in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147.

150. The method of claim 149, wherein the mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147 is one or more of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding the viral protein.

151. The method of any one of claims 146 to 149, wherein the CMV vector is rhesus CMV strain 68-1.

152. 152. The method of any one of claims 146 to 151, wherein the CMV vector does not express an active UL82 protein or an orthologue thereof.

153. 153. The method of claim 152, wherein the CMV vector does not express active UL82 protein, or an ortholog thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL82.

154. 154. The method of claim 153, wherein the mutation in the nucleic acid sequence encoding UL82 is one or more of a point mutation, a frameshift mutation, a truncation mutation, and a deletion of all of the nucleic acid sequence encoding UL82.

155. 155. The method of any one of claims 145 to 154, wherein the tumor-associated antigen is derived from prostate cancer, kidney cancer, mesothelioma, breast cancer, and cervical cancer.

156. 156. The method of any one of claims 145 to 155, wherein the tumor-associated antigen is prostatic acid phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.

157. 157. The method of any one of claims 145 to 156, wherein the effective amount comprises an amount effective to induce a CD8+ T cell response against the tumor-associated antigen in the subject.

158. 157. The method of claim 156, wherein at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the CD8+ T cells are restricted by MHC-I or an orthologue thereof.

159. The method of claim 156, further comprising identifying a CD8+ TCR from the CD8+ T cells induced by the CMV vector, wherein the CD8+ TCR recognizes an MHC-I / tumor antigen-derived peptide complex.

160. The method of claim 158 or 159, wherein the CD8+ TCR is identified by DNA or RNA sequencing.

161. The method of any one of claims 145 to 159, wherein the subject is a human.

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