OA20580A - Modulation of T cell responses by UL18 of human cytomegalovirus. - Google Patents

Modulation of T cell responses by UL18 of human cytomegalovirus. Download PDF

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OA20580A
OA20580A OA1202200063 OA20580A OA 20580 A OA20580 A OA 20580A OA 1202200063 OA1202200063 OA 1202200063 OA 20580 A OA20580 A OA 20580A
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seq
mhc
recombinant hcmv
cells
hcmv vector
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OA1202200063
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Daniel MALOULI
Klaus J. FRUEH
Scott G. Hansen
Louis J. Picker
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Oregon Health & Science University
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Publication of OA20580A publication Critical patent/OA20580A/en

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Abstract

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

Description

MODULATION OF T CELL RESPONSES BY UL18 OF HUMAN CYTOMEGALOVIRUS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/889,310, filed August 20, 2019, which is hereby incorporated by reference în its entirety.
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with govemment support under grant numbers AI059457 and AI 128741 awarded by The National Institutes of Health. The govemment has certain rights in the invention.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted sequence listing in ASCII text file (Name 4153_013PC01_Seqlisting_ST25; Size: 11,029 bytes; and Date of Création: August 19, 2020) filed with the application is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
It has been previously demonstrated that strain 68-1 of Rhésus cytomégalovirus (RhCMV) elicits CD8+- T cells that recognize peptides presented by MHC-II and MHC-E instead of conventional MHC-I. This effect was recapitulated in cynomolgus monkey CMV (CyCMV), thus demonstrating that délétion of the RhCMV and CyCMV homologs of HCMV UL128, UL130, UL146, and UL147 is required to enable the induction of MHC-E-restricted CD8+ T cells (WO 2016/130693, WO 2018/075591). In addition, these vectors elicit MHC-II restricted CD8+ T cells. However, insertion of a targeting site for the endothélial cell spécifie micro RNA (miR) 126 into essential viral genes of these vectors éliminâtes the induction of MHC-IIrestricted CD8+ T cells resulting in MHC-E only vectors that exclusively elicit MHC-E restricted CD8+ T cells (WO 2018/075591). In contrast, insertion of the myeloid cell spécifie miR142-3p into 68-1 RhCMV prevents the induction of MHC-E restricted CD8+ T cells resulting in vectors that elicit CD8+ T cells exclusively restricted by MHC-II (WO 2017/087921). Similarly, délétion of the UL40 homolog Rh67 prevents the induction of MHC-E restricted CD8+ T cells resulting in MHC-II-only vectors (WO 2016/130693).
BRIEF SUMMARY OF THE INVENTION
The présent disclosure relates to a recombinant human CMV (HCMV) vector comprising a nucleic acid sequence encoding heterologous antigen, wherein the recombinant HCMV vector does not express UL18.
In some embodiments, the recombinant HCMV vector does not express ULI 28. In some embodiments, the recombinant HCMV vector does not express ULI 30. In some embodiments, the HCMV vector does not express UL128 and UL130.
The présent disclosure also relates to a HCMV vector comprising a nucleic acid sequence encoding a heterologous antigen, wherein the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, and UL147.
In some embodiments, the recombinant HCMV vector does not express UL18 protein, UL128 protein, UL130 protein, ULI 46 protein, and ULI 47 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding ULI 8, ULI28, ULI 30, ULI 46, or ULI47. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, ULI28, ULI 30, ULI46, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of all ofthe nucleic acid sequence encoding the viral protein.
In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US 11, or an ortholog thereof.
In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains target sites for microRNAs expressed in endothélial cells. In some embodiments, the MRE expressed in endothélial cells is is miR126, miR-126-3p, miR-130a, miR-210, miR221/222, miR-378, miR-296, and miR-328.
In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains target sites for microRNAs expressed in myeloid cells. In some embodiments, the MRE expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, and miR-125.
In some embodiments, the heterologous antigen is a pathogen spécifie antigen, a tumor antigen, a tissue spécifie antigen, or a host self-antigen. In some embodiments, the pathogen spécifie antigen is selected from the group consisting of human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavîrus, Plasmodium parasites, and Mycobacterium tuberculosis.
In some embodiments, the pathogen spécifie antigen is an MHC-E supertope. In some embodiments, the MHC-E supertope is a HIV epitope. In some embodiments, the MFIC-E supertope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the tumor antigen is related to a cancer selected from the group consisting of acute myeîogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma (RCC), and genn cell tumors.
In some embodiments, the host self-antigen is an antigen derived from the variable région of a T cell receptor (TCR) or an antigen derived from the variable région of a B cell receptor.
The présent disclosure also relates to a pharmaceutical composition comprising the recombinant HCMV vector and a pharmaceutically acceptable carrier.
The présent disclosure also relates to an immuno génie composition comprising the recombinant HCMV vector and a pharmaceutically acceptable carrier.
The présent disclosure also relates to a method of generating an immune response in a subject to the at least one heterologous antigen, comprising administering to the subject the recombinant HCMV vector in an amount effective to elicit a CD8-H T cell response to the at least one heterologous antigen.
The présent disclosure also relates to use of the recombinant HCMV vector in the manufacture of a médicament for use in generating an immune response in a subject.
The présent disclosure also relates to the recombinant HCMV for use in generating an immune response in a subject.
The présent disclosure also relates to a method of treating or preventing cancer in a subject, comprising administering the recombinant HCMV vector of in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen.
The présent disclosure also relates to the use of the recombinant HCMV vector in the manufacture of a médicament for use in treating or preventing cancer in a subject.
The présent disclosure also relates to the recombinant HCMV vector for use in treating or preventing cancer in a subject.
The présent disclosure also relates to a method of treating or preventing a pathogenic infection in a subject, comprising administering to the subject the recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen.
The présent disclosure also relates to use of the recombinant HCMV vector in the manufacture of a médicament for use in treating or preventing a pathogenic infection in a subject.
The présent disclosure also relates to the recombinant HCMV vector for use in treating or preventing a pathogenic infection in a subject.
The présent disclosure also relates to a method of treating an autoimmune disease or disorder in a subject, comprising administering to the subject the recombinant HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen.
The présent disclosure also relates to use of the recombinant HCMV vector in the manufacture of a médicament for use in treating an auto immune disease or disorder in a subject.
The présent disclosure also relates to the recombinant HCMV vector for use in treating an autoimmune disease or disorder in a subject.
in some embodiments, at least 10% of the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-E or an ortholog thereof. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at Ieast 75%, at least 80%, at least 85%, at least 90%, or at Ieast 95% ofthe CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-E or an ortholog thereof.
In some embodiments, at least 10% of the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-II or an ortholog thereof. In some embodiments, at least
-520%, at least 30%, at least 40%, at least 50%, at least 60% or at least 75% of the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-II or an ortholog thereof.
In some embodiments, fewer than 10%, fewer than 20%, fewer than 30%, fewer than 40%, or fewer than 50% of the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-class la or an ortholog thereof. In some embodiments, at least 10% of the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-class la or an ortholog thereof. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or the CD8+ T cells elicited by the recombinant HCMV vector are restricted by MHC-class la or an ortholog thereof.
In some embodiments, a CD8+ TCR is identified from the CD8+ T cells elicited by the recombinant HCMV vector, wherein the CD8+ TCR recognizes a MHC-II/heteiOlogous antigenderived peptide complex. In some embodiments, a CD8+ TCR is identified from the CD8+ T cells elicited by the HCMV vector, wherein tire CD8+ TCR recognizes a MHC-E/heteroIogous antigen-derived peptide complex. In some embodiments, a CD8+ TCR is identified from the CD 8+ T cells elicited b y the HCMV vector, wherein the CD8+ TCR recognizes a MHC-class la/heterologous antigen-derived peptide complex.
In some embodiments, the CD8+ TCR is identified by DNA or RNA sequencing.
In sonie embodiments, the CD8+ TCR recognizes MHC-II supertopes.
In some embodiments, the CD8+ TCR recognizes MHC-E supertopes. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of LDAWEK.IRLRPGGKK. (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
The présent disclosure also relates to a method of generating TCR-îransgénie CD 8+ T cells that recognize MHC-E-peptide complexes, the method comprising: (a) administering to a first subject a recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-E/peptide complexes; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes a MHC-E/heterologous antigen-derived peptide compiex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encodîng a second CD8+ TCR and a promoter operabiy linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize a MHC-E peptide complexes. In some embodiments, the recombinant EICMV vector does not express UL18, UL128, UL130, UL146 and/or UL147. In some embodiments, the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147, In some embodiments, the mutations în the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothélial cells. In some embodiments, the miRNA expressed în endothélial cells is miRI26, miR-I26-3p, miR130a, miR-210, miR-221/222, miR-378, mîR-296, or miR-328. In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-spécifie antigen, or a host self-antigen. In some embodiments, the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitîs B virus, hepatitîs C virus, papîllomavirus, Plasmodium parasites, or Mycobacterium tuberculosis.
The présent dîsclosure also relates to a method of generating TCR-transgenic CD8+ T cells that recognize MHC-E-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, wherein the set of CD8+ T cells are generated from the recombinant HCMV vector of any one of claims 5-10, 12-13, or 16-17, wherein the first CD8+ TCR recognizes a MHC-E/heterologous antigen-derived peptide compiex; (b) isolating one or more CD 8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3P of the first CD 8+ TCR, thereby generating one or more TCR-transgenic CD 8+ T cells that recognize MHC-E-peptide complexes. In some embodiments, the recombinant HCMV vector does not express UL18, UL128, UL130, UL146 and/or UL147. In some embodiments, the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL I30, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, troncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothélial cells. In some embodiments, the miRNA expressed in endothélial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR296, or miR-328. In some embodiments, the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tîssue-spécifie antigen, or a host self-antigen. In some embodiments, the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, or Mycobacterium iuberculosis.
In some embodiments, the first CD8+ T cell recognizes MHC-E supertopes. In some embodiments the MHC-E supertopes comprise human immunodeficiency virus epitopes. In some embodiments, the MHC-E supertope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24);
QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ TD NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ 1D NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the second CD8+ T cell recognizes MHC-E supertopes. In some embodiments, the MHC-E supertopes comprise human immunodeflciency virus epitopes. In some embodiments, the MHC-E supertope îs at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQTGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVS1L (SEQ ID NO: 24);
QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.
In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.
In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.
The présent disclosure also relates to a method of generating CD8+ T cells that recognize MHC-E peptide complexes, the method comprising: (a) administering to a non-human primate a recombinant rhésus CMV (RhCMV) or cynomolgus CMV (CyCMV) vector déficient for orthologs of UL128, UL130, UL146, and ULI47 and expressing HIV antigens in an amount effective to generate a set of CD8+ T cells that recognize MHC-E in complex with HIV supertope peptides; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first recognizes a MHC-E/supertope peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3« and CDR3p ofthe first CD8+ TCR, thereby generating one or more transfected CD8-h T cells that recognize a MHC20580
E/heterologous antigen-derived peptide compiex. In some embodimentsthe HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14);
KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQA1SPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24);
QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28);
EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32). The présent disclosure also relates to a method of generating CD8+ T cells that recognize MFIC-E peptide complexes, the method comprising: (a) îdentifying a first CD8+ TCR that recognizes a MHC-E/supertope peptide compiex from a set of CD8+ T cells that recognize MHC-E in compiex with the HIV supertope peptides, wherein the set of CD8+ T cells are générâted from a recombinant rhésus (RhCMV) or cynomolgus CMV (CyCCMV) vector déficient for orthologs of UL12S, UL130, UL146, and UL147 and expressing HIV antigens in an amount effective to generate the set of CD8+ T cells; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucieic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucieic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3P ofthe first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-E peptide complexes. In some embodimentsthe HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16);
QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24);
QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28);
EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the first subject is a nonhuman primate and the second subject is a human, and wherein the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3P of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the non-human primate CDRIct, CDR2a, CDR3a, CDRip, CDR2P, and CDR3p ofthe first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises CDRla, CDR2a, CDR3a, CDRlp, CDR2p, and CDR3p ofthe first CD8+ TCR. In some embodiments, the second CD8+ TCR îs a chimeric CD8+ TCR.
In some embodiments, adminîstering the recombinant HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the recombinant HCMV vector to the first subject.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consistîng of acute myelogenous leukemia, chronic myelogenous leukemîa, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin’s lymphoma, multiple myeloma, maügnant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma (RCC), and germ cell tumors.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent a pathogenic infection. In some embodiments, the pathogénie infection is caused by a pathogen selected from the group consistîng of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
In some embodiments, the transfected CD 8+ T cells are administered to the second subject to induce an autoimmune response to the host self-antigen.
The présent disclosure also relates to a method of generating CD8+- T cells that recognize MHC-II-peptide complexes, the method comprising: (a) adminîstering to a first subject the recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-II/peptîde complexes; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes a MHC-II/heterologous antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encodîng 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 CDR3a and CDR3[3 of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize a MHC-II peptide complex. In some embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US 11, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a MRE, wherein the MRE contains a target site for a miRNA expressed in myeloid cells. In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.
The present disclosure also relates to a method of generating CD8+ T cells that recognize MHC-II-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR that recognizes a MHC-II/heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize MHC-II/peptide complexes, wherein the set of CD8+ T cells are generated from the recombinant HCMV vector; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p of the first CD8-I- TCR, thereby generating one or more CD8+ T cells that recognize a MHC-II peptide complexes. In some
- 12 embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, ULI46 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding ULI8, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, UL13Û, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a MRE, wherein the MRE contains a target site for a miRNA expressed in myeloîd cells. In some embodiments, the miRNA expressed în myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, ormiR-125.
In some embodiments, the first CD8+ T cell recognizes MHC-Π supertopes. In some embodiments, the second CD8+ T cell recognizes MHC-Π supertopes.
In some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing. In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.
In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.
In some embodiments, administering the HCMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the HCMV vector to the first subject.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic
- 13 syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, long cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma (RCC), and germ cell tumors.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent a pathogénie infection. In some embodiments, the pathogénie infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papîllomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to induce an autoimmune response to the host self-antigen.
The présent disclosure also relates to a method of generating CD8+ T cells that recognize MHC-I-peptide complexes, the method comprising: (a) administering to a first subject the recombinant HCMV vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-I/peptide complexes; (b) identifying a first CD8+ TCR from the set of CD8+ T cells, wherein the first CD8+ TCR recognizes a MHC-I/heterologous antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3P of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize a MHC-I peptide complex. In some embodiments, the recombinant HCMV vector comprises a nucleic acid sequence encoding heterologous antigen. In some embodiments, the recombinant HCMV vector does not express UL18. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express UL128 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and ULI47. In some embodiments, the recombinant HCMV vector does not express a UL18 protein, ULI28 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding ULI8, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, ULI30, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the
- 14recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US11, or an ortholog thereof.
The présent disclosure also relates to a method of generating CD8+ T cells that recognize MHC-l-peptide complexes, the method comprising: (a) identifyîng a first CD8+ TCR that recognizcs a MHC-I/heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize a MHC-I/heterologous antigen-derived peptide complex, wherein the set of CD8+ T cells are generated from the recombinant HCMV vector of any one of daims 1-11; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3P of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-I peptide complexes.
in some embodiments, the first CD8+ TCR is identified by DNA or RNA sequencing.
In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR îs identical to the nucleic acid sequence encoding the first CD 8+ TCR.
In some embodiments, the first subject is a human. In some embodiments, the second subject is a human.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent cancer. In some embodiments, the cancer is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkm's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma (RCC), and germ cell tumors.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to treat or prevent a pathogenic infection. In some embodiments, the pathogenic infection is caused by a pathogen selected from the group consisting of human immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
In some embodiments, the transfected CD8+ T cells are administered to the second subject to induce an autoimmune response to the host self-antigen.
In some embodiments, the pathogen spécifie antigen is selected from the group consisting of human immunodeficiency virus, sîmian immunodeficiency virus, herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
In some embodiments, the tumor antigen îs related to a cancer selected from the group consisting of acute myelogenous ieukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma (RCC), and germ cell tumors.
In some embodiments, the host self-antigen is an antigen derived from the variable région of a T cell receptor (TCR) or an antigen derived from the variable région of a B cell receptor.
The présent disclosure also relates to a method of treating or preventing a pathogenic infection in a subject, the method comprising administering a CD8+ T cell to the subject.
The présent disclosure also relates to the use of the CD8+ T in the manufacture of a médicament for use în treating or preventing a pathogenic infection in a subject.
The présent disclosure also relates to the CD8+ T cell for use in treating or preventing a pathogenic infection in a subject.
The présent disclosure also relates to a method of treating or preventing cancer in a subject, the method comprising administering a CD8+ T cell to the subject.
The présent disclosure also relates to use of the CD8+ T cell in the manufacture of a médicament for use in treating or preventing cancer în a subject.
The présent disclosure also relates to the CD8+ T cell for use in treating or preventing cancer in a subject.
The présent disclosure also relates to a method of treating an autoimmune disease or disorder, the method comprising administering a CD8+ T cell to the subject.
The présent disclosure also relates to use ofthe CD8+ T cell in the manufacture of a médicament for use in treating an autoimmune disease or disorder.
The présent disclosure also relates to the CD8+ T cell for use în treating an autoimmune disease or disorder.
The présent disclosure also relates to a method of inducing an autoimmune response to a host self-antigen, the method comprising administering a CD8+ T cell to the subject.
The présent disclosure also relates to a human immunodeficiency virus MHC-E supertope between 9 and 15 amino acids in length that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO; 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEP1DKELYPLAS (SEQ ID NO: 25); KQEP1DKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIE1CGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the recombinant HCMV vector comprises a nucleic acid encoding one or more human immunodeficiency virus antigens. In some embodiments, the recombinant HCMV vector does not express UL128. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express ULI28 and UL130. In some embodiments, the recombinant HCMV vector does not express UL146 and UL147. In some embodiments, the recombinant HCMV vector does not express UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiments, the mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UE82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US 11, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothélial cells. In some embodiments, the miRNA expressed in endothélial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR296, or miR-328. In some embodiments, the recombinant HCMV vector further comprises a
- 17 nucleic acid sequence encoding a MRE, wherein the MRE contains a target site for a miRNA expressed in myeloid cells. In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
Figure 1 shows the average frequencies of CD4+ or CD8+ T cells responding to SIVantigen-derived peptide pools in the indicated cohorts. T cell frequencies were determîned in peripheral blood mononuclear cells (PBMC) at the indicated time points by întracellular cytokine staining (ICS) for IFNy or TNFa in the presence of pools of overlapping (by 1 IA) 15mer peptides representing the SIV antigens. Cohort 1 was immunized with three MHC-E only 68-1 RhCMV vectors carrying récognition sites for mtr 126 in the 3' untranslated région ofthe essential genes R11108 (UL79) and Rhl56 (IE2) and expressing the SIV antigens SlVgag, SIVretanef (fusion of rev, tat, and nef), or the 5' segment of SIVpol. Cohort 2 was immunized withthree MHC-II only 68-1 RhCMV vectors deleted for Rh67 (UL40) and expressing the SIV antigens SlVgag, SIVretanef, or the 5' segment of SIVpol. Cohort 3 was immunized with three MHC-II only 68-1 RhCMV vectors carrying récognition sites for mir!42 in the 3' untranslated région of the essential genes Rhl08 (UL79) and Rhl 56 (IE2) and expressing the SIV antigens SlVgag, SIVretanef, or the 5' segment of SIVpol. Cohort 4 (control cohort) was immunized with three 68-1 RhCMV vectors expressing the SIV antigens SlVgag, SIVretanef, or the 5’ segment of SIVpol.
Figure 2 shows SlVgag-specific CD8+ T cell responses in PBMC obtained from three Rhésus macaques (RM) in each of the indicated cohorts measured in the presence of indîvîdual peptides. Peptides resultîng în spécifie CD8+ T cell responses are indicated by a box, with the color of the box desîgnating MHC restriction as determîned by blocking with the anti-pan-MHCI mAb W6/32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP.
Figure 3 shows plasma viral load after repeated limîtîng dose SIVmac239 challenge of RM in cohorts 1,2, and 3 (left panel) and SlVvîf spécifie CD8+ T cell responses of RM in cohorts 1, 2, and 3 (right panel). Animais that controlled SIV infection (RM controllers) are shown in white boxes and non-controllers are shown in black boxes. One animal in cohort 2 initially controlled SIV infection, but control was lost upon déplétion of CD8+ T cells consistent with this RM being a spontaneous elîte controller.
Figure 4 shows an immunoblot of the SIV supertope fusion construct. Telomerized rhésus fibroblasts (TRF) were infected, or non-infected, with the indicated RhCMV constructs
- 18and lysâtes of infected cells were electrophoretically separated prior to immunoblotting. The SIV supertope-containing fusion protein was visualized with an anti-HA antibody whereas viral protein IE1, Rhl07, and Rhl08 were detected using spécifie antibodies. The protein band observed in mock-infected or uninfected TRF lysâtes with IE antibodies is non-specific.
Figure 5A shows the average frequencies of CD8+ T cells responding to SlV-antigen derived peptides in PBMC of cohort 5 animais (n=8). Cohort 5 was immunized with a 68-1 RhCMV vector carrying récognition sites for mirl26 in the 3’ untranslated région of the essential genes RhIOS (UL79) and RhI56 (IE2) and expressing the MHC-E supertope fusion protein. T cell frequencies were determined in peripheral blood mononuclear cells (PBMC) at the indicated time points by intraceliular cytokine staining (ICS) for IFNy or TNFa in the presence of pools of individual I5mer peptides representîng the SIV supertopes. Figure 5B shows the frequencies of CD8+ T cells responding to spécifie MHC-E restricted supertopes in individual RM. MHC-E restricted supertopes (Gag69 and Gagl20) and other MHC-E restricted Gag epitopes are shown .
Figure 6 shows SIV plasma viral load after repeated limiting dose SIVmac239 challenge of RM in cohort 5 (left panel) and SlVvif spécifie T cell responses (right panel). RM controllers are shown in white boxes and non-controliers are shown in black boxes. SlVvif-specifïc responses demonstrate take of SIV infection in controller animais.
Figure 7A shows the frequencies of CD8+ T cells responding to SIV antigen peptide pools in RM inoculated with 68-i RhCMV expressing SlVgag (n=2), 68-1 RhCMV expressing ULI8 and SIVretanef (n=2), or 68-1 RhCMV expressing UL18 and SIVpol (n=2). Figure 7B shows the frequencies of CD8+ T cells responding to MHC-E restricted supertopes in each RM. Figure 7C shows the frequencies of CD8+ T cells responding to MFIC-II restricted supertopes in each RM.
Figure 8 shows SIVpol spécifie CD8+ T cells responses în PBMC obtained from three RM inoculated with 68-1 RhCMV expressing UL18 and SIVpol. CD8+ T cell responses were measured in the presence of individual peptides. Peptides resulting în spécifie CD8+ T cell responses are indicated by a box, with the color of the box designating MHC restriction as determined by blocking with the anti-pan-MHC-I-mAb W6/32, the MHC-E blockîng peptide VL9 and the MFIC-II blocking peptide CLIP. Ali peptide responses were blocked with W6/32 but not by VL9 peptide or CLIP peptide. Thus, CD8+ T cells are exclusively restricted b y MHCI.
Figure 9A shows a dot plot depicting the frequencies of CD8+ T cells producing IFNy or TNFa in response to SIVpol peptides from an RM inoculated with 68-1 RhCMV expressing ULI8 and SIVpol. Figure 9B shows a dot plot depicting the frequencies of CD8+ T cells producing IFNy or TNFa in response to SIVpol peptides from an RM inoculated with 68-1 RhCMV expressing the UL18 D196S mutant and SIVpol. The frequencies of CD8+ T cells responding to pools of overlapping 15mer peptides comprising SIVpol or the MHC-E restricted supertope peptide SIVpol41 or the MHC-n-restricted supertope peptide SIVpol90 is shown. Whereas intact UL18 prevents the induction of supertope responses, this is not observed for the D196S mutant ofULI8.
Figure 10 shows an immunoblot of human MRC5 fibroblasts uninfected or înfected with HCMV-TR3 (Caposio P. et al, 2019. Characterization of a live-attenuated HCMV-based vaccine platfonn. Sci Rep 9:19236) or with a HCMV-TR3-based vectors in which UL18 was replaced with a HIVgag, HIVnef and HIVpol fusion protein. In addition, the UL18-deleted vector lacked UL128, UL130, UL146 and UL147 since previous work has shown that these genes inhibit MHC-E restricted CD8+ T cell responses (U.S. Patent No, 10,532,099). In addition, the p24 fragment of HIVgag was added for control. The upper blot was probed with antibodies to the HIVgag protein. The lower blot was probed with antibodies to the HCMV pp65 protein.
Figure 11 shows HIV gag, nef and pol-specific CD8+ T cells responses in PBMC obtained from RM inoculated the UL18-deleted vector (Fig. 11, n=2). CD8+ T cell responses were measured on day 56 post-vaccination using overlapping peptide pools corresponding to each portion of the antigen.
DETAILED DESCRIPTION OF THE INVENTION
I. Terms
Unless otherwise noted, technical terms are used according to conventional usage.
Ail publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 62/889,310 fded August 20, 2019, are hereby incorporated by reference in their entirety for ail purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.
Although methods and materials similar or équivalent to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to
-20be limiting. In order to facilitate revîew of the various embodiments of the disclosure, the following explanations of spécifie terms are provided.
Unless the context requires otherwise, throughout the présent spécification and claims, the word “comprise” and variations thereof, such as “comprises” and “comprising,” are to be construed in an open, inclusive sense, that is, as “including, but not lirnited to”. “Consisting of” shah mean excluding more than trace éléments of other ingrédients and substantial method steps disclosed herein. The term “consisting essentially of ’ lîmits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characterîstics of a claimed invention. For example, a composition consisting essentially of the éléments as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. Similarly, a proteîn consîsts essentially of a particular amino acid sequence when the protein includes additional amino acids that contribute to at most 20% of the length ofthe protein and do not substantialiy affect the activity of the protein (e.g., alters the activity of the protein by no more than 50%). Embodiments defined b y each of the transitional terms are within the scope of this invention.
Antigen: As used herein, the terms antigen or immunogen are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once 20 administered to a subject (either directly or by administering to the subject a nucléotide sequence or vector that encodes the protein) the protein is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
Antigen-specific T cell: A CD8+ or CD4+ lymphocyte that recognizes a particular antigen. Generally, antigen-specific T cells specifically bind to a particular antigen presented by
MHC molécules, but not other antigens presented by the same MHC.
Administration: As used herein, the term administration means to provide or give a subject an agent, such as a composition comprising an effective amount of a CMV vector comprising an exogenous antigen by any effective route. Exemplary routes of administration include, but are not lirnited to, injection (such as subeutaneous, intramuscular, intradermal, 30 intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Effective amount: As used herein, the term effective amount refers to an amount of an agent, such as a CMV vector comprising a heterologous antigen or a transfected CDS+ T cell that recognizes a MHC-E/heterologous antigen-derived peptide complex, a MHC-II/heterologous
-21 antigen-derived peptide complex, or a MHC-l/heterologous antigen-derived peptide complex, that is suffi ci ent to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or disease or induce an immune response to an antigen. In some examples, an effective amount is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or dîsease. An effective amount may be a therapeutically effective amount. including an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with infections disease or cancer.
Heterologous antigen: As used herein, the term heterologous antigen refers to any protein or fragment thereof that is not derived from CMV. Heterologous antigens may be pathogen-specific antigens, tumor virus antigens, tumor antigens, host self-antigens, or any other antigen.
Hyperproliferative disease: A dîsease or disorder characterized by the uncontrolled prolifération of cells. Hyperproliferative diseases include, but are not limited to malignant and non-malignant tumors.
Immune tolérance: As used herein immune tolérance refers to a State of unresponsiveness of the immune System to substances that hâve the potential to induce an immune response. Self-tolerance to an individual's own antigens, for exampie, tumor antigens, is achieved through both central tolérance and peripheral tolérance mechanisms.
Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence, such as an N-terminal repeat, such that the peptide will bind an MHC molecuie and induce a cytotoxic T lymphocyte (CTL) response, or a B cell response (for example antibody production) against the antigen from which the immunogenic peptide is derived.
In some embodiments, immunogenic peptides are identifîed using sequence motifs or other methods, such as neural net or polynomial déterminations known in the art. Typicaily, algorithms are used to déterminé the binding threshold of peptides to select those with scores that give them a high probability of binding at a certain affînity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide. Within the context of an immunogenic peptide, a conserved residue is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. In some embodiments, a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide.
-22MicroRNA: As used herein, the term microRNA refers to a major class of biomolecules involved in control of gene expression. For example, in human heart, liver or brain, miRNAs play a roie in tissue spécification or cell lîneage decisions în addition, miRNAs influence a variety of processes, including early development, cell prolifération and ceil death, and apoptosis and fat metabolism. The large number of miRNA genes, the diverse expression patterns, and the abondance of potential miRNA targets suggest that miRNAs may be a significant source of genetic diversity.
A mature miRNA is typically an 8-25 nucléotide non-coding RNA that régulâtes expression of an mRNA including sequences complementary to the miRNA. These small RNA molécules are known to control gene expression by regulating the stability and/or translation of mRNAs. For example, miRNAs bind to the 3' UTR of target mRNAs and suppress translation. MiRNAs may also bind to target mRNAs and médiate gene silencing through the RNAi pathway. MiRNAs may also regulate gene expression by causîng chromatin condensation.
A miRNA silences translation of one or more spécifie mRNA molécules by binding to a miRNA récognition element (MRE,) which is defined as any sequence that dîrectly base pairs with and înteracts with the miRNA somewhere on the mRNA transcript. Often, the MRE is présent in the 3' untranslated région (UTR) of the mRNA, but it may also be présent in the coding sequence or in the 5' UTR. MREs are not necessarily perfect compléments to miRNAs, usually having only a few bases of complementarity to the miRNA and often containing one or more mismatches within those bases of complementarity. The MRE may be any sequence capable of being bound by a miRNA sufficiently that the translation of a gene to which the MRE is operably linked (such as a CMV gene that is essential or augmenting for growth in vivo) is repressed by a miRNA silencing mechamsm such as the RISC.
Mutation: As used herein, the term mutation refers to any différence in a nucleic acid or polypeptide sequence from a normal, consensus, or wild type sequence. A mutant is any protein or nucleic acid sequence comprising a mutation. In addition, a cell or an organism with a mutation may also be referred to as a mutant. Some types of coding sequence mutations include point mutations (différences in individual nucléotides or amino acids); silent mutations (différences in nucléotides that do not resuit in an amino acid changes); délétions (différences in which one or more nucléotides or amino acids are missing, up to and including a délétion of the entire coding sequence of a gene); frameshift mutations (différences in which délétion of a number of nucléotides indivisible by 3 results în an alteration of the amino acid sequence). A mutation that results in a différence in an amino acid may also be called an amino acid
-23 substitution mutation. Amino acid substitution mutations may be described by the amino acid change relative to wild type at a particular position in the amino acid sequence.
Nucléotide sequences or nucleic acid sequences: The tenus nucléotide sequences and nucleic acid sequences refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids. The nucleic acid may be si ngle-strand ed, or partially or completel y double stranded (duplex). Duplex nucleic acids may be homo duplex or heteroduplex.
Operably Linked: As the term operably linked is used herein, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in such a way that it has an effect upon the second nucleic acid sequence. Operably linked DNA sequences may be contiguous, or they may operate at a distance.
Promoter: As used herein, the term promoter may refer to any of a number of nucleic acid control sequences that directs transcription of a nucleic acid. Typically, a eukaryotic promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element or any other spécifie DNA sequence that îs recognîzed by one or more transcription factors. Expression by a promoter may be further modulated by enhancer or repressor éléments. Numerous examples of promoters are available and well known to those of ordinary skîll in the art. A nucleic acid comprising a promoter operably linked to a nucleic acid sequence that codes for a particular polypeptide may be termed an expression vector.
Recombinant; As used herein, the term recombinant with reference to a nucleic acid or polypeptide refers to one that has a sequence that is not natural 1 y occurring or has a sequence that is made by an artîficial combination of two or more otherwise separated segments of sequence, for example a CMV vector comprising a heterologous antigen. This artifîcial combination is often accomplished by Chemical synthesis or, more commonly, by the artifîcial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. A recombinant polypeptide may also refer to a polypeptide that has been made using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism that is not the natural source of the polypeptide (for example, nucleic acids encoding polypeptides that form a CMV vector comprising a heterologous antigen).
Pharmaceutically acceptable carriers: As used herein, a pharmaceutically acceptable carrier ofuse is conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature
-24of the carrier will dépend on the particular mode of administration being employed. For instance, parentéral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluîds such as water, physiological saline, balanced sait solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnésium stéarate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wettîng or emulsîfying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolauraie.
Polynucleotide: As used herein, the tenu polynucleotide refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleîc acid (DNA). A polynucleotide is made up of four bases; adenine, cytosine, guanine, and thymine/uracil (uracil is used in RNA). A coding sequence from a nucleic acid is indicative of the sequence of the protein encoded by the nucleic acid.
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 polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by Chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for ex ample, disulfide bond formation, glycosylation, lipîdation, acétylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
Orthologs of proteins are typically characterized by possession of greater than 75% sequence îdentity counted over the full-length alignaient with the amino acid sequence of spécifie protein using AL1GN set to default parameters. Proteins with even greater simîlarity to a reference sequence will show increasing percentage identifies when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In addition, sequence identity can be compared over the full length of particular domains of the disclosed peptides.
Sequence idcntity/similarity: As used herein, the identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or simiiarity between the sequences. Sequence identity may be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence simiiarity may be measured în terms of percentage identity or simiiarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the
-25 sequences are. Polypeptides or protein domains thereof that hâve a sîgnificant amount of sequence identity and also function the same or sîmilarly to one another (for example, proteins that serve the same fonctions în different species or mutant forms of a protein that do not change the function of the protein or the magnitude thereof) may be called homologs.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv Appl Math 2, 482 (1981); Needleman & Wunsch, J Mol Biol 48, 443 (1970); Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988); Higgins & Sharp, Gene 73, 237-244 (1988); Higgins & Sharp, CABIOS5, 151-153 (1989); Corpet étal. Nue Acids Res 16, 10881-10890 (1988); Huang étal, Computer App Biosci 8, 155-165 (1992); and Pearson et al, Meth Mol Bio 24,307-331 (1994). In addition, Altschul et al, J Mol Biol 215, 403-410 (1990), présents a detaiîed considération of sequence alignment methods and homology calculations.
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, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information may be found at the NCBI web site.
BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those régions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucléotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an artîculated length (such as 100 consecutive nucléotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1166 matches when aligned with a test sequence having 1154 nucléotides is 75.0 percent identical to the test sequence (1166=1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide région that aligna with
-2620 consecutive nucléotides from an identified sequence as foilows contains a région that shares 75 percent sequence identity to that identified sequence (that is, 15^20*100^75).
For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-Iength alignaient with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr database, swissprot database, and patented sequences database. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, Comput Appl Biosci 10, 67-70 (1994.) Other programs use SEG. In addition, a manual alignaient may be perfonned. Proteins writh even greater similarity will show increasing percentage identifies when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a protein.
When aligning short peptides (fewer than around 30 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a protein. When less than the entire sequence îs being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short Windows of 10-20 amino acids, and may possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determinîng sequence identity over such short Windows are described at the NCBI web site.
One indication that two nucleic acîd molécules are closely related îs that the two molécules hybridize to each other under stringent conditions, as described above. Nucleic acid sequences that do not show a hîgh degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy ofthe genetic code. Changes în a nucleic acid sequence may be made using this degeneracy to produce multiple nucleic acid molécules that ail encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity to a nucleic acid that encodes a protein.
Subject: As used herein, the term subject refers to a living multi-cellular vertebrate organisais, a category that includes both human and non-human mammals.
-27Supertope: 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, Le., in the presence or absence of given MHC-I, MHCII, or MHC-E alleles.
Treatment: As used herein, the term treatment refers to an intervention that améliorâtes a sîgn or symptom of a disease or pathologîcal condition. As used herein, the ternis treatment, treat, and treating, with reference to a disease, pathologîcal condition or symptom, also refers to any observable bénéficiai effect of the treatment. The bénéficiai effect may be evidenced, for example, by a delayed onset of clinical symptoms ofthe disease in a susceptible subject, a réduction in severity of some or ail clinical symptoms of the disease, a slower progression of the disease, a réduction in the number of relapses of the disease, an improvement in the overall heaith or well-being of the subject, or by other parameters well known în the art that are spécifie to the particular disease. A prophylactic treatment îs a treatment administered to a subject who does not exhibit signs of a disease or exhîbits only early signs, for the purpose of decreasing the risk of developing pathology. A therapeutic treatment is a treatment administered to a subject after signs and symptoms of the disease hâve developed.
Vaccine: An îmmunogénie composition that can be administered to a mammal, such as a human, to confer immunity, such as active immunity, to a disease or other pathologîcal condition. Vaccines can be used prophylactically or therapeutically. Thus, vaccines can be used reduce the likelihood of developing a disease (such as a tumor or pathologîcal infection) or to reduce the severity of symptoms of a disease or condition, limit the progression of the disease or condition (such as a tumor or a pathologîcal infection), or limit the récurrence of a disease or condition (such as a tumor). In particular embodiments, a vaccine is a replication-deficient CMV expressing a heterologous antigen, such as a tumor associated antigen derived from a tumor of the lung, prostate, ovary, breast, colon, cervix, liver, kidney, bone, or a melanoma.
Vector; Nucleic acid molécules of particular sequence can be incorporated into a vector that is then introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replîcate în a host cell, such as an origin of réplication. A vector may also include one or more selectable marker genes and other genetic éléments known in the art, including promoter éléments that direct nucleic acid expression. Vectors can be viral vectors, such as CMV vectors. Viral vectors may be constructed from wild type or attenuated virus, including réplication déficient virus.
II. Methods for the Modulation of T cell Responses by UL18 of HCMV
Disclosed herein are methods for the modulation of T cell responses by UL18 of HCMV. The methods involve administering an effective amount of at least one recombinant HCMV vector comprising at least one heterologous antigen to a subject, wherein the HCMV vector does not express UL18.
In some embodiments, the method further comprises generating an immune response to the at least one heterologous antigen, comprising administering to the subject the HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating or preventing cancer in a subject, comprising administering the HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating or preventing a pathogenic infection in a subject, comprising administering the HCMV vector in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the method further comprises treating an autoimmune disease or disorder in a subject, comprising administering to the subject the HCMV vector în an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen.
In some embodiments, the UL18-decificent HCMV vector also does not express an UL] 28, UL130, UL146, or UL147 protein due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, or UL147. In addition, any of the UL18-deficient HCMV vectors can be déficient for US11, and/or UL82 protein due to the presence of a mutation in the nucleic acid sequence encoding US11, and/or UL82. The mutation may be any mutation that results in a lack of expression of active proteins. Such mutations may include point mutations, frameshift mutations, délétions of less than ali of the sequence that encodes the protein (truncation mutations), or délétions of ail of the nucleic acid sequence that encodes the protein, or any other mutations.
In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147 and expresses UL40 and US28.
In some embodiments, the HCMV vector comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE). In some embodiments, the HCMV vector lacks UL18, UL128, UL130, UL146, and UL147 (and optionally UL82) and expresses UL40 and US28 and the MRE contains target sites for microRNAs expressed in endothélial cells. Examples of such miRNAs expressed in endothélial cells are miR126, mi R-12 6-3 p, mi R-130a, miR-210, miR-221/222, miR-378, miR-296, and miR-328. In some embodiments, the HCMV vector lacks
-29UL18 and the MRE contains target sites for microRNAs expressed in myeloid cells. Examples of such miRNAs expressed in myeloid cells are miR-142-ep, miR-223, miR-27a, mîR-652, miR155, miR-146a, miR-132, iniR-21, and miR-125.
The MRE may be any miRNA récognition element that silences expression in the presence of a miRNA expressed by endothélial cells. The MRE may be any miRNA récognition element that silences expression in the presence of a miRNA expressed by myeloid cells. Such an MRE may be the exact complément of a miRNA. Altemativeiy, other sequences may be used as MREs for a given miRNA. For example, MREs may be predîcted from sequences. In one example, the miRNA may be searched on the website microRNA.org (www.microma.org). In tum, a lîst of mRNA targets of the miRNA will be listed. For each listed target on the page, 'alignment details' may be accessed and putative MREs accessed.
One of ordinary skill in the art may select a validated, putative, or mutated MRE sequence from the literature that would be predîcted to induce silencing in the presence of a miRNA expressed in a myeloid cell such as a macrophage. One exampie involves the above referenced website. The person of ordinary skill in the art may then obtain an expression construct whereby a reporter gene (such as a fluorescent protein, enzyme, or other reporter gene) has expression driven by a promoter such as a constitutively active promoter or cell spécifie promoter. The MRE sequence may then be introduced into the expression construct. The expression construct may be transfected into an appropriate cell, and the cell transfected with the miRNA of interest. A lack of expression of the reporter gene indicates that the MRE silences gene expression in the presence of the miRNA.
In some embodiments, the heterologous antigen may be a pathogen spécifie antigen, a tumor antigen, a tumor spécifie antigen, or a host self-antigen. In some embodiments, the host self-antigen is derîved from the variable région of a T cell receptor (TCR) or an antigen derived from the variable région of a B cell receptor.
The pathogen spécifie antigen may be derived from, for example, human îmmunodeficiency virus, simian immunodeficîency virus, herpes simplex virus type 1, herpes simplex virus type 2 , hepatîtis B virus, hepatîtis C virus, papillomavirus, Plasmodium parasites, Clostridium tetani, and Mycobacterium tuberculosis.
Tumor antigen s are relatively restricted to tumor cells and can be any protein that induces an immune response. However, many tumor antigens are host (self) proteins and thus are typically not seen as antigenic by the host immune System. Tumor antigens can also be abnormally expressed by cancer cells. Tumor antigens can also be germline/testis antigens expressed in cancer cells, cell lineage différentiation antigens not expressed in adult tissue, or
- 30 antigens overexpressed in cancer cells. Tumor antigens include, but are not limited to, prostatic acidic phosphatase (PAP); Wilms tumor suppressor protein (WT1); Mesothelin (MSLN); Her-2 (HER2); human papilloma virus antigen E6 of strain HPV16; human papilloma virus antigen E7 of strain HPV16; human papilloma virus antigen E6 of strain HPV18; Human papilloma virus antigen E7 of strain HPV18; a fusion protein of human papilloma virus E6 and E7 from HPV16 and HPV18; mucin 1 (MUCI); LMP2; epidermal growth factor receptor (EGFR); p53; New York esophagus 1 (NY-ESO-1); prostate spécifie membrane antigen (PSMA); GD2, carcinoembryonic antigen (CEA); melanoma antigen a/melanoma antigen recognized by T cells I (MelanA/MARTl); Ras; gpl00, Protéinase 3 (PR1), Bcr-abl; Survivin; prostate spécifie antigen (PSA); human telomerase reverse transcriptase (hTERT); EphA2; ML-IAP; alphafetoprotein (AFP); EpCAM; ERG; NA 17; PAX3; ALK; Androgen receptor (AR); Cyclin Bl; MYCN; RhoC; tyrosine related protein 2 (TRP-2); GD3; Fucosyl GM1; PSCA; sLe(a); CYP1B1; P LC Al; GM3; BORIS; Tn; GloboH; Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETV6-AML); NY-BR-1 ; RGS5; squamous antigen rejecting tumor or 3 (SART3); STn; Carbonic anhydrase IX; PAX5; OY-TES1 ; Sperm protein i 7; LCK; HMWMAA; AKAP-4; SSX2; B7H3; Legumain; Tie 2; Page4; VEGFR2; MAD-CT-1; FAP; PDGFR; MAD-CT-2; Fosrelated antigen 1; TAG-72; 9D7; EphA3; Telomerase; SAP-1; BAGE family; CAGE family; GAGE family; MAGE family; SAGE family; XAGE family; preferentially expressed antigen of melanoma (PRAME); melanocortin 1 receptor (MC1R); β-catenin; BRCA1/2; CDK4; chronic myelogenous leukemia 66 (CML66); TGF-β. In certain embodiments, the host self-antigens include prostatic acidic phosphatase, Wilms tumor suppressor protein, mesothelin, or Her-2.
In some embodiments the tumor antigen is derived from a cancer. The cancer includes, but is not limited to, Acute lymphoblastic leukemia; Acute myeloid leukemia; Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cérébral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Braîn tumor, cérébral astrocytoma/malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous System lymphoma, primary; Cerebellar astrocytoma, childhood; Cérébral astrocytoma/Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders;
Colon Cancer; Cutaneous T -cell lymphoma; Desmoplastic small round cell tumor; Endométrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Genn cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retmoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastro intestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cérébral Astrocytoma; Glioma, Childhood Visual Pathway and Hypo thaï amie; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer;
Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancréas); Kaposi sarcoma; Kidney cancer (rénal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T -Cell; Lymphoma, Hodgkin; Lymphomas, Non-Hodgkin (an old classification of ail lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease;
Macroglobulinemia, Waldenstrim; Malîgnant Fibrous Hîstiocytoma of Bone/Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothehoma, Adult Malîgnant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasîa Syndrome, Childhood;
Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoîdes; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Aduit Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histîoeytoma of bone; Ovarian cancer; Ovarian épithélial cancer (Surface épithélial-stromal tumor); Ovarian germ cell tumor; Ovarian low malîgnant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pîneal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituîtary adenoma; Plasma cell
- 32neoplasia/Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Rénal cell carcinoma (kidney cancer); Rénal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, chîldhood; Salîvary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue;
Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma;
Squamous cell carcinoma-see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, ehildhood; T -Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular 10 cancer; Throat cancer; Thymoma, ehildhood; Thymoma and Thymie carcinoma; Thyroid cancer;
Thyroid cancer, ehildhood; Transitional cell cancer of the rénal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, ehildhood; Ureter and rénal pelvis, transitional cell cancer; Uréthral cancer; Uterine cancer, endométrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, ehildhood; Vulvar cancer; Waldenstrom macro globulinemia; and Wilms tumor (kidney cancer.) In some embodiments, the pathogen spécifie antigen is a MHC-E supertope. In some embodiments, the MHC-E supertope is a HIV epîtope. In some embodiments, the MHC-E supertope is at Ieast 10%, at least 20%, at least 30%, at least 40%, at least 50%, ai least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to
LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO; 18);
NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20);
STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22);
IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31 ); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).In some embodiments, one or more of the MHC-E supertopes are used to generate a fusion protein. The fusion protein may contain one or more of the MHC-E supertopes, in any order.
In some embodiments, the HCMV vector is administered in an amount effective to elicit a CD8+ T cell response to the at least one heterologous antigen. In some embodiments, the CD8+ T cell response elicîted by the vector is characterized by having at least 10% of the CD8+
- 33 T cells directed against epitopes presented by MHC-E. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD8+ T cells are restricted by MHC-E. In some embodiments, the CD8+ T cells restricted by MHC-E recognized peptides 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 method further comprises identifying a CD8+ T cell receptor (TCR) from the CD8+ T cells elicited from the HCMV vector.
The TCR can be identified by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes a MHC-E/heterologous antigen-derived peptide complex. In some embodiments, the CD8+ TCR recognizes MHC-E supertopes. In some embodiments, the MHCE supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the method fiirther comprises the use of supertope peptides to identify a MHC-E restricted CD8+ T cell receptor (TCR) from CD8+ T cells elicited by a nonhuman primate CMV, such as rhésus or cynomolgus macaque CMV (RhCMV or CyCMV), that is defective in expression of orthologs of UL128, UL130, UL146 and UL147 (and optionally UL82) and expresses orthologues of UL40 and US28. MHC-E restricted CD+ T cells would be elicited in rhésus macaques with RhCMV or in cynomolgus macaques with CyCMV.
In some embodiments, the CD8+ T cell response elicited by the HCMV vector is characterized by having at least 10% of the CD8+ T cells directed against epitopes presented by MHC-II. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%
-34ofthe CD8+ T cells are restricted by MHC-II. In some embodiments, the CD8+ T cells restricted by MHC-II recognized peptides 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.
In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from the CD8+ T cells elicited from the HCMV vector. The TCR can be identifîed by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes a MHCIl/heterologous antigen-derived peptide complex. In some embodiments, the CDS+ TCR recognizes MHC-II supertopes.
In some embodiments, the CD8+ T cell response elieited by a UL18-deficient HCMV vector that also lacks US 11 is characterized by having at least 10% of the CD8+ T cells directed against epitopes presented by MHC-Ia. In further examples, at least 15%, at least 20%, at least 30%, ai least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95% or at least 95% ofthe CD8+ T cells are restricted by MHC-Ia.
In some embodiments, the method further comprises identifying a CD8+ T cell receptor (TCR) from the CD8+ T cells elicited from the UL18 and US11-déficient HCMV vector. The TCR can be identifîed by DNA or RNA sequencing. In some embodiments, the CD8+ TCR recognizes a MHC-Ia/heterologous antigen-derived peptide complex.
Also disclosed herein is a method of generating CD8+ T cells that recognize MHC-E peptide complexes. This method in volves administering to a first subject a HCMV vector în an amount effective to generate a set of CD8+ T cells that recognize MHC-E/peptide complexes. The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and does not express: an UL18 protein, an UL128 protein, an UL130 protein, an UL146 protein, and an UL147 protein. The vector might also lack an UL82 protein. In some embodiments, the HCMV vector expresses UL40 and US28. In some embodiments, the HCMV vector does not express an UL18, UL138, UL130, UL146, and UL147 protein and comprises a nucleic acid sequence encoding UL40, US28, and a microRNA (miRNA) récognition element (MRE). In some embodiments, the MRE contains target sites for mîcroRNAs expressed in endothélial cells. Examples of such miRNAs expressed in endothélial cells are miR126, mîR126-3p, miR-130a, miR-210, miR-221/222, mîR-378, miR-296, and miR-328.
The antigen may be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable région of a T cell receptor or a B cell receptor.
This method further comprises: identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognîzes a MHC-E/heterologous antigenderived peptide complet. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, this method may further comprise transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3a and CDR3p of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize a MHC-E/ heterologous antigenderived peptide complex. The one or more CD 8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the CD8+ T cells elicited by the HCMV vector, wherein the CD8+ T cell receptor recognîzes a MHC-E/heterologous antigen-derived peptide complex. In some embodiments, the method further comprises identifying a MHC-E restricted CD8+ T cell receptor from CD8+ T cells elicited by a non-human primate CMV, such as rhésus or cynomolgus macaque CMV (RhCMV or CyCMV), that is defective in expression of orthologs ofUL128, LJL13Ü, ULI46, and UL147 and expresses orthologues of UL40 and US28. MHC-E restricted CD8+ T cells would be elicited in rhésus macaques with RhCMV or in cynomolgus macaques with CyCMV. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8-I- T cell receptor that recognîzes MHCE supertopes. In some embodiments, the MHC-E supertope is a human immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO; 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); OKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
Also disclosed here în is a method of generating TCR-transgenic CD8+ T cells that recognize MHC-E-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR from a set of CD8+ T cells, wherein the set of CD8+ T cells are generated from the recombinant HCMV vector, wherein the first CD8+ TCR recognizes a MHC-E/heterologous antigen-derived peptide complex; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD 8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p of the first CD8+ TCR, thereby generating one or more TCR-transgenic CD8+ T cells that recognize MHC-E-peptide complexes.
Also disclosed is a TCR-transfected CD8+ T cell that recognizes MHC-E-peptide complexes prepared by a process comprising the steps of: (1) administering to a first subject a HCMV vector (deleted for UL18, UL128, UL130, UL146, UL147, and, in some embodiments, UL82; expressing UL40 and US28; and, in some embodiments, expressing a nucleic acid sequence encoding a microRNA récognition element) in an amount effective to generate a set of CD8+ T cells that recognize MHC-E/peptide complexes, wherein the recombinant HCMV vector comprises at least one heterologous antigen; (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes a MHC-E/heterologous antigen-derived peptide complex; (3) isolating one or more CD8+ T cells from the first subject or a second subject; and (4) transfecting the one or more CD8+T cells isolated from the first or second subject with an expression vector, thereby creatîng a transfected T cell that recognizes MHC-E peptide complexes wherein the transfected CD 8+ T cells generate an immune response to the MHC-E/heterologous antigen-derive peptide complex.
In some embodiments, this method may further comprise transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3a and CDR3p of the first CD8+ T cell receptor, thereby generating one or more transected CD8+ T cells that recognize a MHC-E/heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the first and/or second CD8+ T cell receptors are identified by RNA or DNA sequencing. In some embodiments, the first and/or second CD8+ T cell receptor recognizes MHC-E supertopes. In some embodiments, the MHC-E supertope is a human
-37immunodeficiency virus epitope. In some embodiments, the MHC-E supertope is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); OKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the first and/or second subject is a human or nonhuman primate. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In some embodiments, the first subject is a nonhuman primate and the second subject is a human, and wherein the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3p of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the nonhuman primate CDR la, CDR2a, CDR3a, CDRIp, CDR2p, and CDR3p ofthe first CD 8+ TCR. In some embodiments, the second CD8+ TCR comprises the CDR la, CDR2a, CDR3a, CDRlp, CDR2P, and CDR3p ofthe first CD8+ TCR.
Also disclosed herein are methods of treating a disease, such as cancer, a pathogenic infection, or an immune disease or disorder, the method comprising administering the transfected T cell that recognizes MHC-E peptide complexes to the first or second subject. Also disclosed herein are methods of inducing an immune response to a host self-antigen or tissue-specific antigen, the method comprising administering the transfected T cell that recognizes MHC-E peptide complexes to the first or second subject.
The cancer, incîudes but is not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelioma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, breast
-38cancer, long cancer, ovarîan cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcinoma, and germ cell tumors.
The pathogenic infection, includes but is not limited to, human immunodeficiency virus, herpes simplex virus type I, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, 5 papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
Also disclosed here are methods of generating CD8+ T cells that recognize MHC-E peptide complexes, the method comprising: (a) identifyîng a first CD8+ TCR that recognizes a MHC-E/supertope peptide complex from a set of CD8+ T cells that recognize MHC-E in complex with the HIV supertope peptides, wherein the set of CD8+ T cells are generated from a 1Ü recombinant rhésus (RhCMV) or cynomolgus CMV (CyCCMV) vector déficient for orthologs of ULI28, UL130, UL146, and UL147 and expressing HIV antigens in an amount effective to generate the set of CD8+ T cells; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-E peptide complexes.
Also disclosed herein is a method of generating CD8+ T cells that recognize MHC-II peptide complexes. This method involves administering to a first subject (or animal) a CMV 20 vector in an amount effective to generate a set of CD8+ T cells that recognize MHC-II/peptide complexes. The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and does not express: an UL18 protein, an UL128 protein, an UL130 protein, an UL146 protein, or an UL147 protein, and, in some embodiments, an UL82 protein.
In some embodiments, the UL18-deficient HCMV vector also comprises a nucleic acid 25 sequence encoding a microRNA (miRNA) récognition element (MRE). In some embodiments, the MRE contains target sites for microRNAs expressed in myeloid cells. Examples of such mîRNAs expressed in myeloid cells are miR-142-ep, miR-223, miR-27a, miR-652, mîR-155, miR-146a, miR-132, miR-21, and miR-125.
The antigen may be any antigen, including a pathogen-specific antigen, a tuinor virus 30 antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable région of a T cell receptor or a B cell receptor.
This method further comprises: identifyîng a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes a MHC-II/heterologous antigen derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identifîed by
-39DNA or RNA sequencing. In some embodiments, this method may further comprise transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3a and CDR3p of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize a MHC-II/heterologous antigenderived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the method further comprises identîfying a CD8+ T cell receptor from the CD8+ T cells elicited by the HCMV vector, wherein the CD8+- T cell receptor recognizes a MHC-II/heterologous antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes MHC-II supertopes.
Also disclosed are methods of generating CD8+ T cells that recognize MHC-II-peptide complexes, the method comprising: (a) identifying a first CD8+ TCR that recognizes a MHCII/heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize MHC-Il/peptide complexes, wherein the set of CD8+ T cells are generated from the recombinant HCMV vector; (b) isolatîng one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p ofthe first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize a MHC-Π peptide complexes.
Also disclosed is a TCR-iransfected CD8+ T cell that recognizes MHC-II-peptide complexes prepared by a process comprising the steps of; (1) administering to a first subject a UL18-deficient HCMV vector (also deleted for LJLI28, UL130, LJL146, or UL147 (or combinations thereof), and, in some embodiments UL82; and/or expressing a nucleic acid encoding a microRNA récognition element) în an amount effective to generate a set of CD8+ T cells that recognize MHC-II/peptide complexes, wherein the recombinant CMV vector comprises at least one heterologous antigen; (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes a MHC-II/heterologous antigen-derived peptide complex; (3) isolatîng one or more CD8+ T cells from the first subject or a second subject; and (4) transfecting the one or more CD8+T cells isolated from the first or second subject with an expression vector, thereby creating a transfected T cell that recognizes
-40MHC-II peptide complexes wherein the transfected CDS+ T cells generate an immune response to the MHC-II/heterologous antîgen-derive peptide complex.
In some embodiments, this method may further comprise transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3a and CDR3p of the first CD 8+ T cell receptor, thereby generating one or more transected CD8+ T cells that recognize a MHC-II/heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the first and/or second CD8+ T cell receptors are identified by RNA or DNA sequencing. In some embodiments, the first and/or second CD8+ T cell receptor recognizes MHC-II supertopes.
In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the first and/or second subject is a human or nonhuman primate. In some embodiments, the second CD8+ TCR is a chimeric CD8+ TCR. In some embodiments, the fist subject is a nonhuman primate and the second subject is a human, and wherein the second CD8+ TCR is a chimeric nonhuman primate-human CD8+ TCR comprising the non-human primate CDR3a and CDR3p of the first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the nonhuman primate CDRla, CDR2a, CDR3a, CDRlp, CDR2p, and CDR3p ofthe first CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the CDRla, CDR2a, CDR3a, CDR1 p, CDR2p, and CDR3p ofthe first CD8+ TCR.
Also disclosed herein are methods of treating a disease, such as cancer, a pathogenic infection, or an immune disease or disorder, the method comprising administering the transfected T cell that recognizes MHC-II peptide complexes to the first or second subject. Also disclosed herein are methods of inducing an immune response to a host self-antigen or tissue-spécifie antigen, the method comprising administering the transfected T cell that recognizes MHC-II peptide complexes to the first or second subject.
The cancer, includes but is not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, mesothelioma, malignant mesothelîoma, kidney cancer, cervical cancer, oropharyngeal cancer, anal cancer, penile cancer, vaginal cancer, vulvar cancer, breast
-41 cancer, lung cancer, ovarîan cancer, prostate cancer, pancreatie cancer, colon cancer, rénal cell carcinoma, and gémi cell tumors.
The pathogenic infection, includes but is not lirnited to, human immunodeficiency virus, herpes simplex virus type I, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavîrus. Plasmodium parasites, and Mycobacterium tuberculosis.
Also disclosed herein is a method of generating CD8+ T cells that recognize MHC-Ia peptide complexes. This method involves administering to a first subject a UL18-deficîent CMV vector that also lacks an US 11 protein in an amount effective to generate a set of CD8+ T cells that recognize MHC-Ia/peptide complexes. The CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and does not express: an US11 protein and an UL18 protein. The vector might also lack an UL128 protein, an UL130 protein, or an UL146 protein, an UL147 protein, and/or an UL82 protein. The antigen may be any antigen, including a pathogen-specific antigen, a tumor virus antigen, a tumor antigen, or a host self-antigen. In some embodiments, the host self-antigen is an antigen derived from the variable région of a T cell receptor or a B cell receptor.
This method further comprises: identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes a MHC-Ia/heterologous antigen-derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identifîed by DNA or RNA sequencing. In some embodiments, this method may further comprise transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3a and CDR3p of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize a MHCIa/heterologous antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the CD8+ T cells elicited by the CMV vector, wherein the CD8+ T cell receptor recognizes a MHC-Ia/heterologous antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identifîed by RNA or DNA sequencing.
Also disclosed are methods of generating CD8+ T cells that recognize MHC-I-peptîde complexes, the method comprising: (a) identifying a first CD8+ TCR that recognizes a MHCI/heterologous antigen-derived peptide complex from a set of CD8+ T cells that recognize a MHC-I/heterologous antigen-derived peptide complex, wherein the set of CD8+ T cells are
-42generated from the recombinant HCMV vector; (b) isolating one or more CD8+ T cells from a second subject; and (c) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises CDR3a and CDR3p of the first CD8+ TCR, thereby generating one or more CD8+ T cells that recognize MHC-I peptide complexes.
Also disclosed is a transfected CD8+ T cell that recognizes MHC-la-peptide complexes prepared by a process comprising the steps of: (1) administering to a first subject a CMV vector defective for US 11 and UL18 (additionally the vector might be defective for UL128, UL130, UL146, UL147, and/or UL82; expressing UL40 and/or US28) in an amount effective to generate a set of CD8+ T cells that recognize MHC-Ia/peptide complexes, wherein the recombinant CMV vector comprises at least one heterologous antigen; (2) identifying a first CD8+ T cell receptor from the set of CD8+ T cells, wherein the first CD8+ T cell receptor recognizes a MHCla/heterologous antigen-derivcd peptide complex; (3) isolating one or more CD8+ T cells from the first subject or a second subject; and (4) transfecting the one or more CD8+T cells isolated from the first or second subject with an expression vector, thereby creating a transfected T cell that recognizes MHC-Ia peptide complexes wherein the transfected CD8+ T cells generate an immune response to the MHC-Ia/heterologous antigen-derive peptide complex.
In some embodiments, this method may further comprise transfecting the one or more CD8+ T celis with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, wherein the second CD8+ T celi receptor comprises CDR3a and CDR30 of the first CD8+ T cell receptor, thereby generating one or more transected CD8+ T cells that recognize a MHC-la/heteroiogous antigen-derîved peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
In some embodiments, the first and/or second CD8+ T cell receptors are identified by RNA or DNA sequencing.
In some embodiments, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In some embodiments, the second CD8-H TCR is a chimeric CD8+ TCR. In some embodiments, the second CD8+ TCR comprises the CDR lot, CDR2a, CDR3a, CDRlp, CDR2p, and CDR3p of the first CD 8+ TCR.
Also disclosed herein are methods of treating a disease, such as cancer, a pathogenic infection, or an immune disease or disorder, the method comprising administering the transfected
-43 T cell that recognizes MHC-Ia peptide complexes to the first or second subject. Also disclosed herein are methods of inducing an immune response to a host self-antigen or tissue-specific antigen, the method comprising administering the transfected T cell that recognizes MHC-la peptide complexes to the first or second subject.
The cancer, includes but is not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastîc leukemia, chronic lympboblastic leukemia, acute lymphoblastîc 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, rénal cell carcinoma, and germ cell tumors.
The pathogénie infection, includes but îs not limited to, human immunodeficiency virus, herpes simplex virus type I, herpes simplex virus type 2, hepatitîs B virus, hepatitîs C virus, papillomavîrus, Plasmodium parasites, and Mycobacterium tuberculosis.
III. HIV Supertope Constructs
Also disclosed are human immunodeficiency virus antigens between 9 and 15 amino acids in length and that is at least 90%, at least 95%, or 100% identical to the amino acid sequence of LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID NO: 15); KAQQAAADT (SEQ JD NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19); VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); 1VRMYSPVSILDIRQ (SEQ ID NO: 23); RMYSPVSIL (SEQ ID NO: 24); QKQEPIDKELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or ILDLWVYHTQGYFPD (SEQ ID NO: 32).
In some embodiments, the recombinant HCMV vector comprises a nucleic acid encoding one or more human immunodeficiency virus antigens. In some embodiments, the recombinant HCMV vector does not express ULI28. In some embodiments, the recombinant HCMV vector does not express UL130. In some embodiments, the recombinant HCMV vector does not express ULI28 and ULI30. In some embodiments, the recombinant HCMV vector does not express ULI46 and ULI47. In some embodiments, the recombinant HCMV vector does not
-44express UL1S protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequence encoding UL18, UL128, UL130, UL146, or UL147. In some embodiinents, the mutations in the nucleic acid sequence encoding U LJ 8, UL128, UL130, UL146, or UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acîd sequence encoding US28, or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express UL82 (pp71), or an ortholog thereof. In some embodiments, the recombinant HCMV vector does not express US 11, or an ortholog thereof. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a microRNA (miRNA) récognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothélial cells. In some embodiments, the miRNA expressed in endothélial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, mîR-378, miR296, or miR-328. In some embodiments, the recombinant HCMV vector further comprises a nucleic acid sequence encoding a MRE, wherein the MRE contains a target site for a miRNA expressed in myeloid cells. In some embodiments, the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR-21, or miR-125.
The CMV vectors disclosed herein may be used as an immunogenîc or vaccine composition contaîning the recombinant CMV virus or vector, and a pharmaceutically acceptable carrier or diluent. An immunologie composition contaîning the recombinant CMV virus or vector (or an expression product thereof) elicits an immunological response—local or systemic. The response can, but need not be, protective. A vaccine composition elicits a local or systemic protective or therapeutic response. Accordingly, the term iinmunogénie composition includes a vaccine composition (as the former term may be a protective composition).
The recombinant CMV vectors disclosed herein may be used in methods of inducing an immunological response in a subject comprising administering to the subject an immunogenîc, immunological or vaccine composition comprising the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.
The recombinant CMV vectors disclosed herein may be used in therapeutic compositions contaîning the recombînant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. The CMV vectors disclosed herein may be prepared by inserting DNA comprising a sequence that encodes the tumor antigen into an essential or non-essential région of the CMV
-45 genome. The method may further comprise deleting one or more régions from the CMV genome. The method may comprise in vivo recombination, Thus, the method may comprise transfecting a cell with CMV DNA in a cell-compatible medium in the presence of donor DNA comprising the heterologous DNA flanked by DNA sequences homologous with portions of the CMV genome, whereby the heterologous DNA is introduced into the genome of the CMV, and optionally then recovering CMV modified by the in vivo recoinbination. The method may also comprise cleaving CMV DNA to obtain cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMV DNA to obtain hybrîd CMV-heterologous DNA, transfecting a cell with the hybrid CMV -heterologous DNA, and optionally then recovering CMV modified by the presence of the heterologous DNA Since in vivo recombination is comprehended, the method accordingly also provides a plasmid comprising donor DNA not naturally occurring in CMV encoding a polypeptide foreign to CMV, the donor DNA is within a segment of CMV DNA that would otherwise be co-linear with an essential or non-essentîal région of the CMV genome such that DNA from an essential or nonessential région of CMV is flanking the donor DNA The heterologous DNA may be inserted into CMV to generate the recombinant CMV in any orientation that yields stable intégration of that DNA, and expression thereof, when desired.
The DNA encoding the heterologous antigen in the recombinant CMV vector may also include a promoter. The promoter may be from any source such as a herpes virus, including an endogenous cytomégalovirus (CMV) promoter, such as a human CMV (HCMV), rhésus macaque CMV (RhCMV), murine, or other CMV promoter. The promoter may also be a nonviral promoter such as the EFla promoter. The promoter may be a truncated transcriptionally active promoter which comprises a région transactivated with a transactivating protein provided by the virus and the minimal promoter région of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter may be composed of an association of DNA sequences corresponding to the minimal promoter and upstream regulatory sequences. A minimal promoter is composed of the CAP site plus ATA box (minimum sequences for basic level of transcription; unregulated level of transcription); upstream regulatory sequences are composed of the upstream element(s) and enhancer sequence(s).
Further, the term truncated indicates thaï the full-length promoter is not completely présent,
i.e., that some portion of the full-length promoter has been removed. And, the truncated promoter may be derived from a herpesvirus such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. There may be up to a 40% and even up to a 90% réduction in size, from a full-length promoter, based upon base pairs. The promoter may also be a modified non-viral promoter. As to HCMV promoters, reference is made to U.S. Pat. Nos. 5,168,062 and 5,385,839. As to transfecting cells
-46with plasmîd DNA for expression therefrom, reference is made to Feigner et al. (1994), J Biol. Chem. 269, 2550-2561. And, as to direct injection of plasmîd DNA as a simple and effective method of vaccination against a variety of infections diseases reference is made to Science, 259:1745-49, 1993. It is therefore within the scope of this disclosure that the vector may be used by the direct injection of vector DNA.
Also disclosed is an expression cassette that may be inserted into a recombinant virus or plasmîd comprising the truncated transcriptionally active promoter. The expression cassette may further include a functional truncated polyadenylation signal; for instance an SV40 polyadenylation signal which is truncated, yet functional. Considering that nature provided a larger signal, it is indeed surprising that a truncated polyadenylation signal is functional. A truncated polyadenylation signal addresses the insert size limit problems of recombinant vîruses such as CMV. The expression cassette may also include heterologous DNA with respect to the virus or system into which it is inserted; and that DNA may be heterologous DNA as described herein.
As to antigens for use in vaccine or immunological compositions, see also Stedman's Medical Dîctîonary (24th édition, 1982, e.g., définition of vaccine (for a lîst of antigens used in vaccine formulations); such antigens or epitopes of interest from those antigens may be used. As to tumor antigens, one skilled in the art may select a tumor antigen and the coding DNA therefor from the knowledge ofthe amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, etc.) and the codon dîctîonary, without undue expérimentation.
One method to déterminé T epitopes of an antigen involves epitope mapping. Overlapping peptides of the tumor antigen are generated by oligo-peptide synthesis. The individual peptides are then tested for their ability to induce T cell activation. This approach has been particularly useful in mapping T cell epitopes since the T cell recognîzes short linear peptides complexed with MHC molécules.
An immune response to a tumor antigen is generated, in general, as follows: T cells recognize proteins only when the protein has been cleaved into smaller peptides and is presented in a complex called the major hîstocompatibility complex (MHC) located on another cell's surface. There are two classes of MHC complexes—class I and class II, and each class is made up of many different alleles. Different species, and individual subjects hâve different types of MHC complex alleles; they are said to hâve a different MHC type. One type of MHC class I molécule is called MHC-E (HLA-E in humans, Mamu-E in RM, Qa-lb in mice). Unlike other MHC-I molécules, MHC-E is highly conserved within and between mammalian species.
-47It is noted that the DNA comprising the sequence encoding the tumor antigen may itself include a promoter for driving expression in the CMV vector or the DNA may be limited to the codîng DNA of the tumor antigen. This construct may be placed in such an orientation relative to an endogenous CMV promoter that it is operably linked to the promoter and is thereby expressed. Further, multiple copies of DNA encoding the tumor antigen or use of a strong or eariy promoter or eariy and late promoter, or any combination thereof, may be done so as to amplify or increase expression. Thus, the DNA encoding the tumor antigen may be suitably posîtioned with respect to a CMV endogenous promoter, or those promoters may be translocated to be inserted at another location together with the DNA encoding the tumor antigen. Nucleic acids encoding more than one tumor antigen may be packaged in the CMV vector.
Further disclosed are pharmaceutical and other compositions containing the disclosed CMV vectors. Such pharmaceutical and other compositions may be formulated so as to be used in any administration procedure known in the art. Such pharmaceutical compositions may be via a parentéral route (intradermal, intraperitoneal, intramuscuiar, subcutaneous, intravenous, or others). The administration may also be via a mucosal route, e.g., oral, nasal, génital, etc.
The disclosed pharmaceutical compositions may be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical arts. Such compositions may be administered in dosages and by techniques well known to those skilled in the medical arts takîng into considération such factors as the breed or species, âge, sex, weight, and condition of the particular patient, and the route of administration. The compositions may be administered alone, or may be co-administered or sequentially administered with other CMV vectors or with other immunological, antigenic or vaccine or therapeutic compositions. Such other compositions may include purified native antigens or epitopes or antigens or epîtopes from the expression by a recombinant CMV or another vector system; and are administered taking into account the aforementîoned factors.
Examples of compositions include liquid préparations for orifice, e.g., oral, nasal, anal, génital, e.g., vaginal, etc., administration such as suspensions, syrups or élixirs; and, préparations for parentéral, subcutaneous, intraperitoneal, intradermal, intramuscuiar or intravenous administration (e.g., injectable administration) such as stérile suspensions or émulsions. In such compositions the recombinant may be in admixture with a suitable carrier, diluent, or excipient such as stérile water, physiologîcal saline, glucose or the like.
Antigenic, immunological or vaccine compositions typically may contain an adjuvant and an amount of the CMV vector or expression product to elieit the desired response. In human applications, alum (aluminum phosphate or aluminum hydroxîde) is a typical adjuvant. Saponin
-48and its purified component Quil A, Freund's complété adjuvant and other adjuvants used in research and veterinary applications hâve toxieitîes which limit their potential use in human vaccines. Chemically defîned préparations such as muramyl dipeptide, monophosphoryllipid A, phospholipid conjugales such as those described by Goodman-Snitkoff et al., J Immunol. 147:410-415 (1991), encapsulation of the protein within a proteoliposome as described by Miller et al., J Exp. Med. 176:1739-1744 (1992), and encapsulation of the protein in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) may also be used.
The composition may be packaged in a single dosage form for immunization by parentéral (e.g., intramuscular, intradermal or subcutaneous) administration or orifice administration, e.g., perlingual (e.g., oral), intragastric, mucosal including intraoral, intraanal, intravagînal, and the like administration. And again, the effective dosage and route of administration are determined by the nature of the composition, by the nature of the expression product, by expression level if recombinant CMV is directly used, and by known factors, such as breed or species, âge, sex, weight, condition and nature of host, as well as LD50 and other screening procedures which are known and do not require undue expérimentation. Dosages of expressed product may range from a few to a few hundred micrograms, e.g., 5 to 500 pg. The CMV vector may be admînistered in any suitable amount to achieve expression at these dosage levels. In nonlimiting examples: CMV vectors may be admînistered in an amount of at least 1Û2 pfu; thus, CMV vectors may be admînistered în at least this amount; or in a range from about IO2 pfu to about 107 pfu. Other suitable carriers or diluents may be water or a buffered saline, with or without a preservative. The CMV vector may be lyophilized for resuspension at the tîme of administration or may be in solution. About may mean within 1%, 5%, 10% or 20% of a defîned value.
It should be understood that the proteins and the nucieic acids encoding them of the présent disclosure may differ from the exact sequences illustrated and described herein. Thus, the disclosure contemplâtes délétions, additions, truncations, and substitutions to the sequences shown, so long as the sequences fonction in accordance with the methods of the disclosure. In this regard, substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four familles: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, and histidine; (3) nonpolar-- alanine, valîne, leucine, îsoleucine, proline, phenylalanine, méthionine, and tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonably predictable that an isolated replacement of leucine with îsoleucine or
-49valine, or vice versa; an aspartate with a glutamate or vice versa; a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structural 1 y related amino acid, will not hâve a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the proteins described but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the scope of the disclosure.
The nucléotide sequences of the présent disclosure may be codon optimized, for example the codons may be optimized for use in human cells. For example, any viral or bacterial sequence may be so altered. Many viruses, including HIV and other lentiviruses, use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the tumor antigen may be achieved as described în Andreetal., J Virol. 72:1497-1503,1998.
Nucléotide sequences encoding functionally and/or antigenically équivalent variants and dérivatives of the CMV vectors and the glycoproteins included therein are contemplated. These functionally équivalent variants, dérivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that resuit in conservative substitutions of amino acid residues, one or a few amino acid délétions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide. Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan. In some embodiments, the variants hâve 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 the antigen, epîtope, immunogen, peptide or polypeptide of interest.
Sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithme. A nonlimiting example of a mathematical algorîthm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.
Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the
-50ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a ΡΑΜΙ20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying régions of local sequence similarity and alignment îs the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 198S; S5: 2444-2448.
Advantageous for use according to the présent dîsclosure is the WU -BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 exécutable programs for several UNIX platforms may be downloaded. This program is based on WUBLAST version 1.4, which in tum is based on the public domain NCB1 -BLAST version 1.4 (Altschul & Gîsh, 1996, Local alignment statistics, Doolittïe ed., Methods in Enzymology 266: 460- 480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States, 1993; Nature Genetîcs 3: 266-272; Karlin & Altschul, 1993; Proc. Natl. Acad. Sci. USA 90; 5873-5877; ail of which are incorporated by reference herein).
The various recombinant nucléotide sequences and antîbodies and/or antigens of the dîsclosure are made using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, Molecular Cloning: A Laboratory Manual, second édition (Sambrook et al. 1989).
Any vector that allows expression of the viruses of the présent dîsclosure may be used in accordance with the présent dîsclosure. In certain embodiments, the disclosed viruses may be used in vitro (such as using cell-free expression Systems) and/or in cultured cells grown în vitro in order to produce the encoded heterologous antigen (e.g., tumor virus antigens, HIV antigens, tumor antigens, and antîbodies) which may then be used for various applications such as in the production of proteinaceous vaccines. For such applications, any vector that allows expression of the virus in vitro and/or in cultured cells may be used.
For the disclosed tumor antigens to be expressed, the protein coding sequence of the tumor antigen should be operably linked to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be operably linked when they are covalenily linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The nucleic acid control sequence may be any nucleic acid element, such as, but not limited to pro mot ers, enhancers, IRES, introns, and other éléments described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. The term promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the disclosure lead to the expression of the encoded protein. The expression of the transgenes of the présent disclosure may be under the control of a constitutive promoter or of an inducible promoter, which initiâtes transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tétracycline, hormones such as ecdysone, or heavy metals. The promoter may also be spécifie 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 may be used for expression ofthe transgenes of the disclosure. For example, suitable promoters and/or enhancers may be selected from the Eukaryotîc Promoter Database (EPDB).
The vectors used in accordance with the présent disclosure may contain a suitable gene regulatory région, such as a promoter or enhancer, such that the antigens of the disclosure may be expressed.
The CMV vectors described herein may contain mutations that may prevent host to host spread, thereby rendering the virus unable to infect immunocompromised or other subjects that could face complications as a resuit of CMV infection. The CMV vectors described herein may also contain mutations that resuit in the présentation of immunodominant and nonimmunodominant epitopes as well as non-canonical MHC restriction. However, mutations in the CMV vectors described herein do not affect the ability of the vector to reinfect a subj ect that has been previously infected with CMV. Such CMV mutations are described in, for example, US Patent Publications 2013-013676S; 2010-0142S23; 2014-014I03S; and PCT application publication WO 2014/13S209, ail of which are incorporated by reference herein.
The disclosed CMV vectors may be admînistered in vivo, for example where the aim is to produce an immunogenic response, including a CD8+ immune response, including an immune response characterized by a high percentage of the CD8+ T cell response being restricted by MHC-E, MHC-II, or MHC-I (or a homolog or ortholog thereof). For example, in some examples it may be desired to use the disclosed CMV vectors in a laboratory animal, such as rhésus macaques for preclinical testing of immunogenic compositions and vaccines using RhCMV. In other examples, it will be désirable to use the disclosed CMV vectors in human subjects, such as în clînical trials and for actual clinical use ofthe immunogenic compositions using HCMV.
For such in vivo applications the disclosed CMV vectors are admînistered as a component of an immunogenic composition further comprising a pharmaceuticaliy acceptable carrier. In some embodiments, the immunogenic compositions of the disclosure are useful to stimulate an immune response against the heterologous antigen, including a tumor antigen, a
-52tumor virus antigen, or a host self-antigen and may be used as one or more components of a prophylactic or therapeutic vaccine against tumor antigens, tumor virus antigens, or host self antigens for the prévention, amelioration or treatment of cancer. The nucleic acids and vectors of tbe disclosure are particularly useful for providing genetic vaccines, i.e., vaccines for delivering the nucleic acids encoding the antigens of the disclosure to a subject, such as a human, such that the antigens are then expressed in the subject to elicit an immune response.
Immunization schedules (or regimens) are well known for animais (including humans) and may be readily determined for the particular subject and immunogenic composition. Hence, the immunogens may be administered one or more times to the subject. Preferably, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to severai weeks, and is often 2, 4, 6, or 8 weeks. For humans, the interval is typically from 2 to 6 weeks. In a particularly advantageous embodiment of the présent disclosure, the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, weeks, 66 weeks, 68 weeks, or 70 weeks. The immunization régimes typically hâve from 1 to administrations of the immunogenic composition, but may hâve as few as one or two or four. The methods of inducing an immune response may also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunization may supplément the initial immunization protocol. The présent methods also include a variety of prime-boost regimens. In these methods, one or more priming îmmunizations are followed by one or more boosting immunizations. The actual immunogenic composition may be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens may also be varied. For example, if an expression vector is used for the priming and boosting steps, it may either be of the same or different type (e.g., DNA or bacterial or viral expression vector). One useful prime-boost regîmen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization. It should also be readily apparent to one of skill in the art that there are severai permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the disclosure to provide priming and boosting regimens. CMV vectors may be used repeatedly while expressing different antigens derived from different pathogens.
-53Examples
EXAMPLE 1 : PROTECTION AGAINST SIV BY INDUCTION OF MHC-E RESTRICTED CD8+ T CELLS
In several studies it was demonstrated that strain 68-1 derived RhCMV vectors expressing SIV antigens control and ultimately eliminate infection by highly pathogénie SIVmac239 (Hansen 2019. A live-attenuated RhCMV/SIV vaccine shows long-term efficacy against heterologous SIV challenge. Science Translational Medicine 1 l:eaaw2607; Hansen 2013. Immune clearance of highly pathogenic SIV infection. Nature 502:100-4). This protection correlated with the ability of strain 68-1 RhCMV to elicit MHC-II and MHC-E restricted CD8+ T cells Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science 351:714-20; Hansen. Cytomégalovirus Vectors Violate CD8+ T Cell Epitope Récognition Paradigms. Science 340:1237874-1237874) However, it was not known whether MHC-Π and/or MHC-E restricted CD8+ T cells are necessary for this protection.
Therefore, the ability to specifically program CD8+ T cells that are restricted exclus!vely by MHC-E or MHC-II enabled the examination of whether MHC-E or MHC-II restricted CD8+ T cells are responsible for the unique protection against SIVmac239. Four rhésus macaque (RM) cohorts were inoculated with different 68-1 RhCMV strains as described below.
Cohort 1: Nine RM were inoculated with three 68-1 RhCMV MHC-E only vectors each carrying three récognition sites for mirl 26 in the 3' untranslated région of the essentîal genes R1H08 (UL79) and R11156 (IE2) and expressing (one insert per vector) the SIV antigens SlVgag, SIVretanef (fusion of rev, tat, and nef), and the 5' segment of SIVpol, respectively.
Cohort 2: 15 RM were inoculated with three 68-1 RhCMV MHC-II only vectors deleted for Rh67 (UL40) and expressing (one insert per vector) the SIV antigens SlVgag, SIVretanef (fusion of rev, tat, and nef) and the 5' segment of SIVpol, respectively.
Cohort 3: 12 RM were inoculated with three 68-1 RhCMV MHC-Π only vectors each carrying three récognition sites for mirl42 in the 3' untranslated région of the essential genes Rhl08 (UL79) and Rhl56 (IE2) and expressing (one insert per vector) the SIV antigens SlVgag, SIVretanef (fusion of rev, tat, and nef) and the 5' segment of SIVpol, respectively.
Cohort 4: (control cohort) 15 RM were inoculated with three 68-1 RhCMV vectors expressing (one insert per vector) the SIV an tî gens SlVgag, SIVretanef (fusion of rev, tat, and nef) and the 5' segment of SIVpol, respectively.
-54Average frequencies of CD4+ or CD8+ T cells respondîng to SlV-antigen derived peptide pools were quantifted. T cell frequencies were determined in peripheral blood mononuclear cells (PBMC) at the indicated time points b y intracellular cytokine staîning for IFNy or TNFa in the presence of pools of overlapping (by HA) 15mer peptides representing the S1V antigens. Each of the RM developed robust CD4+ and CDS+ T cell responses to each of the SIV antigens (Fig. 1).
Next, the MHC-restriction of the SlVgag-specific CD8+ T cell responses was analyzed. SlVgag-specific CD8+ T cell responses in PBMC obtained from three RM in each of the indicated cohorts were measured in the presence of individual peptides. MHC restriction was determined by blocking with the anti-pan-MHC-I mAb W6/32, the MHC-E blocking peptide VL9, and the MHC-II blocking peptide CLIP. Whereas ail peptide responses in cohort 1 animais were blocked by VL9 peptide, peptide responses in cohorts 1 and 3 were blocked by CLIP peptide (Fig. 2). Thus, CD8+ T cells in cohort i are exclusively restricted by MHC-E whereas CD8+ T cells in cohorts 2 and 3 are exclusively restricted by MHC-II. CD8+ T cell responses in cohort 4 animais (not shown) are restricted by both MHC-II and MHC-E as previously reported (Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatîbility complex E. Science 351:714-20; Hansen 2013. Cytomégalovirus Vectors Violate CD8+ T Cell Epitope Récognition Paradigms. Science 340:1237874-1237874).
To détermine whether MHC-E or MHC-II-restricted CD8+ T cells were responsible for protection, cohorts 1, 2, and 3 were challenged by repeated, limiting dose intra-rectal inoculation of SIVmac239. RM were challenged weekly untîl the first plasma viral load (pvl) or SlVvif responses were detected (with the start of infection designated as the previous challenge). Since the vaccine vectors do not express SlVvif, the development of de novo SlVvif responses are proof for infection in the absence of détectable SIV plasma viral load. RM were considered controllers (white boxes) if plasma viremia was never observed or became undetectable within 2 weeks of the initial positive pvl and was then maintained below threshold for at least 4 of the subséquent 5 weeks, in contrast to non-controllers (black boxes), which once infected, manifested continuous viremia with a typical peak and plateau pattern.
Ail animais in cohorts 2 and 3 developed systemic, progressive SIV viremia suggesting that MHC-II restricted CD8+ T cells were unable to provide protection against SIVmac239 infection (Fig. 3). In contrast, 6/9 (67%) of cohort 1 animais vaccinated with 68-1 RhCMV/SIV/miR126 vectors stringently controlled infection with SIVmac239. These data demonstrate that MHC-E restricted CD8+ T cell responses provided protection against highly virulent SIV.
li was previously demonstrated that strain 68-1 derived RhCMV vectors elicit CD8+ T cell responses that display an unusually high epitope density (= number of peptides recognized by T cells within a given antigen) (Hansen. 2013. Cytomégalovirus Vectors Violate CD8+ T Cell Epitope Récognition Paradigms. Science 340:1237874-1237874). It was further shown that some of these MHC-E and MHC-II epitopes, so called supertopes, are recognized in every animal (Hansen 2016. Broadly targeted CD8(+) T cell responses restricted by major histocompatibility complex E. Science 351:714-20).. Supertopes hâve not been described for classical epitopes, presented by MHC-I molécules, and thus represent a unique feature of CMV-based vectors. To détermine whether supertopes alone could account for the protection observed with MHC-E only RhCMV vectors described above, an artificial fusion protein was generated consisting of supertope sequences from individual SIV antigens (Table 1, 15mer and minimal supertope peptide sequences are underlined).
Table I. MHC-E supertopes in each SIV antigen
Antigen MHC Restriction Peptide Sequence SEQ ID NO:
SIVrev MHC-E RRWRRR WQQLLALADRIYS FPDP 1
SlVtat MHC-E TSSASNKPISNRTRHCQPE 2
SlVnef MHC-E ISMRRSRPSGDLRQRLLRA 3
SlVnef MHC-E EKLAYRKQNMDDIDEEDDD 4
SlVnef MHC-E AQTSQWDDPWGEVLAWKFD 5
SlVnef MHC-E YVRYPEEFGSKSGLSEEEV 6
SIVpol MHC-E GGIGGFINTKEYKNVEIEVLGKR 7
SIVpol MHC-E NTPTFAIKKKDKNKWRMLIDFRE 8
SIVpol MHC-E WMGYELWPTKWKLQKIELP 9
SlVgag MHC-E LGLQKCVRMYNPTNILDVK 10
SlVgag MHC-E YMQLGKQQREKQRESREKPYKEV 11
The sequence of the artificial fusion protein îs as follows (HA-epitope tag is underlined): MRRWRRRWQQLLALADRIYSFPDPTSSASNKPISNRTRHCQPEISMRRSRPSGDLRQRLL RAEKLAYRKQNMDDIDEEDDDAQTSQWDDPWGEVLAWKFDYVRYPEEFGSKSGLSEE EVGGIGGFINTKEYKNVEIVLGKRNTPTFA1KKKDKNKWRMLIDFREWMGYELWPTKW KLQKIELPLGLQKCVRMYNPTNILDVKYMQLGKQQREKQRESREKPYKEVYPYDVPDY AD (SEQ ID NO: 12). Immunoblotting was performed to demonstrate the expression of the SIV supertope fusion construct by probing with an anti-H A antibody (Fig. 4).
The SIV MHC-E supertope fusion protein was inserted into 68-1 RhCMV containing mirl26 targeting sites with the goal to focus the CD8+ T cell responses onto a small set of MHC20580
-56E restricted epitopes. The resulting construct was inoculated into 8 RM (Cohort 5). T cell frequencies were determined in peripheral blood mononuclear cells (PBMC) at the indicated time points by intracellular cytokine staining for IFNy or TNFa in the presence of pools of individual 15mer peptides representing the SIV supertopes (Fig, 5). CD8+ T cells were responsive to SlV-antigen derived peptides (Fig. 5A). The CD8+ T cells were responsîve to the MHC-E-restricted supertopes Gag69 and Gag 120 but not other MHC-E-restricted Gag epitopes that are commonly recognized by CD8+ T cells from RM immunized with 68-1 RhCMV/gag vectors expressing whole SlVgag inserts (Fig. 5B). These results show that ail animais elicited SlV-specific CD8+ T cell responses that were excïusively directed to supertopes,
To détermine whether MHC-E supertope-restricted CD8+ T cells would be able to replicate the protection observed with MHC-E-only vectors, cohort 5 was challenged by repeated low dose intra-rectal inoculation of SIVmac239 as described above. RM were challenged weekly until the first plasma viral load (pvl) or SIVvif responses were detected (with the start of infection désignâted as the previous challenge). RM were considered controllers (boxes) if pvl became undetectable within 2 weeks ofthe initial positive pvl and was then maintained below threshold for at least 4 ofthe subséquent 5 weeks, in contrast to noncontrollers (black boxes), which once infected, manifested continuons viremia with a typîcal peak and plateau pattern.
Importantly, 5/7 (71%) of animais vaccinated with a single 68-1 RhCMV/SIV/miR126 vector expressing the supertope-fusion protein controlled infection with SIVmac239 (Fig. 6). These data indicate that CD8+ T cells spécifie for MHC-E supertopes are responsible for protection against highly pathogenic SIV.
In order to design HIV-based supertope antigens, HIV supertopes were mapped b y insertîng HIV antigens into 68-1 RhCMV and inoculating RM. Table 2 contains a list of HIV supertopes identified. The optimal minimal peptide sequence is underlîned.
Table 2. List of HIV supertopes.
Antigen Peptide MHCRestriction Peptide Sequence (15mer) (optimal minimal peptide sequence is underlîned) SEQ ID NOs: (full peptide sequence, optimal minimal peptide sequence)
HIVgag 4 E LDAWEKIRLRPGGKK 13, 14
29 E KKAOQAAADTGNSSQ 15, 16
36 E QMVHQAISPRTLNAW 17, 18
47 E NTMLNTVGGHQAAMQ 19, 20
61 E STLQEQIGWMTNN PP 21,22
69 E IVRMYSPVSILDIRQ 23,24
119 E OKQEPIDKELYPLAS 25,26
HIVpol 14 E SFSFPQITLWQRPLV 27
28 E VRQYDQILIEICGKK 28
81 E EPFRKQNPDIVIYQL 29
148 E YVDGAANRETK.LGKA 30
180 E EEHEKYSNWRAMAS 31
HIVnef 28 E ILDLWVYHTQGYFPD 32
EXAMPLE 2: EXPRESSION OF UL18 PREVENTS THE INDUCTION OF MHC-E AND MHC-II RESTRICTED CD8+ T CELLS
To déterminé the impact of UL18 on the ability of strain 68-1 RhCMV veetors to elicit MHC-II and MHC-E restricted CD8+ T cell responses two RhCMV constructs were generated:
Construct 1: 68-1 RhCMV containing an expression cassette for the 5' fragment of SIVpol under control of the EF1 a promoter in RhCMV gene Rh211 as a vector backbone. UL18 was inserted by replacing the gene Rhl3.1, thus UL18 would be expressed instead of Rhl3.1. The UL18 sequence inserted corresponds to UL18 ofthe HCMV TR isolate.
Construct 2: 68-1 RhCMV in which the gene Rhl07 (homolog of HCMV UL78) was replaced with a fusion protein of SIV rev, tat, and nef (SIVrtn) as a vector backbone. UL18 was inserted by replacing the gene Rhl3.1.
5x106 plaque forming units (PFU) of construct 1 were inoculated into three RhCMV seropositive RM and the same amount of construct 2 was inoculated into two RhCMVseropositive RM on day 0. For control, RM were inoculated with 68-1 RhCMV expressing SlVgag under control of the EF1 a promoter.
On day 7, day 14, and biweekly after that, PBMC were isolated from two RM and the CD8+ T cell responses to the SIV antigens elicited by construct 1, 2, or control were measured by intracellular cytokine staining (ICS) for IFNy and TNFa using overlapping 15mer peptide pools that covered SIVpol, SIVrtn or SlVgag, respectively. To specifically detect CD8+ T cells that recognized peptides in the context of MHC-E or MHC-II it was advantageous that supertopes within each SIV antigen are shared by ail animais (Hansen Science 2013, Hansen Science 2016). Thus, each of the supertope peptides was tested individually by ICS in PBMC of the respective RM.
Frequencies of CD8+ T cells responding to SIV antigen peptide pools thus representing total antigen-specific responses in two animais from each group were analyzed (Fig. 7A).
-58Frequencies of CD8+ T cells responding to MHC-E restricted supertopes and MHC-II restricted supertopes were also analyzed for the same two animais (Figs. 7B, 7C).
Ail animais developed CD8+ T cell responses to the SIV antigen expressed by the RhCMV vector used for inoculation. However, supertope responses were only observed for 68-1 RhCMV/SIVgag whereas both vectors expressing UL18 did not elicit T cells recognizing supertopes. These results thus indicated that UL18 prevented the induction of MHC-E and MHC-Π restricted CD8+ T cells.
Next, MHC-restriction mapping was performed to further déterminé which MHC molécules were responsible for the elicitation of SlVpol-specifîc responses in the three animais that received UL18 expressing 68-1 RhCMV/SIVpoi. SIVpol-spécifie CD8+ T cell responses in PB MC obtained from three RM inoculated with construct 1 were measured in the presence of individual peptides. The CD8+ T cell responses to individual peptides within SIVpol were measured in the presence of spécifie reagents that either block MHC-I, MHC-II, or MHC-E présentation (MHC-I and MHC-E is blocked with antibody W6/32, MHC-II is blocked with HLA-DR-specific antibody and CLIP peptide, MHC-E is blocked with VL9 peptide).
The results shown in Figure 8 reveal that the stimulation of CD8+ T cells by each individual peptide was înhibîted by pan-MHC-I inhibitory antibody W6/32, but not by MHC-E spécifie peptide VL9 or MHC-II spécifie antibodies and CLIP peptide. Thus, ail CD8+ T cell epitopes are restricted b y MHC-I. In contrast, CD8+ T cells from animais inoculated with 68-1 RhCMV expressing SIV antigens recognize ail peptides in the context of MHC-II or MHC-E (Hansen Science 2013, Hansen Science 2016).
These results show that UL18 reprogrammed the CD8+ T cell response most likely by preventing the induction of MHC-11 and MHC-E restricted CD8+ T cells. UL18 is known to engage the host inhibitory receptor LIR-1 (Yang Z, Bjorkman PJ. 2008. Structure of UL18, a peptide-binding viral MHC mimic, bound to a host inhibitory receptor. Proc Natl Acad Sci U S A 105:10095-100; Chapman TL, Heikeman AP, Bjorkman PJ. 1999. The inhibitory receptor L1R-1 uses a common binding interaction to recognize class I MHC molécules and the viral homolog UL18. Immunity 11:603-13). A possible mechanism for this reprogramming is, therefore, that by engaging inhibitory Leukocyte inhibitory receptors (LIRs) on T cells, UL18 prevents the direct priming of CD8+ T cells by 68-1 RhCMV (direct priming refers to T cells being primed b y infect ed cells). In the absence of direct priming, CD8+ T cells are elicited by cross-priming, i.e., îndîrectly by non-infected cells (e.g., dendritic cells) presenting antigen obtained from infected cells. Up to now, UL 18 has not been implicated in preventing T cell priming. These results are thus unexpected and unprecedented.
-59To détermine whether the interaction with the inhibitory receptor LIR1 is responsible for the ability of LJL18 to prevent the induction of MHC-II and MHC-E restricted CD8+ T cells the coding région of UL18 in construct 1 described above was mutated so that the amino acid aspartate at position 196 in the alpha-3 domain would be replaced with serine (D196S). Prevîous structural studies hâve shown that this aspartate is învolved in binding of UL18 to LIR1 (Yang Z, Bjorkman PJ. 2008. Structure of ULI8, a peptide-binding viral MHC mimic, bound to a host inhibitory receptor. Proc Natl Acad Sci U S A 105:10095-100). Moreover, this residue is conserved in ail LIR1 binding HLA-molécules but absent in HLA-like molécules that do not bind LIR 1. The D196S mutant of UL18 was inserted into 68-1 RhCMV expressing SIVpol and the resulting construct was inoculated into two RM. On day 91, PBMC were isolated and the CD8+ T cell responses to the SIVpol was measured by ICS for IFNy and TNFa using overlapping 15mer peptide pools that covered SIVpol or the SIVpol MHC-E supertope peptide Pol41 (GFINTKEYKNVEIEV; SEQ TD NO: 33) or MHC-II supertope Pol90 (LPQGWKGSPAIFQYT; SEQ ID NO: 34). In contrast to animais inoculated with 68-1 RhCMV expressing intact UL18 (Figure 9A), T cell responses to both SIVpol supertopes were observed in animais inoculated with 6S-1 RhCMV expressing the D196S mutant of UL18 (Figure 9B). These results thus indicated that UL18 needs to engage the LIR1 receptor to prevent induction of MHC-E and MHC-II restricted CD8+ T cells.
UL18 is considered to play a rôle in the évasion of NK cells (Prod'homme 2007. The human cytomégalovirus MHC class I homolog LJL18 inhibits LIR-1+ but activâtes LIR-1- NK cells. J hnmunol 178:4473-81). Since NK cell évasion can be crucial for vector fnnction (Sturgill 2016. Natural Killer Cell Evasion Is Essential for Infection by Rhésus Cytomégalovirus. PLoS Pathog 12:eI005868) it was conceivable that délétion of UL18 from HCMV-based vectors would prevent their ability to elicit immune responses to heterologous antigens. To détermine whether UL18-deIeted HCMV is able to elicit T cell responses to an inserted antigen, UL18 was replaced with an HIV antigen, thereby deleting UL18 and using the endogenous ULI8 promoter to drive expression of a HIVgag/nef/ pol fusion protein. Additionally, the genes UL128,UL130, UL146, and UL147 were also deleted from the UL18-deleted vector, since the products of these genes were previously shown to inhibit MHC-E and MHC-II restricted CD8+ T cell responses (U.S. Patent No. 10,532,099). As vector backbone we used HCMV TR3 (Caposio. 2019. Characterization of a live-attenuated HCMV-based vaccine platform. S ci en ti fie Reports 9: 19236). Expression of the HIV fusion protein in the resulting viral vector, (HCMV TR3 AUL18/HIVfusionAUL128-130AUL146-147) was confirmed by immunoblot of human fibroblasts (Figure 10).
-60The UL18-deleted HCMV vector was also inoculated into a RM and the immune response to the HIV antigens was determined in P BMC by ICS on day 56 post-inoculation. As shown in Figure 11, the vector elicited CD8+ T cell responses to HIVgag, HIVnef and HIVpol in RM as demonstrated by using overlapping peptide pools comprising each of these antigens.
Therefore, we conclude that HCMV vectors lacking UL18 retain their ability to elicit T cell responses to heterologous antigens.

Claims (28)

1, A recombinant HCMV vector comprising a nucleic acid sequence encoding heterologous antigen, wherein the recombinant HCMV vector does not express UL18, UL128, UL130, UL146, andUL147.
2, The recombinant HCMV vector of claim 1, wherein the recombinant HCMV vector does not express a UL18 protein, UL128 protein, UL130 protein, UL146 protein, and UL147 protein, or orthologs thereof, due to the presence of one or more mutations in the nucleic acid sequences encoding UL18, UL128, UL130, ULI46, and UL147.
3. The recombinant HCMV vector of claim 2, wherein the mutations in the nucleic acid sequences encoding UL18, UL128, UL130, UL146, and UL147 are selected from the group consisting of point mutations, frameshift mutations, truncation mutations, and délétion of ail of the nucleic acid sequence encoding the viral protein.
4. The recombinant HCMV vector of any one of claims 1-3, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding UL40, or an ortholog thereof.
5. The recombinant HCMV vector of any one of claims 1 -4, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding US28, or an ortholog thereof.
6. The recombinant HCMV vector of any one of claims 1-5, wherein the recombinant HCMV vector does not express UL82 (pp7I), or an ortholog thereof.
7. The recombinant HCMV vector of any one of claims 1-6, wherein the recombinant HCMV vector does not express US11, or an ortholog thereof.
8. The recombinant HCMV vector of any one of claims 1-6, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding a mîcroRNA
- 62 (miRNA) récognition element (MRE), wherein the MRE contains a target site for a miRNA expressed in endothélial cells.
9. The recombinant HCMV vector of claim 8, wherein the miRNA expressed in endothélial cells is miR126, miR-126-3p, miR-130a, miR-210, miR-221/222, miR-378, miR-296, or miR-328.
10. The recombinant HCMV vector of any one of claims 1-7, wherein the recombinant HCMV vector further comprises a nucleic acid sequence encoding a MRE, wherein the MRE contains a target site for a miRNA expressed in myeloid cells.
11. The recombinant HCMV vector of claim 10, wherein the miRNA expressed in myeloid cells is miR-142-3p, miR-223, miR-27a, miR-652, miR-155, miR-146a, miR-132, miR21, or miR-125.
12. The recombinant HCMV vector of any one of claims 1-11, wherein the heterologous antigen is a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen.
13. The recombinant HCMV vector of claim 12, wherein the pathogen-specific antigen is human immunodeficiency virus (HIV), herpes simplex virus type 1, herpes simplex virus type 2, hepatitis B virus, hepatitis C virus, papillomavîrus, Plasmodium parasites, or Mycobacterium tuberculosis.
14. The recombinant HCMV vector of any one of claims 1-6 and 8-9, wherein the pathogenspecific antigen is an MHC-E supertope.
15. The recombinant HCMV vector of claim 14, wherein pathogen-specific antigen comprises a HIV epitope.
16. The recombinant HCMV vector of claim 15, wherein the HIV epitope is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to LDAWEKIRLRPGGKK (SEQ ID NO: 13); DAWEKIRLR (SEQ ID NO: 14); KKAQQAAADTGNSSQ (SEQ ID
-63 NO: 15); KAQQAAADT (SEQ ID NO: 16); QMVHQAISPRTLNAW (SEQ ID NO: 17); HQAISPRTL (SEQ ID NO: 18); NTMLNTVGGHQAAMQ (SEQ ID NO: 19);
VGGHQAAMQ (SEQ ID NO: 20); STLQEQIGWMTNNPP (SEQ ID NO: 21); STLQEQIGW (SEQ ID NO: 22); IVRMYSPVSILDIRQ (SEQ ID NO: 23);
RMYSPVSIL (SEQ ID NO: 24); QKQEPIDK.ELYPLAS (SEQ ID NO: 25); KQEPIDKEL (SEQ ID NO: 26); SFSFPQITLWQRPLV (SEQ ID NO: 27); VRQYDQILIEICGKK (SEQ ID NO: 28); EPFRKQNPDIVIYQL (SEQ ID NO: 29); YVDGAANRETKLGKA (SEQ ID NO: 30); EEHEKYSNWRAMAS (SEQ ID NO: 31); or 1LDLWVYHTQGYFPD (SEQ ID NO: 32).
17. The recombinant HCMV vector of claim 12, wherein the tumor antigen is related to acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, rénal cell carcînoma (RCC), or germ cell tumors.
18. The recombinant HCMV vector of claim 12, wherein the host self-antigen is an antigen derived from the variable région of a T cell receptor (TCR) or an antigen derived from the variable région of a B cell receptor.
19. A pharmaceutical composition comprising the recombinant HCMV vector of any one of claims 1-18 and a pharmaceutically acceptable carrier.
20. An îmmunogenic composition comprising the recombinant HCMV vector of any one of claims 1-18 and a pharmaceutically acceptable carrier.
21. Use of the recombinant HCMV vector of any one of claims 1-18 in the manufacture of a médicament for use in generating an immune response in a subject.
22. The recombinant HCMV vector of any of claims 1-18 for use in generating an immune response in a subject.
23. Use of the recombinant HCMV vector of claim 12 or claim 17 în the manufacture of a médicament for use in treating or preventing cancer in a subject.
24. The recombinant HCMV vector of claim 12 or claim 17 for use in treating or preventing cancer in a subject.
5
25. Use of the recombinant HCMV vector of any one of claims 12-16 in the manufacture of a médicament for use in treating or preventing a pathogenîc infection in a subject.
26. The recombinant HCMV vector of any one of claims 12-16 for use in treating or preventing a pathogenîc infection in a subject.
27. Use of the recombinant HCMV vector of claim 12 or claim 18 in the manufacture of a 10 médicament for use in treating an autoimmune disease or disorder in a subject.
28. The recombinant HCMV vector of claim 12 or claim 18 for use in treating an autoimmune disease or disorder in a subject.
OA1202200063 2019-08-20 2020-08-19 Modulation of T cell responses by UL18 of human cytomegalovirus. OA20580A (en)

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