WO2016130693A1 - Methods and compositions useful in generating non canonical cd8+ t cell responses - Google Patents

Methods and compositions useful in generating non canonical cd8+ t cell responses Download PDF

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Publication number
WO2016130693A1
WO2016130693A1 PCT/US2016/017373 US2016017373W WO2016130693A1 WO 2016130693 A1 WO2016130693 A1 WO 2016130693A1 US 2016017373 W US2016017373 W US 2016017373W WO 2016130693 A1 WO2016130693 A1 WO 2016130693A1
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WIPO (PCT)
Prior art keywords
antigen
mhc
cells
cmv vector
vector
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PCT/US2016/017373
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English (en)
French (fr)
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WO2016130693A9 (en
Inventor
Klaus Frueh
Louis Picker
Scott Hansen
Jonah SACHA
Daniel MALOULI
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Oregon Health and Science University
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Oregon Health and Science University
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Priority to US15/549,814 priority Critical patent/US20180298404A1/en
Priority to CN201680014682.9A priority patent/CN108064304A/zh
Priority to EP16749813.8A priority patent/EP3256595A4/en
Priority to JP2017560883A priority patent/JP6816031B2/ja
Priority to EP21205026.4A priority patent/EP4036239A1/en
Priority to CA2976245A priority patent/CA2976245A1/en
Priority to MX2017010027A priority patent/MX2017010027A/es
Priority to KR1020177025544A priority patent/KR20170136512A/ko
Priority to AU2016219317A priority patent/AU2016219317A1/en
Priority to HK18106974.7A priority patent/HK1247638A1/zh
Priority to SG11201706454VA priority patent/SG11201706454VA/en
Application filed by Oregon Health and Science University filed Critical Oregon Health and Science University
Priority to CN202111437865.0A priority patent/CN114317611A/zh
Priority to EA201791806A priority patent/EA201791806A1/ru
Priority to HK18113013.6A priority patent/HK1254041A1/zh
Priority to BR112017017092A priority patent/BR112017017092A2/pt
Publication of WO2016130693A1 publication Critical patent/WO2016130693A1/en
Publication of WO2016130693A9 publication Critical patent/WO2016130693A9/en
Priority to IL253935A priority patent/IL253935B/en
Anticipated expiration legal-status Critical
Priority to US16/738,178 priority patent/US11091779B2/en
Ceased legal-status Critical Current

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Definitions

  • the field is the use of CMV vectors in immunization. More specifically, the field is the generation of CD8 + immune responses characterized by non-canonical MHC restriction. Still more specifically, the field is the generation of T cells, including CD8 + with receptors that are restricted by MHC-E.
  • Rhesus Cytomegalovirus (RhCMV) vaccine vectors expressing Simian Immunodeficiency Virus (SIV) proteins provide protection from pathogenic SIV (Hansen, S.G. et ai, Not Med 15, 293 (2009); Hansen, S.G. et ai, Nature 473, 523 (2011); both of which are incorporated by reference herein).
  • SIV Simian Immunodeficiency Virus
  • RhCMV-protected macaques exhibited periodic low-level "blips" of viremia, CD4 + memory T cell depletion was not observed, SIV-specific antibody responses did not develop, and subsequently, over time, viral nucleic acid became barely quantifiable while replication competent virus disappeared from the tissues of protected animals. These events did not occur in spontaneous SIV elite controllers and DNA prime/Ad5 boost vaccinated controllers (Hansen, S.G. et ai, Nature 502, 100 (2013); incorporated by reference herein).
  • the method involves administering to the subject an effective amount of a CMV vector.
  • the CMV vector comprises a first nucleic acid that encodes the at least one heterologous antigen, a second nucleic acid sequence that encodes at least one active UL40 protein, or a homolog or ortholog thereof, and a third nucleic acid sequence that encodes at least one US28 protein, or a homolog or ortholog thereof.
  • the CMV vector does not express an active UL128 protein, or an ortholog thereof, and does not express an active UL130 protein, or an ortholog thereof, and at least 10% of the CD8 + T cells generated by the vector are restricted by MHC-E or a homolog thereof.
  • the third nucleic acid sequence encodes two through five active US28 proteins, or homologs or orthologs thereof.
  • the heterologous antigen can be any antigen, including a pathogen-specific antigen derived from, for example, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), herpes simplex virus, hepatitis B or C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
  • HAV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • herpes simplex virus hepatitis B or C virus
  • papillomavirus Plasmodium parasites
  • Plasmodium parasites Plasmodium parasites
  • Mycobacterium tuberculosis Mycobacterium tuberculosis
  • the heterologous antigen can be a tumor antigen including, for example, a tumor antigen related to acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplasia syndrome, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, malignant melanoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, renal cell carcinoma ( CC), and germ cell tumors.
  • the heterologous antigen can be a tissue-specific antigen or a host self-antigen including, for example, an antigen derived from the variable region of a T cell receptor (TCR), an antigen derived from the variable region of a B cell receptor, a sperm antigen, or an egg antigen.
  • the vector does not encode (1) an active UL40 protein (or an ortholog thereof) and/or an active US28 protein (or an ortholog thereof), (2) an active UL128 protein (or an ortholog thereof), and (3) an active UL130 protein (or an ortholog thereof), giving rise to MHC-II "supertope" restricted CD8 + T cells but not HLA-E restricted CD8 + T cells.
  • a human or animal cytomegalovirus vector that includes a first nucleic acid sequence that encodes (1) at least one heterologous protein antigen, (2) a second nucleic acid sequence that encodes at least one active UL40 protein, or a homolog or ortholog thereof, and (3) a third nucleic acid sequence that encodes at least one active US28 protein, or a homolog or ortholog thereof.
  • the vector does not express active UL128 and UL130 proteins, or orthologs thereof.
  • the third nucleic acid sequence encodes two through five active US28 proteins, or homologs or orthologs thereof.
  • a human or animal cytomegalovirus vector that (1) does not express an active UL128 protein (or an ortholog thereof), (2) does not express an active UL130 protein (or an ortholog thereof), and (3) does not express an active UL40 protein (or an ortholog thereof) and/or an active US28 protein (or an ortholog thereof).
  • Also disclosed herein is a method of generating CD8 + T cells that recognize MHC-E- peptide complexes.
  • This method involves administering to a first subject a CMV vector that encodes (1) at least one heterologous antigen, (2) at least one active UL40 protein (or an ortholog or homolog thereof), and (3) at least one active US28 gene (or an ortholog or homolog thereof), in an amount effective to generate a set of CD8 + T cells that recognize MHC-E/peptide complexes.
  • the CMV vector does not encode active UL128 and UL130 proteins, or orthologs thereof.
  • the CMV vector encodes two through five active US28 proteins or orthologs or homologs thereof.
  • the heterologous antigen can be any antigen, including a pathogen-specific antigen, a tumor antigen, a self-antigen, or a tissue-specific antigen.
  • the self-antigen is an antigen derived from the variable region of a T or B cell receptor.
  • this method may further comprise identifying a first CD8 + T cell receptor from the set of CD8 + T cells, wherein the first CD8 + TCR recognizes a MHC- E/heterologous antigen-derived peptide complex.
  • the first CD8 + T cell receptor is identified by DNA or RNA sequencing.
  • this method may further comprise transfecting one or more T cells isolated from the first subject or a second subject 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 second CD8 + T cell receptor, wherein the second CD8 + T cell receptor comprises CDR3a and CDR3 of the first CD8 + T cell receptor, thereby generating one or more transfected CD8 + T cells that recognize a MHC-E/heterologous antigen-derived peptide complex.
  • this method may further comprise administering the transfected CD8 + T cells to the first or second subject to treat a disease, such as cancer, a pathogenic infection, or an autoimmune disease or disorder. In some embodiments, this method may further comprise administering the transfected CD8 + T cells to the first or second subject to induce an autoimmune response to a self-antigen or a tissue-specific antigen.
  • a disease such as cancer, a pathogenic infection, or an autoimmune disease or disorder.
  • this method may further comprise administering the transfected CD8 + T cells to the first or second subject to induce an autoimmune response to a self-antigen or a tissue-specific antigen.
  • 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 CMV vector in an amount effective to generate a set of CD8 + T cells that recognize MHC-E/peptide complexes, wherein the CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen, a second nucleic acid sequence encoding at least one active UL40 protein, or an ortholog or homolog thereof, and a third nucleic acid sequence encoding at least one active US28 protein, or an ortholog or homolog thereof, and wherein the CMV vector does not express active UL128 and UL130 proteins, or orthologs thereof; (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
  • the heterologous antigen can be any antigen, including a pathogen-specific antigen or a tumor antigen.
  • the third nucleic acid sequence of the CMV vector encodes two through five active US28 proteins, or orthologs or homologs thereof.
  • methods of treating a disease such as cancer, a pathogenic infection, or an autoimmune disease or disorder, the method comprising administering the transfected CD8 + T cell that recognizes MHC-E-peptide complexes to the first or second subject.
  • Figure 1A is a set of flow cytometry plots of peripheral blood mononuclear cells (PBMCs) from a strain 68-1 RhCMV/gag-vaccinated macaque (either Rh22034 or Rh21826).
  • PBMCs peripheral blood mononuclear cells
  • RhCMV strain 68-1 does not express gene products from the Rhl3, Rh60, Rhl57.5 and 157.4 (HCMV RL11, UL36, UL128 and UL130, respectively) open reading frames.
  • PBMCs were evaluated for peptide-specific CD8 + T cell recognition using flow cytometric intracellular cytokine staining (ICS) to detect I FN- ⁇ and/or TNF-a production (response frequencies of CD8 + T cells shown in each quadrant) following incubation with the indicated antigen presenting cells that were pulsed with the peptide shown.
  • ICS flow cytometric intracellular cytokine staining
  • the parental, MHC-I negative K562 cells were used as negative controls and also transfected to express the MHC-I molecule indicated, while autologous B-lymphoblastoid cell lines (BLCL) were used as the positive control.
  • Figure IB is a set of flow cytometry plots (left panel) and a bar graph (right panel) of CD8 + T cells in PBMC from a strain 68-1 RhCMV/gag vector-vaccinated macaque (Rh22034 and Rh21826) showing IFN- ⁇ and/or TNF-a production (response frequencies of CD8 + T cells shown in each quadrant) following incubation with antigen presenting cells (autologous BLCL or K562 transfectant expressing only Mamu-E) that were pulsed with Gag 273 _ 287 (SIVmac239 Gag 15-mer #69).
  • antigen presenting cells autologous BLCL or K562 transfectant expressing only Mamu-E
  • the antigen presenting cells were incubated with the Gag 15-mer indicated along with either no additional peptide (no blocking) or in the presence of the Mamu-E binding peptide Rh67 8 _i6 VL9 (Rh67 VL9) or the Mamu-A*002:01 binding peptide Gag 7 i- 7 9 GY9 (SIVgag GY9).
  • the right panel is a comparison of peptide blocking conditions on IFN- ⁇ and/or TNF-a production from CD8 + T cells from four strain 68-1 RhCMV/gag vector-vaccinated macaques incubated with autologous BLCL or a Mamu-E transfectant pulsed with Gag 273 _ 287 (SIVmac239 Gag 15-mer #69). Data are normalized to the response observed with no peptide blocking.
  • Figure 1C is a set of flow cytometry plots (left panel) and a bar graph (right panel) of
  • antigen presenting cells autologous BLCL or K562 transfectant expressing only Mamu-E
  • the antigen presenting cells were incubated with the Gag 15-mer indicated along with either no additional peptide (no blocking) or in the presence of the Mamu-E binding peptide Rh67 8 _i6 VL9 (Rh67 VL9) or the Mamu-A*002:01 binding peptide Gag 7 i_ 79 GY9 (SIVgag GY9).
  • the right panel is a comparison of peptide blocking conditions on IFN- ⁇ and/or TNF-a production from CD8 + T cells from four strain 68-1 RhCMV/gag vector-vaccinated macaques incubated with autologous BLCL or a Mamu-E transfectant pulsed with Gag 477 _ 4 gi (SIVmac239 Gag 15-mer #120). Data are normalized to the response observed with no peptide blocking.
  • Rh60, Rhl57.5, and Rhl57.4 HCMV UL36, UL128, and UL130, respectively
  • MHC-I mAb W6/32
  • MHC-E Rh67 VL9
  • MHC-II mAb G46-6
  • blockade and classified as MHC-I blocked (boxes with white fill), fully MHC-E blocked (boxes with grey fill), partially MHC-E blocked (boxes with horizontal hatch fill), MHC-II blocked (boxes with black fill), or indeterminate (boxes with vertical hatch fill).
  • the minimal number of independent MHC-E blocked epitopes potentially contained within these reactive peptides in each macaque is designated at right (see Methods).
  • macaques 22063 and 22624 were vaccinated with BAC-derived RhCMV/gag while macaques 21826, 22034, 22436, and 22607 were vaccinated with non-BAC derived RhCMVgag(L).
  • Figure 2B is a set of flow cytometry plots of CD8 + T cells in PBMC from a MamuAl*001:01+ strain 68-1 RhCMV/gag vector-vaccinated macaque showing IFN- ⁇ and/or TNF-a production (response frequencies of CD8 + T cells shown in each quadrant) following incubation with antigen presenting cells (autologous BLCL or K562 transfectant expressing only MamuAl*001:01 or Mamu-E) that were pulsed with the Gag 69 - 8 3 (Gag #18) peptide alone (no blocking), or in the presence of MHC-E-binding Rh67 8 -i6 VL9 or Mamu-A*01-binding Gagi 8 i-i89 CM9 peptide.
  • antigen presenting cells autologous BLCL or K562 transfectant expressing only MamuAl*001:01 or Mamu-E
  • Figure 2C is a set of flow cytometry plots of CD8 + T cells in PBMC from a MamuAl*001:01- strain 68-1 RhCMV/gag vector-vaccinated macaque incubated with antigen presenting cells as described for Figure 2B.
  • Figure 3A is a set of two plots showing bulk surface MHC-I (measured by mAb W6/32) on the surface of productively SIV-infected (CD4 " Gag p27 + ) or uninfected (CD4 + Gagp27 " ) CD4+ T cell targets. Representative flow cytometry plots are shown on the left panel while the right panel depicts the mean fluorescent intensity (MFI) of bulk MHC-I staining in SIV infected versus uninfected CD4 + T cells derived from a total of 16 unrelated rhesus macaques.
  • MFI mean fluorescent intensity
  • Figure 3B is a set of two plots showing MHC-E (measured by mAb 4D12) on the surface of productively SIV-infected (CD4 " Gag p27 + ) or uninfected (CD4 + Gagp27 ⁇ ) CD4 + T cell targets. Representative flow cytometry plots are shown on the left panel while the right panel depicts the MFI of MHC-E staining in SIV infected versus uninfected CD4 + T cells derived from a total of 16 unrelated rhesus macaques.
  • Figure 3C is a plot showing the phenotype of MHC-E restricted CD8 + T cells responding to Gag 2 73-287 (69) or Gag 4 77- 4 gi (120) peptide stimulation. Percentages were calculated by examining the number of IFN- ⁇ and/or TNF-a producing cells expressing each marker.
  • Figure 4A is a set of representative flow cytometry plots of CD8 + T cells isolated from macaques vaccinated with either strain 68-1 RhCMV/gag, MVA/gag, strain 68-1.2 RhCMV/gag, or infected with SIV, showing IFN- ⁇ and/or TNF-a production from CD8 + T cells following incubation with autologous SIVmac239-infected CD4 + T cells alone (no block), or in the presence of the MHC-II binding Class ll-associated invariant chain peptide (CLIP) plus the pan- MHC-I blocking mAb W6/32 (W6/32 + CLIP), or Rh67 8 _i 6 VL9 plus CLIP (VL9 + CLIP).
  • CLIP MHC-II binding Class ll-associated invariant chain peptide
  • Figure 4C is a set of flow cytometry plots illustrating the recognition of SIV-infected cells by CD8 + T cell lines (CL) specific for either the MHC-E restricted Gag 47 7_ 4 gi Gag #120 epitope (top row) or the Mamu-A*001:01 restricted Gagi 8 i-i89 CM9 epitope (bottom row). CLs were incubated with uninfected or SIV-infected CD4+ T cells (from Rh22607) in the presence of the blocking conditions indicated.
  • Figure 5 shows the percentage of CD8 + T cells in PBMC from a rhesus macaque inoculated with a Rh67 (UL40)-deleted 68-1 RhCMV expressing SIVgag showing IFN- ⁇ and/or TNF-a production following incubation with overlapping peptides corresponding to SIVgag at the indicated time points.
  • the central panel shows that CD8 + T cells in PBMC from the same animal do not respond to the Mamu-E-restricted peptides Gag 2 7 3 - 28 7 (Gag69) or Gag 47 7_ 4 gi (Gagl20).
  • the right panel shows the percentage of CD8 + T cells in PBMC from the same animal responding to MHC-II restricted peptides (Gag53 and Gag73).
  • MHC-II restricted peptides correspond to so-called supertopes, i.e. these peptides are presented by many different MHC-II alleles and hence elicit responses in most animals.
  • Figure 7A is a set of plots showing surface staining of MHC-II, MHC-la, MHC-E, or MHC-F by cell lines transfected with single Mamu-D molecules.
  • Figure 7B is a table showing genotyping of the indicated rhesus macaque (RM) individuals. Individuals were Mamu-A, -B, and -E genotyped by Roche/454 pyrosequencing. Grey shading indicates alleles selected for MHC-I transfectant generation. Where multiple alleles are listed, the bolded allele was produced.
  • RM rhesus macaque
  • Figure 7C is a set of two plots wherein one MHC-la or MHC-lb allele was transfected into a parental (MHC-I negative) cell line (.221 cells or K562, respectively).
  • a parental (MHC-I negative) cell line (.221 cells or K562, respectively).
  • Cells were stained with a cross-reactive human MHC-I monoclonal antibody (W6/32) for 15 minutes at room temperature to assess MHC-I expression.
  • Cells were washed once with IX PBS supplemented with 10% fetal bovine serum, fixed with 2% paraformaldehyde, collected on a LSRII flow cytometer, and analyzed with FlowJo.
  • MHC-l-expressing B-lymphoblastoid cells (BLCL) served as a positive control, while the MHC-I negative parental cell lines were used as a negative control.
  • BLCL B-lymphoblastoid cells
  • Figure 8A is a set of plots showing representative flow data of a restriction assay from Rh22607 for Gag 120.
  • Figure 8B is a table showing PBMC from the 4 indicated RM (#s 21826, 22436, 22034, and 22607; Mamu-I alleles shown in Fig. 7B) were incubated with autologous B lymphoblastoid cells (BLCL), MHC-l-null .221 or K562 cells, or the indicated single Mamu-I transfectants pulsed with the indicated SIVgag peptides and were then analyzed for CD8 + T cell responses by flow cytometric ICS (see Fig. 1).
  • BLCL autologous B lymphoblastoid cells
  • MHC-l-null .221 or K562 cells or the indicated single Mamu-I transfectants pulsed with the indicated SIVgag peptides and were then analyzed for CD8 + T cell responses by flow cytometric ICS
  • Figure 9 is a set of flow cytometry plots of MHC-I vs. MHC-E blockade studies.
  • Figure 10A is a set of flow cytometry plots showing PBMC from strain 68-1 RhCMV/gag vector-vaccinated macaques were stimulated with Gag 2 7 3 - 2 87 (SIVmac239 Gag 15-mer #69) and flow cytometric ICS was performed.
  • CD8 + T cells responding to these MHC-E bound Gag peptides were identified via IFN- ⁇ and TNF-a and then compared against the remaining cells in PBMC for expression of the markers indicated. Numbers in black indicate the overall percentage of cells in PBMC that are positive for the marker indicated, while the numbers in gray indicate the percentage of IFN- ⁇ and TNF-a producing cells that are positive.
  • Figure 10B is a set of flow cytometry plots showing PBMC from strain 68-1 RhCMV/gag vector-vaccinated macaques were stimulated with Gag 4 77_ 4 gi (SIVmac239 Gag 15-mer #120) and flow cytometric ICS was performed.
  • CD8 + T cells responding to these MHC-E bound Gag peptides were identified via IFN- ⁇ and TNF-a and then compared against the remaining cells in PBMC for expression of the markers indicated. Numbers in black indicate the overall percentage of cells in PBMC that are positive for the marker indicated, while the numbers in gray indicate the percentage of IFN- ⁇ and TNF-a producing cells that are positive.
  • Figure 11 collectively shows MHC restriction of strain 68-1 RhCMV/SIVgag-elicited CD8 +
  • Figure 11A is a set of plots showing results from flow cytometric intra-cellular cytokine staining (ICS) analysis of PBMC from a representative strain 68-1 RhCMV/SIVgag-vaccinated macaque (Rh22034; of 4 similarly analyzed).
  • PBMC from vaccinated macaques were stimulated with the indicated 15mer peptide epitopes pulsed onto the surface of the indicated MHC-I transfectants or control cells with CD8 + T cell recognition determined by detection of IFN- ⁇ and/or TNF-a production by flow cytometric ICS assay (response frequencies of gated CD8 + T cells shown in each quadrant).
  • ICS flow cytometric intra-cellular cytokine staining
  • the parental MHC-l-negative .221 and K562 cells were used as negative controls, while autologous B-lymphoblastoid cells (BLCL) were used as the positive control.
  • the MHC-I molecules tested included both those expressed by Rh22034.
  • Figure 11B is a set of plots showing results from flow cytometric ICS analysis of additional macaque and human MHC-E molecules not expressed by Rh22034 similar to that of Figure 11A.
  • Figure 11C is a set of plots showing phenotypic analysis of PBMC from RM treated with the same strain 68-1 RhCMV/SIVgag vector-vaccinated macaque as shown above (representative of 4 similarly analyzed) were stimulated with autologous BLCL pulsed with either SIVgag 2 73-287(69) or SIVgag 4 77- 4 gi(120), and responding CD3 + lymphocytes (IFN- ⁇ and TNF- a-producing; gate shown in left plot) were phenotyped by flow cytometric ICS assay with responding cells and non-responding cells within the designated gates indicated in grey and black, respectively, in each plot (and their relative % within the rectangular regions shown in each plot indicated in the same colors).
  • Figure 11D is a set of plots of the results where single MHC-E transfectants were pre- incubated with canonical MHC-E-binding peptide VMAPRTLLL (VL9) or a control non-MHC-E binding peptide (SIVgag GY9) prior to pulsing with the indicated SIVgag 15mer peptide epitope.
  • VL9 canonical MHC-E-binding peptide
  • SIVgag GY9 a control non-MHC-E binding peptide
  • Flow cytometric ICS assays were conducted as described above using PBMC from strain 68-1 RhCMV/SIVgag-vaccinated macaques, and the following MHC-E transfectants: Mamu-E*02:04 for SIVgag 273 -287(69), SIVgag 38 5- 3 9 9 (97), and SIVgag 433 - 4 47(109) and Mamu-E*02:ll for SIVgag 257 - 27i(65) and SIVgag 477 - 9 i(120).
  • Figure 12 collectively shows that MHC-E restriction is limited to CD8 + T cell responses elicited by ARhl57.5/.4 RhCMV vectors.
  • Peptides resulting in above background CD8 + T cell responses are indicated by a box, with the fill of the box designating MHC restriction as determined by blocking with the anti-pan-MHC-l mAb W6-32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP.
  • MHC-la-, MHC-E-, and MHC-II- restriction was based on >90% response blocking by W6-32 alone (boxes with white fill), W6-32 and VL9 alone (boxes with grey fill), and CLIP alone (boxes with black fill), respectively, with responses not meeting these criteria labeled indeterminate (boxes with vertical hatch fill).
  • the minimal number of independent epitopes in these MHC restriction categories is shown at right for each macaque.
  • Figure 12B is a table showing CD8 + T cell responses to SIVpol and the M. tuberculosis proteins Ag85B, ESAT-6, and RpfA epitope-mapped as described above in macaques vaccinated with strain 68-1 RhCMV vectors expressing these proteins.
  • Figure 12C is a set of plots (right), another set of plots (middle), and a bar graph (right) showing that analysis of SIV-infected CD4 + cell recognition by CD8 + cells isolated from macaques vaccinated with strain 68-1 RhCMV/gag, MVA/gag, strain 68-1.2 RhCMV/gag vectors, or infected with SIV.
  • the flow profiles at left show IFN- ⁇ and TNF-a production following CD8 + T cell incubation with autologous SIVmac239-infected CD4 + T cells alone (no block), or in the presence of the pan-MHC-l-blocking mAb W6/32 plus the MHC-ll-binding CLIP peptide (anti- MHC-I + CLIP), or MHC-E-binding peptide VL9 plus CLIP (VL9 + CLIP). All plots are gated on live, CD3 + , CD8 + cells.
  • the bar graph at right shows the results from all studied macaques.
  • Figure 13A is a plot showing a comparison of the total number of distinct MHC E- (grey) vs.
  • the horizontal bars indicate median values.
  • Figure 13B is a plot showing a comparison of the density (epitope number per 100 amino acids of protein length) of MHC E-restricted epitopes recognized by circulating CD8 + T cells in individual macaques vaccinated with strain 68-1 RhCMV vectors expressing each of the indicated antigens (note: RhCMV IE1 responses were evaluated in CMV naive macaques administered 68-1 RhCMV/gag). The horizontal bars indicate median values for each group.
  • Figure 13C is a bar graph of an analysis of the breadth of MHC-E-restricted SIVgag epitope-specific CD8 + T cell responses across 125 overlapping (11 amino acid overlap), consecutive SIVgag 15mer peptides in 42 strain 68-1 RhCMV/gag vector-vaccinated macaques. Note that 109/125 15mers (87%) were recognized by MHC-E-restricted CD8 + T cells in at least 1 macaque.
  • Figure 13D (left) is a sequence LOGO indicating the frequency of each amino acid in a given position (relative to their background frequency in SIVmac239 Gag; see methods) by the height of the letter, based on 11 optimal, MHC-E-restricted SIVgag 9mer peptide epitopes recognized by CD8+ T cells in strain 68-1 RhCMV vector-vaccinated macaques.
  • the sequence LOGO is colored according to enrichment (letters with grey fill or hatched letters) or underrepresentation (letters with white fill) among 551 peptides eluted from HLA-E in a TAP- deficient setting by Lampen MH et ai, Mol Immunol 53, 126-131 (2013); incorporated by reference herein.
  • Amino acids enriched in the 2 nd and C-terminal anchor positions among the 551 Lampen et al. peptides were rare among our 11 optimal SIVgag peptides (right), while those that were significantly underrepresented were enriched.
  • Figure 14 is a plot showing that the SIVgag 276 -28 4 and SIVgag 482 -49o epitopes are recognized by CD8 + T cells in all strain 68-1 RhCMV/gag-vaccinated rhesus macaques.
  • the CD8 + T cell response to the indicated SIVgag 9mer peptides was determined in 120 strain 68-1 RhCMV/gag-vaccinated RM using flow cytometric ICS, using peptide-specific induction of TNF-a and/or IFN- ⁇ within CD3 + /CD8 + T cells as the response read-out. All macaques manifested detectable responses to these supertopic epitopes after background subtraction. The response frequencies shown have been memory-corrected to reflect the frequency of epitope- responding cells with the CD8 + , CD95 hlgh memory subset. Horizontal bars indicate median values.
  • Figure 15 collectively shows the validation of transfected cell lines expressing single
  • Figure 15A is a table showing the results where four strain 68-1 RhCMV/SIVgag- vaccinated macaques were Mamu-A, -B, and -E genotyped by Roche/454 pyrosequencing. Grey shading indicates alleles selected for MHC-I transfectant generation. Where multiple alleles are listed, a transfectant expressing the bolded allomorph was produced.
  • Figure 15B is a set of two plots showing expression of single MHC-I molecules.
  • MHC-la or MHC-lb alleles were transfected into a parental (MHC-I negative) cell line (.221 cells or K562 cells) and stained with pan-MHC-l monoclonal antibody (W6/32).
  • MHC-l-expressing B- lymphoblastoid cells (BLCL) served as a positive control, while the MHC-l-negative parental cell lines were used as negative controls.
  • Figure 16A and 16B collectively show a comprehensive analysis of the MHC-la and MHC- lb specificity of RhCMV/SIVgag-induced CD8 + T cell response in 4 macaques.
  • Figure 16A is a set of plots showing representative flow cytometric ICS profiles of MHC restriction analysis of the SIVgag 43 3- 447 (109) response using PBMC from Rh22034.
  • the TNF-a vs. IFN- ⁇ flow profiles shown were gated on CD3 + , CD8 + lymphocytes, with the fraction of cells in each quadrant indicated in the figure.
  • Figure 16B is a table showing PBMC from the 4 indicated macaques (MHC-typing shown in Fig.
  • Figure 17 is a table showing that classical MHC-la allomorphs capable of presenting
  • SIVgag peptides to strain 68-1 RhCMV/SIVgag-elicited CD8 + T cells are not the restricting MHC alleles for these T cell responses.
  • a cohort of 20 strain 68-1 RhCMV/SIVgag vector-vaccinated macaques were MHC-typed for the presence of Mamu-Al*001:01 and -Al*002:01 and tested for CD8 + T cell responses specific for SIVgag 6 g-83(18), SIVgagi 2 g-i43(33), and SIVgagig 7 - 2 n(50).
  • Figures 18A and 18B collectively show that strain 68-1 RhCMV/SIVgag-elicited CD8 + T cells recognize peptide in the context of both rhesus macaque and human MHC-E molecules.
  • Figure 18A is a set of plots showing PBMCs from strain 68-1 RhCMV/SIVgag vector- vaccinated macaques [Rh21826: SIVgag 89 -io3(23), SIVgagi 29 _i4 3 (33), SIVgag 257 -27i(65), SIVgag 473 _ 487(119); Rh22034: SIVgag 61 _ 75 (16), SIVgag 69 _ 83 (18), SIVgag 271 _ 287 (69), SIVgag 38 5- 399 (97), SIVgag 477 _ 49 i(120); Rh22436: SIVgagi 97 _ 2 n(30), SIVgagi 97 _ 2 n(50)] were evaluated for peptide-specific CD8 + T cell recognition using flow cytometric ICS to detect IFN- ⁇ and/or TNF-a production (response frequencies of CD8 + T cells shown
  • Figure 18B is an amino acid alignment of the al and a.2 regions of human and rhesus macaque MHC-E molecules expressed by transfectants represented in Fig. 18A, with the key B and F pocket residues indicated with grey shading. All of the B and F pocket residues interacting with bound peptide are conserved between HLA-E*01:03, Mamu-E*02:04, and Mamu-E*02:ll, while substitutions exist in these residues in Mamu-E*02:20, the most disparate of the MHC-E molecules studied here. Despite harboring substitutions in both B and F pocket residues compared to the other allomorphs, Mamu-E*02:20 is able to bind and present the identical peptides.
  • Figure 19 is a plot showing that strain 68-1 RhCMV/SIVgag-elicited, supertope-specific CD8+ T cells exhibit a conventional CD8a + T cell phenotype.
  • the figure summarizes the phenotypic analysis of MHC-E-restricted CD8 + T cells responding to SIVgag 273 _ 287 (69) or SIVgag477_ 49 i(120) peptide stimulation in four 68-1 RhCMV/SIVgag-vaccinated macaques (Rh21826, Rh22034, Rh22436, Rh22607).
  • the figure shows the percentages of peptide- responding CD3 + T cells (IFN-v + and TNF-a + ) that express the designated phenotypes (see flow cytometric profiles in Fig. 11C).
  • Figure 20A is a set of plots of single MHC-E transfectants pre-incubated with canonical MHC-E-binding peptide VMAPRTLLL (VL9) or control peptide prior to pulsing with the indicated SIVgag 15-mer peptide epitope.
  • VMAPRTLLL VMAPRTLLL
  • SIVgag 15-mer peptide epitope Flow cytometric ICS was conducted as described for Fig.
  • Rh21826 for SIVgag 89 - io 3 (23), SIVgagi 29 _i 43 (33), SIVgagi 97 - 2 ii(50), and SIVgag 473 - 487 (H9) responses; Rh22034 for SIVgag 6 i- 75 (16) and SIVgag 6 g- 83 (18) responses; Rh22436 for the SIVgagn 7 _i 3 i(30) response.
  • MHC-E transfectants were utilized: Mamu-E*02:04 for the SIVgag 69 _ 83 (18) and SIVgag 89 _io 3 (23) responses; Mamu-E*02:ll for the SIVgag 6 i_ 75 (16), SIVgagii 7 _ m (30), SIVgagi 29 _ i 43 (33), SIVgagi 97 _ 2 n(50), and SIVgag 473 _ 487 (119) responses.
  • Figure 20B is a plot where the indicated antigen-presenting cells were pre-incubated with increasing concentrations of VL9 prior to pulse with the SIVgag 4 77_ 4 gi(120) SIVgag 15-mer or optimal Mamu-Al*001:01-restricted Gag-CM9 or Tat-SL8 peptides. These antigen-presenting cells were then incubated with the indicated effectors for flow cytometric ICS analysis, as described for Fig. 20A.
  • Rh22436 is a 68-1 RhCMV/SIVgag-vaccinated RM, while Rh27002 is SIV- infected.
  • VL9 peptide progressively block the ability of MHC-E-expressing antigen-presenting cells to activate SIVgag 47 7- 4 gi(120)-specific CD8 + T cells from a strain 68-1 RhCMV/gag vector-vaccinated macaque, but have no effect on conventionally MHC-la-restricted CD8+ T cells specific for Gag-CM9 or Tat-SL8.
  • Figure 21 shows formal truncation analysis for 8 additional MHC-E-restricted 15 peptide epitopes using peptide-specific CD8 + T cell expression of TNF-a and/or IFN- ⁇ by flow cytometric ICS as response readout.
  • CD8 + T cell responses to amino terminal and carboxy terminal truncations of the parent 15mer were initially determined to define optimal peptide length and the amino- and carboxy-termini of the core epitope (top panel, with grey shading indicating the terminal amino acids of the most stimulatory amino- and carboxy-terminal-truncated peptides).
  • the optimal 9mer implied by this truncation approach was then confirmed by analysis of the 7 consecutive 9mers that make up each 15mer (bottom panel). The 9mers shaded in grey in each of the bottom panels represent the optimal epitope for each parent 15mer.
  • FIG. 22A and 22B collectively show dose response of MHC-E-restricted CD8 + T cells to optimal 9mers pulsed on human and rhesus macaques MHC-E transfectants.
  • Mamu-E*02:04, Mamu-E*02:20 and HLA-E*01:03 transfectants were pulsed with the indicated concentration of the optimal SIVgag 9mer peptide epitopes SIVgag 476 _ 484 , SIVgag 25 9- 2 67, SIVgag 276 _ 284 , or SIVgag 482 _ 490 (see fig.
  • Figure 22A is a set of plots showing a representative analysis of the dose response to SIVgag 476 -484 in Rh22607.
  • Figure 22B is a set of plots showing the dose response (mean ⁇ SEM response frequencies) for CD8 + T cells responding to SIVgag 476 - 4 84, SIVgag 25 g-267, SIVgag 276 -284, SIVgag 482 - 4 9 0 with response frequencies normalized to the response observed with the transfectant pulsed with 10 ⁇ peptide dose.
  • Figure 23 is a chart of genomic differences between RhCMV vector strains 68-1, 68-1.2 and ARhl57.4/.5 68-1.2.
  • the Rhl57.5 (UL128), Rhl57.4 (UL130) and Rhl57.6 (UL131A) genes are encoded on the 2 nd strand in reverse orientation.
  • RhCMV 68-1 acquired distinctive fibroblast adaptations.
  • the Rhl57.5 (UL128) ORF and most of exon 2 of the Rhl57.4 (UL130) ORF were deleted and the adjacent genomic region inverted, resulting in loss of the pentameric receptor complex that mediates viral entry into non-fibroblasts.
  • RhCMV 68-1 RhCMV Fibroblast adaptation of strain 68-1 RhCMV also resulted in insertion of an additional thymidine in the Rh61/Rh60 (UL36) gene, resulting in a frame shift mutation and a premature stop codon.
  • RhCMV 68-1.2 a functional pentameric complex was restored by insertion of Rhl57.5 (UL128) and exon 2 of Rhl57.4 (UL130) from RhCMV strain 180.92 into RhCMV 68.1 right after the first exon of Rhl57.4 (U L130), and the Rh61/Rh60 (UL36) mutation was reverted to wild type configuration.
  • Rhl57.5/.4 UL128/UL130
  • Rhl57.4 UL130
  • Rhl57.5 and Rhl57.4 were specifically re-deleted from strain 68-1.2 by homologous recombination starting 50 bp upstream of the Rh 157.6 (UL131A) stop codon up to the Rhl57.5 (UL128) stop codon, leaving the Rh61/Rh60 (UL36) repair intact.
  • Figure 24 collectively shows differential utilization of MHC-E vs. MHC-la by CD8 + T cells elicited by strain 68-1 vs. strain 68-1.2 RhCMV/gag vectors.
  • Figure 24A shows representative flow cytometric response profiles (IFN- ⁇ vs. TNF-a on gated CD3 + , CD8 + T cells) of MHC-l-dependent, SIVgag epitope-specific CD8 + T cells elicited by the strain 68-1 (Rhl57.4/.5-deleted) RhCMV/gag vectors, with and without blocking with the pan anti-MHC-l-blocking mAb W6-32 or the MHC-E-blocking VL9 peptide.
  • Figure 24B shows representative flow cytometric response profiles (IFN- ⁇ vs. TNF-a on gated CD3 + , CD8 + T cells) of MHC-l-dependent, SIVgag epitope-specific CD8 + T cells elicited by the strain 68-1.2 (Rhl57.4/.5-intact) RhCMV/gag vectors, with and without blocking with the pan anti-MHC-l-blocking mAb W6-32 or the MHC-E-blocking VL9 peptide (see Fig. 20). Note that the VL9 peptide only blocks all MHC-l-dependent responses elicited by the strain 68-1 RhCMV vector.
  • Figure 25 is a restriction analysis of epitope-specific CD8 + T cell responses elicited by RhCMV/gag vectors (strains 68.1 and 68-1.2), MVA/gag vector, and by controlled SIV infection.
  • CD8 + T cell responses to SIVgag were epitope-mapped using flow cytometric ICS to detect recognition of 125 consecutive 15mer gag peptides (with an 11 amino acid overlap) in additional macaques (over the 6 animals from each group shown in Fig. 12A) vaccinated with the indicated SIVgag expressing viral vectors or infected with SIVmac239 itself (SIVmac239 controller macaques).
  • Peptides resulting in above background CD8 + T cell responses are indicated by a box, with the fill of the box designating MHC restriction, as determined by blocking with the anti-pan-MHC-l mAb W6-32, the MHC-E blocking peptide VL9 and the MHC-II blocking peptide CLIP.
  • MHC-la-, MHC-E-, and MHC-ll-restriction was based on >90% response blocking by W6-32 alone (boxes with white fill), W6-32 and VL9 alone (boxes with grey fill), and CLIP alone (boxes with black fill), respectively, with responses not meeting these criteria labeled indeterminate (boxes with vertical hatch fill).
  • Figure 26 is a chart showing epitope mapping of CD8 + T cell responses to the RhCMV Immediate Early-1 (I El) protein in natural (wild type) RhCMV infection and both primary and secondary infection with the strain 68-1 RhCMV/gag vector.
  • CD8 + T cell responses to RhCMV IE1 were epitope-mapped using flow cytometric ICS to detect recognition of 137 consecutive 15mer IE1 peptides (with an 11 amino acid overlap) in 1) macaques that were naturally infected with wildtype (colony circulating) RhCMV (top panel), 2) RhCMV naive macaques inoculated with the strain 68-1 RhCMV/gag vector (middle panel), and 3) naturally wild type RhCMV- infected macaques that were superinfected with the strain 68-1 RhCMV/gag vector (bottom panel).
  • MHC-la-, MHC-E-, and MHC-ll-restriction was based on >90% response blocking by W6-32 alone (boxes with white fill), W6-32 and VL9 alone (boxes with grey fill), and CLIP alone (boxes with black fill), respectively, with responses not meeting these criteria labeled indeterminate (boxes with vertical hatch fill).
  • FIG. 27A is a validation of the specificity of MHC-E-specific mAb 4D12 in rhesus macaques. Histograms showing surface staining of single MHC-la or MHC-lb transfectants by the pan-MHC-l mAb W6/32 (top row) versus the MHC-E-specific mAb 4D12 (bottom row).
  • Figure 27B shows the surface expression of total MHC-I as determined by staining with mAb W6/32
  • Figure 27C shows the surface expression of total MHC-I, as determined by staining with mAb 4D12, on productively SIV-infected and uninfected CD4 + T cells in the same cultures, with SIV-infected cells recognized by intracellular expression of Gag Ag and CD4 down-regulation (Gag + /CD4 low ), and uninfected cells recognized by lack of Gag reactivity and high levels of surface CD4 expression (Gag ⁇ /CD4 hlgh ).
  • the left panels show representative flow cytometric histograms.
  • the right panels depict the MFI of total MHC-I or specific MHC-E staining in SIV infected versus uninfected CD4 + T cells derived from a total of 16 unrelated macaques. P values were determined by the paired Student's T test.
  • the asterisk (*) indicates the Gag-45 15mer peptide which includes the Mamu-Al*001:01-restricted immunodominant SIVgagi 8 i_i 89 (CM9) epitope.
  • Selection of monkeys for this cohort was largely unbiased with respect to MHC-la allomorphs, except for preferential selection of Mamu-Al*001:01 (expressed by 19 of the 30 macaques), accounting for the high frequency of monkeys responding to the Gag45 15mer.
  • the frequency of monkeys with MHC-l-restricted CD8 + T cells reactive to any of the other Gag 15mers is relatively low (only two 15mers with 40% recognition and none >40%), compared to the MHC-E-restricted CD8 + T cell responses elicited by the strain 68-1 RhCMV/gag vector (19 epitopes with >40% recognition frequency, including 2 universal supertopes; Fig. 3C).
  • all but one of the 125 consecutive SIVgag 15mers are recognized by MHC-la-restricted CD8 + T cells in at least one macaque, and all but 13 SIVgag 15mers are targeted in 2 or more macaques.
  • the MHC-E-restricted CD8 + T cells elicited in 42 macaques by the strain 68-1 RhCMV/gag vector failed to recognize 16 of 125 SIVgag 15mers.
  • the MHC-E-restricted CD8 + T cell responses elicited by strain 68-1 RhCMV vectors are remarkably broad for a functionally monomorphic restricting element, they are not as broad as responses supported by an entire population of polymorphic MHC-la molecules, perhaps accounting for the evolutionary dominance of the MHC-la-restricted antigen presentation system.
  • Figure 29 is a set of three plots.
  • the left panel shows the percentage of CD8 + T cells in PBMC from a rhesus macaque inoculated with a Rh214 to Rh220-deleted 68-lRhCMV expressing SIVgag showing IFN- ⁇ and/or TNF-a production following incubation with overlapping peptides corresponding to SIVgag at the indicated time points.
  • the gene region Rh214 to Rh220 encodes five genes with homology to human cytomegalovirus (HCMV) US28: Rh214, Rh215, Rh216, Rh218, Rh220 (D. Malouli et al., J Viro ⁇ 86, 8959 (2012); incorporated by reference herein).
  • the center panel shows that CD8 + T cells in PBMC from the same animal do not respond to the Mamu-E-restricted peptides Gag 2 7 3 _ 28 7 (Gag69) or Gag 4 77- 4 gi (Gagl20).
  • the right panel shows the percentage of CD8 + T cells in PBMC from the same animal responding to MHC-II restricted peptides (Gag53 and Gag73).
  • the MHC-II peptides correspond to so-called supertopes, i.e. these peptides are presented by many different MHC-II alleles and hence elicit responses in most animals.
  • MHC-I mAb W6/32
  • MHC-E Rh67 VL9
  • MHC-II mAb G46-6 blockade and classified as MHC-I blocked (boxes with white fill), MHC-E blocked (boxes with grey fill), MHC-II blocked (boxes with black fill), or indeterminate (boxes with hatch fill). Note that all peptides are restricted by MHC-II demonstrating the need for Rh214-220 to elicit HLA-E specific CD8 + T cell responses.
  • the present invention provides novel recombinant CMV vectors including, but not limited to, recombinant CMV vectors comprising nucleic acids encoding at least one heterologous protein antigen, at least one active UL40 protein, and at least one active US28 protein, but that do not express active UL128 and UL130 proteins.
  • the present invention also provides recombinant CMV vectors including, but not limited to, recombinant CMV vectors comprising nucleic acids encoding at least one heterologous antigen, but that do not express (1) an active UL40 protein and/or an active US28 protein, (2) an active UL128 protein, and (3) an active UL130 protein.
  • Methods of using the novel recombinant CMV vectors such as methods of generating an immune response to at least one heterologous antigen in a subject, methods of generating CD8 + T cells that recognize MHC-E-peptide complexes, and methods of treating disease, are further provided.
  • 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 administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • HCMV vector comprising an exogenous antigen by any effective route.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Cancer A disease or condition in which abnormal cells divide without control and are able to invade other tissues. Cancer cells may spread to other body parts through the blood and lymphatic systems. Cancer is a term for many diseases. There are more than 100 different types of cancer in humans. Most cancers are named after the organ in which they originate.
  • a cancer that begins in the colon may be called a colon cancer.
  • the characteristics of a cancer are not limited to the organ in which the cancer originates.
  • a cancer cell is any cell derived from any cancer, whether in vitro or in vivo.
  • Cancer also includes malignant tumors characterized by abnormal or uncontrolled cell growth.
  • Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • Metalstatic disease or “metastasis” refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
  • the "pathology” of cancer includes all phenomena that compromise the wellbeing of the subject. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • an effective amount refers to an amount of an agent, such as a CMV vector comprising a heterologous antigen or a transfected CD8+ T cell that recognizes a MHC-E/heterologous antigen-derived peptide complex, that is sufficient 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.
  • an "effective amount” is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease.
  • An effective amount can be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with infectious disease, cancer, or autoimmune disease.
  • a mutation is any difference in a nucleic acid or polypeptide sequence from a normal, consensus or "wild type" sequence.
  • a mutant is any protein or nucleic acid sequence comprising a mutation.
  • 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 (differences in individual nucleotides or amino acids); silent mutations (differences in nucleotides that do not result in an amino acid changes); deletions (differences in which one or more nucleotides or amino acids are missing, up to and including a deletion of the entire coding sequence of a gene); frameshift mutations (differences in which deletion of a number of nucleotides indivisible by 3 results in an alteration of the amino acid sequence.
  • a mutation that results in a difference in an amino acid may also be called an amino acid 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.
  • an "inactivating mutation” is any mutation in a viral gene which finally leads to a reduced function or to a complete loss of function of the viral protein.
  • nucleotide sequences or 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 can be single-stranded, or partially or completely double stranded (duplex).
  • Duplex nucleic acids can be homoduplex or heteroduplex.
  • a recombinant nucleic acid or polypeptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence, for example a CMV vector comprising a heterologous antigen and/or made replication deficient by the mutation of one or more genes.
  • This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant polypeptide can 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).
  • a replication deficient CMV is a virus that once in a host cell, cannot undergo viral replication, or is significantly limited in its ability to replicate its genome and thus produce virions.
  • replication-deficient viruses are dissemination-deficient, i.e. they are capable of replicating their genomes, but unable to infect another cell either because virus particles are not released from the infected cell or because non-infectious viral particles are released.
  • replication-deficient viruses are spread-deficient, i.e. infectious virus is not secreted from the infected host are therefore the virus is unable to spread from host to host.
  • a replication-deficient CMV is a CMV comprising a mutation that results in a lack of expression of one or more genes essential for viral replication ("essential genes") or required for optimal replication (“augmenting genes”).
  • essential genes essential for viral replication
  • augmenting genes essential and augmenting genes have been described in the art (in particular US 2013/0136768, which is incorporated by reference herein) and are disclosed herein.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • 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 can 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 example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • Sequence identity/similarity 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 similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage identity or similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and also function the same or similarly to one another (for example, proteins that serve the same functions in different species or mutant forms of a protein that do not change the function of the protein or the magnitude thereof) can be called "homologs.”
  • 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 can be found at the NCBI web site.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • 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.
  • 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-length alignment 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 alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a protein.
  • the alignment is be 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.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
  • 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 refers to a living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
  • treatment refers to an intervention that ameliorates a sign or symptom of a disease or pathological condition.
  • treatment also refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
  • a prophylactic treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits 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 have developed.
  • CMV vectors capable of repeatedly infecting an organism.
  • the CMV vectors comprise a nucleic acid sequence that encodes a heterologous protein antigen and lack expression of active UL128 and UL130 proteins, or orthologs thereof (homologous genes of CMVs that infect other species).
  • the heterologous antigen can be any antigen, including a pathogen-specific antigen derived from, for example, HIV, SIV, herpes simplex virus, hepatitis B or C virus, papillomavirus, Plasmodium parasites, and Mycobacterium tuberculosis.
  • the heterologous antigen can be a tumor antigen including, for example, a tumor antigen 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, renal cell carcinoma (RCC), and germ cell tumors.
  • the CMV vectors also lack an active UL40 protein (or an ortholog thereof) and/or an active US28 protein (or an ortholog thereof).
  • the heterologous antigen ca n be a tissue-specific antigen or a host self-antigen including, for example, an antigen derived from the variable region of a T cell receptor, an antigen derived from the variable region of a B cell receptor, a sperm antigen, or an egg antigen.
  • the vector does not express an active UL128, UL130, US28 or UL40 protein due to the presence of a mutation in the nucleic acid sequence encoding UL128, UL130, or UL40 (or orthologs thereof).
  • the mutation may be any mutation that results in a lack of expression of active UL128, UL130, US28 or UL40 protein.
  • Such mutations ca n include point mutations, frameshift mutations, deletions of less than all of the sequence that encodes the protein (truncation mutations), or deletions of all of the nucleic acid sequence that encodes the protein, or any other mutations.
  • the vector does not express an active UL128, UL130, US28 or UL40 protein (or an ortholog thereof) due to the presence of a nucleic acid sequence in the vector that comprises an antisense or RNAi sequence (siRNA or miRNA) that inhibits the expression of the UL128, UL130, or UL40 protein (or an ortholog thereof).
  • a nucleic acid sequence in the vector that comprises an antisense or RNAi sequence (siRNA or miRNA) that inhibits the expression of the UL128, UL130, or UL40 protein (or an ortholog thereof).
  • Mutations and/or antisense and/or RNAi can be used in any combination to generate a CMV vector lacking active UL128, UL130, US28 or UL40 (or an ortholog thereof).
  • the CMV vector can comprise additional inactivating mutations known in the art to provide different immune responses, such as an inactivating US11 mutation or an inactivating UL82 (pp71) mutation, or any other inactivating mutation.
  • the CMV vector may also comprise at least one inactivating mutations in one or more viral genes encoding viral proteins known in the art to be essential or augmenting for viral dissemination (i.e. spread from cell to cell) in vivo.
  • Such inactivating mutations may result from point mutations, frameshift mutations, truncation mutations, or a deletion of all of the nucleic acid sequence encoding the viral protein.
  • Inactivating mutations include any mutation in a viral gene which finally leads to a reduced function or to a complete loss of function of the viral protein.
  • the methods involve administering an effective amount of a CMV vector to the subject.
  • the CMV vector is characterized by having a nucleic acid sequence that encodes at least one heterologous antigen and a nucleic acid sequence that does not express an active UL128 protein (or an ortholog thereof), does not express an active UL130 protein (or an ortholog thereof), and expresses at least one active UL40 protein and at least one active US28 protein.
  • the at least one active UL40 protein and the at least one active US28 protein can be orthologs or homologs of UL40 and US28.
  • the CD8 + T cell response elicited by this vector is characterized by having at least 10% of the CD8 + T cells directed against epitopes presented by MHC-E. In further examples, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95% or at least 95% of the CD8 + T cells are restricted by MHC-E. In some embodiments, the CMV vector expresses two to five active US28 proteins or orthologs or homologs thereof.
  • 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-E/heterologous antigen-derived peptide complex.
  • the CD8 + T cell receptor is identified by RNA or DNA sequencing.
  • the CMV vector is characterized by having a nucleic acid sequence that does not express active UL128, UL130, and UL40 proteins, and this vector can be used to elicit CD8 + T cells recognizing MC-II supertopes either together with HLA-E-restricted CD8 + T cells (elicited by one or more additional vectors containing intact US28 and UL40) or without HLA-E restricted CD8 + T cells (elicited by one or more additional vectors lacking a functional UL40 or US28 protein).
  • the CMV vector is characterized by having a nucleic acid sequence that does not express active UL128, UL130, and US28 proteins, and this vector can be used to elicit CD8 + T cells recognizing MC-II supertopes either together with HLA-E-restricted CD8 + T cells (elicited by one or more additional vectors containing intact US28 and UL40) or without HLA-E restricted CD8 + T cells (elicited by one or more additional vectors lacking a functional UL40 or US28 proteins).
  • the CMV vector is characterized by having a nucleic acid sequence that does not express active UL128, UL130, US28, and UL40 proteins, and this vector can be used to elicit CD8 + T cells recognizing MC-II supertopes either together with HLA-E-restricted CD8 + T cells (elicited by one or more additional vectors containing intact US28 and UL40) or without HLA-E restricted CD8 + T cells (elicited by one or more additional vectors lacking a functional UL40 or US28 proteins).
  • Also disclosed herein is a method of generating CD8 + T cells that recognize MHC-E- peptide complexes.
  • This method involves administering to a first subject (or animal) a CMV vector that encodes at least one heterologous antigen and an active UL40 protein, or a homolog or ortholog thereof, to generate a set of CD8 + T cells that recognize MHC-E/peptide complexes.
  • the CMV vector does not encode active UL128 and UL130 proteins, or orthologs thereof, and the heterologous antigen can be any antigen, including a pathogen-specific antigen, a tumor antigen, a tissue-specific antigen, or a host self-antigen.
  • the host self-antigen is an antigen derived from the variable region 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- E/heterologous antigen-derived peptide complex; and 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 CDR3 of the first CD8 + TCR, thereby creating one or more transfected CD8 + T cells that recognize MHC-E-peptide complexes.
  • the one or more CD8 + T cells for transfection with the expression vector may be isolated from the first subject or a second subject.
  • this method may further comprise administering the one or more transfected T cells to the first or second subject to treat a disease such as cancer, a pathogenic infection, or an autoimmune disease or disorder.
  • this method may further comprise administering the one or more transfected T cells to the first or second subject to induce an autoimmune response to a tissue-specific antigen or a host self-antigen.
  • 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 CMV vector in an amount effective to generate a set of CD8 + T cells that recognize MHC-E/peptide complexes, wherein the CMV vector comprises a first nucleic acid sequence encoding at least one heterologous antigen and further comprises a second nucleic acid sequence encoding an active UL40 protein, and wherein the CMV vector does not express active UL128 and UL130 proteins, or orthologs thereof; (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
  • the heterologous antigen can be any antigen, including a pathogen-specific antigen, tissue-specific antigen, a host self-antigen, or a tumor antigen.
  • the first CD8 + T cell receptor is identified by RNA or DNA sequencing. Also disclosed herein are methods of treating a disease, such as cancer, a pathogenic infection, or an autoimmune 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 autoimmune 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 methods involve administering an effective amount of a second CMV vector, the second CMV vector comprising a nucleic acid sequence that encodes a second heterologous antigen to the subject.
  • This second vector can be any CMV vector, including a CMV vector with an active UL128 protein (or a homolog or ortholog thereof)) and/or an active UL130 protein (or a homolog or ortholog thereof).
  • the second CMV vector can comprise a second heterologous antigen.
  • the second heterologous antigen can be any heterologous antigen, including a heterologous antigen identical to the heterologous antigen in the first CMV vector.
  • the second CMV vector can be administered at any time relative to the administration of the first CMV vector including before, concurrently with, or after the administration of the first CMV vector. This includes administration of the second vector any number of months, days, hours, minutes or seconds before or after the first vector.
  • Human or animal CMV vectors when used as expression vectors, are innately nonpathogenic in the selected subjects such as humans.
  • the CMV vectors have been modified to render them non-pathogenic (incapable of host-to-host spread) in the selected subjects.
  • a heterologous antigen can be any protein or fragment thereof that is not derived from CMV, including cancer antigens, pathogen-specific antigens, model antigens (such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin), tissue-specific antigens, host self-antigens, or any other antigen.
  • cancer antigens pathogen-specific antigens
  • model antigens such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin
  • tissue-specific antigens such as lysozyme, keyhole-limpet hemocyanin (KLH), or ovalbumin
  • host self-antigens or any other antigen.
  • Pathogen-specific antigens can be derived from any human or animal pathogen.
  • the pathogen may be a viral pathogen, a bacterial pathogen, or a parasite
  • the antigen may be a protein derived from the viral pathogen, bacterial pathogen, or parasite.
  • the parasite may be an organism or disease caused by an organism.
  • the parasite may be a protozoan organism, a protozoan organism causing a disease, a helminth organism or worm, a disease caused by a helminth organism, an ectoparasite, or a disease caused by an ectoparasite.
  • the antigen can be a protein derived from cancer.
  • the cancers include, but are not limited 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, renal cell carcinoma ( CC), and germ cell tumors.
  • the antigen can be a host self-antigen.
  • Host self-antigens include, but are not limited to, antigens derived from the variable region of a T cell receptor or from the variable region of a B cell receptor.
  • the antigen can be a tissue-specific antigen. Tissue-specific antigens include, but are not limited to, sperm antigens or egg antigens.
  • the CMV vectors disclosed herein can be used as an immunogenic, immunological or vaccine composition containing the recombinant CMV virus or vector, and a pharmaceutically acceptable carrier or diluent.
  • An immunological composition containing 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.
  • An immunogenic composition containing the recombinant CMV virus or vector (or an expression product thereof) likewise elicits a local or systemic immunological response which can, but need not be, protective.
  • a vaccine composition elicits a local or systemic protective response.
  • the terms "immunological composition” and "immunogenic composition” include a "vaccine composition” (as the two former terms can be protective compositions).
  • the CMV vectors disclosed herein can be used in methods of inducing an immunological response in a subject comprising administering to the subject an immunogenic, immunological or vaccine composition comprising the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.
  • subject includes all animals, including non-human primates and humans, while “animal” includes all vertebrate species, except humans; and “vertebrate” includes all vertebrates, including animals (as "animal” is used herein) and humans.
  • a subset of "animal” is "mammal”, which for purposes of this specification includes all mammals, except humans.
  • the CMV vectors disclosed herein can be used in therapeutic compositions containing the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.
  • the CMV vectors disclosed herein can be prepared by inserting DNA comprising a sequence that encodes the heterologous antigen into an essential or non-essential region of the CMV genome.
  • the method can further comprise deleting one or more regions from the CMV genome.
  • the method can comprise in vivo recombination.
  • the method can 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 recombination.
  • the method can also comprise cleaving CMV DNA to obtain cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMV DNA to obtain hybrid CMV-heterologous DNA, transfecting a cell with the hybrid CMV-heterologous DNA, and optionally then recovering CMV modified by the presence of the heterologous DNA.
  • 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-essential region of the CMV genome such that DNA from an essential or nonessential region of CMV is flanking the donor DNA.
  • the heterologous DNA can be inserted into CMV to generate the recombinant CMV in any orientation that yields stable integration of that DNA, and expression thereof, when desired.
  • the DNA encoding the heterologous antigen in the recombinant CMV vector can also include a promoter.
  • the promoter can be from any source such as a herpes virus, including an endogenous CMV promoter, such as a HCMV, RhCMV, murine CMV (MCMV), or other CMV promoter.
  • the promoter can also be a non-viral promoter such as the EFla promoter.
  • the promoter can be a truncated transcriptionally active promoter which comprises a region transactivated with a transactivating protein provided by the virus and the minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived.
  • the promoter can 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 TATA 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).
  • upstream regulatory sequences are composed of the upstream element(s) and enhancer sequence(s).
  • truncated indicates that the full- length promoter is not completely present, i.e., that some portion of the full-length promoter has been removed.
  • the truncated promoter can be derived from a herpesvirus such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE.
  • the promoter can be up to a 40% and even up to a 90% reduction in size, from a full-length promoter, based upon base pairs.
  • the promoter can also be a modified non-viral promoter.
  • HCMV promoters reference is made to U.S. Pat. Nos. 5,168,062 and 5,385,839.
  • transfecting cells with plasmid DNA for expression therefrom reference is made to Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561.
  • direct injection of plasmid DNA as a simple and effective method of vaccination against a variety of infectious diseases reference is made to Science, 259:1745-49, 1993. It is therefore within the scope of this invention that the vector can be used by the direct injection of vector DNA.
  • an expression cassette that can be inserted into a recombinant virus or plasmid comprising the truncated transcriptionally active promoter.
  • the expression cassette can 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 viruses such as CMV.
  • the expression cassette can also include heterologous DNA with respect to the virus or system into which it is inserted; and that DNA can be heterologous DNA as described herein.
  • antigens for use in vaccine or immunological compositions see also Stedman's Medical Dictionary (24th edition, 1982, e.g., definition of vaccine (for a list of antigens used in vaccine formulations); such antigens or epitopes of interest from those antigens can be used.
  • heterologous antigens one skilled in the art can select a heterologous antigen and the coding DNA therefor from the knowledge of the 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 dictionary, without undue experimentation.
  • T epitope mapping One method to determine T epitopes of an antigen involves epitope mapping. Overlapping peptides of the heterologous antigen are generated by oligo-peptide synthesis. The individual peptides are then tested for their ability to bind to an antibody elicited by the native protein or to induce T cell or B cell activation. This approach has been particularly useful in mapping T-cell epitopes since the T cell recognizes short linear peptides complexed with MHC molecules.
  • An immune response to a heterologous 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 histocompatibility complex (MHC)" located on another cell's surface.
  • MHC major histocompatibility complex
  • MHC complexes-class I and class I I There are two classes of MHC complexes-class I and class I I, and each class is made up of many different alleles. Different species, and individual subjects have different types of MHC complex alleles; they are said to have a different MHC type.
  • MHC-E HLA-E in humans, Mamu-E in RM, Qa-lb in mice.
  • the DNA comprising the sequence encoding the heterologous antigen can itself include a promoter for driving expression in the CMV vector or the DNA can be limited to the coding DNA of the heterologous antigen.
  • This construct can be placed in such an orientation relative to an endogenous CMV promoter that it is operably linked to the promoter and is thereby expressed.
  • multiple copies of DNA encoding the heterologous antigen or use of a strong or early promoter or early and late promoter, or any combination thereof, can be done so as to amplify or increase expression.
  • the DNA encoding the heterologous antigen can be suitably positioned with respect to a CMV-endogenous promoter, or those promoters ca n be translocated to be inserted at another location together with the DNA encoding the heterologous antigen.
  • N ucleic acids encoding more than one heterologous antigen can be packaged in the CMV vector.
  • compositions containing the disclosed CMV vectors can be formulated so as to be used in any administration procedure known in the art.
  • Such pharmaceutical compositions can be via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or others).
  • the administration can also be via a mucosal route, e.g., oral, nasal, genital, etc.
  • compositions can be prepared in accordance with standard techniques well known to those skilled in the pharmaceutical arts. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the breed or species, age, sex, weight, and condition of the particular patient, and the route of administration.
  • the compositions ca n be administered alone, or can be co-administered or sequentially administered with other CMV vectors or with other immunological, antigenic or vaccine or therapeutic compositions.
  • Such other compositions can include purified native antigens or epitopes or antigens or epitopes from the expression by a recombinant CMV or another vector system; and are administered taking into account the aforementioned factors.
  • compositions include liquid preparations for orifice, e.g., oral, nasal, anal, genital, e.g., vaginal, etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
  • parenteral subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
  • the recombinant may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • Antigenic, immunological or vaccine compositions typically ca n contain an adjuvant and an amount of the CMV vector or expression product to elicit the desired response.
  • alum aluminum phosphate or aluminum hydroxide
  • Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines.
  • Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. I mmunol.
  • 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.) can also be used.
  • lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, N.H.) can also be used.
  • the composition may be packaged in a single dosage form for immunization by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or orifice administration, e.g., perlingual (e.g., oral), intragastric, mucosal including intraoral, intraanal, intravaginal, and the like administration.
  • parenteral i.e., intramuscular, intradermal or subcutaneous
  • orifice administration e.g., perlingual (e.g., oral), intragastric, mucosal including intraoral, intraanal, intravaginal, and the like administration.
  • 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, age, sex, weight, condition and nature of host, as well as LD 50 and other screening procedures which are known and do not require undue experimentation.
  • Dosages of expressed product can range from a few to a few hundred micrograms, e.g., 5 to 500 ⁇ g.
  • the CMV vector ca n be administered in any suitable amount to achieve expression at these dosage levels.
  • CMV vectors can be administered in an amount of at least 10 2 pfu; thus, CMV vectors can be administered in at least this amount; or in a range from about 10 2 pfu to about 10 7 pfu.
  • Other suitable carriers or diluents can be water or a buffered saline, with or without a preservative.
  • the CMV vector ca n be lyophilized for resuspension at the time of administration or can be in solution. "About" can mean within 1%, 5%, 10% or 20% of a defined value.
  • the proteins and the nucleic acids encoding them of the present invention ca n differ from the exact sequences illustrated and described herein.
  • the invention contemplates deletions, additions, truncations, and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention.
  • substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic-aspartate and glutamate; (2) basic-lysine, arginine, histidine; (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • nucleotide sequences of the present invention can be codon optimized, for example the codons can be optimized for use in human cells. For example, any viral or bacterial sequence can be so altered.
  • viruses including HIV and other lentiviruses
  • Nucleotide sequences encoding functionally and/or antigenically equivalent variants and derivatives of the CMV vectors and the glycoproteins included therein are contemplated. These functionally equivalent variants, derivatives, 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 result in conservative substitutions of amino acid residues, one or a few amino acid deletions 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.
  • the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, 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.
  • sequence identity may be determined using any of a number of mathematical algorithms.
  • a nonlimiting example of a mathematical algorithm 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 ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
  • WU-BLAST Woodington University BLAST
  • WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.wustl.edu/blast/executables.
  • This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et ol. (1990), supra; Gish & States, 1993; Nature Genetics 3: 266-272; Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); all of which are incorporated by reference herein).
  • vectors The nucleotide sequences of the present invention may be inserted into “vectors.”
  • vehicle The term “vector” is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art.
  • vector is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.
  • any vector that allows expression of the viruses of the present invention can be used in accordance with the present invention.
  • the disclosed viruses can be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro in order to produce the encoded heterologous antigen (e.g., pathogen-specific antigens, HIV antigens, tumor antigens, and antibodies) which may then be used for various applications such as in the production of proteinaceous vaccines.
  • the encoded heterologous antigen e.g., pathogen-specific antigens, HIV antigens, tumor antigens, and antibodies
  • any vector that allows expression of the virus in vitro and/or in cultured cells may be used.
  • the protein coding sequence of the heterologous antigen should be "operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a 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.
  • nucleic acid control sequence can be any nucleic acid element, such as, but not limited to promoters, enhancers, I RES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I I and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the transgenes of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter can also be specific to a particular cell-type, tissue or organ. Many suita ble promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention.
  • suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
  • the disclosure relates to a recombinant viral vector expressing a heterologous protein antigen.
  • the antigen is an HIV antigen.
  • the HIV antigens include, but are not limited to, the HIV antigens discussed in U.S. Pub. Nos. 2008/0199493 Al and 2013/0136768 Al, both of which are incorporated by reference herein. HIV, nucleic acid or immunogenic fragments thereof, may be utilized as an HIV protein antigen.
  • the HIV nucleotides discussed in U.S. Pub. Nos. 2008/0199493 Al and 2013/0136768 Al can be used.
  • Any antigen recognized by an HIV antibody can be used as an HIV protein antigen.
  • the protein antigen can also be an SIV antigen.
  • the SIV antigens discussed in U.S. Pub. Nos. 2008/0199493 Al and 2013/0136768 Al can be used.
  • the vectors used in accordance with the present invention can contain a suitable gene regulatory region, such as a promoter or enhancer, such that the antigens of the invention can be expressed.
  • antigens of the invention in vivo in a subject, for example in order to generate an immune response against an HIV-1 antigen and/or protective immunity against HIV-1
  • expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen.
  • it may be desired to express the antibodies and/or antigens in a laboratory animal, such as for pre-clinical testing of the HIV-1 immunogenic compositions and vaccines of the invention.
  • the CMV vectors described herein can contain mutations that can prevent host to host spread, thereby rendering the virus unable to infect immunocompromised or other subjects that could face complications as a result of CMV infection.
  • the CMV vectors described herein can also contain mutations that result in the presentation of immunodominant and non- immunodominant epitopes as well as non-canonical MHC restriction.
  • mutations in the CMV vectors described herein do not affect the ability of the vector to re-infect a subject that has been previously infected with CMV.
  • Such CMV mutations are described in, for example, US Patent Publications 2013-0136768; 2010-0142823; 2014-0141038; and PCT application publication WO 2014/138209, all of which are incorporated by reference herein.
  • the disclosed CMV vectors can be administered 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 Class I I and/or MHC-E (or a homolog or ortholog thereof).
  • 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 Class I I and/or MHC-E (or a homolog or ortholog thereof).
  • MHC Class I and/or MHC-E or a homolog or ortholog thereof.
  • the disclosed CMV vectors are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the invention are useful to stimulate an immune response against the heterologous antigen, including a pathogen-specific antigen and may be used as one or more components of a prophylactic or therapeutic vaccine against HIV-1 for the prevention, amelioration or treatment of AIDS.
  • the nucleic acids and vectors of the invention are particularly useful for providing genetic vaccines, i.e. vaccines for delivering the nucleic acids encoding the antigens of the invention to a subject, such as a human, such that the antigens are then expressed in the subject to elicit an immune response.
  • I mmunization schedules are well known for animals (including humans) and can be readily determined for the particular subject and immunogenic composition.
  • the immunogens can be administered one or more times to the subject.
  • there is a set time interval between separate administrations of the immunogenic composition typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.
  • the interval is typically from 2 to 6 weeks.
  • the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.
  • the immunization regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as one or two or four.
  • the methods of inducing an immune response can also include administration of an adjuvant with the immunogens.
  • booster immunization can supplement the initial immunization protocol.
  • the present methods also include a variety of prime-boost regimens. In these methods, one or more priming immunizations are followed by one or more boosting immunizations.
  • the actual immunogenic composition ca n 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 ca n also be varied.
  • an expression vector is used for the priming and boosting steps, it can either be of the same or different type (e.g., DNA or bacterial or viral expression vector).
  • One useful prime-boost regimen 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 several permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the invention to provide priming and boosting regimens. CMV vectors can be used repeatedly while expressing different antigens derived from different pathogens.
  • Example 1 Induction of MHC-E Restricted CD8 + T cells by Rhesus Cytomegalovirus Vaccine Vectors lacking UL128 and UL130 but containing UL40 and US28 genes.
  • RhCMV/SIV vectors drive an alternate SIV- specific CD8 + T cell response that is completely distinct from the canonical responses engendered by conventional vaccine modalities and even from SIV infection itself (Hansen, S.G. et ai, Science 340, 1237874 (2013), incorporated by reference herein).
  • a panel of MHC-I transfectants expressing either a single "classical” (i.e. polymorphic) MHC-la or non-classical MHC-l b allele was developed from a cohort of four strain 68-1 RhCMV/gag-vaccinated macaques mounting strong RhCMV/gag-induced CD8 + T cell responses (Fig. 7).
  • a previously described MHC restriction assay Hansen et al. Science (2013), supra
  • MHC-E HLA-E in humans, Mamu-E in rhesus macaques, and Qa-l b in mice
  • MHC-E is a highly monomorphic, non-classical MHC-l b molecule expressed in nearly every nucleated cell in the body, with particularly high expression in immune system cells (N. Lee et al., Proc Natl Acad Sci USA 95, 5199 (1998) and S. Coupel et al., Blood 109, 2806 (2007), both of which are incorporated by reference herein).
  • HLA class I alleles J.
  • MHC-E was also identified as the restricting allele for the remaining MHC-I blocked CD8 +
  • MHC-E T cells in RhCMV/gag-vaccinated macaques (Fig. 1A).
  • Fig. 1A T cells in RhCMV/gag-vaccinated macaques.
  • MHC-E repetitively binds and presents only a single 9-mer peptide derived from the leader sequence of MHC-la molecules for presentation to NK cells.
  • MHC-E binds a completely separate set of highly diverse CD8 + T cell epitopes whose binding motif do not match that of the dominant MHC-la leader peptides (Lampen et al., supra and C.C. Oliviera et al., J Exp Med 207, 207 (2010); both of which are incorporated by reference herein).
  • MHC-E ability of MHC-E to disengage the leader peptide and subsequently present an alternate peptide repertoire to CD8 + T cells suggests that the alternate MHC-l-restricted CD8 + T cell response is due largely, if not entirely, to presentation by MHC-E.
  • HLA-E restricted CD8 + T cells have recently been discovered against several human pathogens including CMV (G. Pietra et al., Proc Natl Acad Sci U S A 100, 10896 (2003); incorporated by reference herein); EBV (Jorgensen PB et al., PLoS One 7, e46120 (2012); incorporated by reference herein); Salmonella typhi (R. Salerno-Goncalves, et al., J Immunol 173, 5852 (2004); incorporated by reference herein); and Mycobacterium tuberculosis (A. S. Heinzel et al., J Exp Med 196, 1473 (2002) and SA Joosten et al.
  • CMV CMV
  • EBV Jorgensen PB et al., PLoS One 7, e46120 (2012); incorporated by reference herein
  • Salmonella typhi R. Salerno-Goncalves, et al., J Immunol 17
  • HCMV encodes the glycoprotein UL40 (the RCMV homolog is Rh67), that contains the exact 9-mer peptide (VMAPRTLLL, Rh67 8 -i 6 VL9) derived from classical MHC-la leader sequences.
  • VMAPRTLLL the exact 9-mer peptide
  • Rh67 the exact 9-mer peptide
  • the VL9 peptide specifically binds the MHC-E peptide binding groove with extremely high affinity (P. Tomasec et al., Science 287, 1031 (2000); incorporated by reference herein).
  • Antigen presenting cells were pre-incubated with either the Rh67-derived VL9 peptide to block binding of the Gag 2 7 3 _ 28 7 and Gag 4 77- 4 gi peptides to MHC-E, or with an irrelevant Mamu- A*002:01 (A*02)-binding Gag 7 i_ 79 GY9 peptide.
  • MHC-E The contribution of MHC-E to the overall Gag-specific CD8 + T cell response elicited by RhCMV/gag vectors was compared to that of a conventional Modified Vaccinia Ankara (MVA/gag) vector and native SIV infection.
  • Flow cytometric ICS using blocking monoclonal antibodies (mAbs) specific for MHC-I or MHC-II along with the MHC-E blocking Rh67 8 _i 6 VL9 peptide was used to assess the restriction of each epitope-specific response found in a cohort of 25 macaques: 6 vaccinated with strain 68-1 RhCMV/gag, 9 with strain 68-1.2 RhCMV/gag, 7 with MVA/gag, and 8 SIV-infected macaques.
  • MHC-E-blocked CD8 + T cell responses were found only in macaques vaccinated with strain 68-1 RhCMV/gag.
  • RhCMV 68-1 lost the ability to express gene products from the Rhl3, Rh60, Rhl57.5, and Rhl57.4 (HCMV RL11, UL36, UL128, and UL130, respectively) open reading frames (D. Malouli et al., J Virol 86, 8959 (2012) and WO 2014/138209; incorporated by reference herein).
  • Rh60 can be excluded as the gene mediating this inhibitory effect because it is present in the non-BAC derived RhCMV/gag(L) vector (Hansen, S,G, et al., Science 328, 102 (2010); incorporated by reference herein) which induces MHC-E restricted CD8 + T cells (Fig. 2A).
  • RhCMV/gag-induced, W6/32-blocked CD8 + T cell response recognized peptide in the context of MHC-E
  • these incompletely VL9-blocked peptides were recognized in the context of classical MHC-la alleles, such as Gag 6g _ 83 (Gag 15-mer #18) presented by Mamu-A*001:01 (A*01) in Rh22607 (Fig.
  • MHC-E restricted CD8 + T cells participate in the immune response against SIV.
  • HIV and SIV evade CD8 + T cell recognition by Nef-mediated down regulation of the classical MHC class I molecules from the cell surface (O. Schwartz, et ai, Nat Med 2, 338 (1996); K. L. Collins et ai, Nature 391, 397 (1998); both of which are incorporated by reference herein).
  • Nef is unable to down regulate HLA-E and its surface expression actually increases with HIV infection (J. Natterman et ai, Antivir Ther 10, 95, (2005); incorporated by reference herein).
  • the surface phenotype of these cells was examined, and little, if any, NKG2A/NKG2C expression (Figs. 3C and 10) was found. Furthermore, the MHC-E restricted CD8 + T cells exhibited a conventional CD3 + , CD8a + , TCRv6 ⁇ , NKG2A/C " phenotype suggesting that these T cells recognized MHC-E-bound peptides via CD8- stabilized TCRa interactions.
  • CD8 + T cell recognition of infected cells was fully restored when the MHC-I blocking mAb W6/32 was replaced by the MHC-E blocking Rh67 8 _i 6 VL9 peptide in all cases except for CD8 + T cells isolated from strain 68-1 RhCMV/SIV-vaccinated macaques. This suggests that MHC-E restricted CD8 + T cells recognized SIV infected cells.
  • Gag 4 77- 4 gi Gag #120
  • MHC-E restricted CD8 + T cell line This line was tested for the ability to respond to autologous SIV-infected CD4 T cells.
  • a classically MHC-restricted (Mamu-A*001:01 restricted) Gagi 8 i i89 CM9 CD8 + T cell line was also assessed.
  • Both Gag-specific CD8 + T cell lines specifically recognized SIV-infected cells, and recognition was blocked when targets were pre-incubated with the pan-MHC-l blocking mAb W6/32.
  • MHC-E restricted CD8 + T cell line was unable to recognize SIV-infected cells when targets were pre-incubated with the MHC-E binding peptide Rh67 8 -i6 VL9 (Fig. 4C). Cumulatively, these data indicate that MHC-E restricted CD8 + T cells specifically recognize SIV-derived peptide epitopes on the surface of infected cells.
  • CD8 + T cells that recognize peptide antigen in the context of the non-classical MHC-E molecule.
  • CD8 + T cells represent a new cellular immune response for vaccine development and may be particularly effective given the unique immunobiology of MHC-E.
  • HLA-E expression is up regulated, and the increase of MHC-E expression occurs within the first 24 hours of infection at the portal of viral entry (J. Natterman et al., Antivir Ther 10, 95 (2005) and L. Shang et ai, J Immunol 193, 277 (2014); both of which are incorporated by reference herein).
  • HCMV encodes the glycoprotein UL40 (the RCMV homolog is Rh67), that contains the 9-mer peptide (VMAPRTLLL, Rh67 8 -i 6 VL9) binding the MHC-E peptide binding groove with extremely high affinity (P. Tomasec et al., Science 287, 1031 (2000); incorporated by reference herein). Since the in vitro data indicated that VL9 competes for binding with antigen-derived peptides the possibility that deletion of Rh67 (UL40) from the genome of RhCMV 68-1 would further increase the frequency of HLA-E restricted CD8 + T cells in vivo was considered. To examine this possibility, Rh67 (UL40) was deleted from the 68-1 RhCMV/gag vector.
  • the resulting recombinant virus was inoculated into an animal that was naturally infected with RhCMV.
  • PBMC were obtained, and the frequency of CD8 + T cells responding to total SIVgag as well as MHC-E was measured by intracellular cytokine staining using specific peptides.
  • SIVgag responses to total SIVgag were detectable beginning at day 14 post-inoculation.
  • CD8 + T cells responded to MHC-ll-restricted "supertope" peptides Gag53 and Gag73. Contrary to our expectations however, there was no increase of T cell responses to HLA-E-specific supertopes.
  • RhCMV encodes additional genes that are required for the induction of HLA-E and/or MHC-ll-restricted T cell responses by RhCMV lacking UL128-130, gene regions that are non-essential for growth in vitro were deleted from RhCMV 68-1, and the T cell response upon inoculation of rhesus macaques was monitored. While most deletion mutants did not affect T cell specificities, it was observed that deletion of the gene region Rh214-Rh220 eliminated the ability of RhCMV 68-1 to elicit MHC-E restricted responses, whereas MHC-II restricted CD8 + T cell responses were still observed (Figs. 29 and 30).
  • Rh214-Rh220 region encodes five copies of genes that are homologous to (i.e., homologs of) HCMV US28: Rh214, Rh215, Rh216, Rh218 and Rh220 (also known as RhUS28.4, RhUS28.3, RhUS28.2, RhUS28.1, and RhUS28.5, respectively, M. E. Penfold et al. J Virol 77: 10404 (2003) incorporated by reference herein).
  • Rh217 and Rh219 are not considered to represent functional genes based on a series of previously described criteria (D. Malouli et al., J Virol 86, 8959 (2012) incorporated by reference herein).
  • HCMV US28 encodes a G-protein coupled receptor that binds to CC-chemokines (J.L Gao and P. M. Murphy J Biol Chem 269: 28539 (1993)) and chemokine binding was confirmed for at least one of the five RhCMV homologues (M. E. Penfold et al. J Virol 77: 10404 (2003)).
  • CC-chemokines J.L Gao and P. M. Murphy J Biol Chem 269: 28539 (1993)
  • RhCMV homologues M. E. Penfold et al. J Virol 77: 10404 (2003).
  • a requirement of US28 for the induction of MHC-E restricted T cell responses was unexpected. This surprising result therefore suggests that vectors lacking US28, UL128, and UL130 induce MHC-II restricted CD8 + T cells, including MHC-II restricted supertopes, but not MHC-E-restricted CD8 + T cells. Induction of MHC-E restricted CD
  • Rhesus macaques A total of 46 purpose-bred male or female rhesus macaques (RM) (Macaca mulatto) of Indian genetic background were used in the experiments reported in this example, including 9 RM vaccinated with strain 68-1 RhCMV/gag, RM vaccinated with strain 68-1.2 RhCMV/gag, 1 RM inoculated with Rh67-deleted 68-1 RhCMV/gag, 7 RM vaccinated with MVA/gag, 19 unvaccinated RM with SIV infection, and 6 unvaccinated RM naturally infected with colony-circulating strains of RhCMV.
  • RM rhesus macaques
  • RM All RM were used with the approval of the Oregon National Primate Research Center Institutional Animal Care and Use Committee, under the standards of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. RM used in these experiments were free of cercopithicine herpesvirus 1, D-type simian retrovirus, and simian T-lymphotrophic virus type 1. Selected RM were MHC-l-genotyped by deep sequencing.
  • amplicons of Mamu class I sequences were generated via amplification of cDNA by PCR using high-fidelity PhusionTM polymerase (New England Biolabs) and a pair of universal MHC-l-specific primers with the following thermocycling conditions: 98°C for 3min, (98 °C for 5s, 57 °C for Is, 72 °C for 20s) for 23 cycles, and 72°C for 5 min.
  • Each PCR primer contained a unique 10 bp Multiplex Identifier (MID) tag along with an adaptor sequence for 454 Sequencing 1 (5'-GCCTCCCTCGCGCCATCAG-MID-GCTACGTGGACGACACG-3'; 5'- GCCTTGCCAGCCCGCTCAG-MID-TCGCTCTGGTTGTAGTAGC-3'). Resulting amplicons span 190 bp of a highly polymorphic region within exon two.
  • the primary cDNA-PCR products were purified using AMPure XP magnetic beads (Beckman Coulter Genomics). Emulsion PCR and pyrosequencing procedures were carried out with Genome Sequencer FLX instruments (Roche/454 Life Sciences) as per the manufacturer's instructions. Data analysis was performed using a Labkey database in conjunction with Geneious-Pro ® bioinformatics software (Biomatters Ltd.) for sequence assembly.
  • RhCMV/SIV Vectors The construction, characterization, and administration of strain 68- 1-derived RhCMV/SIV have been previously described in detail (Hansen et al. (2009), supra; Hansen et al. (2011), supra; Hansen et al. Nature (2013), supra; Hansen et al. Science (2013), supra; Hansen et al. (2010), supra). All recombinant viruses used in this study were derived from strain RhCMV 68-1 BAC. Due to tissue culture adaptation, RhCMV 68-1 constructs contain a deletion of ORF 157.5 and most of ORF Rhl57.4 encoding homologs of HCMV UL128 and UL130, respectively (Hansen, S.G. et al., J Virol 77, 6620 (2003); incorporated by reference herein).
  • Rh67 was deleted from RhCMV 68- 1 by BAC recombineering. Briefly, Rh67 was replaced with a FRT-flanked Kanamycin-resistance gene-containing PCR fragment by homologous recombination, followed by excision of the KanR-gene using FLP recombinase. Virus was recovered in rhesus fibroblasts and characterized for antigen expression and loss of Rh67(UL40).
  • the SIVgag expression cassette was inserted into Rh211 of RhCMV 68-1.2, a recombinant virus in which Rh61/Rh60 (UL36), Rhl57.4 (UL130), and Rhl57.5 (UL128) had been repaired (A. E. Lilja and T. Shenk, Proc Natl Acad Sci U.S.A. 105, 19950 (2008); incorporated by reference herein). All of the recombinant viruses were characterized and confirmed by restriction digest, and antigen inserts, including their flanking regions, were sequence verified. Expression of SIV antigens was verified by immunoblot. Additionally, adjacent gene expression was verified by RT-PCR. Other Vaccines
  • MVA/gag was constructed by insertion of codon-optimized, full-length SIVmac239 gag gene into the MVA shuttle vector, pLW44, under the control of MH5, an early/late vaccinia promoter, to generate the recombinant plasmid, pJV7. Flanking sequences within pLW44 directed insertion of the recombinant construct into the thymidine kinase locus by homologous recombination. Chicken embryonic fibroblast cells were transfected with pJV7 followed by infection with MVA strain 1974 to generate recombinant virus expressing SIVmac239gag (SIVgag expression confirmed by immunoblot).
  • Recombinant virus was plaque-purified and amplified in large-scale culture. Viral stocks were purified over a 24-40% sucrose gradient followed by pelleting through a 36% sucrose cushion with the pellet then suspended in 1 mM Tris-CI, pH 9.0. For MVA/gag vaccination, RM were administered 10 8 plaque-forming units of this vector via intramuscular injection.
  • Antigens and Antigen-Presenting Cells Sequential 15-mer peptides (overlapping by 11 amino acids) comprising the SIVgag protein were obtained from the NI H AIDS Reagent Program. Synthesis of specific 9-14-mer peptides within these proteins was performed by Genscript (Piscataway, NJ). All peptides are identified by the position of their inclusive amino acids from the n-terminus (e.g., Gag xx _ yy ). Consecutive 15-mers are also designated by their position starting from the n-terminal 15-mer (e.g., Gagi_i 5 is 15mer #1; Gag 5 _ig is 15mer #2, etc.).
  • BLCL Autologous B- lymphoblastoid cell lines
  • Mammalian expression vectors for Mamu class I molecules were generated by ligating each allele into pCEP4 Kpnl/Notl or Hindll l/Notl restriction sites. Plasmids were cloned in DH5a E. coli (Life Technologies, Grand Island, NY), sequence confirmed, and electroporated into MHC-l-negative K562, 721.221, or RMA-S (K.
  • MHC-I transfectants and BLCL were pulsed with Gag peptide of interest at a final concentration of 10 ⁇ for 90 minutes then washed three times with warm PBS and once with warm RIO to remove unbound peptide before combining with freshly isolated PBMC at an effector:target ratio of 10:1.
  • Mamu-E transfectants were incubated at 27° C for 3 hours prior to use in assays and maintained at 27° C throughout peptide incubation until combined with effectors.
  • Autologous SIV-infected target cells were generated by isolation of CD4 + T cells from PBMC with CD4 microbeads and LS columns (Miltenyi Biotec), activation with a combination of IL-2 (vendor), Staphylococcus enterotoxin B (vendor), and anti-CD3 (NHP Reagent Resource), anti-CD28, and anti-CD49d mAbs (BD Biosciences), and spinoculation with sucrose-purified SIVmac239, followed by 3-4 days of culture. Prior to use in T cell assays, SIV-infected target cells were purified using CD4 microbeads and LS columns (Miltenyi Biotec), as previously described (J. B.
  • Infected cell preparations were >95% CD4 + T cells and >50% SIV-infected following enrichment and were used at an effector:target ratio of 40:1 (PBMC and isolated CD8 + T cells) or 8:1 (T cell line effectors).
  • PBMC and isolated CD8 + T cells or 8:1 (T cell line effectors).
  • uninfected, activated CD4 + T cells served as negative control APCs (uninfected targets from SIV + RM were cultured with tenofovir (NIH AIDS Reagent Program, concentration)).
  • SIV-infected CD4 + T cells were generated as described above without post-infection purification and stained for surface MHC-I (clone W6/32), MHC-E (clone 4D12; anti-mouse IgGl M1-14D12), CD3, CD4 and intracellular SIV Gag p27 capsid.
  • T Cell Assays Mononuclear cell preparations for immunologic assays were obtained from blood with Ficoll-Paque (GE Healthcare). Purified CD8 + T cells (>90% pure) were obtained from PBMC using CD8 microbeads and LS columns (Miltenyi Biotec). Epitope-specific T cell lines were prepared by stimulation of PBMC with irradiated, peptide-pulsed BLCL and subsequent culture in media containing IL-2 (vendor), with re-stimulation performed weekly. SIV-specific CD8 + T cell responses were measured by flow cytometric ICS.
  • effector T cells monoclear cells, isolated CD8 + T cells, or T cell lines
  • antigen peptide, peptide-pulsed APCs, or SIV-infected CD4 + T cells
  • mAbs co-stimulatory monoclonal antibodies
  • CD28 and CD49d CD28 and CD49d
  • Brefeldin A Sigma-Aldrich
  • Stimulated cells were fixed, permeabilized, and stained as previously described (Sacha et ai, The Journal of Immunology, 178, 2746-2754 (2007); incorporated by reference herein) and flow cytometric analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star), gating first on small lymphocytes followed by progressive gating on CD3 + , then CD4 ⁇ /CD8a + T cell subsets. Antigen specific response frequencies for resulting CD4 ⁇ /CD8a + populations were determined from intracellular expression of TNF-a and IFN- ⁇ . For epitope deconvolution experiments, strict response criteria were used to prevent false positives.
  • conjugated Abs were used in these studies: a) from BD Biosciences, L200 (CD4; AmCyan), SP34-2 (CD3; PacBlu), SKI (CD8a; TruRed, AmCyan), 25723.11 (IFNg; APC, FITC), 6.7 (TNF; APC), b) from Beckman Coulter, L78 (CD69; PE).
  • Example 2 Generation of CD8 + T cells specific for peptides of interest in the context of MHC-E T cell receptors recognizing antigen-derived peptides of interest in the context of classical, polymorphic MHC-la molecules can be used to transfect autologous T cells for immunotherapy of disease, such as cancer or infectious disease.
  • a major obstacle to this approach is the MHC-la diversity in the human population that limits the use of a given TCR to MHC-la matched patients.
  • TCR recognizing antigen-derived peptides of interest e.g., tumor antigen-derived peptides and pathogen-derived peptides
  • MHC-matching becomes obsolete, and the resulting TCR can be used in all patients.
  • CD8 + T cells recognizing MHC-E/peptide complexes are rare in nature, and there is not currently a reliable method to generate such T cells directed against antigens of interest, such as tumor antigens, pathogen-derived antigens, tissue-specific antigens, or host self-antigens.
  • antigens of interest such as tumor antigens, pathogen-derived antigens, tissue-specific antigens, or host self-antigens.
  • the method described herein is based upon the finding that a rhesus cytomegalovirus (RhCMV) lacking the genes Rhl57.5 and Rhl57.4 (homologs of HCMV UL128 and UL130) elicits MHC-E- restricted CD8 + T cells in rhesus monkeys at a frequency of about 1 peptide epitope per 30-40 amino acids of protein sequence.
  • RhCMV rhesus cytomegalovirus
  • CD8 + T cells directed against individual peptides presented by MHC-E can be generated.
  • the MHC-E/peptide-recognizing TCRs can be identified by any of a number of methods but generally rely on sequencing the alpha and beta chains either directly by PCR from the cDNA of single cells, clonally expanded single cells, or deep sequencing pools of peptide specific CD8 + T cells.
  • the sequence may be derived indirectly by expanding the RNA template by first creating a whole transcriptome library for a single cell, clonally expanded single cell, or pool of peptide specific CD8 + T cells.
  • Peptide specific variable sequences may be generated by rapid amplification of cDNA ends (RACE) or switching mechanism at 5'end of RNA template (SMART) protocols performed on the mRNA.
  • RACE rapid amplification of cDNA ends
  • SMART RNA template
  • PCR anchored in flanking constant regions or similarly from whole transcriptome libraries of single peptide reactive CD8 + cells can be sequenced directly or deep sequenced for their respective TCR variable regions.
  • Validated combinations of alpha and beta chains derived from the TCR sequence of individual or pools of peptide reactive CD8 + T-cells can further be synthesized or cloned.
  • the resulting TCR constructs can then be transfected into T cells that can in turn be administered to patients as a therapy (e.g., cancer therapy or infectious disease therapy).
  • a therapy e.g., cancer therapy or infectious disease therapy.
  • Example 3 Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex-E
  • MHC-E Major histocompatibility complex
  • Rhl57.5/.4 gene-deleted RhCMV vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8a/ + T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens.
  • MHC-E is up- regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity
  • MHC-E-restricted CD8 + T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.
  • Adaptive cellular immunity against intracellular pathogens is the primary responsibility of CD8 + T cells that recognize short (8-10mer) pathogen-derived peptide epitopes presented by highly polymorphic MHC-la molecules on the surface of infected cells (Neefjes J et al., Nat Rev Immunol 11, 823 (2011) and Nikolich-Zugich J et al., Microbes Infect 6, 501 (2004); both of which are incorporated by reference herein).
  • MHC-la allomorphs vary considerably in their peptide binding properties, and therefore the particular pathogen-derived peptides targeted by pathogen-specific CD8 + T cells is largely determined by the peptide binding specificity of the limited number of MHC-la allomorphs expressed by the infected individual (Yewdell JW, Immunity 25, 533 (2006); incorporated by reference herein) Consequently, the epitopes recognized by CD8 + T cells responding to the same pathogen are highly diverse across individuals.
  • This recognition heterogeneity is important, as the nature of epitopes targeted by CD8 + T cell responses can have an enormous influence on the ability of the individual to clear or control various intracellular pathogens, in particular agents like HIV with a high intrinsic capacity for mutational immune escape (Nikolich Switzerlandich (2004), supra and Goulder, P.J. and Watkins, D.I. Nat Rev Immunol 8, 619 (2008); incorporated by reference herein).
  • this MHC-la polymorphism-mediated response diversity allows large populations to survive emerging pathogens because of the high likelihood that at least some members of the population will have MHC-la allomorphs that support effective CD8 + T cell responses (Nikolich-Zugich (2004), supra and Prugnolle F et al., Curr Biol 15, 1022 (2005); incorporated by reference herein).
  • this biology inevitably results in certain individuals within a population being highly susceptible to a given pathogen, even when vaccinated, which hampers efforts to develop universally effective vaccines based on CD8 + T cell responses (Goulder and Watkins (2008), supra and Picker, L.J. et ai, Ann Rev Med 63, 95 (2012); incorporated by reference herein)
  • RhCMV/SIV vectors provide profound protection against highly pathogenic SIV challenge, resulting in stringent control and ultimate clearance of infection (Hansen et al. (2011), supra and Hansen et al. Nature (2013), supra).
  • MHC-E is known to avidly bind canonical VMAPRTL(LVI)L peptides and other closely related 9mer peptides that are derived from positions 3-11 of MHC-la leader sequences for presentation to NKG2A (and to a lesser extent, NKG2C) molecules on NK cells (Lee, N. et al., J Immunol 160, 4951 (1998); Braud, V.M. et al., Nature 391, 795 (1998); Sullivan, L.C. et al., Tissue Antigens 72, 415 (2008); and van Hall, T. et al., Microbes Infect 12, 910 (2010); all of which are incorporated by reference herein).
  • This highly conserved interaction delivers a predominately inhibitory signal to NK cells when cells express normal levels of MHC-la.
  • this inhibitory signal is reduced, facilitating NK cell activation in response to virally-infected or neoplastic cells (Lodoen, M.B. and Lanier, L.L Nat Rev Microbiol 3, 59 (2005) and Wieten L et al., Tissue Antigens 84, 523 (2014); both of which are incorporated by reference herein).
  • CD8 + T cells can also express NKG2A and/or NKG2C (Arlettaz L et al., Eur J Immunol 34, 3456 (2004); incorporated by reference herein), phenotypic analysis of MHC-E-dependent, strain 68-1 RhCMV/SIVgag vector-elicited CD8 + T cells revealed the vast majority of responding cells were CD8a/ + , TCR ⁇ / ⁇ " T cells that lack both NKG2A and NKG2C expression (Figs. 11C and 19).
  • each of the parent 15mers studied could be truncated to an optimal 9mer peptide that was common among different strain 68-1 RhCMV/SIVgag vector-vaccinated monkeys with responses to the parent 15mer (Fig. 21) (Hansen et al. Science (2013), supra).
  • These optimal 9mers could trigger CD8 + T cells from these monkeys when pulsed on Mamu-E transfectants at doses less than 1 nM (Fig. 22), functional avidities that are comparable to T cell recognition of classically MHC-la-restricted epitopes (O'Connor DH et al., Nat Med 8, 493 (2002); incorporated by reference herein).
  • MHC-E-restricted CD8 + T cell responses have been previously identified in HCMV, Hepatitis C virus, Mycobacterium tuberculosis, and Salmonella enterica infections, typically involving epitopes that are structurally related to the canonical MHC-la leader sequence peptides, but foreign to the host (Sullivan (2008), supra; van Hall (2010), supra; Pietra G et al., J Biomed Biotechnol 2010, 907092 (2010); and Caccamo N et al., Eur J Immunol 45, 1069 (2015); all of which are incorporated by reference herein).
  • Rhl57.5/.4- deficient RhCMV vectors to elicit MHC-E- and MHC-ll-restricted CD8 + T cells is not limited to SIVgag-specific responses. Similar mixtures of MHC-E- and MHC-l l-restricted, antigen-specific CD8 + T cell responses were observed with strain 68-1 (Rhl57.5/.4-deficient) RhCMV vectors encoding SIVpol97-441, M. tuberculosis proteins (Ag85B, ESAT6 and RpfA), as well as intrinsic RhCMV proteins such as the I mmediate Early-1 (I El) protein (Figs. 12B and 26).
  • strain 68-1 RhCMV vectors uniquely elicit CD8 + T cell responses that are either MHC-I I or MHC-E-restricted, and that this unusual immunobiology is a specific consequence of deletion of the RhCMV Rhl57.5/.4 genes, which are orthologs of the HCMV UL128/UL130 genes and encode 2 components of the pentameric receptor complex involved in CMV infection of non-fibroblasts (Lilja AE and Shenk T, Proc Natl Acad Sci U.S.A. 105, 19950 (2008); incorporated by reference herein). Moreover, these data confirm that at least some of the epitopes recognized by these MHC-E-restricted CD8+ T cells are naturally processed and presented by cells infected by SIV, a heterologous (non-CMV) pathogen.
  • MHC-E-restricted epitopes The density of MHC-E-restricted epitopes ( ⁇ 4 independent MHC-E-restricted epitopes per 100 amino acids of protein length) is similar among all strain 68-1 RhCMV vector-elicited CD8 + T cell responses, regardless of nature of the antigen analyzed (Fig. 13B). Notably, among the same 42 strain 68-1 RhCMV/SIVgag vector-vaccinated macaques, 109 of the 125 overlapping SIVgag 15mer peptides (87%) were recognized by MHC- E-restricted CD8 + T cells in at least one macaque (Fig. 13C).
  • MHC-E has previously been shown to bind a broader array of peptides than the canonical leader sequence peptides (van Hall (2010), supra and Lampen et ai, supra), the extent of epitope diversity and breadth observed is highly surprising, especially given the limited polymorphism of MHC-E and the observation that the presentation of all MHC-E-restricted epitopes tested to date is independent of this limited sequence polymorphism as well as the sequence difference between Mamu-E and HLA-E (Figs. 11B, 18 and 22). These data suggest that MHC-E-mediated epitope presentation [e.g., MHC-E peptide binding) is even more diverse than previously believed.
  • the VL9 peptide of UL40 was shown to be loaded onto nascent MHC-E chains by a TAP-independent mechanism, and therefore functions to stabilize and up-regulate MHC-E expression in HCMV-infected cells in the face of virus-mediated TAP inhibition and profound MHC-la down-regulation mediated by the HCMV US2-11 gene products (Lodoen & Lanier (2005), supra and Prod'79 (2012), supra). A similar function is likely for RhCMV Rh67 (Richards (2011), supra). MHC-E up-regulation is therefore thought to be a key viral strategy for evading the NK cell response to infected cells that lack MHC-la expression.
  • MHC-E binding VL9 peptide might act as a chaperone that facilitates stable high expression of MHC-E and delivery to an endosomal compartment that would facilitate peptide exchange, analogous to the invariant chain- associated CLIP peptide and MHC-II. Consistent with such a peptide exchange mechanism, MHC-E peptide loading has been directly demonstrated in the M. tuberculosis phagolysosome (Grotzke JE et al., PLoS Pathog 5, el000374 (2009); incorporated by reference herein).
  • CMV is not the only intracellular pathogen to up-regulate MHC-E expression.
  • Hepatitis C also encodes an MHC-E-binding peptide which up-regulates MHC-E expression (Natterman J et al., Am J Pathol 166, 443 (2005); incorporated by reference herein), and both HIV and SIV up- regulate MHC-E by an uncharacterized mechanism in concert with MHC-la down-regulation (Natterman J et al., Antivir Ther 10, 95 (2005); incorporated by reference herein) (Fig. 27).
  • Rhl57.5/.4 gene-deleted RhCMV vectors are able to bypass the intrinsic constraint of MHC-E-restricted CD8 + T cell priming. Although the mechanism by which this bypass is accomplished remains to be elucidated, the ability of these vectors to strongly elicit broad, diverse and MHC-la haplotype-independent CD8 + T cell responses offers the opportunity to develop MHC-E-restricted, CD8 + T cell-targeted vaccines that exploit MHC-E up-regulation, an intrinsic vulnerability in the immune-evasion strategies of many highly adapted persistent pathogens.
  • an MHC-E-restricted CD8 + T cell response-targeted vaccine would elicit largely similar responses in all or most vaccinees, potentially providing for efficacy in all individuals regardless of MHC genotype.
  • MHC-E Evolution may have disfavored MHC-E as a primary restricting molecule for CD8 + T cells in modern mammals in lieu of the polymorphic MHC-la system, but if HCMV vectors are able to recapitulate in humans the biology of Rhl57.5/.4 gene-deleted RhCMV vectors in macaques (or if alternative, non-CMV-based strategies to elicit broadly targeted MHC-E-restricted CD8 + T cell responses can be developed), vaccinologists may be able to resurrect this dormant MHC-E-based adaptive immune system to attack pathogens with novel immune responses that they are not adapted to effectively evade.
  • Vaccines The construction, characterization, and administration of 1) the strain 68-1 RhCMV vectors expressing SIV Gag and 5'-Pol, 2) the strain 68-1.2 RhCMV vector expressing SIV Gag, 3) the MVA and Adenovirus 5 (Ad5) vectors expressing SIV Gag, and 4) the SIV Gag- encoding DNA + IL-12 vaccine have been previously reported (Hansen et al. Science (2013), supra; Hansen et al. (2011), supra; Hansen et al. Nature (2013), supra; and Hansen et al. (2009), supra).
  • a strain RhCMV 68-1 expressing a fusion protein of the M A strain RhCMV 68-1 expressing a fusion protein of the M.
  • tuberculosis gene products RpfA, RpfC and RpfD driven by an MCMV IE promoter and inserted into the 5' region of Rh211 was provided by Aeras (Rockville, MD, USA).
  • recombination primers flanking the target region forward mutagenesis primer 5' - A A A ACT ATA ATC A AC A ACTCTATACCTTTGTTTTG CTG ATG CTA TTGCGT-3' and reverse mutagenesis primer 5 '- ATTTTTCG ATA A A A A A ATC AC AG C A AAC ATACTG GTTTTACACACTTTAT-3'
  • Rhl57.6 and Rhl57.4 (UL130) open reading frames (ORFs) overlap in RhCMV, the deletion was constructed in a fashion that retained the end of the Rhl57.6 (UL131A) ORF plus additional 50 bp to ensure expression of the encoded protein.
  • Mini plasmid R6K-kan-F5 was used to amplify a kanamycin resistance cassette flanked by alternative (F5) FRT sites using the forward primer binding site (5'- GAAAAGTGCCACCTGCAGAT-3') and reverse primer binding site (5'-CAGGAACACTTAACGGCTGA- 3'), which were added to the 3' end of the mutagenesis primers.
  • E/T homologous recombination in E. coli strain SW105 (Warming S et al., Nucleic Acids Res 33, e36 (2005); incorporated by reference herein) was performed as published elsewhere (Muyrers JP et al., Nucleic Acids Res 27, 1555 (1999); incorporated by reference herein).
  • Rhesus Macaques A total of 207 purpose-bred male or female rhesus macaques (Macaca mulatto) of Indian genetic background were used in the experiments reported in this example, 88 of which were also studied in a previous report (Hansen et al. Science (2013), supra).
  • Macaques used in these experiments were free of cercopithicine herpesvirus 1, D-type simian retrovirus, and simian T- lymphotrophic virus type 1. Selected macaques were MHC-I genotyped by deep sequencing, as described (Wiseman (2009), supra).
  • amplicons of Mamu class I sequences were generated via amplification of cDNA by PCR using high-fidelity PhusionTM polymerase (New England Biolabs) and a pair of universal MHC-l-specific primers with the following thermocycling conditions: 98° C for 3 minutes, (98° C for 5 seconds, 57° C for 1 second, 72° C for 20 seconds) for 23 cycles, and 72° C for 5 minutes.
  • Each PCR primer contained a unique 10 bp Multiplex Identifier (MI D) tag along with an adaptor sequence for 454 SequencingTM (5'- GCCTCCCTCGCGCCATCAG-MID-GCTACGTGGACGACACG-3'; 5'-GCCTTGCCAGCCCGCTCAG-MI D- TCGCTCTGGTTGTAGTAGC-3'). Resulting amplicons span 190 bp of a highly polymorphic region within exon two.
  • the primary cDNA-PCR products were purified using AMPure XP magnetic beads (Beckman Coulter Genomics). Emulsion PCR and pyrosequencing procedures were carried out with Genome Sequencer FLX instruments (Roche/454 Life Sciences) as per the manufacturer's instructions. Data analysis was performed using a Labkey database in conjunction with Geneious-Pro ® bioinformatics software (Biomatters Ltd.) for sequence assembly.
  • Antigens and Antigen-Presenting Cells The synthesis of sequential 15-mer peptides
  • All peptides are identified by the position of their inclusive amino acids from the N-terminus (e.g., Gag xx _ yy ). Consecutive 15mers are also designated by their position starting from the N-terminal 15mer (e.g., Gagl-15 (1) is 15mer #1; Gag5-19 (2) is 15mer #2, etc.). Unless otherwise specified, these peptides were used in T cell assays at 2 ⁇ / ⁇ . Autologous B-lymphoblastoid cell lines (BLCL) were generated by infecting rhesus macaque PBMC with Herpesvirus papio, as previously described (Hansen et al. Science (2013), supra).
  • BLCL Autologous B-lymphoblastoid cell lines
  • Mammalian expression vectors for Mamu class I molecules were generated by ligating each allele into pCEP4 Kpnl/Notl or Hindlll/Notl restriction (Ulbrecht M et al., J Immunol 164, 5019 (2000); incorporated by reference herein) sites. Plasmids were cloned in DH5a E. coli (Life Technologies), sequence confirmed, and electroporated into MHC-l-negative K562, 721.221, or RMAS cells (Anderson KS et al., J Immunol 151, 3407 (1993); incorporated by reference herein) using Nucleofector ll/Kit C (Lonza).
  • Transfectants were maintained on drug selection (Hygromycin B) and routinely confirmed for surface expression of MHC-I by staining with pan-MHC-l antibody clone W6/32. Throughout use in T cell assays, mRNA from MHC-I transfectants was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen), amplified by RT-PCR using primer pairs flanking a highly polymorphic region within exon 2, and sequence confirmed.
  • MHC-I transfectants and BLCL were pulsed with peptides of interest at a final concentration of 10 ⁇ for 90 minutes then washed three times with warm PBS and once with warm RPMI 1640 media with 10% fetal calf serum to remove unbound peptide before combining with freshly isolated PBMC at an effector:target ratio of 10:1.
  • Mamu-E transfectants were incubated at 27° C for >3 hours prior to use in assays and maintained at 27° C throughout peptide incubation until combined with effectors.
  • Autologous SIV-infected target cells were generated by isolation of CD4 + T cells from PBMC with CD4 microbeads and LS columns (Miltenyi Biotec), activation with a combination of IL-2 (NIH AIDS Reagent Program), Staphylococcus enterotoxin B (Toxin Technologies Inc.), and anti-CD3 (NHP Reagent Resource), anti-CD28, and anti-CD49d mAbs (BD Biosciences), and spinoculation with sucrose-purified SIVmac239, followed by 3-4 days of culture.
  • IL-2 NIH AIDS Reagent Program
  • Staphylococcus enterotoxin B Toxin Technologies Inc.
  • anti-CD3 NHS Reagent Resource
  • anti-CD28, and anti-CD49d mAbs BD Biosciences
  • SIV infected target cells Prior to use in T cell assays, SIV infected target cells were purified using CD4 microbeads and LS columns (Miltenyi Biotec), as previously described (Sacha JB et al, J Immunol 178, 2746 (2007); incorporated by reference herein). Infected cell preparations were >95% CD4 + T cells and >50% SIV-infected following enrichment and were used at an effector:target ratio of 40:1 (PBMC and isolated CD8+ T cells) or 8:1 (T cell line effectors).
  • MHC association (MHC-la, MHC-E, MHC-II) of a response was determined by pre-incubating isolated mononuclear cells, antigen-presenting cells or SIV-infected CD4 + cells for 1 hour at room temperature (prior to adding peptides or combining effector and target cells and incubating per the standard ICS assay) in the presence of the following blockers: 1) the pan anti-MHC-l mAb W6/32 (10 mg/ml), 2) the MHC-l l-blocking CLIP peptide (MHC-l l-associated invariant chain, amino acids 89-100; 20 ⁇ ), and 3) the MHC-E blocking VL9 peptide (VMAPRTLLL; 20 ⁇ ), alone or in combination.
  • the Mamu-Al*001:01-binding peptide CM9 (CTPYDI NQM; 20 ⁇ ), or the Mamu-Al*002:01-binding peptide GY9 (GSEN LKSLY; 20 ⁇ ) were used as blocking controls. Stimulated cells were fixed, permeabilized and stained as previously described (Hansen et al. Science (2013), supra), and flow cytometric analysis was performed on an LSR-I I instrument (BD Biosciences). Analysis was done using FlowJo software (Tree Star).
  • Antibodies The following conjugated antibodies were used in these studies: a) from BD
  • Biosciences L200 (CD4; AmCyan), SP34-2 (CD3; PacBlu), SKI (CD8a; TruRed, AmCyan), 25723.11 (IFN- ⁇ ; APC, FITC), 6.7 (TNF; APC), MAbll (TNF; Alexa700), b) from Beckman Coulter, L78 (CD69; PE), 2ST8.5H7 (CD8 ; PE), Z199 (NKG2A/C or CD159a/c; PE), c) from Biolegend, W6/32 (pan- MHC-I; PE), OKT-4 (CD4; PE-Cy7), Bl (TCRy/ ⁇ ; Alexa647), d) from Miltenyi Biotec, M-T466 (CD4; APC), e) from eBiosciences, M1-14D12 (mouse IgGl; PE-Cy7).
  • the amino acid composition of each position in the 11 optimal peptides was compared to the amino acid frequencies found in SIVmac239 Gag (GenBank accession #M33262), the insert strain used for the vaccine.
  • SIVmac239 Gag GenBank accession #M33262
  • the full set of 551 eluted peptides previously published in Lampen et al. was used.
  • the peptides in Lampen et al. varied in length, between 8 and 13 amino acids; 9 was the most common length. They had used a motif searching algorithm to explore amino acid enrichment and under-representation among 315 9 mers in their eluted set (Fig.
  • the sequence LOGO shown in Fig. 13D indicates the frequency of each amino acid in a given position (relative to their background frequency in SIVmac239 Gag) by the height of the letter, based on 11 optimal, MHC-E-restricted SIVgag 9mer peptide epitopes recognized by CD8+ T cells in strain 68-1 RhCMV vector-vaccinated macaques.
  • the sequence LOGO in Fig. 13D is colored according to enrichment (boxes with grey fill or hatched boxes) or underrepresentation (boxes with white fill) among 551 peptides eluted from HLA-E in a TAP- deficient setting by Lampen et al. As shown in the right panel of Fig.

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EA201791806A1 (ru) 2017-12-29
JP2018510650A (ja) 2018-04-19
BR112017017092A2 (pt) 2018-06-26
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US20180298404A1 (en) 2018-10-18
EP4036239A1 (en) 2022-08-03
JP6816031B2 (ja) 2021-01-20
HK1247638A1 (zh) 2018-09-28
IL253935A0 (en) 2017-10-31
EP3256595A4 (en) 2018-09-26
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