WO2011133870A2 - Vaccines comprising adenovirus vectors and signaling lymphocyte activating molecule-associated protein (sap) - Google Patents

Vaccines comprising adenovirus vectors and signaling lymphocyte activating molecule-associated protein (sap) Download PDF

Info

Publication number
WO2011133870A2
WO2011133870A2 PCT/US2011/033591 US2011033591W WO2011133870A2 WO 2011133870 A2 WO2011133870 A2 WO 2011133870A2 US 2011033591 W US2011033591 W US 2011033591W WO 2011133870 A2 WO2011133870 A2 WO 2011133870A2
Authority
WO
WIPO (PCT)
Prior art keywords
vector
cells
polypeptide
seq
interest
Prior art date
Application number
PCT/US2011/033591
Other languages
French (fr)
Other versions
WO2011133870A3 (en
Inventor
Andrea Amalfitano
Yasser Aldhamen
Seregin Sergey
Original Assignee
Andrea Amalfitano
Yasser Aldhamen
Seregin Sergey
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Andrea Amalfitano, Yasser Aldhamen, Seregin Sergey filed Critical Andrea Amalfitano
Publication of WO2011133870A2 publication Critical patent/WO2011133870A2/en
Publication of WO2011133870A3 publication Critical patent/WO2011133870A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention provides replication incompetent adenovirus vectors containing a heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further encodes at least a portion of a polypeptide of interest.
  • SAP SLAM family receptor adaptor protein
  • the invention further provides DNA sequences that encode the invention's adenoviral vectors, as well as packaging cells that produce the invention's adenoviral vectors.
  • the invention's compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP).
  • compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, such as in vaccination applications.
  • the invention's compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus.
  • SAP SLAM family receptor adaptor protein
  • Adenoviral based gene delivery vectors remain one of the most promising vaccine platforms for use against numerous pathogens, including HIV.
  • Recent vaccine trials utilizing first generation Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections.
  • Adenovirus based vaccines can induce potent cellular immune responses to HIV derived antigens (as compared to DNA or other virus based vaccine platforms), but these responses still do not reach levels noted in so called long-term non-progressors.
  • the invention provides a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
  • the vector further comprises c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest.
  • the SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein selected from the group consisting of human EAT-2 (SEQ ID NO:01) and mouse EAT-2 (SEQ ID NO:29).
  • the DNA sequence encoding a replication defective adenovirus lacks adenovirus El gene coding sequence.
  • the polypeptide of interest comprises an antigen polypeptide.
  • the antigen polypeptide comprises Human immunodeficiency virus GAG sequence AMQMLKETI (SEQ ID NO: 06).
  • the antigen polypeptide comprises Plasmodium falciparum circumsporozoite antigen NYDNAGTNL (SEQ ID NO:05).
  • the invention also provides a purified DNA sequence comprising, in operable combination, a) a nucleic acid sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), h one embodiment, the DNA sequence further comprises, in operable combination, c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest.
  • the polypeptide of interest comprises an antigen polypeptide.
  • the SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein SEQ ID NO:01.
  • the invention further provides a composition comprising any one or more of the replication incompetent recombinant adenovirus vectors described herein (such as a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further comprises c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest), and a
  • SAP SLAM family receptor adaptor protein
  • the invention provides a method for vaccinating a mammalian subject, comprising a) providing i) a first replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and ii) a second vector comprising a nucleotide sequence that encodes an antigen polypeptide, and b) administering an immunologically effective amount of the first vector and the second vector to the subject under conditions for producing an immune response to the antigen polypeptide, h one embodiment, the subject is at risk of disease.
  • a first replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b)
  • the subject is at risk of infection by a pathogen.
  • the immune response comprises an adaptive immune response.
  • the immune response comprises an innate immune response.
  • the immune response comprises an increase in cytolytic activity of CD8+ T cells that specifically bind to the polypeptide of interest.
  • the invention also provides a method for vaccinating a mammalian subject, comprising a) providing a pharmaceutically acceptable composition comprising any one or more of the vectors disclosed herein, (such as a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and c) a second heterologous nucleic acid sequence that encodes an antigen polypeptide, and b) administering an immunologically effective amount of the composition to the subject under conditions for producing an immune response to the polypeptide of interest.
  • a pharmaceutically acceptable composition comprising any one or more of the vectors disclosed herein, (such as a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a)
  • FIG. 1 Systemic administration of EAT-2 expressing adenovirus vector induces cytokine and chemokines responses.
  • Plasma was harvested at 6h after virus injection.
  • Cytokine induction was evaluated using a multiplexed bead array based quantitative system. The bars represent mean ⁇ SD.
  • Statistical analysis was completed using One Way ANOVA with a Student-Newman- euls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference .
  • FIG. 2 Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo.
  • PBMCs and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars represent mean ⁇ SD.
  • Statistical analysis was completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference. * denotes p ⁇ 0.05, ** denotes p ⁇ 0.01, *** p ⁇ 0.001, # denotes p ⁇ 0.001 over mock.
  • FIG. 3 HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and Ad-EAT2 co-immunization in Balb/c mice after intramuscular injection.
  • Mice were co- immunized intramuscularly in the tibialis anterior with equivalent viral particles of Ad- HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (total of lxlO 7 vps mixed prior to injection).
  • PBMCs peripheral blood mononuclear cells
  • mice were sacrificed and PBMCs (B) or splenocytes (C) were harvested and stained with a PE-conjugated H2-Kd-AMQMLKETI tetramer complex together with an APC-conjugated anti-CD3 and FITC-conjugated anti-CD8 antibodies.
  • the bars represent mean ⁇ SD for six mice per group (pool of two for PBMCs) for virus injected and three mice for naive animals.
  • Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference. * denotes p ⁇ 0.05, ** denotes p ⁇ 0.01.
  • splenocytes were harvested and stimulated ex vivo with the immunodominant peptides (AMQMLKETI: SEQ ID NO:06) for Balb/c and QBI# 804796 (EAMSQVTNSATMMQ) for C57B1/6.
  • Spot forming cells SFCs
  • Data are presented as mean ⁇ SD.
  • Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference. * denotes p ⁇ 0.05, *** p ⁇ 0.001. Representative data from two independent experiments are shown.
  • FIG. 5 Analysis of the breadth of Gag-responses after intramuscular injection of Ad-HIV/Gag and Ad-EAT2 in Balb/c mice: Balb/c mice were co-immunized intramuscularly with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (total dose of l xlO 7 vps mixed prior to injection). At 14 d.p.i., animals were terminally sacrificed, and splenocytes were harvested and stinmlated ex vivo with 15mer HIV-Gag derived peptides QBI#804724, (S LYNT VATLYCVHQR: SEQ ID NO:07),
  • QBI#804753 (GHQ AAMQMLKETINE : SEQ ID NO:08), QBI#804754
  • FIG. 6 Analysis of T cell epitope responses of Balb/c and C57B1/6 mice to HIV-
  • mice Gag in Ad-HIV/Gag and Ad-EAT2 co-injected mice.
  • Ad-GFP Ad-EAT2
  • splenocytes were equivalently pooled and IL-2 ELISPOT analysis was carried out by stimulating individual wells ex vivo with a pool of 2-4 15mer peptides overlapped by 11 , not including peptides included in Figure 4 and 5.
  • SFCs per million splenocytes are shown. The minimal threshold response is indicated by the line above 10.
  • FIG. 7 Ad-HIV/Gag and Ad-EAT2 co-immunization increases the frequency of HIV-Gag specific CD8 + T cells.
  • Ad-GFP Ad-GFP
  • Ad-EAT2 1 x 10 7 total vps for Balb/c and 1 ⁇ 10 9 total vps for C57B1/6 mice.
  • the mice were sacrificed.p.i., the mice were sacrificed and lymphocytes were isolated from spleen. Multiparameter flow cytometry was used to determine the total frequency of cytokine-producing CD8 + T cells.
  • mice were co-immunized with equivalent viral particles of Ad-HIV/Gag mixed with either Ad- GFP or Ad-EAT2 (1 x 10 7 total vps for Balb/c and 1 10 9 total vps for C57B1/6 mice).
  • Ad-HIV/Gag mixed with either Ad- GFP or Ad-EAT2 (1 x 10 7 total vps for Balb/c and 1 10 9 total vps for C57B1/6 mice).
  • syngeneic splenocytes were pulsed with either an irrelevant peptide (NYD-pep) and stained with 1 ⁇ (CFSE Low ) or with the HIV-Gag specific peptides (AMQ peptide for
  • FIG. 10 Systemic administration of EAT-2 expressing adenovirus vector induces cytokine and chemokines responses.
  • Plasma was harvested at 6h after virus injection. Cytokine induction was evaluated using a multiplexed bead array based quantitative system. The bars represent mean ⁇ SD.
  • Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference .
  • FIG. 11 Ad-EAT2 -mediated activation of innate and adaptive immune cells in vivo.
  • CD69 expression by PBMCs (A and B) and splenocytes (C and D) derived NK, NKT, CD3 + CD8 + T cells, CD3 + CD8 " T cells, and B cells was evaluated 48h after virus injection.
  • PBMCs and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars represent mean ⁇ SD.
  • Statistical analysis was completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference.
  • Figure 12 IFNy production from NK cells 6 and 48 hours after Ads injection.
  • Splenocytes were harvested and incubated at 37°C for 5 hours in the presence of Golgi plug. IFNy intracellular staining was performed and cells were sorted on a LSRII flow cytometer. The bars represent mean ⁇ SD.
  • Statistical analysis was completed using One Way ANOVA with a student- Newman- Keuls post-hoc test, p ⁇ 0.05 was deemed a statistically significant difference. * denotes p ⁇ 0.05, *** p ⁇ 0.001.
  • FIG. 13 Cellular immune responses after CD8 + T cells depletion in Ad-HIV/Gag and Ad-EAT2 co-immunized mice.
  • splenocytes from vaccinated Balb/c mice (1 x 10 7 total vps) were equivalently pooled (N-6 mice per treatment) and CD8+ cells were depleted using magnetic beads.
  • 5xl0 5 splenocytes were added to each well and stimulated with the immunodominant peptide AMQMLKETI (SEQ ID NO:06).
  • A A representative flow cytometric analysis before and after CD8 T cell depletion is shown.
  • Figure 14 Exemplary Homo sapiens EAT-2 (SH2 domain containing IB (SH2D1B)) (GenBank Accession No.: NM_053282.4) polypeptide sequence (SEQ ID NO:01) (A) encoded by nucleotide sequence (SEQ ID NO:02).
  • Figure 15 Exemplary human immunodeficiency virus type 1 (HXB2) GAG polypeptide sequence (SEQ ID NO:03) (GenBank Accession No.: 03455).
  • Figure 16 FACS analysis of (A) CD80, (B) CD86, (C) MHC-II, and (D) CD40.
  • Figure 17 FACS analysis of (A) CD80, (B) CD86, (C) MHC-II, and (D) CD40.
  • Figure 18 Ad-EAT2 versus Ad-GFP MOI of 20,000 (same cells but infected with virus).
  • Figure 19 Amino acid sequence for malaria CSPMI (SEQ ID NO:07), CSP T-cell epitope (SEQ ID NO:09), CSP B-Cell epitope (SEQ ID NO: 10), and CSP 156 (SEQ ID NO: l l).
  • Figure 20 Nucleic acid sequence (SEQ ID NO:08) encoding malaria CSP.
  • FIG. 21 Exemplary Mus musculus 2B4 (CD244) (GenBank Accession
  • Figure 23 Exemplary Mus musculus CD84 (GenBank Accession No.: NM_013489) polypeptide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID
  • FIG. 24 Exemplary Mus musculus Ly9 (CD229) (GenBank Accession No.:
  • NM_008534 polypeptide sequence (SEQ ID NO: 19) (A) encoded by nucleotide sequence (SEQ ID NO:20).
  • FIG. 25 Exemplary Mus musculus Lyl08 (NTB-A) (GenBank Accession No.:
  • polypeptide sequence (SEQ ID NO:21) (A) encoded by nucleotide sequence (SEQ ID NO:22).
  • Figure 26 Exemplary Mus musculus SLAM (CD150) (GenBank Accession No.: NM_013730) polypeptide sequence (SEQ ID NO:23) (A) encoded by nucleotide sequence (SEQ ID NO:24).
  • Figure 27 Exemplary Mus musculus SAP (SH2D1 A) (GenBank Accession No.: NM_011364) polypeptide sequence (SEQ ED NO:25) (A) encoded by nucleotide sequence (SEQ ID NO:26).
  • Figure 28 Exemplary Mus musculus ERT (SH2Dlb2) (GenBank Accession No.: NM_001033499) polypeptide sequence (SEQ ID NO:27) (A) encoded by nucleotide sequence (SEQ ID NO:28).
  • Figure 29 Exemplary Mus musculus EAT-2 (SH2 domain protein 1B1 (Sh2dlbl)) (GenBank Accession No.: NM_012009.4) polypeptide sequence (SEQ ID NO:29) (A) encoded by nucleotide sequence (SEQ ID NO:30).
  • recombinant DNA molecule refers to a DNA molecule that is comprised of DNA sequences joined together by means of molecular biological techniques.
  • recombinant protein or “recombinant polypeptide” as used herein refers to a protein molecule that is expressed using a recombinant DNA molecule.
  • portion when made in reference to a nucleic acid sequence refers to a fragment of that sequence.
  • the fragment may range in size from an exemplary 5, 10, 20, 50, and/or 100 contiguous nucleotide residues to the entire nucleic acid sequence minus one nucleic acid residue.
  • a nucleic acid sequence comprising "at least a portion of a nucleotide sequence comprises from five (5) nucleotide residue of the nucleotide sequence to the entire nucleotide sequence.
  • portion when used in reference to a protein (as in a "portion of a given protein") refers to fragments of that protein.
  • the fragments may range in size from an exemplary 4, 10, 20, 30, and/or 50 contiguous amino acid residues to the entire amino acid sequence minus one amino acid residue.
  • a polypeptide sequence comprising "at least a portion of an amino acid sequence” comprises from four (4) contiguous amino acid residues of the amino acid sequence to the entire amino acid sequence.
  • mutant mutation refers to a mutation that is introduced by means of molecular biological techniques. This is in contrast to mutations that occur in nature.
  • recombinant virus refers to a virus that contains a recombinant DNA molecule, recombinant protein and/or recombinant mutation, as well as progeny of that virus.
  • mutation and “modification” refer to a deletion, insertion, or substitution.
  • a “deletion” is defined as a change in a nucleic acid sequence or amino acid sequence in which one or more nucleotides or amino acids, respectively, is absent.
  • An “insertion” or “addition” is that change in a nucleic acid sequence or amino acid sequence that has resulted in the addition of one or more nucleotides or amino acids, respectively.
  • substitution in a nucleic acid sequence or an amino acid sequence results from the replacement of one or more nucleotides or amino acids, respectively, by a molecule that is a different molecule from the replaced one or more nucleotides or amino acids.
  • a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine.
  • a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.
  • substitution of an amino acid may be conservative or non-conservative.
  • Constant substitution refers to the replacement of that amino acid with another amino acid that has a similar hydrophobicity, polarity, and/or structure.
  • the following aliphatic amino acids with neutral side chains may be conservatively substituted one for the other: glycine, alanine, valine, leucine, isoleucine, serine, and threonine.
  • Aromatic amino acids with neutral side chains that may be conservatively substituted one for the other include phenylalanine, tyrosine, and tryptophan. Cysteine and methionine are sulphur-containing amino acids that may be conservatively substituted one for the other. Also, asparagine may be conservatively substituted for glutamine, and vice versa, since both amino acids are amides of dicarboxylic amino acids. In addition, aspartic acid (aspartate) my be conservatively substituted for glutamic acid (glutamate) as both are acidic, charged (hydrophilic) amino acids. Also, lysine, arginine, and histidine my be conservatively substituted one for the other since each is a basic, charged (hydrophilic) amino acid.
  • Non- conservative substitution is a substitution other than a conservative substitution.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological and/or immunological activity may be found using computer programs well known in the art, for example, DNAStarTM software.
  • a "variant” or “homolog” of a polypeptide sequence of interest or nucleotide sequence of interest refers to a sequence that has identity of at least 65% with the an amino acid sequence of interest or nucleotide sequence of interest, including identity of 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%, and/or at least 99%.
  • homologous genomic nucleotide sequences within the scope of the invention include orthologs and paralogs.
  • the term "ortholog” refers to a gene in different species that evolved from a common ancestral gene by speciation. In some embodiments, orthologs retain the same function.
  • paralog refers to genes related by duplication within a genome. In some embodiments, paralogs evolve new functions. In further embodiments, a new function of a paralog is related to the original function. Variants of a polypeptide sequence of interest may contain a mutation.
  • expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression (i.e., transcription and/or translation) of the operably linked coding sequence in a particular host organism.
  • Expression vectors are exemplified by, but not limited to, plasmid, phagemid, shuttle vector, cosmid, virus, chromosome, mitochondrial DNA, plastid DNA, and nucleic acid fragment.
  • Nucleic acid sequences used for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • pathogenic and "virulent” when in reference to a microorganism refer to the ability of the microorganism to produce an infectious disease in another organism (e.g., mammal).
  • purified and “isolated” and grammatical equivalents thereof as used herein, refer to the reduction in the amount of at least one undesirable contaminant (such as protein and/or nucleic acid sequence) from a sample. Thus, purification results in an
  • enrichment i.e., an increase in the amount of a desirable composition, such as a virus, protein and/or nucleic acid sequence in the sample.
  • a desirable composition such as a virus, protein and/or nucleic acid sequence in the sample.
  • packaged adenoviral compositions of the invention maybe purified using methods known in the art (e.g., U.S. Patent 6,946,126 to Amalfitano et al.)
  • operable combination and “operably linked” when in reference to the relationship between nucleic acid sequences and/or amino acid sequences refers to linking the sequences such that they perform their intended function.
  • operably linking a promoter sequence to a nucleotide sequence of interest refers to linking the promoter sequence and the nucleotide sequence of interest in a manner such that the promoter sequence is capable of directing the transcription of the nucleotide sequence of interest resulting in an mRNA that directs the synthesis of a polypeptide encoded by the nucleotide sequence of interest.
  • transfect refers to any mechanism by which a vector may be incorporated into a host cell.
  • a successful transfection results in the capability of the host cell to express any operative genes carried by the vector.
  • Transfections may be stable or transient.
  • One example of a transient transfection comprises vector expression within a cell, wherein the vector is not integrated within the host cell genome.
  • a stable transfection comprises vector expression within a cell, wherein the vector is integrated within the host cell genome.
  • Subject and “animal” interchangeably refer to any multicellular animal, preferably a mammal, e.g., humans, non-human primates, murines, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.).
  • mammalian subjects include mouse, rat, guinea pig, hamster, ferret and chinchilla.
  • Subject in need of reducing one or more symptoms of a disease includes a subject that exhibits and/or is at risk of exhibiting one or more symptoms of the disease.
  • subjects may be at risk based on family history, genetic factors, environmental factors, etc. This term includes animal models of the disease.
  • a protein containing epitope A or free, imlabelled A
  • SLAM and "SLAM family receptor” interchangeably refer a receptor that is expressed by a wide range of immune cells. 13 ' 18 ' 19 .
  • SLAM family receptors are exemplified by mouse 2B4 (CD244) (GenBank Accession No.: NM_018729.2, SEQ ID NO: 13 encoded by SEQ ID NO:14 of Figure 21), mouse CRACC (Slamf?) (GenBank Accession No.:
  • NMJB0710 SEQ ID NO:21 encoded by SEQ ID NO:22 of Figure 25
  • mouse SLAM CD150: (GenBank Accession No.: NM_013730, SEQ ID O:23 encoded by SEQ ID NO:24 of Figure 26).
  • the SLAM family receptors control multiple innate and adaptive immune responses through association with an intracellular signaling SLAM family receptor adaptor protein, described below
  • SLAM family receptor adaptor protein and "SAP” are used interchangeably to refer to an intracellular protein that associates with SLAM, and are exemplified by
  • EAT-2 (also referred to as “SH2dlbl,” “EAT-2A,” “Eat2,” and “Eat2a”) is a protein containing a SRC (MIM 190090) homology-2 (SH2) domain, and that regulates signal transduction through interaction with SLAM family receptors expressed on the surface of antigen-presenting cells.
  • SRC MIM 190090
  • EAT-2 is exemplified by human sequences (SEQ ID NO:01 encoded by SEQ ID NO: 02, GenBank Accession No.: NM_053282.4 of Figure 14), and by mouse sequences (SEQ ID NO:29 encoded by SEQ ID NO:30, GenBank Accession No.: NM_012009.4 of Figure 29).
  • SAP is exemplified by mouse SAP (SH2D1A): NM_011364 (SEQ ID NO:25 encoded by SEQ ID NO:26 of Figure 27)
  • ERT is exemplified by mouse ERT
  • any molecule e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein
  • cell e.g., cell, and/or phenomenon (e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean that the quantity of molecule, cell and/or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis.
  • the quantity of molecule, cell and/or phenomenon in the first sample is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity of the same molecule, cell and/or phenomenon in a second sample.
  • the quantity of molecule, cell, and/or phenomenon in the first sample is lower by any numerical percentage from 5% to 100%, such as, but not limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, and from 90% to 100% lower than the quantity of the same molecule, cell and/or phenomenon in a second sample.
  • any molecule ⁇ e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein
  • cell and/or phenomenon ⁇ e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean that the quantity of the molecule, cell and/or phenomenon in the first sample is higher than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis.
  • the quantity of the molecule, cell and/or phenomenon in the first sample is at least 10%> greater than, at least 25% greater than, at least 50% greater than, at least 75% greater than, and/or at least 90% greater than the quantity of the same molecule, cell and/or phenomenon in a second sample.
  • altered when in reference to the level of any molecule ⁇ e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein), cell, and/or phenomenon ⁇ e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean reduced or increased level of the molecule, cell, and/or phenomenon.
  • polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein
  • phenomenon e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.
  • references herein to any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range.
  • reference herein to a range of "at least 50" includes whole numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc.
  • reference herein to a range of "less than 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc.
  • reference herein to a range of from “5 to 10" includes each whole number of 5, 6, 7, 8, 9, and 10, and each fractional number such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, etc.
  • the invention provides replication incompetent adenovirus vectors containing a heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further encodes a polypeptide of interest.
  • SAP SLAM family receptor adaptor protein
  • the invention further provides DNA sequences that encode, as well as packaging cells that produce, the invention's adenoviral vectors.
  • the invention's compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP).
  • the invention's compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, such as in vaccination applications.
  • the invention's compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus.
  • SAP SLAM family receptor adaptor protein
  • data herein provide evidence supporting an important new strategy to improve the efficacy of vaccines in general, and that of Ad based vaccines specifically, by activating the SLAM family receptors system simultaneously with target antigen vaccination.
  • the inventors confirmed the utility of augmenting the exemplary SLAM family receptors signaling by utilizing an Ad vaccine genetically engineered to overexpress the SLAM family receptors adaptor molecule EAT-2, as it improved the cellular immune responses elicited by a second Ad vaccine expressing a target antigen, in this instance the exemplary HIV derived Gag protein.
  • compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP).
  • SAP SLAM family receptor adaptor protein
  • compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, including in vaccination applications.
  • Vaccine applications include, for example, eliciting immune responses to pathogen-derived antigens (such as those from the HIV virus, for example HIV- gag) as well as disease associated antigens, such as cancers (such as Carcino-embryonic antigen (CEA), her2neu, etc.), etc..
  • pathogen-derived antigens such as those from the HIV virus, for example HIV- gag
  • disease associated antigens such as cancers (such as Carcino-embryonic antigen (CEA), her2neu, etc.), etc.
  • compositions and methods may be used for enhancing an adaptive cellular and/or humoral immune response to a vaccine against an antigen by co-administering (in the same preparation or in separate preparations) a vector that expresses a SLAM family receptor adaptor protein (SAP) as an adjuvant, and another vector that expresses the antigen.
  • SAP SLAM family receptor adaptor protein
  • Expression of the SLAM family receptor adaptor protein (SAP) by the vector enhances the subject's imnrune response to the antigen.
  • compositions and methods may be used for enhancing an adaptive cellular and/or humoral immune response to a vaccine against an antigen by administering a single vector that expresses both a SLAM family receptor adaptor protein (SAP) and the antigen.
  • SAP SLAM family receptor adaptor protein
  • compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus.
  • SAP SLAM family receptor adaptor protein
  • SAPs SLAM Family Receptor Adaptor Proteins
  • rAd recombinant adenovirus
  • TLR Toll Like Receptor
  • the SLAM family of receptors is composed of six distinct members named SLAM (CD150), 2B4 (CD244), Ly9, CD84, NTB-A (natural killer, T and B cell antigen; Lyl08 in the mouse) and CRACC (CD2-like receptor activating
  • cytotoxic cells cytotoxic cells
  • SAP SLAM-associated protein
  • ERT ERT is however a non-functional pseudo-gene in humans.
  • the SAP adaptor regulates SLAM family receptors signaling by recruiting the protein tyrosine kinase Fyn.
  • EAT-2 and ERT transduce SLAM family receptors initiated signals in a process that involves phosphorylation of tyrosine residues directly located in their short carboxyl-terminal tails .
  • SLAM SLAM
  • DCs dendrictic cells
  • SLAM receptor a proinflammatory costimulatory molecule during the activation of dendrictic cells (DCs) and macrophages.
  • DCs dendrictic cells
  • SLAM receptor was also found to be responsible for triggering the
  • IL-6 and IL-12 production of IL-6 and IL-12 by mouse peritoneal macrophages .
  • macrophages derived from SLAM-deficient mice show a marked reduction in secretion of IL-12, TNF, and nitric oxide 22.
  • EAT-2 is the only known SLAM-associated adaptor protein expressed in DCs and macrophages, EAT-2 potentially mediates SLAM induced proinflammatory cytokine expression in these cell types 19 .
  • APCs Ad transduced antigen presenting cells
  • Ad vaccine mediated EAT-2 expression enhanced the cellular immune responses to an HIV-Gag antigen expressed from a co-administered Ad vaccine, as multiply confirmed by Tetramer-based flow cytometry, ELISPOT, and in vivo CTL assays. Since both mice and humans express highly conserved EAT-2 adaptor proteins, the inventors' results suggest that human vaccination strategies that specifically facilitate SLAM family receptors signaling may provide a more effective vaccine against HIV specifically, as well as numerous other vaccine targets in general.
  • NK cells In addition to NK cells, the inventors also observed increased activation of NKT cells after Ad mediated transduction of the EAT-2 gene. Several reports have shown that enhancing the activation of NKT cells can positively influence the initial activation of DCs and/or NK cells, thereby increasing DC-dependent adaptive (cellular) immune responses 45_49 . The inventors' data suggests that harnessing this potential capability is of great interest in designing next generation vaccines.
  • EAT-2 is the only SLAM family receptors adaptor molecule currently known to be expressed in APCs 19 .
  • Ad-EAT2 transduced DCs may directly facilitate induction of antigen specific adaptive immune responses observed in this work.
  • EAT-2 in a variety of alternative cell types may also play an important role.
  • Various studies in mouse models and non-human primates report that improving the breadth of the cellular immune responses elicited by a vaccine to a target antigen is positively correlated with an improved ability of the vaccine to induce protective immiinity 4 ' 50 .
  • the inventors' results demonstrate that administering Ad-HIV/Gag with Ad-EAT2 increased the breadth of the cellular immune responses to the HIV-Gag antigen, which correlated with a significantly improved in vivo cytolytic activity of HIV/Gag specific CD8 + T cells generated after Ad-HIV/Gag and Ad- EAT2 co-immunization.
  • the inventors observed similar results in both C57BL/6 and Balb/c mice, (two mouse strains that can bias adaptive immune responses to a Thl or Th2 response, respectively) indicating that the adjuvant effect of Ad-EAT-2 is not specifically limited by significant immuno-genetic background differences of the host animal, at least in this species.
  • the inventors' findings suggest that enhancing SLAM family receptors signaling by overexpressing EAT-2 during antigen vaccination can serve to improve the ability of a vaccine to stimulate the innate immune system, and subsequently induce improved, antigen specific adaptive immune responses.
  • NK cells represent a subset of innate immune cells that have been shown to play an important role in bridging innate and adaptive immune responses, by influencing DC function ' , providing signals for augmenting Thl immune responses " , and inducing tumor- specific CTLs 40 .
  • NK cell-mediated cytotoxicity of antigen- expressing target cells induces robust antigen-specific adaptive immune responses 41 .
  • EAT-2 has been shown to be indispensable in activating NK cell cytotoxicity, by acting as a downstream adaptor protein facilitating signaling from SLAM family receptor CRACC in mice 42 ' 43 or NTB-A in humans 44 .
  • NK cell activation the latter of which could further drive the activation and/or maturation of DCs and bias the HIV-Gag specific immune profile towards a Thl response .
  • Adenovirus Vectors Comprising SLAM Family Receptor Adaptor Proteins (SAPs)
  • the invention provides replication incompetent recombinant adenovirus vectors comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
  • SAP SLAM family receptor adaptor protein
  • the invention's vectors that encode a SLAM family receptor adaptor protein (SAP) may be used as immune enhancing adjuvants by co-administration with a second vector encoding an antigen.
  • the SAP (as exemplified by EAT-2 SEQ ID NO:01).
  • adenovirus refers to a double- stranded DNA adenovirus of animal origin, such as avian, bovine, ovine, murine, porcine, canine, simian, and human origin.
  • Avian adenoviruses are exemplified by serotypes 1 to 10, which are available from the ATCC, such as, for example, the Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), and K-l 1 (ATCC VR-921) strains, or else the strains designated as ATCC VR-831 to 835.
  • Bovine adenoviruses are illustrated by those available from the ATCC (types 1 to 8) under reference numbers ATCC VR-313, 314, 639-642, 768 and 769.
  • Ovine adenoviruses include the type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340).
  • Murine adenoviruses are exemplified by FL (ATCC VR-550) and E20308 (ATCC VR-528).
  • Porcine adenovirus (5359) may also be used.
  • Adenoviruses of canine origin include all the strains of the CAVI and CAV2 adenoviruses (for example, Manhattan strain or A26/61 (ATCC VR-800) strain).
  • Simian adenoviruses are also contemplated, and they include the adenoviruses with the ATCC reference numbers VR-591-594, 941-943, and 195-203.
  • adenoviruses of which there greater than fifty (50) serotypes are known in the art, are also contemplated, including Ad2, Ad3, Ad4, Ad5, Adl 1, Ad 14, Ad7, Ad9, Adl2, Adl6, Adl7, Ad21, Ad26, Ad34, Ad35, Ad 40, Ad48, Ad49, Ad50 (e.g., U.S. Patent No. 7,300,657 to Pau, U.S. Patent No. 7,468,181 to Vogels, and U.S. Patent No. 6,136,594 to Dalemans).
  • Ad5 adenovirus 5
  • vector refers to an agent that contains and/or transfers genetic material from one cell to another, including for example viruses, bacteriophages, and plasmids.
  • a vector may be used to transfer, introduce and/or insert exogenous modified genetic material (as recombinant DNA) into the genome of a recipient (host) cell. Delivery of genetic material by a "viral vector” is termed “transduction,” with the infected cells described as “transduced.” This process can be performed inside a living organism (in vivo) or in cell culture (in vitro).
  • Viral based gene transfer/vectors include, for example, adenovirus, adeno-associated virus, retroviruses, alphaviruses, lentiviruses, vaccinia viruses, baculo viruses, fowlpox and herpesviruses.
  • Adenovirus may be used as a "viral vector" for gene therapy and vaccination since its DNA does not integrate into the host cell genome, does not replicated during host cell division and it infects a wide range of dividing and non- dividing cells.
  • One strategy for manipulating the tropism of Ad vectors is to insert receptor- binding ligands into the major capsid proteins (fiber, penton base and hexon) at exposed positions, in order to recruit alternative receptors on target cells.
  • a potential location for inserting targeting ligands is the HI loop of the fiber knob, in the hypervariable region 5 of hexon loop LI and in the RGD-motif loop of penton base.
  • oligopeptide ligands may be added to the C-terminus of the adenovirus fiber protein HI loop.
  • the adenovirus capsid is innately pro-inflammatory, as it activates the complement system. This is due, at least in part, to the fact that adenoviruses are a common human pathogen. The proinflammatory nature of the adenovirus capsid often results in the triggering of a rapid immune response when adenoviruses are used a viral vectors.
  • the invention's vectors are preferably replication incompetent.
  • "Replication incompetent,” “replication defective,” “replication attenuated” are used interchangeably to refer to a virus and/or viral vector that has a reduced level of replication compared to wild type virus and/or to a viral vector containing wild type virus nucleotide sequences.
  • Methods for producing replication incompetent adenoviral vectors are known in the art (e.g., U.S. Patent Nos.
  • a replication incompetent adenovirus viras and/or adenoviral vector (a) lacks (i.e., has a deletion of) adenovirus El gene coding sequence, (b) lacks adenovirus El gene coding sequence and E2b gene coding sequence (c) lacks adenovirus El gene coding sequence and adeno viras E4 gene coding sequence, (d) lacks adenovirus El gene coding sequence and adenovirus E2a gene coding sequence, and/or (e) lacks adenovirus El gene coding sequence and adenovirus EIVa2 gene coding sequence.
  • the replication incompetent adenovirus virus and/or adenoviral vector is a gutted adenovirus.
  • gutted and “fully deleted” are used interchangeably in reference to a viral vector, and refer to a viral vector (e.g., plasmid, viras, naked DNA) that lacks all the coding sequences that are otherwise present in a wild-type virus.
  • Gutted vectors may contain non-coding viral sequences, e.g., terminal repeat sequences, and packaging sequences.
  • a gutted adenovirus vector lacks all adenovirus coding sequences and optionally contains adenovirus terminal repeat sequences and/or packaging sequences (e.g., U.S.
  • Gutted vectors are preferred in certain embodiments since they do not express viral vector proteins and hence do not induce an adverse immune or toxic response in a cell.
  • the invention's vectors contain nucleotide sequenced encoding at least a portion of a SLAM family receptor adaptor protein (SAP).
  • SAP SLAM family receptor adaptor protein
  • the invention's vectors may further comprise a second heterologous nucleic acid sequence that encodes a polypeptide of interest. This may be desirable in applications (e.g., vaccination) that utilize a single vector that expresses both an immune enhancer as well as an antigen.
  • the invention's compositions and methods may be used for expression of, delivery of, and/or vaccination against, a polypeptide of interest.
  • polypeptide of interest refers to any polypeptide sequence, nucleotide sequence, and molecule, respectively, the manipulation of which may be deemed desirable for any reason, by one of ordinary skill in the art.
  • Illustrative polypeptides of interest include endogenous polypeptides, heterologous polypeptides, pathogen derived antigens, disease associated antigens, and reporter sequences.
  • heterologous DNA includes a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to wliich it is not ligated in nature, or to which it is ligated at a different location in nature.
  • Heterologous DNA also includes a nucleotide sequence, which is naturally found in the cell or virus into which it is introduced and which contains some modification relative to the naturally-occurring sequence.
  • heterologous DNA encodes heterologous RNA and protein of interest that are not normally produced by the cell and/or virus into which it is introduced.
  • polypeptides of interest include proteins, and portions thereof, that are expressed as glycoproteins, membrane proteins and portions thereof, soluble proteins and portions thereof, epitopes and portions thereof, and the like.
  • the polypeptide of interest comprises a membrane protein (U.S.
  • a “membrane protein” refers to a protein that is at least partially embedded in the lipid bilayer of a cell, virus and the like.
  • Membrane proteins contain a cytoplasmic domain, a transmembrane domain, and an ectodomain.
  • the protein of interest comprises an ectodomain of a membrane protein.
  • ectodomain when in reference to a membrane protein refers to the portion of the protein that is exposed on the extracellular side of a lipid bilayer of a cell, virus and the like. Methods for determining the ectodomain of a protein are known in the art (Singer (1990); High et al. (1993), and McVector software, Oxford Molecular).
  • Exemplary ectodomains include, but are not limited to those described in U.S. Patent Nos. 7,262,270; 7,253,254; 7,250,171 ; 7,223,390; 7,189,403; 7,122,347; 7,119,165; 7,101,556; 7,067,110; 7,060,276; 7,029,685; 7,022,324; 6,946,543; 6,939,952; 6,713,066; 6,699,476; 6,689,367; 6,566,074; 6,531,295; 6,417,341; 6,248,327; 6,140,059; 5,851,993; 5,847,096; 5,837,816; 5,674,753; and 5,344,760. Additional examples of ectodomains include ectodomains of membrane type 1 proteins, type 2 proteins, and type 3 proteins (U.S. Patent Application US 2009/0068221 to Morrison).
  • the protein of interest comprises a soluble protein.
  • soluble protein refers to a protein that is not embedded in the lipid bilayer of a cell, virus and the like. Examples of soluble proteins are known in the art (U.S. Patent Application US 2009/0068221 to Morrison).
  • the polypeptide of interest comprises an antigen.
  • antigen means "antigen,” “immunogen,” “antigenic,” “immunogenic,” “antigenically active,”
  • immunological and “immunologically active” when made in reference to a molecule, refer to any substance that is capable of inducing a specific humoral immune response (including eliciting a soluble antibody response) and/or cell-mediated immune response (including eliciting a CTL response).
  • the antigen is exemplified by Human
  • Immunodeficiency virus gag protein (SEQ ID NO:03), malaria CSP M (SEQ ID NO:07; Figure 19), CSP T-cell epitope (SEQ ID NO:09; Figure 19), CSP B-Cell epitope (SEQ ID NO:10; Figure 19), CSPi 56 (SEQ ID NO:l 1 ; Figure 19), malaria CSP encoded by SEQ ID NO:08 of Figure 20, and Pseudomonas antigen.
  • the polypeptide of interest is an antigen that comprises an epitope.
  • epitope and “antigenic determinant” refer to a structure on an antigen, which interacts with the binding site of an antibody or T cell receptor as a result of molecular complementarity.
  • An epitope may compete with the intact antigen, from which it is derived, for binding to an antibody.
  • secreted antibodies and their corresponding membrane-bound forms are capable of recognizing a wide variety of substances as antigens, whereas T cell receptors are capable of recognizing only fragments of proteins which are complexed with MHC molecules on cell surfaces.
  • Antigens recognized by immunoglobulin receptors on B cells are subdivided into three categories: T-cell dependent antigens, type 1 T cell-independent antigens; and type 2 T cell-independent antigens.
  • T-cell dependent antigens type 1 T cell-independent antigens
  • type 2 T cell-independent antigens type 2 T cell-independent antigens.
  • An antigenic determinant may compete with the intact antigen (i.e. , the immunogen used to elicit the immune response) for binding to an antibody.
  • Exemplary epitopes include, without limitation YPYDVPDYA (SEQ ID NO: 12) (U.S. Patent
  • EphrinA2 epitopes from renal cell carcinoma and prostate cancer U.S. Patent No. 7,297,337), hepatitis C virus epitopes (U.S. Patent Nos. 7,238,356 and 7,220,420), vaccinia virus epitopes (U.S. Patent No. 7,217,526), dog dander epitopes (U.S. Patent No. 7,166,291), human papilloma virus (HPV) epitopes (U.S. Patent Nos. 7,153,659 and 6,900,035), Mycobacterium tuberculosis epitopes (U.S. Patent Nos.
  • the polypeptide of interest is "pathogen derived,” meaning expressed by a pathogen (e.g., bacteria, virus, parasite, protozoan, fungus, etc.), such as Herpes virus, Neisseria gonorrhea, Treponema, Escherichia coli, Respiratory Syncytial virus, tuberculosis, Streptococcus, Chlamydia, and Ebola virus.
  • a pathogen e.g., bacteria, virus, parasite, protozoan, fungus, etc.
  • pathogen e.g., bacteria, virus, parasite, protozoan, fungus, etc.
  • Herpes virus e.g., bacteria, virus, parasite, protozoan, fungus, etc.
  • a pathogen e.g., bacteria, virus, parasite, protozoan, fungus, etc.
  • Herpes virus e.g., bacteria, virus, parasite, protozoan
  • HIV Human Immunodeficiency virus
  • gag protein including the HXB2 strain gag protein (Genbank Accession #K03455) disclosed in the below Examples
  • HIV Gag protein antigen such as HIV Gap protein immunodominant peptide AMQMLKETI (SEQ ID NO:06), HIV Pol protein antigen, HIV Nef protein antigen, malaria CSP f n antigen (SEQ ID NO:07, Figure 19), CSP antigen encoded by DNA sequence SEQ ID NO:08 ( Figure 20), malaria CSP T cell epitope (SEQ ID NO:09; EYLNKIQNSLSTEWSPCSVT; Figure 19), malaria CSP B Cell epitope (SEQ ID NO: 10; N ANPN ANPN ANPN ANPN ANP ;
  • Pseudomonas refers to a gram-negative, rod-shaped and polar-flagella bacteria of the proteobacteria genus. Most Pseudomonas species are naturally resistant to penicillin and the majority of related beta-lactam antibiotics. This low antibiotic susceptibility is attributable, at least in part, to the concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Infectious species in animals, including humans, are Pseudomonas aeruginosa, Pseudomonas oryzihabitans, and plecoglossicida.
  • Pseudomonas aeruginosa is recognized as an emerging opportunistic pathogen of clinical relevance since epidemiological studies indicate that antibiotic resistance is increasing in clinical isolates. P. aeruginosa flourishes in hospital environments, and is a particular problem in this environment since it is the second most common infection in hospitalized patients (i.e. nosocomial infections).
  • "Pseudomonas antigen” includes serogroup 011 O-antigen (Dean et al. (1999) J. Bacteriol. 181(14):4275-84; Deana et al. (2006) FEMS Micorbilogy Letters 187:59 - 630);
  • Pseudomonas PcRv antigen U.S. Patent Application No. 20090191241
  • Pseudomonas exotoxin A translocation domain II U.S. Patent No. 7,595,054, issued 9/29/09
  • the polypeptide of interest is "disease associated,” meaning a polypeptide whose level (e.g., presence, absence, increase, and/or decrease relative to a control, etc.) that is correlated with disease and/or with risk of disease based on family history, genetic factors, environmental factors, etc.
  • Disease associated polypeptides of interest are exemplified by those encoded by, but not limited to, the adenosine deaminase (ADA) gene (GenBank Accession No. Ml 3792) associated with adenosine deaminase deficiency with severe combined immune deficiency; alpha- 1 -antitrypsin gene (GenBank Accession No.
  • Ml 1465) associated with alphal -antitrypsin deficiency
  • beta chain of hemoglobin gene (GenBank Accession No. NM_000518) associated with beta thalassemia and Sickle cell disease
  • receptor for low density lipoprotein gene (GenBank Accession No. D 16494) associated with familial hypercholesterolemia
  • lysosomal glucocerebrosidase gene (GenBank Accession No. K02920) associated with Gaucher disease
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • NP phosphorylase
  • Ml 8533, M17154, and Ml 8026 associated with muscular dystrophy
  • the utrophin (also called the dystrophin related protein) gene (GenBank Accession No. NM_007124) whose protein product has been reported to be capable of functionally substituting for the dystrophin gene
  • the human cystic fibrosis transmembrane conductance regulator (CFTR) gene (GenBank Accession No.M28668) associated with cystic fibrosis.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the disease associated polypeptide of interest is a cancer derived antigen such as carcino-embryonic antigen (CEA) and her2neu antigen.
  • CEA carcino-embryonic antigen
  • her2neu antigen such as her2neu antigen
  • the polypeptide of interest that is expressed by the vectors of the invention may include a reporter polypeptide sequence.
  • Reporter sequence and “marker sequence” are used interchangeably to refer to DNA, RNA, and/or polypeptide sequence that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.
  • Exemplary reporter genes include, for example, ⁇ -glucuronidase gene, green fluorescent protein (GFP) gene, E. coli ⁇ -galactosidase (LacZ) gene, Halobacterium ⁇ - galactosidase gene, E.
  • the reporter sequence comprises GFP, produced by the adenovirus vector (Ad-GFP/rEA) co-expressing GFP and EAT-2. This was constructed using the pAd-Track shuttle.
  • the invention further provides cells that are susceptible to, and useful for packaging the, invention's adenoviral vectors. Without limiting the type of cell, these cells include 293 (Adenovirus), A549 (Adenovirus), HEL (Adenovirus), HEL-299 (Adenovirus), HEp-2 (Adenovirus), HFL-Chang (Adenovirus), HNK (Adenovirus), Hs27 (HFF) (Adenovirus), KB (Adenovirus), LLC-MK2 (Adenovirus), MDCK (Adenovirus), MRC-5 (Adenovirus), MRHF (Adenovirus), NCI-H292 (Adenovirus), pRK (Adenovirus), RK1 (Adenovirus), R-Mix
  • Adenovirus (Adenovirus), Vero (Adenovirus), WI 38 (Adenovirus), HeLa (Adenovirus 3), and HeLa S-4 (Adenovirus 5). These cells are available from Diagnostic Hybrids, Inc., Athens, OH.
  • Additional adenovirus packaging cells are known in the art, including those described in U.S. Patent Nos. US 7582290 to Rodriguez et al., 6602707 and 7105346 to Fallaux et al., 7026164 and 7074618 to Yuanhao et al., 6492160 to Linschoren et al., 6033908 to
  • compositions may be used as vaccines.
  • the inventing provides a composition comprising (a) a replication incompetent recombinant adenovirus vector as described herein and (b) a pharmaceutically acceptable carrier.
  • vaccine refers to a pharmaceutically acceptable preparation that may be administered to a host to induce a humoral immune response (including eliciting a soluble antibody response) and/or cell -mediated immune response (including eliciting a CTL response).
  • compositions that contain molecules that are capable of administration to or upon a subject and that do not substantially produce an undesirable effect such as, for example, adverse or allergic reactions, dizziness, gastric upset, toxicity and the like, when administered to a subject.
  • pharmaceutically acceptable molecule does not substantially reduce the activity of the invention's compositions.
  • Pharmaceutical molecules include, but are not limited to excipients and diluents. Vaccines may contain
  • adjuvant refers to any compound which, when injected together with an antigen, non-specifically enhances the immune response to that antigen.
  • exemplary adjuvants include Complete Freund's Adjuvant, Incomplete Freund's Adjuvant, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI fowl adjuvant (MPL; RIBI
  • carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex;
  • glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
  • Exemplary "diluents” include water, physiological saline solution, human serum albumin, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylene diamine-tetra- acetic acid, buffers such as acetates, citrates or phosphates and agents for adjusting the osmolarity, such as sodium chloride or dextrose.
  • antibacterial agents such as benzyl alcohol
  • antioxidants such as ascorbic acid or sodium bisulphite
  • chelating agents such as ethylene diamine-tetra- acetic acid
  • buffers such as acetates, citrates or phosphates
  • agents for adjusting the osmolarity such as sodium chloride or dextrose.
  • carriers include liquid carriers (such as water, saline, culture medium, saline, aqueous dextrose, and glycols) and solid carriers (such as carbohydrates exemplified by starch, glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid and glutathione, and hydrolyzed proteins.
  • liquid carriers such as water, saline, culture medium, saline, aqueous dextrose, and glycols
  • solid carriers such as carbohydrates exemplified by starch, glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid and glutathione, and hydrolyzed proteins.
  • excipient refers herein to any inert substance (e.g., gum arabic, syrup, lanolin, starch, etc.) that forms a vehicle for delivery of an antigen.
  • excipient includes substances that, in the presence of sufficient liquid, impart to a composition the adhesive quality needed for the preparation of pills or tablets.
  • compositions may be administered in an immunologically effective amount.
  • immunologically effective amount refers to, in one embodiment, an amount of the composition that delays, reduces, palliates, ameliorates, stabilizes, prevents and/or reverses one or more symptoms of the disease compared to in the absence of the composition of interest. Examples include, without limitation, tumor size and/or tumor number in cancer disease, glucose levels in blood and/or urine in diabetes, standard biochemical kidney function tests in kidney disease, etc.
  • the terms also include, in another embodiment, an amount of the composition that reduces infection by a pathogen
  • dosages can be readily determined by clinical trials and depend, for example, on the route of administration, patient weight (e.g. milligrams of drug per kg body weight).
  • delaying symptoms refers to increasing the time period between exposure to the immunogen or virus and the onset of one or more symptoms of the exposure.
  • eliminating symptoms refers to 100% reduction of one or more symptoms of exposure to the immunogen or virus.
  • “pharmaceutically effective amount,” “therapeutically effective amount,” “immunologically effective,” and “protective amount” will depend on the route of administration, the type of subject being treated, and the physical characteristics of the specific subject under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical, veterinary, and other related arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors that those skilled in the art will recognize. The dosage amount and frequency are selected to create an effective level of the compound without substantially harmful effects.
  • Methods of administering a pharmaceutically effective amount of the invention's compounds are well known in the art and include, without limitation, intramuscular administration, intradermal administration, subcutaneous administration, aerosol
  • Administration may be parenteral, oral, intraperitoneal, intranasal, topical, etc.
  • Parenteral routes of administration include, for example, subcutaneous, intravenous, intramuscular, intrastemal injection, and infusion routes.
  • compositions of the invention may be administered before, concomitantly with, and/or after manifestation of one or more symptoms of a disease or condition.
  • the invention's compositions may be administered before, concomitantly with, and/or after administration of another type of drug or therapeutic procedure (e.g., surgery).
  • another type of drug or therapeutic procedure e.g., surgery.
  • the invention's compounds may be administered before, concomitantly with, and/or after administration of antibiotics and/or antivirals.
  • the composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for delivery by injection, h a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • an immunologically-effective amount of the invention's compositions produces a) an innate immune response against the polypeptide of interest and/or b) an adaptive immune response against the polypeptide of interest, and/or c) an altered transcriptome response, when compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
  • a control e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)
  • immunologically-effective amount and “immunologically- effective amount” refer to that amount of a molecule that elicits and/or increases production of an immune response (including production of specific antibodies and/or induction of a TCL response) in a host upon vaccination.
  • immunologically-effective amount refers to that amount of a molecule that elicits and/or increases production of an immune response (including production of specific antibodies and/or induction of a TCL response) in a host upon vaccination.
  • an immunologically-effective amount of the invention's compositions produces an innate immune response against the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
  • a control e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)
  • Innate immune response and “innate immune system” refers to the number and/or biological activity of cells, cytokines and/or chemokines (e.g., IL-12, MCP-1, G.CSF, IL-6, ⁇ -lbeta, RANTES) (e.g. in the plasma space) that defend the host from infection by other organisms, in a non-specific manner.
  • cytokines and/or chemokines e.g., IL-12, MCP-1, G.CSF, IL-6, ⁇ -lbeta, RANTES
  • the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
  • Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.
  • the major functions of the vertebrate innate immune system include recruiting immune cells to sites of infection and inflammation, through the production of chemical factors, including specialized chemical mediators, called cytokines.
  • An additional function is activation of the complement cascade to identify bacteria, activate cells and to promote clearance of dead cells or antibody complexes.
  • a further function is the identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells.
  • Yet another function includes activation of the adaptive immune system through a process known as antigen presentation.
  • Innate immune responses following administration of the invention's adenoviral compositions may be measured by methods known in the art and those disclosed herein, including, detecting influx of cytokines and chemokines into the plasma space, and/or detecting an increase in activation of cells of the innate immune system, such as NK cells, Dendritic Cells, T cells, B-cells and/or other antigen presenting cells..
  • the innate immune response comprises a) an increase in the level of one or more splenocyte cell that is "specific" for the polypeptide of interest, and that expresses one or more of CD3, CD8, NK1.1 and CD69, and b) a reduction in the level of antibody that specifically binds with the polypeptide of interest.
  • the innate immune response comprises an increase in the level of at least one of IL-12, MCP- 1, G-CSF, IL-6, MlP-lbeta, and RANTES (e.g., in the plasma space) of the subject compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
  • a control e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
  • SAP SLAM family receptor adaptor protein
  • the innate immune response comprises an increased number of CD69 positive lymphocytes that are specific for the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
  • the CD69 positive lymphocytes are exemplified by natural killer (NK) cells and natural killer T (NKT) cells.
  • NK Natural killer
  • NK cytotoxic lymphocytes that constitute a major component of the innate immune system, and play a major role in the rejection of tumors and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis or necrosis.
  • NK cells are CD3 - NK1.1 + lymphocyte cells.
  • NKT Natural killer T cells
  • CD3+ NK1.1+ lymphocyte cells CD3+ NK1.1+ lymphocyte cells.
  • the inventor's data demonstrate that an exemplary Adenovirus vector expressing the exemplary SLAM family receptor adaptor protein (SAP) EAT-2, induces greater in vivo activation of innate immune cells (Example 2 and 3).
  • SAP SLAM family receptor adaptor protein
  • an immunologically-effective amount of the invention's compositions produces an adaptive immune response against the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
  • a control e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)
  • Adaptive immune response and “adaptive immune system” refers to the number and/or biological activity of highly specialized, systemic cells and molecules that eliminate or prevent pathogenic challenges.
  • the adaptive or “specific” immune system is activated by the "non-specific” and evolutionarily older innate immune system.
  • the adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. It is adaptive immunity because the body's immune system prepares itself for future challenges.
  • the major functions of the adaptive immune system include the recognition of specific "non-self antigens in the presence of "self, during the process of antigen
  • Another function includes the generation of responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells. Yet a further function includes the development of immunological memory, in which each pathogen is
  • Adaptive immune responses following administration of the invention's compositions comprise non-specific cellular mediated immune (CMI) responses, such as those mediated by interferon gamma (IFNy) (IENgamma).
  • CMI non-specific cellular mediated immune
  • IFNy interferon gamma
  • the adaptive immune response comprises an increase in the level of IFNy produced by one or more cell that is specific for the polypeptide of interest, wherein the cell is selected from CD3+/CD8+ T-cell, CD3+/CD8- T cell, CD3- NK1.1+ natural killer cell, and/or CD3+/NK1.1+ natural killer T cell.
  • the inventors' data show that an exemplary Adenovirus vector expressing the exemplary SLAM family receptor adaptor protein (SAP) EAT-2, induces greater in vivo activation of adaptive immune cells (Example 4) including when co-administered with and Adenovirus expressing an HIV antigen (Example 5).
  • SAP SLAM family receptor adaptor protein
  • the methods of the invention produce an immune response that comprises an increase in cytolytic activity of CD8+ T cells that specifically bind to the antigenic polypeptide of interest.
  • Cytolytic activity refers to cell killing as determine by, for example, a CTL assay as described herein in Example 1.
  • CTLs Cytotoxic T lymphocytes
  • SAP SLAM family receptor adaptor protein
  • the Ad-GFP virus was purified as previously described 51 .
  • the Open Reading Frame (ORF) of EAT-2 gene (Genbank Accession # NM_053282.4) was excised using primers flanked by Xhol andXbal restriction endonucleases (NEB, Ipswich, MA) from a plasmid (Open Biosystem, Huntsville, USA) and subcloned into the pAdTrack Shuttle vector which contains a CMV expression cassette.
  • the resulting pAdTrack-EAT-2 shuttle plasmid was linearized with Pmel restriction enzyme and homologously recombined with the pAdEasyl Ad5 vector genome as previously described 2 yielding pAd-EAT2.
  • HEK293 cells were transfected with Pad linearized plasmid and viable virus was obtained and amplified after several rounds of expanding infection.
  • Ad-EAT2 virus was purified using a CsCl 2 gradient as previously described 53 .
  • the titer obtained was approximately 2.3> ⁇ 10 12 vp/ml.
  • Direct sequencing and restriction enzyme mapping were carried out to confirm the integrity of the EAT-2 sequence.
  • To construct Ad-HIV/Gag the HXB2 Gag gene (Genbank Accession #K03455) was blunt end sub-cloned into the EcoRV site of pShuttle-CMV. Restriction digests and sequencing were used to confirm the sequence integrity and correct orientation of the resulting shuttle (pShuttle-CMV Gag). Recombination and viral propagation was completed as described above. EAT-2 and Gag expression was verified using Western blotting.
  • mice All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • Adult male C57BL/6 or Balb/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
  • Ad5 vectors were injected intravenously (via the retro-orbital sinus, total volume 200 ⁇ ) or intramuscularly (into the tibialis anterior of the right hindlimb, total volume 20 ⁇ ) into 8 weeks old male mice after performing proper anesthesia with isofluorane.
  • a total of 7.5 x 10 10 vps in 200 ⁇ of PBS was injected per mouse intravenously.
  • Several groups of mice were analyzed in this work:
  • Ad-GFP Ad-GFP
  • control groups injected with conventional Ad Ad-GFP
  • mice were injected with lxlO 7 or lx 10 9 ps/mouse (BALB/c or C57B1/6),
  • Ad-HIV/Gag+ Ad-GFP control group co-immunized with first generation Ad5-based vaccine
  • Ad-HIV/Gag+ Ad-EAT2 experimental group, co-immunized with Ad-HIV/Gag and
  • Cytokine and chemokine analysis A mouse 7-plex multiplex based assay was used to determine the indicated cytokine/ chemokine plasma concentrations per the manufacturer's instructions (Bio-Rad, Hercules, CA) via Luminex 100 technology (Luminex, Austin, TX) essentially as previously described 5 .
  • ELISPOT analysis Splenocytes were harvested from individual mice and RBCs were lysed using ACK lysis buffer (Invitrogen, Carlsbad, CA). 96-well Multiscreen high protein binding Immobilon-P membrane plates (Millipore, Billerica, MA) were pre-treated with ethanol, coated with mouse anti-IFNy or IL-2 capture antibodies, incubated overnight, and blocked prior to the addition of 5x 10 5 splenocytes/well.
  • Ex vivo stimulation included the incubation of splenocytes in 100 of media alone (unstimulated) or media containing 4 ⁇ g/ml of Gag specific peptides (Gag-AMQMLKETI: SEQ ID NO:06 constructed by Genscript, Piscataway, NJ) or QBI# 304796 for 24 hours in a 37°C, 5% C0 2 incubator. 15mer Gag specific peptides were obtained from the ⁇ AIDS Reagent and Reference Program Cat# 8117 Lot# 9.
  • Splenocytes were stained with various combinations of the following antibodies: PE-CD69, (3 ⁇ ), FITC-CD8a, APC-CD3, APC-Cy7-CD3, Alexa Floure700-CD8a, PerCpCy5.5-CD19, PE-Cy7-NK1.1, PE-Cy7-TNF , APC-IFNy (4 ⁇ g/ml) (All obtained from BD Biosciences, San Diego, CA), and PerCpCy5.5-IL-2 (4 ⁇ g/ml) (BioLegend, San Diego, CA).
  • Cells were incubated on ice with the appropriate antibodies for 30 minutes, washed, and sorted using an LSR ⁇ instrument and analyzed using FlowJo software.
  • intracellular cytokines staining cells were surface stained, fixed with 2% formaldehyde (Polysciences, Warrington, PA), permeabilized with 0.2% Saponin (Sigma- Aldrich, St. Louis, MO), and stained for intracellular cytokines. Large cells and debris were excluded in the forward- and side-scatter plot, to minimize background levels of staining caused by nonspecific binding of antibodies; we initially stained the cells with CD 16/32 FcR ⁇ / ⁇ antibody.
  • ViViD violet fluorescent reactive dye
  • PBMCs were isolated using Lympholyte-Mammal (Cedarlane, Burlington NQ.Tetramer staining of PBMCs was completed using a PE conjugated MHC-I tetramer folded with the AMQMLKETI peptide (SEQ ID NO:06) generated at the NIH Tetramer Core Facility.
  • CD8 + T cells were depleted from pooled splenocytes preparations using MACS beads and LS columns per the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).
  • EAT-2-expressing Ad vectors enhance Ad vector induced innate immune responses in vivo.
  • Ad-EAT2 adenovirus vector specifically designed to express the SLAM family receptors adaptor protein EAT-2. These EAT-2 expressing Ad vectors were fully viable, grew to high titers, and were purified and quantified as done for conventional Ad vaccines 5 ' 23 .
  • Ad-EAT2 was systemically administered into C57BL/6 mice and measured the levels of cytokines and chemokines in the plasma 6 hours post injection (h.p.i.).
  • Systemic administration of the Ad-EAT2 into C57BL/6 mice resulted in induction of significantly higher plasma levels of RANTES and MCP-1 at 6 h.p.i. as directly compared to Ad-GFP injected control mice (Fig. 1).
  • IL-6, IL-12p40, ⁇ - ⁇ , KC, and G-CSF were also significantly induced by Ad-EAT2 vectors; however, these levels were not statistically different between Ad-EAT2 and Ad-GFP injected mice (Fig. 10).
  • EAT-2 expressing Ad induces greater activation of innate immune cells.
  • Ads enhance innate immune cell effector functions 4 ⁇ 26 .
  • EAT-2 lymphocyte activation marker CD69 as well as IFN- ⁇ production in various immune cells shortly after administration of Ad-EAT2 into C57BL/6 mice.
  • Our results confirm that Ads, in general, significantly induce a rapid activation of NK and NKT cells in both PBMCs and spleens, as measured by the presence of increased percentages of CD69 expressing NK and NKT cells (Fig. 2a and b).
  • Ad-EAT2 Since SLAM family receptors are expressed in various innate and adaptive immune cells 13 and the activation of T cell and/or B cells can be initiated or accentuated by innate immune system activation 9 , we sought to analyze adaptive immune cell responses shortly after administration of Ad-EAT2. Our results first confirmed that Ad vector administration induces a rapid activation of CD3 + CD8 + T-cells CD3 + CD8 ⁇ T-cells, and B cells. For example, injection of Ad-EAT2 resulted in significantly higher numbers of splenic CD69 expressing CD3 + CD8 + T cells (p ⁇ 0.01), CD3 + CD8 " T cells (p ⁇ 0.01), and B cells (p ⁇ 0.001) at 6 h.p.i (Fig.
  • Ad vector expressing EAT-2 enhances T cell responses to a co-administered antigen.
  • Simultaneous administration of vaccines with adjuvants can stimulate the innate immune system to significantly improve the adaptive immune responses to a co-administered antigenic target 27"30 .
  • an Ad-based vector expressing the HIV-1 clade B Gag protein (HXB2) along with either the Ad-GFP, or the Ad- EAT2 vectors.
  • HXB2 HIV-1 clade B Gag protein
  • PBMCs p ⁇ 0.05
  • splenocytes p ⁇ 0.05
  • Ad- EAT2 co-immunization resulted in significantly (pO.001) increased numbers of Gag-specific IFN- ⁇ secreting splenocytes (Fig. 4a, and b, respectively).
  • Fig. 4a, and b We also observed significantly increased numbers of IL-2 secreting splenocytes derived from Balb/c mice co-immunized with Ad-HIV/Gag + Ad-EAT2 as compared to Ad-HIV/Gag+ Ad-GFP co-immunized mice (p ⁇ 0.01) (Fig.
  • CD8 + T cells derived from Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to control mice.
  • TNFa or IL-2 single positive cells we also observed increased numbers of CD8 + T cells that express TNFa or IL-2 derived from Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to Ad-HIV/Gag and Ad-GFP co-immunized mice; however, these trends were not statistically significant.
  • Memory CD8 + T cells from Ad-EAT2 co-immunized mice exhibit Gag-specific cytotoxicity in vivo.
  • CD8 + cytotoxic T lymphocytes provides a critical assessment as to the functional capacity of antigen-specific CD8 + T cells to kill cells displaying an antigen derived peptide.
  • CTL cytotoxic T lymphocytes
  • mice 14 days after vaccination, the two groups of mice were then injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic splenocytes pulsed with the Gag derived peptides AMQMLKETI (SEQ ID NO:06) or QBI# 304796.
  • CFSE carboxyfluorescein succinimidyl ester
  • RAW 264.7 cells were passed into 12-well plates 1 day before the transfection at a concentration of 1 ⁇ 1 ⁇ 5 cells/well.
  • Cells were used for transfection with Calcium phosphate transfection reagent following the manufacturer's instructions in DMEM with 10% FBS.
  • Cells were then either un-transfected or transfected with 500ng, lug, or 2ug of P-Shuttle- CMV plasmid or P-Shuttle-CMV plasmid that expresses EAT-2 gene. 24 hours after transfection, media were changed and cell were re-incubated for another 48 hours.
  • Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nature medicine 13, 843-850 (2007).
  • NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 171, 5140-5147 (2003).

Abstract

The invention provides replication incompetent adenovirus vectors containing a heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further encodes a polypeptide of interest. The compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein. Furthermore, the compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response for vaccination applications.

Description

VACCINES COMPRISING ADENOVIRUS VECTORS AND SIGNALING LYMPHOCYTE ACTIVATING MOLECULE-ASSOCIATED PROTEIN (SAP)
FIELD OF INVENTION
The invention provides replication incompetent adenovirus vectors containing a heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further encodes at least a portion of a polypeptide of interest. The invention further provides DNA sequences that encode the invention's adenoviral vectors, as well as packaging cells that produce the invention's adenoviral vectors. The invention's compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP). The invention's compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, such as in vaccination applications. The invention's compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus. BACKGROUND
Although vaccines have provided a great impact on human health and disease, conventional vaccine technologies cannot elicit the robust immune responses required to eradicate some of the most pathogenic diseases to affect humanity, such as HIV- AIDS.
Adenoviral based gene delivery vectors remain one of the most promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing first generation Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections. In humans, Adenovirus based vaccines can induce potent cellular immune responses to HIV derived antigens (as compared to DNA or other virus based vaccine platforms), but these responses still do not reach levels noted in so called long-term non-progressors.
Most recently, a human clinical trial demonstrated a prophylactic vaccine to HIV1. However, the results of that trial were controversial, and combined with recent disappointing results in the STEP trial, suggest that a more potent cellular immunity inducing vaccine are needed to provide a greater efficacy in preventing HIV infection, and/or generating more potent T-cell responses to antigens, such as the HIV-Gag antigen. Thus there remains a need for new compositions and methods that increase the potency of vaccine platforms.
SUMMARY OF THE INVENTION
The invention provides a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP). In one embodiment, the vector further comprises c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest. In an alternative embodiment, the SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein selected from the group consisting of human EAT-2 (SEQ ID NO:01) and mouse EAT-2 (SEQ ID NO:29). In an alternative embodiment, the DNA sequence encoding a replication defective adenovirus lacks adenovirus El gene coding sequence. In a particular embodiment, the polypeptide of interest comprises an antigen polypeptide. Preferably, in one embodiment, the antigen polypeptide comprises Human immunodeficiency virus GAG sequence AMQMLKETI (SEQ ID NO: 06). In an alternative embodiment, the antigen polypeptide comprises Plasmodium falciparum circumsporozoite antigen NYDNAGTNL (SEQ ID NO:05).
The invention also provides a purified DNA sequence comprising, in operable combination, a) a nucleic acid sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), h one embodiment, the DNA sequence further comprises, in operable combination, c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest. In a particular embodiment, the polypeptide of interest comprises an antigen polypeptide. In one embodiment, the SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein SEQ ID NO:01.
The invention further provides a composition comprising any one or more of the replication incompetent recombinant adenovirus vectors described herein (such as a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further comprises c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest), and a
pharmaceutically acceptable carrier. Moreover, the invention provides a method for vaccinating a mammalian subject, comprising a) providing i) a first replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and ii) a second vector comprising a nucleotide sequence that encodes an antigen polypeptide, and b) administering an immunologically effective amount of the first vector and the second vector to the subject under conditions for producing an immune response to the antigen polypeptide, h one embodiment, the subject is at risk of disease. In another embodiment, the subject is at risk of infection by a pathogen. In one embodiment, the immune response comprises an adaptive immune response. In an alternative embodiment, the immune response comprises an innate immune response. In yet a further embodiment, the immune response comprises an increase in cytolytic activity of CD8+ T cells that specifically bind to the polypeptide of interest.
The invention also provides a method for vaccinating a mammalian subject, comprising a) providing a pharmaceutically acceptable composition comprising any one or more of the vectors disclosed herein, (such as a replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and c) a second heterologous nucleic acid sequence that encodes an antigen polypeptide, and b) administering an immunologically effective amount of the composition to the subject under conditions for producing an immune response to the polypeptide of interest. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : Systemic administration of EAT-2 expressing adenovirus vector induces cytokine and chemokines responses. C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 <1010 vps of either Ad-GFP or Ad-EAT2 vectors. Plasma was harvested at 6h after virus injection. Cytokine induction was evaluated using a multiplexed bead array based quantitative system. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman- euls post-hoc test, p<0.05 was deemed a statistically significant difference . * denotes p<0.05, ** denotes p<0.01, *** denotes pO.001. Figure 2: Ad-EAT2-mediated activation of innate and adaptive immune cells in vivo. C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 x 1010 vps of either Ad-GFP or Ad-EAT2. CD69 expression by PBMCs (A) and splenocytes (B) derived NK, NKT, CD3+CD8+, CD3+CD8", and B cells was evaluated 6h after virus injection.
PBMCs and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, ** denotes p<0.01, *** p<0.001, # denotes p<0.001 over mock.
Figure 3: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and Ad-EAT2 co-immunization in Balb/c mice after intramuscular injection. Mice were co- immunized intramuscularly in the tibialis anterior with equivalent viral particles of Ad- HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (total of lxlO7 vps mixed prior to injection). At 6 d.p.i, peripheral blood mononuclear cells (PBMCs) (A) were collected from the immunized mice and stained with a PE-conjugated H2-Kd-AMQMLKETI tetramer complex (A). At 14 d.p.i., mice were sacrificed and PBMCs (B) or splenocytes (C) were harvested and stained with a PE-conjugated H2-Kd-AMQMLKETI tetramer complex together with an APC-conjugated anti-CD3 and FITC-conjugated anti-CD8 antibodies. The bars represent mean ± SD for six mice per group (pool of two for PBMCs) for virus injected and three mice for naive animals. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, ** denotes p<0.01.
Figure 4: HIV-Gag specific cellular immune responses elicited by Ad-HIV/Gag and Ad-EAT2 co-immunization in Balb/c and C57B1/6 mice after intramuscular injection: Balb/c mice (n=6) (A) or C57B16 mice (n=4) (B) were co-immunized intramuscularly in the tibialis anterior with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad- EAT2 (total of 1 107 vps for Balb/c mice and total of 1 x 109 vps for C57B1/6 mice mixed prior to injection). At 14 d.p.i., splenocytes were harvested and stimulated ex vivo with the immunodominant peptides (AMQMLKETI: SEQ ID NO:06) for Balb/c and QBI# 804796 (EAMSQVTNSATMMQ) for C57B1/6. Spot forming cells (SFCs) were quantified using and ELISPOT reader. Data are presented as mean ± SD. Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, *** p<0.001. Representative data from two independent experiments are shown. Figure 5: Analysis of the breadth of Gag-responses after intramuscular injection of Ad-HIV/Gag and Ad-EAT2 in Balb/c mice: Balb/c mice were co-immunized intramuscularly with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (total dose of l xlO7 vps mixed prior to injection). At 14 d.p.i., animals were terminally sacrificed, and splenocytes were harvested and stinmlated ex vivo with 15mer HIV-Gag derived peptides QBI#804724, (S LYNT VATLYCVHQR: SEQ ID NO:07),
QBI#804753 (GHQ AAMQMLKETINE : SEQ ID NO:08), QBI#804754
(AMQMLKETINEE A AE : SEQ ID NO:09), QBI#804769 (PVGEIY1 RWIILGLN: SEQ ID NO: 10), QBI#804779 (VDRFYKTLRAEQ ASQ : SEQ ID NO:l l), QBI# 804800
(GNFRNQRKTVKCFNC: SEQ ID NO:12), or pool of three peptides (BQI# 804790, 804808, and 804826), and IFNy (A) and IL-2 (B) ELISPOT assays were completed. Bars represent mean ± SD. Statistical analysis was completed using Two-Way ANOVA with a Bonferroni post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, ** denotes p<0.01, ***p<0.001, # denotes pO.001 over mock.
Figure 6: Analysis of T cell epitope responses of Balb/c and C57B1/6 mice to HIV-
Gag in Ad-HIV/Gag and Ad-EAT2 co-injected mice. Balb/c (n=6) (A) or C57BL/6 (n=4) (B) mice were co-immunized with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad-EAT2 (l lO7 total vps for Balb/c and l xlO9 total vps for C57B1/6 mice). At 14 d.p.i. splenocytes were equivalently pooled and IL-2 ELISPOT analysis was carried out by stimulating individual wells ex vivo with a pool of 2-4 15mer peptides overlapped by 11 , not including peptides included in Figure 4 and 5. SFCs per million splenocytes are shown. The minimal threshold response is indicated by the line above 10.
Figure 7: Ad-HIV/Gag and Ad-EAT2 co-immunization increases the frequency of HIV-Gag specific CD8+ T cells. Balb/c (n=6) (A) or C57BL/6 (n=4) (B) mice were co- immunized with equivalent viral particles of Ad-HIV/Gag mixed with either Ad-GFP or Ad- EAT2 (1 x 107 total vps for Balb/c and 1 χ 109 total vps for C57B1/6 mice). At 14 d.p.i., the mice were sacrificed and lymphocytes were isolated from spleen. Multiparameter flow cytometry was used to determine the total frequency of cytokine-producing CD8+ T cells. (A) Representative example of the gating strategy used to define the frequency of cytokine producing CD8+ T cells. Gate were set based on negative control (nai e) and placed consistently across samples. (B and C) the total frequency of CD8+ T cells expressing IFNy is shown. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05. Figure 8: The frequency of CD8+ T cells expressing both IFNy and TNFa from Balb/c (A) or C57B1/6 (B) vaccinated mice. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05.
Figure 9: Increased cytolytic activity of the Gag-specific T-cell in vivo in Ad-
HIV/Gag and Ad-EAT2 co-immunized mice. Balb/c (n=6) (A) or C57BL/6 (n=4) (B) mice were co-immunized with equivalent viral particles of Ad-HIV/Gag mixed with either Ad- GFP or Ad-EAT2 (1 x 107 total vps for Balb/c and 1 109 total vps for C57B1/6 mice). At 14 d.p.i. syngeneic splenocytes were pulsed with either an irrelevant peptide (NYD-pep) and stained with 1 μΜ (CFSELow ) or with the HIV-Gag specific peptides (AMQ peptide for
Balb/c and QBI# 304796 for C57B1/6 mice) and labeled with ΙΟμΜ (CFSEHigh). Five hours after adoptive transfer into either Naive or immunized mice, splenocytes were harvested and sorted using a LSRII flow cytometer. % CFSE positive cells were quantified using FlowJo software. % specific killing = l-((% CFSEmglV % CFSE1 ) immunized (% CFSEHigh / % CFSELow) non-immunized)- Representative figure of two combined independent experiments is shown for Balb/c mice.
Figure 10: Systemic administration of EAT-2 expressing adenovirus vector induces cytokine and chemokines responses. C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 x 1010 vps of either Ad-GFP or Ad-EAT2. Plasma was harvested at 6h after virus injection. Cytokine induction was evaluated using a multiplexed bead array based quantitative system. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference . * denotes p<0.05, ** denotes p<0.01, *** denotes pO.001.
Figure 11 : Ad-EAT2 -mediated activation of innate and adaptive immune cells in vivo.
C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 x 1010 vps of either Ad-GFP or Ad-EAT2. CD69 expression by PBMCs (A and B) and splenocytes (C and D) derived NK, NKT, CD3+CD8+ T cells, CD3+CD8" T cells, and B cells was evaluated 48h after virus injection. PBMCs and Splenocytes were harvested, stained and sorted on a LSRII flow cytometer. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, ** denotes p<0.01, *** denotes pO.001. Figure 12: IFNy production from NK cells 6 and 48 hours after Ads injection.
C57BL/6 mice (n=4) were either mock injected, or intravenously injected with 7.5 x 1010 vps of either Ad-GFP or Ad-EAT2 for 6 h.p.i. (A) or 48 h.p.i. (B). Splenocytes were harvested and incubated at 37°C for 5 hours in the presence of Golgi plug. IFNy intracellular staining was performed and cells were sorted on a LSRII flow cytometer. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a student- Newman- Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, *** p<0.001.
Figure 13: Cellular immune responses after CD8+ T cells depletion in Ad-HIV/Gag and Ad-EAT2 co-immunized mice. At 14 d.p.i., splenocytes from vaccinated Balb/c mice (1 x 107 total vps) were equivalently pooled (N-6 mice per treatment) and CD8+ cells were depleted using magnetic beads. 5xl05 splenocytes were added to each well and stimulated with the immunodominant peptide AMQMLKETI (SEQ ID NO:06). (A) A representative flow cytometric analysis before and after CD8 T cell depletion is shown. Spots from CD8+ un-depleted cells and CD8+ depleted cells were quantified using an automated ELISPOT reader (B and C). %SFC that are CD8- = (#SFCs CD 8 dep / #SFCs CD8+)*100 (D). The bars represent mean ± SD. Statistical analysis was completed using student's t-test.
Figure 14: Exemplary Homo sapiens EAT-2 (SH2 domain containing IB (SH2D1B)) (GenBank Accession No.: NM_053282.4) polypeptide sequence (SEQ ID NO:01) (A) encoded by nucleotide sequence (SEQ ID NO:02).
Figure 15: Exemplary human immunodeficiency virus type 1 (HXB2) GAG polypeptide sequence (SEQ ID NO:03) (GenBank Accession No.: 03455).
Figure 16: FACS analysis of (A) CD80, (B) CD86, (C) MHC-II, and (D) CD40. Figure 17: FACS analysis of (A) CD80, (B) CD86, (C) MHC-II, and (D) CD40. Figure 18 : Ad-EAT2 versus Ad-GFP MOI of 20,000 (same cells but infected with virus).
Figure 19: Amino acid sequence for malaria CSPMI (SEQ ID NO:07), CSP T-cell epitope (SEQ ID NO:09), CSP B-Cell epitope (SEQ ID NO: 10), and CSP 156 (SEQ ID NO: l l).
Figure 20: Nucleic acid sequence (SEQ ID NO:08) encoding malaria CSP.
Figure 21 : Exemplary Mus musculus 2B4 (CD244) (GenBank Accession
No.: NM_018729.2) polypeptide sequence (SEQ ID NO:13) (A) encoded by nucleotide sequence (SEQ ID NO: 14). Figure 22: Exemplary Mus musculus CRACC (Slamf7) (GenBank Accession No.: NM_144539) polypeptide sequence (SEQ ID MO: 15) (A) encoded by nucleotide sequence (SEQ Γΰ NO: 16).
Figure 23: Exemplary Mus musculus CD84 (GenBank Accession No.: NM_013489) polypeptide sequence (SEQ ID NO: 17) (A) encoded by nucleotide sequence (SEQ ID
NO: 18).
Figure 24: Exemplary Mus musculus Ly9 (CD229) (GenBank Accession No.:
NM_008534) polypeptide sequence (SEQ ID NO: 19) (A) encoded by nucleotide sequence (SEQ ID NO:20).
Figure 25: Exemplary Mus musculus Lyl08 (NTB-A) (GenBank Accession No.:
NM_030710) polypeptide sequence (SEQ ID NO:21) (A) encoded by nucleotide sequence (SEQ ID NO:22).
Figure 26: Exemplary Mus musculus SLAM (CD150) (GenBank Accession No.: NM_013730) polypeptide sequence (SEQ ID NO:23) (A) encoded by nucleotide sequence (SEQ ID NO:24).
Figure 27: Exemplary Mus musculus SAP (SH2D1 A) (GenBank Accession No.: NM_011364) polypeptide sequence (SEQ ED NO:25) (A) encoded by nucleotide sequence (SEQ ID NO:26).
Figure 28: Exemplary Mus musculus ERT (SH2Dlb2) (GenBank Accession No.: NM_001033499) polypeptide sequence (SEQ ID NO:27) (A) encoded by nucleotide sequence (SEQ ID NO:28).
Figure 29: Exemplary Mus musculus EAT-2 (SH2 domain protein 1B1 (Sh2dlbl)) (GenBank Accession No.: NM_012009.4) polypeptide sequence (SEQ ID NO:29) (A) encoded by nucleotide sequence (SEQ ID NO:30).
DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below. Further definitions appear throughout the text.
The term "recombinant DNA molecule" as used herein refers to a DNA molecule that is comprised of DNA sequences joined together by means of molecular biological techniques.
The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule that is expressed using a recombinant DNA molecule.
As used herein the term "portion" when made in reference to a nucleic acid sequence refers to a fragment of that sequence. The fragment may range in size from an exemplary 5, 10, 20, 50, and/or 100 contiguous nucleotide residues to the entire nucleic acid sequence minus one nucleic acid residue. Thus, a nucleic acid sequence comprising "at least a portion of a nucleotide sequence comprises from five (5) nucleotide residue of the nucleotide sequence to the entire nucleotide sequence.
The term "portion" when used in reference to a protein (as in a "portion of a given protein") refers to fragments of that protein. The fragments may range in size from an exemplary 4, 10, 20, 30, and/or 50 contiguous amino acid residues to the entire amino acid sequence minus one amino acid residue. Thus, a polypeptide sequence comprising "at least a portion of an amino acid sequence" comprises from four (4) contiguous amino acid residues of the amino acid sequence to the entire amino acid sequence.
The term "recombinant mutation" refers to a mutation that is introduced by means of molecular biological techniques. This is in contrast to mutations that occur in nature.
The term "recombinant virus" refers to a virus that contains a recombinant DNA molecule, recombinant protein and/or recombinant mutation, as well as progeny of that virus.
The terms "mutation" and "modification" refer to a deletion, insertion, or substitution. A "deletion" is defined as a change in a nucleic acid sequence or amino acid sequence in which one or more nucleotides or amino acids, respectively, is absent. An "insertion" or "addition" is that change in a nucleic acid sequence or amino acid sequence that has resulted in the addition of one or more nucleotides or amino acids, respectively. A "substitution" in a nucleic acid sequence or an amino acid sequence results from the replacement of one or more nucleotides or amino acids, respectively, by a molecule that is a different molecule from the replaced one or more nucleotides or amino acids. For example, a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine. Alternatively, a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.
Substitution of an amino acid may be conservative or non-conservative. "Conservative substitution" of an amino acid refers to the replacement of that amino acid with another amino acid that has a similar hydrophobicity, polarity, and/or structure. For example, the following aliphatic amino acids with neutral side chains may be conservatively substituted one for the other: glycine, alanine, valine, leucine, isoleucine, serine, and threonine.
Aromatic amino acids with neutral side chains that may be conservatively substituted one for the other include phenylalanine, tyrosine, and tryptophan. Cysteine and methionine are sulphur-containing amino acids that may be conservatively substituted one for the other. Also, asparagine may be conservatively substituted for glutamine, and vice versa, since both amino acids are amides of dicarboxylic amino acids. In addition, aspartic acid (aspartate) my be conservatively substituted for glutamic acid (glutamate) as both are acidic, charged (hydrophilic) amino acids. Also, lysine, arginine, and histidine my be conservatively substituted one for the other since each is a basic, charged (hydrophilic) amino acid. "Non- conservative substitution" is a substitution other than a conservative substitution. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological and/or immunological activity may be found using computer programs well known in the art, for example, DNAStar™ software.
A "variant" or "homolog" of a polypeptide sequence of interest or nucleotide sequence of interest refers to a sequence that has identity of at least 65% with the an amino acid sequence of interest or nucleotide sequence of interest, including identity of 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%, and/or at least 99%. Thus, homologous genomic nucleotide sequences within the scope of the invention include orthologs and paralogs. The term "ortholog" refers to a gene in different species that evolved from a common ancestral gene by speciation. In some embodiments, orthologs retain the same function. The term "paralog" refers to genes related by duplication within a genome. In some embodiments, paralogs evolve new functions. In further embodiments, a new function of a paralog is related to the original function. Variants of a polypeptide sequence of interest may contain a mutation.
The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression (i.e., transcription and/or translation) of the operably linked coding sequence in a particular host organism. Expression vectors are exemplified by, but not limited to, plasmid, phagemid, shuttle vector, cosmid, virus, chromosome, mitochondrial DNA, plastid DNA, and nucleic acid fragment. Nucleic acid sequences used for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The terms "pathogenic" and "virulent" when in reference to a microorganism, such as virus, bacteria, parasite, etc. refer to the ability of the microorganism to produce an infectious disease in another organism (e.g., mammal).
The terms "purified" and "isolated" and grammatical equivalents thereof as used herein, refer to the reduction in the amount of at least one undesirable contaminant (such as protein and/or nucleic acid sequence) from a sample. Thus, purification results in an
"enrichment," i.e., an increase in the amount of a desirable composition, such as a virus, protein and/or nucleic acid sequence in the sample. For example, packaged adenoviral compositions of the invention maybe purified using methods known in the art (e.g., U.S. Patent 6,946,126 to Amalfitano et al.)
The terms "in operable combination" and "operably linked" when in reference to the relationship between nucleic acid sequences and/or amino acid sequences refers to linking the sequences such that they perform their intended function. For example, operably linking a promoter sequence to a nucleotide sequence of interest refers to linking the promoter sequence and the nucleotide sequence of interest in a manner such that the promoter sequence is capable of directing the transcription of the nucleotide sequence of interest resulting in an mRNA that directs the synthesis of a polypeptide encoded by the nucleotide sequence of interest.
The term "transfect" or "transfecting" as used herein, refers to any mechanism by which a vector may be incorporated into a host cell. A successful transfection results in the capability of the host cell to express any operative genes carried by the vector. Transfections may be stable or transient. One example of a transient transfection comprises vector expression within a cell, wherein the vector is not integrated within the host cell genome. Alternatively, a stable transfection comprises vector expression within a cell, wherein the vector is integrated within the host cell genome.
"Subject" "and "animal" interchangeably refer to any multicellular animal, preferably a mammal, e.g., humans, non-human primates, murines, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.). Thus, mammalian subjects include mouse, rat, guinea pig, hamster, ferret and chinchilla.
"Subject in need of reducing one or more symptoms of a disease, e.g., infection with a pathogen, includes a subject that exhibits and/or is at risk of exhibiting one or more symptoms of the disease. For Example, subjects may be at risk based on family history, genetic factors, environmental factors, etc. This term includes animal models of the disease.
The term "specifically binds" and "specific binding" when made in reference to the binding of antibody to a molecule (e.g., peptide) or binding of a cell (e.g., T-cell) to a peptide, refer to an interaction of the antibody or cell with one or more epitopes on the molecule where the interaction is dependent upon the presence of a particular structure on the molecule. For example, if an antibody is specific for epitope "A" on the molecule, then the presence of a protein containing epitope A (or free, imlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. "SLAM" and "SLAM family receptor" interchangeably refer a receptor that is expressed by a wide range of immune cells. 13' 18' 19. SLAM family receptors are exemplified by mouse 2B4 (CD244) (GenBank Accession No.: NM_018729.2, SEQ ID NO: 13 encoded by SEQ ID NO:14 of Figure 21), mouse CRACC (Slamf?) (GenBank Accession No.:
NM_144539, SEQ ID NO:15 encoded by SEQ ID NO:16 of Figure 22), mouse CD84:
GenBank Accession No.: NM_013489, SEQ ID NO:17 encoded by SEQ ID NO:18 of Figure 23, mouse Ly9 (CD229): (GenBank Accession No.NM_008534, SEQ FD NO:19 encoded by SEQ ID NO:20 of Figure 24), mouse Lyl08 (NTB-A): (GenBank Accession No.:
NMJB0710, SEQ ID NO:21 encoded by SEQ ID NO:22 of Figure 25), and mouse SLAM (CD150): (GenBank Accession No.: NM_013730, SEQ ID O:23 encoded by SEQ ID NO:24 of Figure 26). The SLAM family receptors control multiple innate and adaptive immune responses through association with an intracellular signaling SLAM family receptor adaptor protein, described below
"SLAM family receptor adaptor protein" and "SAP" are used interchangeably to refer to an intracellular protein that associates with SLAM, and are exemplified by
EAT-2, SAP, ERT. 13' 18' 19 In particular embodiments, "EAT-2" (also referred to as "SH2dlbl," "EAT-2A," "Eat2," and "Eat2a") is a protein containing a SRC (MIM 190090) homology-2 (SH2) domain, and that regulates signal transduction through interaction with SLAM family receptors expressed on the surface of antigen-presenting cells. EAT-2 is exemplified by human sequences (SEQ ID NO:01 encoded by SEQ ID NO: 02, GenBank Accession No.: NM_053282.4 of Figure 14), and by mouse sequences (SEQ ID NO:29 encoded by SEQ ID NO:30, GenBank Accession No.: NM_012009.4 of Figure 29). In some embodiments, SAP is exemplified by mouse SAP (SH2D1A): NM_011364 (SEQ ID NO:25 encoded by SEQ ID NO:26 of Figure 27), and ERT is exemplified by mouse ERT
(SH2Dlb2): NM_001033499 (SEQ ID NO:27 encoded by SEQ ID NO:28 of Figure 28).
The terms "reduce," "inhibit," "diminish," "suppress," "decrease," and grammatical equivalents (including "lower," "smaller," etc.) when in reference to the level of any molecule (e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein), cell, and/or phenomenon (e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean that the quantity of molecule, cell and/or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the quantity of molecule, cell and/or phenomenon in the first sample is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity of the same molecule, cell and/or phenomenon in a second sample. In another embodiment, the quantity of molecule, cell, and/or phenomenon in the first sample is lower by any numerical percentage from 5% to 100%, such as, but not limited to, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, and from 90% to 100% lower than the quantity of the same molecule, cell and/or phenomenon in a second sample.
The terms "increase," "elevate," "raise," and grammatical equivalents (including
"higher," "greater," etc.) when in reference to the level of any molecule {e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein), cell, and/or phenomenon {e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean that the quantity of the molecule, cell and/or phenomenon in the first sample is higher than in the second sample by any amount that is statistically significant using any art-accepted statistical method of analysis. In one embodiment, the quantity of the molecule, cell and/or phenomenon in the first sample is at least 10%> greater than, at least 25% greater than, at least 50% greater than, at least 75% greater than, and/or at least 90% greater than the quantity of the same molecule, cell and/or phenomenon in a second sample. This includes, without limitation, a quantity of molecule, cell, and/or phenomenon in the first sample that is at least 10%) greater than, at least 15% greater than, at least 20% greater than, at least 25% greater than, at least 30% greater than, at least 35% greater than, at least 40% greater than, at least 45%> greater than, at least 50% greater than, at least 55% greater than, at least 60% greater than, at least 65% greater than, at least 70% greater than, at least 75% greater than, at least 80%) greater than, at least 85% greater than, at least 90% greater than, and/or at least 95% greater than the quantity of the same molecule, cell and/or phenomenon in a second sample.
The terms "altered," "different," "changed," and grammatical equivalents when in reference to the level of any molecule {e.g., polypeptide sequence such as those disclosed herein, and/or nucleic acid sequence such as those encoding any of the polypeptides described herein), cell, and/or phenomenon {e.g., binding to a molecule, affinity of binding, expression of a nucleic acid sequence, transcription of a nucleic acid sequence, enzyme activity, antibody activity, innate immune response, adaptive immune response, transcriptome response, etc.) in a first sample relative to a second sample, mean reduced or increased level of the molecule, cell, and/or phenomenon.
Reference herein to any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range. To illustrate, and without limitation, reference herein to a range of "at least 50" includes whole numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a further illustration, reference herein to a range of "less than 50" includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc. In yet another illustration, reference herein to a range of from "5 to 10" includes each whole number of 5, 6, 7, 8, 9, and 10, and each fractional number such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, etc.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides replication incompetent adenovirus vectors containing a heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP), and optionally further encodes a polypeptide of interest. The invention further provides DNA sequences that encode, as well as packaging cells that produce, the invention's adenoviral vectors. The invention's compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP). The invention's compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, such as in vaccination applications. The invention's compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus.
For example, data herein provide evidence supporting an important new strategy to improve the efficacy of vaccines in general, and that of Ad based vaccines specifically, by activating the SLAM family receptors system simultaneously with target antigen vaccination. More specifically, in this work the inventors confirmed the utility of augmenting the exemplary SLAM family receptors signaling by utilizing an Ad vaccine genetically engineered to overexpress the SLAM family receptors adaptor molecule EAT-2, as it improved the cellular immune responses elicited by a second Ad vaccine expressing a target antigen, in this instance the exemplary HIV derived Gag protein.
The invention's compositions and methods are useful for enhancing a subject's immune response to a polypeptide of interest by expression, in one or more vectors, of the polypeptide of interest and a SLAM family receptor adaptor protein (SAP).
The invention's compositions and methods are also useful for induction of an innate immune response and/or an adaptive cellular and/or humoral immune response, including in vaccination applications. Vaccine applications include, for example, eliciting immune responses to pathogen-derived antigens (such as those from the HIV virus, for example HIV- gag) as well as disease associated antigens, such as cancers (such as Carcino-embryonic antigen (CEA), her2neu, etc.), etc.. For example, the invention's compositions and methods may be used for enhancing an adaptive cellular and/or humoral immune response to a vaccine against an antigen by co-administering (in the same preparation or in separate preparations) a vector that expresses a SLAM family receptor adaptor protein (SAP) as an adjuvant, and another vector that expresses the antigen. Expression of the SLAM family receptor adaptor protein (SAP) by the vector enhances the subject's imnrune response to the antigen.
Alternatively, the invention's compositions and methods may be used for enhancing an adaptive cellular and/or humoral immune response to a vaccine against an antigen by administering a single vector that expresses both a SLAM family receptor adaptor protein (SAP) and the antigen.
The invention's compositions and methods are also useful for delivering a heterologous polypeptide to a cell, and/or for producing a SLAM family receptor adaptor protein (SAP) by adenoviral expression of the protein, and /or for producing a replication incompetent adenovirus.
For further clarity, the invention is further described under A) Discussion Of Results
In Exemplary Embodiments Described In The Examples, B) Adenovirus Vectors Comprising SLAM Family Receptor Adaptor Proteins (SAPs), C) Polypeptide Of Interest, D) Cells, E) Vaccines, F) Innate Immune Response To The Invention's Compositions, and G) Adaptive Immune Response To The Invention's Compositions.
A) Discussion Of Results In Exemplary Embodiments Described In The Examples
Replication-incompetent, recombinant adenovirus (rAd) based vaccines have been shown to elicit superior cell mediated immune responses against HIV-1 derived antigens in both pre-clinical and clinical studies 2"4. A number of studies have shown that Ad based vaccines induce multiple innate immune responses through Toll Like Receptor (TLR) dependent and independent mechanisms 5"8. Since innate immune responses significantly contribute to the ability of vaccines to generate adaptive immune responses 9-11, the inventors evaluated the potential of simultaneously activating the innate immune system with both a rAd, as well via through manipulation of the signaling lymphocyte activating molecule (SLAM) family receptors pathway. The SLAM family of receptors is composed of six distinct members named SLAM (CD150), 2B4 (CD244), Ly9, CD84, NTB-A (natural killer, T and B cell antigen; Lyl08 in the mouse) and CRACC (CD2-like receptor activating
1 9
cytotoxic cells) . These receptors are expressed mainly in cells of the hemopoietic lineages, including innate and adaptive immune cells 13'14. All SLAM family receptors members except 2B4, (which interacts with CD48), are self ligands and thus are triggered by homophilic interactions through their respective extracellular domains 14"19. The SLAM-associated protein (SAP) family of adaptors is compassed of three members named SAP, EAT-2, and EAT-2-related transducer (ERT; ERT is however a non-functional pseudo-gene in humans). These adaptors associate with high affinity and high specificity with phosphorylated tyrosine- based motifs (ITSM) in the cytoplasmic domain of SLAM family receptors to either augment or inhibit SLAM family receptors induced intracellular signaling in a variety of immune cells12. The SAP adaptor regulates SLAM family receptors signaling by recruiting the protein tyrosine kinase Fyn. In contrast, EAT-2 and ERT transduce SLAM family receptors initiated signals in a process that involves phosphorylation of tyrosine residues directly located in their short carboxyl-terminal tails .
Various reports indicate a possible role for SLAM (CD150) receptor acting as a proinflammatory costimulatory molecule during the activation of dendrictic cells (DCs) and macrophages. For example, antibody-mediated ligation of human SLAM in CD40L-activated DCs augmented the secretion of pro-inflammatory cytokines such as IL-12 and IL-8, but not IL-10 21. Furthermore, SLAM receptor was also found to be responsible for triggering the
22
production of IL-6 and IL-12 by mouse peritoneal macrophages . In addition, macrophages derived from SLAM-deficient mice show a marked reduction in secretion of IL-12, TNF, and nitric oxide 22. Since EAT-2 is the only known SLAM-associated adaptor protein expressed in DCs and macrophages, EAT-2 potentially mediates SLAM induced proinflammatory cytokine expression in these cell types19.
The inventors hypothesized that simultaneous expression of EAT-2 by an Ad-based vaccine system would augment SLAM family receptors signaling in Ad transduced DCs and macrophages. This would generate significantly augmented innate immune responses resulting in improved APC functions in vivo and consequently improve the adaptive immune response generated against a co-expressed target antigen.
In particular, the inventors hypothesized that overexpression of the SLAM family receptors adaptor EAT-2 using an Ad-based vector would facilitate SLAM family receptors signaling in Ad transduced antigen presenting cells (APCs) resulting in improved induction of cell mediated immune responses to exemplary HIV antigens expressed by co- administered Ad vectors . The inventors confirmed that Ad5 vectors expressing EAT-2 are viable, and facilitate bystander activation of NK, NKT, B, CD4+, and CD8+ T cells early after their administration. Ad vaccine mediated EAT-2 expression enhanced the cellular immune responses to an HIV-Gag antigen expressed from a co-administered Ad vaccine, as multiply confirmed by Tetramer-based flow cytometry, ELISPOT, and in vivo CTL assays. Since both mice and humans express highly conserved EAT-2 adaptor proteins, the inventors' results suggest that human vaccination strategies that specifically facilitate SLAM family receptors signaling may provide a more effective vaccine against HIV specifically, as well as numerous other vaccine targets in general.
Date herein confirmed that inducing SLAM family receptors signaling by an adenovirus that expresses the SLAM family receptors adaptor molecule EAT-2 generates stronger cellular immune responses to a co-expressed antigen than that generated by current adenovirus vaccines. The inventors' results highlight for the first time targeting of an important immune regulatory pathway harnessed for the development of novel approaches to improve vaccination efficacy.
Data herein also show that co-vaccination with Ad-EAT2 results in the induction of increased numbers and function of HIV-Gag-specific CD8+ T cells (Examples 5-7)
In addition to NK cells, the inventors also observed increased activation of NKT cells after Ad mediated transduction of the EAT-2 gene. Several reports have shown that enhancing the activation of NKT cells can positively influence the initial activation of DCs and/or NK cells, thereby increasing DC-dependent adaptive (cellular) immune responses 45_49. The inventors' data suggests that harnessing this potential capability is of great interest in designing next generation vaccines.
Previous reports have shown that SLAM family receptors derived signaling can also
21 22
contribute to the increased activity of APCs ' . The inventors note that EAT-2 is the only SLAM family receptors adaptor molecule currently known to be expressed in APCs 19.
Without intending to limit the invention to any mechanism, this may suggest Ad-EAT2 transduced DCs may directly facilitate induction of antigen specific adaptive immune responses observed in this work. However it is also possible that the expression of EAT-2 in a variety of alternative cell types may also play an important role. Various studies in mouse models and non-human primates report that improving the breadth of the cellular immune responses elicited by a vaccine to a target antigen is positively correlated with an improved ability of the vaccine to induce protective immiinity 4' 50. The inventors' results demonstrate that administering Ad-HIV/Gag with Ad-EAT2 increased the breadth of the cellular immune responses to the HIV-Gag antigen, which correlated with a significantly improved in vivo cytolytic activity of HIV/Gag specific CD8+ T cells generated after Ad-HIV/Gag and Ad- EAT2 co-immunization. In addition, the inventors observed similar results in both C57BL/6 and Balb/c mice, (two mouse strains that can bias adaptive immune responses to a Thl or Th2 response, respectively) indicating that the adjuvant effect of Ad-EAT-2 is not specifically limited by significant immuno-genetic background differences of the host animal, at least in this species. In conclusion, the inventors' findings suggest that enhancing SLAM family receptors signaling by overexpressing EAT-2 during antigen vaccination can serve to improve the ability of a vaccine to stimulate the innate immune system, and subsequently induce improved, antigen specific adaptive immune responses.
NK cells represent a subset of innate immune cells that have been shown to play an important role in bridging innate and adaptive immune responses, by influencing DC function ' , providing signals for augmenting Thl immune responses " , and inducing tumor- specific CTLs 40. In addition, it has been shown recently that NK cell-mediated cytotoxicity of antigen- expressing target cells induces robust antigen-specific adaptive immune responses 41. EAT-2 has been shown to be indispensable in activating NK cell cytotoxicity, by acting as a downstream adaptor protein facilitating signaling from SLAM family receptor CRACC in mice 42' 43 or NTB-A in humans 44. Shedding light on the underlying mechanisms operating after the Ad mediated transduction of the EAT-2 gene, the inventors noted enhanced NK cell activation the latter of which could further drive the activation and/or maturation of DCs and bias the HIV-Gag specific immune profile towards a Thl response .
B) Adenovirus Vectors Comprising SLAM Family Receptor Adaptor Proteins (SAPs)
The invention provides replication incompetent recombinant adenovirus vectors comprising a DNA sequence that contains, in operable combination, a) DNA sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP). The invention's vectors that encode a SLAM family receptor adaptor protein (SAP) may be used as immune enhancing adjuvants by co-administration with a second vector encoding an antigen. In another embodiment, the SAP (as exemplified by EAT-2 SEQ ID NO:01).
As used herein, the term "adenovirus" refers to a double- stranded DNA adenovirus of animal origin, such as avian, bovine, ovine, murine, porcine, canine, simian, and human origin. Avian adenoviruses are exemplified by serotypes 1 to 10, which are available from the ATCC, such as, for example, the Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), and K-l 1 (ATCC VR-921) strains, or else the strains designated as ATCC VR-831 to 835. Bovine adenoviruses are illustrated by those available from the ATCC (types 1 to 8) under reference numbers ATCC VR-313, 314, 639-642, 768 and 769. Ovine adenoviruses include the type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340). Murine adenoviruses are exemplified by FL (ATCC VR-550) and E20308 (ATCC VR-528). Porcine adenovirus (5359) may also be used. Adenoviruses of canine origin include all the strains of the CAVI and CAV2 adenoviruses (for example, Manhattan strain or A26/61 (ATCC VR-800) strain). Simian adenoviruses are also contemplated, and they include the adenoviruses with the ATCC reference numbers VR-591-594, 941-943, and 195-203. Human adenoviruses, of which there greater than fifty (50) serotypes are known in the art, are also contemplated, including Ad2, Ad3, Ad4, Ad5, Adl 1, Ad 14, Ad7, Ad9, Adl2, Adl6, Adl7, Ad21, Ad26, Ad34, Ad35, Ad 40, Ad48, Ad49, Ad50 (e.g., U.S. Patent No. 7,300,657 to Pau, U.S. Patent No. 7,468,181 to Vogels, and U.S. Patent No. 6,136,594 to Dalemans). In one preferred embodiment, the adenovirus is adenovirus 5 (Ad5).
As used herein, the term "vector" refers to an agent that contains and/or transfers genetic material from one cell to another, including for example viruses, bacteriophages, and plasmids. A vector may be used to transfer, introduce and/or insert exogenous modified genetic material (as recombinant DNA) into the genome of a recipient (host) cell. Delivery of genetic material by a "viral vector" is termed "transduction," with the infected cells described as "transduced." This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viral based gene transfer/vectors include, for example, adenovirus, adeno-associated virus, retroviruses, alphaviruses, lentiviruses, vaccinia viruses, baculo viruses, fowlpox and herpesviruses. Adenovirus may be used as a "viral vector" for gene therapy and vaccination since its DNA does not integrate into the host cell genome, does not replicated during host cell division and it infects a wide range of dividing and non- dividing cells. One strategy for manipulating the tropism of Ad vectors is to insert receptor- binding ligands into the major capsid proteins (fiber, penton base and hexon) at exposed positions, in order to recruit alternative receptors on target cells. A potential location for inserting targeting ligands is the HI loop of the fiber knob, in the hypervariable region 5 of hexon loop LI and in the RGD-motif loop of penton base. In addition, oligopeptide ligands may be added to the C-terminus of the adenovirus fiber protein HI loop. The adenovirus capsid is innately pro-inflammatory, as it activates the complement system. This is due, at least in part, to the fact that adenoviruses are a common human pathogen. The proinflammatory nature of the adenovirus capsid often results in the triggering of a rapid immune response when adenoviruses are used a viral vectors.
The invention's vectors are preferably replication incompetent. "Replication incompetent," "replication defective," "replication attenuated" are used interchangeably to refer to a virus and/or viral vector that has a reduced level of replication compared to wild type virus and/or to a viral vector containing wild type virus nucleotide sequences. Methods for producing replication incompetent adenoviral vectors are known in the art (e.g., U.S. Patent Nos. 7,300,657 to Pau, 7,468,181 to Vogels, 6,136,594 to Dalemans, 5,994, 132 to Chamberlain et al., 6,797,265 to Amalfitano et al., 7,563,617 to Hearing et al., and 6,262,035 to Campbell et al.). For example, in one embodiment, a replication incompetent adenovirus viras and/or adenoviral vector (a) lacks (i.e., has a deletion of) adenovirus El gene coding sequence, (b) lacks adenovirus El gene coding sequence and E2b gene coding sequence (c) lacks adenovirus El gene coding sequence and adeno viras E4 gene coding sequence, (d) lacks adenovirus El gene coding sequence and adenovirus E2a gene coding sequence, and/or (e) lacks adenovirus El gene coding sequence and adenovirus EIVa2 gene coding sequence.
In a further embodiment, the replication incompetent adenovirus virus and/or adenoviral vector is a gutted adenovirus. The term "gutted" and "fully deleted" are used interchangeably in reference to a viral vector, and refer to a viral vector (e.g., plasmid, viras, naked DNA) that lacks all the coding sequences that are otherwise present in a wild-type virus. Gutted vectors may contain non-coding viral sequences, e.g., terminal repeat sequences, and packaging sequences. For example, a gutted adenovirus vector lacks all adenovirus coding sequences and optionally contains adenovirus terminal repeat sequences and/or packaging sequences (e.g., U.S. Patent Nos. 5,994,132 to Chamberlain et al., 6,797,265 to Amalfitano et al., 7,563,617 to Hearing et al., 6,262,035 to Campbell et al). Gutted vectors are preferred in certain embodiments since they do not express viral vector proteins and hence do not induce an adverse immune or toxic response in a cell. The invention's vectors contain nucleotide sequenced encoding at least a portion of a SLAM family receptor adaptor protein (SAP).
C) Polypeptide Of Interest
In one embodiment, the invention's vectors may further comprise a second heterologous nucleic acid sequence that encodes a polypeptide of interest. This may be desirable in applications (e.g., vaccination) that utilize a single vector that expresses both an immune enhancer as well as an antigen. The invention's compositions and methods may be used for expression of, delivery of, and/or vaccination against, a polypeptide of interest.
A "polypeptide of interest," "nucleotide sequence of interest," and "molecule of interest" refer to any polypeptide sequence, nucleotide sequence, and molecule, respectively, the manipulation of which may be deemed desirable for any reason, by one of ordinary skill in the art. Illustrative polypeptides of interest include endogenous polypeptides, heterologous polypeptides, pathogen derived antigens, disease associated antigens, and reporter sequences.
The terms "endogenous" and "wild type" refer to a sequence that is naturally found in the cell and/or virus into which it is introduced so long as it does not contain some modification relative to the naturally-occurring sequence. The term "heterologous" refers to a sequence, which is not endogenous to the cell and/or virus into which it is introduced. For example, heterologous DNA includes a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to wliich it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence, which is naturally found in the cell or virus into which it is introduced and which contains some modification relative to the naturally-occurring sequence.
Generally, although not necessarily, heterologous DNA encodes heterologous RNA and protein of interest that are not normally produced by the cell and/or virus into which it is introduced.
Examples of polypeptides of interest include proteins, and portions thereof, that are expressed as glycoproteins, membrane proteins and portions thereof, soluble proteins and portions thereof, epitopes and portions thereof, and the like.
In one embodiment, the polypeptide of interest comprises a membrane protein (U.S.
Patent Application US 2009/0068221 to Morrison). A "membrane protein" refers to a protein that is at least partially embedded in the lipid bilayer of a cell, virus and the like.
Membrane proteins contain a cytoplasmic domain, a transmembrane domain, and an ectodomain. In a particular embodiment, the protein of interest comprises an ectodomain of a membrane protein. The term "ectodomain" when in reference to a membrane protein refers to the portion of the protein that is exposed on the extracellular side of a lipid bilayer of a cell, virus and the like. Methods for determining the ectodomain of a protein are known in the art (Singer (1990); High et al. (1993), and McVector software, Oxford Molecular).
Exemplary ectodomains include, but are not limited to those described in U.S. Patent Nos. 7,262,270; 7,253,254; 7,250,171 ; 7,223,390; 7,189,403; 7,122,347; 7,119,165; 7,101,556; 7,067,110; 7,060,276; 7,029,685; 7,022,324; 6,946,543; 6,939,952; 6,713,066; 6,699,476; 6,689,367; 6,566,074; 6,531,295; 6,417,341; 6,248,327; 6,140,059; 5,851,993; 5,847,096; 5,837,816; 5,674,753; and 5,344,760. Additional examples of ectodomains include ectodomains of membrane type 1 proteins, type 2 proteins, and type 3 proteins (U.S. Patent Application US 2009/0068221 to Morrison).
hi one embodiment, the protein of interest comprises a soluble protein. The term "soluble protein" refers to a protein that is not embedded in the lipid bilayer of a cell, virus and the like. Examples of soluble proteins are known in the art (U.S. Patent Application US 2009/0068221 to Morrison).
In one embodiment, the polypeptide of interest comprises an antigen. The terms "antigen," "immunogen," "antigenic," "immunogenic," "antigenically active,"
"immunologic," and "immunologically active" when made in reference to a molecule, refer to any substance that is capable of inducing a specific humoral immune response (including eliciting a soluble antibody response) and/or cell-mediated immune response (including eliciting a CTL response). In one embodiment the antigen is exemplified by Human
Immunodeficiency virus gag protein (SEQ ID NO:03), malaria CSPM (SEQ ID NO:07; Figure 19), CSP T-cell epitope (SEQ ID NO:09; Figure 19), CSP B-Cell epitope (SEQ ID NO:10; Figure 19), CSPi56 (SEQ ID NO:l 1 ; Figure 19), malaria CSP encoded by SEQ ID NO:08 of Figure 20, and Pseudomonas antigen.
In one embodiment, the polypeptide of interest is an antigen that comprises an epitope. The terms "epitope" and "antigenic determinant" refer to a structure on an antigen, which interacts with the binding site of an antibody or T cell receptor as a result of molecular complementarity. An epitope may compete with the intact antigen, from which it is derived, for binding to an antibody. Generally, secreted antibodies and their corresponding membrane-bound forms are capable of recognizing a wide variety of substances as antigens, whereas T cell receptors are capable of recognizing only fragments of proteins which are complexed with MHC molecules on cell surfaces. Antigens recognized by immunoglobulin receptors on B cells are subdivided into three categories: T-cell dependent antigens, type 1 T cell-independent antigens; and type 2 T cell-independent antigens. Also, for example, when a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e. , the immunogen used to elicit the immune response) for binding to an antibody. Exemplary epitopes include, without limitation YPYDVPDYA (SEQ ID NO: 12) (U.S. Patent
Application US 2009/0068221 to Morrison), EphrinA2 epitopes from renal cell carcinoma and prostate cancer (U.S. Patent No. 7,297,337), hepatitis C virus epitopes (U.S. Patent Nos. 7,238,356 and 7,220,420), vaccinia virus epitopes (U.S. Patent No. 7,217,526), dog dander epitopes (U.S. Patent No. 7,166,291), human papilloma virus (HPV) epitopes (U.S. Patent Nos. 7,153,659 and 6,900,035), Mycobacterium tuberculosis epitopes (U.S. Patent Nos. 7,037,510 and 6,991,797), bacterial meningitis epitopes (U.S. Patent No. 7,018,637), malaria epitopes (U.S. Patent No. 6,942,866), and type 1 diabetes mellitus epitopes (U.S. Patent No. 6,930,181).
In some embodiments, the polypeptide of interest is "pathogen derived," meaning expressed by a pathogen (e.g., bacteria, virus, parasite, protozoan, fungus, etc.), such as Herpes virus, Neisseria gonorrhea, Treponema, Escherichia coli, Respiratory Syncytial virus, tuberculosis, Streptococcus, Chlamydia, and Ebola virus. Pathogen derived antigens are exemplified by Human Immunodeficiency virus (HIV) gag protein (including the HXB2 strain gag protein (Genbank Accession #K03455) disclosed in the below Examples), HIV Gag protein antigen such as HIV Gap protein immunodominant peptide AMQMLKETI (SEQ ID NO:06), HIV Pol protein antigen, HIV Nef protein antigen, malaria CSPf n antigen (SEQ ID NO:07, Figure 19), CSP antigen encoded by DNA sequence SEQ ID NO:08 (Figure 20), malaria CSP T cell epitope (SEQ ID NO:09; EYLNKIQNSLSTEWSPCSVT; Figure 19), malaria CSP B Cell epitope (SEQ ID NO: 10; N ANPN ANPN ANPN ANPN ANPN ANP ;
Figure 19), malaria CSP156 (SEQ ID NO:l l ; Figure 19), and Pseudomonas antigen.
The term "Pseudomonas" refers to a gram-negative, rod-shaped and polar-flagella bacteria of the proteobacteria genus. Most Pseudomonas species are naturally resistant to penicillin and the majority of related beta-lactam antibiotics. This low antibiotic susceptibility is attributable, at least in part, to the concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Infectious species in animals, including humans, are Pseudomonas aeruginosa, Pseudomonas oryzihabitans, and plecoglossicida. Pseudomonas aeruginosa is recognized as an emerging opportunistic pathogen of clinical relevance since epidemiological studies indicate that antibiotic resistance is increasing in clinical isolates. P. aeruginosa flourishes in hospital environments, and is a particular problem in this environment since it is the second most common infection in hospitalized patients (i.e. nosocomial infections). "Pseudomonas antigen" includes serogroup 011 O-antigen (Dean et al. (1999) J. Bacteriol. 181(14):4275-84; Deana et al. (2006) FEMS Micorbilogy Letters 187:59 - 630);
Pseudomonas PcRv antigen (U.S. Patent Application No. 20090191241); Pseudomonas exotoxin A translocation domain II (U.S. Patent No. 7,595,054, issued 9/29/09), and
Pseudomonas antigens described in U.S. Patent 7,371,394, issued May 13, 2008.
In one embodiment, the polypeptide of interest is "disease associated," meaning a polypeptide whose level (e.g., presence, absence, increase, and/or decrease relative to a control, etc.) that is correlated with disease and/or with risk of disease based on family history, genetic factors, environmental factors, etc. Disease associated polypeptides of interest are exemplified by those encoded by, but not limited to, the adenosine deaminase (ADA) gene (GenBank Accession No. Ml 3792) associated with adenosine deaminase deficiency with severe combined immune deficiency; alpha- 1 -antitrypsin gene (GenBank Accession No. Ml 1465) associated with alphal -antitrypsin deficiency; beta chain of hemoglobin gene (GenBank Accession No. NM_000518) associated with beta thalassemia and Sickle cell disease; receptor for low density lipoprotein gene (GenBank Accession No. D 16494) associated with familial hypercholesterolemia; lysosomal glucocerebrosidase gene (GenBank Accession No. K02920) associated with Gaucher disease; hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (GenBank Accession No. M26434, J00205, M27558, M27559, M27560, M27561, M29753, M29754, M29755, M29756, M29757) associated with Lesch-Nyhan syndrome; lysosomal arylsulfatase A (ARSA) gene (GenBank Accession No. NM_000487) associated with metachromatic leukodystrophy; ornithine transcarbamylase (OTC) gene (GenBank Accession No. NM_000531) associated with ornithine transcarbamylase deficiency; phenylalanine hydroxylase (PAH) gene (GenBank Accession No. NM_000277) associated with phenylketonuria; purine nucleoside
phosphorylase (NP) gene (GenBank Accession No. NM_000270) associated with purine nucleoside phosphorylase deficiency; the dystrophin gene (GenBank Accession Nos.
Ml 8533, M17154, and Ml 8026) associated with muscular dystrophy; the utrophin (also called the dystrophin related protein) gene (GenBank Accession No. NM_007124) whose protein product has been reported to be capable of functionally substituting for the dystrophin gene; and the human cystic fibrosis transmembrane conductance regulator (CFTR) gene (GenBank Accession No.M28668) associated with cystic fibrosis. In a particular
embodiment, the disease associated polypeptide of interest is a cancer derived antigen such as carcino-embryonic antigen (CEA) and her2neu antigen.
In an alternative embodiment, the polypeptide of interest that is expressed by the vectors of the invention may include a reporter polypeptide sequence. "Reporter sequence" and "marker sequence" are used interchangeably to refer to DNA, RNA, and/or polypeptide sequence that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. Exemplary reporter genes include, for example, β-glucuronidase gene, green fluorescent protein (GFP) gene, E. coli β-galactosidase (LacZ) gene, Halobacterium β- galactosidase gene, E. coli luciferase gene, Neuropsora tyrosinase gene, Aequorin (jellyfish bioluminescenece) gene, human placental alkaline phosphatase gene, and chloramphenicol acetyltransferase (CAT) gene. Reporter genes are commercially available, such as from Clontech, Invitrogen, and Promega. It is not intended that the present invention be limited to any particular detection system or label. In one embodiment, the reporter sequence comprises GFP, produced by the adenovirus vector (Ad-GFP/rEA) co-expressing GFP and EAT-2. This was constructed using the pAd-Track shuttle.
D) Cells
The invention further provides cells that are susceptible to, and useful for packaging the, invention's adenoviral vectors. Without limiting the type of cell, these cells include 293 (Adenovirus), A549 (Adenovirus), HEL (Adenovirus), HEL-299 (Adenovirus), HEp-2 (Adenovirus), HFL-Chang (Adenovirus), HNK (Adenovirus), Hs27 (HFF) (Adenovirus), KB (Adenovirus), LLC-MK2 (Adenovirus), MDCK (Adenovirus), MRC-5 (Adenovirus), MRHF (Adenovirus), NCI-H292 (Adenovirus), pRK (Adenovirus), RK1 (Adenovirus), R-Mix
(Adenovirus), Vero (Adenovirus), WI 38 (Adenovirus), HeLa (Adenovirus 3), and HeLa S-4 (Adenovirus 5). These cells are available from Diagnostic Hybrids, Inc., Athens, OH.
Additional adenovirus packaging cells are known in the art, including those described in U.S. Patent Nos. US 7582290 to Rodriguez et al., 6602707 and 7105346 to Fallaux et al., 7026164 and 7074618 to Yuanhao et al., 6492160 to Linschoren et al., 6033908 to
Moerkapelle et al., 7285265 and 6974695 and 7344883 to Vogels et al. E) Vaccines
The invention's compositions may be used as vaccines. In one embodiment, the inventing provides a composition comprising (a) a replication incompetent recombinant adenovirus vector as described herein and (b) a pharmaceutically acceptable carrier.
The term "vaccine" refers to a pharmaceutically acceptable preparation that may be administered to a host to induce a humoral immune response (including eliciting a soluble antibody response) and/or cell -mediated immune response (including eliciting a CTL response).
The terms "pharmaceutically acceptable," "pharmaceutical" and "physiologically tolerable" refer to a composition that contains molecules that are capable of administration to or upon a subject and that do not substantially produce an undesirable effect such as, for example, adverse or allergic reactions, dizziness, gastric upset, toxicity and the like, when administered to a subject. Preferably also, the pharmaceutically acceptable molecule does not substantially reduce the activity of the invention's compositions. Pharmaceutical molecules include, but are not limited to excipients and diluents. Vaccines may contain
pharmaceutically acceptable adjuvants, diluents, carriers, and/or excipients.
The term "adjuvant" as used herein refers to any compound which, when injected together with an antigen, non-specifically enhances the immune response to that antigen. Exemplary adjuvants include Complete Freund's Adjuvant, Incomplete Freund's Adjuvant, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI fowl adjuvant (MPL; RIBI
Immunochemical Research, Inc.), potassium alum, aluminum phosphate, aluminum hydroxide, QS21 (Cambridge Biotech), Titer Max adjuvant (CytRx), and Quil A adjuvant. Other compounds that may have adjuvant properties include binders such as
carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex;
glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.
Exemplary "diluents" include water, physiological saline solution, human serum albumin, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylene diamine-tetra- acetic acid, buffers such as acetates, citrates or phosphates and agents for adjusting the osmolarity, such as sodium chloride or dextrose.
Exemplary "carriers" include liquid carriers (such as water, saline, culture medium, saline, aqueous dextrose, and glycols) and solid carriers (such as carbohydrates exemplified by starch, glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid and glutathione, and hydrolyzed proteins.
The term "excipient" refers herein to any inert substance (e.g., gum arabic, syrup, lanolin, starch, etc.) that forms a vehicle for delivery of an antigen. The term excipient includes substances that, in the presence of sufficient liquid, impart to a composition the adhesive quality needed for the preparation of pills or tablets.
The invention's compositions may be administered in an immunologically effective amount. As used herein the terms "immunologically effective amount," "pharmaceutically effective amount," "therapeutically effective amount," and "protective amount" of a composition with respect to a disease (such as pathogen infection) refer to, in one embodiment, an amount of the composition that delays, reduces, palliates, ameliorates, stabilizes, prevents and/or reverses one or more symptoms of the disease compared to in the absence of the composition of interest. Examples include, without limitation, tumor size and/or tumor number in cancer disease, glucose levels in blood and/or urine in diabetes, standard biochemical kidney function tests in kidney disease, etc. The terms also include, in another embodiment, an amount of the composition that reduces infection by a pathogen
(e.g., HIV, malaria parasite, Pse domonas species), regardless of whether disease symptoms are altered (i.e., increased or reduced).
Specific "dosages" can be readily determined by clinical trials and depend, for example, on the route of administration, patient weight (e.g. milligrams of drug per kg body weight). The term "delaying" symptoms refers to increasing the time period between exposure to the immunogen or virus and the onset of one or more symptoms of the exposure. The term "eliminating" symptoms refers to 100% reduction of one or more symptoms of exposure to the immunogen or virus.
As used herein, the actual amount, i.e., "dosage," encompassed by the term
"pharmaceutically effective amount," "therapeutically effective amount," "immunologically effective," and "protective amount" will depend on the route of administration, the type of subject being treated, and the physical characteristics of the specific subject under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical, veterinary, and other related arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors that those skilled in the art will recognize. The dosage amount and frequency are selected to create an effective level of the compound without substantially harmful effects.
Methods of administering a pharmaceutically effective amount of the invention's compounds are well known in the art and include, without limitation, intramuscular administration, intradermal administration, subcutaneous administration, aerosol
administration, oral administration, and/or sub-lingual administration. Administration may be parenteral, oral, intraperitoneal, intranasal, topical, etc. Parenteral routes of administration include, for example, subcutaneous, intravenous, intramuscular, intrastemal injection, and infusion routes.
The compositions of the invention may be administered before, concomitantly with, and/or after manifestation of one or more symptoms of a disease or condition. Also, the invention's compositions may be administered before, concomitantly with, and/or after administration of another type of drug or therapeutic procedure (e.g., surgery). For example, in the case of pathogen infection, the invention's compounds may be administered before, concomitantly with, and/or after administration of antibiotics and/or antivirals.
The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for delivery by injection, h a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
In one embodiment, further discussed below, an immunologically-effective amount of the invention's compositions (e.g., vaccines) produces a) an innate immune response against the polypeptide of interest and/or b) an adaptive immune response against the polypeptide of interest, and/or c) an altered transcriptome response, when compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
In one embodiment, "immunogenically-effective amount" and "immunologically- effective amount" refer to that amount of a molecule that elicits and/or increases production of an immune response (including production of specific antibodies and/or induction of a TCL response) in a host upon vaccination. Γ) Innate Immune Response To The Invention's Compositions
In one embodiment, an immunologically-effective amount of the invention's compositions (e.g., vaccines) produces an innate immune response against the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
"Innate immune response" and "innate immune system" refers to the number and/or biological activity of cells, cytokines and/or chemokines (e.g., IL-12, MCP-1, G.CSF, IL-6, ΜΓΡ-lbeta, RANTES) (e.g. in the plasma space) that defend the host from infection by other organisms, in a non-specific manner. Thus, the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.
The major functions of the vertebrate innate immune system include recruiting immune cells to sites of infection and inflammation, through the production of chemical factors, including specialized chemical mediators, called cytokines. An additional function is activation of the complement cascade to identify bacteria, activate cells and to promote clearance of dead cells or antibody complexes. A further function is the identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells. Yet another function includes activation of the adaptive immune system through a process known as antigen presentation.
Innate immune responses following administration of the invention's adenoviral compositions may be measured by methods known in the art and those disclosed herein, including, detecting influx of cytokines and chemokines into the plasma space, and/or detecting an increase in activation of cells of the innate immune system, such as NK cells, Dendritic Cells, T cells, B-cells and/or other antigen presenting cells..
Thus in one embodiment, the innate immune response comprises a) an increase in the level of one or more splenocyte cell that is "specific" for the polypeptide of interest, and that expresses one or more of CD3, CD8, NK1.1 and CD69, and b) a reduction in the level of antibody that specifically binds with the polypeptide of interest. In a particular embodiment, the innate immune response comprises an increase in the level of at least one of IL-12, MCP- 1, G-CSF, IL-6, MlP-lbeta, and RANTES (e.g., in the plasma space) of the subject compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP). In yet another embodiment, the innate immune response comprises an increased number of CD69 positive lymphocytes that are specific for the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)). In a particular embodiment, the CD69 positive lymphocytes are exemplified by natural killer (NK) cells and natural killer T (NKT) cells. "Natural killer" ("NK") cells cytotoxic lymphocytes that constitute a major component of the innate immune system, and play a major role in the rejection of tumors and cells infected by viruses. The cells kill by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis or necrosis. NK cells are CD3 - NK1.1 + lymphocyte cells.
"Natural killer T" ("NKT") cells are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. Many of these cells recognize the non-polymorphic CD Id molecule, an antigen-presenting molecule that binds self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. NKT cells are
CD3+ NK1.1+ lymphocyte cells.
For example, the inventor's data demonstrate that an exemplary Adenovirus vector expressing the exemplary SLAM family receptor adaptor protein (SAP) EAT-2, induces greater in vivo activation of innate immune cells (Example 2 and 3). G) Adaptive Immune Response To The Invention's Compositions
In one embodiment, an immunologically-effective amount of the invention's compositions (e.g., vaccines) produces an adaptive immune response against the polypeptide of interest, compared to a control (e.g., administering a vector that lacks the first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP)).
"Adaptive immune response" and "adaptive immune system" refers to the number and/or biological activity of highly specialized, systemic cells and molecules that eliminate or prevent pathogenic challenges. The adaptive or "specific" immune system is activated by the "non-specific" and evolutionarily older innate immune system. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. It is adaptive immunity because the body's immune system prepares itself for future challenges. The major functions of the adaptive immune system include the recognition of specific "non-self antigens in the presence of "self, during the process of antigen
presentation. Another function includes the generation of responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells. Yet a further function includes the development of immunological memory, in which each pathogen is
"remembered" by a signature antibody. These memory cells can be called upon to quickly eliminate a pathogen should subsequent infections occur.
Adaptive immune responses following administration of the invention's compositions comprise non-specific cellular mediated immune (CMI) responses, such as those mediated by interferon gamma (IFNy) (IENgamma). Thus in one embodiment, the adaptive immune response comprises an increase in the level of IFNy produced by one or more cell that is specific for the polypeptide of interest, wherein the cell is selected from CD3+/CD8+ T-cell, CD3+/CD8- T cell, CD3- NK1.1+ natural killer cell, and/or CD3+/NK1.1+ natural killer T cell. The inventors' data show that an exemplary Adenovirus vector expressing the exemplary SLAM family receptor adaptor protein (SAP) EAT-2, induces greater in vivo activation of adaptive immune cells (Example 4) including when co-administered with and Adenovirus expressing an HIV antigen (Example 5).
Importantly also, the methods of the invention produce an immune response that comprises an increase in cytolytic activity of CD8+ T cells that specifically bind to the antigenic polypeptide of interest. "Cytolytic activity" refers to cell killing as determine by, for example, a CTL assay as described herein in Example 1.
For example, data herein show that "Cytotoxic T lymphocytes" ("CTLs") derived from mice co-immunized with an exemplary Adenovirus vector expressing the exemplary SLAM family receptor adaptor protein (SAP) EAT-2, together with an Adenovirus expressing an HIV antigen, had a significantly higher ability to eliminate the adoptively transferred splenocytes pulsed with the antigen peptides as compared to control mice
(Example 7). Data herein also show that co-vaccination with Ad-EAT2 results in the induction of increased numbers and function of HIV-Gag-specific CD8+ T cells (Figures 5-7). EXPERIMENTAL
The following examples serve to illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. EXAMPLE 1
Methods Used in The Examples Below
Vector construction: The Ad-GFP virus was purified as previously described 51. The Open Reading Frame (ORF) of EAT-2 gene (Genbank Accession # NM_053282.4) was excised using primers flanked by Xhol andXbal restriction endonucleases (NEB, Ipswich, MA) from a plasmid (Open Biosystem, Huntsville, USA) and subcloned into the pAdTrack Shuttle vector which contains a CMV expression cassette. The resulting pAdTrack-EAT-2 shuttle plasmid was linearized with Pmel restriction enzyme and homologously recombined with the pAdEasyl Ad5 vector genome as previously described 2 yielding pAd-EAT2. HEK293 cells were transfected with Pad linearized plasmid and viable virus was obtained and amplified after several rounds of expanding infection. Ad-EAT2 virus was purified using a CsCl2 gradient as previously described 53. The titer obtained was approximately 2.3>< 1012 vp/ml. Direct sequencing and restriction enzyme mapping were carried out to confirm the integrity of the EAT-2 sequence. To construct Ad-HIV/Gag, the HXB2 Gag gene (Genbank Accession #K03455) was blunt end sub-cloned into the EcoRV site of pShuttle-CMV. Restriction digests and sequencing were used to confirm the sequence integrity and correct orientation of the resulting shuttle (pShuttle-CMV Gag). Recombination and viral propagation was completed as described above. EAT-2 and Gag expression was verified using Western blotting.
Animal Procedures: All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC). Adult male C57BL/6 or Balb/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Ad5 vectors were injected intravenously (via the retro-orbital sinus, total volume 200 μΐ) or intramuscularly (into the tibialis anterior of the right hindlimb, total volume 20 μΐ) into 8 weeks old male mice after performing proper anesthesia with isofluorane. A total of 7.5 x 1010 vps in 200 μΐ of PBS was injected per mouse intravenously. Several groups of mice (in C57BL/6 background) were analyzed in this work:
Mock, PBS injected mice, baseline
Ad-GFP, control groups injected with conventional Ad
Ad-EAT2 experimental groups, injected with EAT-2 overexpressing Ad Control and experimental mice were sacrificed at different times after mock or virus treatment: 6 hours post injection (h.p.i.) and 48 h.p.i. (N=4 for virus injected groups, N=4 for Mock injected groups, unless otherwise specified).
For intramuscular injections the following groups of mice were injected with lxlO7 or lx 109 ps/mouse (BALB/c or C57B1/6),
Naive - un-injected mice, baseline
Ad-HIV/Gag+ Ad-GFP, control group co-immunized with first generation Ad5-based vaccine
Ad-HIV/Gag+ Ad-EAT2, experimental group, co-immunized with Ad-HIV/Gag and
EAT-2 expressing Ad-based vaccine.
Control and experimental mice were sacrificed at day 14 (N=6 for virus injected groups, N=3 for Naives, unless otherwise specified). Plasma and tissue samples were collected and processed at the indicated time points in accordance with Michigan State University
Institutional Animal Care and Use Committee. All procedures with recombinant Ads were performed under BSL-2, and all vector treated animals were maintained in ABSL-2 conditions. Care for mice was provided in accordance with PHS and AAALAC standards.
Cytokine and chemokine analysis: A mouse 7-plex multiplex based assay was used to determine the indicated cytokine/ chemokine plasma concentrations per the manufacturer's instructions (Bio-Rad, Hercules, CA) via Luminex 100 technology (Luminex, Austin, TX) essentially as previously described 5.
ELISPOT analysis: Splenocytes were harvested from individual mice and RBCs were lysed using ACK lysis buffer (Invitrogen, Carlsbad, CA). 96-well Multiscreen high protein binding Immobilon-P membrane plates (Millipore, Billerica, MA) were pre-treated with ethanol, coated with mouse anti-IFNy or IL-2 capture antibodies, incubated overnight, and blocked prior to the addition of 5x 105 splenocytes/well. Ex vivo stimulation included the incubation of splenocytes in 100 of media alone (unstimulated) or media containing 4μg/ml of Gag specific peptides (Gag-AMQMLKETI: SEQ ID NO:06 constructed by Genscript, Piscataway, NJ) or QBI# 304796 for 24 hours in a 37°C, 5% C02 incubator. 15mer Gag specific peptides were obtained from the ΝΓΗ AIDS Reagent and Reference Program Cat# 8117 Lot# 9.
Staining of plates was completed per the manufacturer's protocol. Spots were counted and photographed by an automated ELISPOT reader system (Cellular Technology, Cleveland, OH). Ready-set Go ΓΤΝγ and IL-2 mouse ELISPOT kits purchased from eBioscience (San Diego, CA). Cell staining and flow cytometry: Splenocytes were stained with various combinations of the following antibodies: PE-CD69, (3 μ^πύ), FITC-CD8a, APC-CD3, APC-Cy7-CD3, Alexa Floure700-CD8a, PerCpCy5.5-CD19, PE-Cy7-NK1.1, PE-Cy7-TNF , APC-IFNy (4 μg/ml) (All obtained from BD Biosciences, San Diego, CA), and PerCpCy5.5-IL-2 (4 μg/ml) (BioLegend, San Diego, CA). Cells were incubated on ice with the appropriate antibodies for 30 minutes, washed, and sorted using an LSR Π instrument and analyzed using FlowJo software. For intracellular cytokines staining, cells were surface stained, fixed with 2% formaldehyde (Polysciences, Warrington, PA), permeabilized with 0.2% Saponin (Sigma- Aldrich, St. Louis, MO), and stained for intracellular cytokines. Large cells and debris were excluded in the forward- and side-scatter plot, to minimize background levels of staining caused by nonspecific binding of antibodies; we initially stained the cells with CD 16/32 FcR ΠΙ/ΙΙ antibody. In addition we included the violet fluorescent reactive dye (ViViD, Invitorgen) as a viability marker to exclude dead cells from the analysis 54. Blood was isolated by retro-orbital bleeds and PBMCs were isolated using Lympholyte-Mammal (Cedarlane, Burlington NQ.Tetramer staining of PBMCs was completed using a PE conjugated MHC-I tetramer folded with the AMQMLKETI peptide (SEQ ID NO:06) generated at the NIH Tetramer Core Facility. CD8+ T cells were depleted from pooled splenocytes preparations using MACS beads and LS columns per the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). % CD8" SFC = SFC CD8-dep/SFC CD8+). Depletion was verified using FACS analysis using APC-CD8a, and Pacific Blue-CD4 antibodies (BD Biosciences, San Diego, CA). FACS analyses revealed >96% depletion of CD8+ T cells (Fig. 13). In vivo CTL Assay: Balb/c or C57B1/6 mice were co- vaccinated with equivalent doses of Ad-HrV/Gag with either Ad-GFP or Ad-EAT2 (totaling 1 χ 107 vps for Balb/c and IX 109 vps for C57B1/6 mice). At 14 days, syngeneic splenocytes were isolated and either pulsed with an irrelevant peptide specific to the Plasmodium falciparum circumsporozoite antigen
( YDNAGTNL: SEQ ID NO:05) or with the HIV-Gag immunodominant AMQMLKETI peptide (SEQ ID NO:06) or QBI# 304796 for 1 hour at 37°C. Irrelevant peptide pulsed cells were subsequently stained with ΙμΜ CFSE (CFSELow) while Gag-peptides pulsed cells were stained with 10μΜ CFSE (CFSEHigh). Naive and immunized mice were injected with equivalent amount of both CFSELow and CFSEHlgh stained cells via the retro-orbital sinus. After 5 hours, mice were terminally sacrificed and splenocytes were recovered and sorted on an LS II flow cytometer. FlowJo software was used to determine percentages of CFSE stained cells. % Specific killing = l-((% CFSEHigh/ % CFSELow) i_ized (% CFSEHigh / %
CFSE ) non-immunized) · Statistical analysis: Statistically significant differences in toxicities associated with innate immune responses were determined using One Way ANOVA with a Student-Newman-Keuls post-hoc test (p value < 0.05). For ELISPOT analysis, a two way ANOVA was used followed by Bonferroni post hoc test (P < 0.05). For multiparameter flow cytometry, a One Way ANOVA with a Student-Newman-Keuls post-hoc test was used. For in vivo CTL assay, a One Way ANOVA with a Student-Newman-Keuls post-hoc test was used. All graphs in this paper are presented as Mean ± SD. GraphPad Prism software was utilized for statistical analysis.
EXAMPLE 2
EAT-2-expressing Ad vectors enhance Ad vector induced innate immune responses in vivo.
We constructed an adenovirus vector specifically designed to express the SLAM family receptors adaptor protein EAT-2. These EAT-2 expressing Ad vectors were fully viable, grew to high titers, and were purified and quantified as done for conventional Ad vaccines 5' 23. We systemically administered Ad-EAT2 into C57BL/6 mice and measured the levels of cytokines and chemokines in the plasma 6 hours post injection (h.p.i.). Systemic administration of the Ad-EAT2 into C57BL/6 mice resulted in induction of significantly higher plasma levels of RANTES and MCP-1 at 6 h.p.i. as directly compared to Ad-GFP injected control mice (Fig. 1). The levels of IL-6, IL-12p40, ΜΙΡ-Ιβ, KC, and G-CSF were also significantly induced by Ad-EAT2 vectors; however, these levels were not statistically different between Ad-EAT2 and Ad-GFP injected mice (Fig. 10).
EXAMPLE 3
Administration of EAT-2 expressing Ad induces greater activation of innate immune cells.
Ads enhance innate immune cell effector functions 4~26. To evaluate the phenotype of immune cells following systemic injection of an Ad expressing EAT-2, we analyzed the expression of the lymphocyte activation marker CD69 as well as IFN-γ production in various immune cells shortly after administration of Ad-EAT2 into C57BL/6 mice. Our results confirm that Ads, in general, significantly induce a rapid activation of NK and NKT cells in both PBMCs and spleens, as measured by the presence of increased percentages of CD69 expressing NK and NKT cells (Fig. 2a and b). Importantly, injection of Ad-EAT2 resulted in significantly higher numbers of NK and NKT cells expressing CD69 derived from PBMCs or splenocytes at 6 h.p.i. (Fig. 2a and b). At 48 h.p.i., CD69 expression on NK cells remained significantly higher (p<0.05) in PBMCs derived from Ad-EAT2 injected mice as compared to Ad-GFP injected mice (Fig. 11a) but by 48 h.p.i., these differences had dissipated (Fig. 11c).
As compared to mock injected mice, treatment with either Ad-EAT2 or Ad-GFP also induced significantly elevated numbers of IFNy+ NK cells at both 6 and 48 h.p.i. (p<0.001 and p<0.05, respectively) (Fig. 12). The number of NKT cells expressing CD69 were also significantly increased (p<0.05) in PBMCs derived from Ad-EAT2 injected, as compared to the Ad-GFP injected control mice, at 48 h.p.i. (Fig. 11a). CD69 expression on splenic NKT cells isolated from both Ad-EAT2 and Ad-GFP injected mice were both significantly
(p<0.05) increased over mock injected mice (Fig. 1 lc).
EXAMPLE 4
Administration of EAT-2 expressing Ads induces greater activation of adaptive immune cells.
Since SLAM family receptors are expressed in various innate and adaptive immune cells13 and the activation of T cell and/or B cells can be initiated or accentuated by innate immune system activation 9 , we sought to analyze adaptive immune cell responses shortly after administration of Ad-EAT2. Our results first confirmed that Ad vector administration induces a rapid activation of CD3+CD8+ T-cells CD3+CD8~ T-cells, and B cells. For example, injection of Ad-EAT2 resulted in significantly higher numbers of splenic CD69 expressing CD3+CD8+ T cells (p<0.01), CD3+CD8" T cells (p<0.01), and B cells (p<0.001) at 6 h.p.i (Fig. 2a and b) as compared to the numbers of these cells being induced by the control Ad vector. At 48 h.p.i., the percentage of CD69 expressing CD3+CD8+ T cells, CD3+CD8" T cells, and B cells derived from Ad-EAT2 and Ad-GFP injected mice remained significantly higher (p<0.001) over mock injected mice; however, no statistical differences were observed between the two viruses at this time point (Fig. l id). We also evaluated the percentage of CD69 expressing cells in PBMCs from the same animals. At 6 h.p.i., we observed a significantly (p<0.05) higher percentage of CD69 expressing total lymphocytes in Ad-EAT2 injected mice compared to Ad-GFP injected mice. However, when analyzing specific lymphocyte subsets, we observed a significant (p<0.05) percentage of CD69 expressing CD3+CD8~ T cells isolated from Ad-EAT2 injected mice as compared to cells isolated from Ad-GFP injected mice. By 48 h.p.i., statistically significant differences were resolved (Fig. l ib). EXAMPLE 5
Ad vector expressing EAT-2 enhances T cell responses to a co-administered antigen.
Simultaneous administration of vaccines with adjuvants can stimulate the innate immune system to significantly improve the adaptive immune responses to a co-administered antigenic target 27"30. To investigate whether the enhanced innate immune profile promoted by Ad mediated expression of EAT-2 could influence the adaptive immune responses to a coadministered antigen, we co-immunized Balb/c or C57BL/6 mice with an Ad-based vector expressing the HIV-1 clade B Gag protein (HXB2) along with either the Ad-GFP, or the Ad- EAT2 vectors. We performed initial dose curve studies to identify the lowest dose of Ad- HIV/Gag that generated detectable Gag-specific cellular immune responses. As a result, we identified an Ad-HIV/Gag dose of 5.0 xlO6 vps/mouse for Balb/c mice, and 5.0 xlO8 vps for C57BL/6 mice as the most relevant experimental doses for these studies. Six days after Balb/c mice were intramuscularly injected with 5xl06 of Ad-HIV/Gag mixed with equivalent amounts of either Ad-GFP or Ad-EAT2 , we were able to detect heightened Gag specific tetramer-positive CD8+ T cells in PBMCs derived from mice co-immunized with Ad- HIV/Gag + Ad-EAT2 as compared to Ad-HIV/Gag+ Ad-GFP co-immunized mice (Fig. 3a ). At 14 d.p.L, PBMCs (p<0.05) and splenocytes (p<0.05) derived from mice co-immunized with Ad-HIV/Gag + Ad-EAT2 had higher numbers of Gag-specific tetramer-positive CD8+ T cells as compared to the respective cell populations isolated from control mice (Fig. 3b and c). Following ex vivo stimulation with the immunodominant Gag peptides AMQMLKETI (QBI# 304754: SEQ ID NO:06), for Balb/c mice) or QBI# 304796 (for C57BL/6 mice), Ad- EAT2 co-immunization resulted in significantly (pO.001) increased numbers of Gag-specific IFN-γ secreting splenocytes (Fig. 4a, and b, respectively). We also observed significantly increased numbers of IL-2 secreting splenocytes derived from Balb/c mice co-immunized with Ad-HIV/Gag + Ad-EAT2 as compared to Ad-HIV/Gag+ Ad-GFP co-immunized mice (p<0.01) (Fig. 4a). In addition, we observed increased numbers of Gag-specific IFN-γ and IL- 2 co-secreting splenocytes exposed to different HIV-Gag specific peptides QBI# 304742, 304769, 304779, 304800, and a peptide pool (304790, 403808, and 304826) (Fig. 5a and b). In the analysis of the potential effect of EAT-2 overexpression on the breadth of the T cell repertoire responding to various Gag derived peptides, we stimulated splenocytes preparations derived from the immunized mice ex vivo with peptide pools containing 2-4 Gag specific 15mer peptides that spanned the entire HIV-Gag protein sequence. We observed an increased breadth as to the number of HIV Gag-specific peptides that triggered cellular responses from splenocytes derived from the Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to splenocytes from the Ad-HIV/Gag and Ad-GFP co-immunized mice exposed to the same peptide pools (Fig.6 a and b). CD8 T cell depletion studies suggested that the majority of these T cell responses were primarily due to CD8+ T cells, (Fig. 13).
EXAMPLE 6
Assessment of Gag-specific T cell cytokine responses by multiparameter flow cytometry
The increased presence of antigen specific, polyfunctional T cells correlate with improved vaccine-induced protective immunity, and form the basis for a long lived memory response 31~33. To evaluate the cytokine responses of the Gag-specific CD8+ T cells generated after Ad-HIV/Gag and Ad-EAT2 co-immunization, six-color flow cytometry was used to enumerate the frequency of CD8+ T cells producing IFNy, TNFa, and/or IL-2 after ex vivo stimulation. We observed statistically higher numbers of Gag-specific IFNy-positive (p<0.05) (Fig. 7a and b) and IFNy/TNFa-double positive (p<0.05) (Fig. 8a) CD8+ T cells derived from Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to control mice. When evaluating TNFa or IL-2 single positive cells, we also observed increased numbers of CD8+ T cells that express TNFa or IL-2 derived from Ad-HIV/Gag and Ad-EAT2 co-immunized mice as compared to Ad-HIV/Gag and Ad-GFP co-immunized mice; however, these trends were not statistically significant.
EXAMPLE 7
Memory CD8+ T cells from Ad-EAT2 co-immunized mice exhibit Gag-specific cytotoxicity in vivo.
The direct measurement of in vivo functionality of CD8+ cytotoxic T lymphocytes (CTL) provides a critical assessment as to the functional capacity of antigen-specific CD8+ T cells to kill cells displaying an antigen derived peptide. To evaluate the cytolytic activity of the Gag-specific CD8+ T lymphocytes generated after Ad-HIV/Gag and Ad-EAT2 co- immunization in vivo, mice were co-immunized with Ad-HIV/Gag+ Ad-EAT2 or Ad- HIV/Gag+ Ad-GFP. 14 days after vaccination, the two groups of mice were then injected with carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic splenocytes pulsed with the Gag derived peptides AMQMLKETI (SEQ ID NO:06) or QBI# 304796. The elimination of peptide-pulsed splenocytes (CFSE1^11) was examined by a flow cytometry- based CTL assay 34. Our data demonstrated that CTLs derived from mice co-immunized with Ad-HIV/Gag and Ad-EAT2 had a significantly higher ability to eliminate the adoptively transferred splenocytes pulsed with the Gag peptides as compared to control mice p<0.01 for C57B1/6 and p<0.05 for Balb/c mice) (Fig. 8a and b).
EXAMPLE 8
RAW 264.7 cells were passed into 12-well plates 1 day before the transfection at a concentration of 1 χ 1 θ5 cells/well. Cells were used for transfection with Calcium phosphate transfection reagent following the manufacturer's instructions in DMEM with 10% FBS. Cells were then either un-transfected or transfected with 500ng, lug, or 2ug of P-Shuttle- CMV plasmid or P-Shuttle-CMV plasmid that expresses EAT-2 gene. 24 hours after transfection, media were changed and cell were re-incubated for another 48 hours. Cells were then harvested, washed with FACS buffer, and stained with antibodies specific for molecules associated with antigen presenting cell function including: CD40 (FITC), CD80 (APC), CD86 (V450) and MHC-II (Alexa-fiuor700). Data were sorted on a LSR II flow cytometer. FlowJo software were used for data analysis. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a student- Newman-Keuls post-hoc test, p<0.05 was deemed a statistically significant difference. * denotes p<0.05, ** denotes p<0.01, *** p<0.001 statistically different from untransfected cells. GraphPad Prism software was used for statistical analysis. The results are showin in Figures 16-18.
REFERENCES
1. Rerks-Ngarm, S. et al. Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand. The New England journal of medicine (2009). 2. Barouch, D.H. & Nabel, GJ. Adenovirus vector-based vaccines for human immunodeficiency virus type 1. Human gene therapy 16, 149-156 (2005).
3. Catanzaro, A.T. et al. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 candidate vaccine delivered by a replication-defective recombinant adenovirus vector. The Journal of infectious diseases 194, 1638-1649 (2006). 4. Liu, J. et al. Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457, 87-91 (2009).
5. Appledorn, D.M. et al. Adenovirus vector-induced innate inflammatory mediators, MAPK signaling, as well as adaptive immune responses are dependent upon both TLR2 and TLR9 in vivo. J Immunol 181, 2134-2144 (2008).
6. Hartman, Z.C. et al. Adenovirus infection triggers a rapid, MyD 88 -regulated transcriptome response critical to acute-phase and adaptive immune responses in vivo.
Journal of virology 81, 1796-1812 (2007).
7. Nociari, M., Ocheretina, O., Schoggins, J.W. & Falck-Pedersen, E. Sensing infection by adenovirus: Toll-like receptor-independent viral DNA recognition signals activation of the interferon regulatory factor 3 master regulator. Journal of virology 81, 4145-4157 (2007).
8. Muruve, D.A. et al. The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response. Nature 452, 103-107 (2008).
9. Iwasaki, A. & Medzhitov, R. Toll-like receptor control of the adaptive immune responses. Nature immunology 5, 987-995 (2004).
10. Pulendran, B. & Ahmed, R. Translating innate immunity into immunological memory: implications for vaccine development. Cell 124, 849-863 (2006).
11. van Duin, D., Medzhitov, R. & Shaw, A.C. Triggering TLR signaling in vaccination. Trends in immunology 27, 49-55 (2006). 12. Veillette, A., Dong, Z., Perez-Quintero, L.A., Zhong, M.C. & Cruz-Munoz, M.E. Importance and mechanism of 'switch' function of SAP family adapters. Immunological reviews 232, 229-239 (2009).
13. Veillette, A. Immune regulation by SLAM family receptors and SAP-related adaptors. Nature reviews 6, 56-66 (2006). 14. Veillette, A., Dong, Z. & Latour, S. Consequence of the SLAM-SAP signaling pathway in innate-like and conventional lymphocytes. Immunity 27, 698-710 (2007). 15. Mavaddat, N. et al. Signaling lymphocytic activation molecule (CDwl50) is homophilic but self-associates with very low affinity. The Journal of biological chemistry 275, 28100-28109 (2000).
16. Kumaresan, P.R., Lai, W.C., Chuang, S.S., Bennett, M. & Mathew, P.A. CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function. Molecular immunology 39, 1-8 (2002).
17. Flaig, R.M., Stark, S. & Watzl, C. Cutting edge: NTB-A activates NK cells via homophilic interaction. J Immunol 172, 6524-6527 (2004).
18. Ma, C.S., Nichols, K.E. & Tangye, S.G. Regulation of cellular and humoral immune responses by the SLAM and SAP families of molecules. Annual review of immunology 25,
337-379 (2007).
19. Calpe, S. et al. The SLAM and SAP gene families control innate and adaptive immune responses. Advances in immunology 97, 177-250 (2008).
20. Veillette, A. NK cell regulation by SLAM family receptors and SAP -related adapters. Immunological reviews 214, 22-34 (2006).
21. Bleharski, J.R., Niazi, K.R., Sieling, P.A., Cheng, G. & Modlin, R.L. Signaling lymphocytic activation molecule is expressed on CD40 ligand- activated dendritic cells and directly augments production of inflammatory cytokines. J Immunol 167, 3174-3181 (2001).
22. Wang, N. et al. The cell surface receptor SLAM controls T cell and macrophage functions. The Journal of experimental medicine 199, 1255-1264 (2004).
23. Seregin, S.S. et al. CRl/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies. Gene therapy 16, 1245-1259 (2009).
24. Zhu, J., Huang, X. & Yang, Y. A critical role for type I IFN-dependent NK cell activation in innate immune elimination of adenoviral vectors in vivo. Mol Ther 16, 1300- 1307 (2008). 25. Muruve, D.A. The innate immune response to adenovirus vectors. Human gene therapy 15, 1157-1166 (2004).
26. Hensley, S.E. & Amalfitano, A. Toll-like receptors impact on safety and efficacy of gene transfer vectors. Mol Ther 15, 1417-1422 (2007). 27. Huang, Y. et al. Enhancement of HIV DNA vaccine immunogenicity by the NKT cell ligand, alpha-galactosylceramide. Vaccine 26, 1807-1816 (2008).
28. Gonzalez- Aseguinolaza, G. et al. Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines. The Journal of experimental medicine 195, 617-624 (2002). 29. Wille-Reece, U. et al. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Thl and CD8+ T cell responses in nonhuman primates. Proceedings of the National Academy of Sciences of the United States of America 102, 15190-15194 (2005).
30. Wille-Reece, U. et al. Toll -like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. The
Journal of experimental medicine 203, 1249-1258 (2006).
31. Darrah, P.A. et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nature medicine 13, 843-850 (2007).
32. Berts, M.R. et al. HIV nonprogressors preferentially maintain highly functional HIV- specific CD8+ T cells. Blood 107, 4781-4789 (2006).
33. Zimmerli, S.C. et al. HIV- 1 -specific IFN-gamma/IL-2- secreting CD8 T cells support CD4-independent proliferation of HIV- 1 -specific CD8 T cells. Proceedings of the National Academy of Sciences of the United States of America 102, 7239-7244 (2005).
34. Yang, Y., Huang, C.T., Huang, X. & Pardoll, D.M. Persistent Toll-like receptor signals are required for reversal of regulatory T cell-mediated CD 8 tolerance. Nature immunology 5, 508-515 (2004). 35. Degli-Esposti, M.A. & Smyth, M.J. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nature reviews 5, 112-124 (2005).
36. Vitale, M. et al. NK-dependent DC maturation is mediated by TNFalpha and
IFNgamma released upon engagement of the NKp30 triggering receptor. Blood 106, 566-571 (2005).
37. Mailliard, R.B. et al. Dendritic cells mediate NK cell help for Thl and CTL responses: two-signal requirement for the induction of NK cell helper function. J Immunol 111, 2366-2373 (2003).
38. Piccioli, D., Sbrana, S., Melandri, E. & Valiante, N.M. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. The Journal of experimental medicine
195, 335-341 (2002).
39. Martin-Fontecha, A. et al. Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nature immunology 5, 1260-1265 (2004).
40. Kelly, J.M. et al. Induction of tumor- specific T cell memory by NK cell-mediated tumor rejection. Nature immunology 3, 83-90 (2002).
41. Krebs, P. et al. NK-cell -mediated killing of target cells triggers robust antigen- specific T-cell-mediated and humoral responses. Blood 113, 6593-6602 (2009).
42. Cruz-Munoz, M.E., Dong, Z., Shi, X., Zhang, S. & Veillette, A. Influence of CRACC, a SLAM family receptor coupled to the adaptor EAT-2, on natural killer cell function. Nature immunology 10, 297-305 (2009).
43. Tassi, I. & Colonna, M. The cytotoxicity receptor CRACC (CS-1) recruits EAT-2 and activates the PI3K and phospholipase Cgamrna signaling pathways in human NK cells. J Immunol 175, 7996-8002 (2005).
44. Eissmann, P. & Watzl, C. Molecular analysis of NTB-A signaling: a role for EAT-2 in NTB-A-mediated activation of human NK cells. J Immunol 111, 3170-3177 (2006).
45. Fujii, S., Shimizu, K., Smith, C, Bonifaz, L. & Steinman, R.M. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. The Journal of experimental medicine 198, 267-279 (2003).
46. Hermans, IF. et al. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 171, 5140-5147 (2003).
47. Cerundolo, V., Silk, J.D., Masri, S.H. & Salio, M. Harnessing invariant NKT cells in vaccination strategies. Nature reviews 9, 28-38 (2009).
48. Hermans, IF. et al. Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells. J Immunol 178, 2721-2729 (2007). 49. Dondji, B. et al. Intradermal NKT cell activation during DNA priming in
heterologous prime-boost vaccination enhances T cell responses and protection against Leishmania. European journal of immunology 38, 706-719 (2008).
50. Kong, W.P. et al. Expanded breadth of the T-cell response to mosaic human immunodeficiency virus type 1 envelope DNA vaccination. Journal of virology 83, 2201- 2215 (2009).
51. Appledorn, D.M. et al. Complex interactions with several anus of the complement system dictate innate and humoral immunity to adenoviral vectors. Gene therapy 15, 1606- 1617 (2008).
52. He, T.C. et al. A simplified system for generating recombinant adenoviruses.
Proceedings of the National Academy of Sciences of the United States of America 95, 2509- 2514 (1998).
53. Ng, P. & Graham, F.L. Construction of first-generation adenoviral vectors. Methods in molecular medicine 69, 389-414 (2002).
54. Perfetto, S.P. et al. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. Journal of immunological methods 313, 199-208 (2006). All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiment, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and in fields related thereto are intended to be within the scope of the following claims.

Claims

We claim:
1. A replication incompetent recombinant adenovirus vector comprising a DNA sequence that contains, in operable combination,
a) DNA sequence encoding a replication defective adenovirus, and
b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
2. The vector of Claim 1, further comprising c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest.
3. The vector of Claim 1 , wherein said SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein selected from the group consisting of human EAT-2 (SEQ ID NO:01) and mouse EAT-2 (SEQ ID NO:29).
4. The vector of Claim 1, wherein said DNA sequence encoding a replication defective adenovirus lacks adenovirus El gene coding sequence.
6. The vector of Claim 2, wherein said polypeptide of interest comprises an antigen polypeptide.
7. The vector of Claim 6, wherein said antigen polypeptide comprises Human immunodeficiency virus GAG sequence AMQMLKETI (SEQ ID NO:06).
8. The vector of Claim 6, wherein said antigen polypeptide comprises Plasmodium falciparum circumsporozoite antigen NYDNAGTNL (SEQ ID NO:05).
9. A purified DNA sequence comprising, in operable combination,
a) a nucleic acid sequence encoding a replication defective adenovirus, and b) a first heterologous nucleic acid sequence that encodes at least a portion of a SLAM family receptor adaptor protein (SAP).
10. The DNA sequence of Claim 9, further comprising, in operable combination, c) a second heterologous nucleic acid sequence that encodes a polypeptide of interest.
11. The DNA sequence of Claim 10, wherein said polypeptide of interest comprises an antigen polypeptide.
12. The DNA sequence of Claim 9, wherein said SLAM family receptor adaptor protein (SAP) comprises EAT-2 protein SEQ ID NO:01.
13. A composition comprising (a) a replication incompetent recombinant adenovirus vector selected from the group consisting of the vector of Claim land the vector of Claim 2, and (b) a pharmaceutically acceptable carrier.
14. A method for vaccinating a mammalian subject, comprising
a) providing
i) a first vector of Claim 1 , and
ii) a second vector comprising a nucleotide sequence that encodes an antigen polypeptide, and
b) administering an immunologically effective amount of said first vector and said second vector to said subject under conditions for producing an immune response to said antigen polypeptide.
15. The method of Claim 14, wherein said subject is at risk of disease.
16. The method of Claim 14, wherein said subject is at risk of infection by a pathogen.
17. The method of Claim 14, wherein said immune response comprises an adaptive immune response.
18. The method of Claim 14, wherein said immune response comprises an innate immune response.
19. The method of Claim 14, wherein said immune response comprises an increase in cytolytic activity of CD8+ T cells that specifically bind to said polypeptide of interest.
20. A method for vaccinating a mammalian subject, comprising
a) providing a pharmaceutically acceptable composition comprising a vector of Claim 6, and
b) administering an immunologically effective amount of said composition to said subject under conditions for producing an immune response to said polypeptide of interest.
PCT/US2011/033591 2010-04-22 2011-04-22 Vaccines comprising adenovirus vectors and signaling lymphocyte activating molecule-associated protein (sap) WO2011133870A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32688010P 2010-04-22 2010-04-22
US61/326,880 2010-04-22

Publications (2)

Publication Number Publication Date
WO2011133870A2 true WO2011133870A2 (en) 2011-10-27
WO2011133870A3 WO2011133870A3 (en) 2011-12-22

Family

ID=44834828

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/033591 WO2011133870A2 (en) 2010-04-22 2011-04-22 Vaccines comprising adenovirus vectors and signaling lymphocyte activating molecule-associated protein (sap)

Country Status (1)

Country Link
WO (1) WO2011133870A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11130787B2 (en) 2020-06-11 2021-09-28 MBF Therapeutics, Inc. Alphaherpesvirus glycoprotein d-encoding nucleic acid constructs and methods

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086837A1 (en) * 2000-12-21 2002-07-04 Jack Gauldie Acne vaccine
US20040241181A1 (en) * 2001-06-22 2004-12-02 Ertl Hildeghund C. J. Methods of inducing a cytotoxic immune response and recormbinant simian adenovirus compositions useful therein
US20090110695A1 (en) * 2006-03-27 2009-04-30 Menzo Jans Emko Havenga Compositions Comprising a Recombinant Adenovirus and an Adjuvant
US20100015654A1 (en) * 2005-05-16 2010-01-21 Institut De Recherches Cliniques De Montreal/ I.R.C.M. Negative regulation of NK cell functions by EAT-2, a sap-related adaptor expressed in innate immune cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086837A1 (en) * 2000-12-21 2002-07-04 Jack Gauldie Acne vaccine
US20040241181A1 (en) * 2001-06-22 2004-12-02 Ertl Hildeghund C. J. Methods of inducing a cytotoxic immune response and recormbinant simian adenovirus compositions useful therein
US20100015654A1 (en) * 2005-05-16 2010-01-21 Institut De Recherches Cliniques De Montreal/ I.R.C.M. Negative regulation of NK cell functions by EAT-2, a sap-related adaptor expressed in innate immune cells
US20090110695A1 (en) * 2006-03-27 2009-04-30 Menzo Jans Emko Havenga Compositions Comprising a Recombinant Adenovirus and an Adjuvant

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALDHAMEN ET AL.: 'Expression of the SLAM family of receptors adapter EAT-2 as a novel strategy for enhancing beneficial immune responses to vaccine antigens.' J IMMUNOL. vol. 186, no. 2, 13 December 2010, pages 722 - 732 *
CALPE ET AL.: 'The SLAM and SAP gene families control innate and adaptive immune responses.' ADV IMMUNOL vol. 97, 2008, pages 177 - 250 *
CANNONS ET AL.: 'SAP regulates T cell-mediated help for humoral immunity by a mechanism distinct from cytokine regulation.' J EXP MED vol. 203, no. 6, 05 June 2006, pages 1551 - 1565 *
DATABASE GENBANK [Online] 04 November 2001 'Homo sapiens EWS/FLI1 activated transcript 2 protein mRNA, complete cds.' Database accession no. AF403479 *
DUPRE, L. ET AL.: 'SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells.' BLOOD. vol. 105, no. 11, 27 January 2005, pages 4383 - 4389 *
VEILLETTE, A.: 'Immune regulation by SLAM family receptors and SAP-related adaptors.' NAT REV IMMUNOL. vol. 6, January 2006, pages 56 - 66 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11130787B2 (en) 2020-06-11 2021-09-28 MBF Therapeutics, Inc. Alphaherpesvirus glycoprotein d-encoding nucleic acid constructs and methods

Also Published As

Publication number Publication date
WO2011133870A3 (en) 2011-12-22

Similar Documents

Publication Publication Date Title
ES2404689T3 (en) Chimeric vaccines comprising the lumenal domain of LAMP-1 or LAMP-2
Zhou et al. PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice
US20210236623A1 (en) T-cell inducing vaccine composition combination and uses thereof
JP7454645B2 (en) HIV vaccine and its production and use methods
US11773153B2 (en) LAMP constructs
JP7319368B2 (en) Adenovirus and methods for using adenovirus
CA3170513A1 (en) Treatment of covid-19 and methods therefor
Neukirch et al. The potential of adenoviral vaccine vectors with altered antigen presentation capabilities
Cabrera-Mora et al. A recombinant chimeric Ad5/3 vector expressing a multistage plasmodium antigen induces protective immunity in mice using heterologous prime-boost immunization regimens
US20240108703A1 (en) Improved LAMP Constructs Comprising Cancer Antigens
WO2023023940A1 (en) Immunogen for inducing broad-spectrum anti-coronavirus t cell vaccine and use thereof
WO2011133870A2 (en) Vaccines comprising adenovirus vectors and signaling lymphocyte activating molecule-associated protein (sap)
Collett et al. Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates
EP1613731A2 (en) Modified adenoviral e1a constructs and methods of use thereof
EP3793595A1 (en) Improved lamp constructs comprising allergens
RU2782528C1 (en) Adenoviruses and methods for adenovirus application
US20240075129A1 (en) Anti COVID-19 Therapies targeting nucleocapsid and spike proteins
EP4118210A1 (en) Treatment of covid-19 and methods therefor
WO2023201201A1 (en) Bicistronic lamp constructs comprising immune response enhancing genes and methods of use thereof
WO2022133255A2 (en) Immunogenic compositions
AU2010226945A1 (en) Vaccination regimen

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11772769

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11772769

Country of ref document: EP

Kind code of ref document: A2