WO2004056391A1 - A method of immunisation and agents useful for same - Google Patents

A method of immunisation and agents useful for same Download PDF

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Publication number
WO2004056391A1
WO2004056391A1 PCT/AU2003/001705 AU0301705W WO2004056391A1 WO 2004056391 A1 WO2004056391 A1 WO 2004056391A1 AU 0301705 W AU0301705 W AU 0301705W WO 2004056391 A1 WO2004056391 A1 WO 2004056391A1
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Prior art keywords
hiv
vector
antigens
plasmid vector
recombinant
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PCT/AU2003/001705
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French (fr)
Inventor
Stephen J. Kent
Damian F. Purcell
David B. Boyle
Alistair Ramsay
Scott Thomson
Ian A Ramshaw
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The University Of New South Wales
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Priority claimed from AU2002953556A external-priority patent/AU2002953556A0/en
Priority claimed from AU2003905067A external-priority patent/AU2003905067A0/en
Application filed by The University Of New South Wales filed Critical The University Of New South Wales
Priority to AU2003287785A priority Critical patent/AU2003287785A1/en
Publication of WO2004056391A1 publication Critical patent/WO2004056391A1/en

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—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
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to a method of eliciting or otherwise inducing an immune response to a virus and agents for use therein. More particularly, the present invention relates to a method of eliciting or otherwise inducing an immune response to HIV and to agents for use therein.
  • the method of the present invention is predicated on the application of a novel prime-boost vaccination regime utilising, respectively, a CpG optimised plasmid vector and a viral vector, both of which encode two or more substantially non-pathogenic HIV antigens.
  • the present invention is useful, inter alia, as a prophylactic and/or therapeutic treatment regime for viral pathogen infections of mammals such as, for example, HIV infection.
  • a related aspect of the present invention is directed to the optimised vectors for use in this method.
  • HIV-1 human immunodeficiency virus type 1
  • CTL cytotoxic T lymphocytes
  • Th T-helper lymphocytes
  • HIV-1 vaccine regimens employed only non-replicating compounds such as recombinant HIV-1 proteins. Vaccination of humans or non-human primates with recombinant proteins of HIV-1 or the simian immunodeficiency virus (SIN, a simian homologue of HIV-1) generated specific antibody responses but did not generally induce protective immunity in animal studies and resulted in significant numbers of breakthrough HIV-1 infections in small human trials.
  • SIN simian immunodeficiency virus
  • HIV-1 vaccine strategies attempting to induce both enhanced T cell responses and antibody responses have primarily focussed on recombinant vaccinia (rVV) and avian pox viruses (canarypoxviruses and fowlpoxviruses) genetically engineered to express HIV-1 proteins boosted by recombinant HIV-1 proteins (Leong, K.H., AJ. Ramsay, M.J. Morin, H.L. Robinson, D.B. Boyle, and A. Ramshaw. 1995. In Vaccines 95. F. Brown, H. Chanock and E. ⁇ orrby, editors. Cold Spring Harbour Laboratory Press. 327-331).
  • rVV recombinant vaccinia
  • avian pox viruses canarypoxviruses and fowlpoxviruses
  • poxvirus vectors have the theoretical advantage that expression of foreign genes from within the infected host cells allows the loading of MHC class I molecules with immunogenic peptides and the stimulation of CTL responses.
  • vaccinations of humans and outbred non-human primates with poxvirus vectors expressing HIV-1 or SIV antigens and recombinant HIV-1 or SIV proteins despite being theoretically attractive, have induced detectable HIV-1 or SIV specific CTL responses in only a minority of recipients (Fleury, B., G. Janvier, G. Pialoux, F. Buseyne, M. ⁇ . Robertson, J. Tartaglia, E. Paoletti, M.P. Kieny, J.L.
  • Th2 cells Recombinant protein vaccinations, while facilitating a strong antibody response, primarily stimulate a particular subset of Th cells called Th2 cells, defined by their secretion of the cytokines IL-4, IL-5, and IL-10. Th2 cells and the cytokines they secrete may counteract any protective cell-mediated immunity (Kent et al. 1997, supra; Sharma, D.P., AJ.
  • Intramuscular (IM) or epidermal injection of purified plasmid DNA can induce immune responses to encoded antigens (Tang, D.C., M. De Vit, and S.A. Johnston. 1992, Nature 356:152-154).
  • Plasmid DNA vaccines which are simple and inexpensive to produce, have the potential to revolutionise or re-energise many vaccine development fields, including that of HIV-1.
  • IM injection of DNA encoding HIV-1 proteins into 2 chimpanzees generated HIV-1 specific CTL responses in one the animals and induced some protection from non-pathogenic HIV-1SF 2 infection in both animals (Boyer, J.D., K. Ugen, B. Wang, M. Agadjanyan, L. Gilbert, M. Bagarazzi, M.
  • fowlpox vaccines which undergo an abortive infection in mammalian hosts, are safer since wild type vaccinia virus can cause a lethal infection in immunodeficient humans.
  • pathogens such as HIV-1 where T cell mediated responses may be required for protective efficacy, avoiding a marked enhancement of antibody/Th2 responses, as typically observed following recombinant protein boosting, may facilitate enhanced Thl/CTL responsiveness and therefore be desirable.
  • nucleotide and amino acid sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography.
  • Each nucleotide and amino acid sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (eg. ⁇ 210>1, ⁇ 210>2, etc).
  • the length, type of sequence (DNA, protein, etc) and source organism for each nucleotide and amino acid sequence is indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide and amino acid sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO:l, SEQ ID NO:2, etc.).
  • sequence identifier referred to in the specification correlates to the information provided in numeric indicator field ⁇ 400> in the sequence listing, which is followed by the sequence identifier (eg. ⁇ 400>1, ⁇ 400>2, etc). That is SEQ ID NO:l as detailed in the specification correlates to the sequence indicated as ⁇ 400>1 in the sequence listing.
  • One aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to a virus, said method comprising sequentially administering to said mammal:
  • Another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal:
  • Still another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal: (i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
  • a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • RNAseH, Tat, Rev and/or Env which Gag, RT, Pro, RNAseH, Tat, Rev and/or Env have been rendered substantially non-pathogenic
  • a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • said recombinant viral vector dose is administered four weeks later than said recombinant plasmid vector dose.
  • a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal: (i) two doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
  • the second recombinant plasmid vector dose is administered three weeks after the first dose
  • the first recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose
  • the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
  • the second recombinant plasmid vector dose is administered four weeks after the first dose
  • the third recombinant plasmid vector dose is administered four weeks after the second dose
  • the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose
  • the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
  • Yet another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIN, said method comprising sequentially administering to said mammal one or more doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding two or more antigens thereof of said HIV, which antigens have been rendered substantially non-pathogenic, and one or more optimised CPG motifs for a time and under conditions sufficient to elicit or induce an immune response to said antigens.
  • Still another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
  • Yet still anther aspect of the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
  • Still yet another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
  • the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
  • the present invention is directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
  • This aspect of the present invention is still further directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
  • Still another aspect of the present invention is directed to a nucleic acid construct or functional derivative thereof which construct comprises a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
  • the present invention is directed to a nucleic acid construct comprising a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
  • compositions in particular pharmaceutical compositions, comprising the nucleic acid constructs, plasmid vectors and/or viral vectors hereinbefore described.
  • Figure 1 is a schematic representation of a plasmid Map of pHIS-HIV-B
  • Figure 2 is a schematic representation of a plasmid Map of pHIS-HIV-AE
  • Figure 3 is a schematic representation of the interrelationship of pHIS-HIV-AE and pHIS- HIV-B nucleotide sequences.
  • Figure 4 is a representation of the full Vaccine Aligns: Comparison of B (SEQ ID NO:3) and AE (SEQ ID NO:4) Subtype Vaccine Sequences
  • Figure 5 a representation of the 5' primer: 75-mer XhoI/FPV early late promoter PE/L/gag 1-26 (SEQ ID NO:5)
  • Figure 6 a representation of the 3' primer: 58-mer Xhol/pox virus early transcription termination signal T 5 NT/complement of pol 3427-3458 (SEQ ID NO:6)
  • Figure 7 a representation of the 34bp Sequence Inserted (SEQ ID NO:7)
  • Figure 8 a representation of the plasmid pKGlO and pKG-gag/pol(m)
  • Figure 9 a representation of the plasmids for rFPV-HIV-AE
  • Figure 10 is a schematic representation of the splicing and construction of 5' vaccine end B CLADE.
  • Figure 11 is a schematic representation of the sequence of pHIS-HIVB (SEQ ID NO: 1).
  • Figure 12 is a diagrammatic representation of the sequence comparison of vaccines and controls.
  • Figure 13 a representation of the constructed recombinant FPV-HIV-AE vaccines
  • Figure 14 is a graphical representation of rFPV-HIV-B immunogenicity in prime/boost vaccination in mice.
  • Figure 15 is a graphical representation of IFN ⁇ ELISPOT to inactivated HIV-1 following 2nd FPV boost
  • Figure 16 is a graphical representation of IFN ⁇ ELIspot to Inactivated HIV-1 (left) and HIV-1 gag (right) at Challenge
  • Figure 17 is a graphical representation of SIV Gag-specific Response by IFN ⁇ ELISPOT
  • Figure 18 is a graphical representation of the control of pathogenic SHIV challenge by DNA/FPV prime/boost immunisation
  • Figure 19 is a schematic representation of the first macaque study.
  • Figure 20 is a schematic representation of the schedule/dose of macaque experiment.
  • Figure 21 is a graphical representation of proliferation - one week post-first FPV boost.
  • Figure 22 is a graphical representation of proliferation - one week post-final immunisation.
  • Figure 23 is a graphical representation of the comparison of 1 and 2 FPV boosts across groups.
  • Figure 24 is a graphical representation of IFNg ELISPOT to overlapping gag peptides following 2 nd FPV boost by vaccine group.
  • Figure 14 is a schematic representation of DNA vaccine construct plasmid maps.
  • Figure 25 is an image of immature pseudoviral particles budding from HeLa cells transfected with the DNA vaccines pHIS-HIV-B and pHIS-HIV-AE.
  • Figure 26 is an image of HIV-1 protein expression from DNA vaccines in vitro measured by Western blot with HIV patient serum.
  • Figure 27 is an image of HIV-1 protein expression from fowlpox vaccines in vitro measured by Western blot probed with HIV patient serum.
  • Figure 28 is a graphical representation demonstrating the lack of HIV reverse transcriptase activity from DNA vaccines by ultra-sensitive real time PCR-enhanced RT assay TM- PERT.
  • TM-PERT assay used the reverse transcriptase associated with ultracentrifuge purified particles to produce cDNA from an MS-2 phage template provided. The resulting cDNA was detected by a real time PCR assay using am MS-2 specific TaqMan probe and primers. A dilution series standard AMV RT was assayed in parallel to determine the units of RT activity in the samples. Limit of detection for the TM-PERT assays is lxlO "10 units AMV RT.
  • Figure 29 is a schematic representation of mutations made to the crucial zinc finger motifs in nucleocapsid.
  • Figure 30 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles produced by B clade DNA vaccine is equivalent to replication defective HIV RNA packaging mutants, as measured by slot blot.
  • Figure 31 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles produced by B clade DNA vaccine is equivalent to replication defective HIV RNA packaging mutants. Viral particles were standardised for p24 antigen, the RNA was extracted and quantified by the modified HIV-1 NucliSens RNA amplification assay. Residual RNA detected likely reflects gross over-expression of g g RNA following transfection.
  • pDB653 is a replication defective reference packaging mutant (B clade).
  • Figure 32 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles derived from the recombinant fowlpox vaccine is 10-fold less efficient than in wild type HIV-1 NL(AD8). Viral particles were standardised for p24 antigen, the RNA was extracted and quantified by the modified HIV-1 NucliSens RNA amplification assay. rFPV-59 recombinant fowlpox containing a non-mutanted HIV Gag was assayed in parallel.
  • Figure 33 is a graphical representation demonstrating that pHIS-HIV-B and pHIS-HIV- AE vaccine derived pseudoviral particles are NOT able to establish a productive infection in susceptible primary cells during a cell free infection.
  • 2x10 6 PHA activated PBMC were infected with 20ng of p24 of purified viral or pseudoviral particles for 2 hr/37°C, then washed in PBS to remove free viral particles.
  • Cells were maintained in culture for greater than 50 days with the regular addition of lxl 0 6 feeder cells.
  • p24 antigen concentration was measured in the culture supernatant.
  • Figure 34 is a graphical representation demonstrating that pHIS-HIV-B and pHIS-HIV- AE DNA vaccine are NOT able to establish a productive infection in HIV-1 susceptible T cell lines during a cell to cell infection.
  • HeLa cells were transfected with the DNA vaccine and wild type constructs, then at peak antigen production co-cultured with the HIV-1 susceptible call line PM1.
  • Evidence of replication was defined as a doubling of p24 antigen concentration in the cell culture supernatant after the addition of the susceptible cells.
  • Figure 35 is a graphical representation demonstrating that lack of retrovirus replication in HeLa cells infected with BMP grade rFPV-HIV-B, using the ultra-sensitive TM-PERT assay.
  • rFPV-HIV-B vaccines were manufactured at GMP grade in primary chicken epithelial cells that also produce significant levels of RT from endogenous avian retroviruses. The presence of this RT activity is well known and is not considered a safety risk due to the lack of pathogenesis from other widely used human vaccines (measles, mumps, rubella, yellow fever). No replication competent retrovirus could be derived from this RT activity in long term HeLa cell culture, with RT activity falling below the limit of detection (lxlO "9 units AMV RT) and not reappearing even after 70 days of culture.
  • Figure 36 is a schematic representation of the regimen for the non-human primate toxicology study to assess dose toxicity profile and biodistribution of the DNA and rFPV vaccines.
  • Figure 37 summarises the absence of adverse effects in the non-human primate toxicology study.
  • Figure 38 is a schematic representation of the regimen for the rabbit toxicology study to assess the integration of the plasmid DNA and rFPN vaccines.
  • Figure 39 summarises the significant levels of integration not detected in rabbit toxicology study.
  • a very low level of potential integration of D ⁇ A vaccine detected only one of 6 animals early after vaccination. Even if all detected D ⁇ A is assumed to be integrated, calculated integration rate is 1250 times lower than spontaneous mutation rate per human gene. Risk of integration is considered to be minimal.
  • the present invention is predicated, in part, on the development of a consecutive immunisation strategy involving priming with an optimised plasmid vector and boosting with an optimised recombinant fowlpox virus vector, both encoding common HIV antigens mutated to remove active sites and the former additionally co-expressing CpG immunostimulatory molecules.
  • the application of this methodology has been determined to result in a significant boosting of plasmid vector vaccine primed HIV specific T lymphocyte responses following recombinant fowlpox virus vector boosting. This determination has now facilitated the development of compositions and methodology for application, ter alia, in relation to the prophylactic or therapeutic treatment of viral infections, in particular HIV infections, wherein the generation of an effective T cell mediated protective response is desirable.
  • one aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to a virus, said method comprising sequentially administering to said mammal:
  • virus should be understood as a reference to any virus or to homologues or mutants thereof.
  • the subject virus is the human immunodeficiency virus (herein referred to by the abbreviation "HLV").
  • HBV human immunodeficiency virus
  • HIV should be understood as a reference to any HIV strain and to homologues and mutants thereof.
  • homologues in this context should be understood as a reference to HIV strains which naturally occur in a species other than a species undergoing treatment in accordance with the method of the present invention.
  • the present invention is preferably directed to the treatment of humans utilising HIV derived antigens, it should be understood that the present invention extends to the development of methodology directed to the induction of HIV immunity in humans utilising simian immunodeficiency virus antigens.
  • Reference to "mutants” should be understood as a reference to forms of HIV which differ, either structurally or functionally, from the wild type form from which they are derived. In general, such mutants are characterised by nucleic acid deletions, insertions or substitutions. Such mutants are known to develop, for instance, in the context of the development of resistance to drug treatment.
  • said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
  • the present invention therefore more particularly provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal:
  • a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • said HIV is HIV-1 subtype AE, subtype B or subtype C.
  • Reference to "eliciting or otherwise inducing” and "immune response" to an HIV antigen should be understood as stimulating or facilitating stimulation of a specific immune response.
  • This specific immune response may be a T cell and/or a humoral response which is directed to any one or more of the peptides or epitopes, respectively, of the HIV antigen encoded by the nucleotide sequence comprising the recombinant plasmid or viral vector of the present invention.
  • the immune response is a Thl and CTL response.
  • the induction of HIV specific T cell responses are widely seen as critical to the development of effective immunity to HIV.
  • Th2-antibody responses are believed to facilitate enhanced Thl/CTL responsiveness.
  • Thl/CTL-type response even where an immune response is skewed to a Thl/CTL-type response, some degree of antibody generation may nevertheless occur.
  • the method of the present invention is predicated on the determination that the sequential administration of an optimised recombinant plasmid vector followed by an optimised recombinant viral vector, as herein defined, can induce an enhanced and sustained Thl/CTL response directed to HIV.
  • plasmid should be understood as a reference to independently replicating pieces of cytoplasmic DNA. These linear or circular DNA molecules are found in both prokaryotes and eukaryotes capable of autonomous replication. Plasmids are largely described in the context of those derived from bacteria and protozoa.
  • plasmids suitable for use in the method of the present invention include, but are not limited to, pHIS-HIVB, pHIS-HIV-AE or any other CpG optimised plasmid.
  • Reference to the subject plasmid being "recombinant" should be understood as a reference to the vector having been modified relative to its wild type form. This may occur for instance in the context of the insertion of additional nucleic acid segments (such as those which encode the HIV antigens of interest) and/or due to the modification of the plasmid sequence itself, such as would occur in the context of nucleotide optimisation.
  • the population of plasmids which are utilised in the context of the method of the present invention may be naturally occurring plasmids which have been appropriately modified (for example plasmids which have been harvested from cultured bacterial broths) or plasmids which have been otherwise synthetically or recombinantly generated.
  • Reference to a "viral vector” should be understood as a reference to any viral vector which can achieve efficient MHC I loading such as an avipox viral vector (for example fowlpox viral vectors or canarypox viral vectors), a vaccinia viral vector an adenovirus or an alphavirus replicon.
  • the subject viral vectors which are utilised in the present invention may comprise the whole or part of any virus or derivative thereof.
  • Reference to the subject viral vector being "recombinant" in the context of the present invention should be understood to have the same meaning as detailed above in relation to the term "plasmid”.
  • the subject viral vector is a recombinant fowlpox viral vector.
  • a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • viral vectors in the context of the present invention provides a means of achieving antigen expression within host cells and therefore achieving MHC class 1 molecule 28
  • processing of the antigen by an antigen presenting cell may result in the production of one or more peptides which are co-expressed with MHC class II and which stimulate specific T helper cells, in particular Thl cells.
  • MHC class II and which stimulate specific T helper cells, in particular Thl cells.
  • the processing and co-expression of these peptides also occurs in the context of MHC class I and thereby leads to the stimulation of one or more specificities of T cytotoxic cells.
  • the subject antigen may also comprise one or more epitopes to which some degree humoral immune response may be directed.
  • Said epitope may be a linear epitope or a conformational epitope. Where the epitope is a linear epitope, folding of the expressed antigen into its native conformation may not be required to achieve the stimulation of the specific humoral response directed to that epitope.
  • reference to stimulating a response to an HIV "antigen” should be understood as a reference to the stimulation of specific immune cells (eg. T cells) which are directed to one or more sites of the HIV antigen.
  • antigens suitable for use in the present invention include, but are not limited to, one or more of the molecules encoded by the HIV viral genes gag, RT, Pro, RNAseH, rev, tat, vpu, env, nef, vif, vpv and/or pol.
  • said plasmid and/or viral vectors express 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more antigens. In a most preferred embodiment, seven vectors express at least 5 antigens.
  • each gene is given the same name, but appears in non-italicised text with the first letter capitalised.
  • the subject plasmid vector and viral vector incorporate nucleic acid molecules encoding shared antigens.
  • shared is meant that at least some of the subject antigens are expressed by both the plasmid vector and the viral vector. Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 antigens are shared.
  • the subject plasmid vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef while the recombinant fowlpox virus vector incorporates nucleic acid molecules encoding Gag, RT, Pro, RNAseH, Tat, Rev, Env and.
  • reference to these antigens includes reference to derivatives, such as fragments, thereof. - 27
  • the method of the present invention can be applied utilising any suitable viral vector
  • the fowlpox viral vectors are particularly suitable for use since they undergo an abortive infection in mammalian hosts and are therefore safer than other types of viral vectors, such as vaccinia virus, which can cause a lethal infection in immunodeficient humans.
  • the design of the subject vaccine protocol which is based on the prime-boost regime utilising, respectively, a plasmid vector and a recombinant fowlpox virus vector, has been rendered exceptionally efficient due to the determination that both types of vectors should be optimised to express multiple HIV antigens and, further, that the plasmid vectors should express optimised CpG motifs. Still further, since some HIV antigens correspond to active enzymes and binding sites, which can represent a safety hazard in HIV negative subjects, it is necessary to mutate these genes in order to render them harmless.
  • HIV antigen In terms of the HIV antigens which are co-expressed by the vectors of the invention, reference to “HIV antigen” should be understood as a reference to any component of HIV or derivative thereof, such as any component of any one or more HIV genes. Said component may be a peptide, polypeptide or protein.
  • the antigen may comprise one or more sites in respect of which a specific immune response is
  • the subject plasmid vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Tat, Rev, Vpu, Env and/or nef while the recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Pol, Tat, Rev and/or Env.
  • a method for eliciting or otherwise inducing in a mammal an immune response directed to HIN-1 comprising sequentially administering to said mammal:
  • Env and/or Pol which Gag, RT, Pro, RNAseH, Tat, Rev and/or Env have been rendered substantially non-pathogenic
  • a method for eliciting or otherwise inducing in a mammal an immune response directed to HIV-1 comprising sequentially administering to said mammal: (i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and/or Env, which Gag, RT, Rev, Tat, Vpu and/or Env have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
  • said HIV is HIV-1 subtype AE, subtype B or subtype C.
  • said immune response is a Thl/CTL response.
  • references to the subject antigens being "substantially non-pathogenic” should be understood to mean that the subject antigen, when expressed in the mammal undergoing treatment, will not present a significant safety hazard in HIN-negative subjects. This may occur either due to the subject antigen inherently not posing a hazard or due to the nucleic acid molecule encoding the subject antigen having been mutated in order to eliminate or at least reduce one or more hazards.
  • reference to “substantially" non- pathogenic should be understood to indicate that the subject antigen is not necessarily entirely non-pathogenic but exhibits a level of pathogenicity which is acceptable in the context of an immunisation protocol.
  • the relative level of risk which is acceptable in the context of any individual treatment can be determined by the immunologist of skill in the art.
  • the subject gag and pol genes are preferably modified in order to reduce the risks associated with the expression of their naturally occurring forms.
  • the pol gene is modified and the env gene truncated.
  • the subject gag modification blocks gag mediated binding/packaging of the HIV/RNA template.
  • the specific modification which achieves this objective is the deletion, from the expression product, of amino acids 392-395 and 413-416 (the first and second regions in the first and second zinc finger domains).
  • the subject pol modification is required in order to block Rt, RNaseH and Integrase activity. This is achieved via deletion of amino acid 65-74, 113- 115 and 183-186 of the RT expression product. Mutation of Gin 478 to Glu 478 inactivates RNase H while deletion from 30 amino acids into the Integrase coding region to 87 base pairs 5' of the Tat start codon remove Integrase functioning. Env truncation blocks its effect on the T cell population through CD4 engagement and, further, reduces the possibility of an infective particle forming. In short, deletion of 581 base pairs starting from the first Bglll site and replacement of this fragment with a short piece of synthetic DNA containing stop codons in all three frames achieves this objective.
  • gag is modified to block Gag mediated binding/packaging of the HIV RNA template
  • said pol is modified to block RT/RNaseH and integrase activity
  • nef is truncated to block immunomodulatory activity.
  • modifying CpG motifs can be utilised to achieve improved immunogenicity.
  • Unmethylated CpG dinucleotides such as those found in bacterial oligonucleotides, are recognised as a danger signal by the vertebrate immune system.
  • CpG motifs comprising a nuclease resistant phosphorothioate backbone are potent Thl adjuvants. Accordingly, the modification of a vaccine to introduce an appropriate unmethylated CpG pattern can provide both adjuvant like functional activity and skewing of the immune response towards a Thl type response.
  • said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said recombinant fowlpox virus is rFPV-HIV-B or rFPV-HIV-AE.
  • the pHIS-HIV- B vector exhibits defective RT, cannot yield replication competent virus, does not package HIV-1 RNA efficiently and produces modest levels of pseudoviral particles.
  • the rFPV- HIV-B vector similarly does not package HIV RNA efficiently and cannot produce modest levels of pseudoviral particles. However, this vector does exhibit high levels of RT activity.
  • the vectors of the present invention express the specific features detailed herein (for example that the plasmid vector encodes two or more viral antigens and one or more optimised CpG motifs and the recombinant fowlpox virus vector encodes two or more viral antigens), this is not necessarily intended as a limitation in relation to the molecules which comprise these vectors.
  • the subject recombinant fowlpox virus vector may express genes in addition to those specifically detailed herein, such as additional HIV genes.
  • Derivatives should be understood to include fragments, parts, portions, equivalents, analogs, mutants, homologues, mimetics from natural, synthetic or recombinant sources including fusion proteins.
  • Derivatives may be derived from insertion, deletion or substitution of amino acids.
  • Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins.
  • the derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • the vector or derivative thereof may be fused to a molecule to facilitate its entry into a cell.
  • Derivatives of nucleic acid sequences may be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
  • Equivalents should be understood to include reference to molecules which can act as a functional analog or agonist. Equivalents may not necessarily be derived from the subject molecule but may share certain conformational similarities. Alternatively, equivalents may be designed to mimic certain immunological and physiochemical properties of the subject molecule. Equivalents may be detected following, for example, natural product screening. Equivalents also include peptide mimics. Homologues contemplated herein include, but are not limited to, molecules derived from different species. Fragments include portions which are effective in achieving the object of the present invention.
  • the nucleic acid molecule suitable for use in the present invention may be DNA or RNA.
  • Preferably said nucleic acid molecule is DNA.
  • Reference to the HIV antigen encoded by a nucleic acid molecule is a reference to the expression product of said nucleic acid molecule.
  • the method of the present invention is based on a prime-boost regime.
  • the rationale behind such a vaccination strategy is the use of the plasmid vector immunisation step to elicit a low level but persistent immunity which effectively primes for a greatly enhanced T cell response following boosting with another vaccine vector.
  • the boosting vaccine is preferably one which results in expression of the vaccine antigen from within host cells therefore leading to MHC-1 loading in an efficient manner.
  • This type of vaccine strategy when combined with the uniquely optimised vectors of the present invention, results in a greatly enhanced HIV T cell mediated response.
  • the present invention is predicated on the administration of the subject recombinant plasmid vector (being the "priming” step) prior to administration of the recombinant fowlpox virus vector (being the "boosting” step). Still further, it has been determined that the method of the present invention is effective where the priming step comprises the administration of one or more sequential doses of the recombinant plasmid vector and the boosting step comprises the administration of one or more sequential doses of the recombinant fowlpox virus vector.
  • a prime-boost immunisation system comprising two DNA vaccinations three weeks apart and two recombinant fowlpox vaccinations four weeks apart and four weeks after the DNA vaccine achieves a previously unattainable level of T cell responsiveness. Still further, it has been determined that even one plasmid vector dose vaccination followed by one recombinant fowlpox vector dose boost in the context of a minimum interval between the two vaccines of as little as 4 weeks similarly achieves the development of a previously unattainable level of T cell responsiveness.
  • preferred vaccination schedules include, but are not limited to:
  • a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • said recombinant viral vector dose is administered four weeks later than said recombinant plasmid vector dose.
  • a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal:
  • the second recombinant plasmid vector dose is administered three weeks after the first dose
  • the first recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose
  • the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
  • the recombinant plasmid vector dose is administered four weeks after the first dose and the recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose.
  • a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 comprising sequentially administering to said mammal: (i) Three doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
  • the second recombinant plasmid vector dose is administered four weeks after the first dose
  • the third recombinant plasmid vector dose is administered four weeks after the second dose
  • the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose
  • the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
  • said viral vector is a fowlpox virus vector.
  • said HIV is HIV-1 subtype AE, HIV-1 subtype B or HIV-1 subtype C.
  • the subject immune response is preferably a Thl/CTL response.
  • said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env and/or Pol.
  • said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, Pol, RT, Tat, Vpu and/or Env and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol.
  • said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said viral vector is rFPV-HIV-B or rFPV-HIV-AE
  • said immune response is a Thl/CTL response.
  • the inventors have determined that the unique vector design disclosed herein also facilitates the use of the plasmid vector in isolation.
  • yet another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal one or more doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding two or more antigens thereof of said HIV, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs for a time and under conditions sufficient to elicit or induce an immune response to said antigens.
  • said sequential doses are two doses of the plasmid vector 4 weeks apart.
  • said sequential doses are three sequential doses and said antigen is two or more of all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef. In yet another preferred embodiment, said sequential doses are three sequential doses and said antigen is two or more of all, part or a modified form of Gag, RT, Rev, Tat, Vpu and/or Env.
  • said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
  • said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE.
  • an “effective amount” means an amount necessary at least partly to attain the desired immune response, or to prevent or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to stimulate T cells, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • mammal should be understood to include all mammals including primates (e.g. humans, monkeys), livestock animals (e.g. sheep, cows, horses, donkeys, goats, pigs), laboratory test animals (e.g. mice, rats, guinea pigs, rabbits, hamsters), companion animals (e.g. dogs, cats), and captive wild animals (e.g. kangaroos, deer, foxes).
  • said animal is a primate and even more preferably a human.
  • Administration of the subject vectors in the form of a pharmaceutical composition may be performed by any convenient means.
  • the vectors are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal.
  • a broad range of doses may be applicable. Considering a patient, for example, from about 0.1 ⁇ g to about 1 mg of construct may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the plasmid vector is administered at 1 mg/dose and the fowlpox virus vector is administered at 5 x IO 7 pfu.
  • the constructs may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules).
  • the subject constructs are administered intramuscularly and, to the extent that a mucosal immune response is sought, orally, intranasally, intrarectally or intravaginally.
  • administration of the vaccine to the mucosal surfaces is particularly desirable since HIV-1 is predominantly transmitted sexually.
  • a further aspect of the present invention relates to the use of the invention in relation to disease conditions.
  • the present invention is particularly useful in relation to the therapeutic or prophylactic treatment of HIV infection and HIV-induced AIDS.
  • the method of the present invention is applied in the context of inducing immunity to HIV in HIV negative individuals.
  • the method of the present invention is directed to inducing an immune response for the purpose of alleviating or preventing the onset of symptoms associated with viral infection, and in particular HIV infection.
  • Reference herein to "symptoms" associated with viral infection should be understood to extend to both the infection itself as well as the physical and/or physiological consequences of such infection.
  • another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
  • the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
  • said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
  • said immune response is a Thl/CTL response.
  • said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env and/or Pol.
  • said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, Pol, RT, Tat, Vpu and/or Env and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol.
  • said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
  • a single dose recombinant viral vector is administered four weeks later than a single dose recombinant plasmid vector.
  • dual doses of recombinant plasmid vector are administered three weeks apart and dual doses of the recombinant viral vector are administered four weeks apart with the first viral recombinant vector dose being administered four weeks after the second recombinant plasmid vector dose is administered.
  • dual doses of recombinant plasmid vector followed by a single dose of recombinant viral vector are administered four week apart.
  • a mammal undergoing treatment may be a human or animal in need of therapeutic or prophylactic treatment for a disease condition or a potential disease condition.
  • treatment and prophylaxis are to be considered in its broadest context.
  • treatment does not necessarily imply that a mammal is treated until total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract a disease condition.
  • treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylaxis” may be considered as reducing the severity of onset of a particular condition. “Treatment” may also reduce the severity of an existing condition or the frequency of acute attacks (for example, reducing the severity of initial infection).
  • the modulatory agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules.
  • coadministered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules, These molecules may be administered in any order.
  • compositions of the invention contain a pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, vector, vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention.
  • the effective amount of an agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest.
  • the effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent o symptoms) of the condition being treated.
  • the effective amount of agent per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art.
  • the dosage of the therapeutic agents which will be most suitable for prophylaxis or treatment will also vary with the form of administration, the particular agent chosen and the physiological characteristics of the particular patent under treatment. The dose is administered at least once. Subsequent doses may be administered as indicated.
  • mRNA or protein expression levels may be determined. In many instances it will be sufficient to assess the expression level in serum or plasma obtained from such an individual. Decisions as to whether to administer another dose or to change the amount of the composition administered to the individual may be at least partially based on the expression levels.
  • unit dose refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (e.g., nucleic acid, virus stock or host cell) calculated to produce the desired effect in association with the required diluent.
  • active material e.g., nucleic acid, virus stock or host cell
  • the titers of the virus stocks to be administered to a cell or animal will depend on the application and on type of delivery (e.g., in vivo or ex vivo).
  • the virus stocks can be concentrated using methods such as centrifugation.
  • the titers to be administered ex vivo are preferably in the range of 0.001 to 1 infectious unit/cell.
  • Another method of generating viral stocks is to cocultivate stable cell lines expressing the virus with the target cells. This method has been used to achieve better results when using traditional retroviral vectors because the cells can be infected over a longer period of time and they have the chance to be infected with multiple copies of the vector.
  • nucleic acid constructs, vectors, vector systems, virus stocks, or cells which have been transduced or transfected ex vivo the dose is to be determined by dose escalation, with the upper dose being limited by the onset of unacceptable adverse effects.
  • Preliminary starting doses may be extrapolated from experiments using lentiviral vectors in animal models, by methods known in the art, or may be extrapolated from comparisons with known retroviral (e.g., adenoviral) doses.
  • small dosages will be used initially and, if necessary, will be increased until the optimum effect under the circumstances is reached.
  • Exemplary dosages are within the range of 10 8 up to approximately 5 x 10 15 particles.
  • Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
  • the agents of the invention are generally administered with a physiologically acceptable carrier or vehicle therefor.
  • the physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the nucleic acids are sufficiently soluble to retain their activity to deliver a pharmaceutically or therapeutically effective amount of the compound.
  • the pharmaceutically or therapeutically effective amount and method of administration of an agent of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art.
  • a therapeutically effective amount of a nucleic acid of the invention is one sufficient to prevent, or attenuate the severity, extent or duration of the deleterious effects of the condition being treated without causing significant adverse side effects.
  • the route(s) of administration useful in a particular application are apparent to one of ordinary skill in the art.
  • Routes of administration of the agents of the invention include, but are not limited to, parenteral, and direct injection into an affected site.
  • Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous.
  • the route of administration of the agents of the invention is typically parenteral and is preferably intramuscular, subcutaneous, intradermal, intraocular, intracranial, intranasal, and the like. See, e.g. WO99/04026 for examples of formulations and routes of administration.
  • compositions of the agents described above suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions.
  • pharmaceutically acceptable sterile isotonic solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into a joint or other area.
  • the dosage administered will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
  • the present invention relates to the use of:
  • a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non- pathogenic, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a mammalian disease condition characterised by infection with said virus.
  • said virus is HIV and, even more preferably, HIV-1, HIV-1 subtype AE, subtype B or subtype C.
  • said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated names encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env .
  • said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and Env and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and Pol.
  • said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
  • the present invention should also be understood to extend to the vectors described herein and to vaccine compositions comprising said vectors.
  • the present invention is directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs.
  • This aspect of the present invention is still further directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
  • Still another aspect of the present invention is directed to a nucleic acid construct or functional derivative thereof which construct comprises a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
  • the present invention is directed to a nucleic acid construct comprising a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
  • said virus is HIV and, even more preferably, HIV-1, HIV-1 subtype AE, subtype B or subtype C.
  • said recombinant plasmid vector incorporates nucleic acid molecules encoding Gag, RT, Pro, RNaseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
  • said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and Env and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding Gag, Tat, Rev, Env and Pol.
  • said plasmid is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
  • compositions in particular pharmaceutical compositions, comprising the nucleic acid constructs, plasmid vectors and/or viral vectors hereinbefore described.
  • the present invention is directed to the vaccine and pharmaceutical composition, hereinbefore described, when used in the methods hereinbefore described.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example for the purpose of inducing a mucosal immune response, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • Still another aspect of the present invention provides a method of inducing, enhancing or otherwise stimulating an immune response, in a mammal, to HIV said method comprising administering to said mammal an effective amount of a vaccine as hereinbefore defined in accordance with the methodology herein defined sufficient to induce, enhance or otherwise stimulate an immune response to one or more HIV antigens.
  • said immune response correlates to the induction of long term immunity.
  • the method of the present invention is useful in the treatment and/or prophylaxis of HIV infection and HIV-induced AIDS.
  • the vaccine of the present invention may be administered to subjects known to be infected with HIV in order to induce an immune response against HIV thereby preventing the onset of AIDS.
  • the method of the present invention may be used to reduce serum viral load, to alleviate AIDS symptoms or to induce immunity in mammals thought to be at risk of HIV infection.
  • the method of the present invention may be particularly useful either early in HIV infection to prevent the establishment of a viral reservoir or for a period after exposure to a possible source of HIV infection.
  • the vaccines of the present invention may be coadministered with known antiviral compounds or molecules.
  • known antiviral compounds or molecules include, but are not limited to, reverse transcriptase inhibitors (for example, Zidovudine or 3TC) or protease inhibitors (for example, Indinavir).
  • coadministered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the vaccine and the known antiviral compound or molecules.
  • the vaccine and the known antiviral compound or molecule may be administered in any order.
  • the vaccines which are used in the phase I/IIa clinical trials consist of a DNA prime(pHIS- HIV-B or pHIS-HIV-AE) followed by a rFPV boost (rFPV-HIV-B or rFPV-HIV-AE).
  • the DNA vaccines described in this example encode CpG immunostimulatory oligonucleotide sequences and both the DNA and rFPV vaccines express multiple HIV genes mutated to remove active functional sites of the HIV genes where these are deemed to be a safety concern.
  • the provirus DNA that was selected for the DNA construct pNL(AD8) was based on the well characterised B sub-type isolate pNL4.3, but has the env from the CCR5-tropic HIV- 1-ADA strain and was obtained from the NIH reagent repository. Approximately 65% of the provirus is included in the final DNA construct. After the HIV-1 genome was modified, it was inserted into the DNA vaccination vector pHIS-64. This kanamycin resistant plasmid vector contains a cytomegalovirus (CMV) promoter, the bovine growth hormone polyA signal and human optimised CpG immunostimulatory sequences.
  • CMV cytomegalovirus
  • a synthetic intron CMV splice donor + Ig variable region splice acceptor (Eaton DL, Wood Wl, Eaton D, Hass PE, Hollingshead P, Wion K, Mather J, Lawn RM, Vehar GA and Gorman C. 1986. Biochemistry 25:8343-8347) was cloned immediately before the joined HIV sequences in pHIS-64 to increase expression of the HIV genes.
  • the provirus DNA that was selected for the AE sub-type DNA construct was the well characterised isolate p93TH253 obtained from NIH reagent repository. This is an early Thai isolate from 1993 and is reasonable representation of South East Asian AE strains. Approximately 65% of the provirus is included in the final DNA construct.
  • the DNA vector pHIS-54 see Figure 2). This kanamycin resistant plasmid vector contains a CMV promoter, the bovine growth hormone polyA signal, gene and human optimised CpG immunostimulatory sequences.
  • the DNA vaccine expresses the following HIV-1 genes; modified gag, modified RT, rev, tat, vpu, truncated env.
  • gag modifications to block gag mediated binding/packaging of the HIV RNA template ° deleted gag 392-395AAs "CFNC” (first zipper) ° deleted gag 413-416AAs “CWKC” (second zipper)
  • the pHIS-HIV- ⁇ E and the pHIS-HIV-B DNA vaccines share a high degree of sequence identity. They use the same vector, pHIS, that has the immuno-reactogenic human CpG sequences and have an almost identical construction except that the HIN-B env has a stop codon before the deletion of the middle third of the env nucleotide sequence, and HIV- AE does not.
  • pHIS-HIV-B expresses only the first third of the Env protein
  • pHIS-HIN- lE expresses the first and last third of the Env protein ( Figure 3).
  • the middle portion of the Env protein that contains the receptor-binding domains is deleted from BOTH of these constructs.
  • the nucleotide sequence of the two HIV inserts share 85% nucleotide sequence identity, and about the same level of protein identity across the common expressed protein regions.
  • the rFPV vaccines were constructed by the sequential insertion of the corresponding D ⁇ A at sites in the viral genome of the parent FPV strain, FPV-M3.
  • rFPV-HIV-B containing g ⁇ g/pol (mutated)
  • rFPV- HIV-A ⁇ containing g ⁇ g/pol (mutated), env (mutated) and t ⁇ t/rev
  • Construction of the rFPV-HIV-B vaccine a rFPV expressing mutated gag/pol from HIV- 1NL43 was constructed using long range PCR to amplify the gag/pol (m) from the pHIS- HIV-B vaccine with the addition of pox virus specific sequences at each end. These sequences are described in Figure 5 and Figure 6.
  • PCR product 3533bp was cloned into pGEM- Teasy to make pGT -gag/pol (mutated) and the sequence of the entire insert confirmed.
  • the primers used added the rFPV early/late promoter and an early transcription terminator to the genes during the PCR.
  • Xhol restriction endonuclease sites were added to the ends of the promoter gag/pol PCR product.
  • the promoter gag/pol PCR product (as a Xhol fragment) was then cloned into plasmid pKGlO (Sail digested) which contains flanking sequences of FPV F6, 7, 9 gene regions.
  • the FPV flanking sequences were then used to recombine the plasmid with fowlpox strain FPV M3.
  • the pKGlOa vector used for construction of recombinant fowl pox viruses by transient dominant selection was constructed by a series of manipulations and contains:
  • ° plasmid sequences derived from pBlueScript including an E. coli origin of replication and an ampicillin resistance gene.
  • Recombinants were selected on the basis of transient dominant co-expression of E. coli gpt and beta-galactosidase genes.
  • the final recombinant was plaque purified and stocks of rFPV-HIV-B grown in chicken embryo skin cells.
  • the transient dominant selection results in the loss of the marker genes and the rFPV-HIV-B only carries the FPV promoter gag/pol (m) genes inserted within the FPV 6, 7, 9 region of the genome.
  • the rFPV-HIV- ⁇ E vaccine (denoted FPV-117 during construction) was constructed by the sequential insertion of DNA at 3 sites in the viral genome of FPV-M3: F6,7,9 using transfer plasmid pKGlOa, REV site using ⁇ CH34, and TK ORF X using pAF09 (refer to Figure 9).
  • HIV sequences were inserted into the transfer plasmid as a promoter-gene- terminator cassette where the promoter was fowlpox PE/L and the terminator was T 5 NT.
  • HIV AEe77v(m) was inserted into FPV-M3 at the REV site using pCH34-AEe «v(m) to produce construct FPV-090.
  • HIV AEgagpol(m) was inserted using pK.G ⁇ -AEgagpol( ) into the F6,7,9 site of both FPV-M3 and FPV-090 to generate FPV-091 and FPV-092, respectively.
  • FPV-092 was used as a parent for the insertion of a synthetic gene encoding AE tat and rev linked by 5 His residues (A porcine hyperplasia) at the intergenic site between TK and ORF X via transfer plasmid pAF09 to generate FPV-117 ( Figure 8).
  • the first and second insertions were carried out using transient dominant selection (markers Ecog ⁇ t/j3-gal are lost from the recombinant virus) while the third insertion used dominant selection, with retention of the markers in the final construct.
  • AEgagpol(m) and AEe «v(m) were those present in the DNA vaccine construct pHIS-HIV- AE as noted above, with additional modifications to AEe «v(m) to remove a pox virus transcription termination sequence without changes to the amino acid coding.
  • Tat and rev sequences are present in the DNA vaccine as alternate open reading frames to env.
  • the sequence of HIV components of all transfer plasmids was confirmed prior to virus construction.
  • the vaccines were manufactured according to the standards of Good Manufacturing Practice, as defined by the Australian Code of Good Manufacturing Practice for Therapeutic Goods (1990) (rFPV) and US FDA's 21 CFR cGMP regulations and the EU directive 91/356/EEC (DNA).
  • the pHIS-HIN-B vaccine was manufactured by Qiagen within their GMP-facility (Strathmann Biotech GmbH, Hannover, Germany).
  • RBC research cell bank
  • GLP Good Laboratory Practice
  • RT activity - vaccine produces a defective RT enzyme that can not support viral replication, recombination or mutation
  • Replication competency - vaccine does not yield replication competent virus From this research cell bank, a master and working cell bank were manufactured under GMP conditions from which the pHIS-HIV-B vaccine was produced.
  • the stability protocol covers both the "long term" stability of the vaccine at the indicated temperature (-80°C) and then stability at accelerated conditions (-20°C, and +5°C :t 3°C).
  • pHIS-HIV-B Prior to manufacture by Qiagen, pHIS-HIV-B was made as follows.
  • FIG. 10 is a schematic outline of the strategy used for the 5' half.
  • the 3' end was constructed by first cloning the 3' half separately into a shuttle plasmid using convenient restriction enzyme sites. A short fragment was then PCR amplified and cloned back into the template plasmid to create the required deletion. The two modified HIV-1 fragments were then cloned stepwise into pHIS-HIVB.
  • the pHIS-HIVB plasmid expresses high levels of Gag and combined with its greater yield during bacterial amplification has been selected to be the D ⁇ A vaccine component of the B clade HIV vaccine.
  • the rFPV-HIV-B vaccine was manufactured in a new clean room facility dedicated to rFPV vaccine manufacture.
  • the recombinant virus was amplified in the presence of mycophenolic acid selection. Plaquing was then carried out without mycophenolic acid. PCR with primers complementary to sequences flanking the insertion site were used to confirm the absence of wild type parent virus.
  • MSS master seed stock
  • the working seed stock was prepared from the MSS under GMP conditions.
  • the WSS was screened for bacteria, yeasts and mould by growth promotion studies after direct inoculation of growth media.
  • the rFPV-HIV-B vaccine was manufactured from the WSS under GMP conditions. Quality control testing of the rFPV-HIV-B vaccine final product included:
  • the rFPV-HIV-B vaccine was placed on stability testing.
  • the stability protocol covers both the "long term" stability of the vaccine at the indicated temperature (-80°C) and then stability at accelerated conditions (-20°C, 2-8°C, and l8°C).
  • the DNA vaccines are presented in a 3mL glass vial sealed with a rubber stopper and over- sealed with a plastic flip-off top.
  • Each 3mL vial in the treatment group contains either 1 mg/niL of DNA or 1.5 mg/mL in a volume of 1.2mL of 0.9% saline.
  • the solution is colourless and transparent.
  • the DNA matched-placebo vaccines containing diluent (0.9% saline) are also presented in a 3mL glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top.
  • the rFPV vaccines are presented in a 2mL amber glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top. Each contains either 5x10 7 pfu/mL or 2x10 8 of rFPV in a volume of 1.2 mL of Phosphate Buffered Saline (PBS) 10% glycerol.
  • PBS Phosphate Buffered Saline
  • the FPV matched-placebo vaccines containing diluent are also presented in a 2mL amber glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top .
  • the pHIS-HIV and rFPV vaccines are stable when stored at -80°C for up to 18 and 9 months, respectively.
  • the pHIS-HIV vaccine has been stored for up to 18 months and the integrity of the vaccine confirmed by agarose gel electrophoresis and spectrophotometric analysis.
  • the DNA and rFPV vaccines and matched placebos must be stored at -80°C. Vials must be defrosted immediately prior to use. Vials may be stored for up to 72 hours at -20°C without loss of stability.
  • E. coli strain DH5 ⁇ was transformed with pHIS-HIV-B according to standard molecular cloning methods and a subsequent production clone was selected. A master and working cell bank were prepared.
  • An inoculum was prepared using the working cell bank.
  • a shake flask containing growth media (soya peptone, yeast extract, NaCl, K 2 HPO 4 (3H 2 O), KH 2 PO 4 , MgSO 4 (7H 2 O), pH to 7.0 with 1 M NaOH) was inoculated and incubated at 37°C under shaking to the desired optical density (OD).
  • An endotoxin removal (ER) buffer (which does not contain animal derived ingredients) was added to the cleared lysate to remove lipo-polysaccharides.
  • the cleared lysate and ER buffer mix was then loaded onto an anion exchange resin column which had been pre-equilibrated with a QBT buffer (750mM NaCl, pH 7.0,
  • the pHIS-HIV-B was eluted from the column in a shallow gradient using QN buffer (1.6M NaCl, pH 7.0, 50mM MOPS, 15% isopropanol) and the plasmid DNA precipitated by addition of 100% (v/v) isopropanol.
  • the precipitate was collected by centrifugation, the pellet washed with 70% (v/v) ethanol, centrifuged and the plasmid pellet air dried.
  • Step 1 Primary Cell Preparation i. SPF chicken eggs were transferred into a sterile class 3.5 area and the embryos removed and placed in a bottle with phosphate-buffered saline
  • PBS pre-warmed to 37°C.
  • ii After collecting approximately 15 embryos per bottle, the PBS was poured off and the embryos washed again with another volume of PBS. iii. The embryos were washed again with Hanks Balanced Salt Solution (BSS).
  • BSS Hanks Balanced Salt Solution
  • a working enzyme solution containing Hanks BSS and the enzyme collagenase reconstituted in PBS, was added to each bottle with the embryos. The solution was stirred at 37°C to allow the collagenase to digest the embryo tissue v.
  • Growth media (GM) was prepared using specified quantities of foetal bovine serum (FBS), HEPES, L-Glutamine, Streptomycin and Minimum
  • Step 2 First Passage Cells i. After 3 to 5 days the cells were passaged, ii. The media was poured off and the cells washed with PBS. iii. Trypsin PBS at 37°C was added and the bottle placed on the roller unit for 5 minutes. iv. The roller bottles were swirled to dislodge the remaining cells. v. FBS was added to the cell suspension to stop the enzyme action. vi. The cell suspension was poured into centrifuge tubes and spun at 2000rpm, room temperature for 5 to 10 minutes, vii. The cell pellet was then resuspended in 10ml of GM and a cell count performed. viii. Roller bottles were seeded with approximately 1 xl 08 cells per roller, ix. The monolayer became confluent within 3 to 5 days.
  • Step 3 Cell Infection i. Using stock virus of known titre (WSS), a specified amount of virus, based on the multiplicity of infection (moi), was added to trypsin and incubated at 37°C for 30 minutes, ii. GM containing FBS was added to stop the action of the trypsin. iii. The amount of virus required to infect the roller bottles was calculated. iv. The media was poured off the cells and the cells washed with PBS. v. The calculated amount of virus, in PBS, was added to the roller bottles and incubated at 37°C for 60 minutes, vi. GM without FBS or antibiotics was then added to the roller bottles and the bottles incubated at 37°C for a further 3 to 5 days until cytopathic effect (cpe) became visible.
  • WSS multiplicity of infection
  • Step 4 Virus Harvest i.
  • the roller bottles were swirled to dislodge the cell monolayer. ii. Keeping the virus from each roller bottle separate, the viral supernatant containing the cells was poured into centrifuge tubes and spun at 3,000rpm for 20 minutes at 4°C. iii. After centrifugation the supernatant was poured off and the pellet resuspended in PBS. iv. The centrifuge step was repeated and the PBS poured off. v. The pellets were resuspended in lOmls of injectable grade PBS 10%
  • Fowlpox vaccine Dispensing, Labelling and Packaging
  • Vaccine Dispensing i.
  • the required final concentration of the vaccine was 5xl0 7 pfu/ml for dispensing and was calculated as per the instructions in the master batch record, ii.
  • IV bags containing PBS with 10% glycerol were prepared and sterilised at 121 °C for 30 minutes and then sterility tested.
  • iii. Using a sterile syringe a pre-determined volume of viral titre was injected via the IV bag injection port into the IV bag.
  • An IV infusion set was connected to the IV bag and a syringe filler assembled aseptically. v.
  • Placebo Dispensing i. Sterile phosphate buffered saline solution containing 10% glycerol (v/v) in an IV bag was dispensed as the placebo. ii. An IV infusion set was connected to the IV bag and a syringe filler assembled aseptically. iii. 1.2ml of placebo was dispensed into 2ml vials. A stopper was placed on top of each vial and a flip off top (FOTs) placed onto the rubber stopper and crimped. The rest of the dispensing process for the placebo was the same as that described for the fowl pox batch steps v - xi.
  • FOTs flip off top
  • pHIS-HIV and rFPV vaccines were assessed for safety and efficacy using in vitro and in vivo techniques. Detailed below is a summary of the testing performed on each vaccine.
  • the pHIS-HIV-B vaccine was compared to a wild type HIV-1 virion, a nucleocapsid reference mutant and a RNA packaging site mutant (both mutants were replication defective).
  • Results of the modified HIV-1 "QT NucliSensTM" quantitative RNA amplification assay demonstrated that the pHIS-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the wild type virion.
  • RNA slot blot hybridisation demonstrated that the pHIS-HIV-B vaccine packaged RNA to levels that were equal to the nucleocapsid reference mutant and RNA packaging site mutant.
  • the residual levels of RNA reflect the high cellular levels of HIV RNA relative to other RNA during these DNA-transfection experiments.
  • the mutations introduced into the vaccine significantly decreased the potential for packaging and transmission of HIV genes.
  • the pHIS-HIV-B vaccine was compared to a reference RT mutant and a nucleocapsid mutant (that has defective endogenous RT activity) in different assays. Results of the "conventional" RT assay demonstrated that:
  • the HIV pseudoviral particles produced by the pHIS-HIV-B vaccine did not have any RT activity above background ii) the absence of RT activity was the same as the reference RT mutant and the nucleocapsid mutant.
  • the HIV pseudoviral particles produced by the pHIS-HIV-B vaccine do not have any RT activity, ii) this activity was considerably below that of the reference RT mutant which was found to have low level activity with this sensitive assay.
  • the mutations introduced into the vaccine produced a defective RT enzyme that would not support viral replication, recombination or mutation.
  • HIV pseudoviral particles that assemble from the modified gag, modified RT, Pro, rev, tat, vpu and truncated env expressed by the pHIS-HIV-B vaccine were not able to replicate in HIV susceptible cells.
  • Cell free pseudoviral particles derived from the pHIS-HIV-B vaccine were not able to replicate in pooled primary human peripheral blood mononuclear cells from HIV- negative, but otherwise susceptible donors.
  • Direct introduction and expression of the pHIS-HIV-B plasmid in an HIV- susceptible T-cell line did not initiate any infection with a replication competent virus expressing HIV p24 antigen.
  • the pHIS-HIV-AE vaccine was compared to a wild type HIV-1 clade B virion, a nucleocapsid reference mutant and a RNA packaging site mutant (both mutants were replication defunct).
  • Results of QT NucliSensTM quantitative RNA amplification assay demonstrated that the pHIS- nucleocapsid HIV-AE vaccine and pHIS-HIV-B vaccine pseudoviral particles packaged HIV- 1 RNA at a similar rate and that this was seven-fold lower than that of the HIV-1 B-clade wild type virion.
  • the residual levels of RNA reflect the high cellular levels of HIV RNA relative to other RNA during these DNA-transfection experiments. However, since the RNA packaged with pseudoviral particles is replication incompetent, it is considered that this does not pose a safety risk.
  • RT Reverse transcriptase
  • the pHIS- HIV- AE vaccine was compared to a reference RT mutant and a nucleocapsid mutant (that has defective endogenous RT activity) in different assays.
  • the HIV pseudoviral particles produced by the pHIS-HIV-AE vaccine did not have any RT activity above background ii) the absence of RT activity was the same as the reference RT mutant and the nucleocapsid mutant.
  • the HIV pseudoviral particles produced by the pHIS-HIV-AE vaccine do not have any RT activity ii) this activity was considerably below that of the reference RT mutant which was found to have low level activity with this sensitive assay.
  • the mutations introduced into the vaccine produced a defective RT enzyme that would not support viral replication, recombination or mutation.
  • RT, Pro, rev, tat, vpu and truncated env expressed by the pHIS-HIV-AE vaccine were not able to replicate in HIV susceptible cells
  • ii) Cell free pseudoviral particles derived from the pHIS-HIV-AE vaccine were not able to replicate in pooled primary human peripheral blood mononuclear cells from HIN- negative, but otherwise susceptible donors
  • iii) Direct introduction and expression of the pHIS-HIV-AE plasmid in an HIV- susceptible T-cell line did not initiate any infection with a replication competent virus expressing HIV p24 antigen.
  • the rFP V- HIN- B vaccine was compared to an empty FPV vector and to a FPV vaccine with non- mutated gag.
  • Results of the modified HIV-1 QT NucliSensTM quantitative RNA amplification assay demonstrated that the rFPV-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the empty vector and the gag non- mutated vaccine.
  • the mutations introduced into the vaccine function to significantly increase the safety profile of the FPV vector.
  • results of a "conventional" RT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine do not have any RT activity above background.
  • results of a highly sensitive and quantitative TM-PERT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine had relatively high levels of RT activity, but that this was due to RT derived from the chicken epithelial cells used to manufacture the vaccine.
  • the presence of chicken cell derived RT is well known and is not considered a safety risk in human vaccines due to the lack of pathogenesis from other widely used vaccines (measles, mumps, rubella and yellow fever) also prepared in chicken cell substrates that share the presence of the endogenous chicken retroviral RT.
  • the rFPV- HIV- AE vaccine was compared to an empty FPV vector and to a FPV vaccine with non- mutated gag.
  • Results of the modified HIV-1 QT NucliSensTM quantitative RNA amplification assay demonstrated that the rFPV-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the empty vector and the gag non- mutated vaccine.
  • the mutations introduced into the vaccine function to significantly increase the safety profile of the FPV vector.
  • RT Reverse transcriptase activity
  • results of a "conventional" RT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine do not have any RT activity above background.
  • results of a highly sensitive and quantitative TM-PERT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine had relatively high levels of RT activity, but that this was due to RT derived from the chicken epithelial cells used to manufacture the vaccine.
  • chicken cell derived RT is well known and is not considered a safety risk in human vaccines due to the lack of pathogenesis from other widely used vaccines (measles, mumps, rubella and yellow fever) also prepared in chicken cell substrates that share the presence of the RT.
  • gag expression was assessed using a commercial HIV-1 gag antigen.
  • Western immunoblot analysis of the proteins produced in pHIS-HIV-B transfected 293 T cells showed three major bands present; 1) the precursor gag protein at p55, 2) the p39 fragment of gag, and 3) the core protein of gag, p24.
  • the concentration (mg pHIS-HIV-B/mL) of the final filled product was determined by direct UV photometry.
  • the absorption of the nucleotide bases at 260nm was used to quantitate the pHIS-HIV-B in solution. Due to the specific extinction coefficient of double stranded pHIS-HIV-B, a plasmid preparation with absorption of 1 corresponds to a concentration of 50 ⁇ g/mL.
  • the concentration of the pHIS-HIV-B was shown to be 0.95 ⁇ g/mL.
  • pHIS-HIV-B vaccine To assess expression of functional tat by the pHIS-HIV-B vaccine, tat-transactivation function upon an L-TR-enhanced green fluorescent protein (LTR-EGFP) reporter plasmid, was used as the method to quantify the expression of tat protein by the vaccine. Results of the assay demonstrated that the pHIS-HIV-B vaccine expresses functional tat protein with equal efficiency to a wild type proviral plasmid that is known to express tat
  • Elisa capture was used to determine the level of ⁇ 24 expression in transiently transfected cells. High levels of p24 were expressed by the transiently transfected 293 T cells and at similar levels to the pHIS-HIV-B vaccine.
  • a commercial HIV-1 gag antigen was used to assess HIV-1 gag expression.
  • the Western immunoblot analysis of the proteins produced in pHIS-HIV-AE transfected 293 T cells showed three major bands present; 1) the precursor gag protein at p55, 2) the p39 fragment of gag, and 3) the core protein of gag, p24.
  • the concentration (mg pHIS-HIV-AE/mL) of the final filled product is determined by direct UV photometry.
  • tat-transactivation function upon an L-TR-enhanced green fluorescent protein (LTR-EGFP) reporter plasmid is used as the method to quantify the expression of tat protein by the vaccine.
  • a plaque assay was performed to determine the final concentration of the rFPV-HIV-B vaccine.
  • the titre of the rFPV-HIV-B vaccine (batch no. V921105.1) was shown to be 7.57 log 10 pfu/mL.
  • the control reference virus which has a specification of 7.30 log 10 pfu/mL + 0.5 logio, was assayed concurrently and was shown to be within specification. Therefore, the results of the titration are considered to be valid.
  • PCR was performed on rFPV-HIV-B to confirm the genome arrangement at 3 sites in the viral genome. The PCR products generated demonstrated;
  • rFPV- HIV-B vaccine is indistinguishable from the wild-type virus
  • rFPV-HIV-B vaccine is indistinguishable from the virus used to generate the master seed stock.
  • a plaque assay is performed to determine the final concentration of the rFPV-HIV-AE vaccine. This assay is performed under GLP on GMP manufactured vaccine. • Expression of gag/pol
  • PCR is performed on rFPV-HIV-B to confirm the genome arrangement at 3 sites in the viral genome.
  • mice pHIS-HIV-B priming followed by rFPV boosting can induce antibody and cell mediated responses to gag/pol.
  • results in Table 3 are represented as the highest dilution with significant optical density (OD) above background (2 standard deviations), with the first tube (1) starting at 1/10 with doubling dilutions, tube 2 (1/20), tube 3 (1/40).
  • Table 4 Vaccinia virus titrations after D ⁇ A or rFPV alone and after prime/boost
  • the primary objective of this study was to assess the immunogenicity of the D ⁇ A/rFPV prime/boost regimen in non-human primates. Twenty eight colony bred juvenile pigtail macaques (M. nemestrina) free from HIV- 1/SrV/SRV infection, were randomised to one of the following groups:
  • a further 12 animals received 2xDNA and 2xrFPV in combinations of intranasal or intramuscular injections to assess the route of administration. These data are not presented here.
  • the pHIS-HIV-B vaccine was delivered 1 mg IM (l.Omg/mL with 0.5mL IM to the left and right quadriceps).
  • the rFPV-HIV-B vaccine was delivered 5x10 7 PFU IM (0.5mL [2.5x10 7 PFU] to the left and right quadriceps).
  • HlV-specific IFN ⁇ production from T cells was quantified by the ELIspot technique both 5 weeks following the final rFPV boost (to whole inactivated HIV- 1) and on the day of challenge (to both whole inactivated HIV-1 [Figure 15] and a single pool of HIV-1 gag 15mer overlapping peptides [Figure 16]).
  • Control animals had a peak HIV-1 viral load at week 2 following challenge of 3.3 logio copies/mL and all 4 control animals still had detectable plasma HIV- 1 RNA by 5 weeks post infection.
  • the group vaccinated with DNA/FPV had a mean peak VL at week 2 of 2.7 log 10 copies/mL and was undetectable ( ⁇ 1.3 log 10 copies/mL) in all 6 DNA/FPV vaccinated animals.
  • a time-weighted area-under-the-curve analysis across the 4 groups demonstrated significant protection from challenge in the DNA/FPV regimen compared to both the control arm (P ⁇ 0.01).
  • a second boosting FPV vaccination did not appear to substantially enhance the T cell immunogenicity of the prime/boost regimens over a single FPV boosting vaccination.
  • the primary objective of this study was to assess the immunogenicity and protective capability of the DNA/rFPV prime/boost regimen in a SHIV challenge model.
  • SHIV-specific DNA and rFPV constructs were created based on the backbone DNA and rFPV for the B and AE sub-type vaccines. Challenge was performed with SHIVmn229 (III8- derived) LR. >500 M.I.D., with 2 doses over 2 days.
  • a macaque study is conducted to assess the immunogenicity of the pHIS-HIV-AE and rFPV-HIV-AE prime boost regimen.
  • Immunoassay data showed positive humoral and cellular responses in Group 2 animals receiving high dose of DNA vector and rFPV virus vaccine.
  • Two intramuscular injections of the pHIS-HIV-B vaccine followed by one injection of rFPV- HIV-B vaccine to male and female Cynomolgus monkeys were well tolerated.
  • the pHIS-HIV- B vaccine was found predominantly in the muscle of the injection site. Specific humoral and cellular immune responses to the vaccines were evident in the animals receiving high dose of DNA and rFPV vaccines.
  • a total of 26 rabbits (3/sex/group for controls and 5/sex/group for the vaccine groups) were randomised to one of 3 treatment groups: group 1 - saline/PBS glycerol, group 2 - 2 injections of pHIS-HIV-B (2mg/mL) at week 0 and 4, group 3 - 2 injections of pHIS-HIV- B followed by 1 injection of rFPV-HIV-B (2mg/mL of DNA at week 0 and 4, followed by lxl0 8 pfu/mL FPV at week 8).
  • Two groups of 5 rabbits/sex were treated intramuscularly with 2 mg DNA vector (pHIS-HIV-B) on Study Day (SD) 1 and 29, and one of these groups was also treated with 10 8 pfu rFPV-HIV-B on SD 57.
  • An additional control group consisting of 3 rabbits/sex were treated with saline on SD 1 and 29 and with phosphate buffered saline (PBS)/10% glycerol on SD 57.
  • Parameters evaluated included mortality, clinical observations, local reactogenicity, body weights, and DNA vector integration at the site of injection. All animals survived until scheduled termination. Minimal, light pink erythema and minimal oedema were noted at the injection sites in a few animals across all groups.
  • the rate of mutation was estimated to be 8.44 x IO "9 mutations per gene, which is approximately 1200 times less than the rate of spontaneous mutation of 1.5 x 10 '5 mutations per gene reported by Cole and Skopek in 1994 and Nichols, et a/., in 1995. Therefore, the potential for integration of the pHIS-HIN-B plasmid vector into rabbit genomic D ⁇ A is considered to be extremely low.
  • Juvenile macaques (M. nemestrin ⁇ ) were free from HIV-1/SIV/SRV infection, housed under PC3 conditions and anaesthetised with Ketamine (10 mg/kg IM) prior to procedures. The studies were approved by the institutional Animal Experimentation and Ethics Committees. Six groups of macaques were studied as noted in Figure 19. The vaccinations were well tolerated with no significant loss of activity or weight of the macaques following vaccinations (not shown). DNA vaccines were delivered lmg IM to the left and right quadriceps. rFPV was delivered IM (5 x IO 7 PFU) to the left and right quadriceps of macaques.
  • Lymphoproliferative responses were assessed by standard 3 H-thymidine incorporation assay as described (Kent, S.J., A. Woodward, and A. Zhao. 1997, J Infect Dis 176:1188- 1197). Briefly, macaque PBMC in triplicate wells at 10 5 cells/well were stimulated for 6 d with 10 ⁇ g/ml of recombinant HIV-1 p55 or control antigen also produced in baculovirus cultures, or whole inactivated HIV-1MN or control microvesicles produced in the same cell line in media containing 5% autologous heat-inactivated serum and pulsed with 3 H- thymidine for 18h before ⁇ -counting. Proliferation is expressed as stimulation index (SI, mean 3 H-thymidine incorporation of cells stimulated with antigen/mean incorporation in absence of antigenic stimulation).
  • SI stimulation index
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • IFN ⁇ production from T cells can be quantified by the ELISPOT technique.
  • animals receiving rFPV vaccines alone or animals tested immediately after the DNA vaccine prime modest levels of IFN ⁇ producing T cells were observed.
  • animals receiving DNA prime and FPV boost vaccinations had markedly elevated numbers of HIV-1 gag-specific IFN ⁇ producing cells, consistent with the combination of DNA and FPV vaccines being highly stimulatory for HlV-specific T cells.
  • Immature pseudoviral particles were observed to but from HeLa cells transfected with the DNA vaccines pHIS-HIV-B and pHIS-HIV-AE ( Figure 25). Pseudoviral particle formation indicates efficient expression of Gag. Immature and defective particle morphology was seen with both B and AE clade vaccines, consistent with safety mutations in vaccines. Inefficient budding of pseudoviral particles was produced by pHIS-HLV-B.
  • the AE vaccine also expresses high levels of Gag and Pol and truncated Env ( Figure 26).
  • the rFPV-HIV-B vaccine expresses high levels of HIV Gag and Pol in human cells.
  • the rFPV-HIV-AE Gag-Pol also expresses high levels of HIV Gag and Pol.
  • the AE Env and at-Rev were also expressed by final rFPV-AE vaccine ( Figure 27). Reverse transcriptase activity
  • HIV genome More than two thirds of the HIV genome was deleted from each of the vaccine constructs.
  • human DNA contains numerous copies of endogenous retroviral elements that could potentially supply components that might restore replication competence.
  • pHIS-HIV-B and rFPV-HIV-B vaccines show impressive immunogenicity in mice and pigtailed macaques (see Oral presentation by S.J. Kent).
  • HIV clade-B DNA/rFP V prime boost vaccines appear safe and have been approved for phase I human clinical trial by the Australian Therapeutic Goods Administration.
  • H ⁇ V-l human immunodeficiency virus type 1
  • AVAC host-range- restricted canarypox vector

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Abstract

The present invention relates generally to a method of eliciting or otherwise inducing an immune response to a virus and agents for use therein. More particularly, the present invention relates to a method of eliciting or otherwise inducing an immune response to HIV and to agents for use therein. The method of the present invention is predicated on the application of a novel prime-boost vaccination regime utilising, respectively, a CpG optimised plasmid vector and a viral vector, both of which encode two or more substantially non-pathogenic HIV antigens. The present invention is useful, inter alia, as a prophylactic and/or therapeutic treatment regime for viral pathogen infections of mammals such as, for example, HIV infection. A related aspect of the present invention is directed to the optimised vectors for use in this method.

Description

A METHOD OF IMMUNISATION AND AGENTS USEFUL FOR SAME
FIELD OF THE INVENTION
The present invention relates generally to a method of eliciting or otherwise inducing an immune response to a virus and agents for use therein. More particularly, the present invention relates to a method of eliciting or otherwise inducing an immune response to HIV and to agents for use therein. The method of the present invention is predicated on the application of a novel prime-boost vaccination regime utilising, respectively, a CpG optimised plasmid vector and a viral vector, both of which encode two or more substantially non-pathogenic HIV antigens. The present invention is useful, inter alia, as a prophylactic and/or therapeutic treatment regime for viral pathogen infections of mammals such as, for example, HIV infection. A related aspect of the present invention is directed to the optimised vectors for use in this method.
BACKGROUND OF THE INVENTION
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
A safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) infection is urgently needed to curb the HIV-1 pandemic. Much circumstantial evidence suggests that HIV-specific T cell responses may facilitate protective immunity. Individuals exposed to HIV-1 but who do not become persistently infected develop HIV-1 specific cytotoxic T lymphocytes (CTL) and T-helper (Th) lymphocytes without the generation of systemic HIV-1 antibodies, although mucosal HIV-1 antibodies have also been detected (Rowland- Jones, S., J. Sutton, K. Ariyoshi, T. Dong, F. Gotch, S. McAdam, D. Whitby, S. Sabally, A. Gallimore, T. Corrah, M. Takiguchi, T. Schultz, A. McMichael, and H. Whittle. 1995, Nature Med 1 :59-64; Mazzoli, S., D. Trabattoni, S. Lo Caputo, S. Piconi, C. Ble, F. Meacci, S. Ruzzante, A. Salvi, F. Semplici, R. Longhi, M.L. Fusi, N. Tofani, M. Biasin, M.L. Villa, F. Mazzotta, and M. Clerici. 1997, Nαtwre Med 3:1250-1257). The generation of CTL and Th responses, but not antibodies, temporally correlate with the control of acute HIN-1 viremia in humans and macaques. The induction of HIN-1 specific CTL and Th responses is widely seen as critical to the success of an HIN-1 vaccine.
Early candidate HIV-1 vaccine regimens employed only non-replicating compounds such as recombinant HIV-1 proteins. Vaccination of humans or non-human primates with recombinant proteins of HIV-1 or the simian immunodeficiency virus (SIN, a simian homologue of HIV-1) generated specific antibody responses but did not generally induce protective immunity in animal studies and resulted in significant numbers of breakthrough HIV-1 infections in small human trials. Subsequent HIV-1 vaccine strategies attempting to induce both enhanced T cell responses and antibody responses have primarily focussed on recombinant vaccinia (rVV) and avian pox viruses (canarypoxviruses and fowlpoxviruses) genetically engineered to express HIV-1 proteins boosted by recombinant HIV-1 proteins (Leong, K.H., AJ. Ramsay, M.J. Morin, H.L. Robinson, D.B. Boyle, and A. Ramshaw. 1995. In Vaccines 95. F. Brown, H. Chanock and E. Νorrby, editors. Cold Spring Harbour Laboratory Press. 327-331). The use of recombinant poxvirus vectors has the theoretical advantage that expression of foreign genes from within the infected host cells allows the loading of MHC class I molecules with immunogenic peptides and the stimulation of CTL responses. Unfortunately, vaccinations of humans and outbred non-human primates with poxvirus vectors expressing HIV-1 or SIV antigens and recombinant HIV-1 or SIV proteins, despite being theoretically attractive, have induced detectable HIV-1 or SIV specific CTL responses in only a minority of recipients (Fleury, B., G. Janvier, G. Pialoux, F. Buseyne, M.Ν. Robertson, J. Tartaglia, E. Paoletti, M.P. Kieny, J.L. Excler, and Y. Riviere. 1996. J Infect Dis 174:734-738; Egan, M.A., W.A. Pavlat, J. Tartaglia, E. Paoletti, K.J. Weinhold, M.L. Clements, and R.F. Siliciano. 1995. J Infect Dis 171:1623- 1627; Kent, S.J., S.L. Hu, L. Corey, W.R. Morton, and P.D. Greenberg. 1996. J Virol 70:4941-4947; Corey, L., M.J. McElrath, K. Weinhold, T. Matthews, D. Stablein, B. Graham, M. Keefer, D. Schwartz, and G. Gorse. 1998. J Infect Dis 177:301-309; Gallimore, A., M. Cranage, N. Cook, N. Almond, J. Bootman, E. Rud, P. Silvera, M. Dennis, T. Corcoran, J. Stott, and et al. 1995. Nature Med 1:1167-1173). Further, poxvirus-based regimens have demonstrated limited protective efficacy in SIN/macaque studies, and have failed to prevent cases of HIV-1 infection in small human clinical trials (Gallimore et al., 1995, supra; Daniel, M.D., G.P. Mazzara, M.A. Simon, P.K. Sehgal, T. Kodama, D.L. Panicali, and R.C. Desrosiers. 1994. AIDS Res Hum Retroviruses 10:839- 851; Kent, S.J., P.D. Greenberg, M.C. Hoffman, R.E. Akridge, and M.J. McElrath. 1997. J Immunol 158:807-815). Considerable scope exists to improve the ability of poxvirus vectors to induce CTL responses and provide protective immunity.
Recombinant protein vaccinations, while facilitating a strong antibody response, primarily stimulate a particular subset of Th cells called Th2 cells, defined by their secretion of the cytokines IL-4, IL-5, and IL-10. Th2 cells and the cytokines they secrete may counteract any protective cell-mediated immunity (Kent et al. 1997, supra; Sharma, D.P., AJ.
Ramsay, D J. Maguire, M.S. Rolph, and LA. Ramshaw. 1996, J Virol 70:7103-7107). In response to many pathogens and vaccines, humoral and cell-mediated immunity are mutually antagonistic, that is the immune system supports either a strong Thl response (associated with IL-2 and IFN-γ production and enhanced CTL responses) or a strong Th2 response at least at the partial expense of the other. Although arguably desirable, it may not be feasible for an HIV-1 vaccine regimen to induce both strong, sustained antibody and CTL responses (Salk, J., P.A. Bretscher, PL. Salk, M. Clerici, and G.M. Shearer. 1993. Science 260:1270-1272). A vaccine regimen that reproducibly induces predominantly Thl/CTL responses to HIV-1 could potentially generate stronger T cell responses than one that endeavours to induce both antibody and Thl/CTL responses.
Intramuscular (IM) or epidermal injection of purified plasmid DNA can induce immune responses to encoded antigens (Tang, D.C., M. De Vit, and S.A. Johnston. 1992, Nature 356:152-154). Plasmid DNA vaccines, which are simple and inexpensive to produce, have the potential to revolutionise or re-energise many vaccine development fields, including that of HIV-1. IM injection of DNA encoding HIV-1 proteins into 2 chimpanzees generated HIV-1 specific CTL responses in one the animals and induced some protection from non-pathogenic HIV-1SF2 infection in both animals (Boyer, J.D., K. Ugen, B. Wang, M. Agadjanyan, L. Gilbert, M. Bagarazzi, M. Chattergoon, P. Frost, A. Javadian, .V. Williams, Y. Refaeli, R. Ciccarelli, D. McCallus, L. Coney, and D.B. Weiner. 1997, Nature Med 3 :526-532). When IM HIV- 1 DNA vaccination of 2 macaques has been boosted by recombinant protein vaccination, protection of the 2 macaques from non- pathogenic SHINHXB2 infection was observed (Letvin, Ν.L., D.C. Montefiori, Y. Yasutomi, H.C. Perry, M.E. Davies, C. Lekutis, M. Alroy, D.C. Freed, C.L Lord, L.K. Handt, M.A. Liu, and J.W. Shiver. 1997, Proc Natl Acad Sci USA 94:9378-9383). Although the antibody response was enhanced approximately 100-fold by recombinant protein boosting of macaques primed with IM DNA, the HIV-1 specific CTL precursor levels were augmented <2 fold by the recombinant protein boosting and remained at a low level [<15 CTLs/106 peripheral blood mononuclear cells (PBMC)] (Letvin et al. 1997, supra). DNA vaccines alone have resulted in only very limited protection from pathogenic SIV or non- pathogenic SHIVHXB2 infection of macaques (Lu, S., J. Arthos, D.C. Montefiori, Y.
Yasutomi, K. Manson, F. Mustafa, E. Johnson, J.C. Santoro, J. Wissink, J.I. Mullins, J.R. Haynes, N.L. Letvin, M. Wyand, and H.L. Robinson. 1996, J Virol 70:3978-3991; Boyer, J.D., B. Wang, K.E. Ugen, M. Agadjanyan, A. Javadian, P. Frost, K. Dang, R.A. Carrano, R. Ciccarelli, L. Coney, W.V. Williams, and D.B. Weiner. 1996, JMedPrimatol 25:242- 250). Thus, although both DNA and avipox vectors show promise as HIV-1 vaccine candidates, considerable potential exists for novel strategies designed to enhance the T cell immunogenicity and efficacy of both DNA and avipox vaccine vectors.
There have previously been utilised consecutive immunisation strategies involving priming by DNA vaccination and boosting with rFPV vectors encoding common influenza antigens in attempts to generate improved specific immune responses (Leong et al. 1995, supra). The rationale behind this vaccine strategy was that DNA immunization, which elicits low- level but persistent immunity, may prime for greatly enhanced T cell responsiveness following boosting with another vaccine vector, such as rFPV, that expresses vaccine antigens from within host cells and therefore loads MHC class I molecules efficiently. Recombinant fowlpox vectors may have advantages over rVV for use as a boosting vaccine vector. Firstly, fowlpox vaccines, which undergo an abortive infection in mammalian hosts, are safer since wild type vaccinia virus can cause a lethal infection in immunodeficient humans. Secondly, for pathogens such as HIV-1 where T cell mediated responses may be required for protective efficacy, avoiding a marked enhancement of antibody/Th2 responses, as typically observed following recombinant protein boosting, may facilitate enhanced Thl/CTL responsiveness and therefore be desirable.
However, current prime-boost protocols are inefficient. Further, in terms of HIV prophylaxis, another issue that is not resolved with regard to successful HIV vaccines is the delineation of the regions of the HIV genome which are protective. Although many of the 9 HIV genes have been shown to stimulate immune responses, expression of precisely which of these genes is sufficient to stimulate protective immunity is currently unknown.
Accordingly, there is an urgent ongoing need to develop methods of inducing and maintaining immunity to viral pathogens such as HIV. In work leading up to the present invention, there has been developed a novel prime-boost vaccination strategy, which achieves levels of T cell immunity previously unattainable, based on the sequential administration of a CpG optimised plasmid vector, encoding multiple substantially non- pathogenic HIV antigens, followed by a recombinant fowlpox virus vector also expressing multiple substantially non-pathogenic HIV antigens. The inventors have still more surprisingly determined that the use of these uniquely optimised vectors facilitates the application of an abbreviated immunisation schedule which thereby provides a significantly more useful and effective vaccine regime. SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The subject specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide and amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (eg. <210>1, <210>2, etc). The length, type of sequence (DNA, protein, etc) and source organism for each nucleotide and amino acid sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO:l, SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>1, <400>2, etc). That is SEQ ID NO:l as detailed in the specification correlates to the sequence indicated as <400>1 in the sequence listing.
One aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to a virus, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and (ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens or of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
Another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said HIV, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
Still another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal: (i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV- 1 , which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
Yet more particularly, there is provided a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens. In still another aspect there is provided a method for eliciting or otherwise inducing in a mammal an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef, which Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro,
RNAseH, Tat, Rev and/or Env, which Gag, RT, Pro, RNAseH, Tat, Rev and/or Env have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to these antigens.
In still yet another aspect there is provided a method for eliciting or otherwise inducing in a mammal an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and/or Env, which Gag, RT, Rev, Tat, Vpu and/or Env have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and (ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol, which Gag, Pol, Tat, Rev, and/or Env have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of these antigens.
In a further aspect there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) a single dose of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) a single dose of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic,
wherein said recombinant viral vector dose is administered four weeks later than said recombinant plasmid vector dose.
In another further aspect there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal: (i) two doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) two doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered non-pathogenic,
wherein the second recombinant plasmid vector dose is administered three weeks after the first dose, the first recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
In still another further aspect there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) sequentially administering to said mammal two doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one dose of an effective amount of the recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered non-pathogenic wherein the recombinant plasmid vector dose is administered four weeks after the first dose and the recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose.
In yet another further aspect there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) Three doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) Two doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic
wherein said the second recombinant plasmid vector dose is administered four weeks after the first dose, the third recombinant plasmid vector dose is administered four weeks after the second dose, the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
Yet another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIN, said method comprising sequentially administering to said mammal one or more doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding two or more antigens thereof of said HIV, which antigens have been rendered substantially non-pathogenic, and one or more optimised CPG motifs for a time and under conditions sufficient to elicit or induce an immune response to said antigens.
Still another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which viral vector is incorporates nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said virus.
Yet still anther aspect of the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, said one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which fowlpox virus vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV, which antigens have been rendered substantially non- pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said HIV or associated with said HIV-induced AIDS.
Still yet another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which viral vector is incorporates nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said virus. More particularly, the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, said one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which fowlpox virus vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV, which antigens have been rendered substantially non- pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said HIV or associated with said HIV-induced AIDS.
In a related aspect the present invention is directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
This aspect of the present invention is still further directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
Still another aspect of the present invention is directed to a nucleic acid construct or functional derivative thereof which construct comprises a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
In yet another aspect the present invention is directed to a nucleic acid construct comprising a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
Yet another aspect of the present invention is directed to compositions, in particular pharmaceutical compositions, comprising the nucleic acid constructs, plasmid vectors and/or viral vectors hereinbefore described.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic representation of a plasmid Map of pHIS-HIV-B
Figure 2 is a schematic representation of a plasmid Map of pHIS-HIV-AE
Figure 3 is a schematic representation of the interrelationship of pHIS-HIV-AE and pHIS- HIV-B nucleotide sequences.
Figure 4 is a representation of the full Vaccine Aligns: Comparison of B (SEQ ID NO:3) and AE (SEQ ID NO:4) Subtype Vaccine Sequences
Figure 5 a representation of the 5' primer: 75-mer XhoI/FPV early late promoter PE/L/gag 1-26 (SEQ ID NO:5)
Figure 6 a representation of the 3' primer: 58-mer Xhol/pox virus early transcription termination signal T5NT/complement of pol 3427-3458 (SEQ ID NO:6)
Figure 7 a representation of the 34bp Sequence Inserted (SEQ ID NO:7)
Figure 8 a representation of the plasmid pKGlO and pKG-gag/pol(m)
Figure 9 a representation of the plasmids for rFPV-HIV-AE
Figure 10 is a schematic representation of the splicing and construction of 5' vaccine end B CLADE.
Figure 11 is a schematic representation of the sequence of pHIS-HIVB (SEQ ID NO: 1).
Figure 12 is a diagrammatic representation of the sequence comparison of vaccines and controls. Figure 13 a representation of the constructed recombinant FPV-HIV-AE vaccines
Figure 14 is a graphical representation of rFPV-HIV-B immunogenicity in prime/boost vaccination in mice.
Figure 15 is a graphical representation of IFNγ ELISPOT to inactivated HIV-1 following 2nd FPV boost
Figure 16 is a graphical representation of IFNγ ELIspot to Inactivated HIV-1 (left) and HIV-1 gag (right) at Challenge
Figure 17 is a graphical representation of SIV Gag-specific Response by IFNγ ELISPOT
Figure 18 is a graphical representation of the control of pathogenic SHIV challenge by DNA/FPV prime/boost immunisation
Figure 19 is a schematic representation of the first macaque study.
Figure 20 is a schematic representation of the schedule/dose of macaque experiment.
Figure 21 is a graphical representation of proliferation - one week post-first FPV boost.
Figure 22 is a graphical representation of proliferation - one week post-final immunisation.
Figure 23 is a graphical representation of the comparison of 1 and 2 FPV boosts across groups.
Figure 24 is a graphical representation of IFNg ELISPOT to overlapping gag peptides following 2nd FPV boost by vaccine group. Figure 14 is a schematic representation of DNA vaccine construct plasmid maps.
Figure 25 is an image of immature pseudoviral particles budding from HeLa cells transfected with the DNA vaccines pHIS-HIV-B and pHIS-HIV-AE.
Figure 26 is an image of HIV-1 protein expression from DNA vaccines in vitro measured by Western blot with HIV patient serum.
Figure 27 is an image of HIV-1 protein expression from fowlpox vaccines in vitro measured by Western blot probed with HIV patient serum.
Figure 28 is a graphical representation demonstrating the lack of HIV reverse transcriptase activity from DNA vaccines by ultra-sensitive real time PCR-enhanced RT assay TM- PERT. TM-PERT assay used the reverse transcriptase associated with ultracentrifuge purified particles to produce cDNA from an MS-2 phage template provided. The resulting cDNA was detected by a real time PCR assay using am MS-2 specific TaqMan probe and primers. A dilution series standard AMV RT was assayed in parallel to determine the units of RT activity in the samples. Limit of detection for the TM-PERT assays is lxlO"10 units AMV RT.
Figure 29 is a schematic representation of mutations made to the crucial zinc finger motifs in nucleocapsid.
Figure 30 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles produced by B clade DNA vaccine is equivalent to replication defective HIV RNA packaging mutants, as measured by slot blot.
Figure 31 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles produced by B clade DNA vaccine is equivalent to replication defective HIV RNA packaging mutants. Viral particles were standardised for p24 antigen, the RNA was extracted and quantified by the modified HIV-1 NucliSens RNA amplification assay. Residual RNA detected likely reflects gross over-expression of g g RNA following transfection. pDB653 is a replication defective reference packaging mutant (B clade).
Figure 32 is an image indicating that HIV-1 RNA encapsidation into purified psuedoviral particles derived from the recombinant fowlpox vaccine is 10-fold less efficient than in wild type HIV-1 NL(AD8). Viral particles were standardised for p24 antigen, the RNA was extracted and quantified by the modified HIV-1 NucliSens RNA amplification assay. rFPV-59 recombinant fowlpox containing a non-mutanted HIV Gag was assayed in parallel.
Figure 33 is a graphical representation demonstrating that pHIS-HIV-B and pHIS-HIV- AE vaccine derived pseudoviral particles are NOT able to establish a productive infection in susceptible primary cells during a cell free infection. 2x106 PHA activated PBMC were infected with 20ng of p24 of purified viral or pseudoviral particles for 2 hr/37°C, then washed in PBS to remove free viral particles. Cells were maintained in culture for greater than 50 days with the regular addition of lxl 06 feeder cells. p24 antigen concentration was measured in the culture supernatant.
Figure 34 is a graphical representation demonstrating that pHIS-HIV-B and pHIS-HIV- AE DNA vaccine are NOT able to establish a productive infection in HIV-1 susceptible T cell lines during a cell to cell infection. HeLa cells were transfected with the DNA vaccine and wild type constructs, then at peak antigen production co-cultured with the HIV-1 susceptible call line PM1. Evidence of replication was defined as a doubling of p24 antigen concentration in the cell culture supernatant after the addition of the susceptible cells.
Figure 35 is a graphical representation demonstrating that lack of retrovirus replication in HeLa cells infected with BMP grade rFPV-HIV-B, using the ultra-sensitive TM-PERT assay. rFPV-HIV-B vaccines were manufactured at GMP grade in primary chicken epithelial cells that also produce significant levels of RT from endogenous avian retroviruses. The presence of this RT activity is well known and is not considered a safety risk due to the lack of pathogenesis from other widely used human vaccines (measles, mumps, rubella, yellow fever). No replication competent retrovirus could be derived from this RT activity in long term HeLa cell culture, with RT activity falling below the limit of detection (lxlO"9 units AMV RT) and not reappearing even after 70 days of culture.
Figure 36 is a schematic representation of the regimen for the non-human primate toxicology study to assess dose toxicity profile and biodistribution of the DNA and rFPV vaccines.
Figure 37 summarises the absence of adverse effects in the non-human primate toxicology study.
Figure 38 is a schematic representation of the regimen for the rabbit toxicology study to assess the integration of the plasmid DNA and rFPN vaccines.
Figure 39 summarises the significant levels of integration not detected in rabbit toxicology study. A very low level of potential integration of DΝA vaccine detected only one of 6 animals early after vaccination. Even if all detected DΝA is assumed to be integrated, calculated integration rate is 1250 times lower than spontaneous mutation rate per human gene. Risk of integration is considered to be minimal.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated, in part, on the development of a consecutive immunisation strategy involving priming with an optimised plasmid vector and boosting with an optimised recombinant fowlpox virus vector, both encoding common HIV antigens mutated to remove active sites and the former additionally co-expressing CpG immunostimulatory molecules. The application of this methodology has been determined to result in a significant boosting of plasmid vector vaccine primed HIV specific T lymphocyte responses following recombinant fowlpox virus vector boosting. This determination has now facilitated the development of compositions and methodology for application, ter alia, in relation to the prophylactic or therapeutic treatment of viral infections, in particular HIV infections, wherein the generation of an effective T cell mediated protective response is desirable.
Accordingly, one aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to a virus, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens or of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens. Reference to "virus" should be understood as a reference to any virus or to homologues or mutants thereof. In a particularly preferred embodiment, the subject virus is the human immunodeficiency virus (herein referred to by the abbreviation "HLV"). Reference herein to "HIV" should be understood as a reference to any HIV strain and to homologues and mutants thereof. Reference to "homologues" in this context should be understood as a reference to HIV strains which naturally occur in a species other than a species undergoing treatment in accordance with the method of the present invention. For example, although the present invention is preferably directed to the treatment of humans utilising HIV derived antigens, it should be understood that the present invention extends to the development of methodology directed to the induction of HIV immunity in humans utilising simian immunodeficiency virus antigens. Reference to "mutants" should be understood as a reference to forms of HIV which differ, either structurally or functionally, from the wild type form from which they are derived. In general, such mutants are characterised by nucleic acid deletions, insertions or substitutions. Such mutants are known to develop, for instance, in the context of the development of resistance to drug treatment. In a particularly preferred embodiment, said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
The present invention therefore more particularly provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said HIV, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
Still more particularly, there is provided a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incoφorated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
In another preferred embodiment, said HIV is HIV-1 subtype AE, subtype B or subtype C.
Reference to "eliciting or otherwise inducing" and "immune response" to an HIV antigen should be understood as stimulating or facilitating stimulation of a specific immune response. This specific immune response may be a T cell and/or a humoral response which is directed to any one or more of the peptides or epitopes, respectively, of the HIV antigen encoded by the nucleotide sequence comprising the recombinant plasmid or viral vector of the present invention. Preferably, the immune response is a Thl and CTL response. Without limiting the present invention to any one theory or mode of action, the induction of HIV specific T cell responses are widely seen as critical to the development of effective immunity to HIV. Further, and still without limiting the present invention in any way, avoiding a marked enhancement of Th2-antibody responses (as typically observed following recombinant protein boosting) is believed to facilitate enhanced Thl/CTL responsiveness. However, it should nevertheless be understood that even where an immune response is skewed to a Thl/CTL-type response, some degree of antibody generation may nevertheless occur.
The method of the present invention is predicated on the determination that the sequential administration of an optimised recombinant plasmid vector followed by an optimised recombinant viral vector, as herein defined, can induce an enhanced and sustained Thl/CTL response directed to HIV. In this regard, reference to a "plasmid" vector should be understood as a reference to independently replicating pieces of cytoplasmic DNA. These linear or circular DNA molecules are found in both prokaryotes and eukaryotes capable of autonomous replication. Plasmids are largely described in the context of those derived from bacteria and protozoa. Examples of plasmids suitable for use in the method of the present invention include, but are not limited to, pHIS-HIVB, pHIS-HIV-AE or any other CpG optimised plasmid. Reference to the subject plasmid being "recombinant" should be understood as a reference to the vector having been modified relative to its wild type form. This may occur for instance in the context of the insertion of additional nucleic acid segments (such as those which encode the HIV antigens of interest) and/or due to the modification of the plasmid sequence itself, such as would occur in the context of nucleotide optimisation. It should also be understood that the population of plasmids which are utilised in the context of the method of the present invention may be naturally occurring plasmids which have been appropriately modified (for example plasmids which have been harvested from cultured bacterial broths) or plasmids which have been otherwise synthetically or recombinantly generated. Reference to a "viral vector" should be understood as a reference to any viral vector which can achieve efficient MHC I loading such as an avipox viral vector (for example fowlpox viral vectors or canarypox viral vectors), a vaccinia viral vector an adenovirus or an alphavirus replicon. In this regard, it should be understood that the subject viral vectors which are utilised in the present invention may comprise the whole or part of any virus or derivative thereof. Reference to the subject viral vector being "recombinant" in the context of the present invention should be understood to have the same meaning as detailed above in relation to the term "plasmid". Preferably, the subject viral vector is a recombinant fowlpox viral vector.
Yet more particularly, there is provided a method for eliciting or inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
Without limiting the method of the invention to any one theory or mode of action, the use of viral vectors in the context of the present invention provides a means of achieving antigen expression within host cells and therefore achieving MHC class 1 molecule 28
stimulated. For example, processing of the antigen by an antigen presenting cell may result in the production of one or more peptides which are co-expressed with MHC class II and which stimulate specific T helper cells, in particular Thl cells. Most preferably, and in the context of the preferred embodiments defined herein, the processing and co-expression of these peptides also occurs in the context of MHC class I and thereby leads to the stimulation of one or more specificities of T cytotoxic cells. It should be understood that the subject antigen may also comprise one or more epitopes to which some degree humoral immune response may be directed. Said epitope may be a linear epitope or a conformational epitope. Where the epitope is a linear epitope, folding of the expressed antigen into its native conformation may not be required to achieve the stimulation of the specific humoral response directed to that epitope.
Accordingly, it should be understood that reference to stimulating a response to an HIV "antigen" should be understood as a reference to the stimulation of specific immune cells (eg. T cells) which are directed to one or more sites of the HIV antigen. Examples of antigens suitable for use in the present invention include, but are not limited to, one or more of the molecules encoded by the HIV viral genes gag, RT, Pro, RNAseH, rev, tat, vpu, env, nef, vif, vpv and/or pol. Preferably, said plasmid and/or viral vectors express 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more antigens. In a most preferred embodiment, seven vectors express at least 5 antigens. The expression product of each gene is given the same name, but appears in non-italicised text with the first letter capitalised. In a particularly preferred embodiment, the subject plasmid vector and viral vector incorporate nucleic acid molecules encoding shared antigens. By "shared" is meant that at least some of the subject antigens are expressed by both the plasmid vector and the viral vector. Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 antigens are shared.
In one preferred embodiment, the subject plasmid vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef while the recombinant fowlpox virus vector incorporates nucleic acid molecules encoding Gag, RT, Pro, RNAseH, Tat, Rev, Env and. As detailed hereinbefore, reference to these antigens includes reference to derivatives, such as fragments, thereof. - 27
loading. Although the method of the present invention can be applied utilising any suitable viral vector, the fowlpox viral vectors are particularly suitable for use since they undergo an abortive infection in mammalian hosts and are therefore safer than other types of viral vectors, such as vaccinia virus, which can cause a lethal infection in immunodeficient humans.
As detailed hereinbefore, the design of the subject vaccine protocol, which is based on the prime-boost regime utilising, respectively, a plasmid vector and a recombinant fowlpox virus vector, has been rendered exceptionally efficient due to the determination that both types of vectors should be optimised to express multiple HIV antigens and, further, that the plasmid vectors should express optimised CpG motifs. Still further, since some HIV antigens correspond to active enzymes and binding sites, which can represent a safety hazard in HIV negative subjects, it is necessary to mutate these genes in order to render them harmless.
Without limiting the present invention in any way, in terms of the design of successful HIV vaccine, a significant issue is the delineation of the regions of the HIV genome which are immunologically protective. Although many of the nine HIV genes have been shown to stimulate immune responses, expression of the specific genes, or regions of genes which are sufficient to stimulate protective immunity are largely unknown. However, it has now been determined that the expression of multiple HIV genes can achieve the objective of generating protective immunity in an outbred population. In this regard, it should be understood that the number and combination of HIV antigens encoded by the plasmid vector may either differ from the number and combination of HIV antigens encoded by the fowlpox virus vector.
In terms of the HIV antigens which are co-expressed by the vectors of the invention, reference to "HIV antigen" should be understood as a reference to any component of HIV or derivative thereof, such as any component of any one or more HIV genes. Said component may be a peptide, polypeptide or protein. It should be understood that the antigen may comprise one or more sites in respect of which a specific immune response is In another preferred embodiment, the subject plasmid vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Tat, Rev, Vpu, Env and/or nef while the recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Pol, Tat, Rev and/or Env.
Accordingly, in one preferred embodiment, there is provided a method for eliciting or otherwise inducing in a mammal an immune response directed to HIN-1, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef, which Gag, RT, Pro, RNAseH, Rev, Tat, Vpu and Env have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, Pro, Tat, Rev,
Env and/or Pol, which Gag, RT, Pro, RNAseH, Tat, Rev and/or Env have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to these antigens.
In a most preferred embodiment, there is provided a method for eliciting or otherwise inducing in a mammal an immune response directed to HIV-1, said method comprising sequentially administering to said mammal: (i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and/or Env, which Gag, RT, Rev, Tat, Vpu and/or Env have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol, which Gag, Pol, Tat, Rev, and/or Env have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of these antigens.
In another preferred embodiment, said HIV is HIV-1 subtype AE, subtype B or subtype C.
Preferably, said immune response is a Thl/CTL response.
Reference to the subject antigens being "substantially non-pathogenic" should be understood to mean that the subject antigen, when expressed in the mammal undergoing treatment, will not present a significant safety hazard in HIN-negative subjects. This may occur either due to the subject antigen inherently not posing a hazard or due to the nucleic acid molecule encoding the subject antigen having been mutated in order to eliminate or at least reduce one or more hazards. In this regard, reference to "substantially" non- pathogenic should be understood to indicate that the subject antigen is not necessarily entirely non-pathogenic but exhibits a level of pathogenicity which is acceptable in the context of an immunisation protocol. The relative level of risk which is acceptable in the context of any individual treatment can be determined by the immunologist of skill in the art. Without limiting the present invention in any way, and in the context of the preferred embodiment of the present invention, the subject gag and pol genes are preferably modified in order to reduce the risks associated with the expression of their naturally occurring forms. Similarly, the pol gene is modified and the env gene truncated. Still without limiting the invention to any one theory or mode of action, the subject gag modification blocks gag mediated binding/packaging of the HIV/RNA template. The specific modification which achieves this objective is the deletion, from the expression product, of amino acids 392-395 and 413-416 (the first and second regions in the first and second zinc finger domains). The subject pol modification is required in order to block Rt, RNaseH and Integrase activity. This is achieved via deletion of amino acid 65-74, 113- 115 and 183-186 of the RT expression product. Mutation of Gin 478 to Glu 478 inactivates RNase H while deletion from 30 amino acids into the Integrase coding region to 87 base pairs 5' of the Tat start codon remove Integrase functioning. Env truncation blocks its effect on the T cell population through CD4 engagement and, further, reduces the possibility of an infective particle forming. In short, deletion of 581 base pairs starting from the first Bglll site and replacement of this fragment with a short piece of synthetic DNA containing stop codons in all three frames achieves this objective.
Preferably, and in the context of said plasmid vector:
(i) said gag is modified to block Gag mediated binding/packaging of the HIV RNA template;
(ii) said pol is modified to block RT/RNaseH and integrase activity;
(iii) said env is truncated to block its effects on the T cell population through CD4 and
CCR5 engagement and to reduce the possibility of an infective particle forming; and
(iv) said nef is truncated to block immunomodulatory activity. In a related aspect, and without limiting the present invention to any one theory or mode of action, modifying CpG motifs can be utilised to achieve improved immunogenicity. Unmethylated CpG dinucleotides, such as those found in bacterial oligonucleotides, are recognised as a danger signal by the vertebrate immune system. Specifically, CpG motifs comprising a nuclease resistant phosphorothioate backbone are potent Thl adjuvants. Accordingly, the modification of a vaccine to introduce an appropriate unmethylated CpG pattern can provide both adjuvant like functional activity and skewing of the immune response towards a Thl type response. Accordingly, it should be understood that reference to "optimised" CpG motifs in the context of the present invention should be understood as a reference to the use of unmethylated CpG dinucleotides or the removal of neutralising motifs. It should also be understood that determining the concentration of unmethylated CpG dinucleotides to be expressed by any given vector, in order to achieve the objective of the present invention, can be determined as a matter of routine procedure when one considers the teachings provided herein.
Most preferably, said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said recombinant fowlpox virus is rFPV-HIV-B or rFPV-HIV-AE.
Without limiting the present invention to any one theory or mode of action, the pHIS-HIV- B vector exhibits defective RT, cannot yield replication competent virus, does not package HIV-1 RNA efficiently and produces modest levels of pseudoviral particles. The rFPV- HIV-B vector, similarly does not package HIV RNA efficiently and cannot produce modest levels of pseudoviral particles. However, this vector does exhibit high levels of RT activity.
As detailed hereinbefore, it has been determined that the application of a prime-boost vaccination schedule which utilises plasmid and recombinant fowlpox virus vectors incorporating the specific features detailed herein has been surprisingly determined to result in the development of a previously unattainable level of immunity to HIV. Although it is essential that the vectors of the present invention express the specific features detailed herein (for example that the plasmid vector encodes two or more viral antigens and one or more optimised CpG motifs and the recombinant fowlpox virus vector encodes two or more viral antigens), this is not necessarily intended as a limitation in relation to the molecules which comprise these vectors. In particular, it should be understood that the subject recombinant fowlpox virus vector may express genes in addition to those specifically detailed herein, such as additional HIV genes.
Reference to "derivatives" should be understood to include fragments, parts, portions, equivalents, analogs, mutants, homologues, mimetics from natural, synthetic or recombinant sources including fusion proteins. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins.
The derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, the vector or derivative thereof may be fused to a molecule to facilitate its entry into a cell. Derivatives of nucleic acid sequences may be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
Equivalents should be understood to include reference to molecules which can act as a functional analog or agonist. Equivalents may not necessarily be derived from the subject molecule but may share certain conformational similarities. Alternatively, equivalents may be designed to mimic certain immunological and physiochemical properties of the subject molecule. Equivalents may be detected following, for example, natural product screening. Equivalents also include peptide mimics. Homologues contemplated herein include, but are not limited to, molecules derived from different species. Fragments include portions which are effective in achieving the object of the present invention.
The nucleic acid molecule suitable for use in the present invention may be DNA or RNA. Preferably said nucleic acid molecule is DNA. Reference to the HIV antigen encoded by a nucleic acid molecule is a reference to the expression product of said nucleic acid molecule.
The method of the present invention is based on a prime-boost regime. Without limiting the invention to any one theory or mode of action, the rationale behind such a vaccination strategy is the use of the plasmid vector immunisation step to elicit a low level but persistent immunity which effectively primes for a greatly enhanced T cell response following boosting with another vaccine vector. In the context of the present invention, it has been determined that the boosting vaccine is preferably one which results in expression of the vaccine antigen from within host cells therefore leading to MHC-1 loading in an efficient manner. This type of vaccine strategy, when combined with the uniquely optimised vectors of the present invention, results in a greatly enhanced HIV T cell mediated response. In this regard, reference to "sequential" administration should be understood as the administration of vaccine doses in a consecutive fashion. In this regard the present invention is predicated on the administration of the subject recombinant plasmid vector (being the "priming" step) prior to administration of the recombinant fowlpox virus vector (being the "boosting" step). Still further, it has been determined that the method of the present invention is effective where the priming step comprises the administration of one or more sequential doses of the recombinant plasmid vector and the boosting step comprises the administration of one or more sequential doses of the recombinant fowlpox virus vector. Although not intending to limit the scope of the present invention to the specific vaccination regime exemplified herein, it has been determined that a prime-boost immunisation system comprising two DNA vaccinations three weeks apart and two recombinant fowlpox vaccinations four weeks apart and four weeks after the DNA vaccine achieves a previously unattainable level of T cell responsiveness. Still further, it has been determined that even one plasmid vector dose vaccination followed by one recombinant fowlpox vector dose boost in the context of a minimum interval between the two vaccines of as little as 4 weeks similarly achieves the development of a previously unattainable level of T cell responsiveness. Accordingly, the development of the vectors of the present invention, together with the determination of their effectiveness in the context of a prime-boost vaccination regime, has facilitated the development of a significantly shortened vaccination schedule relative to the schedules which have been utilised to date. In this regard, preferred vaccination schedules include, but are not limited to:
(i) a single dose of the plasmid vector followed 4 weeks later by a single dose of the viral vector
(ii) Three doses of the plasmid vector alone 4 weeks apart
(iii) two doses of the plasmid vector followed by one dose of the viral vector wherein each dose is administered 4 weeks apart
(iv) three doses of the plasmid vector followed by 2 doses of the viral vector wherein each dose is administered 4 weeks apart
(v) two doses of the plasmid vector three weeks apart followed by, three weeks later, two doses of the viral vector four weeks apart.
It should be understood that the above-detailed vaccination schedules are not intended to be exhaustive. Rather, in light of the teachings and disclosures provided herein, the person of skill in the art could develop alternative vaccination schedules via routine means such as immunogenicity testing in primates and humans and subsequent protection from HIV exposure. Accordingly, in a most preferred embodiment there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) a single dose of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) a single dose of an effective amount of a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic,
wherein said recombinant viral vector dose is administered four weeks later than said recombinant plasmid vector dose.
In another preferred embodiment there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1 , said method comprising sequentially administering to said mammal:
(i) two doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) two doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered non-pathogenic,
wherein the second recombinant plasmid vector dose is administered three weeks after the first dose, the first recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
In still another preferred embodiment there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal:
(i) sequentially administering to said mammal two doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one dose of an effective amount of the recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered non-pathogenic
wherein the recombinant plasmid vector dose is administered four weeks after the first dose and the recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose.
In yet another preferred embodiment there is provided a method for eliciting or otherwise inducing, in a mammal, an immune response directed to HIV-1, said method comprising sequentially administering to said mammal: (i) Three doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) Two doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic
wherein said the second recombinant plasmid vector dose is administered four weeks after the first dose, the third recombinant plasmid vector dose is administered four weeks after the second dose, the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
Preferably, said viral vector is a fowlpox virus vector.
In another preferred embodiment, said HIV is HIV-1 subtype AE, HIV-1 subtype B or HIV-1 subtype C.
According to these preferred embodiments the subject immune response is preferably a Thl/CTL response.
Even more preferably said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env and/or Pol. Still more preferably, said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, Pol, RT, Tat, Vpu and/or Env and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol.
Most preferably said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said viral vector is rFPV-HIV-B or rFPV-HIV-AE
Still more preferably, said immune response is a Thl/CTL response.
In a related aspect, the inventors have determined that the unique vector design disclosed herein also facilitates the use of the plasmid vector in isolation.
Accordingly, yet another aspect of the present invention provides a method for eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal one or more doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding two or more antigens thereof of said HIV, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs for a time and under conditions sufficient to elicit or induce an immune response to said antigens.
In one preferred embodiment, said sequential doses are two doses of the plasmid vector 4 weeks apart.
In another preferred embodiment, said sequential doses are three sequential doses and said antigen is two or more of all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef. In yet another preferred embodiment, said sequential doses are three sequential doses and said antigen is two or more of all, part or a modified form of Gag, RT, Rev, Tat, Vpu and/or Env.
More preferably, said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
Still more preferably, said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE.
It should be understood that the therapeutic and prophylactic methods and other uses detailed hereinafter in relation to the direct administration of plasmid/viral vector prime boost vaccine schedules extends to, in a related aspect, the administration of two or more sequential doses of plasmid vector alone.
An "effective amount" means an amount necessary at least partly to attain the desired immune response, or to prevent or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual's immune system to stimulate T cells, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
Reference to "mammal" should be understood to include all mammals including primates (e.g. humans, monkeys), livestock animals (e.g. sheep, cows, horses, donkeys, goats, pigs), laboratory test animals (e.g. mice, rats, guinea pigs, rabbits, hamsters), companion animals (e.g. dogs, cats), and captive wild animals (e.g. kangaroos, deer, foxes). Preferably, said animal is a primate and even more preferably a human.
Administration of the subject vectors in the form of a pharmaceutical composition, may be performed by any convenient means. The vectors are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 μg to about 1 mg of construct may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. In a particularly preferred embodiment, the plasmid vector is administered at 1 mg/dose and the fowlpox virus vector is administered at 5 x IO7 pfu.
The constructs may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). Preferably, the subject constructs are administered intramuscularly and, to the extent that a mucosal immune response is sought, orally, intranasally, intrarectally or intravaginally. In this regard, administration of the vaccine to the mucosal surfaces is particularly desirable since HIV-1 is predominantly transmitted sexually.
A further aspect of the present invention relates to the use of the invention in relation to disease conditions. For example, the present invention is particularly useful in relation to the therapeutic or prophylactic treatment of HIV infection and HIV-induced AIDS. In a most preferred embodiment, the method of the present invention is applied in the context of inducing immunity to HIV in HIV negative individuals. In this regard, it should be understood that the method of the present invention is directed to inducing an immune response for the purpose of alleviating or preventing the onset of symptoms associated with viral infection, and in particular HIV infection. Reference herein to "symptoms" associated with viral infection should be understood to extend to both the infection itself as well as the physical and/or physiological consequences of such infection. Accordingly, another aspect of the present invention contemplates a method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which the viral vector is incorporates nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said virus, which antigens have been rendered substantially non- pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said virus.
More particularly, the present invention is directed to a method of therapeutically or prophylactically treating a mammal for a HIV and/or HIV-induced AIDS infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV-1, which antigens have been rendered substantially non-pathogenic, said one or more optimised CpG motifs; and (ii) one or more sequential doses of an effective amount of a recombinant fowlpox virus vector or functional derivative thereof into which fowlpox virus vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said HIV, which antigens have been rendered substantially non- pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said HIV or associated with said HIV-induced AIDS.
Preferably, said HIV is HIV-1, HIV-1 subtype AE, subtype B or subtype C.
Preferably said immune response is a Thl/CTL response.
Even more preferably said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env and/or Pol.
Still more preferably, said plasmid vector is a plasmid vector incorporating nucleic acid molecules encoding all, part or a modified form of Gag, Pol, RT, Tat, Vpu and/or Env and said recombinant fowlpox virus vector incorporates nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and/or Pol.
Most preferably said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
In another most preferred embodiment, a single dose recombinant viral vector is administered four weeks later than a single dose recombinant plasmid vector. In yet another most preferred embodiment, dual doses of recombinant plasmid vector are administered three weeks apart and dual doses of the recombinant viral vector are administered four weeks apart with the first viral recombinant vector dose being administered four weeks after the second recombinant plasmid vector dose is administered.
In still another preferred embodiment, dual doses of recombinant plasmid vector followed by a single dose of recombinant viral vector are administered four week apart.
A mammal undergoing treatment may be a human or animal in need of therapeutic or prophylactic treatment for a disease condition or a potential disease condition.
Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of onset of a particular condition. "Treatment" may also reduce the severity of an existing condition or the frequency of acute attacks (for example, reducing the severity of initial infection).
In accordance with these methods, the modulatory agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules, These molecules may be administered in any order.
The pharmaceutical compositions of the invention contain a pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, vector, vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention. In one embodiment of the invention, the effective amount of an agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest. In another embodiment of the invention, the effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent o symptoms) of the condition being treated. The effective amount of agent per unit dose depends, among other things, on the species of mammal inoculated, the body weight of the mammal and the chosen inoculation regimen, as is well known in the art. The dosage of the therapeutic agents which will be most suitable for prophylaxis or treatment will also vary with the form of administration, the particular agent chosen and the physiological characteristics of the particular patent under treatment. The dose is administered at least once. Subsequent doses may be administered as indicated.
To monitor the response of individuals administered the compositions of the invention, mRNA or protein expression levels may be determined. In many instances it will be sufficient to assess the expression level in serum or plasma obtained from such an individual. Decisions as to whether to administer another dose or to change the amount of the composition administered to the individual may be at least partially based on the expression levels.
The term "unit dose" as it pertains to the inocula refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of active material (e.g., nucleic acid, virus stock or host cell) calculated to produce the desired effect in association with the required diluent. The titers of the virus stocks to be administered to a cell or animal will depend on the application and on type of delivery (e.g., in vivo or ex vivo). The virus stocks can be concentrated using methods such as centrifugation. The titers to be administered ex vivo are preferably in the range of 0.001 to 1 infectious unit/cell. Another method of generating viral stocks is to cocultivate stable cell lines expressing the virus with the target cells. This method has been used to achieve better results when using traditional retroviral vectors because the cells can be infected over a longer period of time and they have the chance to be infected with multiple copies of the vector.
For in vivo administration of nucleic acid constructs, vectors, vector systems, virus stocks, or cells which have been transduced or transfected ex vivo, the dose is to be determined by dose escalation, with the upper dose being limited by the onset of unacceptable adverse effects. Preliminary starting doses may be extrapolated from experiments using lentiviral vectors in animal models, by methods known in the art, or may be extrapolated from comparisons with known retroviral (e.g., adenoviral) doses. Generally, small dosages will be used initially and, if necessary, will be increased until the optimum effect under the circumstances is reached. Exemplary dosages are within the range of 108 up to approximately 5 x 1015 particles.
Inocula are typically prepared as a solution in a physiologically acceptable carrier such as saline, phosphate-buffered saline and the like to form an aqueous pharmaceutical composition.
The agents of the invention are generally administered with a physiologically acceptable carrier or vehicle therefor. The physiologically acceptable carrier is one that does not cause an adverse physical reaction upon administration and one in which the nucleic acids are sufficiently soluble to retain their activity to deliver a pharmaceutically or therapeutically effective amount of the compound. The pharmaceutically or therapeutically effective amount and method of administration of an agent of the invention may vary based on the individual patient, the indication being treated and other criteria evident to one of ordinary skill in the art. A therapeutically effective amount of a nucleic acid of the invention is one sufficient to prevent, or attenuate the severity, extent or duration of the deleterious effects of the condition being treated without causing significant adverse side effects. The route(s) of administration useful in a particular application are apparent to one of ordinary skill in the art. Routes of administration of the agents of the invention include, but are not limited to, parenteral, and direct injection into an affected site. Parenteral routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal and subcutaneous. The route of administration of the agents of the invention is typically parenteral and is preferably intramuscular, subcutaneous, intradermal, intraocular, intracranial, intranasal, and the like. See, e.g. WO99/04026 for examples of formulations and routes of administration.
The present invention includes compositions of the agents described above, suitable for parenteral administration including, but not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for nasal, intravenous, intramuscular, intraperitoneal, subcutaneous or direct injection into a joint or other area.
In providing the agents of the present invention to a recipient mammal, preferably a human, the dosage administered will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history and the like.
In yet another aspect the present invention relates to the use of:
(i) a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non- pathogenic, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a mammalian disease condition characterised by infection with said virus.
Preferably, said virus is HIV and, even more preferably, HIV-1, HIV-1 subtype AE, subtype B or subtype C.
In one preferred embodiment said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated names encoding all, part or a modified form of Gag, RT, Pro, RNAseH, Tat, Rev and/or Env .
In another preferred embodiment said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and Env and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, Tat, Rev, Env and Pol.
Most preferably, said plasmid vector is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
The present invention should also be understood to extend to the vectors described herein and to vaccine compositions comprising said vectors.
Accordingly, in a related aspect the present invention is directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs. This aspect of the present invention is still further directed to a vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
Still another aspect of the present invention is directed to a nucleic acid construct or functional derivative thereof which construct comprises a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
In yet another aspect the present invention is directed to a nucleic acid construct comprising a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
Preferably, said virus is HIV and, even more preferably, HIV-1, HIV-1 subtype AE, subtype B or subtype C.
More preferably said recombinant plasmid vector incorporates nucleic acid molecules encoding Gag, RT, Pro, RNaseH, Rev, Tat, Vpu, Env and/or Nef and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
In another preferred embodiment said recombinant plasmid vector is a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of Gag, RT, Rev, Tat, Vpu and Env and said recombinant viral vector is a recombinant fowlpox virus vector into which is incorporated nucleic acid molecules encoding Gag, Tat, Rev, Env and Pol. Most preferably, said plasmid is pHIS-HIV-B or pHIS-HIV-AE and said fowlpox virus vector is rFPV-HIV-B or rFPV-HIV-AE.
Yet another aspect of the present invention is directed to compositions, in particular pharmaceutical compositions, comprising the nucleic acid constructs, plasmid vectors and/or viral vectors hereinbefore described.
In still another aspect, the present invention is directed to the vaccine and pharmaceutical composition, hereinbefore described, when used in the methods hereinbefore described.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
When the active ingredients are suitably protected they may be orally administered, for example for the purpose of inducing a mucosal immune response, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
Still another aspect of the present invention provides a method of inducing, enhancing or otherwise stimulating an immune response, in a mammal, to HIV said method comprising administering to said mammal an effective amount of a vaccine as hereinbefore defined in accordance with the methodology herein defined sufficient to induce, enhance or otherwise stimulate an immune response to one or more HIV antigens.
Preferably, said immune response correlates to the induction of long term immunity. As detailed hereinbefore, the method of the present invention is useful in the treatment and/or prophylaxis of HIV infection and HIV-induced AIDS. For example, the vaccine of the present invention may be administered to subjects known to be infected with HIV in order to induce an immune response against HIV thereby preventing the onset of AIDS. Alternatively, the method of the present invention may be used to reduce serum viral load, to alleviate AIDS symptoms or to induce immunity in mammals thought to be at risk of HIV infection.
The method of the present invention may be particularly useful either early in HIV infection to prevent the establishment of a viral reservoir or for a period after exposure to a possible source of HIV infection.
In accordance with the method of the present invention, the vaccines of the present invention may be coadministered with known antiviral compounds or molecules. Such compounds or molecules include, but are not limited to, reverse transcriptase inhibitors (for example, Zidovudine or 3TC) or protease inhibitors (for example, Indinavir). By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the vaccine and the known antiviral compound or molecules. The vaccine and the known antiviral compound or molecule may be administered in any order.
Further features of the present invention are more fully described in the following non- limiting examples. EXAMPLE 1 pHIS-HIV and rFPV Boost Vaccine Chemistry
The vaccines which are used in the phase I/IIa clinical trials consist of a DNA prime(pHIS- HIV-B or pHIS-HIV-AE) followed by a rFPV boost (rFPV-HIV-B or rFPV-HIV-AE). The DNA vaccines described in this example encode CpG immunostimulatory oligonucleotide sequences and both the DNA and rFPV vaccines express multiple HIV genes mutated to remove active functional sites of the HIV genes where these are deemed to be a safety concern.
Structure of the DNA vaccines
In designing the DNA vaccines, three key aspects were considered: 1) safety, 2) immunogenicity, and 3) gene expression. To make the DNA vaccines safe for human administration, the genes in the HIV-1 virus genome whose function posed a theoretical risk were either deleted or mutated. The designed mutations were mostly small deletions in sequences important for either the structural integrity or function of each gene. Two of the larger key genes known to be important for T cell mediated immunity are the gag and pol genes of HIV-1. As much of these two genes were incorporated as possible. To express these two genes from a DNA vaccine, rev and the rev responsive element (RRE, and RNA element encoded with the env gene) are required, so these were left intact in the constructs. The tαt and vpu genes, recently recognised as being potentially important T cell targets and closely associated with rev in the HIV-1 genome, were also included intact. The N-terminal 34 amino acids of the nef that contain immunodominant T-cell epitopes were also included. Below is a summary of the specific modifications made to the B subtype and AE sub-type HIV-1 genomes.
Construction of the pHIS-HW-B vaccine
The provirus DNA that was selected for the DNA construct pNL(AD8) was based on the well characterised B sub-type isolate pNL4.3, but has the env from the CCR5-tropic HIV- 1-ADA strain and was obtained from the NIH reagent repository. Approximately 65% of the provirus is included in the final DNA construct. After the HIV-1 genome was modified, it was inserted into the DNA vaccination vector pHIS-64. This kanamycin resistant plasmid vector contains a cytomegalovirus (CMV) promoter, the bovine growth hormone polyA signal and human optimised CpG immunostimulatory sequences.
In the first stage of pHIS-HIV-B construction the 5' and 3' halves of the pNL4.3 provirus were mutated separately. This was achieved by a mixture of PCR splicing by overlap extension with long oligonucleotides and standard cloning. The 3' end was constructed by first cloning the 3' half separately into a shuttle plasmid using convenient restriction enzyme sites. A short fragment was then PCR amplified and cloned back into the template plasmid to create the required deletion. The two modified HIV-1 fragments were then cloned stepwise into pHIS- 64. The 5' fragment was then further modified and inserted into a fowl pox virus vector as described below in the section entitled: Structure of the rFPV vaccines. A synthetic intron CMV splice donor + Ig variable region splice acceptor) (Eaton DL, Wood Wl, Eaton D, Hass PE, Hollingshead P, Wion K, Mather J, Lawn RM, Vehar GA and Gorman C. 1986. Biochemistry 25:8343-8347) was cloned immediately before the joined HIV sequences in pHIS-64 to increase expression of the HIV genes.
Specific Modifications to pHIS-HIV-B
The following modifications were made to the pHIS-HIV-B DNA vaccine:
• gag modifications to block gag mediated binding/packaging of the HIV RNA template ° deleted gag 392-395AAs "CFNC" (first zinc finger motif)
0 deleted gag 413-416AAs "CWKC" (second zinc finger motif)
• pol modifications (also vir and vpr deletion) to block RT, RNaseH and integrase activities deleted RT 65-74Ms "KKDSTKWRKL" (SEQ ID NO:2) ("thumb" region involved in dNTP phosphate binding) deleted RT 113-115Ms "DAY" (involved in dNTP phosphate binding) deleted RT 183-186Ms "YMDD" (critical for Mg2+ A and B site binding and 3' primer terminus binding) mutated Gln478 to Glu478 to inactivate RNase H large deletion from 30MS into integrase coding region to 87bp 5' of the tat start codon to remove integrase function (also removes vif 'and vpr)
• env truncation to block its effects on the T cell population through CD4 and CCR5 engagement and to further reduce the possibility of an infective particle formation.
° deleted 581 bp starting from the first BglH site in env (7031 in the pNL4.3 sequence) and replaced this fragment with a short piece of synthetic DNA containing stop codons in all three frames.
• røe/truncation (also removes all of the overlapping L TR sequences) to block immunomodulatory activities ° deleted all ne/after 34M
An illustration of the final construct is given in Figure 1.
Construction of the pHIS-HIV-AE vaccine
The provirus DNA that was selected for the AE sub-type DNA construct was the well characterised isolate p93TH253 obtained from NIH reagent repository. This is an early Thai isolate from 1993 and is reasonable representation of South East Asian AE strains. Approximately 65% of the provirus is included in the final DNA construct. After the HIV- 1 genome was modified, it was inserted into the DNA vector pHIS-54 (see Figure 2). This kanamycin resistant plasmid vector contains a CMV promoter, the bovine growth hormone polyA signal, gene and human optimised CpG immunostimulatory sequences. The DNA vaccine expresses the following HIV-1 genes; modified gag, modified RT, rev, tat, vpu, truncated env.
Specific Modifications to pHIS-HIV-AE
The following modifications were made to the pHIS-HIV-AE DNA vaccine:
• gag modifications to block gag mediated binding/packaging of the HIV RNA template ° deleted gag 392-395AAs "CFNC" (first zipper) ° deleted gag 413-416AAs "CWKC" (second zipper)
• pol modifications (and vifand vpr deletion) to block RT, RNase and integrase activities:
° deleted RT 65-74AAs "KKDSTKWRKL" ("thumb" region involved in dNTP phosphate binding) ° deleted RT 113-1 ISAAs "DAY" (involved in dNTP phosphate binding) ° deleted RT 183-186AAs "YMDD" (critical for Mg2+ A and 8 binding and 3' primer terminus binding)
° mutated Gln478 to Glu478 to inactivate RNase H
° large deletion from 30MS into integrase coding region to 87bp 5' of the tot start codon to remove integrase function (also removes vifand vpr)
• env modification to block its effects on the T cell population through CD4 engagement and further reduce possibility of an infective particle forming:
° deleted 581 bp starting from the first Bglll site in env (8909 in the p93TH253) so that the end of env was in frame 0 corrected the stop codon at 8036 so that the last 1/3 of env is expressed • nef truncation (also removes all of the overlapping LTR sequences) to block immunomodulatory activities
° deleted all nef after 34AA
In Figure 2, a plasmid map of the resultant pHIS-HIV-AE is given to illustrate the final construct.
Homology ofpHIS-HIV-AE topHIS-HIV-B
The pHIS-HIV-^E and the pHIS-HIV-B DNA vaccines share a high degree of sequence identity. They use the same vector, pHIS, that has the immuno-reactogenic human CpG sequences and have an almost identical construction except that the HIN-B env has a stop codon before the deletion of the middle third of the env nucleotide sequence, and HIV- AE does not. The consequence of this is that pHIS-HIV-B expresses only the first third of the Env protein, whereas pHIS-HIN- lE expresses the first and last third of the Env protein (Figure 3). The middle portion of the Env protein that contains the receptor-binding domains is deleted from BOTH of these constructs. The nucleotide sequence of the two HIV inserts share 85% nucleotide sequence identity, and about the same level of protein identity across the common expressed protein regions.
Full details of the structure of the DΝA pHIS-HIV-Bad pHIS-HIV-AΕ Sub-Type vaccines are provided in Figure 4.
Structure of the rFPV vaccines
The rFPV vaccines were constructed by the sequential insertion of the corresponding DΝA at sites in the viral genome of the parent FPV strain, FPV-M3. Below is a summary of the specific construction details for rFPV-HIV-B (containing gαg/pol (mutated)) and rFPV- HIV-AΕ (containing gαg/pol (mutated), env (mutated) and tαt/rev) vaccines. Construction of the rFPV-HIV-B vaccine, a rFPV expressing mutated gag/pol from HIV- 1NL43 was constructed using long range PCR to amplify the gag/pol (m) from the pHIS- HIV-B vaccine with the addition of pox virus specific sequences at each end. These sequences are described in Figure 5 and Figure 6.
PCR product (3533bp) was cloned into pGEM- Teasy to make pGT -gag/pol (mutated) and the sequence of the entire insert confirmed.
The primers used added the rFPV early/late promoter and an early transcription terminator to the genes during the PCR. In addition, Xhol restriction endonuclease sites were added to the ends of the promoter gag/pol PCR product. The promoter gag/pol PCR product (as a Xhol fragment) was then cloned into plasmid pKGlO (Sail digested) which contains flanking sequences of FPV F6, 7, 9 gene regions. The FPV flanking sequences were then used to recombine the plasmid with fowlpox strain FPV M3.
The pKGlOa vector used for construction of recombinant fowl pox viruses by transient dominant selection was constructed by a series of manipulations and contains:
° 1485bp of FPV sequence PCR amplified from viral DNA (FPV-M3) and including ORFs F6, F7 and F9 [GenBank accession number M17418 nucleotides 13 to 1297]
° 34bp of sequence inserted between residues 835 and 836, as described in Figure 7. ° a cassette containing two markers under pox virus promoters: Ecog t under the vaccinia virus P .5 and β-galactosidase under fowlpox P
° plasmid sequences derived from pBlueScript including an E. coli origin of replication and an ampicillin resistance gene.
A Xhol fragment from pGT -gag/pol (mutated) was inserted at the Sail site in pKGlOa and the resultant plasmid pKG-gag/pol (mutated) is shown in Figure 8(b).
Recombinants were selected on the basis of transient dominant co-expression of E. coli gpt and beta-galactosidase genes. The final recombinant was plaque purified and stocks of rFPV-HIV-B grown in chicken embryo skin cells. The transient dominant selection results in the loss of the marker genes and the rFPV-HIV-B only carries the FPV promoter gag/pol (m) genes inserted within the FPV 6, 7, 9 region of the genome.
The predicted genome configuration with insertion of the promoter-HIVgtfg/^ø/-terminator cassette, was confirmed by PCR and sequencing of a 4,626bp fragment from rFPV-HIV-B using primers within the F6 and F9 ORFs of the fowlpox genome. PCR using rFPV-HIV-B DNA provided no evidence of a fragment at 1071 bp as was generated by wild type (FPV- M3) DNA used as a control.
Construction of the rFPV-HIV-AE vaccine
The rFPV-HIV-^E vaccine (denoted FPV-117 during construction) was constructed by the sequential insertion of DNA at 3 sites in the viral genome of FPV-M3: F6,7,9 using transfer plasmid pKGlOa, REV site using ρCH34, and TK ORF X using pAF09 (refer to Figure 9).
In each case HIV sequences were inserted into the transfer plasmid as a promoter-gene- terminator cassette where the promoter was fowlpox PE/L and the terminator was T5NT. HIV AEe77v(m) was inserted into FPV-M3 at the REV site using pCH34-AEe«v(m) to produce construct FPV-090. HIV AEgagpol(m) was inserted using pK.G\ -AEgagpol( ) into the F6,7,9 site of both FPV-M3 and FPV-090 to generate FPV-091 and FPV-092, respectively. Finally, FPV-092 was used as a parent for the insertion of a synthetic gene encoding AE tat and rev linked by 5 His residues (AEtatHrev) at the intergenic site between TK and ORF X via transfer plasmid pAF09 to generate FPV-117 (Figure 8). The first and second insertions were carried out using transient dominant selection (markers Ecogρt/j3-gal are lost from the recombinant virus) while the third insertion used dominant selection, with retention of the markers in the final construct. The sequences for AEgagpol(m) and AEe«v(m) were those present in the DNA vaccine construct pHIS-HIV- AE as noted above, with additional modifications to AEe«v(m) to remove a pox virus transcription termination sequence without changes to the amino acid coding. Tat and rev sequences are present in the DNA vaccine as alternate open reading frames to env. The absence of splicing in the pox virus system and the need to avoid tandem repeats which would be unstable, required the construction of a synthetic fusion gene to allow expression of both tat and rev in the rFPV. The sequence of HIV components of all transfer plasmids was confirmed prior to virus construction.
Manufacturing
The vaccines were manufactured according to the standards of Good Manufacturing Practice, as defined by the Australian Code of Good Manufacturing Practice for Therapeutic Goods (1990) (rFPV) and US FDA's 21 CFR cGMP regulations and the EU directive 91/356/EEC (DNA).
pHIS-HIV Vaccines
The pHIS-HIN-B vaccine was manufactured by Qiagen within their GMP-facility (Strathmann Biotech GmbH, Hannover, Germany).
A research cell bank (RCB) was manufactured under Good Laboratory Practice (GLP) conditions. Testing and characterisation of the RCB included:
• Gene expression by Western Blot and ELISA of p24 - confirmed gene expression
• Expression of mutated RT gene - no RT expression noted
• Expression of tat - expression confirmed • RΝA encapsidation - vaccine packaged HIN-1 RΝA less efficiently than wild type virion
• RT activity - vaccine produces a defective RT enzyme that can not support viral replication, recombination or mutation
• Replication competency - vaccine does not yield replication competent virus From this research cell bank, a master and working cell bank were manufactured under GMP conditions from which the pHIS-HIV-B vaccine was produced.
Quality control testing of the pHIS-HIN-B vaccine final product included:
• Sterility testing according to the EP 20024th Ed. and USP 25 - Complied
• Presence of E. coli host DΝA, endotoxin (according to EP 2002 4th Ed.), protein and RΝA - conformed with specifications
• Purity testing by UV photometry - conformed with specification • Ratio oc/ccc topoisomers - result recorded
• Sequencing of Plasmid (MCB and final vaccine) - identical to research cell bank
• Appearance - conformed with specification
Following manufacture of the GMP material, the pHIS-HIV-B vaccine was placed on stability testing. The stability protocol covers both the "long term" stability of the vaccine at the indicated temperature (-80°C) and then stability at accelerated conditions (-20°C, and +5°C :t 3°C).
Prior to manufacture by Qiagen, pHIS-HIV-B was made as follows.
In the first stage of the DΝA construction the 5' and 3' halves of the pΝL4-3 provirus were mutated separately. This was achieved by a mixture of PCR splicing by overlap extension with long oligonucleotides and standard cloning. Figure 10 is a schematic outline of the strategy used for the 5' half. The 3' end was constructed by first cloning the 3' half separately into a shuttle plasmid using convenient restriction enzyme sites. A short fragment was then PCR amplified and cloned back into the template plasmid to create the required deletion. The two modified HIV-1 fragments were then cloned stepwise into pHIS-HIVB. The 5' fragment was then further modified and inserted into a Fowlpox virus vector as described elsewhere. Also a synthetic intron (CMV splice donor + Ig variable region splice acceptor, see Eaton et al (1986) Biochemistry 25 p8343) was cloned immediately before the joined HIV sequences in pHIS-HIVB to create a second DNA vaccine candidate with increased expression of the HIV genes and improved plasmid yield during manufacture. Introducing a synthetic intron increases plasmid yield from bacteria because it separates a cryptic bacterial promoter in the CMV promoter from the slightly toxic HIV sequences. Figures 11 and 12 demonstrate the DNA vaccine candidate constructed pHIS-HIVB followed by its sequence. Testing of this plasmid has involved immuno-fluorescence assays (IF As) p24 antigen assays, immunoblotting for Gag and RT and functional assays for Tat and Rev to analyse in vitro gene expression. The pHIS-HIVB plasmid expresses high levels of Gag and combined with its greater yield during bacterial amplification has been selected to be the DΝA vaccine component of the B clade HIV vaccine.
rFPV-HIV Vaccines
The rFPV-HIV-B vaccine was manufactured in a new clean room facility dedicated to rFPV vaccine manufacture.
The recombinant virus was amplified in the presence of mycophenolic acid selection. Plaquing was then carried out without mycophenolic acid. PCR with primers complementary to sequences flanking the insertion site were used to confirm the absence of wild type parent virus.
• Subsequent to isolation, the recombinant virus was amplified. The master seed stock (MSS) was then prepared from the recombinant virus under GLP conditions. Testing and characterisation of the MSS included: • Sequencing of the inserts by PCR - confirmed sequence of the HIV gag/pol (m) insert
• Gene expression by immunofluorescence staining with a monoclonal antibody to p24 of gag and by Western Blotting - confirmed gene expression
• Adventitious agent screen on 143B cells - free of vaccinia virus
• RΝA encapsidation - vaccine packaged HIV-1 RΝA less efficiently than empty vector and non-mutated vaccine
• RT activity - vaccine associated with high levels of RT activity due to host cell The working seed stock (WSS) was prepared from the MSS under GMP conditions. The WSS was screened for bacteria, yeasts and mould by growth promotion studies after direct inoculation of growth media.
The rFPV-HIV-B vaccine was manufactured from the WSS under GMP conditions. Quality control testing of the rFPV-HIV-B vaccine final product included:
Sterility testing according to the EP 2002 4th Ed. - Complied with specification • Adventitious agent screen on 143B cells - free of vaccinia virus
Mycoplasma testing - no mycoplasma isolated
Gene expression by Western Blot - confirmed protein expression
Titre by plaque assay - complied with specification pH - complied with specification • Extractable volume - complied with specification
Appearance - complied with specification
Sequencing of the inserts by PCR - vaccine sequence is identical to the MSS
Strain verification by PCR - virus used to produce the vaccine is identical to the MSS
Following manufacture of the GMP material, the rFPV-HIV-B vaccine was placed on stability testing. The stability protocol covers both the "long term" stability of the vaccine at the indicated temperature (-80°C) and then stability at accelerated conditions (-20°C, 2-8°C, and l8°C).
Presentation
pHIS-HIV Vaccines
The DNA vaccines are presented in a 3mL glass vial sealed with a rubber stopper and over- sealed with a plastic flip-off top. Each 3mL vial in the treatment group contains either 1 mg/niL of DNA or 1.5 mg/mL in a volume of 1.2mL of 0.9% saline. The solution is colourless and transparent.
The DNA matched-placebo vaccines containing diluent (0.9% saline) are also presented in a 3mL glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top.
rFPV-HIV Vaccines
The rFPV vaccines are presented in a 2mL amber glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top. Each contains either 5x107 pfu/mL or 2x108 of rFPV in a volume of 1.2 mL of Phosphate Buffered Saline (PBS) 10% glycerol.
The FPV matched-placebo vaccines containing diluent (PBS 10% glycerol) are also presented in a 2mL amber glass vial sealed with a rubber stopper and over-sealed with a plastic flip-off top .
Finished Product Specifications
pHIS-HIV and Placebo Finished Product Specifications
The finished product specifications for the DNA vaccines (and matched placebo) are described in Table 1 below:
Table 1 : pHIS-HIV Finished Product Specifications
Figure imgf000067_0001
N/A = Not Applicable
rFP V and Placebo Final Product Specifications
The finished product specifications for the rFPV vaccines (and matched placebo) are described in Table 2 below.
Table 2: FPV-HIV Product Specifications
Figure imgf000067_0002
Stability and Shelf Life
The pHIS-HIV and rFPV vaccines are stable when stored at -80°C for up to 18 and 9 months, respectively. The pHIS-HIV vaccine has been stored for up to 18 months and the integrity of the vaccine confirmed by agarose gel electrophoresis and spectrophotometric analysis.
Storage Conditions
The DNA and rFPV vaccines and matched placebos must be stored at -80°C. Vials must be defrosted immediately prior to use. Vials may be stored for up to 72 hours at -20°C without loss of stability.
EXAMPLE 2 Manufacture of the vaccines
pHIS-HIV Vaccine Manufacture
i. E. coli strain DH5α was transformed with pHIS-HIV-B according to standard molecular cloning methods and a subsequent production clone was selected. A master and working cell bank were prepared.
ii. An inoculum was prepared using the working cell bank. A shake flask containing growth media (soya peptone, yeast extract, NaCl, K2HPO4 (3H2O), KH2PO4, MgSO4 (7H2O), pH to 7.0 with 1 M NaOH) was inoculated and incubated at 37°C under shaking to the desired optical density (OD).
iii. The entire content of the flask was then used to inoculate a 200L fermentor containing medium. Batch fermentation of the inoculum was performed at 37°C. Dissolved oxygen, agitation, pH and aeration were controlled during fermentation. When fermentation broth reached the desired OD, the cells were harvested by batch centrifugation.
iv. Cells were suspended in 50mM Tris, lOmM EDT A buffer (pH 8.0) containing 50μG/ml RNase and lysed by addition of 200mM NaOH containing 1 % SDS. The lysate was neutralised by the addition of 3M potassium acetate (pH5.5) and the crude lysate filtered through a QIAfilter cartridge (0.2μm).
v. An endotoxin removal (ER) buffer (which does not contain animal derived ingredients) was added to the cleared lysate to remove lipo-polysaccharides. The cleared lysate and ER buffer mix was then loaded onto an anion exchange resin column which had been pre-equilibrated with a QBT buffer (750mM NaCl, pH 7.0,
50mM MOPS, 15% isopropanol, 0.15% Triton X-100).
vi. The column was washed with QC buffer (IM NaCl, pH 7.0, 50mM MOPS, 15% isopropanol).
vii. The pHIS-HIV-B was eluted from the column in a shallow gradient using QN buffer (1.6M NaCl, pH 7.0, 50mM MOPS, 15% isopropanol) and the plasmid DNA precipitated by addition of 100% (v/v) isopropanol. The precipitate was collected by centrifugation, the pellet washed with 70% (v/v) ethanol, centrifuged and the plasmid pellet air dried.
viii. The washed precipitate containing the pHIS-HIV-B was resuspended in 0.9% saline to the desired concentration (1 mg/ml) and then aseptically filtered through a
0.2μm filter within a class A area with class B background.
ix. The filtered resuspended pHIS-HIV-B was aliquoted at 1.2mls into 3ml glass vials and sealed with a rubber stopper and flip-off top. rFPV-HIV Vaccine Manufacture
a) Step 1 : Primary Cell Preparation i. SPF chicken eggs were transferred into a sterile class 3.5 area and the embryos removed and placed in a bottle with phosphate-buffered saline
(PBS) pre-warmed to 37°C. ii. After collecting approximately 15 embryos per bottle, the PBS was poured off and the embryos washed again with another volume of PBS. iii. The embryos were washed again with Hanks Balanced Salt Solution (BSS). iv. A working enzyme solution, containing Hanks BSS and the enzyme collagenase reconstituted in PBS, was added to each bottle with the embryos. The solution was stirred at 37°C to allow the collagenase to digest the embryo tissue v. Growth media (GM) was prepared using specified quantities of foetal bovine serum (FBS), HEPES, L-Glutamine, Streptomycin and Minimum
Essential Medium (MEM), vi. After stirring, the supernatant was filtered through a gauze topped glass funnel and FBS added to the cell suspension to stop further enzyme action, vii. The supernatant was poured into centrifuge tubes and spun down into a pellet at room temperature for 5 to 10 minutes at 2000rpm. viii. The cell pellet was resuspended in 10ml of GM and a cell count performed, ix. Roller bottles were seeded with approximately 1 xlO8 cells in 200-250ml of
GM and then incubated at 37°C. x. The media was replaced the next day with fresh GM.
b) Step 2: First Passage Cells i. After 3 to 5 days the cells were passaged, ii. The media was poured off and the cells washed with PBS. iii. Trypsin PBS at 37°C was added and the bottle placed on the roller unit for 5 minutes. iv. The roller bottles were swirled to dislodge the remaining cells. v. FBS was added to the cell suspension to stop the enzyme action. vi. The cell suspension was poured into centrifuge tubes and spun at 2000rpm, room temperature for 5 to 10 minutes, vii. The cell pellet was then resuspended in 10ml of GM and a cell count performed. viii. Roller bottles were seeded with approximately 1 xl 08 cells per roller, ix. The monolayer became confluent within 3 to 5 days.
c) Step 3: Cell Infection i. Using stock virus of known titre (WSS), a specified amount of virus, based on the multiplicity of infection (moi), was added to trypsin and incubated at 37°C for 30 minutes, ii. GM containing FBS was added to stop the action of the trypsin. iii. The amount of virus required to infect the roller bottles was calculated. iv. The media was poured off the cells and the cells washed with PBS. v. The calculated amount of virus, in PBS, was added to the roller bottles and incubated at 37°C for 60 minutes, vi. GM without FBS or antibiotics was then added to the roller bottles and the bottles incubated at 37°C for a further 3 to 5 days until cytopathic effect (cpe) became visible.
d) Step 4: Virus Harvest i. The roller bottles were swirled to dislodge the cell monolayer. ii. Keeping the virus from each roller bottle separate, the viral supernatant containing the cells was poured into centrifuge tubes and spun at 3,000rpm for 20 minutes at 4°C. iii. After centrifugation the supernatant was poured off and the pellet resuspended in PBS. iv. The centrifuge step was repeated and the PBS poured off. v. The pellets were resuspended in lOmls of injectable grade PBS 10%
Glycerol. vi. Each 10ml amount was sonicated (6x15 second bursts at 450 watts) on sterile cooled water, vii. After sonication, the 10ml volumes were pooled and the virus concentrate suspension securely sealed within centrifuge tubes, fully labelled with construct details and batch number and stored at -80°C until the viral titre was determined.
Fowlpox vaccine: Dispensing, Labelling and Packaging
Vaccine Dispensing: i. The required final concentration of the vaccine was 5xl07 pfu/ml for dispensing and was calculated as per the instructions in the master batch record, ii. IV bags containing PBS with 10% glycerol were prepared and sterilised at 121 °C for 30 minutes and then sterility tested. iii. Using a sterile syringe a pre-determined volume of viral titre was injected via the IV bag injection port into the IV bag. iv. An IV infusion set was connected to the IV bag and a syringe filler assembled aseptically. v. In-process testing of fill volume (non destructive and individual weight) and visual inspection for particulates was carried out during the entire filling process, vi. 1.2ml of vaccine was dispensed into 2ml vials loaded into filling trays, vii. Upon completion of filling and inserting stoppers for each tray, FOT's were placed on the stoppers and crimped. viii. Samples from each tray were placed into a plastic sample bag and reserved for sterility testing. All samples reserved for sterility testing were placed at - 80°C. ix. Any units containing solution on the outside of the vial were rejected, as were any units containing defective sealing or incorrectly crimped FOTs. x. The total number of filled vials was calculated and the quantity of vaccine remaining after completion of the dispensing operation reconciled, xi. All trays of vials, stoppers and FOTs transferred for use in the batch were reconciled.
Labelling, Packaging and Quality Control: i. A randomisation code for 30 patients will be pre-prepared and attached to the batch record, ii. For each label the Label Approval Form was completed prior to printing. iii. A sample of the approved labels was attached to the batch record at each section, iv. In-process checks (IPC) were performed in the packaging and labelling area for weight of each vial (>5.8g) and appearance (free from foreign particulate). v. Wearing gloves, the exterior of all vials was wiped with 70% alcohol prior to labelling, vi. Batch documents together with Final Product Results were submitted to
IDT Quality Assurance Department for batch record auditing and subsequent batch release.
f) Placebo Dispensing, Labelling and Packaging
Placebo Dispensing: i. Sterile phosphate buffered saline solution containing 10% glycerol (v/v) in an IV bag was dispensed as the placebo. ii. An IV infusion set was connected to the IV bag and a syringe filler assembled aseptically. iii. 1.2ml of placebo was dispensed into 2ml vials. A stopper was placed on top of each vial and a flip off top (FOTs) placed onto the rubber stopper and crimped. The rest of the dispensing process for the placebo was the same as that described for the fowl pox batch steps v - xi.
Labelling, Packaging and Quality Control of the placebo batch was the same as that described above for the fowl pox batch.
EXAMPLE 3 pHIS-HIV prime and rFPV boost vaccine
Preclinical Testing
The batches of pHIS-HIV and rFPV vaccines were assessed for safety and efficacy using in vitro and in vivo techniques. Detailed below is a summary of the testing performed on each vaccine.
In Vitro Safety Assessment
DNA Vaccines 4.1.1.1 pHIS-HIV-B vaccine
(a) RNA encapsidation
To confirm the effectiveness of the mutation designed to reduce packaging of RNA into pseudoviral particles, the pHIS-HIV-B vaccine was compared to a wild type HIV-1 virion, a nucleocapsid reference mutant and a RNA packaging site mutant (both mutants were replication defective). Results of the modified HIV-1 "QT NucliSens™" quantitative RNA amplification assay demonstrated that the pHIS-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the wild type virion. RNA slot blot hybridisation demonstrated that the pHIS-HIV-B vaccine packaged RNA to levels that were equal to the nucleocapsid reference mutant and RNA packaging site mutant. The residual levels of RNA reflect the high cellular levels of HIV RNA relative to other RNA during these DNA-transfection experiments. Thus, the mutations introduced into the vaccine significantly decreased the potential for packaging and transmission of HIV genes. (b) Reverse transcriptase (RT) activity
To confirm the effectiveness of the mutations designed to inactivate the RT enzyme, the pHIS-HIV-B vaccine was compared to a reference RT mutant and a nucleocapsid mutant (that has defective endogenous RT activity) in different assays. Results of the "conventional" RT assay demonstrated that:
i) the HIV pseudoviral particles produced by the pHIS-HIV-B vaccine did not have any RT activity above background ii) the absence of RT activity was the same as the reference RT mutant and the nucleocapsid mutant.
Results of a highly sensitive and quantitative TM-PERT assay demonstrated that:
i) the HIV pseudoviral particles produced by the pHIS-HIV-B vaccine do not have any RT activity, ii) this activity was considerably below that of the reference RT mutant which was found to have low level activity with this sensitive assay.
Thus the mutations introduced into the vaccine produced a defective RT enzyme that would not support viral replication, recombination or mutation.
(c) Lack of replication competency
To assess the replication competency of the virus-like particles derived from the vaccine, several analyses were performed. Results of these studies confirmed that:
i) HIV pseudoviral particles that assemble from the modified gag, modified RT, Pro, rev, tat, vpu and truncated env expressed by the pHIS-HIV-B vaccine were not able to replicate in HIV susceptible cells. ii) Cell free pseudoviral particles derived from the pHIS-HIV-B vaccine were not able to replicate in pooled primary human peripheral blood mononuclear cells from HIV- negative, but otherwise susceptible donors. iii) Direct introduction and expression of the pHIS-HIV-B plasmid in an HIV- susceptible T-cell line did not initiate any infection with a replication competent virus expressing HIV p24 antigen. iv) a stringent method of initiating infection was employed where HIV- susceptible T- cells were co-cultivated with HeLa cells expressing extremely high levels of HIV pseudoviral particles following transfection with the pHIS-HIV-B plasmid. Even using this technique no replication and expansion of HIV was detected, using the sensitive p24 assay.
Based on these results, it was concluded that the pHIS-HIV-B vaccine does not yield replication competent virus. There was no evidence of genes or endogenous retroviral elements present in the human cells repairing the defects engineered into the pHIS-HIV-B plasmid. Thus, these data support the view that the replication defects engineered into the pHIS-HIV-B vaccine have comprehensively debilitated replication competence.
pHIS-HIV-AE vaccine
(a) RNA encapsidation
The pHIS-HIV-AE vaccine was compared to a wild type HIV-1 clade B virion, a nucleocapsid reference mutant and a RNA packaging site mutant (both mutants were replication defunct). Results of QT NucliSens™ quantitative RNA amplification assay demonstrated that the pHIS- nucleocapsid HIV-AE vaccine and pHIS-HIV-B vaccine pseudoviral particles packaged HIV- 1 RNA at a similar rate and that this was seven-fold lower than that of the HIV-1 B-clade wild type virion. The residual levels of RNA reflect the high cellular levels of HIV RNA relative to other RNA during these DNA-transfection experiments. However, since the RNA packaged with pseudoviral particles is replication incompetent, it is considered that this does not pose a safety risk. (b) Reverse transcriptase (RT) activity
To confirm the effectiveness of the mutation designed to eliminate RT activity, the pHIS- HIV- AE vaccine was compared to a reference RT mutant and a nucleocapsid mutant (that has defective endogenous RT activity) in different assays.
Results of the "conventional" RT assay demonstrated that:
i) the HIV pseudoviral particles produced by the pHIS-HIV-AE vaccine did not have any RT activity above background ii) the absence of RT activity was the same as the reference RT mutant and the nucleocapsid mutant.
Results of a highly sensitive and quantitative TM-PERT assay demonstrated that:
i) the HIV pseudoviral particles produced by the pHIS-HIV-AE vaccine do not have any RT activity ii) this activity was considerably below that of the reference RT mutant which was found to have low level activity with this sensitive assay.
Thus the mutations introduced into the vaccine produced a defective RT enzyme that would not support viral replication, recombination or mutation.
(c) Lack of replication competency
To assess the replication competency of the virus-like particles derived from the vaccine several analyses were performed. Results of these studies confirmed that: i) HIV pseudoviral particles that assemble from the modified gag, modified
RT, Pro, rev, tat, vpu and truncated env expressed by the pHIS-HIV-AE vaccine were not able to replicate in HIV susceptible cells, ii) Cell free pseudoviral particles derived from the pHIS-HIV-AE vaccine were not able to replicate in pooled primary human peripheral blood mononuclear cells from HIN- negative, but otherwise susceptible donors, iii) Direct introduction and expression of the pHIS-HIV-AE plasmid in an HIV- susceptible T-cell line did not initiate any infection with a replication competent virus expressing HIV p24 antigen. iv) a stringent method of initiating infection was employed where HIN- susceptible T- cells were co-cultivated with HeLa cells expressing extremely high levels of HIV pseudoviral particles following transfection with the pHIS-HIV-AE plasmid. Even using this technique no replication and expansion of HIN was detected, using the sensitive p24 assay.
Based on these results, it was concluded that the pHIS-HIN-AE vaccine does not yield replication competent virus. There was no evidence of genes or endogenous retroviral elements present in the human cells repairing the defects engineered into the pHIS-HIV- AE plasmid. Thus, these data support the view that the replication defects engineered into the pHIS-HIV-B vaccine have comprehensively debilitated replication competence.
rFPV Vaccines
(a) rFPV-HIV-B vaccine
• RNA encapsidation
To confirm the effectiveness of mutations designed to reduce RΝA packaging, the rFP V- HIN- B vaccine was compared to an empty FPV vector and to a FPV vaccine with non- mutated gag. Results of the modified HIV-1 QT NucliSens™ quantitative RNA amplification assay demonstrated that the rFPV-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the empty vector and the gag non- mutated vaccine. Thus the mutations introduced into the vaccine function to significantly increase the safety profile of the FPV vector.
• Reverse transcriptase (RT) activity
Results of a "conventional" RT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine do not have any RT activity above background. Results of a highly sensitive and quantitative TM-PERT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine had relatively high levels of RT activity, but that this was due to RT derived from the chicken epithelial cells used to manufacture the vaccine. The presence of chicken cell derived RT is well known and is not considered a safety risk in human vaccines due to the lack of pathogenesis from other widely used vaccines (measles, mumps, rubella and yellow fever) also prepared in chicken cell substrates that share the presence of the endogenous chicken retroviral RT.
(b) rFP V-HIV-AE vaccine
• RNA encapsidation
To confirm the effectiveness of mutations designed to reduce RNA packaging, the rFPV- HIV- AE vaccine was compared to an empty FPV vector and to a FPV vaccine with non- mutated gag. Results of the modified HIV-1 QT NucliSens™ quantitative RNA amplification assay demonstrated that the rFPV-HIV-B vaccine pseudoviral particles packaged HIV-1 RNA 10-fold less efficiently than the empty vector and the gag non- mutated vaccine. Thus the mutations introduced into the vaccine function to significantly increase the safety profile of the FPV vector.
Reverse transcriptase (RT) activity Results of a "conventional" RT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine do not have any RT activity above background. Results of a highly sensitive and quantitative TM-PERT assay demonstrated that the HIV pseudoviral particles produced by the rFPV-HIV-B vaccine had relatively high levels of RT activity, but that this was due to RT derived from the chicken epithelial cells used to manufacture the vaccine. The presence of chicken cell derived RT is well known and is not considered a safety risk in human vaccines due to the lack of pathogenesis from other widely used vaccines (measles, mumps, rubella and yellow fever) also prepared in chicken cell substrates that share the presence of the RT.
In Vitro Characterisation
DNA Vaccines
(a) pHIS-HIV-B vaccine
• p24 gag Expression
Transfection of adherent cells was used to determine the in vitro expression of gene inserts. Bright specific cytoplasmic immunofluorescence was observed in 90-100% of the transiently pHIS-HIN-B transfected 293 T cells, indicating the expression of p24 gag. The negative controls showed no fluorescence.
• ELISA capture assay for p24 gag expression
To determine the level of p24 expression in transiently transfected cells, a p24 ELISA capture assay (Innotest™ HIN antigen mAb, Innogenetics ELISA) was used. High levels of p24 were expressed by the transiently transfected "293" T cells. • Western Immunoblot analysis for HIV-1 gag expression
gag expression was assessed using a commercial HIV-1 gag antigen. Western immunoblot analysis of the proteins produced in pHIS-HIV-B transfected 293 T cells showed three major bands present; 1) the precursor gag protein at p55, 2) the p39 fragment of gag, and 3) the core protein of gag, p24.
• Western Immunoblot for HIV-1 gene expression using HIV-1 positive serum
Expression of multiple HIV-1 gene products was performed in comparison to other pHIS- HIV- B plasmids. The Western Immunoblot profile of pHIS-HIV-B in cell lysates using HIV positive patient serum confirms high level expression of various HIV-1 p55 gag and the processed form p39, p24 and pl7. In the pelleted supernatant from pHIS-HIV-B transfected cells, only the p55 gag product is detected.
• Expression of the mutated RT gene
To assess expression of the mutated, non-functional, HIV-1 RT (pol) gene by the pHIS- HIV-B vaccine, a Western Immunoblot was performed on cell lysates using an RT - specific monoclonal antibody obtained from the NIH AIDS reagent repository. Control mutants were also used. The results showed two bands of RT expression from the pHIS- HIV-B vaccine confirming expression of RT proteins. These bands were at a slightly lower molecular weight compared with the wild type HIV-1 pNL(AD8), consistent with the mutations in the RT genes which remove the functional activity. The mock lanes and the pCMV-dpol plasmid, which has a complete deletion of the RT gene, showed no RT expression. • pHIS-HIV-B concentration
The concentration (mg pHIS-HIV-B/mL) of the final filled product was determined by direct UV photometry. The absorption of the nucleotide bases at 260nm was used to quantitate the pHIS-HIV-B in solution. Due to the specific extinction coefficient of double stranded pHIS-HIV-B, a plasmid preparation with absorption of 1 corresponds to a concentration of 50 μg/mL. The concentration of the pHIS-HIV-B was shown to be 0.95μg/mL.
• Sequencing
Sequencing of pHIS-HIV-B showed it to be 100% identical to the research and master cell bank.
• Expression of tat
To assess expression of functional tat by the pHIS-HIV-B vaccine, tat-transactivation function upon an L-TR-enhanced green fluorescent protein (LTR-EGFP) reporter plasmid, was used as the method to quantify the expression of tat protein by the vaccine. Results of the assay demonstrated that the pHIS-HIV-B vaccine expresses functional tat protein with equal efficiency to a wild type proviral plasmid that is known to express tat
(b) pHIS-HIV-AE vaccine
• Expression of 'the modified HIV-1 genes
Transfection of adherent cells was used to determine the in vitro expression of gene inserts. Bright specific cytoplasmic immunofluorescence was observed in 90-100%) of the transiently pHIS-HIV-AE transfected "293" T cells. The negative controls showed no fluorescence. • ELISA capture assay for p24 gag expression
Elisa capture was used to determine the level of ρ24 expression in transiently transfected cells. High levels of p24 were expressed by the transiently transfected 293 T cells and at similar levels to the pHIS-HIV-B vaccine.
• Western Immunoblot analysis for HIV-1 gag expression
A commercial HIV-1 gag antigen was used to assess HIV-1 gag expression. The Western immunoblot analysis of the proteins produced in pHIS-HIV-AE transfected 293 T cells showed three major bands present; 1) the precursor gag protein at p55, 2) the p39 fragment of gag, and 3) the core protein of gag, p24.
• Expression of the mutated RT gene
To assess expression of the mutated, non-functional, HIV-1 RT (pol) gene by the pHIS- HIV-B vaccine, a Western Immunoblot is be performed on cell lysates using an RT - specific monoclonal antibody obtained from the NIH AIDS reagent repository. Control mutants are also used.
• pHIS-HI V-AE concentration
The concentration (mg pHIS-HIV-AE/mL) of the final filled product is determined by direct UV photometry.
• Sequencing
Sequencing of pHIS-HIV-AE is performed • Expression of tat
To assess expression of functional tat by the pHIS-HIV-AE vaccine, tat-transactivation function upon an L-TR-enhanced green fluorescent protein (LTR-EGFP) reporter plasmid, is used as the method to quantify the expression of tat protein by the vaccine.
rFPV Vaccines
(a) rFPV-HIV-B vaccine
Virus titration
A plaque assay was performed to determine the final concentration of the rFPV-HIV-B vaccine. The titre of the rFPV-HIV-B vaccine (batch no. V921105.1) was shown to be 7.57 log10 pfu/mL. The control reference virus, which has a specification of 7.30 log10 pfu/mL + 0.5 logio, was assayed concurrently and was shown to be within specification. Therefore, the results of the titration are considered to be valid.
Expression of gag/pol
Western Blot analysis of rFPV-HIV-B vaccine was performed to confirm expression of the gag protein by the vaccine. The western blot analysis demonstrated expression of HIV-1 gag protein, as indicated by the presence of the 55kDa precursor protein. It was also confirmed that protein expression had not changed during the manufacturing process from the initial master seed stock through to the working seed stock and batch. Western Blot analysis was also used to demonstrate pol protein expression in human HeLa cells infected with the rFPV- HIV-B vaccine. The expression of pol from the rFPV-HIV-B vaccine was equally efficient as that produced from a FPV vaccine with no safety mutations. • Strain verification
PCR was performed on rFPV-HIV-B to confirm the genome arrangement at 3 sites in the viral genome. The PCR products generated demonstrated;
a) the presence of an insertion at F6, 7, 9 sites in the viral genome, b) the inserted pHIS-HIV-B contains HIV-1 gag and pol sequences, c) at 2 sites not involved in the generation of the recombinant virus, the rFPV- HIV-B vaccine is indistinguishable from the wild-type virus, d) rFPV-HIV-B vaccine is indistinguishable from the virus used to generate the master seed stock.
• Sequence of the HIV inserts
The predicted genome configuration with insertion of the promoter-HIVg g o -terminator cassette, was confirmed by PCR and sequencing of a 4,626bp fragment from rFPV-HIV-B using primers within the F6 and F9 ORFs of the fowl pox genome. PCR using rFPV-HIV- B DNA provided no evidence of a fragment at 1071bp as was generated by wild type (FPV-M3) DNA used as a control.
PCR analysis showed that the sequence of the HIV insertion in the GMP manufactured rFPV- HIV-B vaccine was identical to that of the master seed stock.
(b) rFPV-HIV-AE vaccine
• Virus titration
A plaque assay is performed to determine the final concentration of the rFPV-HIV-AE vaccine. This assay is performed under GLP on GMP manufactured vaccine. • Expression of gag/pol
Western Blot analysis of rFPV-HIV-B vaccine is performed to confirm expression of the gag protein by the vaccine. This assay is performed under GLP on GMP manufactured vaccine.
• Strain verification
PCR is performed on rFPV-HIV-B to confirm the genome arrangement at 3 sites in the viral genome.
• Sequence of the HIV inserts
PCR analysis is performed.
In vivo Characterisation
The DNA and rFPV vaccines were tested for efficacy and safety in several animal species prior to the commencement of the clinical trial. Specific studies are summarised in the following sections.
Efficacy of the DNA vaccines in rodents
(a) pHIS-HIV-B immunogenicity
• Humoral immune response
The objective of this study was to demonstrate in mice that pHIS-HIV-B priming followed by rFPV boosting can induce antibody and cell mediated responses to gag/pol. The results in Table 3, are represented as the highest dilution with significant optical density (OD) above background (2 standard deviations), with the first tube (1) starting at 1/10 with doubling dilutions, tube 2 (1/20), tube 3 (1/40).
Table 3: Antibody titres after DNA priming with or without rFPV boosting
Figure imgf000087_0001
Antibody titres after DNA priming with or without rFPV boosting demonstrated significant immunogenicity of the pHIS-HIV-B vaccine in mice.
Protective capacity
This study assessed the protective capacity with rVN. Titration of vaccinia virus in the ovaries of mice immunised with pHIS-HIN-B and boosted with rFPY-gag/pol (native) showed significant inhibition of virus growth. Only prime-boost immunised mice were able to control the growth of vaccinia virus. Immunisation with DΝA alone or fowlpox virus alone showed no significant effect (Table 4).
Table 4: Vaccinia virus titrations after DΝA or rFPV alone and after prime/boost
Figure imgf000087_0002
(b) pHIS-HIV-AE immunogenicity
A preliminary assessment of the immunogenicity of the pHIS-HIV-AE (rFPV-HJN-AE) and GLP produced construct was assessed in mice using an ELISPOT assay, which showed that the pHIS-HIV-AE and rFPV-HIV-AE prime/boost was immunogenic. The serum antibody responses showed low but significant antibody to p24 in mice.
Efficacy of the rFPV vaccines in rodents
(a) rFPV-HIV-B immunogenicity
The immunogenicity of the HIV-1 expressed by the rFPV-HIV-B vaccine was confirmed in mouse prime/boost vaccination experiments. Effective immunogenicity was demonstrated by a substantial reduction in the level of vaccinia virus recovered from the ovaries of mice that had received the DΝA/rFPV combination (Figure 14).
(b) rFPV-HIV-AE immunogenicity
Refer to Section (b) of "Efficacy of the DΝA vaccines in rodents"
Efficacy of the DNA and rFPV vaccines in Macaques
(a) pHIS-HIV-B and rFP V-HIV-B HIV-1 Challenge
The primary objective of this study was to assess the immunogenicity of the DΝA/rFPV prime/boost regimen in non-human primates. Twenty eight colony bred juvenile pigtail macaques (M. nemestrina) free from HIV- 1/SrV/SRV infection, were randomised to one of the following groups:
Figure imgf000089_0001
A further 12 animals received 2xDNA and 2xrFPV in combinations of intranasal or intramuscular injections to assess the route of administration. These data are not presented here.
The pHIS-HIV-B vaccine was delivered 1 mg IM (l.Omg/mL with 0.5mL IM to the left and right quadriceps). The rFPV-HIV-B vaccine was delivered 5x107 PFU IM (0.5mL [2.5x107 PFU] to the left and right quadriceps).
All macaques remained healthy following the vaccinations and gained weight normally. No large swelling or erythema was observed at the injection sites. Serial analyses of blood cell counts, plasma electrolytes, urea, creatinine, total protein, albumin, bilirubin, liver enzymes, and creatinine kinase did not reveal any adverse effects of vaccination.
HlV-specific IFNγ production from T cells (generally CD 8+ T cells) was quantified by the ELIspot technique both 5 weeks following the final rFPV boost (to whole inactivated HIV- 1) and on the day of challenge (to both whole inactivated HIV-1 [Figure 15] and a single pool of HIV-1 gag 15mer overlapping peptides [Figure 16]).
One week following 2 DNA vaccinations alone (week 4), no significant T cell proliferative activity was detected to either p55 gag protein or whole inactivated HIV-1. In animals receiving only rFPV vaccines, no significant proliferative response was detected above control animals (P>0.05). Animals in the 3 groups receiving DNA prime and FPV boost vaccinations generated significant T cell proliferative activity to both p55 gag protein and whole inactivated HIV-1. The second rFPV boost vaccination did not detectably enhance HlV-specific T cell proliferative activity compared to the response after a single FPV boost (week 7 vs week 11, p>0.05).
Although not a primary goal of these vaccines, weak HIV-1 antibody responses by EIA were detected in the 3 groups animals immunised with the prime/boost regimens only and these were similar across the groups. No HIV antibody responses were detected in animals only administered rFPV alone or control vaccines.
To assess protection from HIV-1, a non-pathogenic intravenous challenge with an HIV- ILAI-based isolate was performed on the animals in the control group and the 3 prime/boost regimens. Control animals had a peak HIV-1 viral load at week 2 following challenge of 3.3 logio copies/mL and all 4 control animals still had detectable plasma HIV- 1 RNA by 5 weeks post infection. The group vaccinated with DNA/FPV had a mean peak VL at week 2 of 2.7 log10 copies/mL and was undetectable (<1.3 log10 copies/mL) in all 6 DNA/FPV vaccinated animals. A time-weighted area-under-the-curve analysis across the 4 groups demonstrated significant protection from challenge in the DNA/FPV regimen compared to both the control arm (P<0.01).
In addition to the main study described above, a small pilot study of 3 macaques (M43, M44, M46) was undertaken to assess adverse responses to the novel vaccines, during which a series of blood tests were undertaken to study haematology, biochemistry, liver function tests and creatine kinase levels. The vaccines did not adversely affect the multiple parameters studied (data not shown).
In summary, this macaque study showed:
• The pHIS-HIV-B prime/rFPV-HIV-B boost immunisation regimen was highly stimulatory for HlV-specific T cells.
• The results were concordant across 2 different T cell assays. • The pHIS-HIV-B or rFPV-HIV-B vaccines alone were not by themselves highly T cell immunogenic, demonstrating the marked effect observed with the prime/boost regimen.
• A second boosting FPV vaccination did not appear to substantially enhance the T cell immunogenicity of the prime/boost regimens over a single FPV boosting vaccination.
• The vaccines were well tolerated.
(b) SHIV Challenge
The primary objective of this study was to assess the immunogenicity and protective capability of the DNA/rFPV prime/boost regimen in a SHIV challenge model.
Thirty colony bred juvenile pigtail macaques (M. nemestrina) free from HIV-1/SIV/SRV infection, were randomised to one of the following groups:
Figure imgf000091_0001
SHIV-specific DNA and rFPV constructs were created based on the backbone DNA and rFPV for the B and AE sub-type vaccines. Challenge was performed with SHIVmn229 (III8- derived) LR. >500 M.I.D., with 2 doses over 2 days.
In SHIV-vaccinated groups, a mean of 797±141 IFNγ spot forming cells/106 PBMC to SIV gag and 1 005+138 to inactivated SIV were induced by vaccination, compared to <30 in controls (Figure 16). Intracellular IFNγ staining analyses, CFSE proliferation studies, epitope mapping studies and in vivo killing studies demonstrated induction of high levels of broadly reactive CD4+ and CD 8+ functional T cell immune responses. The immunogenicity of DNA or FPV vaccines alone and a regimen with only one DNA prime was inferior.
Partial protection from SHTV challenge was observed in the prime/boost group (Figure 18): The mean ± standard error (SE) CD4 T cell counts was 0.5+0.7% in controls vs. 12.3+8.3% in vaccinees, mean ± SE peak viral load was 8.2+0.3 vs. 6.7+0.9 logio copies SHIV RNA/mL), and mean ± SE set point viral load was 6.2+0.6 vs. 4.9+0.4, all p<0.01. The outcome of SHTV challenge across groups was consistent with the immunogenicity results, except that a regimen of 3 DNA vaccines alone was similar to the DNA/FPV prime/boost regimen.
In summary, high level functional SHIV-specific CD4 and CD8 T cell responses were induced by the DNA prime/rFPV boost regimen. Compared to the 2xDNA and rFPV boost, the DNA vaccine alone or single DNA prime, induced inferior T cell immunogenicity. Following challenge with SHIV, partial protective efficacy against virulent SHIV challenge in a pigtail macaque model was observed. The single DNA prime was inferior in protective efficacy, but significant efficacy of DNA vaccination alone was observed despite lower level immunogenicity.
(c) pHIS-HIV-AE and rFPV-HIV-AE Immunogenicity
A macaque study is conducted to assess the immunogenicity of the pHIS-HIV-AE and rFPV-HIV-AE prime boost regimen.
Toxicology Studies
The DNA and rFPV vaccines were also assessed primarily for safety and secondarily for efficacy in the toxicology and integration studies. Details of these studies are provided in the following sections. (a) pHIS-HIV-B and rFP V-HIV-B Toxicology Study
This study was designed to evaluate the toxicity profile and biodistribution of a combined pHIS-HIV-B and rFPV-HIV-B vaccine when administered intramuscularly over an eight week period with an eight- week recovery period to Cynomolgus monkeys. Toxicology, pharmacological, or pathologic observations served as evidence of absorption. This study was conducted in compliance with the Good Laboratory Practice Regulations. A total of 36 monkeys were randomised to one of 3 treatment groups: group 1 saline/PBS glycerol, group 2 - high doses of pHIS-HIV-B and rFPV-HIV-B vaccines, group 3 - therapeutic doses of pHIS- HIV-B and rFPV-HIV-B vaccines. Six monkeys/sex/group were treated intramuscularly with saline or 2 or 0.15 mg pHIS-HIV-B on days 1 and 29, and with phosphate buffered saline/10% glycerol, IO8 pfu or 7.5x106 pfu rFPV-HIV-B on day 57. Treatment had no effect on general health, body weights, local reactogenicity, food consumption, bone marrow, organ weights, organ-to-body weight ratios, and organ-to- brain weight ratios. Additionally, no test article-related effects were noted during all ophthalmologic and electrocardiographic examinations. Evaluation of the clinical pathology parameters revealed that there was a treatment effect on day 58 in the Group 2 animals that caused an increase in the total white blood cell count with a corresponding increase in the individual types of leukocytes. This most likely resulted directly from irritation at the injection sites following rFPV-HIV-B injection. It was transient, as there were no changes from baseline in the white blood cell count on days 64 and 113. Biodistribution data showed that the DNA vector was found predominantly in muscle at the injection site, with small numbers of copies of the DNA vector found in local lymph nodes.
Immunoassay data (anti-HIV-1 ELISA and IFNγ ELISPOT) showed positive humoral and cellular responses in Group 2 animals receiving high dose of DNA vector and rFPV virus vaccine. Cellular infiltrates of lymphocytes and histiocytes, graded as minimal to mild, were microscopically present at the injection sites of Group 2 animals. These local cellular responses were considered as being test article-related. Two intramuscular injections of the pHIS-HIV-B vaccine followed by one injection of rFPV- HIV-B vaccine to male and female Cynomolgus monkeys were well tolerated. The pHIS-HIV- B vaccine was found predominantly in the muscle of the injection site. Specific humoral and cellular immune responses to the vaccines were evident in the animals receiving high dose of DNA and rFPV vaccines.
(b) pHIS-HIV-B and rFPV-HIV-B Integration Study
This study was performed in order to evaluate the integration of pHIS-HIV-B vaccine when administered intramuscularly to New Zealand White rabbits, in the presence or absence of the rFPV-HIV-B vaccine, prior to clinical trials in human patients. The test articles were administered intramuscularly because this is a route of human exposure and at a dose that is 2 times the human dose to represent a worst case scenario. This study was conducted in compliance with GLP Regulations.
A total of 26 rabbits (3/sex/group for controls and 5/sex/group for the vaccine groups) were randomised to one of 3 treatment groups: group 1 - saline/PBS glycerol, group 2 - 2 injections of pHIS-HIV-B (2mg/mL) at week 0 and 4, group 3 - 2 injections of pHIS-HIV- B followed by 1 injection of rFPV-HIV-B (2mg/mL of DNA at week 0 and 4, followed by lxl08pfu/mL FPV at week 8). Two groups of 5 rabbits/sex were treated intramuscularly with 2 mg DNA vector (pHIS-HIV-B) on Study Day (SD) 1 and 29, and one of these groups was also treated with 108 pfu rFPV-HIV-B on SD 57. An additional control group consisting of 3 rabbits/sex were treated with saline on SD 1 and 29 and with phosphate buffered saline (PBS)/10% glycerol on SD 57. Parameters evaluated included mortality, clinical observations, local reactogenicity, body weights, and DNA vector integration at the site of injection. All animals survived until scheduled termination. Minimal, light pink erythema and minimal oedema were noted at the injection sites in a few animals across all groups. No other adverse clinical signs of toxicity were noted throughout the study. There were no adverse effects of test article treatment on body weights. Integration data indicated that one animal in Group 2 (receiving 2 mg DNA) was found to have a detectable amount of plasmid DNA (95 copies/μg DNA) associated with high molecular weight DNA following one round of purification. Due to low yield of DNA, restriction enzyme digestion and a second round of purification could not be performed on this sample. Therefore, it could not be determined if the detected signal was the result of co-migration of concatomerized plasmid DNA or from integration events into host genomic DNA. Assuming the worst case and that all detected plasmid is integrated, at 95 copies/μg DNA, the rate of mutation was estimated to be 8.44 x IO"9 mutations per gene, which is approximately 1200 times less than the rate of spontaneous mutation of 1.5 x 10'5 mutations per gene reported by Cole and Skopek in 1994 and Nichols, et a/., in 1995. Therefore, the potential for integration of the pHIS-HIN-B plasmid vector into rabbit genomic DΝA is considered to be extremely low. Due to the high degree of nucleotide sequence identity between the B-clade and AE-clade vaccines, the bioretention of pHIS- AE at the injection site of 6 pigtailed macaques was first measured at 60 days after the final injection of 3mg of DΝA vaccine. The levels of residual plasmid DΝA measured were below the level of assay quantification and even if all this was assumed to be integrated then this would be significantly below the spontaneous mutation rate.
Intramuscular injections of the DΝA vector (pHIS-HIV-B) with/without rFPV-HIV-B to male and female New Zealand white rabbits were well tolerated. No significant integration of the DNA vector was detected.
EXAMPLE 4 pHIS-HIV PRIME AND rFPV BOOST VACCINE
CLINICAL TRIALS
Study HVDDT -NO1-AI-O5395 (pHIS-HIV-B/FPV-HIV-B)
The phase I/IIa clinical trial is being conducted in Sydney (Protocol No. HVDDT-N01-AI- 05395). A summary of the protocol is provided in Table 5 Table 5: Protocol No. HVDDT -NO1-AI-O5395 Synopsis
Figure imgf000096_0001
Figure imgf000097_0001
The AE Study (pHIS-HIV-AE/rFPV-HIV-AE)
The phase I/IIa AE subtype clinical trial protocol is provided in Table 6. Table 6: The AE Study
Figure imgf000098_0001
Figure imgf000099_0001
EXAMPLE 5
PRELIMINARY MACAQUE ANALYSIS
Macaques and vaccinations strategy
Juvenile macaques (M. nemestrinά) were free from HIV-1/SIV/SRV infection, housed under PC3 conditions and anaesthetised with Ketamine (10 mg/kg IM) prior to procedures. The studies were approved by the institutional Animal Experimentation and Ethics Committees. Six groups of macaques were studied as noted in Figure 19. The vaccinations were well tolerated with no significant loss of activity or weight of the macaques following vaccinations (not shown). DNA vaccines were delivered lmg IM to the left and right quadriceps. rFPV was delivered IM (5 x IO7 PFU) to the left and right quadriceps of macaques. Macaques received prime-boost immunisations 2 DNA vaccinations 3 weeks apart and 2 rFPV vaccinations 4 weeks apart 4 weeks later as noted in Figure 20. Control macaques were administered either empty vaccines (Group 1), FPV vaccines alone (Groups 2 and 3) or DNA vaccines plus FPV vaccines.
T cell proliferative responses
Lymphoproliferative responses were assessed by standard 3H-thymidine incorporation assay as described (Kent, S.J., A. Woodward, and A. Zhao. 1997, J Infect Dis 176:1188- 1197). Briefly, macaque PBMC in triplicate wells at 105 cells/well were stimulated for 6 d with 10 μg/ml of recombinant HIV-1 p55 or control antigen also produced in baculovirus cultures, or whole inactivated HIV-1MN or control microvesicles produced in the same cell line in media containing 5% autologous heat-inactivated serum and pulsed with 3H- thymidine for 18h before β-counting. Proliferation is expressed as stimulation index (SI, mean 3H-thymidine incorporation of cells stimulated with antigen/mean incorporation in absence of antigenic stimulation).
Following DNA vaccination alone, no significant T cell proliferative activity was detected (not shown). Similarly, in animals receiving optimised FPV vaccination only (Groups 2 and 3, Figures 21-23), no significant proliferative activity was detected to either p55 gag protein or whole inactivated HIV. However, in groups receiving DNA prime and FPV boost vaccinations, significant T cell proliferative activity to both p55 gag protein, and whole HIV was detected. The preferred regimen DNA prime/FPV boost showed the greatest immunogenicity.
IFNγ ELISpot responses
PBMC were isolated by density gradient centrifugation (Ficoll-Paque, Amersham Pharmacia, Uppsala Sweden) from heparin-treated blood collected from each animal and stored in liquid nitrogen. PBMC were batch-thawed and stimulated with Aldrithiol-2 inactivated HIV-1 (10 μg/ml), control microvesicles, a set of 122 15mer HIV-1 gag peptides overlapping by 11 aa spanning the gag protein (10 μg/ml; NIH AIDS Research and Reference Reagent Program, MD), or an equivalent concentration of DMSO. Samples were assayed using the monkey IFNγ ELISPOT kit (U-CyTech bv, Utrecht, The
Netherlands), according to the manufacturer's instructions and as previously described (Dale, C.J., A. Zhao, S.L. Jones, D.B. Boyle, LA. Ramshaw, and S.J. Kent. 2000 JMed Primatol 29:240-247). Briefly, 150 μl antigen-stimulated PBMC were counted and incubated in ELISPOT plates pre-coated with anti-IFNγ monoclonal antibodies. 'Spots' were detected using labelled anti-biotin antibodies. Spots were counted on an automated counter (A.I.D., Germany). Results were normalised for the cell counts and expressed as the number of IFNγ spot-forming cells per 10 antigen-stimulated PBMC.
IFNγ production from T cells (generally CD8+ T cells) can be quantified by the ELISPOT technique. In animals receiving rFPV vaccines alone or animals tested immediately after the DNA vaccine prime, modest levels of IFNγ producing T cells were observed. However, animals receiving DNA prime and FPV boost vaccinations had markedly elevated numbers of HIV-1 gag-specific IFNγ producing cells, consistent with the combination of DNA and FPV vaccines being highly stimulatory for HlV-specific T cells. EXAMPLE 6
EVALUATION OF DNA AND RECOMBINANT FOWLPOX PRIME/BOOST
VACCINES EXPRESSING MULTIPLE HIV PROTEINS FROM SUBTYPE B AND
AE STRAINS
Pseudoviral particle morphology
Immature pseudoviral particles were observed to but from HeLa cells transfected with the DNA vaccines pHIS-HIV-B and pHIS-HIV-AE (Figure 25). Pseudoviral particle formation indicates efficient expression of Gag. Immature and defective particle morphology was seen with both B and AE clade vaccines, consistent with safety mutations in vaccines. Inefficient budding of pseudoviral particles was produced by pHIS-HLV-B.
HIV protein expression
Efficient expression of both B and AE clade HIV DNA and rFPV vaccines in HeLa cells yielded 60ng of p24/μg plasmid (0.001 x human dose) and 300ng of ρ24/5xl06 pfu rFPV (1 x human dose) respectively and significant levels of pseudoviral particles with a characteristic immature morphology.
A high level of Gag and Pol expression and a low Gag processing was observed. The AE vaccine also expresses high levels of Gag and Pol and truncated Env (Figure 26).
The rFPV-HIV-B vaccine expresses high levels of HIV Gag and Pol in human cells. The rFPV-HIV-AE Gag-Pol also expresses high levels of HIV Gag and Pol. The AE Env and at-Rev were also expressed by final rFPV-AE vaccine (Figure 27). Reverse transcriptase activity
Three short deletions were introduced into the RT region of Pol targeting the thumb, dNTP -binding and catalytic core motifs to eliminate any chance of the vaccine antigens supporting the reverse transcription of viral or cellular RNA being packaged into pseudoviral particles (Figure 28). Any residual RT activity could foster mutagenesis of cellular or viral or cellular DNA and repair resulting in replication competent virus.
RiY l packaging by pseudovirion
Because the DNA and rFPV vaccines produce modest levels of pseudoviral particles, small deletions were introduced into both of the zinc finger motifs of the nucleocapsid protein (NCp7) (Figure 29). The DNA vaccine constructs needed to retain active splicing for expression of Tat, Rev, Vpu and Env proteins. This preserved part of the HIV RNA packaging sequences in the DNA vectors. In contrast, most of the RNA packaging signal was removed from the rFPV vaccines which cannot undergo splicing (Figures 30-32).
Efficient packaging of HIV RNA would mobilise these sequences and could assist the generation of replication competent virus.
Lack of replication competency
More than two thirds of the HIV genome was deleted from each of the vaccine constructs. However, human DNA contains numerous copies of endogenous retroviral elements that could potentially supply components that might restore replication competence.
Several steps in the normal reproduction cycle of replication competent HIV retro virus have been ablated by targeted mutations. The reverse transcription of the HIV-1 RNA genome to cDNA and generation of the "pre-integration complex" has been eliminated. The Vpr and Vif proteins that functionally support and protect this complex have been deleted. The integration reaction required for successful viral replication has been prevented in the HIV vaccines by near complete elimination of the HIV integrase coding region and integrase-binding αtt sequences within the LTRs. A computer search of the complete human genome found no HIV-related LTR that could provide these elements to the vaccine DNA.
To address the possibility that unforeseen events could rescue replication competent virus from the DNA or rFPV vaccines, an attempt was made to rescue replication competent virus during long term culture of cells exposed to pseudoviral particles or expressing HIV proteins from these vaccines.
Refer Figures 33-35.
Toxicology and integration assays
The DNA and rFPV vaccines were assessed for their safety in toxicology studies in
Cynomolgus macaques using a high dose (2mg/lxl0 pfu) or a therapeutic dose (0.15mg/7.5xl06 p u). Plasmid biodistribution, toxicity profile, pharmacological and pathological examinations were performed under GLP conditions in the monkeys and less extensively in rabbits. A plasmid integration study was performed in rabbits after intramuscular injection of pHIS-HIV-B (2mg) alone or in combination with the rFPV
(lxl 08 pfu). Residual plasmid was measured using quantitative PCR in the high molecular weight DNA fraction. Tests for adventitious agents were negative.
Refer Figures 36-39.
Conclusions
In vitro safety assessment and characterisation of the DNA and recombinant fowlpox clade B HIV vaccines has demonstrated that: a) the pHIS-HIV-B vaccine: expresses gag, modified pol and tat in vitro has a defective RT with no enzyme activity is not able to yield replication competent virus produces modest levels of pseudoviral particles does not package HIV-1 RNA efficiently is able to express gag and tat in vitro but not RT.
b) the rFPV-HIV-B vaccine: • expresses gag and pol in vitro
• produces modest levels of pseudoviral particles
• does not package HIV-1 RNA efficiently.
In vivo safety assessment and characterisation of the clade B vaccines demonstrated that the pHIS-HIV-B and rFPV-HIV-B vaccines were well tolerated in rabbits and Cynomolgus macaques.
The pHIS-HIV-B and rFPV-HIV-B vaccines show impressive immunogenicity in mice and pigtailed macaques (see Oral presentation by S.J. Kent).
HIV clade-B DNA/rFP V prime boost vaccines appear safe and have been approved for phase I human clinical trial by the Australian Therapeutic Goods Administration.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY
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Claims

CLAIMS:
1. A method for eliciting or inducing, in a mammal, an immune response directed to a virus, said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incoφorated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens or of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient to elicit or induce an immune response to one or more of said antigens.
2. A method of therapeutically or prophylactically treating a mammal for a viral infection said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which viral vector is incorporates nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said virus.
3. A method of vaccinating a mammal to a viral pathogen said method comprising sequentially administering to said mammal:
(i) one or more sequential doses of an effective amount of a recombinant plasmid vector or functional derivative thereof into which plasmid is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) one or more sequential doses of an effective amount of a recombinant viral vector or functional derivative thereof into which viral vector is incorporates nucleic acid molecules encoding all, part or a modified form of two or more antigens thereof of said virus, which antigens have been rendered substantially non-pathogenic,
for a time and under conditions sufficient for said immune response to reduce, inhibit or otherwise alleviate any one or more symptoms associated with infection of said mammal by said virus.
4. The method according to any one of claims 1, 2 or 3 wherein said virus is HIN.
5. The method according to claim 4 wherein said HIN is HIN-1.
6. The method according to claim 5 wherein said HIV-1 is HIV-1 subtype AE.
7. The method according to claim 5 wherein said HIV-1 is HIV-1 subtype B.
8. The method according to claim 5 wherein said HIV-1 is HIV-1 subtype C.
9. The method according to any one of claims 1-8 wherein said immune response is a Thl/CTL-type response.
10. The method according to any one of claims 1-10 wherein said plasmid vector antigens are Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said viral vector antigens are Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
11. The method according to any one of claims 1-10 wherein said plasmid vector antigens are Gag, RT, Rev, Tat, Vpu and/or Env and said viral vector antigens are Gag, Tat, Rev, Env and/or Pol.
12. The method according to claim 10 or 11 wherein said gag and RT encoding nucleic acids of said plasmid vector are modified, said env and nef encoding nucleic acids of said plasmid vector are truncated and said gag and pol encoding nucleic acids of said recombinant viral vector are mutated.
13. The method according to claim 12 wherein in the context of said plasmid vector:
(i) said gag is modified to block Gag mediated binding/packaging of the HIV RNA template;
(ii) said pol is modified to block RT/RNaseH and integrase activity;
(iii) said env is truncated to block its effects on the T cell population through CD4 and CCR5 engagement and to reduce the possibility of an infective particle forming; and (iv) said nef is truncated to block immunomodulatory activity.
14. The method according to any one of claims 10-13 wherein said viral vector is a fowlpox virus vector.
15. The method according to claim 11 wherein said plasmid vector is pHIS-HIV-B.
16. The method according to claim 11 wherein said plasmid vector is pHIS-HIV-AE.
17. The method according to claim 11 wherein said fowlpox virus vector is rFPV-HIV- B.
18. The method according to claim 11 wherein said fowlpox virus vector is rFPV-HIV- AE.
19. The method according to claim 11 wherein said plasmid vector is pHIS-HIV-B and said viral vector is rFPV-HIV-B.
20. The method according to claim 11 wherein said plasmid vector is pHIS-HIV-AE and said viral vector is rFPV-HIV-AE.
21. The method according to any one of claims 1-20 wherein a single dose of said recombinant viral vector is administered 4 weeks later than a single dose of said plasmid vector.
22. The method according to any one of claims 1-20 wherein two doses of said plasmid vector and two doses of said viral vector are administered to said mammal, which second recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
23. The method according to any one of claims 1 -20 wherein two doses of said plasmid vector and one dose of said viral vector are administered to said mammal, which recombinant plasmid vector dose is administered four weeks after the first dose and the recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose.
24. The method according to any one of claims 1-20 wherein three doses of said plasmid vector and two doses of said viral vector are administered to said mammal, which second recombinant plasmid vector dose is administered four weeks after the first dose, the third recombinant plasmid vector dose is administered four weeks after the second dose, the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
25. A method of eliciting or inducing, in a mammal, an immune response directed to HIV, said method comprising sequentially administering to said mammal one or more doses of an effective amount of a recombinant plasmid vector or functional derivative thereof, into which plasmid vector is incorporated nucleic acid molecules encoding two or more antigens thereof of said HIV, which antigens have been rendered substantially non- pathogenic, and one or more optimised CpG motifs for a time and under conditions sufficient to elicit or induce an immune response to said antigens.
26. The method according to claim 25 wherein said virus is HIV.
27. The method according to claim 26 wherein said HIV is HIV-1.
28. The method according to claim 27 wherein said HIV-1 is HIV-1 subtype AE.
29. The method according to claim 27 wherein said HIV-1 is HIV-1 subtype B.
30. The method according to claim 27 wherein said HIV-1 is HIV-1 subtype C.
31. The method according to any one of claims 25-30 wherein said immune response is a Thl/CTL-type response.
32. The method according to any one of claims 25-31 wherein said plasmid vector antigens are Gag, RT, Pro, RNaseH, Rev, Tat, Vpu, Env and/or Nef and said viral vector antigens are Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
33. The method according to any one of claims 25-31 wherein said plasmid vector antigens are Gag, RT, Rev, Tat, Vpu and/or Env and said viral vector antigens are Gag,
Tat, Rev, Env and/or Pol.
34. The method according to claim 32 or 33 wherein said gag and RT encoding nucleic acids of said plasmid vector are modified, said env and nef encoding nucleic acids of said plasmid vector are truncated and said gag and pol encoding nucleic acids of said recombinant viral vector are mutated.
35. The method according to claim 34 wherein in the context of said plasmid vector:
(i) said gag is modified to block Gag mediated binding/packaging of the HIV
RΝA template;
(ii) said pol is modified to block RT/RΝAseH and integrase activity;
(iii) said env is truncated to block its effects on the T cell population through
CD4 and CCR5 engagement and to reduce the possibility of an infective particle forming; and
(iv) said nef is truncated to block immunomodulatory activity.
36. The method according to claim 33 wherein said plasmid vector is pHIS-HIN-B.
37. The method according to claim 33 wherein said plasmid vector is pHIS-HIV-AE.
38. Use of:
(i) a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs; and
(ii) a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of a mammalian disease condition characterised by infection with said virus.
39. Use according to claim 38 wherein said virus is HIV.
40. Use according to claim 39 wherein said HIV is HIV-1.
41. Use according to claim 40 wherein said HIV-1 is HIV-1 subtype AE.
42. Use according to claim 41 wherein said HIV-1 is HIV-1 subtype B.
43. Use according to claim 41 wherein said HIV-1 is HIV-1 subtype C.
44. The method according to any one of claims 38-43 wherein said immune response is a Thl/CTL-type response.
45. Use according to any one of claims 38-44 wherein said plasmid vector antigens are Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said viral vector antigens are Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
46. Use according to any one of claims 38-44 wherein said plasmid vector antigens are Gag, RT, Rev, Tat, Vpu and/or Env and said viral vector antigens are Gag, Tat, Rev, Env and/or Pol.
47. The method according to claim 45 or 46 wherein said gag and RT encoding nucleic acids of said plasmid vector are modified, said env and nef encoding nucleic acids of said plasmid vector are truncated and said gag and pol encoding nucleic acids of said recombinant viral vector are mutated.
48. Use according to claim 47 wherein in the context of said plasmid vector:
(i) said gag is modified to block Gag mediated binding/packaging of the HIV RNA template;
(ii) said pol is modified to block RT/RNaseH and integrase activity;
(iii) said env is truncated to block its effects on the T cell population through CD4 and CCR5 engagement and to reduce the possibility of an infective particle forming; and
(iv) said nef is truncated to block immunomodulatory activity.
49. The method according to any one of claims 45-47 wherein said viral vector is a fowlpox virus vector.
50. Use according to claim 46 wherein said plasmid vector is pHIS-HIV-B.
51. Use according to claim 46 wherein said plasmid vector is pHIS-HIV-AE.
52. Use according to claim 46 wherein said fowlpox virus vector is rFPV-HIV-B.
53. Use according to claim 46 wherein said fowlpox virus vector is rFPV-HIV-AE.
54. Use according to claim 46 wherein said plasmid vector is pHIS-HIV-B and said viral vector is rFPV-HIV-B.
55. Use according to claim 46 wherein said plasmid vector is pHIS-HIV-AE and said viral vector is rFPV-HIV-AE.
56. Use according to any one of claims 48-55 wherein a single dose of said recombinant viral vector is administered 4 weeks later than a single dose of said plasmid vector.
57. Use according to any one of claims 48-55 wherein two doses of said plasmid vector and two doses of said viral vector are administered to said mammal, which second recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
58. Use according to any one of claims 48-55 wherein two doses of said plasmid vector and one dose of said viral vector are administered to said mammal, which recombinant plasmid vector dose is administered four weeks after the first dose and the recombinant fowlpox virus vector dose is administered four weeks after the second recombinant plasmid vector dose.
59. Use according to any one of claims 48-55 wherein three doses of said plasmid vector and two doses of said viral vector are administered to said mammal, which second recombinant plasmid vector dose is administered four weeks after the first dose, the third recombinant plasmid vector dose is administered four weeks after the second dose, the first recombinant fowlpox virus vector dose is administered four weeks after the third recombinant plasmid vector dose and the second recombinant fowlpox virus dose is administered four weeks after the first recombinant fowlpox virus vector dose.
60. A vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant plasmid vector or functional derivative thereof into which plasmid vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
61. A vaccine capable of inducing an immune response directed to a virus, said vaccine comprising as the active component a recombinant viral vector or functional derivative thereof, into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic,
62. A nucleic acid construct or functional derivative thereof which construct comprises a plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
63. A nucleic acid construct comprising a recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic.
64. A plasmid vector into which is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of a virus, which antigens have been rendered substantially non-pathogenic, and one or more optimised CpG motifs.
65. A recombinant viral vector or functional derivative thereof into which vector is incorporated nucleic acid molecules encoding all, part or a modified form of two or more antigens of said virus, which antigens have been rendered substantially non-pathogenic.
66. The vaccine of claim 60 or 61 , the nucleic acid construct of claim 62 or 63, the plasmid vector of claim 64 or the viral vector of claim 65 wherein said virus is HIV.
67. The vaccine, nucleic acid construct, plasmid vector or viral vector according to claim 66 wherein said HIV is HIV-1.
68. The vaccine, nucleic acid construct, plasmid vector or viral vector according to claim 67 wherein said HIV-1 is HIV-1 subtype AE.
69. The vaccine, nucleic acid construct, plasmid vector or viral vector according to claim 67 wherein said HIV-1 is HIV-1 subtype B.
70. The vaccine, nucleic acid construct, plasmid vector or viral vector according to claim 67 wherein said HIV-1 is HIV-1 subtype C.
71. The vaccine, nucleic acid construct, plasmid vector or viral vector according to any one of claims 60-70 wherein said immune response is a Thl/CTL-type response.
72. The vaccine, nucleic acid construct or plasmid vector according to any one of claims 60, 62, 64 or 66-70 wherein said plasmid vector antigens are Gag, RT, Pro, RNAseH, Rev, Tat, Vpu, Env and/or Nef and said viral vector antigens are Gag, RT, Pro, RNAseH, Tat, Rev and/or Env.
73. The vaccine, nucleic acid construct or plasmid vector according to any one of claims 60, 62, 64 or 66-70 wherein said plasmid vector antigens are Gag, RT, Rev, Tat, Vpu and/or Env and said viral vector antigens are Gag, Tat, Rev, Env and/or Pol.
74. The vaccine, nucleic acid construct or plasmid vector according to claim 72 or 73 wherein said gag and RT encoding nucleic acids of said plasmid vector are modified, said env and nef encoding nucleic acids of said plasmid vector are truncated and said gag and pol encoding nucleic acids of said recombinant viral vector are mutated.
75. The vaccine, nucleic acid construct or plasmid vector according to claim 74 wherein in the context of said plasmid vector:
(i) said gag is modified to block Gag mediated binding/packaging of the HIV RNA template;
(ii) said pol is modified to block RT/RNaseH and integrase activity;
(iii) said env is truncated to block its effects on the T cell population through CD4 and CCR5 engagement and to reduce the possibility of an infective particle forming; and
(iv) said nef is truncated to block immunomodulatory activity.
76. The nucleic acid construct or viral vector according to claim 61, 63 65 or 66-70 wherein said viral vector is a fowlpox virus vector.
77. The vaccine, nucleic acid construct or plasmid vector according to claim 73 wherein said plasmid vector is pHIS-HIV-B.
78. The vaccine, nucleic acid construct or plasmid vector according to claim 73 wherein said plasmid vector is pHIS-HIV-AE.
79. The nucleic acid construct or viral vector according to claim 73 wherein said fowlpox virus vector is rFPV-HIV-B.
80. The nucleic acid construct or viral vector according to claim 73 wherein said fowlpox virus vector is rFPV-HIV-AE.
81. A pharmaceutical composition comprising the nucleic acid, plasmid vector or recombinant viral vector constructs of any one of claims 62-80.
82. The vaccine, nucleic acid construct, plasmid vector, recombinant viral vector or pharmaceutical composition of any one of claims 60-81 when used in accordance with the method of any one of claims 1-37.
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