WO2003047617A2 - Vaccine - Google Patents

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Publication number
WO2003047617A2
WO2003047617A2 PCT/GB2002/005411 GB0205411W WO03047617A2 WO 2003047617 A2 WO2003047617 A2 WO 2003047617A2 GB 0205411 W GB0205411 W GB 0205411W WO 03047617 A2 WO03047617 A2 WO 03047617A2
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Prior art keywords
composition
vector
fowlpox
genome
replicating
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PCT/GB2002/005411
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French (fr)
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WO2003047617A3 (en
Inventor
Stephen Laidlaw
Mike Skinner
Adrian Hill
Sarah Gilbert
Richard Anderson
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Isis Innovation Limited
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Priority claimed from GB0128733A external-priority patent/GB2382578A/en
Application filed by Isis Innovation Limited filed Critical Isis Innovation Limited
Priority to GB0407935A priority Critical patent/GB2396814B/en
Priority to JP2003548872A priority patent/JP2005517639A/en
Priority to CA002467486A priority patent/CA2467486A1/en
Priority to EP02783253A priority patent/EP1450854A2/en
Priority to AU2002347317A priority patent/AU2002347317B2/en
Publication of WO2003047617A2 publication Critical patent/WO2003047617A2/en
Publication of WO2003047617A3 publication Critical patent/WO2003047617A3/en
Priority to US10/856,118 priority patent/US7273605B2/en

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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/86—Viral vectors
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    • A61K39/0005—Vertebrate antigens
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    • A61K39/12—Viral antigens
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • 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
    • A61P31/04—Antibacterial agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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    • 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
    • C12N15/863—Poxviral vectors, e.g. entomopoxvirus
    • C12N15/8633—Avian poxviral vectors
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
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    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
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    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • A—HUMAN NECESSITIES
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    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to pox viruses.
  • the present invention relates to a fowlpox virus genome which has a modification in one or more wild-type FPV genes, a viral particle comprising such a genome, and its use to deliver a nucleotide of interest ("NOI") to a target cell.
  • NOI nucleotide of interest
  • the present invention also relates to vaccination methods using viral vectors, in particular heterologous prime-boost vaccination regimes employing two different non- replicating viral vector compositions.
  • Poxviruses have been previously exploited as recombinant vectors for the heterologous expression of foreign proteins.
  • recombinant vaccinia virus has been studied as a tool for transient expression of genes in mammalian cells and an experimental recombinant vaccine vector (reviewed by Moss, 1991, Proc Natl Acad Sci USA 93, 11341-8; and Moss, 1996, Proc Natl Acad Sci USA 93, 11341-8).
  • vaccinia virus In common with the other poxviruses, vaccinia virus resides within the cell cytoplasm where it expresses the proteins needed for viral replication. Recombinant vaccinia can, therefore, deliver foreign antigens to the cytoplasm of mammalian cells, thereby allowing them direct access to antigen processing pathways which leads to presentation of antigen derived peptides in association with MHC Class I and Class II molecules on the cell surface (Moss, 1991, Proc Natl Acad Sci USA 93, 11341-8). This property makes vaccinia useful as recombinant vaccines, particularly for stimulating CD8+ and CD4+ T-cell immune responses.
  • vaccinia viruses such as modified vaccinia Ankara (MVA) (Meyer et al, 1991, J Gen Virol 72, 1031-8; Sutter and Moss, 1992, Proc Natl Acad Sci USA 89, 10847-51 ; Sutter et al, 1994, Vaccine 12, 1032-40), which undergoes limited replication in human cells (Blanchard et al, 1998, J Gen Virol 79, 1159-67), and the avipox viruses, such as fowlpox, which do not proliferate in mammalian cells (Somogyi et al, 1993, Virology 197, 439-44).
  • MVA modified vaccinia Ankara
  • Wild-type fowlpox viruses which cause proliferative skin lesions that are rarely lethal in birds, are of commercial concern in the poultry industry.
  • Live attenuated vaccines against fowlpox virus have been produced by multiple passage of the virus in avian cells.
  • Such attenuated fowlpox viruses expressing antigens from poultry pathogens have been extensively exploited as recombinant vaccines for avian use (reviewed by Boyle and Heine, 1993, Immunol Cell Biol 71, 391-7; Paoletti, 1996, Proc Natl Acad Sci USA 93, 11349-53).
  • avipox viruses can express antigens in mammalian cells and can induce a protective immune response against mammalian pathogens (Taylor and Paoletti, 1998, Vaccine 6, 466-8; Taylor et al, 1998a, Vaccine 6, 504-8; Taylor et al, 1988b, Vaccine 6, 497-503), led to the development of recombinant fowlpox viruses as vaccines for use in mammals.
  • non-antigen based immunisation There are also other types of non-antigen based immunisation, which include passive immunisation (the direct administration of antibodies) and non-specific immunisation (such ad by the administration or cytokines or cytokine inhibitors).
  • a vaccine induces the right sort of immune response for the disease.
  • Many known vaccines are useful for generating antibodies, but do not induce significant cell-mediated immune responses.
  • a number of diseases are particularly susceptible to prevention and/or treatment by a T cell immune response.
  • cytolytic CD8+ T cells may protect against or help to clear viral infections.
  • diseases such as tuberculosis, malaria and H.pylori infection there is evidence for a protective role for CD4+ T cells which can secrete IFN ⁇ .
  • Some known viral vaccination strategies are associates with a number of complications and side effects. For example smallpox vaccination can cause generalised vaccinia, eczema vaccinatum, progressive vaccinia, and neurological and cardiac complications (Feery (1977) Med J. Aust 6 180-183; Goldstein et al (1975) Pediatrics 55 342-7)
  • the present inventors have obtained the full genomic sequence of an attenuated fowlpox virus strain (FP9).
  • FP9 lacks (or has modifications in) a number of genes present in wild-type fowlpox virus (FPV).
  • the genome of FP9 is 266 kbp, which is smaller than the genome for FPV-M, a fowlpox vaccine strain which has previously been described as a vector system (Coupar et al (1990) Virology 179 159-167).
  • the present inventors have shown that FP9 is superior to FPV-M is its capacity to elicit CD8+ T-cell immune responses.
  • the present invention provides an attenuated Fowlpox virus genome which has a modified form of one or more of the following wild-type FPV genes (gene nomenclature according to Afonso et al (2000) J. Virol 74 3815-3831):
  • FPV 190 FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260.
  • the present invention also provides a method for attenuating fowlpox comprising modifying one or more of said genes. Preferably said genes are modified as described in more detail below.
  • the present invention also provides an attenuated fowlpox virus genome which is less than 275 kbp in size. There is an upper limit on the total size of genome which can be efficiently packaged by the virus. If the genome itself is small, larger the amount of heterologous sequence it can carry.
  • the attenuated Fowlpox virus genome comprises the sequence shown in SEQ ID No. 1.
  • the attenuated Fowlpox genome of the present invention may also comprise a nucleotide of interest "NOI".
  • the "NOI” may be a therapeutic gene.
  • the present invention also provides a viral particle which comprises such a genome.
  • the genome comprises an NOI
  • the viral particle is capable of delivering the NOI to a target cell.
  • the viral particle may be capable of delivering a pre-expressed protein to a target cell.
  • the present invention also provides a vaccine, priming or boosting agent comprising such a genome or viral particle.
  • the present invention also provides a boosting composition comprising a non- replicating viral vector.
  • the composition is capable of boosting the immune response primed by M. bovis BCG in primates.
  • Prime-boost regimes may be homologous (where the same composition is administered two or more times) or heterologous.
  • the present invention also provides a vaccination kit which comprises: (i) a first composition which comprises an FP9 fowlpox viral particle; and
  • heterologous prime boost regimes using two different non-replicating viral vectors are surprisingly effective at inducing a T cell immune response in primates.
  • the use of non-replicating vectors avoids the adverse reactions associated with replicating virus (such as smallpox, see above).
  • the present invention also provides a vaccination kit which comprises:
  • composition which comprises a first non-replicating viral vector
  • second composition which comprises a second non-replicating viral vector for sequential administration in either order.
  • the vaccination kit comprises: (i) a first composition which comprises a first non-replicating poxvirus vector;
  • the kit may be suitable for administration to a primate subject in order to treat and/or prevent a disease.
  • the first and second compositions are capable of expressing the same antigen.
  • the present invention also provides a vaccination method which comprises the step of administering such a vaccine, priming or boosting composition or kit to a subject. Such administration should elicit a T-cell immune response in the subject.
  • the vaccination method may be used to treat or prevent, for example, diseases caused by or due to a chronic infection such as HIV, malaria, tuberculosis and East Coast Fever.
  • the present invention does not involve the recombinant CR3 gene as described in WO 02/068654 such as in Example 1 of WO 02/068654.
  • Figure 1 Annotated genome of FP9
  • Figure 2 A table showing the genes modified in FP9 during its passage in CEF tissue culture from HP 1.
  • FIG. 3 Comparison of the T-cell immune response elicited by recombinant FP9 and FPV-M
  • Figure 4 Comparison of the CD8+ T-cell immune response elicited by recombinant FP9, MVA and a DNA vaccine encoding PbSCP
  • Fowlpox FP9 can serve as both a priming and boosting agent in heterologous prime-boost immunisation regimes
  • Figure 6 Fowlpox FP9 can elicit both CD4+ and CD 8+ T cell responses against M. tuberculosis Ag85A in Balb/c mice.
  • Figure 7 Fowlpox FP9 elicits a T-cell immune response against M. tuberculosis Ag85A in non-human primates
  • Figure 8 Ex vivo IFN ⁇ ELISPOT responses against FP9 and MVA viral antigens.
  • Figure 9 Ex vivo IFN ⁇ Elispot response in Balb/c mice following aerosol or mucosal priming with M. bovis BCG and boosting with MVA85A.
  • Figure 10 Ex vivo IFN ⁇ Elispot response in Rhesus macaques following aerosol or mucosal priming with M. bovis BCG and boosting with MVA85A and FPAg85A.
  • Figure 11 shows a bar chart.
  • Figure 12 shows a bar chart.
  • Figure 13 shows two bar charts.
  • Figure 14 shows a graph.
  • Figure 15 shows a bar chart.
  • FIG. 16 shows Specific immune responses in BALB/c mice following prime/boost immunisation with pSG2.MEl and FP9.ME1.
  • BALB/c mice were immunised im. with pSG2.MEl (DNA) or iv. with FP9.ME1 (FP9) and boosted 15 days later in a similar manner.
  • Control animals were immunised with identical vectors encoding irrelevant antigens.
  • Thirteen days after the booster immunisation the T cell responses elicited against the tumour epitopes were determined in splenocytes using the IFN ⁇ ELISPOT assay.
  • Columns represent the antigen-specific IFN ⁇ sfc/million splenocytes ⁇ 1 SD for four mice per group.
  • Figure A depicts the summed epitope-specific responses and the response elicited against phytohemagglutinin (PHA) for each group, whereas B depicts the response against individual epitopes.
  • Figure 17. Shows specific immune responses in C57BL/6 mice following prime/boost immunisation with pSG2.MEl and FP9.ME1.
  • C57BL/6 mice were immunised im. with pSG2.MEl (DNA) or iv. with FP9.ME1 (FP9) and boosted 14 days later in a similar manner. Control animals were immunised with identical vectors encoding irrelevant antigens.
  • T cell response elicited against the tumour epitopes were determined in splenocytes using the IFN ⁇ ELISPOT assay.
  • Columns represent the antigen-specific IFN ⁇ sfc/million splenocytes ⁇ 1 SD for four mice per group.
  • Figure A depicts the summed epitope-specific responses and the response elicited against phytohemagglutinin (PHA) as a positive control for each group, whereas B depicts the response against individual epitopes.
  • PHA phytohemagglutinin
  • B depicts the response against individual epitopes.
  • the legend of B indicates immunodominant (DOM) and subdominant (SUB) epitopes that are recognised by CD8+ T cells, as well as those recognised by CD4+ T cells (CD4).
  • Figure 18 shows the mean interferon gamma response to PPD at various weeks after intratracheal M. tuberculosis challenge of cynomolgous macaques.
  • the present invention relates to vaccination regimes using non-replicating viral vectors.
  • viral vectors are known in the art which are capable of delivering an NOI via infection of a target cell.
  • Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, poxviral vectors or parvovirus vectors (see Kestler et ⁇ /1999 Human Gene Ther 10(10):1619-32).
  • retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MMV murine leukemia virus
  • HCV human immunodeficiency virus
  • EIAV equine infectious anaemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • FuSV Fujinami sarcoma virus
  • the present invention provides a vaccine, priming or boosting composition which comprises a non-replicating pox virus vector.
  • the family of poxviruses can be split into two subfamilies, the Chordopoxvirinae and the Entomopoxviriniae.
  • the Chordopoxvirinae (poxviruses of vertebrates) include orthopoxviruses, parapoxviruses, avipoxviruses, caripoxviruses, leporipoxviruses, suipoxviruses, molluscipoxviruses and yatapoxviruses.
  • Table 2 gives some examples of species within each genus of the pox virus family.
  • the present invention provides a vaccination kit which comprises:
  • composition which comprises a first non-replicating viral vector
  • second composition which comprises a second non-replicating viral vector. for sequential administration in either order.
  • the first and/or the second viral vector may be a pox virus vector.
  • compositions may act as a "priming" composition, to be administered first, and the other composition may act as a "boosting” composition, to be administered after an appropriate time interval (such as three weeks).
  • the first and second non-replicating viral vectors should be sufficiently different that no significant cross-reaction occurs.
  • the two viral vectors may be derived from viruses belonging to different familes, for example, a poxviral vector and an adenoviral vector.
  • the two viral vectors may be derived from viruses belonging to the same family (such as pox viruses) but different geni.
  • the first non-replicating poxvirus vector may be an avipox virus vector and the second non-replicating poxvirus vector may be an orthopox virus vector.
  • the two non-replicating viral vectors may even be derived from different species within the same genus, as long as the species are sufficiently different.
  • one of the two non-replicating poxvirus vectors is a fowlpox virus vector (i.e. derived from a fowlpox).
  • the fowlpox virus vector may comprise a genome according to the present invention.
  • the virus vectors used in the present invention should be non-replicating in the cells of the subject (for example in human cells).
  • the term "non-replicating” or “replication- impaired” as used herein means not capable of replication to any significant extent in the majority of normal subject cells. Viruses which are non-replicating or replication- impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. There will generally be one or a few cell types in which the viruses can be grown, such as CEF cells. Replication of a virus is generally measured in two ways: 1) DNA synthesis and 2) viral titre. More precisely, the term “nonreplicating or replication-impaired" as used herein and as it applies to poxviruses means viruses which satisfy either or both of the following criteria: 1) exhibit a 1 log (10 fold) reduction in DNA synthesis compared to the
  • Copenhagen strain of vaccinia virus in MRC-5 cells (a human cell line);
  • wild-type fowlpox viruses cause proliferative skin lesions in birds. It is possible to obtain field isolates of FPV from scab material collected from poultry infections (Boyle et al (1997) Arch. Virol. 142:737-748). The genomic sequence of a pathogenic fowlpox virus is available (Afonso et al (2000) J. Gen. Virol. 74(8) 3815-3831).
  • Live attenuated virus strains may be produced by multiple passage of the virus in avian cells.
  • Various attenuated virus strains of fowlpox virus are known such as FPV M (mild vaccine strain) and FPV S (standard vaccine strain) obtainable from Cyanamid- Websters PtY, Ltd Australia.
  • Pox viruses have evolved strategies for evasion of the host immune response that include the production of secreted proteins that function as soluble receptors for tumour necrosis factor, IL-I p, interferon (IFN)-oc/ andlFN-y, which normally have sequence similarity to the extracellular domain of cellular cytokine receptors (such as chemokine receptors). These viral receptors generally inhibit or subvert an appropriate host immune response, and their presence is associated with increased pathogenicity.
  • IL-I p interferon (IFN)-oc/ andlFN-y
  • FPV genes The genome of a fowlpox virus is composed of a single double-stranded DNA molecule with covelently linked terminal hairpins. Basic information about the sites for restriction endonuclease cleavage is available (Coupar et al (1990) as above).
  • the present inventors have sequenced the virulent precursor of FP9 (HPl) at all positions where the sequence of FP9 differed from this sequence. This method reveals differences which are attributable to lineage variations and those which have accored during tissue culture passage, adaptation and concomittant attenuation.
  • the genes modified in FPl during its passage in CEF tissue culture from HPl is shown in Figure 2.
  • the fowlpox virus genome of the present invention may have a modification in one or more of the following wild-type genes:
  • modification is intended to mean a variation (such as a deletion, substitution or addition) from the wild type sequence.
  • the modified gene sequence may encode a protein with a different amino acid sequence.
  • the amino acid sequence may have one or more amino acid deletions, additions or substitotions when compared to the wild-type sequence.
  • the fowlpox virus genome of the present invention has a modification which results in a non-conservative amino acid substitution in one or more of the following genes:
  • the genome may have a substantial modification in one or more of the wild-type genes.
  • the term "substantial modification” is intended to mean that the gene is modified in such a way that it no longer functions as the wild-type gene.
  • the substantially modified gene may be incapable of encoding a protein, or it may encode a protein which is incapable of or has a greatly reduced capacity to function as the wild-type protein.
  • the modification may be a deletion.
  • the entire gene may be deleted.
  • the modified gene may comprise one or more partial deletions sufficient to remove or greatly reduce the function of the gene.
  • the modification may be a substitution or an addition.
  • a recombination event may have occurred, with the effect that portion of sequence from elsewhere in the genome is incorporated into the gene (with optionally a corresponding loss of wild-type sequence).
  • a partial deletion, substitution or addition may cause a "frame-shift" mutation, resulting in incorrect reading of the downstream sequence.
  • the mutation may result in the creation of a stop codon (a "terminating mutation") such that the downstream sequence is ignored.
  • the mutation may result in the removal of a stop codon, which may result in two genes becoming fused to form a chimaeric gene.
  • the genome has a partial deletion in one or more of the following genes:
  • the genome entirely lacks one or more of the following genes:
  • the genome has a frame-shift mutation in one or more of the following genes:
  • the genome has a termination mutation in one or more of the following genes:
  • the genome has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV098.
  • the genome of FPV-M has been estimated to be in the region of 308 kb (Coupar et al (1990) as above). The smaller the viral genome, the more heterologous DNA can be included.
  • the fowlpox virus genome of the present invention is preferably less than 275 kb in size. More preferably the genome is about 266 kbp.
  • the size of the genome is considered without any heterologous sequences (for example a "nucleotide of interest" (NOI) see below).
  • NOI nucleotide of interest
  • a heterologous sequence for delivery may be incorporated into the fowlpox genome by homologous recombination. For the purposes of size determination, the genome size is considered before such a recombination event.
  • the fowlpox virus genome according to claim 1 comprises the sequence shown in SEQ ID NO. 1.
  • This "sequence" is intended to encompass homologues of the sequence, provided that the fowlox genome as a whole is capable of acting as a vector system (i.e. receiving a heterologous gene by homologous recombination and, when incorporated into a viral particle delivering the heterologous gene to a target cell).
  • homologue means a nucleic acid sequence having a degree of homology with the sequence shown in SEQ ID NO 1.
  • identity can be equated with “identity”.
  • an homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.
  • a homologue will comprise sequences that code for functional portions of an encoded protein (active sites etc.) which are the same as the subject sequence but may differ in other areas.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each base in one sequence is directly compared with the corresponding base in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • % homology preferably % sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • sequences may also have deletions, insertions or substitutions of nucleic acid residues which produce a silent change and result in a functionally equivalent substance.
  • the gene encodes a protein the protein encoded by a homologue may be identical (due to the degeneracy of the genetic code) or functionally equivalent (for example a conservative mutation may appear in the sequence).
  • the homolog encodes a protein having at least 90%, preferably at least 95%, more preferably at least 98% homology to the protein encoded by the SEQ ID NO. 1 gene. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • the present invention also relates to a viral particle comprising such a fowlpox virus genome.
  • the viral particle (or part therof) may be used as a vector system for delivering a nucleotide of interest (NOI) to a target cell.
  • NOI nucleotide of interest
  • the term "viral particle” as used herein includes a viral vector comprising such a particle (or part thereof) capable of delivering an NOI or a pre-expressed protein to a target cell,
  • the NOI may be inserted into the viral genome by homologous recombination using methods known in the art (see for example Schneider et al (1998) Nature Medicine 4(4) 397-402).
  • the fowlpox viral particle of the present invention may be used to deliver a pre-expressed protein to a target cell.
  • a "pre-expressed protein” is a protein translated before it reaches the target cell.
  • the cell in which the viral particle is grown may express a protein which is then incorporated into the viral particle.
  • the cell in which the viral particle is grown may be engineered to express one or more proteins of interest (POI). If the cell is a primary cell (such as a CEF cell) it may be transiently transfected with a nucleotide encoding such a POI. Alternatively, a stably tranfected cell line may be used for growth, such as the quail cell line QT35.
  • a primary cell such as a CEF cell
  • a stably tranfected cell line may be used for growth, such as the quail cell line QT35.
  • the pre-expressed protein is targetted to be incorporated into the viral particle.
  • the term NOI includes any suitable nucleotide sequence, for example, a synthetic RNA DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof.
  • the sequence need not be a coding region. If it is a coding region, it need not be an entire coding region.
  • the RNA/DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation.
  • the sequence is, comprises, or is transcribed from cDNA.
  • the NOI may encode a protein of interest ("POI").
  • POI protein of interest
  • the vector system could be used to examine the effect of expression of a foreign gene on the target cell.
  • the fowlpox virus delivery system could be used to screen a cDNA library for a particular effect on the target cell.
  • the NOI may be capable of blocking or inhibiting the expression of a gene in the target cell.
  • the NOI may be an antisense sequence. The inhibition of gene expression using antisense technology is well known.
  • the NOI or a sequence derived from the NOI may be capable of "knocking out” the expression of a particular gene in the target cell.
  • the NOI may be capable of integrating in the genome of the target cell so as to disrupt expression of the particular gene.
  • the NOI may disrupt expression by, for example, introducing a premature stop codon, by rendering the downstream coding sequence out of frame, or by affecting the capacity of the encoded protein to fold (thereby affecting its function).
  • the NOI may be capable of enhancing or inducing ectopic expression of a gene in the target cell.
  • the NOI or a sequence derived therefrom may be capable of "knocking in” the expression of a particular gene.
  • An NOI delivered by the vector delivery system may be capable of immortalising the target cell.
  • a number of immortalisation techniques are known in the art (see for example Katakura Y et al (1998) Methods Cell Biol. 57:69-91).
  • an NOI delivered by the vector delivery system may be used for selection or marker purposes.
  • the NOI may be a selection gene, or a marker gene.
  • selectable markers have been used successfully in retroviral vectors. These are reviewed in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 444) and include, but are not limited to, the bacterial neomycin and hygromycin phosphotransferase genes which confer resistance to G418 and hygromycin respectively; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine; the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and the bacterial genes which confer resistance to puromycin or
  • the NOI may have or encode a protein which has a therapeutic effect.
  • an NOI delivered by the vector delivery system may be a therapeutic gene - in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may code for a product that is capable of eliciting a therapeutic effect.
  • the NOI may, for example, be or encode one of the following: cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppresser protein and growth factors, membrane proteins, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (such as with an associated reporter group).
  • cytokines cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional
  • the NOI is capable of encoding a disease associated antigen. Exposure to an antigen in the context of a fowlpox vector may provoke or boost an immune responses to the antigen such that an existing or subsequent challenge is dealt with more effectively.
  • the nature of the antigen will depend on the disease. If the disease is caused by an organism or infectious agent (bacteria, virus, protozoa, prion) the antigen may be derivable from this organism or agent. If the disease is caused by tumour(s) the antigen is preferably a tumour associated antigen such as HER-2/neu, MUC01, cancer testis antigens or oncogene(s) or product(s) thereof.
  • a tumour associated antigen such as HER-2/neu, MUC01, cancer testis antigens or oncogene(s) or product(s) thereof.
  • the disease-associated antigen comprises one or more T cell epitopes.
  • the antigen may comprise a complete protein from a disease causing organism (such as Antigen 85A from M. tuberculosis).
  • the antigen may comprise a string of T cell epitopes from one or more proteins from disease-causing organism(s).
  • An example of this is the MEPfTrap polypeptide from P. falciparum which comprises a string of T-cell epitopes from P. falciparum CSP and the Pb9 epitope fused to the P. falciparum TRAP gene.
  • the plasmid pEFL or derivatives thereof is used in the making of fowlpox constructs according to the present invention, preferably the plasmid pEFL29.
  • PEFL 29 has regions of homology which allow it to integrate into FP9 at ORF1. This disrupts ORF-1 but does not delete it.
  • the plasmid adds three genes lac A, lac Y and lac Z to the genome. This disruption is an insertion, and some nucleotides are deleted.
  • the Fowlpox Orfl gene bases deleted from pEFL29 are (capital letters):
  • the genome and/or particle of the present invention may be used in a method to treat and/or prevent a disease in a subject.
  • the genome and/or particle may be employed in a vaccine which is administered to a subject for prophylactic or therapeutic purposes.
  • the vaccine may also comprise an adjuvant (see below).
  • a multiple dose vaccination program may involve repeat doses of the same composition or one or more doses of two or more different compositions.
  • the present invention provides a vaccine pack which comprises a vaccine provided in a manner which facilitates repeat administration.
  • the pack may contain a plurality of vials comprising the correct dose of vaccine ready to be injected into the patient. If the vaccine is to be taken orally, the doses may be present as pills or capsules.
  • a “priming” composition which is administered to the patient first and a “boosting” composition which is administered some time later.
  • the genome and/or viral particle of the present invention may be used in a priming composition and/or a boosting composition.
  • compositions may be employed in heterologous vaccination programs. If the genome/particle of the present invention comprises an NOI (optionally capable of encoding a POI), then preferably the other composition comprises the same NOI or POI.
  • Other compositions include "naked DNA", non- viral vector systems and other viral vector systems.
  • Naked DNA may be linear or circular (far example, a plasmid). It may be provided in a carrier such as a liposome or in a free form.
  • Suitable non-viral vectors for use in the priming composition include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage, whole antigens with adjuvant, and other similar systems.
  • a viral vector system it may be an advantage if it is derived from a different virus (i.e. not fowlpox) to minimise cross-reaction.
  • the vector may be derived from another avipox virus , such as canary pox, or from a different genus of pox viruses (as shown in Table 2). Particularly preferred is an attenuated vaccinia vector system such as MVA or NVYAC.
  • Other suitable viral vectors are vectors based on non-pox viruses, such as adeno vims, herpes virus and Venezuelan equine encephalitis virus (VEE).
  • Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA (Darji A et al 1997 Cell 91: 765-775).
  • the present invention provides a vaccination kit which comprises:
  • the present invention also relates generally to heterologous vaccination regimes using two different non-replicating viral vectors.
  • the present inventors have shown for the first time that heterlogous prime-boost regimes using two different non-replicating viral vectors is efficient in generating an immune response in a primate subject.
  • the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
  • composition which comprises a first non-replicating viral vector
  • second composition which comprises a second non-replicating viral vector to the subject in either order.
  • the subject is a mammal, more preferably the subject is a primate, most preferably the subject is a human.
  • the first and/or the second composition is a poxvirus vector.
  • the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering (i) a first composition which comprises a first non-replicating pox viral vector;
  • the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering
  • composition which comprises a first non-replicating viral vector
  • second composition which comprises a second non-replicating viral vector to the subject in either order.
  • the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering
  • composition which comprises a DNA vaccine
  • second composition which comprises a first non-replicating viral vector
  • composition which comprises a second non-replicating viral vector; to the subject.
  • the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering (i) a first composition which comprises a first non-replicating viral vector;
  • composition which comprises a second non-replicating viral vector
  • composition(s) which comprises a third non-replicating viral vector; to the subject.
  • the invention relates to a method as described above wherein at least one of the composition(s) comprises a poxvirus vector.
  • the invention relates to a method as described above wherein said non-replicating viral vector(s) comprise poxvirus vector(s).
  • the invention relates to a method as described above wherein at least two of the poxvirus vectors are derivable from poxviruses from different genera.
  • the invention relates to a method as described above wherein at least one poxvirus vector is derivable from an avipox virus and at least one poxvirus vector is derivable from an orthopox virus.
  • the invention relates to a method as described above wherein at least one of the vectors is derivable from a fowlpox virus.
  • the invention relates to a method as described above wherein at least one of the vectors is, or is derivable from, FP9.
  • the invention relates to a method as described above wherein one of the vectors comprises a fowlpox virus genome selected from
  • a fowlpox virus genome which has a partial deletion in one or more of the following genes: FPV158, FPV219, FPV222;
  • (ix) a fowlpox virus genome comprising the sequence shown in SEQ ID No. 1, with a deletion of ACTGGTGCGAAGATCTC from ORF1.
  • the invention relates to a method as described above wherein the fowlpox virus genome also comprises a NOI. In another aspect, the invention relates to method as described above wherein the NOI is under the control of a poxvirus promoter.
  • the invention relates to a method as described above wherein the NOI encodes an antigen from P. berghei, P. falciparum, P.cynomolgi, P.vivax, M. tuberculosis or T.parva.
  • the invention relates to a method as described above wherein the composition comprising the viral vector derivable from an avipox vims is administered as a boosting composition.
  • the invention relates to method as described above wherein the composition comprising the viral vector derivable from an avipox vims is administered as a priming composition.
  • the invention relates to method as described above wherein the subject is a primate.
  • the invention relates to method as described above wherein the subject is a human.
  • the invention relates to use of a non-replicating viral vector in a vaccine for an animal, wherein said vector is, or is derivable from, FP9.
  • the invention relates to use as described above wherein said animal is a mammal.
  • the invention relates to use as described above wherein said mammal is a primate.
  • the invention relates to use as described above wherein said primate is a human.
  • the invention relates to use of a non-replicating viral vector in medicine, wherein said vector is, or is derivable from, FP9.
  • the invention in another aspect, relates to a method of eliciting an immune response in a subject comprising administering a composition comprising a non-replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
  • the invention in another aspect, relates to a method of boosting a pre-existing immune response in a subject comprising administering a composition comprising a non- replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
  • the subject is a primate, in particular a human.
  • the vectors are derivable from poxviruses from different genera.
  • one may be derivable from an avipox vims and the other from an orthopox virus (see Table 2).
  • compositions comprises a vector derivable from an avipox vims
  • this composition is administered as a boosting composition.
  • one of the vectors is derivable from a fowlpox virus. More preferably one of the vectors is or is derivable from FP9.
  • one of the vectors may comprise a fowlpox vims genome according to the first aspect of the invention.
  • the vector comprises a genome having the sequence shown in SEQ ID No. 1
  • the FP9 or FP9 derived vector is used as a prime.
  • the FP9 or FP9 derived vector is used as a boost.
  • the first and second compositions are capable of expressing the same antigen.
  • the first and second compositions may be packaged together or individually for separate sale.
  • the kit may comprise other components for mixing with one or both of the compositions before administration (such as diluents, carriers, adjuvants etc.- see below).
  • the kit may also comprise written instructions concerning the vaccination protocol.
  • the present inventors have shown that a non-replicating viral vector according to the present invention is effective at boosting a pre-existing immune response to an antigen.
  • the present inventors have shown for the first time that non- replicating viruses can be used to boost the immune response primed by M. bovis BCG in mice and in primates and melanomas in primates such as humans.
  • the pre-existing immune response may have been generated by a vaccine.
  • vaccine types have been developed and are known in the art, these include living organism, intact but non-living organisms, subcellular fragments, toxoids, DNA- based vaccines and anti-idiotype (see Table 1).
  • the pre-existing response is generated by an attenuated living pathogen, such as BCG.
  • the present invention provides a boosting composition comprising a non-replicating viral vector capable of boosting a pre-existing immune response in a subject.
  • the subject is a primate.
  • the viral vector is a pox viral vector.
  • the viral vector may comprise a genome according to the first aspect of the invention.
  • the present invention also relates generally to multiply heterologous vaccination regimes, such as triply heterologous regimes, using different non-replicating viral vectors.
  • the invention thus provides a triple regime comprising administering to a subject three heterologous compositions.
  • said three compositions each differ from their neighbouring composition.
  • the first composition comprises X
  • the second composition will preferably differ from X.
  • the third composition may be similar or identical to the first composition.
  • all three compositions are different from one another.
  • one of the compositions may be a DNA based composition such as a DNA vaccine.
  • the invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
  • composition which comprises a first non-replicating viral vector
  • composition which comprises a second non-replicating viral vector; to the subject.
  • the invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
  • composition which comprises a first non-replicating viral vector
  • second composition which comprises a second non-replicating viral vector
  • composition which comprises a third non-replicating viral vector; to the subject.
  • the first and the second compositions are heterologous, and the second and third compositions are heterologous. More preferably the first and the second and the third compositions are heterologous. Most preferably said compositions are heterologous with respect to the viral vector component of said compositions.
  • a treatment regimen may involve pDNA priming followed by recombinant MVA boosting followed by recombinant FP9 boosting followed by recombinant adenovirus boosting and so forth. These treatment/immunisation cycles may be advantageously repeated to sustain a therapeutic T cell response in a therapeutic setting.
  • Preferably three or more different viral vectors are used in the triply heterologous regimes of the invention.
  • immunisation is performed using the following vectors: DNA-FP9-MVA-adenovirus-recombinant herpes in any order.
  • each vector expresses the same antigen(s).
  • the vectors are administered in the order DNA-FP9-MVA-adenovirus-recombinant herpes.
  • immunisation orders and regimes are: DNA-FP9-MVA and DNA-MVA-FP9.
  • the vectors are derivable from poxviruses from different genera.
  • one may be derivable from an avipox vims and another from an orthopox vims (see Table 2).
  • one of the vectors is derivable form a fowlpox vims.
  • one of the vectors may comprise a fowlpox vims genome according to the first aspect of the invention.
  • the vector comprises a genome having the sequence shown in SEQ ID No. 1
  • the first and second and third compositions are capable of expressing the same antigen.
  • Example 12 (eg. Figure 11-DDFM) demonstrates the effectiveness of the triple regime.
  • the subject is a primate, in particular a human.
  • the first and second and third compositions may be packaged together or individually for separate sale.
  • the kit may comprise other components for mixing with one or more of the compositions before administration (such as diluents, carriers, adjuvants etc.- see below).
  • the kit may also comprise written instructions concerning the vaccination protocol.
  • the vaccination method or program should elicit a T-cell immune response in the subject.
  • T cells in general can be detected by the present of TCR, CD3, CD2, CD28, CD5 or CD7 (human only).
  • CD4+ T cells and CD8+ T cells can be distinguished by their co-receptor expression (for example, by using anti-CD4 or anti-CD8 monoclonal antibodies). Since CD4+ T cells recognise antigens when presented by MHC class II molecules, and CD8+ recognise antigens when presented by MHC class I molecules, CD4+ and CD8+ T cells can also be distinguished on the basis of the antigen presenting cells with which they will react.
  • CD4+ T cell epitopes there may be one or more CD4+ T cell epitopes and one or more CD8+ T cell epitopes. If the particular epitope has already been characterised, this can be used to distinguish between the two subtypes of T cell, for example on the basis of specific stimulation of the T cell subset which recognises the particular epitope.
  • CD4+ T cells can also be subdivided on the basis of their cytokine secretion profile.
  • the T H 1 subset (sometimes known as "inflammatory CD4 T cells") characteristically secretes IL-2 and IFN ⁇ and mediates several functions associated with cytotoxicity and local inflammatory reactions.
  • T H I cells are capable of activating macrophages leading to cell mediated immunity.
  • the T H 2 subset (sometimes known as "helper CD4 T cells”) characteristically secretes 11-4, IL-5, IL-6 and IL-10, and is thought to have a role in stimulating B cells to proliferate and produce antibodies (humoral immunity).
  • T H I and T H 2 cells also have characteristic expression of effector molecules.
  • T H I cells expressing membrane-bound TNF and T H 2 cells expressing CD40 ligand which binds to CD40 on the B cell.
  • T cell immune response may thus be readily determined, for example using fluorescence activated cell scanning (FACScan).
  • FACScan fluorescence activated cell scanning
  • the target antigen may be characteristic of the target disease. If the disease is an infectious disease, caused by an infectious pathogen, then the target antigen may be derivable from the infectious pathogen.
  • the target antigen may be an antigen which is recognised by the immune system after infection with the disease. Alternatively the antigen may be normally "invisible" to the immune system such that the method induces a non-physiological T cell response. This may be helpful in diseases where the immune response triggered by the disease is not effective (for example does not succeed in clearing the infection) since it may open up another line of attack.
  • Preferred Breast Cancer antigens are MUC-1, HER2, CEA; Preferred Colon cancer antigens: CEA, MUC-1, MAGE-12, mutant P53; Preferred Cervical cancer antigens: human paplioma vims proteins E6 and E7; Preferred EBV-induced B and T cell lymphomas antigens: EBNA1 and 2, LMP 1; Preferred renal cancer antigens: HER-2 neu, RAGE, MUC-1.
  • Preferred HPV antigens are viral proteins El -8, LI and L2 Preferred HS V antigens are viral proteins gM, gH, gK, GG, gD
  • HBV antigens are viral proteins small, middle and large surface antigen, core antigen, polymerase
  • HCV proteins are viral proteins core protein, envelope protein, NS2, NS3,
  • the antigen may be a tumor antigen, for example HER2/neu, MUC-1, MAGE-1,
  • the antigen may be an autoantigen, for example tyrosinase.
  • the antigen is derivable from M. tuberculosis.
  • the antigen may be ESAT6 or MPT63.
  • the antigen is derivable from the malaria-associated pathogen P. Falciparum.
  • the compositions of the present invention may comprise T cell epitopes from more than one antigen.
  • the composition may comprise one or more T cell epitopes from two or more antigens associated with the same disease.
  • the two or more antigens may be derivable from the same pathogenic organism.
  • the composition may comprise epitopes from a variety of sources.
  • the ME-TRAP insert described in the examples comprises T cell epitopes from P. falciparum, tetanus toxoid, M. tuberculosis and M. bovis.
  • the method of the present invention will be useful for treating and/or preventing a number of diseases, especially those which are susceptible to a T-cell mediated immune response.
  • the method of the present invention will be useful in the treatment and/or prevention of diseases which are or are caused by chronic infections, particularly persistent, latent infections.
  • a non-exhaustive list of suitable diseases includes: tuberculosis, HIV, malaria. H. pylori, influenza, hepatitis, CMV, human papilloma vims (HPV), herpes vims-induced diseases and other viral infections, leprosy, non-malarial protozoan parasites such as toxoplasma, and various malignancies such as tumours and/or cancers, infectious disease caused by protozoans: malaria, particularly Plasmodium falciparum and P.
  • the method of the present invention is particularly useful in vaccination strategies to protect against tuberculosis, malaria and East Coast Fever.
  • compositions described herein may be employed as therapeutic or prophylactic vaccines. Whether prophylactic or therapeutic immunisation is the more appropriate will usually depend upon the nature of the disease. For example, it is anticipated that cancer will be immunised against therapeutically rather than before it has been diagnosed, while anti-malaria vaccines will preferably, though not necessarily be used as a prophylactic.
  • the present invention also relates to a pharmaceutical composition such as a vaccine, priming or boosting agent.
  • the pharmaceutical composition may also comprise, for example, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutically acceptable carrier for example, a pharmaceutically acceptable diluent, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions comprising a DNA plasmid vector may comprise granulocyte macrophage-colony stimulating factor (GM-CSF), or a plasmid encoding it, to act as an adjuvant; beneficial effects are seen using GM-CSF in polypeptide form.
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • Adjuvants such as QS21 or SBAS2 (Stoute J A et al. 1997 N Engl J Medicine 226: 86-
  • compositions of the present invention may be used with proteins, peptides or nucleic acids to enhance the induction of T cell responses.
  • the composition may also be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilising agent(s).
  • the pharmaceutical composition could be for veterinary (i.e. animal) usage or for human usage.
  • the composition may be used to treat for example mammals (especially cattle) or birds.
  • the subject is a mammalian subject, in particular a primate (e.g. human) or ungulate (e.g. cow) subject.
  • a primate e.g. human
  • ungulate e.g. cow
  • a therapeutically effective daily intradermal or intramuscular dose of the compositions of the present invention is likely to range from 10 5 -10 10 plaque-forming units (pfu).
  • the physician or veterinary surgeon will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.
  • the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • compositions of the present invention may also be administered by aerosol for inhalation by the subject.
  • compositions of the present invention may also be conveniently administered by injection, such as intradermal and/or intramuscular injection.
  • the compositions may be administered using a suitable devine into the skin or other tissues (for example using a "gene gun" or similar).
  • compositions can be also be administered in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, or subcutaneously.
  • compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
  • compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • compositions of the present invention in sustained release formulations.
  • the present invention provides the use of non-cultured CEF cells to grow an avipox virus.
  • the avipox vims is a fowlpox virus, especially one which comprises a fowlpox genome according to the first aspect of the present invention.
  • non-cultured is used to indicate that the cells are used before they multiply to any significant extent in culture.
  • the cells should be in culture for insufficient time for the number of cells in the population to double. It is routine practice to maintain primary CEF cells in culture for up to one month by regular passage and supplementation of the growth medium.
  • the non-cultured CEF cells of the present invention should be used without being passaged.
  • Primary non-cultured CEFs can be obtained by loading freshly prepared CEFs into tissue culture dishes at a concentration sufficient to give a confluent monolayer without growth. The plate is then used, preferably within 24hours of being plated out.
  • Fowlpox strain FP9 was derived by 438 passages through chicken embryo fibroblasts in tissue culture from the virulent wild-type fowlpox HP-1 strain (Mayr and Malicki (1966) Monbl Veterinarmed 13 1-3) and then plaque purified.
  • Fp9 has been fully sequenced (SEQ ID No 1) and annotated ( Figure 1). FP9 does not contain the REV provims. At 266 kbp the genome of FP9 is somewhat smaller than the estimated size of FPV-M (greater than 300kbp).
  • EXAMPLE 2 Comparison of the immune response elicited by recombinant FP9 and FPV-M
  • Plasmodium berghei circumsporozoite surface protein (PbCSP) was inserted into the terminal 6.8 kbp BamHI fragment (Boursnell et al, 1990a, J Gen Virol 71, 621-8; Boursnell et al, 1990b, Vet Microbiol 23, 305-16; Campbell et al, 1989, J Gen Virol 70, 145-54) of the genome of both viruses by homologous recombinantion (Quingzhong et al, 1994, Vaccine 12, 569-73).
  • the vaccinia p7.5 early-late promoter was used to drive the expression of the inserted genes in both recombinant viruses.
  • the PbCSP protein contains an H-2K d restricted 9 amino acid peptide epitope that can induce a protective CD8+ T-cell resp;onse against liver stage P. berghei infection in Balb/c mice (Plebanski et al, 1998, Eur J Immunol 28, 4345-55; Schneider et al, 1998, Nat Med 4, 397-402).
  • mice Female Balb/c mice were immunised intravenously with 1 x 10 6 PFU of FP9 or FPV- M alone or expressing Plasmodium berghei CSP (PbCSP). Seven days following immunisation, the T-cell immune response elicited in splenocytes was determined using the IFN ⁇ ELSPOT assay. The response against the recombinant antigen, PbCSP was determined using the MHC-class I restricted Pb9 epitope (recognised by CD8+ T- cells). As a positive control, the T-cell response against whole virus was determined by exposing immune splenocytes to those infected with fowlpox vims. Columns in Figure 1 represent the mean IFN- ⁇ spot forming cells (SFC) per million splenocytes + the standard error of the mean for four mice per group.
  • SFC spot forming cells
  • EXAMPLE 3 Prime-boost immunisation regimes with FP9PbCSP and induction of a protective immune response against P. berghei
  • mice Female Balb/c mice were immunised intravenously with 1 x 10 6 PFU of FP9PbCSP (FP9) or MVAPbCSP (MVA), or intramuscularly with 50 ⁇ g of pSG2PbCSP (DNA) and boosted two weeks later with either FP9PbCSP or MVAPbCSP.
  • the CD8+ T-cell response against PbCSP was determined using the MHC-class I restricted Pb9 epitope in an IFN ⁇ ELISPOT assay 14 days following the boost.
  • Columns represent the mean IFN ⁇ spot forming cells (SFC) per million splenocytes + the standard error of the mean for three mice per group ( Figure 4).
  • mice Two weeks following the booster immunisation, animals were challenged by intravenous injection with 2000 P. berghei sporozoites and subsequently monitored for blood stage infection 6 to 14 days after challenge (Table 2). Results are cumulative for four experiments.
  • EXAMPLE 4 Construction of FP9 viruses encoding different foreign recombinant antigens and epitope strings
  • genes encoding whole antigens of both prokaryotic and eukaryotic origin and synthetic genes encoding poly-epitope and poly-protein fusions were inserted into the genome of FP9 by homologous recombination (Table 4). These genes encode antigens which differ widely in structure and are derived from a variety of different pathogens.
  • Each of the recombinant FP9 derivatives listed in Table 4 have been propagated in vitro in chicken embryo fibroblasts to high concentrations, indicating that they are both viable and stable.
  • the vaccinia p7.5 early-later promoter was used to drive expression of the encoded antigens in all of the recombinant vimses.
  • the encoded antigens have been found to be expressed in all cases where this has been determined (Table 4). This includes the largest of these constructs, FP9L 3 SEPTL, which harbours a 9.9 kbp synthetic gene encoding a poly-protein expressed as 6 fused malarial antigens.
  • ND not determined.
  • EXAMPLE 5 Induction of T-cell immune responses against P. falciparum antigens by FP9
  • the MEPfTrap polypeptide which consists of a string of T-cell epitopes from P. falciparum CSP and the Pb9 epitope (Gilbert et al, 1997 Nat Biotechnol 15, 1280-4) fused to the P. falciparum TRAP gene (Robson et al, 1988, Nature 335, 79-82), was developed as an antigen for vaccination against P. falciparum malaria in humans.
  • FP9 encoding MEPfTrap has been constructed (Table 4) and shown to elicit CD8+ T-cell responses in potency tests carried out in Balb/c mice (107+20 IFN ⁇ spot forming cells/million splenocytes).
  • EXAMPLE 6 Induction of CD8+ and CD4+ T cell immune responses against Antigen 85 A of M. tuberculosis by FP9
  • Antigen 85A is a major secreted and protective antigen from M. tuberculosis.
  • the immune response elicited by FP9 encoding Ag85A from M. tuberculosis was initially determined in Balb/c mice ( Figure 6).
  • Antigen 85A contains a 20 amino acid H-2 d MHC class Il-restricted peptide epitope (recognised by CD4+ T-cells) as well as a nine amino acid H-2 d MHC class I-restricted epitope (recognised by CD8+ T-cells) (Huygen et al, 1994).
  • mice Female Balb/c mice were immunised intravenously with 1 x 10 7 PFU of recombinant FP9 encoding antigen 85A (FP9Ag85A) from mycobacterium tuberculosis or, for comparison, 1 x 10 6 PFU of MVA encoding the same antigen.
  • the response against 85 A was determined using the MHC-class II restricted PI 5 epitope (recognised by CD4+ T-cells) and the MHC-class I restricted Pl l epitope (recognised by CD8+ T- cells) in an IFN ⁇ ELISPOT assay six days following immunisation.
  • Columns represent the mean IFN ⁇ spot forming cells (SFC) per million splenocytes + the standard error of the mean for three mice per group.
  • a male Rhesus macaque immunised twice with recombinant MVA encoding antigen 85 A (MVA85A) was boosted (i) 21 and 26 weeks later with 5 x 10 8 PFU of FP9Ag85A.
  • the response against Ag85A was determined in peripheral blood mononucleocytes (PBMCs) using pools of overlapping peptides covering the amino acid sequence of the Ag85A polypeptide in an IFN ⁇ ELISPOT assay. Curves represent the IFN ⁇ spot forming cells (SFC) per million PBMCs for 4 pools of peptides.
  • PBMCs peripheral blood mononucleocytes
  • SFC spot forming cells
  • EXAMPLE 7 - FP9 and MVA do not elicit cross-reactive T-cells against viral antigens
  • MVAPbCSP immune splenocytes recognise naive cells infected with MVA but not FP9 ( Figure 8).
  • IFN ⁇ secreting T-cell response can be observed when FP9PbCSP immune splenocytes are exposed to spleen cells infected with MVAPbCSP and vice versa ( Figure 8).
  • the IFN ⁇ response was determined by exposing splenocytes isolated from immune animals to naive splenocytes pulsed with Pb9 peptide or those infected with FP9, FP9PbCSP, MVA, or MVAPbCSP.
  • Columns ( Figure 8) represent the mean number of IFN ⁇ spot forming cells (sfc) per million splenocytes ⁇ S.E.M. for three mice per group.
  • Wild-type and recombinant poxvimses are usually grown in vitro in cultured chicken embryo fibroblasts (CEFs).
  • CEFs chicken embryo fibroblasts
  • Primary "non-cultured" CEFs can be obtained by loading freshly prepared CEFs into tissue culture dishes at a concentration sufficient to give a confluent monolayer without growth.
  • FP9 and recombinant FP9 forms visible viral plaques and give good yields of vims (Table 5) following infection of monolayers of primary "non-cultured” CEFs.
  • plaque formation and higher yields of attenuated canarypox vims, ALVAC have been obtained using primary "non-cultured” CEFs by the present inventors.
  • Phase lib study is conducted in Gambian adults using the same immunisation regime. Volunteers are monitored for enhanced immune responses and evidence of blood stage infection during the course of the trial, which ns through the malaria season (June to December).
  • phase I studies are also conducted in the Gambia during 2002 using appropriate recombinant FP9 and MVA to vaccinate against Mycobacteria tuberculosis and HIV. Both trials are conducted using volunteers who are infected with the respective pathogens and, therefore, evalute both the capacity of FP9 and a heterologous prime- boost immunisation regime using two non-replicating vimses to serve as a therapeutic vaccine.
  • vaccination according to the present invention produces and/or enhances protective response in humans.
  • EXAMPLE 1 1 - Boosting the T-cell response against Ag85A primed by M. bovis BCG using non-replicating recombinant poxviruses
  • M. bovis BCG is widely administered to children throughout the developing world as a vaccine against tuberculosis caused by M. tuberculosis. Although effective against severe childhood forms of tuberculosis, the protective efficacy of M. bovis BCG against adult forms of the disease is highly variable and thought to wane over time. Since Ag85A is a major secreted antigen of both M. bovis BCG and M. tuberculosis, boosting the immune response elicited against Ag85A by M. bovis BCG immunization is a credible strategy for enhancing the protective efficacy of this vaccine against adult tuberculosis.
  • bovis BCG elicited a variable and short-lived enhancement of the immune response against PPD and Ag85A protein.
  • subsequent boosting with FP9Ag85A significantly enhanced the T-cell immune response against these proteins ( Figure 10) as well as against peptide pools spanning the Ag85A protein sequence.
  • Further experiments determine the nature and location of the T-cell response elicited in the immunized animals.
  • non-replicating vimses can be used to boost the immune response primed by M. bovis BCG in mice and in primates.
  • non-replicating poxvimses may be effective as therapeutic vaccines against persistent latent infection with M. tuberculosis in man.
  • mice were immunized intradermally (i.d.) with M. bovis BCG and boosted 12 weeks later id. with 1 X 10 6 PFU of MVA85A.
  • Control animals were immunized with M. bovis BCG or MVA85A alone.
  • T-cell immune responses were determined against PPD and Ag85A using the P15 (CD4+) and Pll (CD8+) epitopes in an IFN ⁇ ELISPOT assay 12 days following immunisation.
  • Columns ( Figure 9) represent the mean IFN- ⁇ spot forming cells (SFC) per million splenocytes ⁇ the standard error of the mean for three mice per group.
  • Ag85B (which shares approximately 80% homology with Ag85A) or purified protein derivative from M. tuberculosis (PPD) in an IFN ⁇ ELISPOT assay.
  • Curves ( Figure 10) represent the IFN- ⁇ spot forming cells (SFC) per million PBMCs from a single animal.
  • the ME response is predominantly to CD8 T cell epitopes.
  • the T996 response is the the homologous strain of TRAP, the 3D7 response to a heterologous strain of TRAP.
  • FP9-ME.TRAP boosts a DNA primed T cell response. It is also clear that a further immunisation with MVA-ME.TRAP enhances responses induced by the DDF regime.
  • FP9 provides an effective boost to T cell responses in human primates.
  • Malaria-na ⁇ ve human volunteers were immunised with a FP9-MVA prime-boost regime as follows: In this Example there are two cohorts, one of five vaccinees and one of 12 vaccinees.
  • FP9-ME.TRAP was administered intradermally at lxl 0e8 pfu at day 0 FP9-ME.TRAP was administered intradermally at lxl 0e8 pfu at day 21 MVA-ME.TRAP was administered intradermally at a dose of 1.5x10e8 pfu at day 49.
  • Figure 12 shows the calculated reduction in liver stage parasites resulting from vaccination assuming that parasites multiply 8 fold every 48 hours in peripheral blood.
  • the "late FFM" group represents the two rechallengees (see above) and a further volunteer challenged for the first time at 5 months post vaccination (the FFM group were challenged 13-50 days post the last vaccination.
  • results for four vaccinees administered only a single priming immunisation with FP9 (“FM").
  • the significance levels are calculated from log rank test Kaplan-Meier analysis of times to parasitiaemia in vaccines and non- vaccinated challenge controls.
  • fowlpox such as FP9 is an effective prime as well as an effective boost in the methods of the present invention.
  • DNA (2mg i.m.) administered two (DDM) or three (DDDM) times at a 3-4 week intervals followed by MVA-ME.TRAP 1.5x10e8 id (DDM group - In Figure 13 the DDM and DDDM vaccinee subgroups are merged as there was no significant difference between these.)
  • treatment of subjects according to the present invention produces protective immune responses in said subjects.
  • primates are Macaques, animals naturally susceptible to Mycobacterium tuberculosis.
  • EXAMPLE 16 The FP9 strain is more potent than Webster's FPV-M in priming and boosting CD8 T cell reponse.
  • mice Female BALB/c mice (6 - 8 weeks old) were immunised intradermally (id.) with
  • Viruses were diluted to 2 X 10 PFU/ml in pyrogen free PBS, characterised as described in EXAMPLES 1 and 2 of the patent application, and administered id. by bilateral injection of 25ul into each ear of a mouse. Fourteen days after immunisation, the mice were sacrificed by cervical dislocation and the T cell response elicited against the Pb9 epitope of PbCSP and a control epitope from ⁇ -galactosidase were determined using the IFN ⁇ assay as described below:
  • BioRad AP Conjugate Substrate kit (170-6432 ). For one plate:
  • Coating plates coat MAIP multiscreen plates (Millipore MAIPS4510) with rat anti-mouse IFN ⁇ (Mab AN 18) antibody. Dilute to lO ⁇ g/ml in Phosphate
  • Blocking plates Flick off coating antibody and wash plates once with 150 ul of sterile PBS (Sigma P-3813) per well using a multi-channel pipette. Flick off the PBS, add lOOul complete ⁇ -MEM medium per well, and incubate at room temperature for 1+ hour. It is important to keep the plates sterile at this stage.
  • Plate layout may be chosen by the operator according to the needs of the particular experiment.
  • test peptide and control peptide to twice the desired final concentration to naive splenocytes at 10 million/ml in complete ⁇ -MEM medium.
  • Add 50 ⁇ l of control peptide and target cells to columns 1, 5 and 9.
  • Results were calculated as the number of antigen-specific IFN ⁇ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the students T-test (two-sample assuming equal variances) using Microsoft Excel 2000.
  • P 0.002
  • P boosting
  • MVAPbCSP MVAPbCSP
  • FP9PbCSP proved to be more potent than FPV-MPbCSP as a priming and/or boosting agent, and is equally potent as a priming or boosting agent in combination with recombinant MVA.
  • a synthetic gene of 440 bp encoding for a novel polypeptide (ME1) consisting of characterised CD8+ and CD4+ restricted epitopes from lymphocytic choriomeningitis vims (LCMV) antigens and murine tumour (P815 and CT26) antigens was synthesised as follows:
  • T cell epitope string encoding tumour and vims epitopes relevant in murine models of chronic infection (LCMV) and cancer (P815/CT26) is generated as set out below.
  • LCMV-derived epitopes are H-2 -restricted and the cancer epitopes are H-2 -restricted.
  • the order of the epitopes alternates between H-2 d and H-2 b .
  • the epitope string does not contain Apal nor Ascl target sequences.
  • the epitope string does not contain the pox vims early gene transcription termination sequence, TTTTTNT.
  • the ME1 epitope string was ligated into a DNA vaccine vector (PSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.MEl was subsequently recombined into the chromosome of FP9 following established methods to constmct FP9.ME1.
  • This vims was subsequently bulk-purified by propagation in chicken embryo fibroblasts (CEF) and centrifugation through a 30% sucrose cushion. The titre of the vims was confirmed by titration on CEF and X-gal staining, following established methods. Vimses were prepared for immunisation by suspension in pyrogen free PBS at 1 X 10 7 PFU/ml. Plasmid PSG2.ME1 was bulk-purified using a Qiagen Giga column and resuspended at 1 mg/ml in pyrogen free PBS.
  • mice Female BALB/c (H-2 d ) C57BL/6 (H-2 b ) mice (6 - 8 weeks old) were immunised intramuscularily (im.) with PSG2.ME1 or a control plasmid pSG2.Mel3, which contains an irrelevant epitope string.
  • Vimses FP9.ME1 and the empty control vims FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 10 6 PFU. Animals were boosted two weeks after immunisation.
  • mice Fourteen days after boosting, the mice were sacrificed by cervical dislocation and the T cell response elicited against the LCMV, P815, CT26 and control epitopes from ⁇ -galactosidase were determined using the IFN ⁇ assay as described in Example 16.
  • Results were calculated as the number of antigen-specific IFN ⁇ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the students T-test (two-sample assuming equal variances) and ANOVA using Microsoft Excel 2000.
  • ME1 encodes epitopes from LCMV antigens characterised in H-2 b haplotype mice and those from tumour antigens characterised in H-2 d haplotype mice. Accordingly, the capacity of FP9.ME1 to elicit CD4+ and CD8+ T - cell epitopes was tested in both BALB/c (H-2 d ) C57BL/6 (H-2 d ) mice as described in Table 6. The results of these experiments are shown in Table 6.
  • Experiments 1 and 2 were conducted in parallel. Each group contained four 6 - 8 week-old female mice. Vaccines were administered as described in Methods and priming and boosting were 14 - 16 days apart. T- cell responses were determined by the IFN ⁇ ELISPOT assay as in Example 16 using the LCMV, tumour and model epitopes described above. Results relate to the cumulative response against the LCMV and tumour derived epitopes, which are based on observations using the murine malaria and tuberculosis models.
  • mice Female BALB/c (H-2 d ) mice (6 - 8 weeks old) will be injected with pSG2.MEl, FP9.ME1, or control constmcts pSG2.Mel3 and FP9.EFL29 as described above. Animals are boosted two weeks after immunisation. Fourteen days after boosting, the mice are challenged by subcutaneous (sc.) injection in the left flank with 5 X 10 5 CT26 tumour cells as follows:
  • CT26 tumours are amenable to this experimental approach. Although conventional wild-type CT26 cells grow in a laterally spreading fashion in the subcutaneous space, the highly transferable variant, CT26, grows well as a more compact tumor mass, probably related to increased adhesion properties, making for more reproducible measurements.
  • the procedure can be accomplished rapidly by a single operator.
  • the mouse is held in the left hand with the scruff of the neck between the first two fingers and the tail held against the palm with the fourth finger.
  • the syringe is held and the needle inserted just through the skin on the left flank of the mouse.
  • the skin should be gently lifted away from the body of the mouse to ensure that the tip of the needle is within the subcutaneous space. Only then is the 50 ⁇ l volume expelled.
  • mice at least two times a week to examine for tumour growth and to assess general health. Once tumours appear, measure and record the longest and shortest dimensions using callipers.
  • Tumours are first palpable 1 week after implantation. Measurements should be made to the nearest 0.5 mm.
  • Tumour size is determined by measuring the length and the width of the tumour, then taking the average of these measurements.
  • Statistical analysis is performed using GraphPrism Instat, with differences in tumour incidence determined by the Fishers Exact test and differences in tumour size using the students T-test (two-sample assuming unequal variances).
  • CT26 is a colonic carcinoma cell line derived from BALB/c mice.
  • CT26 forms a solid tumour in BALB/c mice approximately 1 week after subcutanteous injection.
  • the MEl epitope string contains a protective CD8+ restricted epitope derived from the MuLV gp70 envelope protein that is expressed by the CT26 tumour (see above). Since the prime/boost immunisation regime using pSG2.MEl followed by FP9.ME1 elicits an enhanced CD8+ T cell immune response against this epitope (see above), we showed that this immunisation regime elicits protection against CT26 challenge in BALB/c mice. Table 7 shows the results.
  • mice Groups of 12 BALB/c mice are primed and boosted 14 days apart as shown. Fourteen days after boosting the animals are challenged by sc. injection with CT26 cells in the left flank. The animals are observed for two weeks, then dissected and tumour presence and size determined as described in Methods.
  • mice Female C57BL/6 (H-2 b ) mice (6 - 8 weeks-old) were injected with PSG2.ME1, FP9.ME1, or control constmcts pSG2.Mel3 and FP9.EFL29 as described above. Animals were boosted two weeks after immunisation. Fourteen days after boosting, 6 animals per group were challenged by intraperitoneal (ip.) injection with 2 X 10 5 PFU LCMV Armstrong (mild infection) and 6 animals iv. with 2 X 10 6 PFU LCMV Clone 13 (severe infection).
  • Viral load in the spleens of challenged animals is determined as follows:
  • LCMV is a well characterised vims that causes chronic and acute infections in mice.
  • the Armstrong strain causes a mild, self-resolving infection
  • the Clone 13 strain causes a severe infection that can develop into a chronic infection.
  • the MEl epitope string was designed to contain two CD4+ epitopes and several CD8+ restricted epitopes from LCMV (see above), some of which have been characterised as protective against LCMV infection. Since prime/boost immunisation using FP9.ME1 elicits a significantly enhanced T cell response against the LCMV epitopes in MEl ( Figure 17), this immunisation regime exhibits protective efficacy against challenge with both the Armstrong and Clone 13 strains of LCMV. Table 8 shows the results.
  • mice Groups of 12 C57BL/6 mice are primed and boosted 14 days apart as shown. Fourteen days after boosting, 6 animals per group will be challenged with LCMV Armstrong (mild infection) or LCMV Clone 13 (severe infection) as described in Methods. The limit of detection of the assay is 1000 pfu/organ (Logio 3.00). Viral loads for groups A, B and C are based on results using 6 animals per group. Viral loads for D, E and F are on the immunogenicity results presented in Figure 17).
  • Figure 15 shows antigen-specific immune responses following heterologous prime/boost immunisation with FP9PbCSP, FPV-MPbCSP and MVAPbCSP.
  • BALB/c mice were immunised id. bi-laterally in the ears with FP9PbCSP (FP9), FPV-MPbCSP (FPV-M), or MVAPbCSP and boosted heterologously 14 days later in a similar manner.
  • Fourteen days after the booster immunisation the T cell response elicited against the Pb9 epitope of PbCSP and a control epitope from ⁇ -galactosidase were determined in splenocytes using the IFN ⁇ ELISPOT assay.
  • Columns represent the mean antigen specific IFN ⁇ sfc/million splenocytes ⁇ 1 SD for four mice per group. P values were determined by the student's T test assuming equal variances using Microsoft Excel 2000.
  • Example 20 Websters' Attenuated Fowlpox is Disctinct from FP9
  • This Example the unique character of FP9 compared to existing fowlpox vectors is demonstrated.
  • the genetic composition of FP9 is determined as above and compared to existing pox vector 'Websters'. Data are presented in Table 9.
  • Example 21 Recombinant FP9 elicits CD4 and CD8 T cell responses against epitopes from viruses and tumours
  • Example 17 further determines whether immunisation with recombinant FP9 alone, or in a prime/boost regime, will elicit T cell responses against CD4+ and CD8+ T cell epitopes from viral and tumour antigens.
  • MEl novel polypeptide
  • LCMV lymphocytic choriomeningitis vims
  • P815 and CT26 murine tumour antigens
  • MEl was ligated into a DNA vaccine vector (pSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.MEl was subsequently recombined into the chromosome of FP9 following established methods to constmct FP9.ME1.
  • This vims was subsequently bulk-purified by propagation in chicken embryo fibroblasts (CEF) and centrifugation through a 30% sucrose cushion. The titre of the virus was confirmed by titration on CEF and X-gal staining, following established methods. Vimses were prepared for immunisation by suspension in pyrogen-free PBS at 1 X 10 7 PFU/ml. Plasmid pSG2.MEl was bulk-purified using a Qiagen Giga column and resuspended at 1 mg/ml in pyrogen free PBS.
  • mice Female BALB/c (H-2 d ) or C57BL/6 (H-2 b ) mice (6 - 8 weeks old) were immunised intramuscularly (im.) with 50 ⁇ g of pSG2.MEl or control plasmid pSG2.Mel3, which contains an irrelevant epitope string.
  • Vimses FP9.ME1 and the empty control vims FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 10 6 PFU. Animals were boosted 14 - 15 days after immunisation.
  • mice Fourteen to 15 days after boosting, the mice were sacrificed by cervical dislocation and the T cell response elicited against the LCMV, P815, CT26 and control epitopes from ⁇ -galactosidase were determined using the IFN ⁇ assay as described in above. Results were calculated as the number of antigen-specific IFN ⁇ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the student's T-test (two-sample assuming unequal variances) using Microsoft Excel 2000. One-sided -values are given in all cases.
  • MEl encodes epitopes from LCMV antigens characterised in H-2 haplotype mice and those from tumour antigens characterised in H-2 d haplotype mice (Appendix II). Accordingly, the capacity of FP9.ME1 to elicit immune responses was tested in both BALB/c (H-2 d ) and C57BL/6 (H- 2 b ) mice.
  • heterologous prime/boost immunisation regimes using pSG2.MEl and FP9.ME1 elicited greatly enhanced immune responses against CD8+ epitopes characterised as dominant and subdominant, and those recognised by CD4+ T cells when compared to the homologous immunisation regimes using pSG2.MEl and FP9.ME1 alone.
  • this prime/boost immunisation regimes is likely to be even more efficacious against LCMV infection in C57BL/6 mice than the homologous prime/boost immunisation regimes (See above).

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Abstract

The present invention relates to a fowlpox virus genome which has modifications in one or more wild-type FPV genes. The present invention also relates to a viral particle comprising such a genome and its use to deliver a nucleotide of interest (NOI) to a target cell. The present invention also relates to vaccination methods, particularly a method which comprises administering a priming composition (which comprises a first non-replicating viral vector) and a boosting composition (which comprises a second non-replicating viral vector) to a subject to treat and/or prevent a disease.

Description

VACCINE
The present invention relates to pox viruses. In particular, the present invention relates to a fowlpox virus genome which has a modification in one or more wild-type FPV genes, a viral particle comprising such a genome, and its use to deliver a nucleotide of interest ("NOI") to a target cell.
The present invention also relates to vaccination methods using viral vectors, in particular heterologous prime-boost vaccination regimes employing two different non- replicating viral vector compositions.
BACKGROUND
Poxviruses
Poxviruses have been previously exploited as recombinant vectors for the heterologous expression of foreign proteins. In particular, recombinant vaccinia virus has been studied as a tool for transient expression of genes in mammalian cells and an experimental recombinant vaccine vector (reviewed by Moss, 1991, Proc Natl Acad Sci USA 93, 11341-8; and Moss, 1996, Proc Natl Acad Sci USA 93, 11341-8).
In common with the other poxviruses, vaccinia virus resides within the cell cytoplasm where it expresses the proteins needed for viral replication. Recombinant vaccinia can, therefore, deliver foreign antigens to the cytoplasm of mammalian cells, thereby allowing them direct access to antigen processing pathways which leads to presentation of antigen derived peptides in association with MHC Class I and Class II molecules on the cell surface (Moss, 1991, Proc Natl Acad Sci USA 93, 11341-8). This property makes vaccinia useful as recombinant vaccines, particularly for stimulating CD8+ and CD4+ T-cell immune responses. Concern about the capacity of vaccinia virus to replicate in mammalian cells has limited its clinical use and led to the search for safer alternatives. These include attenuated vaccinia viruses, such as modified vaccinia Ankara (MVA) (Meyer et al, 1991, J Gen Virol 72, 1031-8; Sutter and Moss, 1992, Proc Natl Acad Sci USA 89, 10847-51 ; Sutter et al, 1994, Vaccine 12, 1032-40), which undergoes limited replication in human cells (Blanchard et al, 1998, J Gen Virol 79, 1159-67), and the avipox viruses, such as fowlpox, which do not proliferate in mammalian cells (Somogyi et al, 1993, Virology 197, 439-44).
Wild-type fowlpox viruses, which cause proliferative skin lesions that are rarely lethal in birds, are of commercial concern in the poultry industry. Live attenuated vaccines against fowlpox virus have been produced by multiple passage of the virus in avian cells. Such attenuated fowlpox viruses expressing antigens from poultry pathogens have been extensively exploited as recombinant vaccines for avian use (reviewed by Boyle and Heine, 1993, Immunol Cell Biol 71, 391-7; Paoletti, 1996, Proc Natl Acad Sci USA 93, 11349-53). In fact, two recombinant fowlpox viruses expressing antigens from Newcastle's disease virus are commercially available for veterinary use in the USA (Paoletti, 1996, Proc Natl Acad Sci USA 93, 11349-53).
The observation that avipox viruses can express antigens in mammalian cells and can induce a protective immune response against mammalian pathogens (Taylor and Paoletti, 1998, Vaccine 6, 466-8; Taylor et al, 1998a, Vaccine 6, 504-8; Taylor et al, 1988b, Vaccine 6, 497-503), led to the development of recombinant fowlpox viruses as vaccines for use in mammals. Most significantly, recombinant fowlpox expressing antigens from HIV have shown promise as vaccines in non-human primates (Dale et al, 2000, JMed Primatol 29, 240-7; Kent et al, 1988, J Virol 72, 10180-8; Kent et al, 2000, Vaccine 18, 2250-6). In addition, recombinant fowlpox vaccines encoding tumour-associated antigens have been evaluated in animals (Grosenbach et al, 2001, Cancer Res 61, 4497-505; Irvine et al, 1997, JNatl Cancer Inst 89, 1595-601; Wang et al, 1995, J Immunol 154, 4685-92) and are presently undergoing human clinical trials. The majority of attenuated fowlpox vaccine strains are not fully defined in terms of their genome organisation and exact sequence. In fact, the genomes of some have recently been found to carry an infectious copy of the provirus of avian reticuloendotheliosis virus (REV) (Hertig et al, 1997, Virology 235, 367-76) which may limit their use as recombinant vectors.
There is an upper limit on genome size for vectors derived from pox viruses. For vaccinia, it is thought that the maximum size of heterologous sequence that can be effectively packaged and delivered is 10% of the size of the genome.
Thus there is a need for an improved vector system, which lacks the capacity to replicate in mammalian cells, but which is better characterised, is better at eliciting T- cell immune responses, has an improved capacity to accommodate and deliver heterologous DNA and/or has improved safety over known attenuated fowlpox vaccine strains.
Vaccination strategies
There are numerous methods known in the art to stimulate an immune response in a subject in order to prevent and/or treat a disease. Examples of antigenic preparations used as vaccines are shown in the following table (Table 1).
Table 1
Figure imgf000004_0001
Figure imgf000005_0001
There are also other types of non-antigen based immunisation, which include passive immunisation (the direct administration of antibodies) and non-specific immunisation (such ad by the administration or cytokines or cytokine inhibitors).
A problem with many of these approaches is that the immune response wanes over time, such that it is no longer effective, for example in controlling or eradicating an infection.
It is important that a vaccine induces the right sort of immune response for the disease. Many known vaccines are useful for generating antibodies, but do not induce significant cell-mediated immune responses. A number of diseases are particularly susceptible to prevention and/or treatment by a T cell immune response. For example, cytolytic CD8+ T cells may protect against or help to clear viral infections. Also, in the case of diseases such as tuberculosis, malaria and H.pylori infection there is evidence for a protective role for CD4+ T cells which can secrete IFNγ.
Some known viral vaccination strategies are associates with a number of complications and side effects. For example smallpox vaccination can cause generalised vaccinia, eczema vaccinatum, progressive vaccinia, and neurological and cardiac complications (Feery (1977) Med J. Aust 6 180-183; Goldstein et al (1975) Pediatrics 55 342-7)
There is thus need for improved vaccination strategies, particularly those capable of stimulating or boosting the T-cell arm of the immune system which cause a minimum of adverse reactions.
SUMMARY OF THE INVENTION
The present inventors have obtained the full genomic sequence of an attenuated fowlpox virus strain (FP9).
FP9 lacks (or has modifications in) a number of genes present in wild-type fowlpox virus (FPV). The genome of FP9 is 266 kbp, which is smaller than the genome for FPV-M, a fowlpox vaccine strain which has previously been described as a vector system (Coupar et al (1990) Virology 179 159-167). The present inventors have shown that FP9 is superior to FPV-M is its capacity to elicit CD8+ T-cell immune responses.
Thus the present invention provides an attenuated Fowlpox virus genome which has a modified form of one or more of the following wild-type FPV genes (gene nomenclature according to Afonso et al (2000) J. Virol 74 3815-3831):
FPV001, FPV018, FPV054, FPV063, FPV066, FPV070, FPV071, FPV093, FPV097/098, FPV115, FPV124, FPV125, FPV127, FPV158, FPV159, FPV160,
FPV 190, FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260.
The present invention also provides a method for attenuating fowlpox comprising modifying one or more of said genes. Preferably said genes are modified as described in more detail below. The present invention also provides an attenuated fowlpox virus genome which is less than 275 kbp in size. There is an upper limit on the total size of genome which can be efficiently packaged by the virus. If the genome itself is small, larger the amount of heterologous sequence it can carry.
In a preferred embodiment, the attenuated Fowlpox virus genome comprises the sequence shown in SEQ ID No. 1.
The attenuated Fowlpox genome of the present invention may also comprise a nucleotide of interest "NOI". The "NOI" may be a therapeutic gene.
The present invention also provides a viral particle which comprises such a genome. Where the genome comprises an NOI, preferably the viral particle is capable of delivering the NOI to a target cell. Alternatively (or in addition) the viral particle may be capable of delivering a pre-expressed protein to a target cell.
The present invention also provides a vaccine, priming or boosting agent comprising such a genome or viral particle.
The present invention also provides a boosting composition comprising a non- replicating viral vector. In a preferred embodiment, the composition is capable of boosting the immune response primed by M. bovis BCG in primates.
For vaccination (and treatment) purposes, multiple-dose procedures are often more effective at generating an immune response than a single administration of vaccine. Prime-boost regimes may be homologous (where the same composition is administered two or more times) or heterologous.
The present invention also provides a vaccination kit which comprises: (i) a first composition which comprises an FP9 fowlpox viral particle; and
(ii) a second composition for simultaneous, separate or sequential administration. The use of viral vectors in heterologous vaccination regimes where either the priming or boosting agent is a DNA-vaccine has previously been recognised (Schneider et al (1998) Nature Medicine 4(4) 397-402; Kent et al (1998) J. Gen. Virol 72(12) 10180-8; Robinson et al (1999) Nature Medicine 5(5): 526-534).
However, the present inventors have shown for the first time that heterologous prime boost regimes using two different non-replicating viral vectors are surprisingly effective at inducing a T cell immune response in primates. The use of non-replicating vectors avoids the adverse reactions associated with replicating virus (such as smallpox, see above).
Thus the present invention also provides a vaccination kit which comprises:
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector for sequential administration in either order.
In a preferred embodiment the vaccination kit comprises: (i) a first composition which comprises a first non-replicating poxvirus vector;
(ii) a second composition which comprises a second non-replicating poxvirus vector for sequential administration in either order.
The kit may be suitable for administration to a primate subject in order to treat and/or prevent a disease.
In the kits of the present invention, preferably the first and second compositions are capable of expressing the same antigen. The present invention also provides a vaccination method which comprises the step of administering such a vaccine, priming or boosting composition or kit to a subject. Such administration should elicit a T-cell immune response in the subject.
The vaccination method may be used to treat or prevent, for example, diseases caused by or due to a chronic infection such as HIV, malaria, tuberculosis and East Coast Fever.
When one or more of the genome, viral particle, vaccine, priming agent/composition, boosting agent/composition, construct or kit (s) of the present invention involve or are at least in part derived from HIV, then said genome, viral particle, vaccine, priming agent/composition, boosting agent/composition, construct or kit (s) are not those disclosed in WO 02/068654 (corresponding to PCT/CU02/00001). In particular, the present invention does not involve the recombinant CR3 gene as described in WO 02/068654 such as in Example 1 of WO 02/068654.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Annotated genome of FP9
Figure 2: A table showing the genes modified in FP9 during its passage in CEF tissue culture from HP 1.
Figure 3 : Comparison of the T-cell immune response elicited by recombinant FP9 and FPV-M
Figure 4: Comparison of the CD8+ T-cell immune response elicited by recombinant FP9, MVA and a DNA vaccine encoding PbSCP
Figure 5: Fowlpox FP9 can serve as both a priming and boosting agent in heterologous prime-boost immunisation regimes Figure 6: Fowlpox FP9 can elicit both CD4+ and CD 8+ T cell responses against M. tuberculosis Ag85A in Balb/c mice.
Figure 7: Fowlpox FP9 elicits a T-cell immune response against M. tuberculosis Ag85A in non-human primates
Figure 8: Ex vivo IFNγ ELISPOT responses against FP9 and MVA viral antigens.
Figure 9: Ex vivo IFNγ Elispot response in Balb/c mice following aerosol or mucosal priming with M. bovis BCG and boosting with MVA85A.
Figure 10: Ex vivo IFNγ Elispot response in Rhesus macaques following aerosol or mucosal priming with M. bovis BCG and boosting with MVA85A and FPAg85A.
Figure 11 shows a bar chart. Figure 12 shows a bar chart. Figure 13 shows two bar charts. Figure 14 shows a graph. Figure 15 shows a bar chart.
Figure 16. shows Specific immune responses in BALB/c mice following prime/boost immunisation with pSG2.MEl and FP9.ME1. BALB/c mice were immunised im. with pSG2.MEl (DNA) or iv. with FP9.ME1 (FP9) and boosted 15 days later in a similar manner. Control animals were immunised with identical vectors encoding irrelevant antigens. Thirteen days after the booster immunisation the T cell responses elicited against the tumour epitopes were determined in splenocytes using the IFNγ ELISPOT assay. Columns represent the antigen-specific IFNγ sfc/million splenocytes ± 1 SD for four mice per group. Figure A depicts the summed epitope-specific responses and the response elicited against phytohemagglutinin (PHA) for each group, whereas B depicts the response against individual epitopes. Figure 17. Shows specific immune responses in C57BL/6 mice following prime/boost immunisation with pSG2.MEl and FP9.ME1. C57BL/6 mice were immunised im. with pSG2.MEl (DNA) or iv. with FP9.ME1 (FP9) and boosted 14 days later in a similar manner. Control animals were immunised with identical vectors encoding irrelevant antigens. Fourteen days after the booster immunisation the T cell response elicited against the tumour epitopes were determined in splenocytes using the IFNγ ELISPOT assay. Columns represent the antigen-specific IFNγ sfc/million splenocytes ± 1 SD for four mice per group. Figure A depicts the summed epitope-specific responses and the response elicited against phytohemagglutinin (PHA) as a positive control for each group, whereas B depicts the response against individual epitopes. The legend of B indicates immunodominant (DOM) and subdominant (SUB) epitopes that are recognised by CD8+ T cells, as well as those recognised by CD4+ T cells (CD4).
Figure 18 shows the mean interferon gamma response to PPD at various weeks after intratracheal M. tuberculosis challenge of cynomolgous macaques.
DETAILED DESCRIPTION OF INVENTION
Viruses and viral vectors
The present invention relates to vaccination regimes using non-replicating viral vectors.
Many viral vectors are known in the art which are capable of delivering an NOI via infection of a target cell. Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, poxviral vectors or parvovirus vectors (see Kestler et Λ/1999 Human Gene Ther 10(10):1619-32).
Examples of retroviruses include but are not limited to: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
A detailed list of retroviruses may be found in Coffin et /("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758- 763).
Poxviruses
In a prefferd embodiment the present invention provides a vaccine, priming or boosting composition which comprises a non-replicating pox virus vector.
The family of poxviruses can be split into two subfamilies, the Chordopoxvirinae and the Entomopoxviriniae. The Chordopoxvirinae (poxviruses of vertebrates) include orthopoxviruses, parapoxviruses, avipoxviruses, caripoxviruses, leporipoxviruses, suipoxviruses, molluscipoxviruses and yatapoxviruses. A review of poxviruses, their structure and organisation, biological and antigenic properties is given in Murphy et al (1995) Virus Taxonomy Springer Verlag, Vienna pp79-87. The following table (Table 2) gives some examples of species within each genus of the pox virus family.
Table 2
Figure imgf000012_0001
Figure imgf000013_0001
The present invention provides a vaccination kit which comprises:
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector. for sequential administration in either order.
The first and/or the second viral vector may be a pox virus vector.
In a preferred embodiment the present invention provides a vaccination kit which comprises:
(i) a first composition which comprises a first non-replicating poxvirus vector;
(ii) a second composition which comprises a second non-replicating poxvirus vector. for sequential administration in either order.
One of the compositions may act as a "priming" composition, to be administered first, and the other composition may act as a "boosting" composition, to be administered after an appropriate time interval (such as three weeks). The first and second non-replicating viral vectors should be sufficiently different that no significant cross-reaction occurs.
The two viral vectors may be derived from viruses belonging to different familes, for example, a poxviral vector and an adenoviral vector. Alternatively the two viral vectors may be derived from viruses belonging to the same family (such as pox viruses) but different geni. For example, the first non-replicating poxvirus vector may be an avipox virus vector and the second non-replicating poxvirus vector may be an orthopox virus vector.
The two non-replicating viral vectors may even be derived from different species within the same genus, as long as the species are sufficiently different.
Distinguishing features of each pox virus genus are known in the art, for example see Murphy et al ( 1995 - as above).
In a preferred embodiment, one of the two non-replicating poxvirus vectors is a fowlpox virus vector (i.e. derived from a fowlpox). For example, the fowlpox virus vector may comprise a genome according to the present invention.
Non-replicating
The virus vectors used in the present invention should be non-replicating in the cells of the subject (for example in human cells). The term "non-replicating" or "replication- impaired" as used herein means not capable of replication to any significant extent in the majority of normal subject cells. Viruses which are non-replicating or replication- impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication. There will generally be one or a few cell types in which the viruses can be grown, such as CEF cells. Replication of a virus is generally measured in two ways: 1) DNA synthesis and 2) viral titre. More precisely, the term "nonreplicating or replication-impaired" as used herein and as it applies to poxviruses means viruses which satisfy either or both of the following criteria: 1) exhibit a 1 log (10 fold) reduction in DNA synthesis compared to the
Copenhagen strain of vaccinia virus in MRC-5 cells (a human cell line);
2) exhibit a 2 log reduction in viral titre in HELA cells (a human cell line) compared to the Copenhagen strain of vaccinia virus.
Fowlpox viruses
As mentioned above, wild-type fowlpox viruses cause proliferative skin lesions in birds. It is possible to obtain field isolates of FPV from scab material collected from poultry infections (Boyle et al (1997) Arch. Virol. 142:737-748). The genomic sequence of a pathogenic fowlpox virus is available (Afonso et al (2000) J. Gen. Virol. 74(8) 3815-3831).
Live attenuated virus strains may be produced by multiple passage of the virus in avian cells. Various attenuated virus strains of fowlpox virus are known such as FPV M (mild vaccine strain) and FPV S (standard vaccine strain) obtainable from Cyanamid- Websters PtY, Ltd Australia.
Pox viruses have evolved strategies for evasion of the host immune response that include the production of secreted proteins that function as soluble receptors for tumour necrosis factor, IL-I p, interferon (IFN)-oc/ andlFN-y, which normally have sequence similarity to the extracellular domain of cellular cytokine receptors (such as chemokine receptors). These viral receptors generally inhibit or subvert an appropriate host immune response, and their presence is associated with increased pathogenicity.
FPV genes The genome of a fowlpox virus is composed of a single double-stranded DNA molecule with covelently linked terminal hairpins. Basic information about the sites for restriction endonuclease cleavage is available (Coupar et al (1990) as above).
The genome of US dept Agric Standard Challenge (virulent) fowlpox strain has been sequenced and the nomenclature used for this strain is followed herein (Afonso et al (2000) J. Virol. 74 3815-1831). This review also lists the 260 ORFs of this fowlpox strain (Table 1) together with their predicted structure and/or function and Accession number.
The present inventors have sequenced the virulent precursor of FP9 (HPl) at all positions where the sequence of FP9 differed from this sequence. This method reveals differences which are attributable to lineage variations and those which have accored during tissue culture passage, adaptation and concomittant attenuation. The genes modified in FPl during its passage in CEF tissue culture from HPl is shown in Figure 2.
The fowlpox virus genome of the present invention may have a modification in one or more of the following wild-type genes:
FPV001, FPV018, FPV054, FPV063, FPV066, FPV070, FPV071, FPV093, FPV097/098, FPV115, FPV124, FPV125, FPV127, FPV158, FPV159, FPV160, FPV190, FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260.
The term "modification" is intended to mean a variation (such as a deletion, substitution or addition) from the wild type sequence.
Where the gene encodes a protein, the modified gene sequence may encode a protein with a different amino acid sequence. For example, the amino acid sequence may have one or more amino acid deletions, additions or substitotions when compared to the wild-type sequence. In a preferred embodiment the fowlpox virus genome of the present invention has a modification which results in a non-conservative amino acid substitution in one or more of the following genes:
FPV018, FPV063, FPV066, FPV093, FPV127, FPV191, FPV207.
Alternatively (or in addition) the genome may have a substantial modification in one or more of the wild-type genes. The term "substantial modification" is intended to mean that the gene is modified in such a way that it no longer functions as the wild-type gene. For example, if the wild-type gene encodes a protein, the substantially modified gene may be incapable of encoding a protein, or it may encode a protein which is incapable of or has a greatly reduced capacity to function as the wild-type protein.
The modification may be a deletion. For example, the entire gene may be deleted. Alternatively, the modified gene may comprise one or more partial deletions sufficient to remove or greatly reduce the function of the gene.
Alternatively the modification may be a substitution or an addition. For example, a recombination event may have occurred, with the effect that portion of sequence from elsewhere in the genome is incorporated into the gene (with optionally a corresponding loss of wild-type sequence).
A partial deletion, substitution or addition may cause a "frame-shift" mutation, resulting in incorrect reading of the downstream sequence. Alternatively (or in addition) the mutation may result in the creation of a stop codon (a "terminating mutation") such that the downstream sequence is ignored.
The mutation may result in the removal of a stop codon, which may result in two genes becoming fused to form a chimaeric gene. In a preferred embodiment, the genome has a partial deletion in one or more of the following genes:
FPV158, FPV219, FPV222.
In another preferred embodiment, the genome entirely lacks one or more of the following genes:
FPV001, FPV124, FPV125, FPV159, FPV160, FPV220, FPV221, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260
In another preferred embodiment, the genome has a frame-shift mutation in one or more of the following genes:
FPV054, FPV070, FPV071, FPV115, FPV 190, FPV207.
In another preferred embodiment, the genome has a termination mutation in one or more of the following genes:
FPV071. FPV239
In another preferred embodiment, the genome has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV098.
Genome size
The genome of FPV-M has been estimated to be in the region of 308 kb (Coupar et al (1990) as above). The smaller the viral genome, the more heterologous DNA can be included.
The fowlpox virus genome of the present invention is preferably less than 275 kb in size. More preferably the genome is about 266 kbp. The size of the genome is considered without any heterologous sequences (for example a "nucleotide of interest" (NOI) see below). In order to use the fowlpox virus as a vector system, a heterologous sequence for delivery may be incorporated into the fowlpox genome by homologous recombination. For the purposes of size determination, the genome size is considered before such a recombination event.
Preferably the fowlpox virus genome according to claim 1 comprises the sequence shown in SEQ ID NO. 1. This "sequence" is intended to encompass homologues of the sequence, provided that the fowlox genome as a whole is capable of acting as a vector system (i.e. receiving a heterologous gene by homologous recombination and, when incorporated into a viral particle delivering the heterologous gene to a target cell).
Homologues
Here, the term "homologue" means a nucleic acid sequence having a degree of homology with the sequence shown in SEQ ID NO 1. Here, the term "homology" can be equated with "identity".
In the present context, an homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Typically, a homologue will comprise sequences that code for functional portions of an encoded protein (active sites etc.) which are the same as the subject sequence but may differ in other areas. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences. % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each base in one sequence is directly compared with the corresponding base in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following nucleic acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical bases, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for nucleic acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package
(University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of nucleic acid residues which produce a silent change and result in a functionally equivalent substance. If the gene encodes a protein, the protein encoded by a homologue may be identical (due to the degeneracy of the genetic code) or functionally equivalent (for example a conservative mutation may appear in the sequence). Preferably the homolog encodes a protein having at least 90%, preferably at least 95%, more preferably at least 98% homology to the protein encoded by the SEQ ID NO. 1 gene. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Figure imgf000022_0001
Fowlpox virus vectors
The present invention also relates to a viral particle comprising such a fowlpox virus genome.
The viral particle (or part therof) may be used as a vector system for delivering a nucleotide of interest (NOI) to a target cell. In this sense, the term "viral particle" as used herein includes a viral vector comprising such a particle (or part thereof) capable of delivering an NOI or a pre-expressed protein to a target cell, The NOI may be inserted into the viral genome by homologous recombination using methods known in the art (see for example Schneider et al (1998) Nature Medicine 4(4) 397-402).
The construction of recombinant poxviruses which express an NOI requires that the insertion of the NOI is made at a site which is nonessential for the replication of the viral genome. Suitable insertion sites for FP9 have been described (Pollitt et al (1998) 17: 5-9; Laidlaw et al 1998 J Virol 72 pp6742) Pre-expressed protein
The fowlpox viral particle of the present invention (or part thereof) may be used to deliver a pre-expressed protein to a target cell. A "pre-expressed protein" is a protein translated before it reaches the target cell. For example, the cell in which the viral particle is grown may express a protein which is then incorporated into the viral particle.
The cell in which the viral particle is grown may be engineered to express one or more proteins of interest (POI). If the cell is a primary cell (such as a CEF cell) it may be transiently transfected with a nucleotide encoding such a POI. Alternatively, a stably tranfected cell line may be used for growth, such as the quail cell line QT35.
Preferably the pre-expressed protein is targetted to be incorporated into the viral particle.
NOI
In the present invention, the term NOI includes any suitable nucleotide sequence, for example, a synthetic RNA DNA sequence, a recombinant RNA/DNA sequence (i.e. prepared by use of recombinant DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including combinations thereof. The sequence need not be a coding region. If it is a coding region, it need not be an entire coding region. In addition, the RNA/DNA sequence can be in a sense orientation or in an anti-sense orientation. Preferably, it is in a sense orientation. Preferably, the sequence is, comprises, or is transcribed from cDNA.
The NOI may encode a protein of interest ("POI"). In this way, the vector system could be used to examine the effect of expression of a foreign gene on the target cell. For example, the fowlpox virus delivery system could be used to screen a cDNA library for a particular effect on the target cell. The NOI may be capable of blocking or inhibiting the expression of a gene in the target cell. For example, the NOI may be an antisense sequence. The inhibition of gene expression using antisense technology is well known.
The NOI or a sequence derived from the NOI may be capable of "knocking out" the expression of a particular gene in the target cell. There are several "knock out" strategies known in the art. For example, the NOI may be capable of integrating in the genome of the target cell so as to disrupt expression of the particular gene. The NOI may disrupt expression by, for example, introducing a premature stop codon, by rendering the downstream coding sequence out of frame, or by affecting the capacity of the encoded protein to fold (thereby affecting its function).
Alternatively, the NOI may be capable of enhancing or inducing ectopic expression of a gene in the target cell. The NOI or a sequence derived therefrom may be capable of "knocking in" the expression of a particular gene.
An NOI delivered by the vector delivery system may be capable of immortalising the target cell. A number of immortalisation techniques are known in the art (see for example Katakura Y et al (1998) Methods Cell Biol. 57:69-91).
An NOI delivered by the vector delivery system may be used for selection or marker purposes. For example, the NOI may be a selection gene, or a marker gene. Many different selectable markers have been used successfully in retroviral vectors. These are reviewed in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 444) and include, but are not limited to, the bacterial neomycin and hygromycin phosphotransferase genes which confer resistance to G418 and hygromycin respectively; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine; the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and the bacterial genes which confer resistance to puromycin or phleomycin. All of these markers are dominant selectable and allow chemical selection of most cells expressing these genes.
In a preferred embodiment, however, the NOI may have or encode a protein which has a therapeutic effect. For example, an NOI delivered by the vector delivery system may be a therapeutic gene - in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may code for a product that is capable of eliciting a therapeutic effect.
The NOI may, for example, be or encode one of the following: cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, a single chain antibody, fusion proteins, enzymes, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumour suppresser protein and growth factors, membrane proteins, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (such as with an associated reporter group).
In one preferred embodiment, the NOI is capable of encoding a disease associated antigen. Exposure to an antigen in the context of a fowlpox vector may provoke or boost an immune responses to the antigen such that an existing or subsequent challenge is dealt with more effectively.
The nature of the antigen will depend on the disease. If the disease is caused by an organism or infectious agent (bacteria, virus, protozoa, prion) the antigen may be derivable from this organism or agent. If the disease is caused by tumour(s) the antigen is preferably a tumour associated antigen such as HER-2/neu, MUC01, cancer testis antigens or oncogene(s) or product(s) thereof.
Preferably the disease-associated antigen comprises one or more T cell epitopes. The antigen may comprise a complete protein from a disease causing organism (such as Antigen 85A from M. tuberculosis). Alternatively, the antigen may comprise a string of T cell epitopes from one or more proteins from disease-causing organism(s). An example of this is the MEPfTrap polypeptide from P. falciparum which comprises a string of T-cell epitopes from P. falciparum CSP and the Pb9 epitope fused to the P. falciparum TRAP gene.
Preferably the plasmid pEFL or derivatives thereof is used in the making of fowlpox constructs according to the present invention, preferably the plasmid pEFL29. PEFL 29 has regions of homology which allow it to integrate into FP9 at ORF1. This disrupts ORF-1 but does not delete it. The plasmid adds three genes lac A, lac Y and lac Z to the genome. This disruption is an insertion, and some nucleotides are deleted. The Fowlpox Orfl gene bases deleted from pEFL29 are (capital letters):
AgagatcccgccagacggggaacctgggtcaacgACTGGTGCGAAGATCTCtgcgtatggatatggtctc eg tgttgttacgtcaagagatgtattcg
Vaccines
The genome and/or particle of the present invention may be used in a method to treat and/or prevent a disease in a subject.
For example, the genome and/or particle may be employed in a vaccine which is administered to a subject for prophylactic or therapeutic purposes. The vaccine may also comprise an adjuvant (see below).
It has been found that multiple dose vaccination (for therapy or disease prevention) is often more effective than single doses. A multiple dose vaccination program may involve repeat doses of the same composition or one or more doses of two or more different compositions. For homologous vaccination programs, the present invention provides a vaccine pack which comprises a vaccine provided in a manner which facilitates repeat administration. For example, the pack may contain a plurality of vials comprising the correct dose of vaccine ready to be injected into the patient. If the vaccine is to be taken orally, the doses may be present as pills or capsules.
In heterologous vaccination programs, there is usually a "priming" composition which is administered to the patient first and a "boosting" composition which is administered some time later. The genome and/or viral particle of the present invention may be used in a priming composition and/or a boosting composition.
A number of other compositions may be employed in heterologous vaccination programs. If the genome/particle of the present invention comprises an NOI (optionally capable of encoding a POI), then preferably the other composition comprises the same NOI or POI. Other compositions include "naked DNA", non- viral vector systems and other viral vector systems.
Naked DNA (or RNA) may be linear or circular (far example, a plasmid). It may be provided in a carrier such as a liposome or in a free form.
Suitable non-viral vectors for use in the priming composition include lipid-tailed peptides known as lipopeptides, peptides fused to carrier proteins such as KLH either as fusion proteins or by chemical linkage, whole antigens with adjuvant, and other similar systems.
If a viral vector system is used, it may be an advantage if it is derived from a different virus (i.e. not fowlpox) to minimise cross-reaction. The vector may be derived from another avipox virus , such as canary pox, or from a different genus of pox viruses (as shown in Table 2). Particularly preferred is an attenuated vaccinia vector system such as MVA or NVYAC. Other suitable viral vectors are vectors based on non-pox viruses, such as adeno vims, herpes virus and Venezuelan equine encephalitis virus (VEE). Suitable bacterial vectors include recombinant BCG and recombinant Salmonella and Salmonella transformed with plasmid DNA (Darji A et al 1997 Cell 91: 765-775).
For heterologous vaccine programs, the present invention provides a vaccination kit which comprises:
(i) a first composition which comprises a fowlpox viral particle; and
(ii) a second composition for simultaneous, separate or sequential administration.
Heterologous vaccination regimes
The present invention also relates generally to heterologous vaccination regimes using two different non-replicating viral vectors.
The present inventors have shown for the first time that heterlogous prime-boost regimes using two different non-replicating viral vectors is efficient in generating an immune response in a primate subject.
In particular, the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector to the subject in either order.
Preferably the subject is a mammal, more preferably the subject is a primate, most preferably the subject is a human.
In a preferred embodiment the first and/or the second composition is a poxvirus vector.
Thus in a preferred embodiment, the present invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering (i) a first composition which comprises a first non-replicating pox viral vector;
(ii) a second composition which comprises a second non-replicating pox viral vector to the subject in either order.
In another aspect, the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector to the subject in either order.
In another aspect, the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a DNA vaccine; (ii) a second composition which comprises a first non-replicating viral vector
(iii) a third composition which comprises a second non-replicating viral vector; to the subject.
In another aspect, the invention relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering (i) a first composition which comprises a first non-replicating viral vector;
(ii) a second composition which comprises a second non-replicating viral vector
(iii) a third composition which comprises a third non-replicating viral vector; to the subject. In another aspect, the invention relates to a method as described above wherein at least one of the composition(s) comprises a poxvirus vector.
In another aspect, the invention relates to a method as described above wherein said non-replicating viral vector(s) comprise poxvirus vector(s).
In another aspect, the invention relates to a method as described above wherein at least two of the poxvirus vectors are derivable from poxviruses from different genera.
In another aspect, the invention relates to a method as described above wherein at least one poxvirus vector is derivable from an avipox virus and at least one poxvirus vector is derivable from an orthopox virus.
In another aspect, the invention relates to a method as described above wherein at least one of the vectors is derivable from a fowlpox virus.
In another aspect, the invention relates to a method as described above wherein at least one of the vectors is, or is derivable from, FP9.
In another aspect, the invention relates to a method as described above wherein one of the vectors comprises a fowlpox virus genome selected from
(i) a fowlpox virus genome which has a modified form of one or more of the following wild-type FPV genes:
FPV001, FPV018, FPV054, FPV063, FPV066, FPV070, FPV071, FPV093, FPV097, FPV098, FPV115, FPV124, FPV125, FPV127, FPV158, FPV159, FPV160, FPV190, FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260;
(ii) a fowlpox virus genome which has a partial deletion in one or more of the following genes: FPV158, FPV219, FPV222;
(iii) a fowlpox virus genome which lacks one or more of the following genes:
FPV001, FPV 124, FPV 125, FPV 159, FPV 160, FPV220, FPV221, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260;
(iv) a fowlpox virus genome which has a frame-shift mutation in one or more of the following genes:
FPV054, FPV070, FPV071, FPV115, FPV190, FPV207;
(v) a fowlpox virus genome which has a termination mutation in one or more of the following genes:
FPV071. FPV239;
(vi) a fowlpox virus genome which has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV098;
(vii) a fowlpox virus genome which is less than 275 kbp in size;
(viii) a fowlpox vims genome comprising the sequence shown in SEQ ID No. 1
(ix) a fowlpox virus genome comprising the sequence shown in SEQ ID No. 1, with a deletion of ACTGGTGCGAAGATCTC from ORF1.
In another aspect, the invention relates to a method as described above wherein the fowlpox virus genome also comprises a NOI. In another aspect, the invention relates to method as described above wherein the NOI is under the control of a poxvirus promoter.
In another aspect, the invention relates to a method as described above wherein the NOI encodes an antigen from P. berghei, P. falciparum, P.cynomolgi, P.vivax, M. tuberculosis or T.parva.
In another aspect, the invention relates to a method as described above wherein the composition comprising the viral vector derivable from an avipox vims is administered as a boosting composition.
In another aspect, the invention relates to method as described above wherein the composition comprising the viral vector derivable from an avipox vims is administered as a priming composition.
In another aspect, the invention relates to method as described above wherein the subject is a primate.
In another aspect, the invention relates to method as described above wherein the subject is a human.
In another aspect, the invention relates to use of a non-replicating viral vector in a vaccine for an animal, wherein said vector is, or is derivable from, FP9.
In another aspect, the invention relates to use as described above wherein said animal is a mammal.
In another aspect, the invention relates to use as described above wherein said mammal is a primate.
In another aspect, the invention relates to use as described above wherein said primate is a human. In another aspect, the invention relates to use of a non-replicating viral vector in medicine, wherein said vector is, or is derivable from, FP9.
In another aspect, the invention relates to a method of eliciting an immune response in a subject comprising administering a composition comprising a non-replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
In another aspect, the invention relates to a method of boosting a pre-existing immune response in a subject comprising administering a composition comprising a non- replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
Preferably the subject is a primate, in particular a human.
Preferably the vectors are derivable from poxviruses from different genera. In particular, one may be derivable from an avipox vims and the other from an orthopox virus (see Table 2).
When one of the compositions comprises a vector derivable from an avipox vims, preferably this composition is administered as a boosting composition.
In a preferred embodiment, one of the vectors is derivable from a fowlpox virus. More preferably one of the vectors is or is derivable from FP9. For example, one of the vectors may comprise a fowlpox vims genome according to the first aspect of the invention. In a highly preferred embodiment, the vector comprises a genome having the sequence shown in SEQ ID No. 1
When the disease is malaria, preferably the FP9 or FP9 derived vector is used as a prime.
When the disease is tuberculosis, preferably the FP9 or FP9 derived vector is used as a boost. Preferably the first and second compositions are capable of expressing the same antigen.
The first and second compositions may be packaged together or individually for separate sale.
The kit may comprise other components for mixing with one or both of the compositions before administration (such as diluents, carriers, adjuvants etc.- see below).
The kit may also comprise written instructions concerning the vaccination protocol.
Boosting compositions
The present inventors have shown that a non-replicating viral vector according to the present invention is effective at boosting a pre-existing immune response to an antigen. In particular, the present inventors have shown for the first time that non- replicating viruses can be used to boost the immune response primed by M. bovis BCG in mice and in primates and melanomas in primates such as humans.
The pre-existing immune response may have been generated by a vaccine. A number of different vaccine types have been developed and are known in the art, these include living organism, intact but non-living organisms, subcellular fragments, toxoids, DNA- based vaccines and anti-idiotype (see Table 1). In a highly preferred embodiment, the pre-existing response is generated by an attenuated living pathogen, such as BCG.
The present invention provides a boosting composition comprising a non-replicating viral vector capable of boosting a pre-existing immune response in a subject.
Preferably the subject is a primate. Preferably the viral vector is a pox viral vector. For example, the viral vector may comprise a genome according to the first aspect of the invention.
Triple and Multiple Regimes
The present invention also relates generally to multiply heterologous vaccination regimes, such as triply heterologous regimes, using different non-replicating viral vectors.
The invention thus provides a triple regime comprising administering to a subject three heterologous compositions. Preferably said three compositions each differ from their neighbouring composition. For example, if the first composition comprises X then the second composition will preferably differ from X. Clearly, in this embodiment, it is possible that the third composition may be similar or identical to the first composition. Preferably all three compositions are different from one another.
In one embodiment, one of the compositions may be a DNA based composition such as a DNA vaccine. Preferably at least the second and third compositions comprise non-replicating viral vectors.
Thus the invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a DNA vaccine;
(ii) a second composition which comprises a first non-replicating viral vector
(iii) a third composition which comprises a second non-replicating viral vector; to the subject.
In a preferred embodiment, the invention provides a method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector
(iii) a third composition which comprises a third non-replicating viral vector; to the subject.
In a preferred embodiment, the first and the second compositions are heterologous, and the second and third compositions are heterologous. More preferably the first and the second and the third compositions are heterologous. Most preferably said compositions are heterologous with respect to the viral vector component of said compositions. For example a treatment regimen may involve pDNA priming followed by recombinant MVA boosting followed by recombinant FP9 boosting followed by recombinant adenovirus boosting and so forth. These treatment/immunisation cycles may be advantageously repeated to sustain a therapeutic T cell response in a therapeutic setting.
Preferably three or more different viral vectors are used in the triply heterologous regimes of the invention.
In a highly preferred embodiment, immunisation is performed using the following vectors: DNA-FP9-MVA-adenovirus-recombinant herpes in any order. Preferably each vector expresses the same antigen(s). Preferably the vectors are administered in the order DNA-FP9-MVA-adenovirus-recombinant herpes.
Further preferred immunisation orders and regimes are: DNA-FP9-MVA and DNA-MVA-FP9.
Preferably the vectors are derivable from poxviruses from different genera. In particular, one may be derivable from an avipox vims and another from an orthopox vims (see Table 2). In a preferred embodiment, one of the vectors is derivable form a fowlpox vims. For example, one of the vectors may comprise a fowlpox vims genome according to the first aspect of the invention. In a highly preferred embodiment, the vector comprises a genome having the sequence shown in SEQ ID No. 1
Preferably the first and second and third compositions are capable of expressing the same antigen.
Example 12 (eg. Figure 11-DDFM) demonstrates the effectiveness of the triple regime.
Preferably the subject is a primate, in particular a human.
The first and second and third compositions may be packaged together or individually for separate sale.
The kit may comprise other components for mixing with one or more of the compositions before administration (such as diluents, carriers, adjuvants etc.- see below).
The kit may also comprise written instructions concerning the vaccination protocol.
T cell responses
The vaccination method or program should elicit a T-cell immune response in the subject.
The nature of a T cell immune response can be characterised by virtue of the expression of cell surface markers on the cells. T cells in general can be detected by the present of TCR, CD3, CD2, CD28, CD5 or CD7 (human only). CD4+ T cells and CD8+ T cells can be distinguished by their co-receptor expression (for example, by using anti-CD4 or anti-CD8 monoclonal antibodies). Since CD4+ T cells recognise antigens when presented by MHC class II molecules, and CD8+ recognise antigens when presented by MHC class I molecules, CD4+ and CD8+ T cells can also be distinguished on the basis of the antigen presenting cells with which they will react.
Within a particular target antigen, there may be one or more CD4+ T cell epitopes and one or more CD8+ T cell epitopes. If the particular epitope has already been characterised, this can be used to distinguish between the two subtypes of T cell, for example on the basis of specific stimulation of the T cell subset which recognises the particular epitope.
CD4+ T cells can also be subdivided on the basis of their cytokine secretion profile. The TH1 subset (sometimes known as "inflammatory CD4 T cells") characteristically secretes IL-2 and IFNγ and mediates several functions associated with cytotoxicity and local inflammatory reactions. THI cells are capable of activating macrophages leading to cell mediated immunity. The TH2 subset (sometimes known as "helper CD4 T cells") characteristically secretes 11-4, IL-5, IL-6 and IL-10, and is thought to have a role in stimulating B cells to proliferate and produce antibodies (humoral immunity).
THI and TH2 cells also have characteristic expression of effector molecules. THI cells expressing membrane-bound TNF and TH2 cells expressing CD40 ligand which binds to CD40 on the B cell.
The type of T cell immune response may thus be readily determined, for example using fluorescence activated cell scanning (FACScan).
Target antigens
The target antigen may be characteristic of the target disease. If the disease is an infectious disease, caused by an infectious pathogen, then the target antigen may be derivable from the infectious pathogen. The target antigen may be an antigen which is recognised by the immune system after infection with the disease. Alternatively the antigen may be normally "invisible" to the immune system such that the method induces a non-physiological T cell response. This may be helpful in diseases where the immune response triggered by the disease is not effective (for example does not succeed in clearing the infection) since it may open up another line of attack.
Preferred Breast Cancer antigens are MUC-1, HER2, CEA; Preferred Colon cancer antigens: CEA, MUC-1, MAGE-12, mutant P53; Preferred Cervical cancer antigens: human paplioma vims proteins E6 and E7; Preferred EBV-induced B and T cell lymphomas antigens: EBNA1 and 2, LMP 1; Preferred renal cancer antigens: HER-2 neu, RAGE, MUC-1.
Preferred HPV antigens are viral proteins El -8, LI and L2 Preferred HS V antigens are viral proteins gM, gH, gK, GG, gD
Preferred HBV antigens are viral proteins small, middle and large surface antigen, core antigen, polymerase
Preferred HCV proteins are viral proteins core protein, envelope protein, NS2, NS3,
NS4 and NS5 region
The antigen may be a tumor antigen, for example HER2/neu, MUC-1, MAGE-1,
MAGE-3 or NY-ESO.
The antigen may be an autoantigen, for example tyrosinase.
In a preferred embodiment of the invention, the antigen is derivable from M. tuberculosis. For example, the antigen may be ESAT6 or MPT63.
In another preferred embodiment of the invention, the antigen is derivable from the malaria-associated pathogen P. Falciparum. The compositions of the present invention may comprise T cell epitopes from more than one antigen. For example, the composition may comprise one or more T cell epitopes from two or more antigens associated with the same disease. The two or more antigens may be derivable from the same pathogenic organism.
Alternatively, the composition may comprise epitopes from a variety of sources. For example, the ME-TRAP insert described in the examples comprises T cell epitopes from P. falciparum, tetanus toxoid, M. tuberculosis and M. bovis.
Target Diseases
The method of the present invention will be useful for treating and/or preventing a number of diseases, especially those which are susceptible to a T-cell mediated immune response.
In particular, the method of the present invention will be useful in the treatment and/or prevention of diseases which are or are caused by chronic infections, particularly persistent, latent infections.
A non-exhaustive list of suitable diseases includes: tuberculosis, HIV, malaria. H. pylori, influenza, hepatitis, CMV, human papilloma vims (HPV), herpes vims-induced diseases and other viral infections, leprosy, non-malarial protozoan parasites such as toxoplasma, and various malignancies such as tumours and/or cancers, infectious disease caused by protozoans: malaria, particularly Plasmodium falciparum and P. vivax, toxoplasma, Theileria parva, Trypanosomas cmzi, by mycobacteria such as tuberculosis and leprosy, bacteria such as Chlamydia pneumoniae and Helicobacter pylori, by viruses such as HIV, EBV, CMV, HBV, HCV, HPV, HSV, RSV, influenza virus and various malignacies such as renal, colorectal, lung, skin (melanoma), liver, ovary, testis, pancreas, uterus, prostate, stomach, head and neck, cervix, breast cancer and various lymphomas, as well as HIV/ AIDS, heptitis B, hepatitis C, malaria, tuberculosis, HPV infection and disease, HSV infection and disease, CMV infection and disease, EBv infection and disease, leishmaniasis, listeriosis, theileria, HTLV infection and disease, pneumococcal disease, staphylococcal disease, lung cancer, breast cancer, colon cancer, melanoma, myeloma, lymphoma, renal cell carcinoma.
The method of the present invention is particularly useful in vaccination strategies to protect against tuberculosis, malaria and East Coast Fever.
The compositions described herein may be employed as therapeutic or prophylactic vaccines. Whether prophylactic or therapeutic immunisation is the more appropriate will usually depend upon the nature of the disease. For example, it is anticipated that cancer will be immunised against therapeutically rather than before it has been diagnosed, while anti-malaria vaccines will preferably, though not necessarily be used as a prophylactic.
Pharmaceutical compositions/Vaccines
The present invention also relates to a pharmaceutical composition such as a vaccine, priming or boosting agent.
The pharmaceutical composition may also comprise, for example, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
In particular, a composition comprising a DNA plasmid vector may comprise granulocyte macrophage-colony stimulating factor (GM-CSF), or a plasmid encoding it, to act as an adjuvant; beneficial effects are seen using GM-CSF in polypeptide form.
Adjuvants such as QS21 or SBAS2 (Stoute J A et al. 1997 N Engl J Medicine 226: 86-
91) may be used with proteins, peptides or nucleic acids to enhance the induction of T cell responses. In the pharmaceutical compositions of the present invention, the composition may also be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilising agent(s).
The pharmaceutical composition could be for veterinary (i.e. animal) usage or for human usage. For veterinary usage, the composition may be used to treat for example mammals (especially cattle) or birds.
Preferably the subject is a mammalian subject, in particular a primate (e.g. human) or ungulate (e.g. cow) subject.
Administration
In general, a therapeutically effective daily intradermal or intramuscular dose of the compositions of the present invention is likely to range from 105-1010 plaque-forming units (pfu).
Typically, the physician or veterinary surgeon will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Transmission of members of the Chordopoxvirinae may occur by aerosol (Murphy et al (1995) as above). The compositions of the present invention may also be administered by aerosol for inhalation by the subject. The compositions of the present invention may also be conveniently administered by injection, such as intradermal and/or intramuscular injection. In addition, the compositions may be administered using a suitable devine into the skin or other tissues (for example using a "gene gun" or similar). Where appropriate, the pharmaceutical compositions can be also be administered in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
It is also possible to administer the compositions of the present invention in sustained release formulations.
Poxvirus growth method
It is known to grow wild-type and recombinant poxviruses in vitro in cultured chicken embryo fibroblasts (CEFs). The present inventors have suφrisingly found that substantially improved plaque formation and virus yield can be obtained for avipox viruses by using non-cultured CEFs.
The present invention provides the use of non-cultured CEF cells to grow an avipox virus.
Preferably the avipox vims is a fowlpox virus, especially one which comprises a fowlpox genome according to the first aspect of the present invention.
The term "non-cultured" is used to indicate that the cells are used before they multiply to any significant extent in culture. Preferably the cells should be in culture for insufficient time for the number of cells in the population to double. It is routine practice to maintain primary CEF cells in culture for up to one month by regular passage and supplementation of the growth medium. The non-cultured CEF cells of the present invention should be used without being passaged.
Primary non-cultured CEFs can be obtained by loading freshly prepared CEFs into tissue culture dishes at a concentration sufficient to give a confluent monolayer without growth. The plate is then used, preferably within 24hours of being plated out.
The invention is further described, for the purposes of illustration only, in the following examples
EXAMPLE 1 : Production and characterisation of fowlpox strain FP9
Fowlpox strain FP9 was derived by 438 passages through chicken embryo fibroblasts in tissue culture from the virulent wild-type fowlpox HP-1 strain (Mayr and Malicki (1966) Zentralbl Veterinarmed 13 1-3) and then plaque purified.
The genome of Fp9 has been fully sequenced (SEQ ID No 1) and annotated (Figure 1). FP9 does not contain the REV provims. At 266 kbp the genome of FP9 is somewhat smaller than the estimated size of FPV-M (greater than 300kbp).
EXAMPLE 2: Comparison of the immune response elicited by recombinant FP9 and FPV-M
To determine whether recombinant FP9 was superior to FPV-M in its capacity to elicit T-cell responses against a recombinant antigen, the Plasmodium berghei circumsporozoite surface protein (PbCSP) was inserted into the terminal 6.8 kbp BamHI fragment (Boursnell et al, 1990a, J Gen Virol 71, 621-8; Boursnell et al, 1990b, Vet Microbiol 23, 305-16; Campbell et al, 1989, J Gen Virol 70, 145-54) of the genome of both viruses by homologous recombinantion (Quingzhong et al, 1994, Vaccine 12, 569-73). The vaccinia p7.5 early-late promoter was used to drive the expression of the inserted genes in both recombinant viruses. The PbCSP protein contains an H-2Kd restricted 9 amino acid peptide epitope that can induce a protective CD8+ T-cell resp;onse against liver stage P. berghei infection in Balb/c mice (Plebanski et al, 1998, Eur J Immunol 28, 4345-55; Schneider et al, 1998, Nat Med 4, 397-402). Suφrisingly, FP9 encoding the PbCSP gene (FP9PbCSP) elicited a significantly higher CD8+ mediated T-cell response in Balb/c mice than that elicited by FPV-M encoding the CSP gene (FPV-MPbCSP), although both viruses induced substantial T-cell responses against viral antigens (Figure 3).
Method
Female Balb/c mice were immunised intravenously with 1 x 106 PFU of FP9 or FPV- M alone or expressing Plasmodium berghei CSP (PbCSP). Seven days following immunisation, the T-cell immune response elicited in splenocytes was determined using the IFNγ ELSPOT assay. The response against the recombinant antigen, PbCSP was determined using the MHC-class I restricted Pb9 epitope (recognised by CD8+ T- cells). As a positive control, the T-cell response against whole virus was determined by exposing immune splenocytes to those infected with fowlpox vims. Columns in Figure 1 represent the mean IFN-γ spot forming cells (SFC) per million splenocytes + the standard error of the mean for four mice per group.
EXAMPLE 3: Prime-boost immunisation regimes with FP9PbCSP and induction of a protective immune response against P. berghei
Comparison of the CD8+ T-cell response elicited by FP9PbCSP with a DNA-vaccine and MVA encoding the PbCSP antigen in Balb/c mice (Figure 4) revealed that the response elicited by recombinant FP9 was significantly higher than that elicited by the DNA-vaccine, although lower than that elicited by recombinant MVA. Nevertheless, FP9PbCSP boosted the CD8+ T-cell response primed by DNA-vaccination, as well as acting as either a priming or boosting agent in combination with MVAPbCSP (Figure 5). Significantly, priming with FP9PbCSP and subsequent boosting with MVAPbCSP induced considerably higher levels of protection against challenge with P. berghei sporozoites than other homologous or heterologous prime-boost immunisation regimes (Table 3).
Figure imgf000046_0001
Table 3. Protection against Plasmodium berghei challenge by prime-boost immunisation regimes using recombinant FP9PbCSP
Method Female Balb/c mice were immunised intravenously with 1 x 106 PFU of FP9PbCSP (FP9) or MVAPbCSP (MVA), or intramuscularly with 50 μg of pSG2PbCSP (DNA) and boosted two weeks later with either FP9PbCSP or MVAPbCSP. The CD8+ T-cell response against PbCSP was determined using the MHC-class I restricted Pb9 epitope in an IFNγ ELISPOT assay 14 days following the boost. Columns represent the mean IFNγ spot forming cells (SFC) per million splenocytes + the standard error of the mean for three mice per group (Figure 4).
Two weeks following the booster immunisation, animals were challenged by intravenous injection with 2000 P. berghei sporozoites and subsequently monitored for blood stage infection 6 to 14 days after challenge (Table 2). Results are cumulative for four experiments.
EXAMPLE 4: Construction of FP9 viruses encoding different foreign recombinant antigens and epitope strings To demonstrate that FP9 can be used as a general system for delivering recombinant antigens, genes encoding whole antigens of both prokaryotic and eukaryotic origin and synthetic genes encoding poly-epitope and poly-protein fusions were inserted into the genome of FP9 by homologous recombination (Table 4). These genes encode antigens which differ widely in structure and are derived from a variety of different pathogens. Each of the recombinant FP9 derivatives listed in Table 4 have been propagated in vitro in chicken embryo fibroblasts to high concentrations, indicating that they are both viable and stable. The vaccinia p7.5 early-later promoter was used to drive expression of the encoded antigens in all of the recombinant vimses. The encoded antigens have been found to be expressed in all cases where this has been determined (Table 4). This includes the largest of these constructs, FP9L3SEPTL, which harbours a 9.9 kbp synthetic gene encoding a poly-protein expressed as 6 fused malarial antigens.
Table 4: Recombinant FP9 viruses constructed to date
Figure imgf000047_0001
Figure imgf000048_0001
a Expression of the recombinant virus was determined either by immunofluorescence or by the presence of an immune response. ND, not determined.
EXAMPLE 5: Induction of T-cell immune responses against P. falciparum antigens by FP9
The MEPfTrap polypeptide, which consists of a string of T-cell epitopes from P. falciparum CSP and the Pb9 epitope (Gilbert et al, 1997 Nat Biotechnol 15, 1280-4) fused to the P. falciparum TRAP gene (Robson et al, 1988, Nature 335, 79-82), was developed as an antigen for vaccination against P. falciparum malaria in humans. FP9 encoding MEPfTrap has been constructed (Table 4) and shown to elicit CD8+ T-cell responses in potency tests carried out in Balb/c mice (107+20 IFNγ spot forming cells/million splenocytes).
EXAMPLE 6: Induction of CD8+ and CD4+ T cell immune responses against Antigen 85 A of M. tuberculosis by FP9
Antigen 85A (Ag85A) is a major secreted and protective antigen from M. tuberculosis. The immune response elicited by FP9 encoding Ag85A from M. tuberculosis was initially determined in Balb/c mice (Figure 6). Antigen 85A contains a 20 amino acid H-2d MHC class Il-restricted peptide epitope (recognised by CD4+ T-cells) as well as a nine amino acid H-2d MHC class I-restricted epitope (recognised by CD8+ T-cells) (Huygen et al, 1994). Immunisation with FP9Ag85A induced both CD4+ and CD8+ T-cell responses against Ag85A, indicating that FP9 will induce CD4+ as well as CD8+ T-cell responses against encoded antigens in mice. In further expiments, FP9Ag85A was found to induce significant boosting of the T-cell response induced by immunisation with MVA85A in Rhesus macaques (Figure 7). Method
Female Balb/c mice were immunised intravenously with 1 x 107 PFU of recombinant FP9 encoding antigen 85A (FP9Ag85A) from mycobacterium tuberculosis or, for comparison, 1 x 106 PFU of MVA encoding the same antigen. The response against 85 A was determined using the MHC-class II restricted PI 5 epitope (recognised by CD4+ T-cells) and the MHC-class I restricted Pl l epitope (recognised by CD8+ T- cells) in an IFNγ ELISPOT assay six days following immunisation. Columns represent the mean IFNγ spot forming cells (SFC) per million splenocytes + the standard error of the mean for three mice per group.
A male Rhesus macaque immunised twice with recombinant MVA encoding antigen 85 A (MVA85A) was boosted (i) 21 and 26 weeks later with 5 x 108 PFU of FP9Ag85A. The response against Ag85A was determined in peripheral blood mononucleocytes (PBMCs) using pools of overlapping peptides covering the amino acid sequence of the Ag85A polypeptide in an IFNγ ELISPOT assay. Curves represent the IFNγ spot forming cells (SFC) per million PBMCs for 4 pools of peptides.
EXAMPLE 7 - FP9 and MVA do not elicit cross-reactive T-cells against viral antigens
There is evidence to suggest that prime-boost immunisation with different but not the same viral vector will elicit enhanced CD8+ T-cell responses against recombinant antigens (Gilbert et al., Vaccine In Press). Recent evidence suggests CD8+ T-cell responses against the viral vector will inhibit boosting when the same recombinant virus is used as a priming and boosting agent (unpublished observation, Eric G. Sheu). Immune splenocytes from mice immunised with FP9PbCSP do not produce IFNγ when exposed to naϊve cells infected with MVA but do when exposed to cells infected with FP9 (Figure 8). Conversly, MVAPbCSP immune splenocytes recognise naive cells infected with MVA but not FP9 (Figure 8). Despite this, an IFNγ secreting T-cell response can be observed when FP9PbCSP immune splenocytes are exposed to spleen cells infected with MVAPbCSP and vice versa (Figure 8). Considered together, these results indicate that recombinant FP9 and recombinant MVA will elicit T-cell responses against an encoded antigen without eliciting T-cells that cross-react with antigens from both vimses.
Further preliminary results indicate that non-replicating canary poxvimses also do not cross-react with MVA. Thus, the avipoxvimses and orthopoxviruses may not elicit cross-reactive T-cells in general making them suitable as priming or boosting agents in the same vaccination regime.
Method Ex vivo IFNγ elispot responses against PbCSP and viral antigens in splenocytes isolated from mice fourteen days after i.v. immunisation with either FP9PbCSP or MVAPbCSP. The IFNγ response was determined by exposing splenocytes isolated from immune animals to naive splenocytes pulsed with Pb9 peptide or those infected with FP9, FP9PbCSP, MVA, or MVAPbCSP. Columns (Figure 8) represent the mean number of IFNγ spot forming cells (sfc) per million splenocytes ± S.E.M. for three mice per group.
EXAMPLE 8 - Growth of avipoxvimses in vitro
Wild-type and recombinant poxvimses are usually grown in vitro in cultured chicken embryo fibroblasts (CEFs). In initial attempts to grow recombinant FP9 by infection of cultured CEF monolayers in our laboratory, we observed erratic viral plaque formation and poor yields of vims (Table 5). In contrast, good plaque formation and consistently high yields of MVA were obtained following infection of similar cultured CEFs (Table 5). Primary "non-cultured" CEFs can be obtained by loading freshly prepared CEFs into tissue culture dishes at a concentration sufficient to give a confluent monolayer without growth. The present inventors have found that FP9 and recombinant FP9 forms visible viral plaques and give good yields of vims (Table 5) following infection of monolayers of primary "non-cultured" CEFs. Similarly, plaque formation and higher yields of attenuated canarypox vims, ALVAC have been obtained using primary "non-cultured" CEFs by the present inventors. Together these studies indicate that the avipoxvimses are more fastidious than MVA, and possibly other orthopoxvimses, in terms of their growth requirements. Without wishing to be bound by theory, the present inventors believe that the replication of avipoxvimses within CEFs may require a specific host molecule that is lost as the CEFs are cultured in vitro. In contrast, MVA, and possibly the orthopoxvimses in general, do not require this host molecule for replication. The main consequence of this observation is that primary non-cultured CEFs are likely to be required for the production of large quantities of recombinant avipoxvimses. Accordingly, in practice, large-scale production of recombinant FP9 for Phase I clinical studies has only been achieved using "non-cultured" CEFs.
Table 5: Approximate yield of vims from cultured and non-cultured CEFs
Figure imgf000051_0001
Cultured CEFs were passaged in culture prior to infection with vims.
2For "non-cultured" CEFs, primary CEFs were loaded at approximately 1.5 X 105 cells/cm2 in tissue culture flasks and grown for 12 - 18 h prior to infection.
EXAMPLE 9 - Induction of a protective immune response against P. falciparum malaria by FP9 in human volunteers
Phase I clinical trials of FP9MEPfTrap (see Example 5):
Healthy adult volunteers were immunised twice with over 3 X 107 PFU of FP9MEPfTrap and subsequently boosted with the same dose of MVAMEPfTrap. Two of five immunised volunteers were protected against challenge with bites from five female anopheles stephensii mosquitoes infected with virulent P. falciparum malaria. In contrast, five out of five malaria naive volunteers succumbed to P. falciparum malaria in the same experiment. Thus, demonstrating for the first time that heterologous prime-boost immunisation of human volunteers with two recombinant non-replicating vimses can elicit a protective immune response against an infectious disease.
Protection was determined by the failure of volunteers to develop blood-stage malaria, demonstrating that poxvirus immunisation blocked the P. falciparum infection in the liver.
Experiments also determine the mechanism of protection, although it is likely that protection is mediated by T-cell responses elicited against the MEPfTrap polypeptide.
EXAMPLE 10 - Clinical trials in a Malaria endemic region
Phase I Trial
To determine whether a heterologous prime-boost immunisation regime using recombinant FP9 and MVA can boost immune responses in a malaria-exposed population, a phase I clinical trial using constmcts encoding the MEPfTrap molecule is conducted in the Gambia.
Adult volunteers (approximately 12) are immunised twice with the FP9MEPfTrap constmct and boosted with a single dose of MVAMEPfTrap. T-cell and antibody immune responses are determined in blood, the primary puφose of the study being to demonstrate whether immunisation with the recombinant non-replicating poxvimses boosts the existing anti-malaria response primed by natural infection.
Phase lib study
Phase lib study is conducted in Gambian adults using the same immunisation regime. Volunteers are monitored for enhanced immune responses and evidence of blood stage infection during the course of the trial, which ns through the malaria season (June to December).
Further studies
Further phase I studies are also conducted in the Gambia during 2002 using appropriate recombinant FP9 and MVA to vaccinate against Mycobacteria tuberculosis and HIV. Both trials are conducted using volunteers who are infected with the respective pathogens and, therefore, evalute both the capacity of FP9 and a heterologous prime- boost immunisation regime using two non-replicating vimses to serve as a therapeutic vaccine.
Thus it is demonstrated that vaccination according to the present invention produces and/or enhances protective response in humans.
EXAMPLE 1 1 - Boosting the T-cell response against Ag85A primed by M. bovis BCG using non-replicating recombinant poxviruses
M. bovis BCG is widely administered to children throughout the developing world as a vaccine against tuberculosis caused by M. tuberculosis. Although effective against severe childhood forms of tuberculosis, the protective efficacy of M. bovis BCG against adult forms of the disease is highly variable and thought to wane over time. Since Ag85A is a major secreted antigen of both M. bovis BCG and M. tuberculosis, boosting the immune response elicited against Ag85A by M. bovis BCG immunization is a credible strategy for enhancing the protective efficacy of this vaccine against adult tuberculosis. This strategy has been pursued by others in murine models by boosting with recombinant Ag85A protein (Brooks et ai, 2001 Infect Immun 69, 2714-7) or a DNA-vaccine encoding this antigen (Tanghe et al., 2001 Infect Immun 69, 3041-7.).
Based on the observation that FP9Ag85A can boost an immune response primed by immunization with MVA85A, the present inventors wished to determine whether poxvirus constmcts could boost the immune response primed by M. bovis BCG. Preliminary experiments conducted in Balb/c mice indicated that both the CD4+ and CD8+ T-cells responses Ag85A could be boosted primed by immunization with M. bovis BCG (Figure 9). Further experiments conducted using Rhesus macaques showed that immunization with M. bovis BCG by either intradermal or aerosol routes will induce T-cell responses against Ag85A. Boosting with MVA85A early after administration of M. bovis BCG elicited a variable and short-lived enhancement of the immune response against PPD and Ag85A protein. However, subsequent boosting with FP9Ag85A significantly enhanced the T-cell immune response against these proteins (Figure 10) as well as against peptide pools spanning the Ag85A protein sequence. Further experiments determine the nature and location of the T-cell response elicited in the immunized animals.
These experiments provide the first example that non-replicating vimses can be used to boost the immune response primed by M. bovis BCG in mice and in primates. In addition, they establish the concept that non-replicating poxvimses may be effective as therapeutic vaccines against persistent latent infection with M. tuberculosis in man.
Method Balb/c mice were immunized intradermally (i.d.) with M. bovis BCG and boosted 12 weeks later id. with 1 X 106 PFU of MVA85A. Control animals were immunized with M. bovis BCG or MVA85A alone. T-cell immune responses were determined against PPD and Ag85A using the P15 (CD4+) and Pll (CD8+) epitopes in an IFNγ ELISPOT assay 12 days following immunisation. Columns (Figure 9) represent the mean IFN-γ spot forming cells (SFC) per million splenocytes ± the standard error of the mean for three mice per group.
Male Rhesus macaques were immunized either intradermally or by aerosol delivery with BCG and boosted twice with 5 X 10 PFU of recombinant MVA85A and twice with 5 X 108 PFU FP9Ag85A. The response against Ag85A was determined in peripheral blood mononucleocytes (PBMCs) using purified Ag85A, recombinant
Ag85B (which shares approximately 80% homology with Ag85A) or purified protein derivative from M. tuberculosis (PPD) in an IFNγ ELISPOT assay. Curves (Figure 10) represent the IFN-γ spot forming cells (SFC) per million PBMCs from a single animal.
EXAMPLE 12 - Fowlpox is an effective boost
Three healthy malaria-naive human volunteers were immunised as follows:
2mg of DNA-ME.TRAP intramuscularly at week 0, 2mg of the same vaccine again intramuscularly at week 3 (day 21), 1 x 10e8 pfu of FP9-ME.TRAP intradermally at week 7, and 1.5xl0e8 pfu of MVA-ME.TRAP intradermally at week 11.
Mean responses measured by ELISPOT in PBMCs are shown in figure 11.
The ME response is predominantly to CD8 T cell epitopes. The T996 response is the the homologous strain of TRAP, the 3D7 response to a heterologous strain of TRAP.
It is clear that FP9-ME.TRAP boosts a DNA primed T cell response. It is also clear that a further immunisation with MVA-ME.TRAP enhances responses induced by the DDF regime.
It is therefore demonstrated that FP9 provides an effective boost to T cell responses in human primates.
The effectiveness of a triple immunisation regime with DNA then FP9 and then MVA is also demonstrated in human primates (DDFM).
Example 13 : Fowlpox is an effective prime
Malaria-naϊve human volunteers were immunised with a FP9-MVA prime-boost regime as follows: In this Example there are two cohorts, one of five vaccinees and one of 12 vaccinees.
FFM regime
FP9-ME.TRAP was administered intradermally at lxl 0e8 pfu at day 0 FP9-ME.TRAP was administered intradermally at lxl 0e8 pfu at day 21 MVA-ME.TRAP was administered intradermally at a dose of 1.5x10e8 pfu at day 49.
All volunteers were challenged with 3D7 strain P. falcipamm sporozoites administered by the bites of five infectious mosquitoes, along with non- vaccinated controls.
In the first cohort 2/5 FFM vaccinees were fully protected from infection. This level of protection is significantly different from 24 pooled non- vaccinated controls challenged at various time points in the same way (P < 0.05, chi-squared test); all the controls were infected.
Two of the protected vaccinees were re-challenged six months later and one was still fully protected.
11 volunteers in the second cohort were challenged identically and these vaccines developed patent malaria parasitaemia significantly later than control vaccines (P < 0.05).
Figure 12 shows the calculated reduction in liver stage parasites resulting from vaccination assuming that parasites multiply 8 fold every 48 hours in peripheral blood. The "late FFM" group represents the two rechallengees (see above) and a further volunteer challenged for the first time at 5 months post vaccination (the FFM group were challenged 13-50 days post the last vaccination.
Also shown are results for four vaccinees administered only a single priming immunisation with FP9 ("FM"). The significance levels are calculated from log rank test Kaplan-Meier analysis of times to parasitiaemia in vaccines and non- vaccinated challenge controls.
Overall there was highly significant protection observed in the FFM vaccines compared to controls (P < 0.01).
Four individuals immunised with a DDMF regime (two DNA does of 2mg i.m. at four week intervals followed by 1.5xl0e8 MVA ME.TRAP 3-4 week later followed by 1.0xl0e8 FP9-ME.TRAP 3-4 weeks later) were also significantly protected (P< 0.05).
Thus it is demonstrated that fowlpox such as FP9 is an effective prime as well as an effective boost in the methods of the present invention.
EXAMPLE 14: Clinical trials of higher dose immunisation regimes
Higher dose prime-boost regimes were studied in rural Gambian adult males who have had life-long exposure to malaria infection.
28 subjects were immunised with ME.TRAP vaccines according to one of the regimes set out below (14 subj ects per group/regime) :
1) DDM regime
DNA (2mg i.m.) administered two (DDM) or three (DDDM) times at a 3-4 week intervals followed by MVA-ME.TRAP 1.5x10e8 id (DDM group - In Figure 13 the DDM and DDDM vaccinee subgroups are merged as there was no significant difference between these.)
2) FFM regime
FP9 (Ixl0e8 i.d.) administered twice at 3 week intervals followed by MVA-ME.TRAP 1.5xl0e8 id (FFM group). Data are presented in Figure 13.
Both groups showed T cell responses to the malaria ME.TRAP insert prior to vaccination. These responses were boosted more by a FP9 immunisation than by a DNA immunisation (compare DD+7 days with FF+ 7 days) indicating that FP9 immunisation boosts naturally primed T cell responses in human primates.
Both DDM and FFM regimes induced high level (> 250/million) T cell responses in these volunteer subjects naturally exposed to malaria.
It is demonstrated that prime-boost regimes (DDM and FFM) are more immunogenic than FP9 alone and MVA alone even though T cell responses in these individuals are primed by natural malaria infection.
It is also demonstrated that the induced responses show good malaria-strain crossreactivity - the magnitude of responses to the vaccine strain (T996) and the 3D7 non- vaccine strain of TRAP are comparable.
Comparable immunogenicity was observed in 11 human primate volunteers immunised with the same FFM regime (see figure 14).
Thus it is demonstrated that treatment of subjects according to the present invention produces protective immune responses in said subjects.
EXAMPLE 15: Immunisation against TB antigens in primates
To assess the possible protective efficacy of prime-boost immunisation against tuberculosis using recombinant FP9 as a boosting agent the following study was performed in primates. In this example the primates are Macaques, animals naturally susceptible to Mycobacterium tuberculosis.
At the biomedical primate centre (BPRC) in the Netherlands three cynomolgous macaques were immunised with BCG intradermally and administered MVA-Ag85A intradermally after 8 weeks and FP9-Ag85A intradermally after a further four weeks. In parallel three other macaques were immunised with BCG alone. Four weeks after the final immunisation all these macaques along with unimmunised macaques were challenged with a large intratracheal dose (of 1000 CFU) of M. tuberculosis. The animals were observed for 28 weeks, immunoassays performed and the animals were sacrificed at 28 weeks post-challenge and an autopsy performed.
At autopsy all non-immunised macaques had macroscopic evidence of tuberculosis; 1/3 BCG immunised macaques had no macroscopic evidence of tuberculosis; 2/3 BCG-MVA-FP9 immunised macaques had no evidence of tuberculosis.
ELISPOT assays post-challenge using PPD and ESAT-6 as mycobacterial antigens showed substantially lower responses in the BCG-MVA-FP9 immunised animals than in challenge controls.
Interferon gamma ELISA assays post-challenge using PPD as the mycobacterial antigen showed substantially lower responses in the BCG-MVA-FP9 immunised animals than in challenge controls immunised with BCG alone or an adjuvant alone (control animals), supporting the view that these BCG-MVA-FP9 prime-boosted macaques were very substantially protected from tuberculosis (figure 18). The figure shows the mean interferon gamma response to PPD at various weeks after intratracheal M. tuberculosis challenge of cynomolgous macaques. BCG-MVA-FP9 animals have the lowest immune responses to PPD indicating the least M. tuberculosis replication after challenge and thus the highest degree of protection. Similar data were obtained using ESAT6 as an antigen. Thus it is demonstrated that primates such as macaques which are prime-boost immunised according to the present invention are very substantially protected from tuberculosis.
EXAMPLE 16: The FP9 strain is more potent than Webster's FPV-M in priming and boosting CD8 T cell reponse.
Objective: To determine whether FP9PbCSP will elicit enhanced antigen-specific T cell responses when compared to FPV-MPbCSP when administered by a clinically relevant route in prime/boost immunisation regimes with MVAPbCSP.
Method
Female BALB/c mice (6 - 8 weeks old) were immunised intradermally (id.) with
FPPbCSP, FPV-MPbCSP or MVAPbCSP and boosted in a similar manner two weeks later. Viruses were diluted to 2 X 10 PFU/ml in pyrogen free PBS, characterised as described in EXAMPLES 1 and 2 of the patent application, and administered id. by bilateral injection of 25ul into each ear of a mouse. Fourteen days after immunisation, the mice were sacrificed by cervical dislocation and the T cell response elicited against the Pb9 epitope of PbCSP and a control epitope from β-galactosidase were determined using the IFNγ assay as described below:
Murine IFNγ ELISPOT protocol
A. Materials
IFN-gamma ELISpot ALP Kit Mabtech 3321-2A 600μg anti-IFN-gamma purified Mab AN 18
50μg anti-IFN-gamma biotinylated Mab R46A2 50μl Streptavidin- Alkaline Phosphatase
Complete α-MEM medium 500ml MEM α-modification Sigma M-4526 50ml FCS [10%] Sigma F-2442 5ml pen/strep [100U penicillin lOOμg strep] Sigma P-0781
10ml L-glutamine [4mM] Sigma G-7513
500μl 2-Mercaptoethanol [50μm] Gibco BRL 31350-010
ACK buffer
8.29g NH4C1 [0.15M] (Sigma A-4514) lg KHCO3[lmM] (Sigma P-9144) 37.2mg Na2EDTA (Sigma ED2SS) 800ml milli-Q water Adjust pH to 7.2-7.4 with HCI (Sigma S-7653) Make up to 1000 ml with water and autoclave
Colour Development Buffer:
BioRad AP Conjugate Substrate kit (170-6432 ). For one plate:
5ml deionised water
200μl of 25x buffer
50μl reagent A
50μl reagent B Mix well and use immediately
Protocol
1. Preparation of Plates:
1.1. Coating plates: coat MAIP multiscreen plates (Millipore MAIPS4510) with rat anti-mouse IFNγ (Mab AN 18) antibody. Dilute to lOμg/ml in Phosphate
Buffered Saline (PBS; Sigma P-3813) and add 50μl per well to MAIP plates. Incubate overnight at 4°C in a humidified chamber
1.2. Blocking plates: Flick off coating antibody and wash plates once with 150 ul of sterile PBS (Sigma P-3813) per well using a multi-channel pipette. Flick off the PBS, add lOOul complete α-MEM medium per well, and incubate at room temperature for 1+ hour. It is important to keep the plates sterile at this stage.
2. Splenocyte preparation:
2.1. Cmsh individual spleens in 2 ml of PBS with the plunger of a 10ml syringe in a 70μm cell strainer (Falcon 352350) contained in a petri dish, add 5 ml of PBS, suspend splenocytes by pipetting, and transfer into a 50 ml tube. Rinse cell strainer and dish with a further 10 ml of PBS and add to the 50 ml tube. Centrifuge at 1500φm for 5 min.
2.2. Remove supernatant, re-suspend cells by tapping tube and add 5ml ACK buffer and mix by inversion. Incubate at room temperature for no longer than 5 minutes. Add 25ml PBS, mix by inversion and centrifuge at 1500φm for 5 min.
2.3. Remove supernatant re-suspend pellet by tapping the tube, add 10ml PBS and vortex. Count using an improved Neubauer haemacytometer by diluting 1 :10 in 0.4% trypan blue solution (Sigma T-8154). Aliquot amount needed for the Elispot and centrifuge at 1500φm for 5 min, resuspend by vortexing in an appropriate volume of complete Alpha MEM medium to give a concentration of 10 million cells/ml.
3. Plate setup:
50μl 50μl 50μl > → -> < > → - < > → ->
1 2 3 4 5 6 7 8 9 10 11 12
Figure imgf000062_0001
Figure imgf000063_0001
C C
Note: Plate layout may be chosen by the operator according to the needs of the particular experiment.
3.1. Flick blocking media from plate and add 50μl of complete alpha MEM medium to columns 3, 4, 7, 8, 11&12.
3.2. Add 150μl of splenocytes to columns 2, 6 and 10 in duplicate. (Up to 12 samples per plate)
3.3. Take 50μl of splenocytes from columns 2, 6 and 10 and transfer to columns 1, 5 and 9 respectively: these are the negative control wells.
3.4. Serially dilute each sample by taking 50μl from columns 2, 6 and 10, to columns 3, 7 and 11, mix well and transfer 50μl to 4, 9 and 12. Discard 50μl after mixing final columns in dilution.
3.5. Add test peptide and control peptide to twice the desired final concentration to naive splenocytes at 10 million/ml in complete α-MEM medium. Add 50μl of control peptide and target cells to columns 1, 5 and 9. Add 50μl test peptide and target cells to remaining columns.
3.6. Incubate plates at 37°C for 18-20 hours.
4. Developing the Assay
4.1. Wash plates twice with PBS containing 0.05% Tween 20 (Sigma PI 379), once with distilled water and twice with PBST. 4.2. Add 50μl/well of biotinylated rat anti-mouse interferon-gamma diluted to 1 μg/ml in PBS. Incubate for 2 hours at room temperature.
4.3. Wash plates four times with PBST, then add 50 μl Streptavidin Alkaline Phosphatase (Mabtech) diluted to 1 μg/ml in PBS. Incubate at room temperature for 1 hour.
4.4. Wash plates four times with PBST, add 50μl/well of colour development buffer
4.5. Incubate at room temperature until spots develop (approx. 10 min). Wash plates well with tap water, peel off plastic bottom and leave to dry overnight on paper towels.
Results
Results were calculated as the number of antigen-specific IFNγ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the students T-test (two-sample assuming equal variances) using Microsoft Excel 2000.
FP9PbCSP elicited a significantly enhanced antigen-specific T cell response against PbCSP compared to FPV-MPbCSP when used as a priming (P = 0.002) or boosting (P = 0.004) agent in combination with MVAPbCSP (Figure 15). Interestingly, there was no significant difference between groups that had been primed with FP9PbCSP and boosted with MVAPbCSP and vice versa.
Thus, FP9PbCSP proved to be more potent than FPV-MPbCSP as a priming and/or boosting agent, and is equally potent as a priming or boosting agent in combination with recombinant MVA.
EXAMPLE 17: Recombinant FP9 protects against viral diseases Objective
To determine whether immunisation with recombinant FP9 alone, or in a prime/boost regime, will elicit T cell responses against CD4+ and CD8+ T cell epitopes from viral and tumour antigens.
Method
A synthetic gene of 440 bp encoding for a novel polypeptide (ME1) consisting of characterised CD8+ and CD4+ restricted epitopes from lymphocytic choriomeningitis vims (LCMV) antigens and murine tumour (P815 and CT26) antigens was synthesised as follows:
ME1 model epitope string:
A T cell epitope string encoding tumour and vims epitopes relevant in murine models of chronic infection (LCMV) and cancer (P815/CT26) is generated as set out below.
Figure imgf000065_0001
Figure imgf000066_0001
To avoid potential immune competition LCMV-derived epitopes are H-2 -restricted and the cancer epitopes are H-2 -restricted. The order of the epitopes alternates between H-2d and H-2b.
Epitope String Sequence
Showing Epitopes:
Cloning site Kozak M [GLNGPDIYKGVYQFKSVEFD] [KAVYNFATCGI] [LPYLGWLVF] [FQPQNGQFI] [GYCGLRGTGV] [SGVENPGGYCL] [SPSYAYHQF] [YTVKYPNL] [TPHPARIGL] [CSANNSHHYI] [SGEGWPYIACRTSIVGRAWE] [DAPIYTNV] [YPYDVPDYA] AA (stop signal)
Amino Acid String:
MGLNGPDIYKGVYQFKSVEFDKAVYNFATCGILPYLGWLVFFQPQNGQFIGYC GLRGTGVSGVENPGGYCLSPSYAYHQFYTVKYPNLTPHPARIGLCSANNSHHY ISGEGWPYIACRTSIVGRAWEDAPIYTNVYPYDVPDYAAA
Flanking nucleotides and amino acids
GGGCCCGCCGCCACCATGG. M G LNGPDIYKGVYQFKSVEFDKAVYNFATCGIL
PYLGWLVFFQPQNGQFIGYCGLRGTGVSGVENPGGYCLSPSYAYHQFYTVKY PNLTP
HPARIGLCSANNSHHYISGEGWPYIACRTSIVGRAWEDAPI
...TAAGGCGCGCC YTNVYPYDVPDYAAA
The epitope string does not contain Apal nor Ascl target sequences.
The epitope string does not contain the pox vims early gene transcription termination sequence, TTTTTNT.
Vector manufacture
The ME1 epitope string was ligated into a DNA vaccine vector (PSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.MEl was subsequently recombined into the chromosome of FP9 following established methods to constmct FP9.ME1.
This vims was subsequently bulk-purified by propagation in chicken embryo fibroblasts (CEF) and centrifugation through a 30% sucrose cushion. The titre of the vims was confirmed by titration on CEF and X-gal staining, following established methods. Vimses were prepared for immunisation by suspension in pyrogen free PBS at 1 X 107 PFU/ml. Plasmid PSG2.ME1 was bulk-purified using a Qiagen Giga column and resuspended at 1 mg/ml in pyrogen free PBS. Female BALB/c (H-2d) C57BL/6 (H-2b) mice (6 - 8 weeks old) were immunised intramuscularily (im.) with PSG2.ME1 or a control plasmid pSG2.Mel3, which contains an irrelevant epitope string. Vimses FP9.ME1 and the empty control vims FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 106 PFU. Animals were boosted two weeks after immunisation. Fourteen days after boosting, the mice were sacrificed by cervical dislocation and the T cell response elicited against the LCMV, P815, CT26 and control epitopes from β-galactosidase were determined using the IFNγ assay as described in Example 16.
Results were calculated as the number of antigen-specific IFNγ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the students T-test (two-sample assuming equal variances) and ANOVA using Microsoft Excel 2000.
Experimental Design and Results
To avoid competition between the viral and tumour epitopes, ME1 encodes epitopes from LCMV antigens characterised in H-2b haplotype mice and those from tumour antigens characterised in H-2d haplotype mice. Accordingly, the capacity of FP9.ME1 to elicit CD4+ and CD8+ T - cell epitopes was tested in both BALB/c (H-2d) C57BL/6 (H-2d) mice as described in Table 6. The results of these experiments are shown in Table 6.
Table 6: Experiments to determine the immunogenicity of the ME1 multiple epitope string
Figure imgf000068_0001
Figure imgf000069_0001
Experiments 1 and 2 were conducted in parallel. Each group contained four 6 - 8 week-old female mice. Vaccines were administered as described in Methods and priming and boosting were 14 - 16 days apart. T- cell responses were determined by the IFNγ ELISPOT assay as in Example 16 using the LCMV, tumour and model epitopes described above. Results relate to the cumulative response against the LCMV and tumour derived epitopes, which are based on observations using the murine malaria and tuberculosis models.
EXAMPLE 18: Recombinant FP9 protects against cancer
Objective
To determine whether immunisation with FP9.ME1 alone, or in a prime/boost regime with pSG2.MEl, will elicit a protective immune response against CT26 tumour challenge in mice. Therefore, demonstrating in principle that recombinant FP9 can elicit antigen-specific T cell responses that will protect against cancer.
Method
Female BALB/c (H-2d) mice (6 - 8 weeks old) will be injected with pSG2.MEl, FP9.ME1, or control constmcts pSG2.Mel3 and FP9.EFL29 as described above. Animals are boosted two weeks after immunisation. Fourteen days after boosting, the mice are challenged by subcutaneous (sc.) injection in the left flank with 5 X 105 CT26 tumour cells as follows:
Growth of CT26 tumours subcutaneously Some experimental questions are best addressed using a measurable solid tumor mass implanted subcutaneously. CT26 cells are amenable to this experimental approach. Although conventional wild-type CT26 cells grow in a laterally spreading fashion in the subcutaneous space, the highly transferable variant, CT26, grows well as a more compact tumor mass, probably related to increased adhesion properties, making for more reproducible measurements.
Materials
CT26 cells
Complete DMEM medium with 10% (w/v) FCS
Sterile PBS
BALB/c mice, 6 to 8 weeks old
1 -ml sterile disposable syringes 16-G and 25-G needles Calipers
Additional reagents and equipment for counting cells.
1. Culture CT26 cells as in complete DMEM media with 10% FCS under
2. Harvest cells and rinse two times with PBS by centrifuging for 5 min at 200 X g, room temperature.
3. Count cells and resuspend in PBS at 2 X 107 cells/ml. Transfer suspension to 1-ml syringe and 16-G needle then change to a 25-G needle.
4. Inoculate BALB/c mice by subcutaneous injection of 50 μl/mouse.
Ensure the cells are uniformly resuspended in the syringe just before injecting by gentle tapping and inversion. The conditions given will result in 106 tumor cells being implanted. A cell concentration of 2 X 105 to 1 X 107 cells has been used successfully.
5. The procedure can be accomplished rapidly by a single operator. The mouse is held in the left hand with the scruff of the neck between the first two fingers and the tail held against the palm with the fourth finger. With the right hand, the syringe is held and the needle inserted just through the skin on the left flank of the mouse. Using the syringe and needle as leverage, the skin should be gently lifted away from the body of the mouse to ensure that the tip of the needle is within the subcutaneous space. Only then is the 50 μl volume expelled.
Examine mice at least two times a week to examine for tumour growth and to assess general health. Once tumours appear, measure and record the longest and shortest dimensions using callipers.
The mean of these two measurements will yield the Mean Tumour Diameter for that mouse at that time point.
Tumours are first palpable 1 week after implantation. Measurements should be made to the nearest 0.5 mm.
Mice are observed for two weeks following challenge for evidence of tumour development, then sacrificed and dissected. Tumour size is determined by measuring the length and the width of the tumour, then taking the average of these measurements. Statistical analysis is performed using GraphPrism Instat, with differences in tumour incidence determined by the Fishers Exact test and differences in tumour size using the students T-test (two-sample assuming unequal variances).
Experimental Design and Expected Results CT26 is a colonic carcinoma cell line derived from BALB/c mice. CT26 forms a solid tumour in BALB/c mice approximately 1 week after subcutanteous injection. The MEl epitope string contains a protective CD8+ restricted epitope derived from the MuLV gp70 envelope protein that is expressed by the CT26 tumour (see above). Since the prime/boost immunisation regime using pSG2.MEl followed by FP9.ME1 elicits an enhanced CD8+ T cell immune response against this epitope (see above), we showed that this immunisation regime elicits protection against CT26 challenge in BALB/c mice. Table 7 shows the results.
Table 7: tumour incidence in immunised BALB/c mice following tumour challenge
Figure imgf000072_0001
Groups of 12 BALB/c mice are primed and boosted 14 days apart as shown. Fourteen days after boosting the animals are challenged by sc. injection with CT26 cells in the left flank. The animals are observed for two weeks, then dissected and tumour presence and size determined as described in Methods.
The table presents tumour incidence based on the immunogenicity data presented in 16. Differences between the average tumour size in each group would also be expected, but are not shown in the table. EXAMPLE 19: Recombinant FP9 protects against Lymphocytic Choriomeningitis vims (LCMV)
Objective:To determine whether immunisation with FP9.ME1 alone, or in a prime/boost regime with PSG2.ME1, will elicit a protective immune response against LCMV challenge in mice.
This demonstrates that recombinant FP9 can elicit antigen-specific CD4+ and CD8+ T cell responses that will protect against viral infection when used in accordance with the present invention.
Method
Female C57BL/6 (H-2b) mice (6 - 8 weeks-old) were injected with PSG2.ME1, FP9.ME1, or control constmcts pSG2.Mel3 and FP9.EFL29 as described above. Animals were boosted two weeks after immunisation. Fourteen days after boosting, 6 animals per group were challenged by intraperitoneal (ip.) injection with 2 X 105 PFU LCMV Armstrong (mild infection) and 6 animals iv. with 2 X 106 PFU LCMV Clone 13 (severe infection).
Animals challenged with LCMV Armstrong were sacrificed 3 days after challenge and those challenged with LCMV Clone 13 were sacrificed 7 days after challenge.
Viral load in the spleens of challenged animals is determined as follows:
Plaque assay for the titration of LCMV
1. Aliquot 3 x 105 Vero cells in 3ml of medium into each well of a six-well plate and culture overnight at 37 °C under 5% CO2 until they form a confluent monolayer. 2. Make serial dilutions of the test samples on ice, starting at 1 : 100 (10 μl sample + 1ml medium) and making subsequent 10-fold dilutions (100 μl + 900 μl medium). 3. Remove the medium from the Vero cells (taking care not to damage the monolayer) and add 500 μl per well of the diluted test sample. Include at least one well per assay to which medium only is added as a negative control. Incubate the plates at 37 °C under 5% CO2 for one hour to allow vims infection.
4. Overlay each well with 3 - 4ml of a 1 : 1 mixture of 1 % agarose in water and 2 x 199 medium containing 10% FBS. Wrap the plates in foil and culture at 37 °C under 5% CO2 incubator for six days.
5. Fix and stain.
Statistical analysis was performed using Excel 2000, with differences in incidence of infection determined by the Chi-squared test and differences in viral load determined using the students T-test (two-sample assuming equal variances). Two-sided P-values are determined in all cases.
Results
LCMV is a well characterised vims that causes chronic and acute infections in mice. The Armstrong strain causes a mild, self-resolving infection, whereas the Clone 13 strain causes a severe infection that can develop into a chronic infection. The MEl epitope string was designed to contain two CD4+ epitopes and several CD8+ restricted epitopes from LCMV (see above), some of which have been characterised as protective against LCMV infection. Since prime/boost immunisation using FP9.ME1 elicits a significantly enhanced T cell response against the LCMV epitopes in MEl (Figure 17), this immunisation regime exhibits protective efficacy against challenge with both the Armstrong and Clone 13 strains of LCMV. Table 8 shows the results.
Table 8: LCMV challenge in C57BL/6 mice
Figure imgf000074_0001
Figure imgf000075_0001
Groups of 12 C57BL/6 mice are primed and boosted 14 days apart as shown. Fourteen days after boosting, 6 animals per group will be challenged with LCMV Armstrong (mild infection) or LCMV Clone 13 (severe infection) as described in Methods. The limit of detection of the assay is 1000 pfu/organ (Logio 3.00). Viral loads for groups A, B and C are based on results using 6 animals per group. Viral loads for D, E and F are on the immunogenicity results presented in Figure 17).
Figure 15 shows antigen-specific immune responses following heterologous prime/boost immunisation with FP9PbCSP, FPV-MPbCSP and MVAPbCSP. BALB/c mice were immunised id. bi-laterally in the ears with FP9PbCSP (FP9), FPV-MPbCSP (FPV-M), or MVAPbCSP and boosted heterologously 14 days later in a similar manner. Fourteen days after the booster immunisation the T cell response elicited against the Pb9 epitope of PbCSP and a control epitope from β-galactosidase were determined in splenocytes using the IFNγ ELISPOT assay. Columns represent the mean antigen specific IFNγ sfc/million splenocytes ± 1 SD for four mice per group. P values were determined by the student's T test assuming equal variances using Microsoft Excel 2000.
Example 20: Websters' Attenuated Fowlpox is Disctinct from FP9 In this Example the unique character of FP9 compared to existing fowlpox vectors is demonstrated. The genetic composition of FP9 is determined as above and compared to existing pox vector 'Websters'. Data are presented in Table 9.
Deletions
Twenty-five deletions were observed in FP9 relative to the virulent US FPV sequence (FPV US; Afonso et al., 2000). Of these, 6 were differences between the US and European lineage and 19 occurred during passage (and concomitant attenuation) that eventually led to FP9.
Of those 19 passage-specific deletion loci, 15 have been examined in Websters (FPV- M) and all show the same sequence as FPV US, not that of FP9.
Insertions
Similarly, 15 insertions distinguish FP9 from FPV US. Of these, only 5 occurred during passage and attenuation.
One of these 5 passage-specific insertion loci has been checked in Websters and it shows the same sequence as FPV US, not FP9.
Base substitutions
77 single base substitutions distinguish FP9 from FPV US. Of these, 25 occurred during passage and attenuation.
11 of these 25 passage-specific substitution sites have been checked in Websters and, in all cases, they have the same sequence as FPV US, not FP9. Another FP9 mutation, which is also found in HPl and is thus a lineage-specific mutant, is also the same as FPV US in Websters. Thus is is shown that Websters attenuated vaccine is clearly genetically distinct from FP9.
Example 20 Table 9
-4 -4
Figure imgf000078_0001
-4 oe
Figure imgf000079_0001
Figure imgf000080_0001
xl25_ H10
Insertion Insertό. 2bp (AT)x4 in FPV US -> as US 104 x5 (f/s)
Figure imgf000081_0001
oe o
Figure imgf000081_0002
Figure imgf000082_0001
Example 21: Recombinant FP9 elicits CD4 and CD8 T cell responses against epitopes from viruses and tumours
Objective
Further to Example 17, this Example further determines whether immunisation with recombinant FP9 alone, or in a prime/boost regime, will elicit T cell responses against CD4+ and CD8+ T cell epitopes from viral and tumour antigens.
Method
A synthetic gene of 440 bp encoding for a novel polypeptide (MEl) consisting of characterised CD8+ and CD4+ restricted epitopes from lymphocytic choriomeningitis vims (LCMV) antigens and murine tumour (P815 and CT26) antigens was synthesised (see above). MEl was ligated into a DNA vaccine vector (pSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.MEl was subsequently recombined into the chromosome of FP9 following established methods to constmct FP9.ME1. This vims was subsequently bulk-purified by propagation in chicken embryo fibroblasts (CEF) and centrifugation through a 30% sucrose cushion. The titre of the virus was confirmed by titration on CEF and X-gal staining, following established methods. Vimses were prepared for immunisation by suspension in pyrogen-free PBS at 1 X 107 PFU/ml. Plasmid pSG2.MEl was bulk-purified using a Qiagen Giga column and resuspended at 1 mg/ml in pyrogen free PBS.
Female BALB/c (H-2d) or C57BL/6 (H-2b) mice (6 - 8 weeks old) were immunised intramuscularly (im.) with 50μg of pSG2.MEl or control plasmid pSG2.Mel3, which contains an irrelevant epitope string. Vimses FP9.ME1 and the empty control vims FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 106 PFU. Animals were boosted 14 - 15 days after immunisation. Fourteen to 15 days after boosting, the mice were sacrificed by cervical dislocation and the T cell response elicited against the LCMV, P815, CT26 and control epitopes from β-galactosidase were determined using the IFNγ assay as described in above. Results were calculated as the number of antigen-specific IFNγ spot forming cells/million splenocytes (sfc/million). Differences between groups were determined by the student's T-test (two-sample assuming unequal variances) using Microsoft Excel 2000. One-sided -values are given in all cases.
Results
To avoid competition between the viral and tumour epitopes, MEl encodes epitopes from LCMV antigens characterised in H-2 haplotype mice and those from tumour antigens characterised in H-2d haplotype mice (Appendix II). Accordingly, the capacity of FP9.ME1 to elicit immune responses was tested in both BALB/c (H-2d) and C57BL/6 (H- 2b) mice.
Immunisation of BALB/c mice using pSG2.MEl and/or FP9.ME1 elicited IFNγ-secreting T cells against the tumour epitopes (H-2 ) (Figure 16), but not against the LCMV epitopes (H-2b). The total frequency of IFNγ-secreting T cells elicited against the tumour epitopes was significantly (P < 0.004) higher following heterologous prime/boost immunisation with pSG2.MEl/FP9.MEl than other heterologous or homologous immunisation regimes (Figure 16). Immunisation with other regimes elicited T cell responses that were significantly higher than immunisation with control constmcts, but not significantly different (P > 0.05) from each other. Importantly, prime/boost immunisation with pSG2.MEl/FP9.MEl elicited substantial T cell responses against the immunodominant epitope from CT26, the MSR epitope from P815 and a model Ld-restricted epitope from beta-galactosidase. Among these epitopes, response against the CT26 epitope was most substantial and exceeded the limit of detection (2000 sfc/million) of the IFN-γ ELISPOT assay used in this experiment. These results indicate that prime/boost immunisation using pSG2.MEl/FP9.MEl is likely to elicit a protective immune response against CT26 colo- rectal carcinoma in BALB/c mice (See above).
Immunisation of C57BL/6 mice using pSG2.MEl and/or FP9.ME1 elicited IFNγ- secreting T cells against the LCMV epitopes (H-2 ), but not against the tumour epitopes (H-2b) (Figure 17). The total frequency of IFNγ-secreting T cells elicited against LCMV epitopes by prime/boost immunisation with pSG2.MEl/FP9.MEl was significantly higher than that elicited by homologous immunisation with FP9.ME1 (P = 0.003) alone or pSG2.MEl (P = 0.016) alone. Interestingly, heterologous prime/boost immunisation regimes using pSG2.MEl and FP9.ME1 elicited greatly enhanced immune responses against CD8+ epitopes characterised as dominant and subdominant, and those recognised by CD4+ T cells when compared to the homologous immunisation regimes using pSG2.MEl and FP9.ME1 alone. Thus, this prime/boost immunisation regimes is likely to be even more efficacious against LCMV infection in C57BL/6 mice than the homologous prime/boost immunisation regimes (See above).
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a first non-replicating viral vector;
(ii) a second composition which comprises a second non-replicating viral vector to the subject in either order wherein at least one of the composition(s) comprises a poxvirus vector derivable from a fowlpox vims.
2. A method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a DNA vaccine; (ii) a second composition which comprises a first non-replicating viral vector (iii) a third composition which comprises a second non-replicating viral vector; to the subject wherein at least one of the composition(s) comprises a poxvirus vector derivable from a fowlpox virus.
3. A method for treating and/or preventing a disease in a subject which comprises the step of administering
(i) a first composition which comprises a first non-replicating viral vector;
(ii) a second composition which comprises a second non-replicating viral vector
(iii) a third composition which comprises a third non-replicating viral vector; to the subject wherein at least one of the composition(s) comprises a poxvirus vector derivable from a fowlpox vims.
4. A method according to any of claims 1-3 wherein at least one of the vectors is, or is derivable from, FP9.
5. A method according to any preceding claim wherein each of said non-replicating viral vector(s) comprises a poxvirus vector.
6 A method according to claim 5 wherein at least two of the poxvims vectors are derivable from poxvimses from different genera.
7. A method according to claim 6 wherein at least one poxvims vector is derivable from an avipox vims and at least one poxvims vector is derivable from an orthopox vims.
8. A method according to any preceding claim wherein one of the vectors comprises a fowlpox vims genome selected from
(i) a fowlpox vims genome which has a modified form of one or more of the following wild-type FPV genes:
FPVOOl, FPV018, FPV054, FPV063, FPV066, FPV070, FPV071, FPV093, FPV097, FPV098, FPV115, FPV124, FPV125, FPV127, FPV158, FPV159, FPV160, FPV190, FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260;
(ii) a fowlpox vims genome which has a partial deletion in one or more of the following genes:
FPV158, FPV219, FPV222;
(iii) a fowlpox vims genome which lacks one or more of the following genes:
FPVOOl, FPV 124, FPV 125, FPV 159, FPV 160, FPV220, FPV221, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260;
(iv) a fowlpox vims genome which has a frame-shift mutation in one or more of the following genes:
FPV054, FPV070, FPV071, FPV115, FPV190, FPV207; (v) a fowlpox vims genome which has a termination mutation in one or more of the following genes:
FPV071. FPV239;
(vi) a fowlpox vims genome which has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV098;
(vii) a fowlpox vims genome which is less than 275 kbp in size;
(viii) a fowlpox vims genome comprising the sequence shown in SEQ ID No. 1
(ix) a fowlpox vims genome comprising the sequence shown in SEQ ID No. 1, with a deletion of ACTGGTGCGAAG ATCTC from ORF 1.
9. A method according to claim 8 wherein the fowlpox vims genome also comprises a NOI.
10. A method according to claim 9 wherein the NOI is under the control of a poxvims promoter.
11. A method according to claim 9 or 10 wherein the NOI encodes an antigen from P. berghei, P. falciparum, P.cynomolgi, P. vivax, M. tuberculosis or T.parva.
12. A method according to claim 7 wherein the composition comprising the viral vector derivable from an avipox virus is administered as a boosting composition.
13. A method according to claim 7 wherein the composition comprising the viral vector derivable from an avipox vims is administered as a priming composition.
14. A method according to any preceding claim wherein the subject is a primate.
15. A method according to claim 14 wherein the subject is a human.
16. Use of a non-replicating viral vector in a vaccine for an animal, wherein said vector is, or is derivable from, FP9.
17. Use according to claim 16 wherein said animal is a mammal.
18. Use according to claim 17 wherein said mammal is a primate.
19. Use according to claim 18 wherein said primate is a human.
20. Use of a non-replicating viral vector in medicine, wherein said vector is, or is derivable from, FP9.
21. A method of eliciting an immune response in a subject comprising administering a composition comprising a non-replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
22. A method of boosting a pre-existing immune response in a subject comprising administering a composition comprising a non-replicating viral vector to said subject wherein said vector is, or is derivable from, FP9.
23. A fowlpox vims genome which has a modified form of one or more of the following wild-type FPV genes:
FPVOOl, FPV018, FPV054, FPV063, FPV066, FPV070, FPV071, FPV093, FPV097, FPV098, FPV115, FPV124, FPV125, FPV127, FPV158, FPV159, FPV160, FPV190, FPV191, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260.
24. A fowlpox vims genome according to claim 23, which has a partial deletion in one or more of the following genes:
FPV158, FPV219, FPV222.
25. A fowlpox vims genome according to claim 23 or 24, which lacks one or more of the following genes:
FPVOOl, FPV 124, FPV 125, FPV 159, FPV 160, FPV220, FPV221, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260
26. A fowlpox vims genome according to any preceding claim, which has a frame- shift mutation in one or more of the following genes:
FPV054, FPV070, FPV071, FPV115, FPV190, FPV207.
27. A fowlpox vims genome according to any preceding claim, which has a termination mutation in one or more of the following genes:
FPV071. FPV239.
28. A fowlpox virus genome according to any preceding claim, which has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV098.
29. A fowlpox virus genome which is less than 275 kbp in size.
30. A fowlpox vims genome according to any preceding claim, comprising the sequence shown in SEQ ID No. 1.
31. A genome according to any preceding claim which also comprises an NOI.
32. A genome according to claim 31 , wherein the NOI is under the control of a poxvims promoter.
33. A genome according to claim 31 or 32, wherein the NOI encodes an antigen from P. berghei, P. falciparum, P.cynomolgi, P. vivax, M. tuberculosis or T.parva.
34. A viral particle which comprises a genome according to any preceding claim.
35. A viral particle which comprises a genome according to any of claims 31 to 33 and which is capable of delivering the NOI to a target cell.
36. A vaccine comprising a viral particle according to claim 34 or 35.
37. A priming or boosting agent comprising a viral particle according to claim 34 or 35.
38. A vaccination kit which comprises:
(i) a first composition which comprises a Fowlpox viral particle according to claim 34 or 35; and (ii) a second composition for simultaneous, separate or sequential administration.
39. A kit according to claim 38, which comprises:
(i) a priming composition which comprises an Fowlpox viral particle according to claim 34 or 35; and
(ii) a boosting composition which comprises a vaccinia viral particle.
40. A vaccination kit which comprises:
(i) a first composition which comprises a first non-replicating viral vector; (ii) a second composition which comprises a second non-replicating viral vector for sequential administration to a primate in either order.
41. A vaccination kit according to claim 40, which comprises:
(i) a first composition which comprises a first non-replicating poxvims vector; (ii) a second composition which comprises a second non-replicating poxvims vector.
42. A kit according to claim 41, wherein the first non-replicating poxvims vector is an avipox vims vector and the second non-replicating poxvims vector is an orthopox vims vector.
43. A kit according to claim 41 or 42 wherein the first non-replicating poxvims vector is a fowlpox vims vector.
44. A kit according to claim 43, wherein the first non-replicating poxvims vector is or is derivable from a viral particle according to claim 34 or 35.
45. A kit according to any of claims 38 to 44, wherein the first and second compositions are capable of expressing the same antigen.
46. A boosting composition which comprises a non-replicating viral vector capable of boosting a pre-existing immune response in a primate subject.
47. A boosting composition according to claim 46, which comprises a fowlpox viral vector.
48. A boosting composition according to claim 47, which is or is derivable from a viral particle according to claim 34 or 35.
49. A vaccination method which comprises the step of administering a vaccine according to claim 36, a priming or boosting composition according to any of claims 37, 46, 47 or 48 or a kit according to any of claims 38 to 45 to a subject.
50. The use of a vaccine according to claim 36, a priming or boosting composition according to any of claims 37, 46, 47 or 48 or a kit according to any of claims 38 to 45 in the manufacture of a medicament for treating and/or preventing a disease in a subject.
51. A method according to claim 49 or a use according to claim 50, which elicits a T- cell immune response in the subject.
52. A method or use according to any or claims 49 to 51 , wherein the disease is or results from a chronic infection such as malaria, tuberculosis or East Coast Fever.
53. The use of non-cultured CEF cells to grow an avipoxvirus.
54. The use according to claim 53, wherein the avipoxvims is a fowlpox vims.
55. The use according to claim 54, wherein the poxvims is a viral particle according to claim 34 or 35.
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GB2402391A (en) * 2003-06-04 2004-12-08 Oxxon Pharmaccines Ltd Fowlpox recombinant genome
JP2013028619A (en) * 2003-06-17 2013-02-07 Mannkind Corp Method to elicit, enhance and sustain immune response against mhc class i-restricted epitope, for prophylactic or therapeutic purpose
ES2313807A1 (en) * 2005-07-30 2009-03-01 Consejo Superior De Investigaciones Cientificas Recombinant vectors based on the modified vaccinia ankara (mva) virus as vaccines against leishmaniasis
WO2007014977A1 (en) * 2005-07-30 2007-02-08 Consejo Superior De Investigaciones Científicas Recombinant vectors based on the modified vaccinia ankara (mva) virus as vaccines against leishmaniasis
WO2008122817A3 (en) * 2007-04-10 2008-12-11 Isis Innovation Immunogenic compositions
WO2008122817A2 (en) * 2007-04-10 2008-10-16 Isis Innovation Immunogenic compositions
WO2011160177A1 (en) 2010-06-23 2011-12-29 Monash University Constrained immunogenic compositions and uses therefor
US10682407B2 (en) 2010-06-23 2020-06-16 Monash University Constrained immunogenic compositions and uses therefor
WO2012016290A1 (en) 2010-08-04 2012-02-09 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Modified hepatitis c virus proteins
US9079950B2 (en) 2010-08-04 2015-07-14 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Modified Hepatitis C virus proteins
US9884109B2 (en) 2010-11-26 2018-02-06 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions and methods
WO2012068637A1 (en) 2010-11-26 2012-05-31 The Macfarlane Burnet Institute For Medical Research And Public Health Compositions and methods
US10561721B2 (en) 2014-09-03 2020-02-18 Bavarian Nordic A/S Methods and compositions for inducing protective immunity against filovirus infection
US10561722B2 (en) 2014-09-03 2020-02-18 Bavarian Nordic A/S Methods and compositions for enhancing immune responses
US11173201B2 (en) 2014-09-03 2021-11-16 Bavarian Nordic A/S Methods and compositions for inducing protective immunity against filovirus infection
US11918639B2 (en) 2014-09-03 2024-03-05 Bavarian Nordic A/S Methods and compositions for inducing protective immunity against filovirus infection
CN104878043A (en) * 2015-06-01 2015-09-02 石河子大学 Orf virus virulence gene VIR-deleted (interferon-resistance gene deleted) mutant and its preparing method and application
US10925956B2 (en) 2016-07-15 2021-02-23 Janssen Vaccines & Prevention B.V. Methods and compositions for inducing protective immunity against a marburg virus infection
US11173204B2 (en) 2017-04-06 2021-11-16 Janssen Vaccines & Prevention B.V. MVA-BN and Ad26.ZEBOV or Ad26.filo prime-boost regimen

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