ZA200402629B - Vaccine. - Google Patents

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ZA200402629B
ZA200402629B ZA200402629A ZA200402629A ZA200402629B ZA 200402629 B ZA200402629 B ZA 200402629B ZA 200402629 A ZA200402629 A ZA 200402629A ZA 200402629 A ZA200402629 A ZA 200402629A ZA 200402629 B ZA200402629 B ZA 200402629B
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vector
virus
composition
replicating
fowlpox
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ZA200402629A
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Stephen Laidlaw
Mike Skinner
Adrian Hill
Sarah Gilbert
Richard Anderson
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Isis Innovation
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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, J Med 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, J Natl 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
Living organisms Natural Vaccinia (for small pox)
TT wen : attenuated Polio (Sabin; oral polio vaccine)
Measles, mumps, rubella, yellow fever 17d
Varicella-zoster (human herpes virus 3)
BCG (for TB)
Intact but non-living | viruses Polio (Salk), rabies, influenza, hepatitis A, organisms typhus
Subcellular Capsular Pneumococcus, meningococcus,
EC I
Recombinant DNA- | Gene cloned and | Hepatitis B (yeast derived)
Genes expressed in
Ee I
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 IFNy. }
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 5 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):
FPVO001, FPV018, FPV054, FPV063, FPV066, FPV070, FPV(071, 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 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: (1) a first composition which comprises an FP9 fowlpox viral particle; and (11) 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: (1) a first composition which comprises a first non-replicating viral vector; (i1) 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 HP1.
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 CD8+ 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 IFNy ELISPOT responses against FP9 and MVA viral antigens.
Figure 9: Ex vivo IFNy Elispot response in Balb/c mice following aerosol or mucosal priming with M. bovis BCG and boosting with MVAS5A.
Figure 10: Ex vivo IFNy Elispot response in Rhesus macaques following aerosol or mucosal priming with M. bovis BCG and boosting with MVASS5A 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.ME1 and FP9.ME1. BALB/c mice were immunised im. with pSG2.MEI1 (DNA) or iv. with FP9.ME]1 (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 IFNy ELISPOT assay. Columns represent the antigen-specific IFNy 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.ME1 (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 IFNy
ELISPOT assay. Columns represent the antigen-specific IFNy 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 (CDA). :
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 ef a/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 er al(“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
Orthopoxvirus buffalopox virus, camelpox virus, cowpox virus, ectromelia virus, : monkeypox virus, rabbitpox virus, raccoonpox virus, teterapox virus, vaccinia virus, variola virus, voleopox virus, skunkpox virus, Uasin ' Gishu disease virus
Parapoxvirus bovine papular stomatis virus, orf virus, parapoxvirus of red deer in
New Zealand, pseudocowpox virus, Auzduk disease virus, chamois contagious ecthyma, sealpox virus
Avipoxvirus canarypox virus, fowlpox virus, juncopox virus, mynahpox virus, pigeonpox virus, psittacinepox virus, quailpox virus, sparrowpox virus, starlingpox virus, turkeypox virus, peacockpox virus and penguinpox virus goatpox virus, lumpy skin disease virus, sheeppox virus
Leporipoxvirus hare fibroma virus, myxoma virus, rabbit fibroma virus, squirrel fibroma virus
Molluscipoxvirus | Molluscum contagiosum virus
Yaba monkey tumor virus
The present invention provides a vaccination kit which comprises: (1) a first composition which comprises a first non-replicating viral vector; (i1) 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: (1) 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. 13
PR . 3 a ee il
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/ andIFN-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 (HPI) 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 FP1 during its passage in CEF tissue culture from HP1 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, FPVO018, FPV054, FPV063, FPV066, FPV(070, 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:
FPVO018, 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:
FPVO054, FPV070, FPV071, FPV115, FPVI190, 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 arelatively 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 ts 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 ef 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 ef 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 BLOSUMS62 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
BLOSUMSG62.
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 oo 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:
ALIPHATIC Non-polar ot
Polar - uncharged
TT ee
Polar - charged
TT mowATe | [wey 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, anti- viral 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, MUCO01, 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 MEP{Trap 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):
AgagatcccgecagacggggaacctgggtcaacgACTGGTGCGAAGATCTC Ctgegtatggatatggtcte «cg tgttgttacgtcaagagatgtaticg
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 virus, 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: (1) 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 1) 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
6) a first composition which comprises a first non-replicating pox viral vector; (i1) 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 (A) 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 (1) a first composition which comprises a first non-replicating viral vector; (i1) 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 5S 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 (1) 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, FPV124, FPV125, FPV159, FPV160, 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:
FPVO054, 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 virus 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 ORF].
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 virus 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 virus 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 virus and the other from an orthopox virus (see Table 2). :
When one of the compositions comprises a vector derivable from an avipox virus, 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 virus 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 5 .
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 (1) 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 (1) a first composition which comprises a first non-replicating viral vector;
(ii) a second composition which comprises a second non-replicating viral vector (ili) 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-MV A-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 virus and another from an orthopox virus (see Table 2). '
In a preferred embodiment, one of the vectors is derivable form a fowlpox virus. For example, one of the vectors may comprise a fowlpox virus 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, CDS 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 Tul subset (sometimes known as “inflammatory CD4 T cells”) characteristically secretes IL-2 and IFNy and mediates several functions associated with cytotoxicity and local inflammatory reactions. Tyl cells are capable of activating macrophages leading to cell mediated immunity. The Ty2 subset (sometimes known as “helper CD4 T cells”) characteristically secretes 11-4, IL-5, IL-6 and 11-10, and is thought to have a role in stimulating B cells to proliferate and produce antibodies (humoral immunity).
Ty! and Ty2 cells also have characteristic expression of effector molecules. Tyl cells expressing membrane-bound TNF and Ty2 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 1s 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 virus 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 E1-8, L1 and L2
Preferred HSV 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 virus (HPV), herpes virus-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 cruzi, 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 etal. 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 10°-10'° 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 surprisingly 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 virus 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.
S
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 provirus. 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-2K¢ 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 ef al, 1998, Eur J Immunol 28, 4345-55; Schneider et al, 1998, Nar Med 4, 397-402). Surprisingly, 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 10° 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 IFNy 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 virus. Columns in
Figure 1 represent the mean IFN-y 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).
Animals Protected Protection (%)
DNA / FP9 5/40 12.5
DNA /MVA 7/40 17.5
FP9/MVA 27/40 67.5
MVA / FP9 15/40 37.5
FP9 / FP9 3/19 15.7
MVA/MVA 3/20 15.0
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 10° PFU of FP9PbCSP (FP9) or MVAPbCSP (MVA), or intramuscularly with 50 pg 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 IFNy ELISPOT assay 14 days following the boost. Columns represent the mean
IFNy 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 viruses. 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;SEPTL, 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 [ComtrutName | Recombinant Anges | Eprsion | Patogen | Dowse
FP9PbLCSP Circumsporozoite Yes Plasmodium Malaria — Rodent pe
FPOMEPfTrap Epitope string fused to | Yes Plasmodium Malaria- Human’
Trombospondin-related Jalciparum adhesive protein (TRAP)
FPIPfLSA-1 Liver Stage Antigen | Yes (LSA1)
FPIL3SEPTL Exp 1, Pfs16, LSAI, Yes
FP9PcCSP CSP ND Plasmodium Malaria- me EE
FP9PcTRAP TRAP
FP9PvCSP CSP ND Plasmodium Malaria- Human and nd i cal
FPOPVTRAP TRAP
FPOAg85A Antigen 85A Yes Mycobacterium | Tuberculosis-
RS SN ha il
Cattle
FPOPfsi6 Pfs16 ND . FP9Tel Epitope string ND ® 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 ef al, 1997 Nat Biotechnol 15, 1280-4) fused to the P. falciparum TRAP gene (Robson er 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 (107420 IFNy spot forming cells/million splenocytes).
EXAMPLE 6: Induction of CD8+ and CD4+ T cell immune responses against Antigen 85A 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-2¢ MHC class [I-restricted peptide epitope (recognised by CD4+ T-cells) as well as a nine amino acid H-2¢ 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 MVASS5A in Rhesus macaques (Figure 7).
Method
Female Balb/c mice were immunised intravenously with 1 x 10’ PFU of recombinant
FP9 encoding antigen 85A (FP9Ag85A) from mycobacterium tuberculosis or, for comparison, 1 x 10° PFU of MVA encoding the same antigen. The response against 85A was determined using the MHC-class II restricted P15 epitope (recognised by
CD4+ T-cells) and the MHC-class I restricted P11 epitope (recognised by CD8+ T- cells) in an IFNy ELISPOT assay six days following immunisation. Columns represent the mean IFNy 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 85A (MVAB8SA) was boosted Wd) 21 and 26 weeks later with 5 x 10® 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 IFNy ELISPOT assay. Curves represent the IFNy 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 ef 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 FPOPbCSP do not produce IFNy when exposed to naive 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 IFNy 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 viruses.
Further preliminary results indicate that non-replicating canary poxviruses also do not cross-react with MVA. Thus, the avipoxviruses 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 IFNy elispot responses against PbCSP and viral antigens in splenocytes isolated from mice fourteen days after i.v. immunisation with either FP9PbCSP or
MVAPbCSP. The IFNy 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 IFNy spot forming cells (sfc) per million splenocytes + S.E.M. for three mice per group.
EXAMPLE 8 - Growth of avipoxviruses in vitro
Wild-type and recombinant poxviruses 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 virus (Table 5). In contrast, good plaque formation and consistently high yields of MV A 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 virus'(Table 5) following infection of monolayers of primary “non-cultured” CEFs. Similarly, plaque formation and higher yields of attenuated canarypox virus, ALVAC have been obtained using primary “non-cultured” CEFs by the present inventors.
Together these studies indicate that the avipoxviruses are more fastidious than MVA, and possibly other orthopoxviruses, in terms of their growth requirements. Without wishing to be bound by theory, the present inventors believe that the replication of avipoxviruses within CEFs may require a specific host molecule that is lost as the
CEFs are cultured in vitro. In contrast, MVA, and possibly the orthopoxviruses 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 avipoxviruses. 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 virus from cultured and non-cultured CEFs
Virus Approximate yield of virus
IR ves [Cao Nor Cured CBR !Cultured CEFs were passaged in culture prior to infection with virus.
For “non-cultured” CEFs, primary CEFs were loaded at approximately 1.5 X 10° cells/cm? 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 FPOMEPfTrap (see Example 5):
Healthy adult volunteers were immunised twice with over 3 X 107 PFU of
FPOMEP{Trap and subsequently boosted with the same dose of MVAMEP{Trap. 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-hoost immunisation of human volunteers with two recombinant non-replicating viruses 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 MEP{Trap 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 constructs encoding the MEP{Trap molecule is conducted in the Gambia.
Adult volunteers (approximately 12) are immunised twice with the FPOMEP{Trap construct and boosted with a single dose of MVAMEP{Trap. T-cell and antibody immune responses are determined in blood, the primary purpose of the study being to demonstrate whether immunisation with the recombinant non-replicating poxviruses boosts the existing anti-malaria response primed by natural infection. : Phase IIb study
Phase IIb 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 runs 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 viruses 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 11 - Boosting the T-cell response against Ag85A primed by M. bovis BCG using non-replicating recombinant poxviruses 20M. 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 al., 2001 Infect Immun 69, 2714-7) or a
DNA-vaccine encoding this antigen (Tanghe ef al., 2001 Infect Immun 69, 3041-7).
Based on the observation that FP9Ag85A can boost an immune response primed by immunization with MVAS5A, the present inventors wished to determine whether poxvirus constructs 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 MVASSA 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 viruses 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 poxviruses 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 10° PFU of MVA85A. Control animals were immunized with
M. bovis BCG or MVASSA alone. T-cell immune responses were determined against
PPD and Ag85A using the P15 (CD4+) and P11 (CD8+) epitopes in an IFNy
ELISPOT assay 12 days following immunisation. Columns (Figure 9) represent the mean IFN-y 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 10° 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 IFNy ELISPOT assay. Curves (Figure 10) represent the IFN-y 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), 1x 10e8 pfu of FP9-ME.TRAP intradermally at week 7, and 1.5x10e8 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-naive 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 1x10e8 pfu at day 0
FP9-ME.TRAP was administered intradermally at 1x10e8 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. falciparum 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 s5 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 1.5x10e8 MVA ME. TRAP 3-4 week later followed by 1.0x10e8 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 subjects 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 siginificant difference between these.) 2) FFM regime
FP9 (1x10e8 i.d.) administered twice at 3 week intervals followed by MVA-ME.TRAP 1.5x10e8 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 MV A 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-MV A-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 ESATG6 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 FPOPbCSP 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 MVAPbBCSP 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 bi- lateral 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 3-galactosidase were determined using the IFNy assay as described below:
Murine IFNy ELISPOT protocol
A. Materials
IFN-gamma ELISpot ALP Kit Mabtech 3321-2A 600pg anti-IFN-gamma purified Mab AN18 50g anti-IFN-gamma biotinylated Mab R46A2 50ul Streptavidin-Alkaline Phosphatase
Complete a-MEM medium 500ml MEM a-modification Sigma M-4526 50ml FCS [10%] Sigma F-2442
5ml pen/strep [100U penicillin 100pg strep] Sigma P-0781 10ml L-glutamine [4mM] Sigma G-7513 500pl 2-Mercaptoethanol [50pm] Gibco BRL 31350-010
ACK buffer 8.29g NH,Cl1 [0.15M] (Sigma A-4514) 1g KHCO;[1mM] (Sigma P-9144) 37.2mg Na,EDTA (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 200ul of 25x buffer 50ul reagent A 50ul 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 IFNy (Mab AN18) antibody. Dilute to 10pug/ml in Phosphate
Buffered Saline (PBS; Sigma P-3813) and add 50pl 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. F lick off the PBS, add 100ul complete o-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. Crush individual spleens in 2 ml of PBS with the plunger of a 10ml syringe in a 70um 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 1500rpm 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 1500rpm 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 1500rpm 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: : 50pl 50ul 50ul a “—> >> “—> > > 1 2 3 4 5 6 7 8 9 10 11 12
HEINE EN EI EE qwel [of feel | Jo]
I ES I EO I
I ES EO II EI EEO I
I I BN EO EO
Ec CE
FE J
C Cc 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 50pl of complete alpha MEM medium to columns 3, 4, 7, 8, 11&12. 3.2. Add 150p1 of splenocytes to columns 2, 6 and 10 in duplicate. (Up to 12 samples per plate) 3.3. Take 50pl 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 50u! from columns 2, 6 and 10, to columns 3, 7 and 11, mix well and transfer 50ul to 4, 9 and 12. Discard 50pl 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/m! in complete o-MEM medium. Add 50pul of control peptide and target cells to columns 1, 5 and 9.Add 50pl 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 P1379), once with distilled water and twice with PBST.
4.2. Add 50pl/well of biotinylated rat anti-mouse interferon-gamma diluted to 1pg/ml in PBS. Incubate for 2 hours at room temperature. 4.3. Wash plates four times with PBST, then add 50 ul Streptavidin Alkaline
Phosphatase (Mabtech) diluted to 1ug/ml in PBS. Incubate at room temperature for 1 hour. 4.4. Wash plates four times with PBST, add 50ul/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 IFNy 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 MVAPDbCSP 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 virus (LCMV) antigens and murine tumour (P815 and CT26) antigens was synthesised as follows:
ME] model epitope string:
AT cell epitope string encoding tumour and virus epitopes relevant in murine models of chronic infection (LCMV) and cancer (P815/CT26) is generated as set out below.
Disease Model Abbr. Epitope MHC CD8/4 | Antigen restrictio a =
Cancer P815 in P1A LPYLGWLVF Lf CD8 P1A
CET
P815 in MSR GYCGLRGTGV K* CD8 MSR
Err
CT26 in CT26 SPSYAYHQF L° CD8 gp70 [os I al
Virus LCMV 2 FQPQNGQFI y CDS NP 396-
RA on Sl i
LCMV 3 SGVENPGGYCL ’ CD8 GP276-
Fl i
J Ml ll +l 212
LCMV 5 CSANNSHHYI Db CD38 GP 92-101
LCMV GLNGPDIYKGVYQFKSV | H-2° CD4 GP61-80 . EFD
LCMV 7 SGEGWPYIACRTSIVGR H-2" CD4 NP309-
AWE 328
HA tag YPYDVPDYA Antibody Tag for detection of expression
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-2% and H-2".
Epitope String Sequence
Showing Epitopes:
Cloning site Kozak M [GLNGPDIYKGVYQFKSVEFD] [KAVYNFATCGI] [LPYLGWLVF] [FQPQNGQFI] [GYCGLRGTGV] [SGVENPGGYCL] [SPSYAYHQF] [YTVKYPNL] [TPHPARIGL] [CSANNSHHYT] [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
YINVYPYDVPDYAAA
The epitope string does not contain Apal nor Ascl target sequences.
The epitope string does not contain the pox virus early gene transcription termination sequence, TTTTTNT.
Vector manufacture
The ME epitope string was ligated into a DNA vaccine vector (PSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.ME1 was subsequently recombined into the chromosome of FP9 following established methods to construct FP9.ME1.
This virus 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. Viruses were prepared for immunisation by suspension in pyrogen free PBS at 1 X 10’ PFU/ml. Plasmid PSG2.ME! was bulk-purified using a Qiagen Giga : column and resuspended at 1 mg/ml in pyrogen free PBS.
Female BALB/c (H-2%) C57BL/6 (H-2") mice (6 — 8 weeks old) were immunised : intramuscularily (im.) with PSG2.MEI1 or a control plasmid pSG2.Mel3, which contains an irrelevant epitope string. Viruses FP9.ME1 and the empty control virus ’ FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 10°
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 B-galactosidase were determined using the IFNy assay as described in Example 16.
Results were calculated as the number of antigen-specific [FNy 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. 15S Experimental Design and Results
To avoid competition between the viral and tumour epitopes, ME! encodes epitopes from LCMV antigens characterised in H-2° haplotype mice and those from tumour antigens characterised in H-2? haplotype mice. Accordingly, the capacity of FP9.MEI1 to elicit CD4+ and CD8+ T — cell epitopes was tested in both BALB/c (H-2%) C57BL/6 (H-2% 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 a5 ego
C57BL/6 A PSG2 FP9.EFL29 0 0
BNHEEEH
C PSG2.ME1 FP9.ME1 <2000 0
D FP9.MEI1 FP9.MEI1 >200 0
E PSG2.ME1 PSG2.ME1 >100 0
2 BALB/c A PSG2 FP9.EFL29 0 0 (H-2% B FP9.ME1 PSG2.ME1 0 >200
C PSG2.ME1 FP9.MEI1 0 <2000
D FP9.ME1 FP9.ME1 0 >200
E PSG2.ME1 PSG2.ME1 0 >100
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 IFNy 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.ME]1 alone, or in a prime/boost regime with pSG2.MEI1, 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-2% mice (6 — 8 weeks old) will be injected with pSG2.MEI,
FP9.MEI, or control constructs 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° 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 transfectable 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 10’ 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 pl/mouse.
Ensure the cells are uniformly resuspended in the syringe just before injecting by gentle tapping and inversion.
The conditions given will result in 10° tumor cells being implanted. A cell : concentration of 2 X 10° to 1 X 10” 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 pl 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 MEI 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.ME1 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
Group Prime Boost Tumour a
A None None 12/12
B pSG2.Mel3 | FP9.EFL29 11/12
D FP9.ME1 pSG2.ME1 8/12
D pSG2.MEl1 FP9.ME1 0/12
E FP9.ME1 FP9.ME1 5/12
F pSG2.ME1 | pSG2.ME1l 8/12
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. n
EXAMPLE 19: Recombinant FP9 protects against Lymphocytic Choriomeningitis virus (LCMV) :
Objective:To determine whether immunisation with FP9. ME] alone, or in a ‘ 5S 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-2°) mice (6 - 8 weeks-old) were injected with PSG2.ME1,
FP9.ME1, or control constructs 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° PFU
LCMV Armstrong (mild infection) and 6 animals iv. with 2 X 10° 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 ul sample + ml medium) and making subsequent 10-fold dilutions (100 pl + 900 pl medium).
3. Remove the medium from the Vero cells (taking care not to damage the monolayer) and add 500 pl 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 virus 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 virus 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 ME] 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. ME] elicits a significantly enhanced T cell response against the LCMV epitopes in ME1 (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
Armstrong Clone 13
A None None 6.26 + 0.33 7.53 +£0.04 c pSG2.Mel3 | FP9.EFL29 524+ 0.15 7.321 0.11
D | pSG2ME1 FP3.ME1 3.00 £0.05 3.00 + 0.05 .
E FP9.MEI FP9.MEL 5.00 + 0.10 6.00 + 0.10
F PSG2MES PSGZ.MEI 4.00 + 0.10 5.00 £ 0.10
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 (Logo 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 MVAPDBCSP 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 B-galactosidase were determined in splenocytes using the IFNy ELISPOT assay. Columns represent the mean antigen specific IFNy 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 HP1 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.
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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 (ME1) consisting of characterised CD8+ and CD4+ restricted epitopes from lymphocytic choriomeningitis virus (LCMV) antigens and murine tumour (P815 and CT26) antigens was synthesised (see above). MEI was ligated into a DNA vaccine vector (pSG2) and the fowlpox shuttle vector pEFL29. Plasmid pEFL29.ME1 was subsequently recombined into the chromosome of FP9 following established methods to construct FP9.MEI1. This virus 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. Viruses were prepared for immunisation by suspension in pyrogen-free PBS at 1 X 10’ 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-2% or C57BL/6 (H-2°) mice (6 — 8 weeks old) were immunised intramuscularly (im.) with 50pg of pSG2.ME1 or control plasmid pSG2.Mel3, which contains an irrelevant epitope string. Viruses FP9.ME1 and the empty control virus
FP9.EFL29 were administered intravenously (iv.) into the tail vein at a dose of 1 X 10°
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 p-galactosidase were determined using the IFNy assay as described in above. Results were calculated as the
8&3 number of antigen-specific IFNy 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 P-values are given , in all cases.
Results
To avoid competition between the viral and tumour epitopes, ME1 encodes epitopes from
LCMV antigens characterised in H-2° haplotype mice and those from tumour antigens characterised in H-2° haplotype mice (Appendix II). Accordingly, the capacity of
FP9.ME]1 to elicit immune responses was tested in both BALB/c (H-2%) and C57BL/6 (H- 2%) mice.
Immunisation of BALB/c mice using pSG2.ME1 and/or FP9.ME]1 elicited IFNy-secreting T cells against the tumour epitopes (H-2%) (Figure 16), but not against the LCMV epitopes (H-2°). The total frequency of IFNy-secreting T cells elicited against the tumour epitopes was significantly (P < 0.004) higher following heterologous prime/boost immunisation with pSG2.ME1/FP9.MET1 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 constructs, but not significantly different (P > 0.05) from each other. Importantly, prime/boost immunisation with pSG2.ME1/FP9.MEI elicited substantial T cell responses against the immunodominant epitope from CT26, the MSR epitope from P815 and a model Li-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-y ELISPOT assay used in this experiment. These results indicate that prime/boost immunisation using pSG2.ME1/FP9.ME]1 is likely to elicit a protective immune response against CT26 colo- rectal carcinoma in BALB/c mice (See above). [J
Immunisation of C57BL/6 mice using pSG2.ME1 and/or FP9.ME] elicited IFNy- secreting T cells against the LCMV epitopes (H-2%), but not against the tumour epitopes 83 i
(H-2°) (Figure 17). The total frequency of IFNy-secreting T cells elicited against LCMV epitopes by prime/boost immunisation with pSG2.ME1/FP9.ME1 was significantly higher than that elicited by homologous immunisation with FP9.ME1 (P = 0.003) alone or : pSG2.ME1 (P = 0.016) alone. Interestingly, heterologous prime/boost immunisation regimes using pSG2.ME1 and FP9.ME] 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.MEI 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.
L}

Claims (18)

1. Use of a non-replicating viral vector in a vaccine for an animal, wherein said vector is, or is derivable from, FP9.
2. Use according to claim 1 wherein said animal is a mammal.
3. Use according to claim 2 wherein said mammal is a primate.
4. Use according to claim 3 wherein said primate is a human.
5. Use of a non-replicating viral vector in medicine, wherein said vector is, or is derivable from, FP9.
6. 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, FPVI91, FPV207, FPV219, FPV220, FPV221, FPV222, FPV 239, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260.
7. A fowlpox virus genome according to claim 6, which has a partial deletion in one or more of the following genes: FPV158, FPV219, FPV222.
8. A fowlpox virus genome according to claim 6 or 7, which lacks one or more of the following genes: FPV001, FPV124, FPV125, FPV159, FPV160, FPV220, FPV221, FPV241, FPV242, FPV243, FPV244, FPV245, FPV246, FPV247, FPV260. Amended sheet 14/04/2005
9. A fowlpox virus genome according to any one of claims 6 to 8, which has a frame-shift mutation in one or more of the following genes: FPV054, FPV070, FPV071, FPV115, FPV190, FPV207.
10. A fowlpox virus genome according to any one of claims 6 to 9, which has a termination mutation in one or more of the following genes: FPVO071, FPV239.
11. A fowlpox virus genome according to any one of claims 6 to 10, which has a chimaeric gene caused by fusion (by deletion) of genes FPV097 and FPV(098.
12. A fowlpox virus genome which is less than 275 kbp in size.
13. A fowlpox virus genome according to any one of claims 6 to 12, comprising the sequence shown in SEQ ID No. 1.
14. A fowlpox virus genome according to any one of claims 6 to 13 which also comprises an NOL
15. A fowlpox virus genome according to claim 14, wherein the NOI is under the control of a poxvirus promoter.
16. A fowlpox virus genome according to claim 14 or 15, wherein the NOI encodes an antigen from P. berghei, P. falciparum, P.cynomolgi, P.vivax, M. tuberculosis or T.parva.
17. A viral particle which comprises a genome according to any one of claims 6 to 16.
18. A viral particle which comprises a genome according to any of claims 14 to 16 and which is capable of delivering the NOI to a target cell.
19. A vaccine comprising a viral particle according to claim 17 or 18. Amended sheet 14/04/2005
20. A priming or boosting agent comprising a viral particle according to claim 17 or 2L A vaccination kit which comprises: ® a first composition which comprises a Fowlpox viral particle according to claim 17 or 18; and (id) a second composition for simultaneous, separate or sequential administration.
22. A vaccination kit according to claim 21, which comprises: )) a priming composition which comprises a Fowlpox viral particle according to claim 17 or 18; and (ii) a boosting composition which comprises a vaccinia viral particle.
23. A vaccination kit which comprises: 63) a first composition which comprises a first non-replicating viral vector; (i1) a second composition which comprises a second non-replicating viral vector for sequential administration to a primate in either order. 24, A vaccination kit according to claim 23, which comprises: 0) a first composition which comprises a first non-replicating poxvirus vector; (ii) a second composition which comprises a second non-replicating poxvirus vector.
25. A vaccination kit according to claim 24, wherein the first non-replicating poxvirus vector is an avipox virus vector and the second non-replicating poxvirus vector is an orthopox virus vector.
26. A vaccination kit according to claim 24 or 25, wherein the first non-replicating poxvirus vector is a fowlpox virus vector. Amended sheet 14/04/2005
WQ 03/047517 PCT/GB02/05411
27. A vaccination kit according to claim 26, wherein the first non-replicating poxvirus vector is or is derivable from a viral particle according to claim 17 or 18.
28. A vaccination kit according to any of claims 21 to 27, wherein the first and second compositions are capable of expressing the same antigen.
29. A boosting composition which comprises a non-replicating viral vector capable of boosting a pre-existing immune response in a primate subject.
30. A boosting composition according to claim 29, which comprises a fowlpox viral vector.
31. A boosting composition according to claim 30, which is or is derivable from a viral particle according to claim 17 or 18.
32. The use of a vaccine according to claim 19, a priming or boosting composition according to any of claims 20, 29, 30 or 31 or a kit according to any of claims 21 to 28 in the manufacture of a medicament for treating and/or preventing a disease in a subject.
33. A use according to claim 32, which elicits a T-cell immune response in the subject.
34. A use according to either of claims 32 to 33, wherein the disease is or results from achronic infection.
35. A use according to claim 34, wherein the chronic infection is malaria, tuberculosis or East Coast Fever.
36. The use of non-cultured CEF cells to grow an avipoxvirus.
37. The use according to claim 36, wherein the avipoxvirus is a fowlpox virus. Amended sheet 14/04/2005
WG 03/047617 PCT/GB02/05411
38. The use according to claim 37, wherein the poxvirus is a viral particle according to claim 17 or 18.
39. Use of a non-replicating viral vector according to claim 1, substantially as herein described with reference to any one of the illustrative examples.
40. A fowlpox virus genome according to claim 6, substantially as herein described with reference to any one of the illustrative examples.
41. A viral particle according to claim 17, substantially as herein described with reference to any one of the illustrative examples.
42. A priming or boosting agent according to claim 20, substantially as herein described with reference to any one of the illustrative examples.
43. A vaccination kit according to claim 21, substantially as herein described with reference to any one of the illustrative examples.
44. A boosting composition according to claim 29, substantially as herein described with reference to any one of the illustrative examples.
45. The use of non-cultured CEF cells according to claim 36, substantially as herein described with reference to any one of the illustrative examples. Amended sheet 14/04/2005
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