WO2004108940A2 - Produits - Google Patents

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Publication number
WO2004108940A2
WO2004108940A2 PCT/GB2004/002387 GB2004002387W WO2004108940A2 WO 2004108940 A2 WO2004108940 A2 WO 2004108940A2 GB 2004002387 W GB2004002387 W GB 2004002387W WO 2004108940 A2 WO2004108940 A2 WO 2004108940A2
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Prior art keywords
nucleic acid
recombinant nucleic
deletion
site
viral genome
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PCT/GB2004/002387
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WO2004108940A3 (fr
Inventor
Michael Anthony Skinner
Michael Keith Howard
Joerg Schneider
Stephen Mark Laidlaw
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INSTITUTE OF ANIMAL HEALTH
Oxxon Therapeutics Ltd
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INSTITUTE OF ANIMAL HEALTH
Oxxon Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • heterologous nucleic acids there is a need for new viral vectors for delivery and expression of heterologous nucleic acids.
  • the heterologous nucleic acids In order to use a viral genome in a vector situation, the heterologous nucleic acids must be incorporated into the viral vector genome.
  • Viral genomes are very compact with little or no redundant genetic material. Indeed, many viruses possess almost no intergenic nucleic acid, and often possess single ORFs encoding multiple promoters, bidirectional promoters and even overlapping ORFs. Therefore, it is readily appreciated that choosing a site for insertion of heterologous nucleic acid sequence is extremely difficult since virtually all of the viral nucleic acid can have essential functions, or even multiple essential functions, which would be unsuitable for insertion/deletion. There is thus a need for new viral acceptor loci ('insertion sites') which can tolerate the disruption which insertion of foreign nucleic acid can entail.
  • the present invention seeks to overcome problem(s) associated with the prior art. Summary of the Invention
  • the invention is based upon the finding that naturally occurring and/or attenuated viral strains can contain deletable genetic material. More in particular, the invention is founded on the surprising finding that viral strains can possess naturally occurring deletions of genetic material without losing viability. Furthermore, it is shown herein that the deletable material may be advantageously replaced by heterologous nucleotide sequence, providing novel viral vectors and viruses.
  • deletion sites are non-essential regions for viral survival.
  • the 'deletion' sites ordinarily comprise tracts of nucleic acid, and are therefore capable of having at least the same quantity of nuleic acid reintroduced to that site without any adverse effects on viral integrity.
  • the invention provides additional advantages in contribution to attenuation of the viral vectors simultaneous with the making of the insertions as described herein.
  • the present invention provides insertion site(s), viral vector nucleic acids(s) comprising heterologous nucleotide sequence inserted at said sites and viruses comprising said nucleic acids.
  • the mvention provides a recombinant nucleic acid comprising at least part of a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion being located at a naturally occurring deletion site of said genome, wherein the insertion site is an insertion site selected from table 2, or a homologue thereof.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion being located at a naturally occurring deletion site of said genome, wherein the insertion site is an insertion site selected from table 2, or a homologue thereof.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 20, 22, 23, 24 or 25 of table 2, or a homologue thereof.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 20, 22 or 24 of table 2, or a homologue thereof.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 2, 8, 9, 10,
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 2, 8, 9, 11, 12, 13, 14, 20, 22 or 24 of table 2, or a homologue thereof.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 8, 9, 11,
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 8, 9, 11,
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 9, 11, 13, 22 or 24 of table 2, or a homologue thereof. In another aspect, the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site selected from deletions 11 or 13 of table 2, or a homologue thereof.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site of a medium or long deletion.
  • the invention relates to a recombinant nucleic acid as described above, wherein the insertion site is an insertion site of a long deletion.
  • the invention relates to a recombinant nucleic acid as described above, wherein the viral genome is not an FP9 viral genome, and the insertion is located at a site homologous to a naturally occurring FP9 deletion site as described above or a naturally occurring mutant insertion site as described below.
  • the viral genome is a canarypox genome or a pigeonpox genome, preferably a canarypox genome.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion site comprising a deletion of a viral gene, wherein the gene is selected from table 2 or table 3, or a homologue thereof.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion site comprising a deletion of a viral gene, wherein the gene is selected from table 2 or table 3, or a homologue thereof, but is not FPN054, 115, 219, 220, 221, 222, 241-247, or type A inclusion protein.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion site comprising a deletion of a viral gene, wherein the gene is selected from table 2 or table 3, or a homologue thereof, but is not FPV001, 054, 115, 171, 219, 220, 221, 222, 241-247, or type A inclusion protein.
  • the invention relates to a recombinant nucleic acid as described above wherein the heterologous nucleotide sequence comprises and is capable of supporting expression of an antigen.
  • the antigen is a cancer related or disease related antigen, preferably the antigen is a cancer or disease antigen selected from table 4.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a Poxvirus.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a Chordopoxvirus.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from an Avipox virus.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a Fowlpox virus.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a Fowlpox virus strain selected from HP-l, US-FPN, FPN-M or FP9.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a Fowlpox virus strain FP9.
  • the invention relates to a recombinant nucleic acid as described above wherein the viral genome is or is derived from a vims mentioned in Table 3.
  • the invention relates to a recombinant poxvirus comprising a nucleic acid molecule as described above.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion being located at a naturally occurring deletion site of said genome.
  • the invention in another aspect, relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion being located at a naturally occuning mutant insertion site of said genome.
  • said insertion site is selected from table 6.
  • the invention relates to a recombinant nucleic acid comprising a viral genome, said viral genome comprising an insertion of a heterologous nucleotide sequence into the viral genome, said insertion being located at a naturally occurring mutant insertion site of said genome, wherein the mutant insertion site is selected from table 6, or a homologue thereof.
  • the mutant insertion site is selected from mutant insertions 1, 3, 5, 6, 10, 11, 13 or 15 of table 6, or a homologue thereof.
  • These sites represent ORFs (rather than intergenic or other genetic regions), thereby advantageously reducing the risk of interference with regulatory regions of the viral genome and advantageously maintaining the disruption of said ORFs which may contribute to the technical character of a viral genome comprising said mutant insertions.
  • the mutant insertion site is selected from mutant insertions 2, 6, 12, 13 or 14 of table 6, or a homologue thereof. These sites represent insertions occurring in FP9 which do not occur in HP-1 or US-FPN and therefore provide advantageous discrimination with regard to the HP-1/US-FPV strains.
  • the mutant insertion site is selected from mutant insertions 6 or 13 of table 6, or a homologue thereof. These sites represent insertions into ORFs occurring in FP9 which insertions do not occur in HP-1 or US-FPN and are therefore advantageous both with respect to the likely neutral effect with regard to regulatory regions, maintenance of the ORF disruption, and combine the advantage of providing differentiation from the HP-1/US-FPN strains.
  • mutant insertion site is mutant insertion 6 of table 6, or a homologue thereof.
  • US-FPN is a wild-type Fowlpox virus which has been sequenced.
  • Webster's strain FPN-M (sometimes refe ⁇ ed to as FPW) is an attenuated form of this virus.
  • HP-1 is a European wild-type Fowlpox virus.
  • FP9 is an attenuated form of this virus. The complete FP9 genome is provided herein.
  • Fowlpox strain FP9 was derived by 438 passages through CEFs in tissue culture from the virulent wild-type Fowlpox HP-1 strain and then plaque purified.
  • 26 deletions are present in FP9 which are not present in US wild-type (US FPN). 7 of these 26 deletions are present in the European wild-type (HP-1).
  • the 19 of these deletions which are absent from both the US and European wild-type fowlpox strains are particularly prefened. Of these 19 deletions, the 15 which are demonstrated to be absent from Webster's strain are especially prefened. The remaining 4/19 deletions are absent from at least US FPN and HP-1.
  • the insertion site(s) according to the invention are preferably the 15/19 deletions which are confirmed to be absent from US wild-type (US FPV), European wild-type (HP-1) and Webster's FPN-M.
  • deletions have been numbered 1-26 (see table 2).
  • the 15 which have been sequenced against Webster's are deletions 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 20, 22 and 24. Their size and the genes they affect are listed in Table 1.
  • the insertion site is or is defined by FP9 deletions 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 20, 22 or 24 of table 2.
  • a virus If a virus is stable when a deletion is present then the virus must be able to function without that particular length of D ⁇ A.
  • a foreign (heterologous) sequence can be inserted into the deletion site. Advantageously this occurs without further destabilizing the virus.
  • a relatively long D ⁇ A sequence may advantageously be inserted according to the present invention so it is prefened to insert it at a point which previously held a long strand D ⁇ A.
  • prefened insertion sites are those defined by medium deletions, more prefened insertion sites defined by large deletions. These are disclosed in table 2, and are discussed in more detail below.
  • This gene is in a non-essential site in Fowlpox virus and thus a prefened insertion site.
  • the D8 and D9 genes represent prefened insertion sites, especially prefened in Fowlpox.
  • This deletion/gene is a prefened insertion site, especially prefened in Fowlpox viruses.
  • Deletion 14 makes a point mutation in FPN171 (early transcription factor VETFL). This is a 3 base-pair deletion spanning 2 codons such that encoded amino acid residues 526-527 RY becomes H (one residue deleted). This gene is the homologue of the vaccinia A7L gene which is essential for virus morphogenesis. Because of the risk of disrupting an essential gene, appropriate care would need to be taken in using this as an insertion site. Deletions 15-17
  • deletions 15-17 are treated as defining a single deletion spanning the first base of deletion 15 to the last base of deletion 17.
  • Deletion 16 shortened an A-type inclusion protein, A25L. This is arguably related to Deletion NI in MNA, but is different from this MNA deletion. This is especially prefened in fowlpox viruses.
  • Deletion 17 shortened an A-type inclusion protein, A25L. This is arguably related to Deletion NI in MNA, but is different from this MNA deletion. This is especially prefened in fowlpox viruses.
  • Deletion 20 lengthens FPN213 TM protein.
  • the function of this protein is not known and it must be borne in mind that elongation of the protein may affect its function in FP9. Therefore, because of the risk of disrupting this gene function, appropriate care would need to be taken in using this as an insertion site.
  • Deletion 10 splits the FPV115 (MIL) Ankyrin repeat in two.
  • MIL is one of the deleted genes in MNA deletion II, but MVA deletion II is different from deletion 10 of the present invention. This is especially prefened in fowlpox/avipox viruses, especially in fowlpox viruses.
  • Deletion 12 23bp This is a deletion of an intergenic region. The deletion is located immediately downstream of the TAA termination codon for the P35 gene, FPV 140 (H3L an IMN membrane associated protein). The H3L gene is non-essential but deletion of this gene may cause reduced plaque size and/or virus yield. There would therefore be a risk that insertion of a cassette into Deletion 12 would interfere with the expression of FPN140. If reduced FPN 140 gene expression produced the same phenotype in FPV as does reduced H3L expression in VACV. These considerations should be taken into account when using deletion 12 as an insertion site.
  • Deletion 2 deletes FPV001 in addition to a promoter from FPV002.
  • the site is diploid. Insertion into the first gene is possible but success rates can be variable as the site is often unstable.
  • the site lies 22 bp upstream of FP9.002, which is non-essential.
  • Deletion 9 does not delete ORFs in FP9. This is an unusual site and a large deletion.
  • the genome structure differs between FP9 and US FPV.
  • Deletion 9 is a prefened insertion site.
  • the FPVUS strain has B22R serpin homologue genes. A number of these are deleted in MVA (although B22R itself is not). Insertion into FPVUS FPV097 is also provided according to the present invention.
  • the site is most prefened for European strains of FPV, most preferably for FP9.
  • Deletion 11 deletes FPV124 (NlR/ ⁇ 28 family) and the 5-prime part of FPV125 (v- type Ig domain, function unknown). Neither of these genes has homologues in vaccinia but they do in the US FPV strain. NIR has been shown to be a virulence factor in ectromelia virus and is in Shope virus.
  • the left end extends into the intergenic region between FP9.123 & FP9.124 but leaves over 120 bp of non-coding sequence upstream of FP9.123 (similar to variola virus Bangladesh B22R).
  • the right end leaves just 80 bp of the end of sequence encoding FP9.125.
  • An insertion might affect expression of Fp9.123 but this protein is believed to be non-essential.
  • the divergent expression of genes flanking this locus is likely to be advantageous to the divergent expression of foreign antigen and/or selectable marker and/or other heterologous sequence inserted at this locus in accordance with the present invention.
  • Deletion 13 2187bp Deletion 13 knocks out FPV159 (NlR/ ⁇ 28 family) and FPV160 (unknown).
  • deletion of FPV150 to FPV163 may be used as an insertion site.
  • FPV150-FPV163 form an insertion site.
  • ankyrin repeat family member may be used as insertion sites.
  • ankyrin repeat genes may be used an insertion site(s) singly or in combination.
  • Deletion 22 deletes FPV219 (Ankyrin), FPV220 (unknown) and FPV221 (A47L) and FPV222 (Ankyrin). As mentioned above when discussing Deletion 13, many Ankyrin genes are unique to FPV.
  • This site is especially prefened for fowlpox viruses, especially FP9.
  • Deletion 24 9334bp Deletion 24 deletes FPV241-FPV246 (all Ankyrin repeat family members) and FPV247 (Efc family). As mentioned above when discussing Deletion 13, many Ankyrin genes are unique to FPV.
  • Deletion 24 is particularly prefened for fowlpox, preferably for FP9.
  • insertion 6 is most prefened as it leads to truncation of FP9.104 protein (unknown function, possible nuclease) by 33 %.
  • This term has its natural meaning in the art, and may also include exogenous homologous material eg. material from the same or a similar viral strain, but recombined into a new position of the target genome.
  • heterologous nucleotide sequence comprises coding sequence capable of being expressed in the target cell. Expression may be produced by use of any suitable promoter known to those skilled in the art.
  • heterologous sequence comprises antigen encoding sequence.
  • the antigen encoded is derived from or associated with a cancer and/or an infectious disease. Examples of prefened cancer(s) and infectious disease(s) are given in Table 4 below.
  • the antigen encoded comprises at least part of one or more of the antigens given in Table 4, preferably the antigen encoded comprises one or more of the antigens given in Table 4.
  • insertion sites according to the present invention may be seen as viral genetic loci which comprise naturally occuning deletion(s), preferably in a viral gene.
  • said deletion(s) are in the coding sequence of a gene, but may equally be in a non-coding (intergenic) region.
  • a non-coding region may be a promoter region.
  • the term 'insertion sites' comprises equivalents thereto in diverse viral species, and/or may include expanded deletions as explained below.
  • insertion sites are comprised in table 3.
  • insertion sites are comprised in table 2.
  • insertion site(s) are individual sites selected from table 2 as explained above in the summary of the invention.
  • genes known to contain a naturally occurring deletion may themselves be wholly deleted and thereby form an insertion site according to the invention. This advantageously permits insertion of a greater quantity of exogenous/heterologous nucleic acid into said insertion site.
  • one reason for doing this is that the presence of a deletion in a particular gene may be an indicator of loss of function and/or redundancy and therefore permit an insertion site to be advantageously expanded according to the present invention.
  • the invention relates to insertion site(s) according to the present invention which are viral genetic loci which comprise naturally occurring mutant insertion(s), preferably in a viral gene.
  • insertion site(s) according to the present invention which are viral genetic loci which comprise naturally occurring mutant insertion(s), preferably in a viral gene.
  • the finding of a naturally occurring mutant insertion at a particular locus may indicate that that locus is tolerant of nucleic acid insertions at that point, and therefore it would be useful as an insertion site according to the present invention.
  • Examples of prefened insertion sites according to this aspect of the invention may be found in table 6 which lists naturally occuning mutant insertion sites which are preferably used as insertion sites according to the present invention.
  • the insertion sites of the present invention may be utilised in any suitable virus.
  • a suitable virus is any virus which possesses a genetic equivalent to one or more of the insertion site(s) of the present invention.
  • the insertion sites are used in poxviruses, preferably in chordopoxviruses, preferably in orthopox and/or in avipox viruses, preferably in avipox viruses, preferably in fowlpox viruses, preferably in fowlpox strain Websters (FPV-M), preferably in fowlpox strain HP-1, most preferably in fowlpox strain FP9.
  • the virus may be a homologue of one or more of these viruses.
  • FP9 is a convenient example of poxviruses generally, in particular of chordopoxviruses, in particular of avipox viruses, and in particular of fowlpox viruses.
  • the BLAST search is carried out using the "Standard nucleotide - nucleotide BLAST" search on the NCBI Nucleotide website: http://www.ncbi.nlm.nih.gov/blast.
  • the relevant genes were located within the genome of the US FPV.
  • the DNA sequence was then used to search the BLAST database. The higher the score, the better the match. Obviously Fowlpox appeared as the best match in each case with a score of approximately 2000 and an Expect value close to zero.
  • the BLAST score is an indicator of equivalence in accordance with table 3.
  • the poxviruses (excluding Fowlpox) were noted and listed in table 3.
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the cunent size one might expect to see 1 match with a similar score simply by chance. This means that the lower the E-value, or the closer it is to "0" the more "significant” the match is. However, keep in mind that searches with short sequences, can be virtually indentical and have relatively high EValue. This is because the calculation of the E-value also takes into account the length of the Query sequence. This is because shorter sequences have a high probability of occuring in the database purely by chance. For more details please see the calculations in the BLAST materials.
  • the Expect value can also be used as a convenient way to create a significance threshold for reporting results. You can change the Expect value threshold on most main BLAST search pages. When the Expect value is increased from the default value of 10, a larger list with more low-scoring hits can be reported.
  • insertion sites may be identified by reference to sequence homology.
  • sequence homology can be regarded as sequence identity.
  • Exemplary values indicating that the equivalent site had been found in diverse species are at least 50%> amino acid sequence identity within avipox viruses, or at least 24% amino acid sequence identity within poxviruses. Further guidance may be obtained from table 3.
  • BLAST scores may be used to identify insertion sites.
  • Exemplary values indicating that the equivalent site had been found in diverse species are at least a blast score of 35, preferably a score of 40, preferably even more, such as a score in the thousands for near perfect matching.
  • Exemplary values may be derived from table 3.
  • the sites identified in this manner indicate the locations of the insertion sites according to the present invention in other viruses such as those mentioned in Table 3.
  • the deleted nucleotides are infe ⁇ ed by comparison of the immediate flanking sequences to the publicly available US-FPV (Afonso et al 2000 J.virol 74(8)pp3815-31).
  • the nucleotide sequence 'sandwiched' between the flanking sequences is the 'deleted' sequence which gives rise to an insertion site according to the present invention.
  • This sequence is then used as a query in a sequence comparison technique such as BLAST and compared with the target viral sequence.
  • these infe ⁇ ed 'deleted' nucleotides may be used in conjunction with nucleotide sequences flanking the deletion as necessary to produce a sufficiently long probe sequence for searching.
  • This comparison shows the equivalent site in the target virus which conesponds to the insertion site of interest. Further fine detailed sequence mapping can easily be carried out eg. by eye level sequence comparison in order to design primers, choose restriction sites and suchlike for the construction of a nucleic acid or virus according to the present invention.
  • insertion sites according to the present invention are defined in viral genome(s) other than FP9 by comparison of the FP9 viral genome to said viral genome(s).
  • Insertion sites in canarypox or pigeonpox are prefened, insertion sites in canarypox are particularly prefened.
  • tables 2 and 3 present numerous examples of insertion sites according to the present invention, and table 3 in particular presents insertion sites from a range of diverse viruses according to the present invention. Furthermore, table 7 presents a wide range of insertion sites according to the present invention, in particular from canarypox. Naturally the reader will appreciate that the teachings regarding the location of insertion sites according to the present invention in genetically diverse viral species apply equally to deletion sites per se, gene deletion sites or mutant insertion sites as described herein.
  • the location of gene deletion sites may equally be applied to the location of gene insertion sites, that is to say viral gene(s) which could be used as insertion sites due to the fact that the naturally occurring mutant insertions occur in those gene(s) which may indicate loss of function and/or redundancy as appropriate and therefore infer suitability as an insertion site according to the present invention.
  • Figures 1, la, 2 and 2a show a plasmid maps.
  • Figure 3 shows a diagram.
  • Figures 4a and 4b show diagrams.
  • Figures 5, 6, 9, 10 , 11 and 12 show plasmid maps.
  • Figures 7 and 8 show bar charts.
  • Figure 7 shows IFN- ⁇ ELIspot responses elicited by FP9.HBs26 following a single and prime boost immunization.
  • Groups of female BALB/c mice (H2 d ) were immunised intravenously (i.v.) with 5x10 6 pfu of FP9.HBs26 or with MVA.HBs, or intramuscularly with 50 ⁇ g pSG2.HBs DNA vaccine as indicated.
  • SFC spot forming cells
  • Figure 8 shows IFN- ⁇ ELIspot responses elicited by FP9.HBs26 and AFOX5.HBs26 following a single and prime boost immunization.
  • Groups of female BALB/c mice (H2 d ) were immunised intravenously with 5x10 6 pfu of FP9.HBs26 or AFOX5.HBs26 deletion mutants as indicated.
  • Fourteen days after immunization the number of IFN- ⁇ SFC/million splenocytes was determined by IFN- ⁇ ELIspot using HBs specific CD8 + epitopes IPQ, WGP, the green fluorescent protein-specific epitope HYL, and the HBs- specific CD4 + epitope TVN.
  • A shows IFN- ⁇ ELIspot responses elicited by FP9.HBs26 and AFOX5.HBs26 following a single and prime boost immunization.
  • sequence listing shows a number of sequences.
  • SEQ ID NOs 1-8 show primers
  • SEQ ID NO 9 shows the sequence of the FP9 viral genome
  • SEQ ID NO 10 co ⁇ esponds to sequence for the basic deletion plasmid pl8dl3 (without foreign genes or promoters).
  • SEQ ID NO 11 conesponds to sequence for the basic deletion plasmid P18d9 (without promoters or foreign genes).
  • SEQ ID Nos 12-21 are explained in the examples.
  • Example 1 Construction of vector plasmid for insertion into Deletion 13.
  • Novel plasmids were constructed to enable the generation of recombinant FP9 fowlpox. Insertion of foreign genes was targeted precisely to to the site of the naturally occurring deletion 13 in the FP9 genome. Sequences of FP9 DNA flanking the site of a 2187bp deletion located at basepair number 180256 of the FP9 genome sequence (SEQ JD NO 9) were amplified by PCR and cloned into the multiple cloning site of plasmid pUC18.
  • the primers for the left 1500bp flank were 5'- CTC GCA TGC ATG ATT ATG TTA TTG AAT GTA TAG-3' (SEQ ID No: 1) and 5'- GTC CTG CAG GTT CGT GCA CCG TAT TCT TCT TTA-3' (SEQ ID NO:2) (sites for restriction enzymes Sphl and Pstl are underlined).
  • the primers for the right 1500bp DNA flank were 5'- GTG TCT AGA AAA GAC AAA CGT GGT TAT AAA CAA-3' (SEQ ID NO:3) and 5'- GTG GGT
  • a Pstl fragment of DNA of approximately 338bp containing a synthetic epitope string of melanoma-associated antigens was cut from the expression plasmid pSG2.Mel3 (Mj Palmowski et al 2002 J. Immunol 168 pages 4391-4398), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site of pl8dl3Z to make the plasmid transfer vector pl8dl3.Mel3.
  • the plasmid pl8dl3.Mel3 that directs the insertion of foreign genes into the site of deletion 13 of the FP9 genome was used to generate the recombinant virus AFOX3.Mel3.
  • the method for making the recombinant FP9 was essentially as described in Y.
  • pl8dl3.HBs The plasmid pl8dl3.HBs that directs the insertion of foreign genes into the site of deletion 13 of the FP9 genome was used to generate the recombinant virus AFOX3.HBs.
  • the method for making the recombinant FP9 was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant virus plaques were screened with the colourigenic substrate, Xgal, as described in MEG Boursnell et al (1990)
  • Novel plasmids were constructed to enable the generation of recombinant FP9 fowlpox. Insertion of foreign genes was targeted precisely to to the site of the naturally occurring deletion 9 in the FP9 genome. Sequences of FP9 DNA flanking the site of a 583 lbp deletion located at basepair number 101431 of the FP9 genome sequence SEQ ID NO 9) were amplified by PCR and cloned into the multiple cloning site of plasmid pUC18. The primers for the left 1500bp flank were 5'- GTG GAG CTC AAC CAT GTA TTT TCA TCA TCT GTA -3' (SEQ ID No:5) and 5'- GTG GGT ACC CAT AAT AGA
  • pEFL29P a transfer plasmid made previously by the replacement of the unique Smal site of pEFL29 (Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573) with a Pmel linker (NEB #1177S), was blunt ended and inserted into the blunted Sail restriction site of pl8d9 to make pl8d9Z.
  • the introduced Escherichia coli LacZ gene is under the control of the fowlpox virus late promoter P4b.
  • This inserted fragment from pEFL29 also contains the vaccinia virus P7.5 early promoter upstream of the unique Pmel site for the insertion and expression of foreign genes in the FP9 genome.
  • a Pstl fragment of DNA of approximately 338bp containing a synthetic epitope string of melanoma-associated antigens was cut from the expression plasmid pSG2.Mel3 (Mj Palmowski et al 2002 J. Immunol 168 pages 4391-4398), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Pmel site of pl8d9Z to make the plasmid transfer vector pl8d9.Mel3.
  • the plasmid pl8d9.Mel3 that directs the insertion of foreign genes into the site of deletion 9 of the FP9 genome was used to generate the recombinant virus AFOX4.Mel3.
  • the method for making the recombinant FP9 was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant virus plaques were screened with the colourigenic substrate, Xgal, as described in MEG Boursnell et al (1990)
  • the invention is applicable to many genetically diverse viruses by identification of the equivalent (homologous) insertion site in said viruses to those disclosed herein with regard to the FP9 genome sequence.
  • the equivalent (homologous) sites are presented from Canarypox, illustrating how a person skilled in the art can follow the teachings presented herein to apply the invention in genetically diverse viruses.
  • Tulman et al disclose the genome sequence of Canarypox virus (Tulman ER, Afonso CL, Lu Z, Zsak L, Kutish GF, Rock DL. "The genome of canarypox virus.” J Virol. 2004 Jan;78(l):353-66.).
  • FP9 and canarypox genomes are compared and the results are presented in Table 7. From this comparison, canarypox sites are identified equivalent to the insertion sites disclosed herein with regard to FP9. Thus, the invention is applied to a genetically diverse virus such as canarypox.
  • Prefened insertion sites are those which are present in FP9 but not in the parent virus HP-1 or the US strain US-FPV. Those marked with an asterix (*) are also absent from Webster's FPV-M, an attenuated form of US-FPV.
  • the terminal non-coding and intergenic regions have no conesponding "Best matching ORF” and are marked with a hyphen (-).
  • ALVAC is an attenuated poxvirus deposited with the ATCC, accession number VR- 2547. The above methodology is applied equally to ALVAC or other related viruses.
  • Novel plasmids were constructed to enable the generation of recombinant FP9 fowlpox. Insertion of foreign genes was targeted precisely to the site of the naturally occurring 'Deletion 11' in the FP9 genome.
  • the synthetic insert was subcloned into pSP72LinkLacZ as an Avrll-Sphl fragment to allow the use of the Notl recognition site within the synthetic sequence.
  • the resulting plasmid was named, pFdell 1, ( Figure 5).
  • the plasmid pSP72.LinkLacZ is based on the commercially available cloning plasmid, pSP72, (Promega Inc.).
  • the standard multiple cloning site is replaced with a synthetic linker containing unique restriction sites and the vaccinia virus P7.5 late-early promoter and PI 1 late promoter a ⁇ anged in a head- to-head orientation.
  • the linker also has the first twenty nucleotides of the Escherichia coli LacZ, (beta-galactosidase), gene as found in the vaccinia virus recombination plasmid pSCl l, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).
  • the synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1 (Invitrogen, Paisley, Scotland), to make pLink.
  • the sequence of the 300 base pair linker is given below (SEQ ID 13).
  • LacZ gene from pSCll is subcloned into pLINK to make pLinkLacZ.
  • the linker and LacZ gene were then subcloned into pSP72 as a Xhol-Bglll fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3 -prime to the PI 1 late promoter to make pSP72LinkLacZ.
  • a double expression cassette containing both the small and middle forms of the Hepatitis B Surface Antigen protein-coding region and a selectable marker was built in a modified form of pSP72LinkLacZ.
  • the Pl l late promoter and the LacZ gene were replaced by digesting pSP72LinkLacZ with Nsil and Sphl to insert a short oligonucleotide linker containing the compact synthetic early-late promoter described in S. Chakrabarti et al. 1997 Biotechniques 23(6) pl094-1097 (SEQ ID 14).
  • a DNA fragment encoding the green fluorescent protein (gfp) present in the commercial plasmid, pQBI25 (Q-BIOgene, Montreal, Quebec) was transfe ⁇ ed into the modified pSP72LinkLacZ such as to place the selectable marker under the control of the compact synthetic promoter.
  • the small and middle forms of the Hepatitis B Surface Antigen protein-coding region were isolated as a Hindlll-Nsil fragment from the expression plasmid pCMVS2.S (M-L Michel et al 1995 Proc Natl Acad Sci USA 92 pages 5307-5311), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site.
  • HBs and selectable marker expression cassettes were then transfened from the modified pSP72LinkLacZ as a Notl fragment into the unique Notl restriction site of pFDell 1 to make pFDell 1.HBs26.
  • the plasmid pFDell l.HBs26 ( Figure 6), that directs the introduction of foreign genes into the site of Deletion 11 of the FP9 genome, was used to generate the recombinant virus AFOX5.HBs26.
  • the method for making the recombinant FP9 was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant virus plaques were screened using an inverted fluorescence microscope to detect gfp-positive plaques.
  • Eight recombinant virus plaques were purified through two rounds of plaque-picking using the gfp selection.
  • AFOX5.HBs26 masters were tested for GFP and HBs protein expression in CEF (chicken embryo fibroblasts) and A549 (human lung carcinoma) by western blot analysis following methods described in O'Brien and Campo, Oncogene 1998 17 p293-301. The levels of protein product appeared to be higher when the cassettes were inserted into the Deletion 11 site rather than the (diploid) control insertion site, FPV002.
  • Viras Growth AFOX5.HBs26 viras growth was tested; a multi step growth curve experiment was performed infecting confluent CEF in triplicates at a multiplicity of infection (moi) of 0.05 plaque forming unit (pfu) per cell.
  • AFOX5.HBs26 showed a growth index comparable to FP9.HBs26, the control viras canying the HBs26 cassette in FP9 ORFl site, (constructed using a shuttle plasmid based on pEFL29 as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569- 573).
  • Plaque Morphology AFOX5.HBs26 viruses present plaques with morphology comparable to the control virus, FP9.HBs 26.
  • the insert expresses the preS2-S antigen of the hepatitis B viras and a g ⁇ (green fluorescent protein) selectable marker.
  • mice immunized with FP9 10 constracts with the g ⁇ and the preS2-S antigen of the hepatitis B virus expression cassette inserted into deletion 11 induce HBs- and g ⁇ -specifrc IFN- ⁇ responses.
  • AFOX5.HBs26 constracts have the capacity to induce HBs- and g ⁇ -specific IFN- ⁇ responses following a single immunization. Observations obtained from prime boost study further demonstrate that AFOX5.HBs26 is 25 immunogenic as a priming reagent.
  • An expression cassette expressing the preS2-S antigen of the hepatitis B viras and the 30 green fluorescent protein (g ⁇ ) was inserted into ORF 1 to generate FP9.HBs26 and into deletion 11 to generate AFOX5.HBs26.
  • Each construct was evaluated in vivo for the capacity to induce HBs-specific IFN- ⁇ secreting CD8 + and CD4 + T cells responses in BALB/c mice as assayed by IFN- ⁇ ELIspot.
  • IFN- ⁇ secreting CD8 + cells reactive to the green fluorescent protein (g ⁇ ) epitope HYL were also determined.
  • the immunogenicity of AFOX5.HBs26 was compared to the same dose of FP9.HBs26.
  • MVA.HBs was used as a booster. This lacks the g ⁇ .
  • viras was sonicated for exactly 1 minute prior to dilution.
  • Recombinant viruses were diluted on the same day as immunization to 5xl0 7 pfu/ml in sterile PBS and kept on wet ice prior to injection. Each dose consisted of lOO ⁇ l of recombinant viras; 5xl0 6 pfu /dose.
  • mice Female BALB/c mice (H2 d ; 6 - 8 weeks) were used in all experiments as described in Table 8.
  • Table 8 Experimental design Experiment 1: Immunogenicity of recombinant Fowlpox virus containing GPT-gfp/HBs in ORFl in a single and a prime boost immunization regime.
  • mice All mice were kept in individually ventilated cages; Thoren racks. Recombinant viras was administered, 5xl0 6 pfu in lOO ⁇ l of PBS intravenously (i.v.) into the tail vein of each mouse.
  • the DNA vaccine plasmid pSG2.HBs was prepared from pSG2.Mel3 (Palmowski, MJ. et al 2002 J Immunol 168 p4391-4398) and was administered to anaesthetised mice by intramuscular (i.m.) injection into both anterior tibialis muscles (50 ⁇ l per muscle).
  • Peptides in bold are epitopes from HBs antigen, whereas HYL peptide is an epitope from the green fluorescent protein.
  • FP9.HBs26 containing the expression cassette inserted into ORFl has the capacity to induce potent HBs-specific IFN- ⁇ secreting T cell responses.
  • FP9 constracts with the expression cassette inserted into deletion 11 have the capacity to induce HBs and g ⁇ -specifrc IFN- ⁇ responses following a single immunization in mice.
  • mice that have been primed with FP9.HBs26 clone 1 and boosted 10 with MVA.HBs was comparable to the strength of the IFN- ⁇ response following a single shot of either FP9.HBs26 alone or a single shot of MVA.HBs alone ( Figure 8B versus Figure 8 A and Figure 8B versus Figure 7A).
  • FP9HBs26 and AFOX5.HBs26 constracts, but not MVA.HBs, express g ⁇ . Therefore, 15 the IFN- ⁇ response against the g ⁇ epitope HYL forms a good indicator whether an immune response against the prime has been elicited.
  • Each of the FP9.HBs26 and AFOX.HBs26 viruses have elicited IFN- ⁇ responses against HYL comparable to the HYL-specifrc LFN- ⁇ response following a single shot immunization ( Figure 8B versus Figure 8A).
  • AFOX5.HBs26 constracts induce HBs- and g ⁇ -specific IFN- ⁇ responses following a single immunization.
  • Prime boost study further supports the finding that AFOX5.HBs26 is immunogenic as a priming reagent.
  • AFOX5.HBs26 constracts have the capacity to induce HBs- and g ⁇ - specific IFN- ⁇ responses following a single immunization.
  • Prime boost study further supports the finding that AFOX5.HBs26 is immunogenic as a priming reagent.
  • Example 10 Constraction of AFOX3.HBs26 (FP9 Deletion 13) Novel plasmids were constmcted to enable the generation of recombinant FP9 fowlpox. Insertion of foreign genes was targeted precisely to the site of the naturally occuning 'Deletion 13' in the FP9 genome.
  • Sequences of FP9 DNA comprising 500bp flanking each side of the site of a 2187 bp deletion located at base pair number 180256 of the FP9 genome sequence (SEQ ID NO 9) were synthesized by GenScript Corporation (120 Centennial Ave. Piscataway, New Jersey 08854, USA); in addition, this synthesis included the recognition sequence for the Notl restriction enzyme, GCGGCCGC, placed between position 180256- 180257, and a 5-prime tem inal Avrll and a 3-prime terminal Sphl recognition site, (SEQ ID 17). The synthetic sequence was provided by GenScript cloned into Smal site of pUC57 (Fermentas Life Sciences, Hanover, MD, USA).
  • the synthetic deletion was subcloned into pSP72LinkLacZ as an Avrll-Sphl fragment to allow the use of the Notl recognition site within the synthetic sequence.
  • the resulting plasmid was named pFDell3, ( Figure 9).
  • the plasmid pSP72.LinkLacZ is based on the commercially available cloning plasmid, pSP72, (Promega Inc.).
  • the standard multiple cloning site is replaced with a synthetic linker containing unique restriction sites and the vaccinia viras P7.5 late-early promoter and PI 1 late promoter ananged in a head-to-head orientation.
  • the linker also has the first twenty nucleotides of the Escherichia coli LacZ, (beta-galactosidase), gene as found in the vaccinia viras recombination plasmid pSCll, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).
  • the synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1 (Invitrogen, Paisley, Scotland), to make pLink.
  • the sequence of the 300 base pair linker is given below (SEQ ID 13).
  • Part of the LacZ gene from pSCl l is subcloned into pLINK to make pLinkLacZ.
  • linker and LacZ gene were then subcloned into pSP72 as a Xhol-Bglll fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3-prime to the PI 1 late promoter to make pSP72LinkLacZ.
  • a double expression cassette containing both the small and middle forms of the Hepatitis B Surface Antigen protein-coding region and a selectable marker was built in a modified form of pSP72LinkLacZ.
  • the small and middle forms of the Hepatitis B Surface Antigen protein-coding region were isolated as a Hindlll-Nsil fragment from the expression plasmid pCMVS2.S (M-L Michel et al 1995 Proc Natl Acad Sci USA 92 pages 5307-5311), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site.
  • HBs and selectable marker expression cassettes were then transfened from the modified pSP72LinkLacZ as a Notl fragment into the unique Notl restriction site of pFDell3 to make pFDell3.HBs26. Constraction of Recombinant Viras AFOX3.HBs26
  • the plasmid pFDell l.HBs26 ( Figure 10), that directs the introduction of foreign genes into the site of Deletion 11 of the FP9 genome, was used to generate the recombinant viras AFOX5.HBs26.
  • the method for making the recombinant FP9 was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant viras plaques were screened using an inverted fluorescence microscope to detect g ⁇ -positive plaques.
  • Two recombinant viras plaques were purified through seven rounds of plaque-picking using the mycophenolic acid selection.
  • AFOX3.HBs26 master was tested for GFP and HBs protein expression in CEFs (chicken embryo fibroblasts) following methods described in O'Brien and Campo, Oncogne 1998 17 p293-301. The levels of expression of the two heterologous proteins from within the Deletion 13 site and the control locus, FPV002, were similar.
  • AFOX3.Hbs26 viras presents plaques with morphology comparable to FP9.HBs26, the control viras ca ⁇ ying the HBs26 cassette in FP9 ORFl site, (Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573).
  • Example 11 Constraction of AFOX10.HBs26 (FP9 insertion 6)
  • Novel plasmids are constructed to enable the generation of recombinant FP9 fowlpox. Insertion of foreign genes is targeted precisely to the site of the naturally occurring 'Insertion 6' in the FP9 genome.
  • Sequences of FP9 DNA comprising 500bp flanking each side of the site of a 2bp insertion located at basepair number 116201 of the FP9 genome sequence (SEQ ID NO 9) are synthesized by Geneart GmbH, (Josef-Engert-Str. 9, D-93053 Regensburg, Germany); in addition, this synthesis includes the recognition sequence for the Notl restriction enzyme, GCGGCCGC, placed between the two bases forming the inserted sequence, and a 5-prime terminal Avrll and a 3-prime terminal Sphl recognition site, (SEQ ID 21).
  • the synthetic sequence provided by Geneart is cloned between the Sad and Kpnl of pCR-script (Stratagene, La Jolla, USA).
  • the synthetic insert is subcloned into pSP72LinkLacZ as an Avrll-Sphl fragment to allow the use of the Notl recognition site within the synthetic sequence.
  • the resulting plasmid is named, pFin6, ( Figure 11).
  • the plasmid pSP72.LinkLacZ is based on the commercially available cloning plasmid, pSP72, (Promega Inc.).
  • the standard multiple cloning site is replaced with a synthetic linker containing unique restriction sites and the vaccinia viras P7.5 late-early promoter and Pl l late promoter ananged in a head-to-head orientation.
  • the linker also has the first twenty nucleotides of the Escherichia coli LacZ, (beta-galactosidase), gene as found in the vaccinia viras recombination plasmid pSCl l, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).
  • the synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1 (Invitrogen, Paisley, Scotland), to make pLink.
  • the sequence of the 300 base pair linker is given below (SEQ ID 13).
  • LacZ gene from pSCl l is subcloned into pLINK to make pLinkLacZ.
  • the linker and LacZ gene were then subcloned into pSP72 as a Xhol-Bglll fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3-prime to the PI 1 late promoter to make pSP72LinkLacZ.
  • a double expression cassette containing both the small and middle forms of the Hepatitis B Surface Antigen protein-coding region and a selectable marker was built in a modified form of pSP72LinkLacZ.
  • the small and middle forms of the Hepatitis B Surface Antigen protein-coding region are isolated as a Hindlll-Nsil fragment from the expression plasmid pCMVS2.S (M-L Michel et al 1995 Proc Natl Acad Sci USA 92 pages 5307-5311), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site.
  • HBs and selectable marker expression cassettes are then transfened from the modified pSP72LinkLacZ as a Notl fragment into the unique Notl restriction site of pFin6 to make pFin6.HBs26. Constraction and Characterisation of Recombinant Viras AFOX10.HBs26
  • the plasmid pFin6.HBs26 ( Figure 12), that directs the introduction of foreign genes into the site of Insertion 6 of the FP9 genome, is used to generate the recombinant viras AFOX10.HBs26.
  • the method for making the recombinant FP9 is essentially as
  • plasmid transfer vector is purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant viras plaques are screened using an inverted fluorescence microscope to detect g ⁇ -positive plaques.
  • Example 12 Insertion of heterologous nucleic acid into Deletion 13 of the Webster (FPW) strain of fowlpox
  • Novel plasmids were constructed to enable the generation of recombinant FPW fowlpox. Insertion of foreign genes in FPW was targeted so as to generate a deletion within the FPW equivalent to the naturally occurring 'Deletion 13' in the FP9 genome.
  • Sequences of FP9 DNA comprising 500bp flanking each side of the site of a 2187 bp deletion located at base pair number 180256 of the FP9 genome sequence (SEQ ID NO 9) were synthesized by GenScript Corporation (120 Centennial Ave. Piscataway, New Jersey 08854, USA); in addition, this synthesis included the recognition sequence for the Notl restriction enzyme, GCGGCCGC, placed between position 180256- 180257, and a 5-prime terminal Avrll and a 3-prime terminal Sphl recognition site, (SEQ ID 17). The synthetic sequence was provided by GenScript cloned into Smal site of pUC57 (Fermentas Life Sciences, Hanover, MD, USA). Constraction of Recombination Plasmid: pFDe!3 ( Figure 9)
  • the synthetic deletion was subcloned into pSP72LinkLacZ as an Avrll-Sphl fragment to allow the use of the Notl recognition site within the synthetic sequence.
  • the resulting plasmid was named pFDell 3, ( Figure 9).
  • the plasmid pSP72.LinkLacZ is based on the commercially available cloning plasmid, pSP72, (Promega Inc.).
  • the standard multiple cloning site is replaced with a synthetic linker containing unique restriction sites and the vaccinia viras P7.5 late-early promoter and Pl l late promoter ananged in a head-to-head orientation.
  • the linker also has the first twenty nucleotides of the Escherichia coli LacZ, (beta-galactosidase), gene as found in the vaccinia virus recombination plasmid pSCl l, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).
  • the synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1 (Invitrogen, Paisley, Scotland), to make pLink.
  • the sequence of the 300 base pair linker is given below (SEQ ID 13).
  • LacZ gene from pSCll is subcloned into pLINK to make pLinkLacZ.
  • the linker and LacZ gene were then subcloned into pSP72 as a Xhol-Bglll fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3-prime to the PI 1 late promoter to make pSP72LinkLacZ.
  • a double expression cassette containing both the small and middle forms of the Hepatitis B Surface Antigen protein-coding region and a marker gene was built in a modified form of pSP72LinkLacZ.
  • the small and middle forms of the Hepatitis B Surface Antigen protein-coding region were isolated as a Hindlll-Nsil fragment from the expression plasmid pCMVS2.S (M-L Michel et al 1995 Proc Natl Acad Sci USA 92 pages 5307-5311), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site.
  • HBs and marker gene expression cassettes were then transfened from the modified pSP72LinkLacZ as a Notl fragment into the unique Notl restriction site of ⁇ FDell3 to make pFDell3.HBs26.
  • the plasmid pFDell 3. HBs26 ( Figure 10), that directs the introduction of foreign genes into the site of Deletion 13 of the FP9 genome, was used to generate the recombinant viras AWOX3.HBs26.
  • the method for making the recombinant FPW was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant viras plaques were screened using an inverted fluorescence microscope to detect g ⁇ -positive plaques.
  • Example 13 Insertion of heterologous nucleic acid into Deletion 11 of the Webster (FPW) strain of fowlpox
  • This example describes the constraction and characterisation of AWOX5.HBs26, comprising the introduction/insertion into the Deletion 11 equivalent (ie. homologue) in the Webster (FPW) strain of fowlpox.
  • Novel plasmids were constructed to enable the generation of recombinant FPW fowlpox. Insertion of foreign genes in FPW was targeted so as to generate a deletion within the FPW equivalent to the naturally occurring 'Deletion 11 ' in the FP9 genome.
  • Sequences of FP9 DNA comprising 500bp flanking each side of the site of a 3426bp deletion located at basepair number 150465 of the FP9 genome sequence (SEQ ID NO 9) were synthesized by Geneart GmbH, (Josef-Engert-Str. 9, D-93053 Regensburg, Germany); in addition, this synthesis included the recognition sequence for the Notl restriction enzyme, GCGGCCGC, placed between position 150465-150466, and a 5- prime terminal Avrll and a 3-prime terminal Sphl recognition site, (SEQ ID 12).
  • the synthetic sequence provided by Geneart was cloned between the Sad and Kpnl of pCR-script (Stratagene, La Jo Ila, USA).
  • the plasmid pSP72.LinkLacZ is based on the commercially available cloning plasmid, pSP72, (Promega Inc.).
  • the standard multiple cloning site is replaced with a synthetic linker containing unique restriction sites and the vaccinia viras P7.5 late-early promoter and Pl l late promoter ananged in a head- to-head orientation.
  • the linker also has the first twenty nucleotides of the Escherichia coli "LacZ, (beta-galactosidase), gene as found in the vaccinia viras recombination plasmid pSCl l, (Chakrabarti et al 1985 Mol Cell Biol 5 p3403-3409).
  • the synthetic linker is synthesized as a series of overlapping oligonucleotides, amplified by PCR, and cloned into the commercial cloning plasmid, pcDNA3.1 (Invitrogen, Paisley, Scotland), to make pLink.
  • the sequence of the 300 base pair linker is given below (SEQ ID 13).
  • LacZ gene from pSCl l is subcloned into pLINK to make pLinkLacZ.
  • the linker and LacZ gene were then subcloned into pSP72 as a Xhol-Bglll fragment such that a complete beta-galactosidase-encoding open reading frame is formed 3-prime to the PI 1 late promoter to make pSP72LinkLacZ.
  • a double expression cassette containing both the small and middle forms of the Hepatitis B Surface Antigen protein-coding region and a marker gene was built in a modified form of pSP72LinkLacZ.
  • the Pl l late promoter and the LacZ gene were replaced by digesting pSP72LinkLacZ with Nsil and Sphl to insert a short oligonucleotide linker containing the compact synthetic early-late promoter described in S. Chakrabarti et al. 1997 Biotechniques 23(6) pl094-1097 (SEQ LD 14).
  • a DNA fragment encoding the green fluorescent protein (g ⁇ ) present in the commercial plasmid, pQBI25 (Q-BIOgene, Montreal, Quebec) was transfened into the modified pSP72LinkLacZ such as to place the marker gene under the control of the compact synthetic promoter.
  • the small and middle forms of the Hepatitis B Surface Antigen protein-coding region were isolated as a Hindlll-Nsil fragment from the expression plasmid pCMVS2.S (M-L Michel et al 1995 Proc Natl Acad Sci USA 92 pages 5307-5311), blunted with Klenow and T4 DNA polymerases, and ligated into the unique Smal site.
  • HBs and marker gene expression cassettes were then transfened from the modified pSP72LinkLacZ as a Notl fragment into the unique Notl restriction site of pFDell 1 to make pFDell 1.HBs26.
  • the plasmid pFDell l.HBs26 ( Figure 6), that directs the introduction of foreign genes into the site of Deletion 11 of the FP9 genome, was used to generate the recombinant viras AWOX5.HBs26.
  • the method for making the recombinant FPW was essentially as described in Y. Qingzhong et al (1994) Vaccine 12 (6) pages 569-573, except that the plasmid transfer vector was purified using a Qiagen Plasmid Maxi Kit according to the manufacturer's protocol, and the recombinant virus plaques were screened using an inverted fluorescence microscope to detect g ⁇ -positive plaques.
  • Recombinant virus plaques were purified through two round of plaque-picking; the plaque morphology and size was as expected for a recombinant fowlpox vector.
  • the DNA extracted from each recombinant was used for testing the identity and the purity of the viras using the PCR reaction.
  • the identity and purity PCRs were performed following the Basic Protocol 15.1.1. in Cu ⁇ ent Protocols Molecular Biology Volume 3, one set of primers (Dell lRF and Dell lLF, SEQ ID 15 and 16) able to recognise both recombinant viras and wt was used. All clones appear to contain the HBs26 cassette.
  • Table 3 List of genes and their occurrence in other poxviruses
  • Prostate PSA prostate specific antigen
  • HPV E1-8 (viral protein)
  • HPV L1 (viral protein)
  • HPV L2 (viral protein)
  • HSV gM viral protein
  • HSV gH viral protein
  • HSV g (viral protein)
  • HSV GG viral protein
  • HSV gD viral protein

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  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un acide nucléique de recombinaison comprenant un génome viral dans lequel a été insérée une séquence nucléotidique hétérologue, l'insertion étant située sur un site naturel de délétion ou d'insertion dudit génome. Dans un mode de réalisation préféré, le site d'insertion est pris dans la table 2, ou dans un homologue de ladite table. L'invention concerne également des virus contenant lesdits acides nucléiques.
PCT/GB2004/002387 2003-06-04 2004-06-04 Produits Ceased WO2004108940A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0312803A GB2402391A (en) 2003-06-04 2003-06-04 Fowlpox recombinant genome
GB0312803.0 2003-06-04

Publications (2)

Publication Number Publication Date
WO2004108940A2 true WO2004108940A2 (fr) 2004-12-16
WO2004108940A3 WO2004108940A3 (fr) 2005-04-21

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PCT/GB2004/002387 Ceased WO2004108940A2 (fr) 2003-06-04 2004-06-04 Produits

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GB (1) GB2402391A (fr)
WO (1) WO2004108940A2 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE1004877A3 (fr) * 1991-05-27 1993-02-16 Solvay Virus de l'avipox recombinant, culture de cellules infectees par ce virus et vaccins pour la volaille derives de ce virus.
WO1996040880A1 (fr) * 1995-06-07 1996-12-19 Syntro Corporation Virus de variole aviaire recombines et leurs utilisations
UA68327C2 (en) * 1995-07-04 2004-08-16 Gsf Forschungszentrum Fur Unwe A recombinant mva virus, an isolated eukaryotic cell, infected with recombinant mva virus, a method for production in vitro of polypeptides with use of said cell, a method for production in vitro of virus parts (variants), vaccine containing the recombinant mva virus, a method for immunization of animals
GB2382578A (en) * 2001-11-30 2003-06-04 Isis Innovation Fowlpox-based vaccine
CA2467486A1 (fr) * 2001-11-30 2003-06-12 Isis Innovation Limited Vaccin
PL218318B1 (pl) * 2002-05-16 2014-11-28 Bavarian Nordic As Rekombinowany wirus krowianki Ankara (MVA), zawierająca go komórka, szczepionka lub kompozycja farmaceutyczna, sposób lub zestaw do jego otrzymywania, sekwencja DNA zawierająca genom tego wirusa oraz sposób wykrywania tego wirusa lub komórek nim zainfekowanych

Also Published As

Publication number Publication date
WO2004108940A3 (fr) 2005-04-21
GB2402391A (en) 2004-12-08
GB0312803D0 (en) 2003-07-09

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