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WO1996016164A1 - Preparations virales, immunogenes et vaccins - Google Patents

Preparations virales, immunogenes et vaccins Download PDF

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Publication number
WO1996016164A1
WO1996016164A1 PCT/GB1995/002740 GB9502740W WO9616164A1 WO 1996016164 A1 WO1996016164 A1 WO 1996016164A1 GB 9502740 W GB9502740 W GB 9502740W WO 9616164 A1 WO9616164 A1 WO 9616164A1
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Prior art keywords
virus
gene
mutant
cells
viral
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PCT/GB1995/002740
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English (en)
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Stephen Charles Inglis
Michael Edward Griffith Boursnell
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Cantab Pharmaceuticals Research Limited
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Priority to AU39308/95A priority Critical patent/AU3930895A/en
Publication of WO1996016164A1 publication Critical patent/WO1996016164A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to viral preparations, immunogens, and vaccines, and in particular to mutant viruses, their culture, vaccines, and their preparation and uses.
  • the invention also provides corresponding complementing recombinant cell lines, including productively infected complementing recombinant cell lines; pharmaceutical compositions containing the defective virus produced thereby; and methods for their use for example as immunogens and vaccines, e.g. as vectors for carrying heterologous nucleic acid, and as materials for stimulating, priming or expanding T-cell populations e.g. cytotoxic T-eelIs specific for the viruses or virally encoded or heterologous- encoded gene products.
  • Vaccine preparations have traditionally taken the form either of "killed” vaccines or of "attenuated live” vaccines. Such traditional forms have been recently augmented by materials and methods based on recombinant DNA techniques. Among these are uses and proposed uses of various subunit (protein antigen) vaccines made by rDNA technique by expression of the wanted materials in host cells unrelated to the pathogen for which the vaccine is to be specific. Also proposed are various forms of disabled infectious virus vaccines made by molecular genetic methods.
  • the prior art includes especially PCT specifications WO 92/05263 (Immunology Ltd: Inglis et al) and WO 94/21807 (Cantab Pharmaceuticals Research: Inglis et al), which describe for example the production and use in vaccines of mutant viruses whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect normal host cells and undergo replication and expression of viral antigen genes in such cells but cannot produce infectious new virus particles.
  • Such virus can be cultured in recombinant cell lines expressing the gene product in respect of which the mutant virus is defective.
  • the prior art also includes studies of immunomodulatory viral genes and gene products.
  • I A York et al . in Cell 77 (May 201994) 525- 535. describe a cytosolic herpes simplex virus protein that inhibits antigen presentation to CD8+ T lymphocytes. This protein is designated immediate- early protein ICP47-
  • ICP47 immediate- early protein
  • HSV HSV can evade detection by CD8+ T lymphocytes when the infection is in human fibroblasts. which can explain the predominance of CD4+ rather than CD8+ HSV- specific cytotoxic T lymphocytes in humans in vivo.
  • ICP47 is a protein expressed in HSV type 1.
  • a protein equivalent to ICP47 exists in HSV type 2 and has been designated IE12 (Marsden et al. J Gen Virol 62: 17-27 (1982)).
  • adenovirus genes normally responsible for downregulating the immune response of an infected subject e.g. relevant genes discussed in G Fejer. I Gyory. J Tufariello. MS Horwitz, J Virol 1994 Sep. 68(9): 5871-81 (showing that human adenoviruses contain a complex transcription region (E3) coding for proteins that inhibit the action of several arms of the immune system against the infected cells).
  • E3 complex transcription region
  • the present invention provides a mutant virus which has a genome defective in respect of a first gene essential for the production of infectious vi us, and in which a second gene, native to the vi us and normally functioning to downregulate a host's immune response against wild-type virus, is structurally or functionally inactivated.
  • the genome of the mutant virus is made defective in respect of the first gene essential for the production of infectious virus by infected host cells, so that the virus can infect normal host cells, but cannot cause production of infectious new virus particles from such normal cells.
  • the second gene is inactivated in order to maximise or enhance the immune response of a vaccinated host to the limited virus replication (e.g. often a single or incomplete round of virus replication) due to the mutant virus. This is advantageous in a vaccine where one wants to stimulate the host's immune system as much as possible.
  • Such mutant virus can be capable of protecting a subject of a susceptible species immunised therewith, against infection by the corresponding wild-type virus. or against a pathogen corresponding to a gene carried by the mutant virus as described below.
  • the mutant virus can be a mutant DNA or RNA virus e.g. a mutant non- retroviral virus, or a DNA virus e.g. doublestranded DNA virus, of a kind that normally causes expression of a gene for downregulating a host immune reponse to virus infection, e.g. as described above.
  • mutant herpesvirus such as herpes simplex virus, e.g. HSV1 or HSV2; other human herpesviruses such as CMV, EBV, VZV. HHV6 and HHV7; and (non-human) animal (veterinary) herpesviruses such as PRV (of pigs), BHV (of cattle). EHV (of horses).
  • the invention also provides corresponding mutant adenovirus, e.g. of type 2 or type 5.
  • the invention can further be applied to any virus where one can identify in the wild-type both an essential gene (ie a gene which plays an essential role in the production of infectious virus and which if absent or defective would significantly reduce the ability of the virus to replicate and produce infective particles) and a gene which normally functions to downregulate the host immune response.
  • mutant viruses which are defective in respect of a first gene essential for the production of infectious virus, such that the virus can infect normal cells and undergo replication and expression of viral antigen in these cells but cannot produce infectious progeny virus, and which is also defecti e in respect of a second gene native to the virus and which normally functions to downregulate a host's immune response against wild-type virus (e.g. downregulation genes as mentioned in references given herein).
  • the first (essential) gene as referred to above, which is inactivated or lacking (preferably deleted in its entirety) in the disabled mutant virus can for example in the case of a herpesvirus be a gene for an essential glycoprotein.
  • a herpesvirus be a gene for an essential glycoprotein.
  • the first gene (essential for production of infectious new virus particles) can for example be deleted entirely, or completely or partially inactivated, e.g. by any mutation that blocks expression, e.g. a point or promoter mutation or an inactivating insertional mutation.
  • Such disabled mutant virus can be propagated on a culture of a complementing host cell line. i.e. a genetic recombinant host cell line that carries and can express a gene that complements the function of the essential viral gene in respect of which the disabled virus is defective.
  • a complementing host cell line i.e. a genetic recombinant host cell line that carries and can express a gene that complements the function of the essential viral gene in respect of which the disabled virus is defective.
  • Such genetically-disabled viruses are replication-competent in complementing host cells of the recombinant host cell line, but they are replication-incompetent, i.e. ultiplication-incompetent, in ordinary (normal) host cells, and do not produce there any infectious progeny virus particles.
  • Replication- incompetence does not exclude that the genetically- disabled virus causes some intracellular viral molecular replication events to take place in normal host cells infected therewith.
  • Replication-incompetence also does not exclude that non-infectious virus-like particles are produced by an infected normal host cell (see e.g. WO 92/05263; Immunology Limited: Inglis et al).
  • the mutation giving rise to the genetic disability is preferably in an essential gene such that intracellular molecular replication of many or even most virally-encoded products such as nucleic acids and proteins does take place in a normal host cell, although no infectious new virus particles are ultimately formed thereby. (Examples are the genes for essential envelope glycoproteins gD. gH and gL as mentioned above.)
  • Normal host cells in the present context, are cells within the normal host range of the parent virus or wildtype precursor of a mutant virus as discussed herein, which have not been made recombinant to carry and express the essential gene of viral origin as was deleted out of the mutant virus (nor an equivalent essential viral gene), and do not express a gene product complementary to the product of the essential gene in respect of which the mutant virus genome is defective: normal cells thus contrast with recombinant complementing cells that have been made to carry a gene that can be expressed so as to complement an artificial deletion or disability in the mutant virus.
  • Defective (replication incompetent) recombinant adenoviruses which are infectious but unable to produce infectious progeny virus in normal (non- recombinant) host cells, as well as complementing recombinant cell lines capable of supporting their growth, are also known per se and can be made for example by adaptation of techniques and the use of genes and gene inactivations as mentioned or cited in specification WO 93/19092 (CNRS and CRC: M Perricaudet et al); or by corresponding adaptation of techniques mentioned in FL Graham et al . J Gen Virol, 36 (1977) 59-72. or T Harrison et al. Virology 77 (1977) 319-329.
  • the mutant virus usually causes replication of some viral components and expression of viral antigen genes in normal host cells, so that the virus, although it can infect normal cells, does not cause production of infectious new virus particles therefrom.
  • the first gene can be such (e.g. herpesvirus gD or gH) that in the absence of its function, on account of the genomic defect, the mutant virus can replicate sufficiently to give rise to production and release, from normal cells in culture or from cells of an infected subject, of non-infectious viral particles.
  • mutant herpesviruses where a gene encoding one of certain essential glycoproteins. such as gH or gD. has been deleted from the virus genome.
  • the present invention also provides in certain embodiments a mutant virus whose genome is defective in respect of a first gene essential for the production of infectious virus and which further carries heterologous genetic material, e.g. encoding an immunogen from a pathogen exogenous to the virus, so that the virus can infect normal cells and undergo replication and expression of heterologous genetic material, e.g. immunogen, but cannot produce infectious new virus particles, and wherein, as discussed above, a second gene native to the virus, which normally functions to downregulate a host immune response against wild-type virus, is inactivated.
  • heterologous genetic material e.g. encoding an immunogen from a pathogen exogenous to the virus
  • the mutant virus can cause expression in host cells of heterologous genetic material, such as an antigen corresponding to another pathogen, eg. a bacterial or viral pathogen.
  • a mutant virus example can be used as an immunogen e.g. to confer immunity against the other pathogen upon a subject of a susceptible species immunised with the mutant virus.
  • the viruses in this type of example can be derived from a similar range of parent virus types as mentioned above.
  • the disabled mutant viruses provided hereby can for example have the property that in a host infected therewith they can establish a latent infection with periodic reactivation, such reactivation leading to production of proteins encoded by the mutant virus in the latently infected cell.
  • the second (immune downregulatory) gene to be functionally or structurally altered in the practice of the present invention is one that is native to the virus and in the corresponding wild-type virus normally functions to downregulate a host immune response against such corresponding wild-type virus.
  • This second (immune downregulatory) gene can in a herpesvirus be a gene encoding ICP47 or IE12 protein or a gene substantially homologous to either of those genes, e.g. as described in P Mavromara-Nazos et al . J Virol, 1986. 60(2): 807-812. Usable degrees of homology can be about 40X or more, eg 50* or 60* or 70* or more. Guidance is given below on identifying homologous genes in order to establish gene deletions of genes homologous with ICP47 in other herpesviruses.
  • the second (immune downregulatory) gene as referred to above can be one of the genes reponsible in adenovirus for downregulating the immune response of an infected subject, for example the E3-gpl9k gene, or another of the relevant genes discussed in G Fejer, I Gyory. et al . J Virol 1994 Sep. 68(9): 5871-81 (showing that human adenoviruses contain a complex transcription region (E3) coding for proteins that inhibit the action of several arms of the immune system against the infected cells).
  • E3-gpl9k gene or another of the relevant genes discussed in G Fejer, I Gyory. et al . J Virol 1994 Sep. 68(9): 5871-81 (showing that human adenoviruses contain a complex transcription region (E3) coding for proteins that inhibit the action of several arms of the immune system against the infected cells).
  • E3-gpl9k complex transcription region
  • Deletion of such an adenovirus gene can be carried out by deleting the E3-gpl9k gene or another of the relevant genes mentioned and identified in the above-cited papers: they can be deleted by readily available adaptations of techniques mentioned in the cited papers or of readily available standard techniques.
  • By adapting such gene-deletion techniques and applying them to the mentioned adenovirus genes a varietyof genetically disabled adenoviruses can be made, lacking a gene that is normally responsible for downregulating a host's immune response, and usually also carrying a foreign antigen gene; they can provide enhanced immune response, e.g. against the foreign antigen.
  • Corresponding immune downregulatory genes can be identified in further adenoviruses where desired, using standard methods related to those exemplified herein for identifying analogues of herpesvirus ICP47 genes.
  • the invention thus provides further examples of suitable mutant viruses which are defective recombinant adenoviruses in which there is a deletion or inactivation of a gene (such as the adenovirus gpl9k (E3-gpl9k) gene) reponsible for downregulating the immune response of a subject infected with corresponding wildtype virus; usually they are vectors also carrying a gene for a foreign antigen against which an immune response is wanted.
  • 'Functional inactivation'. in respect of the second (immune downregulatory) gene means that its normal immune downregulatory gene function is not observed. This can be achieved e.g. by introducing a defect into said second gene so that expression is inhibited or prevented.
  • the second gene can for example be deleted entirely, or completely or partially inactivated, e.g. by any mutation that blocks expression, e.g. a point or promoter mutation or an inactivating insertional mutation.
  • mutant virus which has a genome defective in respect of a gene (such as ICP47) native to the virus and which normally functions to downregulate a host immune response against wild-type virus.
  • a gene such as ICP47
  • alternative embodiments can be made in which a substantially similar effect can be achieved by the delivery of a signal for example through anti-sense RNA to inhibit the expression of the ICP47 protein. Functional inactivation can thus be achieved by arranging to deliver a signal to inhibit the expression of the second gene.
  • RNA sequence which is substantially or fully complementary to all or part of the mRNA from the second gene, and which serves to block translation of the mRNA and production of the protein such as ICP47, can be effective.
  • Methods for providing such "antisense' polynucleotides are known per se.
  • expression of any of the viral genes mentioned herein that normally act to downregulate the host immune response can be disrupted, i.e. inhibited, by the use of complementary antisense polynucleotides (e.g. 'antisense' to prevent mRNA translation, or 'antisense' or 'sense' polynucleotides to prevent DNA transcription), or by the use of suitable ribozy es.
  • complementary antisense polynucleotides e.g. 'antisense' to prevent mRNA translation, or 'antisense' or 'sense' polynucleotides to prevent DNA transcription
  • suitable ribozy es e.g. 'antisense' to prevent mRNA translation, or 'antisense' or 'sense' polynucleotides to prevent DNA transcription
  • a patient can be immunised for prophylactic or therapeutic purposes by the administration of a vaccine comprising a mutant virus which has a genome defective in respect of a gene essential for the production of infectious virus, so that the virus can infect normal cells and undergo replication and expression of viral antigen genes in those cells but cannot produce infectious progeny virus, the genome also having a gene (such as ICP47) native to the virus and which functions to downregulate the patient's immune response against the virus, and a nucleotide sequence that is transcribed to produce an RNA molecule that is at least in part complementary the the mRNA sequence of the gene functioning in the downregulation, wherein the transcribed RNA is of sufficient length to inhibit the translation of the mRNA and thereby inhibit the production of protein encoded by the gene functioning in the downregulation, so that the immune response of the patient to the vaccine is potentiated.
  • a vaccine comprising a mutant virus which has a genome defective in respect of a gene essential for the production of infectious virus, so that the virus can infect normal
  • antisense polynucleotides are known per se, and are readily adaptable to the specificity needed for the present application by using suitable nucleotide sequences, e.g. of at least about 12 nucleotides complementary in sequence to the sequence of a chosen target; by choosing from among known promoters suitable to the cellular environment in which they are to be effective, and other measures well known per se.
  • suitable nucleotide sequences e.g. of at least about 12 nucleotides complementary in sequence to the sequence of a chosen target; by choosing from among known promoters suitable to the cellular environment in which they are to be effective, and other measures well known per se.
  • suitable nucleotide sequences e.g. of at least about 12 nucleotides complementary in sequence to the sequence of a chosen target
  • promoters suitable to the cellular environment in which they are to be effective, and other measures well known per se.
  • techniques for use of antisense RNA to disrupt expression of a target gene are included or cited (in connection
  • ribozymes to disrupt gene expression
  • techniques for making and administering ribozymes (or antisense oligonucleotides) in order to cleave a target mRNA or otherwise disrupt the expression of a target gene are included or cited in specification WO 94/13793 (Apollon: CJ Pachuk et al) (as applied to ribozymes that target certain mRNAs relevant to leukemias).
  • applications to other target specificities are readily accessible by adaptation.
  • mutant virus as described above to immunise a subject of a susceptible species and confer protection against infection by a corresponding wild-type virus.
  • the virus has been made to carry heterologous DNA encoding a protein such as an antigen from another pathogen, an immune response to the other protein can be evoked.
  • a mutant virus as disclosed herein, with an inactivating defect in a gene essential for the production of infectious virus, and wherein a second gene which normally functions to downregulate a host immune response against a virus that carries such a second gene is inactivated can also be used as a safe vector for delivering, to the immune system of an infected host, an antigen normally foreign to the virus.
  • a gene encoding a desired foreign antigen can be inserted in an effective manner by cloning the desired gene next to a viral promoter, to obtain a DNA cassette containing the gene and the promoter; cloning the cassette into a suitable plas id; and co-transferring the plasmid into a complementing cell line along with DNA purified from the mutant virus with its defect in a gene of which the product is provided by the cell line; and screening for recombinant virus.
  • An example of the application of this technique is described, for example, in respect of a gene encoding SIV gpl20 antigen, in PCT specification W092/05263 (Immunology Limited: Inglis et al).
  • Mutant viruses provided by the invention can be used as immunogens, e.g. for prophylactic or therapeutic use in generating an immune response in a subject infected therewith.
  • a mutant virus can be used in the preparation of an immunogen such as a vaccine for therapeutic or prophylactic use.
  • an immunogen such as a vaccine for therapeutic or prophylactic use.
  • a subject of a susceptible species can be given an immunologically effective amount of an immunogenic pharmaceutical preparation such as a vaccine, comprising the mutant virus and a pharmaceutically acceptable carrier.
  • the present invention also provides an immunogen such as a vaccine comprising such a mutant virus, e.g. an immunogenic preparation such as a vaccine comprising such mutant virus together with a pharmaceutically acceptable vehicle such as is used in the preparation of live vaccines; optionally including an adjuvant.
  • an immunogen such as a vaccine comprising such a mutant virus
  • an immunogenic preparation such as a vaccine comprising such mutant virus together with a pharmaceutically acceptable vehicle such as is used in the preparation of live vaccines; optionally including an adjuvant.
  • the second gene is for example the gene encoding ICP47. IE12 or a gene substantially homologous thereto
  • the use of a mutant virus according to the invention can result in an enhanced host CD8+ T lymphocyte immune response directed to the mutant virus.
  • CD8+ T lymphocytes are recognised as constituting a major component of the anti-viral, e.g. anti-HSV. immune response, and CD8+ T lymphocytes can play a major role in clearing the host of virus (DS Sch id et al . Curr Topics Microbiol Immunol. 179: 57. 1992).
  • Mutant viruses defective in ICP47. IE12 or a gene substantially homologous or corresponding in function thereto, e.g. the corresponding genes mentioned above from adenoviruses can be contained more effectively by cytotoxic CD8+ T lymphocytes, rendering them less pathogenic, but can still be of good, enhanced immunogenicity.
  • the invention also provides corresponding productively infected complementing recombinant cell lines; pharmaceutical compositions containing the defective virus, e.g. with usual carriers, vehicles, and adjuvants; and methods for their use for example as immunogens and/or as materials for stimulating, priming or expanding cytotoxic T-cells specific for them.
  • the invention also finds application for example in vitro, in the in- vitro priming or expansion of cytotoxic T cells specific to a viral antigen (or to a heterologous antigen encoded by heterologous nucleic acid introduced into the mutant virus as mentioned above).
  • the invention in a further aspect provides for production in vitro of appropriate cytotoxic T cells (CTLs) capable of controlling the virus infected cells.
  • CTLs cytotoxic T cells
  • cytotoxic T cells are generally directed against peptides derived from foreign proteins which are synthesised within the antigen-presenting cell: inactivated virus or individual proteins are poor at raising cytotoxic T cell responses.
  • the activated cells are then expanded in culture over a period of weeks with further re-stimulation with antigen and a growth factor such as interleukin-2.
  • the invention further provides a method for producing virus- specific cytotoxic T cells which method comprises; (a) isolating a sample of blood ononuclear cells, lymphocytes or T cells from a patient; (b) culturing said sample in vitro in the presence of a mutant virus which is defective in respect of a first gene essential for the production of infectious virus, and which also has had inactivated a gene that normally functions to downregulate a host immune response, as discussed above (and/or culturing in the presence of cells infected by the mutant virus); the mutant virus optionally including a heterologous nucleotide sequence encoding a heterologous antigen; and (c) reinfusing cultured cells into the patient.
  • the primed or expanded T cells can be used for other purposes, e.g. in-vitro diagnostic testing to indicate the extent of their capacity for priming or stimulation by the mutant virus and/or virus-infected cells.
  • Mutant viruses as hereby provided can be manufactured by a method involving the culturing of cells which have been infected with the mutant virus, the cells also expressing a gene which complements the first defective viral gene so as to allow production of infectious virus particles containing the defective genome, and recovering the mutant virus from the culture.
  • FIGS. 1 and 2 are diagrams illustrating the construction of plasmids pIMMB45. pIMMB47+. and pIMMB46 respectively. Construction of Virus with Deletions in gH and ICP47
  • SC16 delta gH mutant (A Forrester et al , J Virol 66: 341- 348. 1992) is a HSV-1 mutant virus having only a part of the gH gene. That part is functionally inactive.
  • the missing part of the gH gene is replaced with an E.coli lacZ gene under the control of the CMV IE promoter, thus enabling the mutant gH defective virus to be identified by its blue colour under an agarose overlay containing the colorigenie substrate Xgal (as described in A Forrester et al. J Virol 66: 341-348.1992).
  • a HSV-1 mutant virus containing the inactivated ICP47 gene (also known as US12) is designated R3631.
  • the R3631 mutant can be obtained by using or adapting the description of P Mavro ara-Nazos et al . J Virol 60(2): 807-12, 1986.
  • the R3631 mutant has the ICP47 (US12) gene deleted and the ICP47 (US12) gene is inactive in the sense that it is deleted.
  • the loss of ICP47 (US12) gene activity also results in the inactivity of the US11 gene which is adjacent to ICP47 (US12).
  • the two mutant HSV-1 viruses (SC16 delta-gH and R3631) are infected into Vero cells at a multiplicity of infection of 5 pfu/cell for each virus. Cells are incubated until an extensive cytopathic effect occurs. The cells are then harvested and virus released by sonication. Released viral particles are then used to infect cells at a suitable multiplicity of infection, such that individual plaques can be picked. Plaques are picked under an agarose overlay containing Xgal so that any recombinants containing the lacZ gene (which replaces part of and thereby inactivates the gH coding sequence), can be identified by their blue colour. Plaques are transferred directly into wells of a 96-well tissue culture dish containing Vero cells.
  • Each plaque is allowed to grow until all the cells in the well are infected. The cells are then harvested and virus released by sonication as before. Half of the harvested plaque is stored at -80°C and the other half is used to make total cell and viral DNA as described in Kintner & Brandt (1994), Journal of Virological Methods 48: 2-3 pp 189-196.
  • Viruses are plaqued out on Vero cells and F6 cells (A Forrester et al . J Virol 66: 341-348. 1992). Any virus lacking the gH gene should grow on F6 cells but not on Vero cells. This will confirm the loss of an active gH gene.
  • gH defective viruses should contain the lacZ gene which was used to replace part of the gH gene. These viruses should appear blue in colour under an agarose overlay containing Xgal.
  • Viruses are grown up and DNA is made from them as described in Kintner & Brandt (1994), Journal of Virological Methods 48: 2-3 pp 189-196.
  • the viral DNA is digested and transferred to a nylon filter ("Molecular Cloning: A Laboratory Manual”. eds. Sambrook, Fritsch and Maniatis.2nd ed.. Cold Spring Harbor Laboratory Press 1989).
  • the viral DNA is hybridised with a radiolabelled oligonucleotide homologous to the US12 gene sequences as above.
  • a restriction digest of control viral DNA from a US12-containing virus is included on the filter.
  • the oligonucleotide will hybridise to the restriction enzyme fragment containing the US12 gene and a band will be visible on an autoradiograph of the filter ("Molecular Cloning: A Laboratory Manual", eds. Sambrook, Fritsch and Maniatis, 2nd ed.. Cold Spring Harbor Laboratory Press 1989).
  • the gH-deleted US12-deleted mutant virus made in this way could have other mutations at a different site.
  • the most appropriate virus to use as a gH-deleted control in later experiments is a gH-deleted US12-deleted mutant virus in which the US12 has been re ⁇ inserted.
  • a virus is usually referred to as a revertant. and will be called the gH-deleted revertant henceforth.
  • the gH-deleted revertant is made by making a recombinant between the gH-deleted US12-deleted mutant virus, and DNA from the US11-US12 region.
  • a suitable plasmid which contains a restriction fragment comprising the US12 sequences that were deleted from HSV-1 strain F in order to make R3631. and also additional flanking DNA sequences of approximately 500 base pairs on either side of the US12 deletion region.
  • the recombination is carried out by transfecting both viral DNA from the gH-deleted US12-deleted mutant virus, and the above mentioned plasmid DNA. in a suitable cell. Homologous recombination takes place between the viral DNA and the plasmid DNA. The process is described in A Forrester et al . J Virol 66: 341-348. 1992.
  • Recombinants containing the replaced US12 gene are identified by probing with a US12-specific oligonucleotide as described above. Recombinant virus is grown up and checked again as described above.
  • the gH-deleted US12-deleted mutant virus can be tested for its efficacy as a vaccine by using a mouse model system as described in H Farrell et al., J. Virol. 68, 927-32, 1994.
  • Groups of mice are vaccinated by scarification of the ear pinna with various doses ranging from 10 2 to 10 6 pfu of gH-deleted mutant virus, US12-deleted mutant virus, the gH-deleted US-12-deleted mutant virus, and the gH revertant.
  • a control group is vaccinated with PBS.
  • mice are challenged in the opposite ear pinna with 10 6 pfu of wild- type HSV-1 (strain SC16).
  • mice Five days post challenge the mice are killed, the challenged ears removed and frozen at -70°C. The ears are homogenised and the amount of infectious challenge virus in each ear is determined by plaque titration. The reduction in virus titres in the vaccinated groups of mice compared to the PBS-treated controls is a measure of the protection afforded by the virus vaccine. It is known that the gH-deleted virus can completely abolish the presence of infectious virus at a vaccinating dose of 5 x 10 5 pfu. whilst even at 5 x 10* pfu a reduction of 1000-fold is observed.
  • the gH- deleted US12-deleted mutant can increase the level of protection as would be evident by observing complete protection from any infectious virus at lower challenge doses than in the gH-deleted mutant-virus vaccinated mice, and a greater reduction of infectious virus titres as compared to the PBS-vaccinated controls.
  • a gH-deleted mutant virus containing an inactive US12 gene can be constructed by replacing normal functional US12 coding sequences in the gH- deleted mutant virus with inactive mutant US12 sequences in a plasmid, using homologous recombination.
  • the mutant US12 sequence differs from normal US12 coding sequences by containing a stop codon in all three reading frames, thus leading the production of a truncated, inactive ICP47 protein.
  • the altered sequence occurs in the 5' section of the US12 gene to avoid disrupting the mRNA start site for the next gene along, namely US11. This site is at genome position 12855.
  • a possible altered sequence is to replace the sequence ATG CGG GTT GGG CCC from 12916-12930 (complementary strand), by the sequence ATG TAA A TGA C TAG T CCC. This will insert stop codons in all reading frames after the fifteenth codon in US12, and introduce a Spe I restriction site into the virus at this point, which can be used for identifying the recombinant virus. This change occurs after the third and final methionine codon in the US12 sequence, which will mean that translation of a truncated protein is highly unlikely. The only product that will probably be made is a short truncated peptide comprising the first 15 amino acids of the US12 protein. This is highly unlikely to have any biological activity.
  • sequences either side of the altered region are copied from the virus by the polymerase chain reaction (PCR) technique. These sequences act as flanking sequences, allowing homologous recombination between the virus and the plasmid to take place.
  • PCR polymerase chain reaction
  • Suitable oligonucleotides for use are shown below, but other oligoncleotides can be used. A person skilled in the art can readily select other oligoncleotides that will be different in their precise positioning, but which will perform the same basic function.
  • a plasmid is constructed in which the altered US12 sequences are flanked, and contiguous with, the flanking sequences. This is achieved by joining the two PCR products together by gene splicing by overlap extension (Horton et al.. Gene 77: 61, 1989). The final spliced product can be cloned into a PCR cloning vector such as pGEM-T (Pro ega Corporation). Recombination is carried out as descibed above, using DNA from the gH-deleted virus, e.g. SC16 delta- gH). and the US12-altered plasmid construct.
  • pGEM-T Pro ega Corporation
  • Recombinant viruses containing the altered sequences ie the mutated US12 gene
  • a revertant virus in which the US12 gene is corrected can be made in a similar manner to that descibed above, except that the sequences used to correct the gene only need to be the correct sequence plus 500 base pairs either side.
  • Production of gH-mutant HSV-2 virus requires a cell line expressing the HS -2 gH and an HSV-2 virus lacking the gH gene.
  • a complementing cell line expressing the HS -2 gH gene is obtained using plasmid pIMCO ⁇ , containing the HSV-2 (strain 25766) gH gene, constructed as follows.
  • Plasmid pIMMB24A is made as follows: an HSV-2 gH gene is constructed from two adjacent BamHI fragments of the HSV-2 strain 25766.
  • the plasmids used are pTW49. containing the approximately 3484 base pair BamHI R fragment and pTW54, containing the approximately 3311 base pair Bam HI S fragment, both cloned into the BamHI site of pBR322.
  • the plasmids can be cloned easily from any strain of HSV-2.
  • the 5' end of the HSV-2 gH gene is excised from pTW54 using BamHI and KpnI. to produce a 2620 base pair fragment which is gel-purified.
  • the 3' end of the HSV-2 gH gene is excised from pTW49 using BamHI and Sail, to produce a 870 base pair fragment which is also gel- purified.
  • the two fragments are cloned into pUC19 which has been digested with Sail and KpnI.
  • This plasmid now contains the entire HSV-2 gH gene.
  • Plasmid pIMMB24A is digested with Ncol and BstXI and the fragment containing the central portion of the gH gene is purified from an agarose gel .
  • the 5' end of the gene is reconstructed from two oligonucleotides (CE 39 AGCTTAGTACTGACGAC and CE40 CATGGTCGTCAGTACTA) which, when hybridised, form a linking sequence bounded by HindiII and Ncol sites.
  • the 3' end of the gene is reconstructed from two oligonucleotides (CE 37 GTGGAGACGCGAATAATCGCGAGC and CE38 GGCCGCTCGCGA ⁇ ATTCGCGTCTCCACAAAA) which, when hybridised, form a linking sequence bounded by BstXI and NotI sites.
  • Plasmid pIMC05 is constructed as follows: A 4.3kb Sst-1 fragment encoding the HSV-1 (HFEM) gH gene and upstream HSV-1 gD promoter (-392 to +11) is excised from the plasmid pgDBrgH (A Forrester et al .. J Virol 1992. 66: 341-348. v.q. H Farrell et al.. J Virol 1994, 68: 927-932) and cloned into pUC119 (J Vieira & J Messing. Methods in Enzymology (1987) 153. 3-11) to produce plasmid pUC119gH.
  • flanking sequences are deleted from pUC119gH. and a Not 1 site is introduced by utagenesis, 87 bp downstream of the gH stop codon.
  • the resulting plasmid. pIMC03. is used to generate a Not 1-Sst 1 fragment which is repaired and ligated into the eucaryotic expression vector pRC/CMV (Invitrogen Corporation), pre-digested with Not 1 and Nru 1 to remove the CMV IE promoter.
  • the resulting plasmid, pIMC05 contains the HSV-1 gH gene under the transcriptional control of the virus inducible gD promoter and BGH (Bovine Growth Hormone) poly A. It also contains the neomycin resistance gene for selection of G418 resistant stable cell lines.
  • the two oligonucleotide linkers mentioned above and the purified Ncol- BstXI gH fragment are cloned in a triple ligation into Hindlll-NotI digested pIMC05. thus replacing the HSV-1 gH gene by the HSV-2 gH gene.
  • the resultant plasmid is designated pIMCO ⁇ .
  • the complementing cells are produced using plasmid pIMC08 in a manner similar to the way in which complementing (CR1) cells were produced using pIMC05 (WO 94/21807; Cantab Pharmaceuticals: Inglis et al).
  • Viral DNA is purified from virus by standard methods. Flanking sequences to either side of the gH gene are amplified from this viral DNA by PCR using Vent DNA polymerase (available from New England Biolabs) which has a lower error rate than Taq DNA polymerase (see Fig 3).
  • the oligonucleotides used for PCR include restriction site recognition sequences, as well as the specific viral sequences (see below).
  • Two vectors are made, one for the first stage and one for the second stage of recombination. For both vectors the right hand flanking sequences start at the same position to the right of the gH gene.
  • the first stage vector has left hand flanking sequences that, in addition to deleting the HSV-2 gH gene, also delete the 3' portion of the viral TK gene.
  • the second stage vector has left hand flanking sequences which restore the complete TK gene, and extend right up to the 5' end of the gH gene, as desired in the final virus.
  • oligonucleotides used are as follows:
  • the two PCR fragments made by oligos MB97-MB96 and by oligos MB57-MB58 are digested with the restriction enzymes appropriate to the sites that have been included in the PCR oligonucleotides.
  • the MB97-MB96 fragment is digested with Hindlll and Hpal.
  • the MB57-MB58 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector pUC119 (J Vieira and J Messing, see above) which has been digested with Hindlll and EcoRI.
  • the resultant plasmid is called pIMMB45 (see Fig 1).
  • the E.coli beta- galactosidase gene under the control of the Cytomegalovirus (CMV) immediate early promoter, is inserted into pIMMB45.
  • CMV Cytomegalovirus
  • the CMV promoter plus beta- galactosidase gene is excised from the plasmid pMVIO using Hindlll. The ends are filled in using the Klenow fragment of DNA polymerase. The fragment is gel-purified.
  • the plasmid pIMMB45 is digested with Hpal. phosphatased with Calf Intestinal Alkaline Phosphatase (CIAP) to abolish self ligation, and gel- purified. The gel-purified fragments are then ligated together to produce the plasmid pIMMB47+ (see Fig 2).
  • the two PCR fragments made by oligos MB94-MB109 and by oligos MB57- MB108 are digested with the restriction enzymes appropriate to the sites that have been included in the PCR oligonucleotides.
  • the MB94-MB109 fragment is digested with HindiI and Hpal.
  • the MB57-MB108 fragment is digested with Hpal and EcoRI. These fragments are then ligated into the vector puC119 which has been digested with Hindlll and EcoRI.
  • the resultant plasmid is called pIMMB46 (see Fig.3).
  • the oligo nucleotides used are as follows:
  • Virus DNA is made from strain HG52-D, which is a plaque-purified isolate of the HSV-2 strain HG52. by the method of Walboomers & Ter Schegget, Virology 74: 256-258 (1976).
  • Virus DNA 2.5 micro g
  • pIMMB47+ plasmid DNA (0.25 micro g) is transfected into CRl cells (see above and W094/21807; Cantab Pharmaceuticals: Inglis et al) using the calcium phosphate precipitation method (Chen & Okayama. Molecular and Cellular Biology, 7, p2745). Recombination takes place within the cells, and a mixture of recombinant and wild type virus is produced.
  • the mixture is plaque-purified three times on CRl cells in the presence of acyclovir (10 micro g/ml), to select for TK-minus virus.
  • a single plaque is then grown up and analysed.
  • the virus is titrated on normal Vero cells and on CRl cells. If the virus is a gH-deleted mutant it should only grow on CRl cells and not on Vero cells. The virus will not grow on the non-complementing Vero cells, but will grow on the CRl complementing cell line, which expresses the HSV-1 gH gene. The virus will also grow on complementing cells which express the HSV-2 gH gene.
  • DNA is made from this TK-minus gH-deleted mutant virus and a recombination is carried out as above with the plasmid pIMMB46. In this case
  • TK-plus recombinants are selected, on a gH-expressing TK-minus BHK cell line. by growth in medium containing methotrexate. thymidine, glycine. adenosine and guanosine. Virus is harvested and grown again under selective conditions twice more before a final plaque purification is carried out on cell line CRl.
  • Virus is grown up and analysed by Southern blotting to confirm it to be the expected structure.
  • a US12-deleted HSV-2 can be made using an equivalent method to that described above (in the section entitled "Construction of a gH-deleted US12- deleted Mutant Virus by Recombination with a Modified Plasmid Copy of the US12 gene").
  • the DNA sequences of the HSV-2 gene differ in detail to those of the HSV-1 gene (about 70*-80* similarity) and the method can be modified accordingly.
  • the DNA sequence of the US12 region is determined by standard methods. Using this information, a strategy for deletion of the US12 gene can be devised which is similar in principle to that of the method given above.
  • Cloning of US12-containing sequences from HSV-2 DNA can be isolated from the virus according to the procedure of JMM Walboomers & Ter Schegget, in J Virol 74: 256-258. 1976).
  • the DNA can be digested with the restriction enzyme Hindlll and the 11th largest band (Hindlll K: see Cortini & Wilkie. J gen Virol 39: 259, 1978) isolated from an agarose gel. This band is approximately 10 kilobases in length.
  • the bands Hindlll J and Hindlll K run almost on top of each other, so they will probably have to be purified together and distinguished later.
  • This fragment can then be ligated into a vector such as pUC19 (Pharmacia PL Biochemicals) digested with Hindlll.
  • the ligation mix is transformed into E. coli cells as described in "Molecular Cloning: A Laboratory Manual”, eds. Sambrook. Fritsch and Maniatis. 2nd ed., Cold Spring Harbor Laboratory Press 1989. Ampicillin resistant colonies are picked.
  • DNA is prepared and digested with Hindlll to excise the HSV-specific insert ("Molecular Cloning: A Laboratory Manual”, eds. Sambrook, Fritsch and Maniatis, 2nd ed.. Cold Spring Harbor Laboratory Press 1989).
  • a clone with an insert of the appropriate size can be selected and sequenced as described below.
  • Hindlll K fragment will contain an EcoRI site, wheras the Hindlll J fragment will not.
  • the ampicillin resistant colonies can be transferred to a nylon membrane and probed with a DNA fragment containing the HSV-1 US12 sequences ("Molecular Cloning: A Laboratory Manual", eds. Sambrook. Fritsch and Maniatis.2nd ed.. Cold Spring Harbor Laboratory Press 1989). Because of the degree of homology between HSV- 1 and HSV-2 (approximately 70*-80*). the HSV-1 probe can be used to detect the HSV-2 US12 gene under standard hybridisation conditions.
  • the DNA sequence of the cloned HSV-2 fragments is determined as described in M Boursnell et al (J gen Virol 68: 57, 1987) and D McGeoch et al. (J gen Virol 69: 1531-1574, 1988), incorporated herein by reference. Briefly, as described in greater detail in the references, the HSV specific insert is isolated from the cloned plasmids, sonicated, and subcloned into the phage cloning vector M13mpl0. Sequencing is then carried out by the dideoxy sequencing method.
  • the DNA sequence obtained for HSV-2 is to be compared to the published sequence of HSV-1 in this region (D McGeoch et al. J Mol Biol 181: 1-13.
  • HSV-2 human immunoglobulins-1
  • ORF open reading frame
  • the percentage match between equivalent genes in HSV-1 and HSV-2 is normally in the 70-80* region (D McGeoch et al . J Gen Virol 68: 19.1987) both in DNA and in amino acid comparisons.
  • the equivalent HSV-2 gene to the HSV-1 US12 will be clearly identifiable because no other pair of genes will give a match anywhere near 70*.
  • HSV-1 mutant which is gH-negative and ICP47 (US12)-negative
  • HSV-2 mutant which is gH-negative and IE 12-negative.

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Abstract

L'invention porte sur un virus mutant comportant un génome (a) défectueux au niveau d'un premier gène, ce qui est essentiel pour la production du virus infectieux. Ledit virus mutant peut ainsi infecter les cellules normales, être capable de réplication et exprimer les gènes de l'antigène viral dans lesdites cellules sans toutefois être capable d'y produire de nouvelles particules virales infectieuses; et (b) inactivé structurellement ou fonctionnellement au niveau d'un deuxième gène natif du virus et dont la fonction normale est de rétro-réguler une réponse immunitaire hôte contre le virus de type sauvage correspondant. Les virus mutants, qui s'obtiennent par culture sur des lignées de cellules complémentaires exprimant le premier gène, s'avèrent utiles comme immunogènes et vaccins, et pour stimuler les lymphocytes T cytotoxiques notamment in vitro.
PCT/GB1995/002740 1994-11-23 1995-11-23 Preparations virales, immunogenes et vaccins WO1996016164A1 (fr)

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US7981669B2 (en) 2003-07-25 2011-07-19 Biovex Limited Viral vectors

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WO1992005263A1 (fr) * 1990-09-25 1992-04-02 Cantab Pharmaceuticals Research Limited Vaccin viral mutant a genome deficient et produit en transcomplementant la lignee cellulaire
WO1993019092A1 (fr) * 1992-03-19 1993-09-30 Centre National De La Recherche Scientifique Adenovirus recombinants defectifs exprimant des proteines caracteristiques du virus epstein-barr
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FARRELL H E ET AL: "Vaccine potential of a herpes simplex virus type 1 mutant with an essential glycoprotein deleted.", JOURNAL OF VIROLOGY 68 (2). 1994. 927-932 *
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MCLEAN C S ET AL: "Long-term protection following vaccination with a gH-deleted herpes simplex virus type 1.", KEYSTONE SYMPOSIUM ON MOLECULAR ASPECTS OF VIRAL IMMUNITY, KEYSTONE, COLORADO, USA, JANUARY 16-23, 1995. JOURNAL OF CELLULAR BIOCHEMISTRY SUPPLEMENT 0 (19A). 1995. 312. *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053505A3 (fr) * 2000-01-21 2001-12-27 Biovex Ltd Souches de virus herpetique
GB2374873A (en) * 2000-01-21 2002-10-30 Biovex Ltd Herpes virus strains
GB2374873B (en) * 2000-01-21 2004-05-26 Biovex Ltd Herpes virus strains
US7223593B2 (en) 2000-01-21 2007-05-29 Biovex Limited Herpes virus strains for gene therapy
US8277818B2 (en) 2000-01-21 2012-10-02 Biovex Limited Herpes virus strains for gene therapy
US8680068B2 (en) 2000-01-21 2014-03-25 Biovex Limited Herpes virus strains
US10301600B2 (en) 2000-01-21 2019-05-28 Biovex Limited Virus strains
US7981669B2 (en) 2003-07-25 2011-07-19 Biovex Limited Viral vectors
US8679830B2 (en) 2003-07-25 2014-03-25 Biovex Limited Viral vectors

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