WO1996016164A1

WO1996016164A1 - Viral preparations, immunogens, and vaccines

Info

Publication number: WO1996016164A1
Application number: PCT/GB1995/002740
Authority: WO
Inventors: Stephen Charles Inglis; Michael Edward Griffith Boursnell
Original assignee: Cantab Pharmaceuticals Research Limited
Priority date: 1994-11-23
Filing date: 1995-11-23
Publication date: 1996-05-30
Also published as: AU3930895A; GB9423663D0

Abstract

The invention provides a mutant virus having a genome which is (a) defective in respect of a first gene, which is 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 new virus particles therefrom, and (b) structurally or functionally inactivated in respect of a second gene, native to the virus and normally functioning to downregulate a host immune response against the corresponding wild-type virus. The mutant viruses are produced by culture on complementing cell lines expressing the first gene: they are of use e.g. as immunogens and vaccines and for stimulating cytotoxic T cells, e.g. in vitro.

Description

VIRAL PREPARATIONS. IMMUNOGENS. AND VACCINES

Field of the invention

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.

Background of the invention

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. In regard to the latter, 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.

Cell lines suitable for the culture of certain disabled viruses of this general type have been described: for example FL Graham et al. J Gen Virol 36 (1977) 59-72, described human cell lines transformed with fragments of adenovirus type 5 DNA, which expressed certain viral genes, and these cell lines were shown to be able to support growth of virus mutants which were defective in respect of those genes and could not grow on normal human cells (T Harrison et al. Virology 77 (1977) 319-329). Specification WO 93/19092 (CNRS and CRC: M Perricaudet et al) describes recombinant defective virus vectors based on such defective adenoviruses. Cells expressing the T-antigen-encoding region of SV40 virus genome (a papovavirus) have also been shown capable of supporting replication of viruses specifically deleted in that region (Y Gluzman, in Cell 23 (1981) 182-195).

The literature also describes cell lines expressing proteins of herpes simplex virus: the gB glycoprotein (W Cai et al . J Virol 61(3) (1987) 714-

721), the gD glycoprotein (MW Ligas and DC Johnson. J Virol 62(5) (1988) 1486-

94) and the Immediate Early Protein ICP4 (NA DeLuca et al . J Virol 56(2)

(1985) 558-70). These too have been shown capable of supporting replication of viruses inactivated in respect of the corresponding genes. Complete or substantial sequence data has been published for several viruses such as Epstein-Barr virus EBV (R Baer et al , Nature 310 (1984 July 19) 207-211), human cytomegalovirus CMV (Weston and Barrel!. J Mol Biol 192

(1986) 177-208, varicella-zoster virus VZV (AJ Davison and Scott, J Gen Virol 67 (1986) 759-816) and herpes simplex virus HSV (D McGeoch et al .. J Gen Virol 69 (1988) 1531-1574). The gH glycoprotein (see A Forrester et al , J Virol 1992, 66, pp341-348) is known to have ho ologues in EBV. CMV and VZV (D J McGeoch et al . Nucl Acids Res (1986) 14(10): 4281-4291; and PJ Desai et al , J Gen Virol 69 (1988) 1147).

The prior art also includes studies of immunomodulatory viral genes and gene products. In particular. 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- The authors comment that by expressing ICP47, 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. The authors comment that the effects of ICP47 "may have important implications in the design of HSV vaccines" and expect that ICP47 may be useful as a small, mobile immune suppressive agent in virus-based gene therapy vectors, in tissue transplantation or in the therapy of certain autoimmune diseases. 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)).

Other animal viruses are also known to evade T lymphocyte recognition by downregulating MHC class I expression or by expressing proteins that alter or block the MHC class 1 antigen presentation pathway (LR Gooding. Cell 71: 5-7. 1992). For example, in cells infected with cytomegalovirus. another member of the herpesvirus family, MHC-mediated presentation of virus antigens is also downregulated (del Val et al. J Exp Med 176: 729-738. 1992). In a further example, an adenovirus gene and its product designated E3/19K have been shown to have an effect in downregulating host immune response to infection by the virus (WA Jefferies et al . J Exp Med.1990 Dec, 172(6): 1653- 4). Also known per se are further 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).

Summary and description of the invention An aim of the present invention is to provide live viral vaccines and immunogens with enhanced immunogenicity but also freedom from liability to spread to non-vaccinated subjects. A further aim is to provide new live viral immunogenic materials and their uses, as described below. Other aims will also be apparent from the present description. 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. Examples include 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). FHV (of cats), or MDV (of poultry) and others. 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. The skilled person can readily adapt the description and details given here below to the preparation of other 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. such as herpes simplex virus gB. gD, gH or gL or their ho ologues in other herpesviruses.

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. 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, or multiplication- incompetence, in this sense, 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. Infection with 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. Examples include 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.

In these embodiments, 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. Such 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, especially where they are based on herpesvirus, 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.

Where the disabled mutant virus is based on adenovirus, 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). Such genes are identified in: TW Her iston et al. Virology 1993 Dec. 197(2): 593-600 (Sequence and functional analysis of the human adenovirus type 7 E3 gpl9 protein from 17 clinical isolates): TW Hermiston et al. J Virol 1993 Sep. 67(9): 5289-98; Wold WS. Gooding LR. Mol Biol Med, 1989 Oct. 6(5): 433-52; or Tollefson AE. Wold WS, J Virol 1988 Jan. 62(1): 33-9 (Identification and gene mapping of a 14.700- molecular-weight protein encoded by region E3 of group C adenoviruses).

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.

The particular description below specifically concerns the construction of a 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. However, since the aim is to prevent viral expression of that gene in the host, 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. For example, co-expression from the virus genome of an 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.

Thus, as alternative to inactivating mutations such as gene deletion, 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. Thus 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. Techniques for use of 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. For example, techniques for use of antisense RNA to disrupt expression of a target gene are included or cited (in connection with a sialidase gene) in specification WO 94/26908 (Genentech: TG Warner et al). Techniques for making and using antisense oligonucleotides capable of binding specifically to mRNA molecules are also included or cited in specification WO 94/29342 (La Jolla Cancer Research Foundation and the Regents of the University of Michigan: R Sawada et al) (in particular connection with mRNA encoding human lamp-derived polypeptides). Techniques for antisense oligonucleotides complementary to target RNA are included or cited in specification WO 94/29444. (Department of Health and Human Services: B Ensoli and R Gallo) (as applied to basic fibroblast growth factor RNA). Techniques for making and using antisense oligonucleotides having a sequence substantially complementary to an mRNA which is in turn complementary to a target nucleic acid, in order to inhibit the function or expression of the target, are included or cited in WO 94/24864 (General Hospital Corporation: HE Blum et al). (as applied to inhibition of hepatitis B viral replication). Applications to other target specificities are readily accessible by adaptation.

Techniques for using ribozymes to disrupt gene expression are also known. For example, 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). Here also, applications to other target specificities are readily accessible by adaptation.

Also provided by the invention is the use of mutant virus as described above to immunise a subject of a susceptible species and confer protection against infection by a corresponding wild-type virus. In the case where 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. Thus, 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. This can be achieved by inserting, into the genome of the virus, nucleic acid sequences coding for such an antigen, in such a way as to cause their expression during infection of host cells by the virus. For example, 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. According to the invention such a mutant virus can be used in the preparation of an immunogen such as a vaccine for therapeutic or prophylactic use. For production of an immune response, e.g. for prophylactic or therapeutic immunisation, 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.

Where 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).

This can be useful for example in dealing with two complications of many immunosuppressiveor cytotoxictreatments, generalised virae ia following virus infections, and expansion of virus-transformed cells as a result of latent virus reactivation. These complications can arise from impairment of the normal mechanism for controlling such infections as a result of the treatment.

Accordingly, the invention in a further aspect provides for production in vitro of appropriate cytotoxic T cells (CTLs) capable of controlling the virus infected cells.

This can be done, by adapting per-se known tehcnique. by isolating peripheral blood mononuclear cells or lymphocytes or T cells prior to treatment of the patient, and stimulating such cells in vitro with a preparation of live virus and/or virus-infected cells. It is necessary to use live rather than killed virus, as 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. However, there is the concern that there may be residual live virus in the cell culture when the CTLs are re-infused into the patient. Use of a disabled virus capable of inducing CTL activity but incapable of producing infectious new virus particles, if inadvertently given along with the in vitro expanded cells. provides an advantage over a known system using replication-competent virus. Hence 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. Alternatively, 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.

Knownmethods applicable to comparable techniques based on replication- competent virus can be used and adapted to implement these uses of mutant viruses according to the present invention.

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.

The invention is further particularly described below in embodiments given by way of example only and not for limitation. Background to the particular description includes the construction and properties of a gH- defective virus, described in A Forrester et al. (1992) J Virol 66: 341-348. in W092/05263 (Immunology Ltd: Inglis et al) and in W094/21807 (Cantab Pharmaceuticals: Inglis et al). Genetic manipulation procedures can be carried out by using or readily adapting standard methods e.g. as described in "Molecular Cloning: A Laboratory Manual", eds. Sa brook, Fritsch and Maniat s, 2nd ed.. Cold Spring Harbor Laboratory Press 1989.

The particular description below is illustrated non-li itatively by reference to the accompanying drawings, in which Figures 1. 2 and 3 are diagrams illustrating the construction of plasmids pIMMB45. pIMMB47+. and pIMMB46 respectively. Construction of Virus with Deletions in gH and ICP47

In order to produce a virus containing both a defective gH gene and a defecti e/deleted ICP47 gene, a recombinant between two separate HSV-1 mutants is made. 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. In the SC16 delta gH mutant, 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.

These viral DNA samples, which are known to come from viruses with a deletion in the gH gene, are now probed with a US12 specific probe to see if they also have a deletion in the US12 gene. The viral DNA, immobilised on a nylon filter. ("Molecular Cloning: A Laboratory Manual", eds. Sambrook, Fritsch and Maniatis. 2nd ed.. Cold Spring Harbor Laboratory Press 1989) is hybridised with a radiolabelled oligonucleotide homologous to the US12 gene sequences (e.g.5" CGTACGCCGACGTACGCG 3'). Control viral DNA is included on the filter. If the virus contains the US12 gene, the oligonucleotide will hybridise to the viral DNA. Several viruses which lack the US12 gene are picked and analysed more thoroughly as described below to confirm that they lack both the gH gene and the US12 gene.

Analysis for Defective gH Gene 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. In addition 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.

Analysis for Deletion of ICP47 (US12) Gene

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. Using a suitable restriction enzyme, 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. If the virus contains the US12 gene, 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).

Using these criteria, recombinant viruses lacking both the gH gene and the US12 gene are selected.

Because it is theoretically possible that 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. Such 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 is created 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.

Testing of Vaccine Potential of gH-deleted US12-deleted Mutant Virus

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 10² to 10⁶ 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. After 3 weeks, mice are challenged in the opposite ear pinna with 10⁶ pfu of wild- type HSV-1 (strain SC16). 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 10⁵ 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.

Alternative method of Construction of a gH-deleted US12-deleted Mutant Virus by Recombination with a Modified Plasmid Copy of the US12 Gene

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. In this example, the mutant US12 sequence differs from normal US12 coding sequences by containing a stop codon in all three reading frames, thus leading the 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.

The original sequence and the altered sequence are shown below:

Original sequence: SEQ ID No: 1

MetSerTrpAlaLeuGluMetAlaAspThrPheLeuAspThrMetArgValGlyProArg ATGTCGTGGGCCCTGGA TGGCGGACACCπCCTGGACACCATCCGGGTTGGGCCCAGG Inside right to mutate

ThrTyrAlaAspValArgAspGlulleAsnLysArgGlyArgGluAspArgGluAlaAla ACGTACGCCGACGTACGCGATGAGATCAATAAAAGGGGGCGTGAGGACCGGGAGGCGGCC Inside left

ArgThrAlaValHisAspProGluArgProLeuLeuArgSerProGlyLeuLeuProGlu AGAACCGCCGTGCACGACCCGGAGCGTCCCCTGCTGCGCTCTCCCGGGCTGCTGCCCGAA

11eAlaProAsnAlaSerLeuGlyValAlaHisArgArgThrGlyGlyThrValThrAsp ATCGCCCCCAACGCATCCπGGGTGTGGCACATCGAAGAACCGGCGGGACCGTGACCGAC

SerProArgAsnProValThrArg*** AGTCCCCGTAATCCGGTAACCCGTTGA

Altered Sequence: SEQ ID No: 2

MetSerTrpAlaLeuGluMetAlaAspThrPheLeuAspThrMet*** *** *** ATGTCGTGGGCCCTGGAAATGGCGGACACCπCCTGGACACCATGTAAπGACTAGTCCCAGG

Inside right 3 stop codons

ThrTyrAlaAspValArgAsp ACGTACGCCGACGTACGCGAT Inside left

In order to produce this mutation, 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. 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.

For PCR of the left hand flanks: Outside left GCTTCGAGATCGTAGTGTCCG (SEQ ID No: 3)

Inside left ATGTAATTGACTAGTCCCAGGACGTACGCCGACGTACGC (SEQ ID No: 4) Spel

(approximately 500 base pairs to left of alteration)

For PCR of the right hand flanks:

Inside right GGGACTAGTCAATTACATGGTGTCCAGGAAGGTGTCCGC

(SEQ ID No: 5) Spel

Outside right CGGCTCGGGATCGGGATCGCA (SEQ ID No: 6) (approximately 500 base pairs to the right of alteration)

(Note: the bases indicated by bold lettering (the first 18 bases in each of seq id 4 & 5), denote added or altered bases, and the other bases act as homologous priming sequences for PCR).

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. Recombinant viruses containing the altered sequences, ie the mutated US12 gene, can then be identified by probing with an oligonucleotide sequence homologous to the mutated sequences, namely TAATTGACTAGTC, as described above. 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.

Testing of the vaccine potential of this virus can be carried out as described above.

Construction of an HSV-2 gH-deleted US12-deleted virus

To construct a Herpes simplex virus type-2 with both gH and US12 deleted, a preliminary step is to make a virus lacking in gH. This is described in W094/21807 and a description also follows.

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.

Complementing cell line

A complementing cell line expressing the HS -2 gH gene is obtained using plasmid pIMCOδ, 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 GGCCGCTCGCGAΠATTCGCGTCTCCACAAAA) 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. In order to facilitate further cloning and minimise homology between vector and virus, certain 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 pIMCOβ. 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).

Construction of plasmids to make the gH-deleted mutant DISC HSV-2 Polymerase chain reaction (PCR) of flanking sequences:

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.

The oligonucleotides used are as follows:

HindiII MB97 TCGAAGCπCAGGGAGTGGCGCAGC SEQ ID No: 7

Hpal MB96 TCAGTJAACGGACAGCATGGCCAGGTCAAG SEQ ID No: 8

Hpal MB57 TCAGπAACGCCTCTGπCCTTTCCCπC SEQ ID No: 9

EcoRI

MB58 TCAGAATTCGAGCAGCTCCTCATGπCGAC SEQ ID No:10 Construction of recombination vectors a) First stage recombination vector pIMMB47+ :-

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).

To allow for easy detection of the first stage recombinants, the E.coli beta- galactosidase gene, under the control of the Cytomegalovirus (CMV) immediate early promoter, is inserted into pIMMB45. 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).

b) The second stage recombination vector, PIMMB46:-

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:

EcoRI MB108 TCAG HCGTTCCGGGAGCAGGCGTGGA SEQ ID No:11

MB109 TCAGπAACTGCACTAGTnTAAπAATACGTATGCCGTCCGTCCCGGCTGCCAGTC

Hpal SEQ ID No:12

Construction of recombinant viruses a) First stage 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) and 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.

b) Second stage.

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.

Construction of an HSV-2 gH-deleted US12-deleted virus

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.

In order to delete the equivalent gene in HSV-2, 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.

For example, in order to determine the sequence of the US12 gene the following procedures can be followed.

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. Digestion of clones with enzymes other than Hindlll will allow discrimination between Hindlll J and Hind III K. For example Cortini & Wilkie (J gen Virol 39: 259. 1978) report that the Hindlll K fragment will contain an EcoRI site, wheras the Hindlll J fragment will not. In an alternative mehod of identifying the relevant fragment, 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.

Sequencing of the HS -2 US12-containing clone

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.

Identification of the HSV-2 US12 gene

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.

1985). Any of a large number of sequence analysis and comparison programs can be used for this purpose, for example the programs written by the University of Wisconsin Genetics Group (Devereux et al.. Nucleic Acids Research 12: 387.

1984) or the Lasergene (Trade Mark) package for the Apple Macintosh (DNASTAR Inc). In brief the sequence is to be scanned for open reading frames (ie stretches of DNA potentially encoding proteins). The open reading frames in the HSV-2 sequence are compared to the HSV-1 US12 open reading frame (ORF). either as DNA or as protein. 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*.

Using this sequence information a US12-deleted virus can be constructed by adaptation of the method described above.

The above description specifically concerns the creation of an HSV-1 mutant which is gH-negative and ICP47 (US12)-negative and an HSV-2 mutant which is gH-negative and IE 12-negative.

The examples, options and embodiments described and mentioned herein are for illustrative purposes only, not for limitation. All documents cited herein are hereby incorporated by reference. The invention disclosed herein is susceptible of many modifications and variations readily accessible to the person skilled in the field concerned, in the light of the content of the foregoing description and appended claims, and such modifications and variations, together with combinations and subco binations of the features mentioned herein and in the appended claims and the cited documents, are included within the scope of this disclosure.

Claims

1. A mutant virus which (a) has a genome defect in that it is inactivated in respect of a first gene essential for the production of infectious new virus particles, and (b) is structurally or functionally inactivated in respect of a second gene native to the virus which normally functions to downregulate a host immune response against the corresponding wild-type virus; e.g. in the form of an immunogenic preparation, for example a vaccine for therapeutic or prophylactic use. e.g. together with a pharmaceutically acceptable vehicle.

2. A mutant virus according to claim 1. wherein the defect in the first gene allows the virus to infect normal cells and undergo replication and expression of viral antigen genes in those cells.

3. A mutant virus which (a) has a genome defect in that it is inactivated in respect of a first gene essential for the production of infectious new virus particles, and which carries genetic material encoding an immunogen from a pathogen exogenous to the virus, such that the virus can infect normal eel1s and undergo some replication and expression of the genetic material encoding said immunogen but cannot produce infectious new virus particles, and (b) is structurally or functionally inactivated in respect of a second gene native to the virus which normally functions to downregulate a host immune response against the corresponding wild-type virus.

4. A mutant virus according to claim 1.2 or 3 wherein defect (a) is such that the mutant virus can cause production and release of non-infectious viral particles from normal host cells infected therewith.

5. A mutant virus according to any preceding claim which is non- retroviral, e.g. a mutant of a double-stranded DNA virus; e.g. a mutant herpesvirus or adenovirus.

6. A mutant virus according to any of claims 1 to 4 which is a mutant of a herpesvirus. for example HSV. e.g. HSV1 or HSV2.

7. A mutant virus according to claim 6 wherein the defect (a) is in respect of a gene encoding an essential viral glycoprotein. e.g. in the viral gH gene.

8. A mutant virus according to any preceding claim wherein the second gene is the ICP47 gene or a functional equivalent, e.g. a herpesviral gene with substantial homology with the ICP47 gene, or the IE12 gene or a herpesviral gene with substantial homology with the IE12 gene.

9. A mutant virus according to any preceding claim which is capable in a species infected therewith of establishing a latent infection with periodic reativation, leading to production of proteins encoded by the virus in the latently infected cell.

10. A mutant virus according to any preceding claim in which defects (a) and (b) each comprise deletion of the viral gene concerned.

11. A mutant virus according to any one of claims 1 to 9 wherein the second gene is inactivated by coexpression from the virus genome of an RNA molecule that is at least in part complementary to the mRNA sequence of said second gene, said RNA molecule being of sufficient length to inhibit translation of the said mRNA.

12. Use of a mutant virus according to any preceding claim in the preparation of an immunogen, for example a vaccine, for therapeutic or prophylactic use in the production of an immune response against the virus or an antigen encoded thereby.

13. Use of a mutant virus according to any of claims 1 to 11 in the priming or stimulation of cytotoxic T cells in vitro.