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• • A—HUMAN NECESSITIES
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  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
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  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
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  • C12N2710/16011—Herpesviridae
  • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
  • C12N2710/16661—Methods of inactivation or attenuation
  • C12N2710/16662—Methods of inactivation or attenuation by genetic engineering

Definitions

• Viral vaccines are traditionally of two sorts.
• the first sort are 'killed' vaccines, which are virus preparations which have been killed by treatment with a suitable chemical such as beta-propriolactone.
• the second type are live 'attenuated' vaccines, which are viruses which have been rendered less pathogenic to the host, either by specific genetic manipulation of the virus genome, or, more usually, by passage in some type of tissue culture system.
• kill vaccines do not replicate in the host, they are usually administered by injection, and hence may generate an inappropriate kind of immune response.
• the Salk vaccine a killed preparation of poliovirus, produces an imunoglobulin (Ig) G antibody response, but does not stimulate the production of IgA in the gut, the natural site of primary infection.
• Ig imunoglobulin
• this vaccine though it can protect the individual from the neurological complications of poliomyelitis, does not block primary infection, and so does not confer "herd immunity”.
• killed viruses do not enter and replicate inside host cells. Hence any beneficial immunological response to non-structural proteins produced during replication is not available. They also often fail to stimulate the production of cytotoxic T cells directed against virus antigens. "Dead" antigens can be picked up by antigen presenting cells and presented to T cells.
• the presentation occurs via MHC Class II molecules and leads to stimulation of T helper cells.
• the T helper cells help B cells to produce specific antibody against the antigen.
• virus antigens In order to stimulate the production of cytotoxic T cells, virus antigens must be processed through a particular pathway inside the infected cell, and presented as broken-up peptide fragments on MHC Class I molecules. This degradation pathway is thought to work most effectively for proteins that are synthesised inside the infected cell, and hence only virus that enters host cells and expresses immunogenic viral protein is capable of generating virus-specific cytotoxic T cells. Therefore, killed vaccines are poor inducers of cytotoxic T cells against virus infection. From this point of view, live attenuated vaccines are more satisfactory.
• live attenuated viruses have been made by deleting an inessential gene or partly damaging one or more essential genes (in which case, the damage is such that the genes are still functional, but do not operate so effectively).
• live attenuated viruses' often retain residual pathogenicity which can have a deleterious effect on the host.
• the attenuation is caused by a specific deletion, there remains the possibility of reversion to a more virulent form. Nevertheless, the fact that some viral protein production occurs in the host means that they are often more effective than killed vaccines which cannot produce such viral protein.
• Live attenuated viruses can also be used as 'vaccine vectors' for other genes, in other words carriers of genes from a second virus (or other pathogen) against which protection is required.
• members of the pox virus family eg. vaccinia virus, are used as vaccine vectors.
• vaccinia virus members of the pox virus family
• adenoviruses For adenoviruses, a human cell line was transformed with fragments of adenovirus type 5 DNA (Graham, Smiley, Russell & Nairn, J. Gen. Virol., 36, 59- 72, 1977). The cell line expressed certain viral genes, and it was found that it could support the growth of virus mutants which had those genes deleted or inactivated
• WO92/05263 published on 2 April 1992 provides a mutant non-retroviral virus whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect normal cells and undergo replication and expression of viral antigen genes in those cells but cannot produce normal infectious virus.
• Mutant non-retroviral viruses in accordance with the teaching of WO92/05263 provide a unique way of combining the efficacy and safety of a killed vaccine with the extra immunological response induced by the in vivo production of viral protein by the attenuated vaccine.
• the invention of WO92/05263 comprises two features. Firstly, a selected gene is inactivated within the virus genome, usually by creating a specific deletion.
• This gene will be involved in the production of infectious virus, but preferably not preventing replication of the viral genome.
• the infected cell can produce more viral protein from the replicated genetic material, and in some cases new virus particles may be produced, but these would not be infectious. This means that the viral infection cannot spread from the site of inoculation.
• a second feature of the invention of WO92/05263 is a cell which provides the virus with the product of the deleted gene, thus making it possible to grow the virus in tissue culture.
• the virus lacks a gene encoding an essential protein, if it is grown in the appropriate host cell, it will multiply and produce complete virus particles which are to outward appearances indistinguishable from the original virus.
• This mutant virus preparation is inactive in the sense that it has a defective genome and cannot produce infectious virus in a normal host, and so may be administered safely in the quantity required to generate directly a humoral response in the host.
• the mutant virus need not be infectious for the cells of the host to be protected and merely operates in much the same way as a conventional killed or attenuated virus vaccine.
• the immunising virus is itself still infectious, in the sense that it can bind to a cell, enter it, and initiate the viral replication cycle and is therefore capable of initiating an infection within a host cell of the species to be protected, and producing therein some virus antigen.
• the immunising virus is itself still infectious, in the sense that it can bind to a cell, enter it, and initiate the viral replication cycle and is therefore capable of initiating an infection within a host cell of the species to be protected, and producing therein some virus antigen.
• WO92/05263 provided in vivo data which showed that intra-epithelial vaccination of mice via the ear with a mutant form (as described above) of HSV-1 gave better protection against later challenge with wild-type HSV-1, than similar vaccination with killed HSV-1.
• a clear protective effect against the establishment of latent infection in the cervical ganglia was also shown for vaccination with the mutant HSV-1.
• mice with DISC HSV-2 provided better protection against replication of the challenge virus w.t. HSV-2 than inactivated DISC HSV-2.
• the present invention provides a pharmaceutical mutant which comprises a mutant non-retroviral virus whose genome is defective in respect of a gene essential for the production of infectious virus such that the virus can infect normal cells and undergo replication and expression of viral antigen genes in those cells but cannot produce normal infectious virus, for prophylactic or therapeutic use in generating an immune response in a subject infected therewith.
• the defect mdy allow the production and release from the cells of non-infectious viral particles.
• the present invention provides a pharmaceutical which comprises a mutant non-retroviral virus whose genome is defective in respect of a gene essential for the production of infectious virus such that the virus can infect normal cells and replicate therein to give rise to the production and release from the cells of non-infectious viral particles.
• the pharmaceutical may be a vaccine capable of protecting a patient immunised therewith against infection or the consequences of infection by a non-retroviral virus.
• the pharmaceutical may be a vaccine capable of protecting a patient immunised therewith against infection or the consequences of infection by the corresponding wild-type virus.
• the pharmaceutical may be a therapeutic capable of treating a patient with an established non-retiroviral virus infection.
• the pharmaceutical may be a therapeutic capable of treating a patient with an infection established by the corresponding wild-type virus.
• the pharmaceutical may be sub-cutaneously, intra ⁇ muscularly, intra-dermally, epithelially-, (with or without scarification), nasally-, vaginally-, or orally- administrable comprising excipients suitable for the selected administration route.
• the mutant may be from a double-stranded DNA virus.
• the mutant may be from a herpes virus.
• the mutant may be from a herpes simplex virus (HSV).
• the mutant may be a type-1 HSV or a type-2 HSV.
• the defect may be in the glycoprotein gH gene.
• the present invention provides a type-2 HSV whose genome is defective in respect of a gene essential for the production of infectious HSV-2 such that the virus can infect normal cells and undergo replication and expression of viral antigens in those cells but cannot produce normal infectious virus, for prophylactic or therapeutic use in generating an immune response in a subject infected with HSV eg HSV-2.
• the mutant HSV-2 defect allows the production and release from the cells of non-infectious virus particles. Also provided is a type-2 HSV whose genome is defective in respect of a gene essential for the production of infectious HSV-2 such that the virus can infect normal cells and replicate therein to give rise to the production and release from the cells of non-infectious viral particles.
• the mutant may be capable of protecting a patient immunised therewith against infection or the consequences of infection with HSV eg infection by the corresponding wild-type virus.
• the mutant may be capable of treating a patient with an established HSV infection eg infection by the corresponding wild-type virus.
• the defect may be in the glycoprotein gH gene.
• the present invention also provides use of a mutant type-1 HSV whose genome is defective in respect of a gene essential for the production of HSV-1 such that the virus can infect normal cells and undergo replication and expression of viral antigen genes in those cells but cannot produce normal infectious virus, for preparation of a pharmaceutical for prophylactic or therapeutic use in generating an immune response in a subject against type-2 HSV infection.
• the use may be in respect of pharmaceuticals for intra-epithelial (with or without scarification), intra- vaginal, intra-nasal or per-oral administration.
• the present invention also provides an assembly comprising a pharmaceutical (for prophylaxis ie a vaccine or for therapy ie a therapeutic) as described above in a container preferably a pre-filled syringe or glass vial/ampoule with printed instructions on or accompanying the container concerning the administration of the pharmaceutical to a patient to prevent or treat conditions caused by HSV infection.
• the printed instructions may concern the prevention or treatment of facial or genital lesions.
• Vaccines containing the mutants as described can be prepared in accordance with methods well known in the art wherein the mutant is combined in admixture with a suitable vehicle.
• suitable vehicles include, for example, saline solutions, or other additives recognised in the art for use in compositions applied to prevent viral infections.
• Such vaccines will contain an effective amount of the mutant as hereby provided and a suitable amount of vehicle in order to prepare a vaccine useful for effective administration to the host.
• Dosage rates can be determined according to known methods. For example, dosage rate may be determined by measuring the optimum amount of antibodies directed against a mutant resulting from administration of varying amounts of the mutant in vaccine preparations. Attention is directed to New Trends and Developments in Vaccines,
• Therapeutics comprising a mutant as herein provided can be formulated according to known methods to provide therapeutically useful compositions, whereby the mutant is combined in admixture with a pharmaceutically acceptable carrier vehicle.
• a pharmaceutically acceptable carrier vehicle Suitable vehicles and their formulation are described in Remington's Pharmaceutical Science by E. W. Martin.
• Such compositions will contain an effective amount of the mutant hereof together with a suitable amount of carrier vehicle in order to prepare therapeutically acceptable compositions suitable for effective administration to the host.
• vaccines are prepared as injectables,
• the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, trehalose, or the like and combinations thereof.
• the vaccine may contain minor amounts of auxiliary substances such as other stabilisers and/or pH buffering agents, which enhance the stability and thus the effectiveness of the vaccine.
• the vaccines may be administered parenterally, by injection, for example, subcutaneously, intraepithelially (with or without scarification). Additional formulations which are suitable for other modes of administration eg oral, vaginal and nasal formulations are also provided.
• Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of trehalose mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
• the compositions may take the form of solutions, suspensions, tablets, pills, capsules sustained release formulations or powders.
• the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically effective.
• the quantity to be administered will have been predetermined from preclinical and clinical ( phase I ) studies to provide the optimum immunological response.
• the vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule.
• a multiple dose schedule is one in which a primary course of vaccination may be with 1-3 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
• the dosage regimen will also, have been determined from preclinical and clinical studies as maintaining the optimum immunological response over time.
• Figure 1 illustrates the DISC virus concept.
• Figure 2 shows clearance of wild-type HSV-1 (w.t. HSV- 1) strain SCI6 virus in the ears of mice vaccinated with either live DISC HSV-1 or inactivated ( ⁇ -propiolactone treated) w.t. HSV-1 (strain SC16). Groups of 4 mice were vaccinated at the doses indicated by scarification of the left ear pinna. Mice were challenged 14 days post- vaccination with 2x10° pfu w.t. HSV-1 strain SC16 in the right ear pinna and virus titres were measured 5 days post challenge. Data are expressed as the geometric means and standard errors of the means.
• Figure 3 shows measurement of titres of neutralising and ELISA antibody to w.t. HSV-1 in mice vaccinated with either w.t. HSV-1 (strain SC16), live DISC HSV-1, killed DISC HSV-1 or PBS. Sera from mice were assayed in the presence of complement for neutralising antibodies to w.t. HSV-1 in a plaque reduction assay. Individual titres are expressed as the reciprocal dilution of sera required to neutralise 50% of the infectivity obtained in the absence of antibody.
• FIG. 4 shows delayed-type hypersensitivity (DTH) responses in mice vaccinated with either w.t. HSV-1 (strain SC16), live DISC HSV-1, killed DISC HSV-1 or PBS.
• Mice were vaccinated in the left ear pinna at the doses indicated 14 days prior to challenge with 10 6 pfu w.t. HSV-1 (strain SC16) in the opposite ear.
• Ear thickness was measured 24 and 48 hours post-challenge and is expressed as the difference between the challenged and vaccinated ear. Data are presented as the means of differences in ear thickness (in ⁇ m) .
• FIG. 5 shows cytotoxic T cell (CTL) responses in mice vaccinated with either live DISC HSV-1, killed DISC HSV-1, MDK (a thymidine kinase negative HSV-1 strain) or PBS.
• CTL cytotoxic T cell
• Mice were immunised twice intraperitoneally three weeks apart and cell suspensions made from spleens 10 days after the second injection. Cells were stimulated in vitro for 4 days before being tested in a CTL assay using 51 Cr- labelled A20/2J as target cells. Data are presented as mean % 51 Cr release from quadruplicate samples at each point. Standard errors of the means are all ⁇ 10%.
• Figure 6 shows clinical symptoms as assessed by erythema score in guinea-pigs post challenge with 10 52 pfu w.t. HSV-2 (strain MS) subsequent to vaccination with doses of 2 x 10 7 pfu DISC HSV-1 at a 3 week interval either by the intra-epithelial or the intra-vaginal route;
• Figure 7 shows clinical symptoms as assessed by total lesion score in guinea-pigs post challenge with 10 5,2 pfu w.t. HSV-2 (strain MS) subsequent to vaccination with doses of 2 x 10 7 pfu DISC HSV-1 at a 3 week interval either by the intra-epithelial or the intra-vaginal route.
• Figure 8 shows post challenge virus w.t. HSV-2 (strain MS) subsequent to vaccination with doses of 2 x 10 7 pfu DISC HSV-1 at a 3 week interval either by the intra-epithelial or the intra-vaginal route.
• Figure 8 shows post
• FIG. 9a shows recurrent disease as the cumulative mean erythema index per animal.
• Fig. 9b shows recurrent disease as cumulative mean number of days with disease per animal.
• Figure 10 shows mean lesion score per animal (guinea- pigs) with w.t. HSV-2 (strain MS) infection and which have been vaccinated via the vaginal, oral or nasal routes with a mock virus preparation, DISC HSV-1 or inactivated DISC HSV-1.
• Figure 11 shows mean erythema score per animal (guinea-pigs) with w.t. HSV-2 (strain MS) infection and which have been vaccinated via the vaginal, oral or nasal routes with a mock virus preparation, DISC HSV-1 or inactivated DISC HSV-1.
• Figure 12 shows the mean log titre of w.t. HSV-2
• strain MS per animal (guinea-pigs) with w.t. HSV-2 (strain MS) infection and which have been vaccinated via the vaginal, oral or nasal routes with a mock virus preparation, DISC HSV-1 or inactivated DISC HSV-1.
• Figure 13 shows recurrent disease following therapeutic vaccination. This is shown as mean cumulative number of days on which disease was observed (disease/days) in groups of guinea-pigs vaccinated with DISC HSV-1 either intra-epithelially or intra-vaginally or with a mock virus preparation intra-vaginally after challenge with w.t. HSV-2 (strain MS). Disease was classified as either presence of one or more lesions or an erythema score of 1 or more.
• Animals were monitored from 4 weeks after initial challenge with w.t. HSV-2 (strain MS) (day o) for 100 days. Animals were vaccinated at Day 0, Day 24 and Day 44 with 2 x 10 7 pfu or equivalent dose as indicated.
• Figure 14 relates to the long-term protective effect in mice of vaccination with DISC HSV-1 against challenge with w.t. HSV-1 (strain SC16).
• the graph shows the mean log titre of w.t. HSV-1 in the ears 5 days post challenge and 223 days post vaccination.
• Figure 15 relates to the long-term protective effect in mice of vaccination with DISC HSV-1 against challenge with w.t. HSV-1 (strain SC16).
• the graph shows neutralising antibody titres days 15, 27, 90, 152 and 218 post vaccination as stated.
• Figure 16 relates to the protective effect in mice of vaccination with DISC HSV-2 against challenge with w.t.
• HSV-2 (strain HG52 ) for vaccinations with live DISC HSV-2, killed DISC HSV-2 and w.t. HSV-2 (strain HG52) at varying doses
• the graph shows mean log titre of w.t. HSV-2 in the ear post challenge.
• Figure 17 illustrates the construction of a single plas id containing the complete HSV-2 gH gene.
• Figure 18 shows the sequence of HSV-2 strain 25766 in the region of the gH gene including a translation of the gH gene in single letter amino acid code.
• Figure 19 shows a comparison of the DNA sequence of
• HSV-1 and HSV-2 strain 25766 in the region of the gH gene are HSV-1 and HSV-2 strain 25766 in the region of the gH gene.
• Figure 20 shows a comparison of the deduced amino acid sequences of the HSV-1 strain 17 and HSV-2 strain 25766 gH proteins.
• Figure 21 shows graphically the level of similarity between the DNA sequences of HSV-1 and HSV-2 in the region of the gH gene (from U GCG program Plotsimilarity) .
• Figure 22 shows graphically the level of similarity between the amino acid sequences of the HSV-1 and HSV-2 gH proteins (from UWGCG program Plotsimilarity).
• Figure 23 shows the construction of pIMMB26; two fragments from the left and right sides of the HSV2 gH gene were amplified by PCR and cloned into pUC119. The four oligonucleotides MB57, MB58, MB59 and MB60 are shown.
• Figure 24 shows the construction of pIMMB45.
• Figure 25 shows construction of the first stage recombination vector pIMMB47+.
• Figure 26 shows construction of the second stage recombination vector pIMMB46.
• Figure 27 shows a restriction map analysis for recombinants HG52-D, TK minus DISC virus, TK plus DISC virus.
• Figure 28 shows Southern blots of BamHI digestions of various viruses, probed with the right-hand flanking sequence as shown in Fig. 27. Lane 5: HG52-D virus, lane 2: TK-minus "first stage” DISC virus and lanes 3, 4, 6, 7 and 8: TK-plus "second stage” DISC viruses.
• Herpes Simplex Virus Deleted in Glycoprotein H (qH-HSV)
• Herpes simplex virus is a large DNA virus which causes a wide range of pathogenic symptoms in man, including recurrent facial and genital lesions', and a rare though often fatal encephalitis. In general, it seems that type 1 HSV (HSV-1) seems to be particularly associated with facial lesions, whilst type 2 HSV (HSV-2) seems to be particularly associated with genital lesions. To some extent infection with HSV can be controlled by chemotherapy using the drug Acyclovir, but as yet there is no vaccine available to prevent primary infection or the consequences of this infection. Thus there is a need both for better therapeutics to treat established HSV infections and for prophylactics to prevent the establishment of HSV infection and/or its associated pathology.
• a difficulty with vaccination against HSV is that the virus generally spreads within the body by direct transfer from cell to cell. Thus humoral immunity is unlikely to be effective, since circulating antibody can only neutralise extracellular virus. Of more importance for the control of virus infection, is cellular immunity, and so a vaccine which is capable of generating both humoral and cellular immunity, but which is also safe, would be a considerable advantage.
• a suitable target gene for inactivation within the HSV genome is the glycoprotein H gene (gH) .
• the gH protein is a glycoprotein which is present on the surface of the virus envelope. This protein is thought to be involved in the process of membrane fusion during entry of the virus into the infected cell. This is because temperature sensitive virus mutants with a lesion in this gene are not excreted from virus infected cells at the non-permissive temperature (Desai et al. , J. Gen. Virol. 69, 1147-1156, 1988). The protein is expressed late in infection, and so in its absence, a considerable amount of virus protein synthesis may still occur.
• HSV2 Herpes Simplex type 2
• pTW49 is the BamHl R fragment of
• pTW54 is the BamHl S Fragment of HSV2 strain 25766 cloned into pBR322.
• the construction of a single plasmid containing the complete gH gene is shown in Figure 17.
• pTW49 was digested with BamHl and Sail, and an 870 base pair (bp) fragment isolated from an agarose gel.
• pTW54 was digested with BamHl and Kpnl and a 2620bp fragment isolated from an agarose gel. The two fragments were ligated together with the plasmid pUC119 cut with Sail and Kpnl, resulting in the plasmid pIMMB24.
• pIMMB24 was digested with Sail and Kpnl.
• the plasmid was digested with Dral (which cuts in the vector sequences), to aid in isolation of the 3490bp insert.
• the 3490bp insert containing the HSV2 sequences was purified from an agarose gel. It was then sonicated, the ends repaired using T4 DNA polymerase and Klenow, and size fractionated on an agarose gel. A fraction containing DNA molecules of approximately 300-600bp in length was ligated into M13mpll cut with S al (Amersham International UK). The ligated mixture was transformed into E.coli strain TGI, and individual plaques were picked. Single- stranded DNA was made from each plaque picked, -and was sequenced using the dideoxy method of sequencing, either with Klenow enzyme or with Sequenase, and using 35 S dATP.
• sequence data was also obtained by sequencing directly from the pIMMB24 plasmid using oligonucleotide primers designed from sequence already obtained. In order to obtain sequence from regions flanking the gH gene, some sequence information was also obtained from the plasmid pTW49. Because of the high G+C ratio of HSV2 DNA, there were several sequence interpretation problems due to 'compressions' on the gels. These have yet to be resolved. In a small number of places therefore, the present sequence represents the best guess as to what the correct sequence is, based on comparisons with the previously published HSVl sequence.
• Oligonucleotides MB57, MB58, MB59 and MB75 were designed to isolate and clone the regions of sequence flanking the HSV2 gH gene. As shown in Figure 23, the oligonucleotides were used in a polymerase chain reaction (PCR) to amplify fragments of DNA from either side of the gene. Restriction sites were included in the oligonucleotides so that the resultant fragments contained these sites at their ends, enabled cloning of the fragments into a suitably cut plasmid. The following oligonucleotides, based on the HSV2 sequence, were used for this purpose:
• such a plasmid allows the skilled person to produce a defective HSV-2 virus lacking precisely the sequences for the gH gene (see below). If these same sequences are cloned into a suitable cell carrying a copy of the gH gene deleted from the HSV-2 genome, this 'complementing cell' can then support the growth of the defective HSV-2 virus by providing the gH protein. Because the sequences have been chosen so that there is no overlap between the sequences in the cell and the sequences in the virus, the possibility of the virus acquiring the gene from the cell by recombination is virtually eliminated.
• HSV-1 gH gene F6 cells, Forrester et al, Journal of Virology, 1992, 66, p. 341-348
• F6 cells Forrester et al, Journal of Virology, 1992, 66, p. 341-348
• CR1 cells use the same promoter and gH gene as F6 cells, but the sequences downstream of the gene are truncated so that there is no overlap of sequences between the final DISC virus and the cell line. This is very useful since it means that homologous recombination cannot occur between the DISC virus and the cell line DNA.
• wild-type gH-plus viruses occur by recombination at about 1 in 10 b viruses.
• Another cell line, CR2 was also made, which expresses the gH gene from the HSV-2 strain 25766. This also supports the growth of a DISC HSV-2 and also has no overlapping sequences between the virus and the cell.
• Viral DNA is purified from virus by standard methods. Flanking sequences to either side of the gH gene are amplified by PCR using Vent DNA polymerase (New England Biolabs ) which has a lower error rate than Taq DNA polymerase (see Fig. 24).
• 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 restor 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 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 which has been digested with Hindlll and EcoRI.
• the resultant plasmid is called pIMMB45 (see Fig. 24).
• 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 a suitable plasmid carrying the promoter and gene using one or mote appropriate restriction enzymes. If necessary, the ends are filled in using the Klenow fragment of DNA polymerase. This is the approach taken by the present applicants. However alternative methodologies will be apparent to those skilled in the art.
• the beta-galactosidase gene may be under the control of the SV40 promoter, in which case, the gene and promoter can be excised from the plasmid pCHHO (Pharmacia PL Biochemicals ) using BamHl and Tthllll, and the ends are filled in using the Klenow fragment of DNA polymerase ( Ecob-Prince, M.S., et al 1993 J. Gen. Virol, 74, p. 985-994). 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. 25).
• 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 Hindlll 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
• the resultant plasmid is called pIIMB46 (see Fig. 26).
• the oligonucleotides used are as follows:
• Virus DNA was made from strain HG52-D, which is a plaque-purified isolate of the HSV-2 strain HG52.
• Virus DNA (2.5 ⁇ g) and pIMMB47+ plasmid DNA (0.25 ⁇ g ) was transtected into CR1 cells u ing the CuP0 precipitation method (Chen & Okayama, Molecular and Cellular Biology, 7, p. 2745). Recombination takes place within the cells, and a mixture of recombinant and wild type virus is produced. The mixture was plaque-purified three times on CRl cells in the presence of acyclovir ( 10 ⁇ g/ml ) , to select for TK- minus virus. A single plaque was then grown up and analysed.
• the virus was titrated on normal Vero cells and on CRl cells. If the virus is a gH-deleted virus, it should only grow on CRl cells and not on Vero cells. Table 1 shows that this is the case. It can be seen that the virus does not grow at all on the non-complementing Vero cells even at the highest virus concentrations, but does grow well on the CRl complementing cell line, which expresses the HSV-1 gH gene. The virus also grows well on CR2 cells which express the HSV-2 gH gene (data not shown).
• Table 1 growth of first stage recombinant virus on complementing (CRl) and non-complementing (Vero) cells.
• TK-minus DISC virus DNA was made from this TK-minus DISC virus and a recombination was carried out as above with the plasmid pIMMB46.
• TK-plus recombinants were selected, on a gH-expressing TK-minus BHK cell line, by growth in medium containing ⁇ iethotrexate, thymidine, glycine, adenosine and guanosine.
• Virus was harvested and grown again under selective conditions twice more before a final plaque purification was carried out on CRl. Virus was grown up and analysed by Southern blotting.
• Virus DNA from the original HG52-D, the TK-minus DISC virus, and the TK- plus DISC virus were digested with BamHl and separated on an agarose gel. The DNA bands were then transferred to nylon membrane by the Southern blotting method, and probed with radiolabelled fragments from the right hand flanking sequences.
• Fig. 27 shows the structures of these viruses, with the expected band sizes after BamHl digestion. The probe used is marked as 'R' beneath a dashed line. The probe should hybridise to a different size band in each of these viruses, as follows:
• Fig. 28 shows that this is the case.
• Lane 5 shows the HG52-D virus
• Lane 2 contains the TK-minus "first stage” DISC virus
• lanes 3, 4, 6, 7 and 8 contain TK-plus "second stage” DISC viruses. This confirms that the DNA structure in each of these viruses is as expected.
• the present application refers to certain strains of HSV-1 and HSV-2. It is not necessary that the general teaching contained herein is put into effect with precisely the mentioned strains. Strains of HSV-1 and HSV-2 having high sequence homology to one another by which the invention may be put into effect are readily available. For example, one source of HSV is the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA. The following are available from ATCC under the indicated accession numbers.
• ATCC American Type Culture Collection
• mice 4-5 week old BALB/c mice were vaccinated with varying doses of DISC HSV-1 or inactivated virus by scarification in the left ear pinna. Virus was inactivated using ⁇ - propiolactone (for further details see WO92/05263 published on 2 April 1992 and incorporated herein by reference).
• the mice were challenged with 2 x 10 6 pfu w.t. HSV-1 (strain SC16) in the opposite ear two weeks after vaccination. The amount of virus present in that ear 5 days post challenge was assayed by plaquing on BHK cells. (See Fig. 2. )
• mice were vaccinated with 5xl0 5 pfu of DISC HSV-1, killed DISC HSV-1, w.t. HSV-1 (strain SC16) or with PBS and serum samples taken at 2 and 14 weeks post vaccination.
• Neutralising antibodies were measured in the presence of complement and expressed as the inverse of the serum dilution which reduced the number of plaques by 50%.
• ELISA antibody titres were measured on plates coated with HSV-1 infected BHK cell lysates and titrated to endpoint. ( See Fig. 3. ) It can be seen from Fig. 3 that no significant differences in antibody titres were observed between animals vaccinated with DISC HSV-1 and an equivalent amount of killed DISC HSV-1.
• the importance of a DTH response in protection against herpes virus infection has been well documented.
• the ability of the DISC HSV-1 to raise a DTH response was investigated by vaccinating groups of mice with DISC HSV-1, killed DISC HSV-1, and live w.t. HSV-1, by scarification of the left ear pinna.
• the DTH responses induced by different doses of the various vaccine preparations thus correlate with their protective effect against challenge virus replication.
• the efficacy of vaccination with low doses of the DISC HSV-1 vaccine may therefore be due to the induction of T cell- mediated immunity.
• Cytotoxic T cells have been shown to be involved in the protection against, and recovery from, primary HSV infection.
• DISC HSV-1 vaccinated mice were therefore studied for the presence of HSV-1 specific cytotoxic T cell activity.
• Cytotoxic T cell activity following immunisation was generated and assayed according to standard procedures eg as exemplified in Martin, S. et al, 1988, J. Virol. 62:
• mice 2265-2273 and Gallichan, W.S. et al, J. Infect. Dis. 168: 633-629. More specifically, groups of female BALB/c mice were immunised intra-peritoneally with 2 x 10 7 pfu of virus (DISC HSV-1; killed DISC HSV-1; MDK a thymidinfe kinase negative HSV-1 strain) on day 0 and the immunisations repeated (same dose and route) after 3 weeks. A group of control mice received 0.1ml of PBS intraperitoneally at the same time points. Ten days after the second immunisation the spleens of the mice were removed and pooled for each group.
• DISC HSV-1 killed DISC HSV-1
• MDK a thymidinfe kinase negative HSV-1 strain
• Spleens were also removed from unim unised BALB/c mice for the preparation of feeder cells ( 16 feeder spleens being sufficient for 4 groups of six effector spleens). All subsequent steps were performed in a laminar flow hood using aseptic technique.
• the spleens were passed through a sterile tea-strainer to produce a single cell suspension in RPMI 1640 medium supplemented with 10% heat inactivated foetal calf serum (effector medium). Debris was allowed to settle and the single cell suspension was transferred to a fresh container.
• the cell suspensions were washed twice in effector medium (1100 rpm, 10 minutes) and then passed through sterile gauze to remove all clumps. The effector spleen cell suspensions were then stored on ice until required.
• Feeder spleen cells were resuspended to 1 x 10 7 cells/ml in effector medium and mitomycin C was added to a final concentration of 20 ⁇ g/ml. The feeder cells were incubated at 37 ⁇ C for 1 hour. Feeder cells were washed four-times in PBS supplemented with 1% FCS and once in PBS with no protein. Live virus ( DK) was added to the mitomycin C treated feeder cell pellet at a concentration of 3 pfu of virus per spleen cell. Following a one hour incubation at 37°C the feeder cells were washed once with effector cell medium.
• DK Live virus
• Effector cells were resuspended to 5 x 10 6 cells/ml, whilst feeder cells were resuspended to 2.5 x 10 6 cells/ml.
• 500 ⁇ l of effector cell suspension and 500 ⁇ l feeder cell suspension were added to the wells of a 24 well plate. The plates were incubated in a humid atmosphere at 37°C (5% C0 2 ) for 4 days.
• the effector and feeder cells were harvested from the 24 well plate.
• the cells were spun down once and the pellet resuspended in effector medium ( 5ml of medium per 2 plates ) .
• the cell suspension was layered onto lymphocyte separation medium and spun at 2500rpm for 20 minutes.
• the live effector cells were harvested from the interface and washed twice, once at 1500 rpm for 15 minutes and once at 1100 rpm for 10 minutes.
• the effector cells were finally resuspended at the required concentration in effector medium and stored on ice until required.
• Uninfected syngeneic A202J target cells A20/2J cells were harvested from tissue culture flasks; 2 x 10 7 cells were added to each of 2 containers (to become infected and uninfected targets ) . The cells were washed with DMEM (with no additions) . To the infected cells live MDK virus was added at lOpfu per cell and an equivalent volume of EMEM was added to the uninfected cells. One mCi of 51Cr was added to each of the universals and the cells were incubated at 37 ⁇ C (in a waterbath) for 1 hour. The target cells were then washed three times (10 minutes, llOOrpm) in target medium (DMEM supplemented with 10% FCS ) and finally resuspended to the required cell concentration in target cell medium.
• target medium DMEM supplemented with 10% FCS
• Both uninfected and infected target cells were resuspended to 1 x 10° cells/ml and 1 x 10 b cells/ml and lOO ⁇ l ( ie to give 1 x 10 s targets/well and 1 x 10 4 targets/well respectively) was plated out into the appropriate wells of a round bottomed 96 well plate. All experimental points were set up in quadruplicate. Each effector cell type was resuspended to 8 x 10 fa cells/ml in effector medium and two-fold dilutions were prepared.
• effector to target ratios were: 8:1, 4:1, 2:1 and 1:1.
• effector to target ratios were 80:1, 40:1, 20:1 and 10:1.
• Maximum chromium release for each target cell type was obtained by adding lOO ⁇ l of 20% Triton X-100 to wells containing target cells only ( ie no effectors).
• the spontaneous release for each target cell type was obtained by the addition of lOO ⁇ l effector cell medium to wells containing target cells only.
• the plates were incubated at 37 ⁇ C for four hours in a humid atmosphere. After this time the plates were spun for four minutes at 1500rpm and lOO ⁇ l of supernatant was removed from each of the wells. The supernatant was transferred to LP2 tubes and radioactivity contained in the tubes was then counted for 1 minute on a gamma' counter. The % specific chromium release was determined using the formula
• HSV-1 specific CTL activity comparable to that produced by infection with the fully replicative MDK virus.
• HSV-1 specific CTL activity was observed in mice immunised with killed DISC HSV-1 or in PBS treated animals, although some non ⁇ specific killing was observed in these animals. The reason for this is not clear, but it could represent a high level of NK cell activity.
• Vaccination of mice with the DISC HSV-1 has thus been shown to induce antibody, CTL and DTH activity against HSV- 1 virus antigens.
• the ability to activate both humoral and cell-mediated immune responses against a broad spectrum of virus proteins may explain the effectiveness of the DISC virus vaccination. LONG-TERM PROTECTION
• the in vivo mouse ear model was used to study long term prophylactic effect of DISC HSV-1
• mice 4-5 week old BALB/c mice were divided into groups containing 6 animals each. The groups were vaccinated as follows:
• Fig. 15 shows the neutralising antibody titres induced by the various vaccinations. This shows that since 2 doses of DISC HSV-1 produce the same titre as two doses of the inactivated DISC HSV-1, the protective effect of DISC HSV-1 cannot be simply explained by antibody induction.
• PROPHYLACTIC EFFECT OF DISC HSV-2 The in vivo mouse ear model was used to study the prophylactic effect of DISC HSV-2.
• mice Six week old BALB/c mice were divided into groups. They were immunised by scarification of the left ear pinna as follows. Group Vaccination Material and Dose
• the DISC HSV-2 was a gH deletion mutant of strain HG52 Three weeks later, all groups were challenged by scarification of the right ear pinna with 5 x 10 4 of w.t. HSV-2 (strain HG52 ) .
• the amount of virus present in the challenged ear (right) 5 days post challenge was assayed by plaquing on BHK cells (see Fig. 16).
• the results as depicted by the figure show that vaccination with DISC HSV-2 at doses of 5 x 10 3 , 5 x 10 4 and 5 x 10 5 pfu provides good protection against challenge with w.t. HSV-2 (strain HG52 ) compared to killed DISC HSV-2.
• better protection was obtained with w.t. HSV-2 at doses of 5 x 10 4 and 5 x lO 5 pfu, but the use of normal live wild type viruses as vaccines is undesirable.
• HSV-2 appears to be closely associated with genital lesions.
• the guinea pig currently provides the best animal model for primary and recurrent genital disease in humans (Stanberry, L.R. et al. J. Inf. Dis. 1982, 146, 397-404). Therefore the applicants have extended the earlier described mouse studies to the guinea pig vaginal model of HSV-2 infection which provides a useful system to assess the i munogenicity of candidate vaccines against genital HSV-2 infection in humans. It permits a comprehensive assessment of primary clinical symptoms following intra- vaginal challenge with HSV-2, and also analysis of the frequency of subsequent recurrences.
• a control group of 21 animals was vaccinated intra-vaginally with a mock virus preparation and a further group of 14 animals was vaccinated intra-epithelially with two equivalent doses of ⁇ -propiolactone-inactivated w.t. HSV-1.
• Vaccinated animals were challenged 3 weeks later with 10 5*2 pfu w.t. HSV-2 virus (strain MS) and monitored for the symptoms of primary and recurrent disease.
• strain MS pfu w.t. HSV-2 virus
• animals were assessed daily over a two week period for symptoms of primary infection.
• Clinical lesions were scored as a direct numerical value, and erythema was scored on a scale of 1-5.
• the vaginal area was also measured as an index of oedema (data not shown). The results are shown in Figs. 6 and 7. Points on the graphs represent mean erythema score per animal per day (Fig. 6) and mean total lesion score per day per animal (Fig. 7).
• DISC HSV-1 was propagated on Vero cells ( F6 ) which had been transfected with the HSV-1 gH gene as described previously in WO92/05263 published on 2 April 1992.
• F6 cells confluent monolayers of F6 cells were infected with DISC HSV-1 at a multiplicity of 0.1 pfu per cell and harvested when 90-100% cpe was observed.
• Cells were harvested with a cell scraper, pelleted by centrifugation and the pellet resuspended in a small volume of Eagles Minimum Essential Medium (EMEM). The suspension was sonicated for 1 minute and stored in aliquots at -70°C. Virus titres were determined on F6 cells.
• EMEM Eagles Minimum Essential Medium
• DISC HSV-1 was inactivated by the addition of ⁇ - propiolactone at a concentration of 0.05% for one hour at room temperature. Inactivation was checked by adding the virus to F6 cells.
• HSV-2 strain MS was propagated and titred on
• Groups of 12 animals were immunised with two doses of 8 x 10 6 pfu DISC HSV-1 or with equivalent doses of inactivated DISC HSV-1, on days 1 and 17 of the experiment. Immunisation was performed with either 0.05 ml of virus intravaginally, with 0.2 ml of virus intranasally or with 0.2 ml virus orally. A control group of 12 animals was vaccinated intravaginally with a mock preparation of virus consisting of sonicated Vero cells. All groups were challenged intravaginally on day 34 with 10 5*2 pfu HSV-2 ( strain MS ) and the experiment blinded by randomisation of the cages by an independent worker.
• the mean lesion score per animal, the mean erythema score and the effect of vaccination on post challenge virus replication for each of the immunisation groups are shown in Figs 10, 11 and 12 respectively.
• vaccination with DISC HSV-1 by the intravaginal route provided a high degree of protection from primary symptoms of infection.
• vaccination with inactivated DISC HSV-1 at an equivalent dose did not lead to any significant protection.
• Intranasal immunisation with DISC HSV-1 resulted in an even higher degree of protection than intravaginal vaccination. This was particularly apparent when looking at the number of days with severe disease, as defined by a lesion score of 6 or more (see table 2). Inactivated DISC HSV-1 gave some protection via the intranasal route, but it was not as effective as vaccination with DISC HSV-1.
• Vaccination via the oral route also led to protection, but to a lesser degree than intranasal or intravaginal vaccination. Again vaccination with DISC HSV-1 virus protected more efficiently than vaccination with inactivated DISC HSV-1.
• Intravaginal vaccination with inactivated virus resulted in clinical disease symptoms similar to those observed in mock-infected guinea-pigs.
• Intranasal vaccination with inactivated DISC HSV-1 gave a significant degree of protection, but not as high as
• Sera were collected from these animals at the end of the 100 day observation period.
• the ELISA and NT antibody titres in the sera were not significantly higher than those recorded post-challenge but before therapeutic treatment and there were no significant differences in titres between the mock-treatment group and the DISC HSV-1 treated groups.
• Days 1-13 covers the period between the two treatments.
• Days 14-27 covers the two week period subsequent to the second treatment.
• Days 1-27 covers the complete period.
• treatment with DISC HSV-2 was effective in alleviating symptoms caused by infection with HSV-2 strain MS.
• Treatment with DISC HSV-2 was more effective than treatment with DISC HSV- 1.

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