GB2322130A - Simian herpes B virus glycoprotein D and its use in recombinant viral vaccines - Google Patents

Abstract

A prophylactic or therapeutic vaccine for use in protecting mammals such as humans or animals is described. The vaccine is based upon the glycoprotein D (gD) of simian herpes B virus. Specifically, the vaccine comprises gD of B virus or a fragment or variant thereof which is capable of producing a protective immune response in a mammal to which it is administered, or a vector which includes a nucleotide sequence which encodes such a protein, fragment or variant.

Description

Virus Vaccine The present invention relates to a virus vaccine, specifically a vaccine to simian herpes B virus, to its preparation and pharmaceutically acceptable formulations and methods of prophylactic and therapeutic methods of treatment using said vaccine.

More than thirty-five different herpesviruses of primates have been isolated and identified (McCarthy & Tosolini, (1975). Proceedings of the Royal Society of Medicine 68, 145-150). Of these, simian herpes B virus poses the greatest threat to the health of monkey handlers (reviewed by Weigler, (1992)Clinical Infectious Diseases 14, 555567). B virus has been designated cercopithecine herpesvirus 1 (CHV1) by the International Committee for the Taxonomy of Viruses (Francki et al, (1991) Fifth Report of the International Committee on Taxonomy of Viruses. Arch. Virol. Suppl. Springer-Verlag, Vienna.) but will be referred to here as B virus, the name most commonly used.

Highly neurotropic in man, infection with this virus is fatal in almost 70% of cases.

Fortunately, symptomatic infection in man, by accidental monkey-bite or from infected tissues or fluids, is rare. Since the discovery of the virus in the 1930's, only 31 symptomatic human cases have been described in the literature (Palmer, (1987) Journal of Medical Primatology 16, 99-130). However, 21 of these patients died from encephalitis. The frequency of asymptomatic human infection is unknown.

Early diagnosis is of paramount importance in treating human cases (Holmes et al, (1990) Annals of Internal Medicine 112, 833-839). The time taken for antibodies to develop to a detectable level delays the identification of B virus infection. Antibody-based detection is further hindered by the close serological relationship between B virus and herpes simplex virus. A PCR-based method for detecting B virus in clinical specimens should facilitate earlier and more specific diagnosis of B virus infection (Slomka et al, (1993) Archives of Virology 131, 89-99).

Acyclovir may be used for therapy in humans if the disease is identified at an early stage. Continuous oral dosing has been shown to be required in order to avoid reactivation of latent virus (Holmes et al, 1990 supra). Passive immunotherapy for post-exposure treatment has not been effective (Boulter et al, (1981) British Medical Journal 283, 1495-1497). A formalinised vaccine has been prepared in rabbit kidney cell cultures and used experimentally to immunize persons against B virus (Hull & Nash,(1960) American Journal of Hygiene 71, 15-18.; Hull et al, (1962) American Journal of Hygiene 76, 239-251). However, the vaccine was a poor immunogen: 20% of the 300 who received it did not respond even after repeated booster doses at 3-6 month intervals (Hull, (1971) Laboratory Animal Sciences 21, 1068-1071). This vaccine was never licensed for general use.

Insufficient knowledge concerning the properties of B virus, particularly with respect to latent infections, precludes the development of an attenuated strain of the virus for vaccination purposes.

The nucleotide sequence of a region of the SHBV genome which includes inter alia, the gD gene has been identified by hybridisation with the gD gene of herpes simplex virus type 1 (HSV-1) (Bennett A.M. et al., J. Gen. Virol. (1992) 73, 2963-2967). The gene encoding glycoprotein D (gD) of simian herpes B virus (SHBV) together with the derived amino acid sequence is shown hereinafter in Figure 1.

Although expression of HSV glycoprotein D in vaccinia virus has yielded promising results, the development of a vaccine against HSV has not so far been successful.

The applicants have found however that glycoprotein D (gD) of simian herpes B virus produces a protective immunogenic response when administered to a mammal.

Hence, the present invention provides a prophylactic or therapeutic vaccine comprising a pep tide having the amino acid sequence shown in Figure 1, or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered, or a vector which includes a nucleotide sequence which encodes said peptide, and is able to express said sequence when administered to a mammal.

Suitable fragments of the peptide have one or more amino acids deleted from the sequence and may be as small as 6 amino acids in length, provided they contain at least one antigenic determinant of the glycoprotein D of B virus.

As used herein, the term "variant" means that the peptide has a sequence which is similar to that of gD of SHBV or fragments thereof, but wherein one or more amino acid residues are different. The changes do not alter the function of the peptide in terms of its ability to produce an immune response which protects against the B virus, for example by producing antibodies which are cross reactive with B virus. For example, peptides which are 60% homologous to the native sequence, suitably more than 80% homologous and preferably more than 90% homologous to the native sequence and which have similar gross biological properties would constitute "variants".

The vaccine may comprise the peptide itself, suitably formulated as a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or excipient. Such compositions form a further aspect of the invention. The compositions may be in a form suitable for mucosal or parenteral application. Mucosal applications include intra-nasal or oral applications.

Suitable carriers are well known in the art and include solid and liquid diluents, for example, water, saline or aqueous ethanol. The liquid carrier is suitably sterile and pyrogen free.

The compositions may be in the form of liquids suitable for infusion or injection, or syrups, suspensions or solutions, as well as solid forms such as capsules, tablets, or reconstitutable powders.

Peptides as described above may be prepared by various means including chemical synthesis, isolation from natural sources followed by any chemical modification if required, or more preferably, using recombinant DNA technology. Thus in a preferred embodiment, there is provided a method for preparing a peptide as described above, which method comprises including nucleotide sequence which encodes said peptide into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture. The host cell may be eukaryotic or prokaryotic, but is conveniently a prokaryotic cell such as E. coli.

In a preferred embodiment however, the vaccine may be in the form of a vector, such as an viral vector, which is adapted to express the peptide of the invention in situ. Such vectors form a further aspect of the invention. The vector may contain the usual expression control functions such as promoters, enhancers and signal sequences, as well as a selection marker in order to allow detection of successful transformants. The selection of these will depend upon the precise nature of the vector chosen and will be known to or readily determinable by a person skilled in the art.

The vaccine may alternatively be in the form of a DNA vaccine where the vector consists of a DNA plasmid which is adapted to express the peptide in situ.

Suitably the vector is a viral vector, for example a vector derived from vaccinia, adenovirus, or herpes simplex virus (HSV) BCG or BCC.

It is suitably attenuated to minimise any harmful effects associated with the virus on the host.

Preferably, the vector is derived from vaccinia virus, as it has many properties which make it a suitable vector for vaccination, including its ability to efficiently stimulate humoral as well as cell-mediated immune responses.

vBVgD was constructed using the WR strain of vaccinia in this work.

Preferably, a more highly attenuated strain of vaccinia which would be more acceptable for use in humans is employed. Such strains include Lister, which was used for wide scale vaccination against smallpox, NYVAC (Tartaglia et al, (1992). AIDS Research and Human Retroviruses 8,1445-1447) which contains specific genome deletions, or MVA (Mayr et al, (1975) Infection 3, 6-14) which is also highly attenuated.

Vaccines based upon viral vectors are suitably formulated for parenteral administration as described above. However, it is possible to formulate such vaccines for oral administration, for example by incorporating the vector into a gut-colonising microorganism such as Salmonella and particularly S. typhimurium.

As illustrated hereinafter, recombinant vaccinia virus was constructed which expressed the gD gene of B virus. The ability of the recombinant virus to elicit protective immune responses against B virus disease was investigated.

A recombinant vaccinia virus has been constructed which expresses B virus gD and has been shown to protect rabbits against lethal infection with B virus, indicating that this forms the basis of an effective vaccine.

In a preferred embodiment, the vaccine of the invention further comprises a further pep tide which comprises a different B virus glycoprotein or immunogenic fragment or variant thereof. The further peptide may be included in the vaccine formulation, or suitably the viral vector component of the vaccine carries a nucleotide sequence which encodes said further peptide and is able to co-express the further peptide with the peptide described above. The further peptide may suitably comprise B virus glycoprotein B or an immunogenic fragment or variant thereof.

In yet another embodiment, the vaccine further comprises a cytokine or an active fragment or variant thereof. Once again, the cytokine may be incorporated into the vaccine formulation, or more suitably, may be co-expressed by the vector. Examples of suitable cytokines include interleukin 2 (IL-2) and interleukin 6 (IL-6).

A particularly suitable cytokine is interleukin 2 (IL-2), which may be expressed from for example a vaccinia virus recombinant.

IL-2 is known to be responsible for the clonal expansion of antigenactivated T cells (Smith, (1984) Reviews in Immunology 2, 319-333).

Alternatively, antibody levels can be enhanced using other cytokines.

For example, expression of IL-6 by vaccinia vectors has been shown to induce a high level of IgG1 (Ruby et al, 1992 Vaccine Research 1, (4), 347-356), and IL-5 and IL-6 induced mucosal IgA responses to coexpressed influenza HA (Ramsay et al, (1994) Reproduction, Fertility and Development 6, 389-392).

An effective vaccine suitably prevents the establishment of latent infections to prevent transmission of the disease during a recurrent episode. B virus causes a recurrent infection in monkeys, and there is evidence that this also occurs in humans. For example, a survivor of the Pensacola outbreak stopped taking the prescribed daily doses of acyclovir and was subsequently re-admitted to hospital with B virus lesions (Holmes et al, 1990 supra). The present studies have shown that B virus was not found in the dorsal root ganglia of the vaccinated rabbits which were screened and hence, it is thought that a vaccine in accordance with the present invention may prevent latent injection.

The vaccine of the present invention may be used to treat humans or animals. In particular it may be given to monkeys, as a veterinary vaccine, to prevent infection from imported animals, or as a prophylactic or therapeutic vaccine for humans, particularly monkey handlers who are at risk from for example imported animals which had not yet been screened, or where a latent B virus infection had not been detected.

The vaccine may be used to immunise animals for the purpose of producing antiserum directed against the peptide of the invention.

Such antiserum could be used in diagnostic tests for B virus.

The vaccine of the invention may be incorporated into a multivalent vaccine in order to increase the benefit-to-risk ratio of vaccination. For example, a vaccine for simultaneous immunisation against Lassa fever virus, the filoviruses and B virus may have more widespread application.

The dosage of the vaccines of the invention will depend upon the nature of the mammal being immunised as well as the precise nature and form of the vaccine. This will be determined by the clinician responsible. However in general, a peptide dosage of 0.1-50pg per dose will be suitable and where the vaccine comprises a virus vector such as a vaccinia virus vectors, dosages of the vector may be in the range of from 105-102p.f.u. per dose, The vaccines of the invention will produce an immune response in test animals including the production of antibodies. These antibodies may be useful in passive vaccination programmes or in diagnosis of B virus disease. For diagnostic purposes, the antibodies may form part of a kit as is conventional in the art.

The following Examples illustrate the invention.

Example 1 Construction of recambinant vaccinia virus exDressina B virus aD The WR strain of vaccinia virus was used for making recombinant virus. Human TK 143B cells, used for transfection and selection of recombinant virus, were obtained from the European Collection of Animal Cell Cultures and were grown in Eagle's medium containing 10% foetal calf serum (FCS; Life Technologies, Bedfordshire, UK) and 25pg/ml of 5-bromodeoxyuridine (Sigma). Stocks of vaccinia viruses were produced and titrated using CV-1 cells grown in Eagle's medium containing 10% FCS.

The strain of B virus used in this work had been designated prototypic B virus (Wall et al, 1989 Virus Research 12, 283-296) and was termed Cyno 2. The prototypic strain was an oral isolate from a cynomolgus monkey (Vizozo, 1975 British Journal of Experimental Pathology 56, 489-494). B virus was inoculated into simian Vero E6 cells grown in medium 199 supplemented with 10% FCS, using a multiplicity of infection of 4-5. Virus was recovered as described, but without sonication (Slomka et al, 1995 Journal of Vi rologi cal Methods 55, 27-35).

The B virus gD gene was subcloned from the plasmid pSK*2.6 (Bennett et al, 1992 supra) into a shuttle vector plasmid, p1107. p1107 was constructed from the plasmid pGS2O (Mackett et al, 1984 Journal of Virology 49, 857-864) by inserting the 19K early vaccinia promoter upstream of the Ecogpt gene. Expression of the Ecogpt gene allows dominant selection of recombinant viruses (Isaacs et al, 1990 Virology 178, 626-630). The 1.8kbp Xho I fragment containing the gD gene was isolated from pSK2.6, recessed 3' ends were filled using the Klenow enzyme, and the fragment was cloned into the Sma I site of p1107. The resulting plasmid was designated pAK2.

pAK2 was used to transfect vaccinia virus-infected HuTK 143B cells using Lipofectin reagent according to the manufacturer's instructions (Life Technologies, Bedfordshire, UK). Recombinant viruses were selected by passaging twice in medium containing mycophenolic acid (25pg/ml), xanthine (250CLg/ml) and hypoxanthine (15pg/ml).

Recombinant viruses were designated vBVgD and were subjected to three rounds of plaque-purification before preparation of large stocks.

Expression of B virus gD was detected by Western Blotting of vBVgDinfected cells using polyclonal antiserum to B virus. The antiserum hybridised to a protein of approximately 57 kilodaltons (kDa). The size of the unmodified protein as predicted from the amino acid sequence data is expected to be 42,646Da. This discrepancy in molecular weight has also been observed with gD homologues in HSV-1, HSV-2 and PRV, and may be accounted for by the addition of N- and 0linked carbohydrate during post-translational processing, or by secondary structure of the protein.

Example 2 Protection studies using vBVaD New Zealand White female rabbits, Albino strain, 1.5-2.0 kg were obtained from Froxfield Farms UK Ltd. Rabbits were divided randomly into four groups. Group 1 (3 rabbits) received a mock vaccination of phosphate buffered saline (PBS). Group 2 (3 rabbits) received 108 PFU of vaccinia strain WR. Groups 3 and 4 (6 rabbits each) received 108 PFU of recombinant virus vBVgD. Group 4 received a booster dose of 108 PFU vBVgD after 4 weeks. The rabbits were immunised by dermal scarification of the upper dorsal region. Whole blood samples were taken from each animal at weekly intervals, serum separated and stored at -700C.

All animals were challenged 9 weeks after the primary immunisation by sub-cutaneous injection of 100 PFU B virus strain Cyno 2 (Vizozo, 1975 supra). The rabbits were subsequently monitored for signs of disease for a four week period.

A summary of the results is presented in Table 1.

Table 1

Group No. Vaccination Primary Secondary Challenge No.

Rabbits Vaccination vaccination Survivors 1 3 PBS 0 weeks None 10 0/31 weeks 2 3 108pfu 0 weeks None 10 O/3 WR weeks 3 6 103pfu 0 weeks None 10 5/62 vBVgD weeks 4 6 10'pfu 0 weeks 4 weeks 10 5/6) vBVgD weeks Notes: All control animals died on day 8 post challenge.

2 One rabbit in this group sustained a vertebral fracture 7 days after immunisation. Euthanasia was undertaken.

3 One rabbit showed reduced intake of water and food on day 17 post-challenge, although classical signs of B virus infection (paralysis at site of inoculation) were not observed. On day 22 slight rear limb paralysis was noted.

The animal died on day 23 post challenge.

All control animals (groups 1 and 2) died of B virus disease on day 8 post-challenge. Of 11 rabbits immunized with vBVgD and challenged with B virus, 10 rabbits survived with no signs of ill health over a 30 day observation period. One immunised rabbit from group 4 remained healthy until day 17 when signs of reduced food intake were observed. On day 22 the animal showed a slight rear limb paralysis but appeared well. However the animal unexpectedly died 24 hours later. The prolonged time to death indicated that some level of protection was attained.

Example 3 Serum immunoglobulin response During the course of the experiment reported in Example 2, the serum immunoglobulin response of the rabbits was monitored as follows: All animals were bled 2 days before challenge. Unvaccinated rabbits, all of which died at 8 days post challenge, were bled at autopsy.

Surviving rabbits were bled at 31 days post challenge. Presence of antibodies to B virus was determined by 3 methods: ELISA, Western blot (WB)and B virus neutralisation as follows.

ELISA for B virus antigen B virus antigen was prepared by infecting twenty 80cm2 flasks of BHK21 cells at a multiplicity of infection of 0.1 and incubating at 370C for 48 hours. Monolayers were washed and scraped into PBS. Cells were pelleted at 2000rpm for 5 mins, and the pellet frozen and thawed three times. B virus infectivity was inactivated by gamma irradiation (3.5 Mega Rads). The cell pellet (21ml) was solubilised by addition of 9ml 33% glycerol, 1.7% sodium deoxycholate, 1.7% NP40.

Aliquots were stored at -700C.

B virus antigen was diluted 1:100 in 10mM Tris-HCl (pH 7.5) and 100C11 was added to each well of a microtitre plate (Grenier). After overnight incubation at 40C, the wells were washed with PBS and incubated with 2001l1 10% FCS in PBS for 1 hr at 370C to inhibit nonspecific binding. The wells were washed and 100curl of diluted rabbit sera was added in diluent (10% FCS, 0.1% Tween 20 in PBS).

Control serum was polyclonal anti-B virus, prepared using gamma irradiated B virus strain Cyno 2 (Cropper et al, 1992 Archives of Virology 123, 267-277) which was inoculated into rabbits as described (Slomka et al, 1995 Journal of General Virology 76, 2161-2168).

After 1 hr incubation at 370C, the wells were washed four times with 0.2% Tween 20 in PBS. Horseradish peroxidase conjugated goat antirabbit immunoglobulin was added in diluent at a dilution of 1:1000 and incubated for 1 hr at 370C. Wells were washed four times as before and finally twice with PBS. TMB solution was added (100p1 per well) and incubated for exactly 2 min at room temperature before addition of 10p1 2M H2SO4 (Harlow and Lane, 1988, Antibodies - A laboratory manual. New York: Cold Spring Harbor Laboratory Press).

An ELISA plate reader (Wellcozyme) was used to take readings at 450nm.

To quantify immunoglobulin responses, sera were diluted two-fold from 1:20 to 1:40960 and tested by ELISA. The highest dilution at which a given sample remained positive (O.D.450 of > 0.270) was noted as the serum titre.

Western blot assay of B virus humoral responses E6 cell lysates (infected with B virus or uninfected control) were prepared for polyacrylamide gel electrophoresis (PAGE) and electroblotted to PVDF membranes (Amersham) as described (Slomka et al, 1995 Journal of General Virology 76, 2161-2168). Rabbit sera was diluted to 1:200 in PBS containing 10% non-fat milk (Marvel) and 0.1% Tween 20. Membranes were incubated with rabbit sera, washed and incubated with anti-rabbit horseradish peroxidase conjugate (Dako).

Immune detection was performed using enhanced chemiluminescence (Amersham) according to the manufacturer's protocol.

Virus neutralisation A known titre of B virus was mixed with diluted rabbit serum (2-fold dilution series from 1:2 to 1:64) to determine the level of neutralizing B virus antibody (Lees et al, 1991 Laboratory Animal Science 41, 360-364).

Results The results of these assays are summarised for the four treatment groups of rabbits.

None of the rabbits in groups 1 (unvaccinated) and 2 (inoculated with vaccinia WR) possessed B virus antibody by any of the 3 assays prior to challenge. Sera obtained at autopsy revealed that 4 animals did not possess antibody to the challenge virus, indicating that death had occurred prior to production of a detectable humoral response to B virus. One rabbit in group 2 (#755) produced antibody which was detected by WB and was directed to proteins which migrated faster than the gD band. A neutralisation titre of 1:4 was also noted for this sample, but no response was detected by ELISA.

All 5 rabbits in group 3 (given one dose of vBVgD) developed an antigD humoral response prior to challenge which was still apparent at 31 days post challenge, as shown by WB and ELISA (Table 2).

TABLE 2 ANTIBODY TITRES IN SERUM FROM RABBITS IMMUNIZED WITH VACCINIA VIRUS EXPRESSING B VIRUS GLYCOPROTEIN D

Rabbit Treatment Pre-challenge 31 days post- Neut. titre at 31 group group titrea challenge' days post L challengeb 758 3C 80 320 < 2 I 759 3 320 320 < 2 I 760 3 160 160 < 2 I 761 3 640 640 < 2 763 3 160 320 2 764 4d 160 160 < 2 765 4 160 160 4 766 4 640 640 8 767 4 160 640 1 2 I 769 4 320 320 8 Results have been expressed as the reciprocal of the serum dilution giving ODiso > 0.27 (mean of negative sera plus 3 standard deviations) Results have been expressed as the reciprocal of the serum dilution giving 50% plaque reduction Group 3 was given one dose of vaccinia recombinant Group 4 was given a second dose of vaccinia recombinant at 4 weeks Antibodies to other B virus proteins were not observed by WB suggesting that B virus replication had not occurred following challenge. No B virus neutralisation response was detected prechallenge. One rabbit in group 3 (#763) developed a weak neutralising response (1:2) at 31 days post-challenge (Table 2).

Although group 4 had received two doses of vBVgD, none had developed neutralising antibody to B virus prior to challenge. Antibody to gD was detected by WB before and after challenge in the 5 surviving rabbits. Antibodies to other B virus proteins were not detectable in the 5 healthy animals at 1 month post challenge, indicating absence of B virus replication. ELISA confirmed that a B virus humoral response had developed prior to challenge and in view of the WB findings it was apparent that this response was directed at gD. The surviving rabbits in group 4 differed serologically from group 3 in their B virus neutralisation response. This was detectable in 4 of 5 rabbits at 31 days post-challenge, with titres ranging from 1:2 to 1:8 (Table 2).

However, the humoral response was distinct in one rabbit (&num;768) from group 4 who developed paralysis and died 23 days after challenge.

This animal had developed anti-gD antibody by 7 days post vaccination, although this response subsequently became undetectable.

ELISA showed the rapid development of an antibody response to B virus soon after the first vaccination, at a time when only one other member of group 4 had developed a weak response. An antibody response to gD was detected by WB at 10 days post challenge, but 6 days later a humoral response to other B virus proteins was observed, providing evidence of B virus replication. Antibody to B virus remained detectable post-challenge by ELISA, while a strong B virus neutralisation titre ( > 1:64) was apparent at the time of autopsy.

Serological responses from the 10 protected rabbits in groups 3 and 4 were quantified by ELISA. Sera drawn 2 days prior to challenge and 1 month post challenge were tested in this manner. The results indicate that the majority (7/10) of the protected rabbits did not experience a quantitative boost in anti-gD antibody following challenge, this including 3 animals which had developed neutralising antibody. The remaining 3/10 rabbits demonstrated an increase in anti-gD titre by up to 4-fold which was not considered to be a significant rise, although 2 of these animals did develop neutralising antibody.

Example 4 Detection of B virus re9lication.

This was effected using the following techniques: A. Virus isolation Swabs were taken at death from inoculation sites in the 6 vaccinated rabbits from groups 1 and 2. Confluent Vero E6 cells in 25cm flasks were inoculated with the swab samples. The cells were incubated for 2 weeks in maintenance medium and examined daily for cytopathic effect.

Only one animal (&num;754) yielded infectious virus (Table 3).

B. "Rot-start PCR of B virus DNA All were tested by modified B virus PCR (188bp amplimer) which had a detection limit of approximately 10 molecules of B virus DNA (i.e. 40 ng of excised 9.6 kbp Bam HI fragment "g", Harrington et al,J. Gen.

Virol.(1992), 73, 1217-1226.

Primers which amplify a 188bp region of the B virus Cyno 2 genome were used to test the rabbit swab specimens (Slomka et al, 1993 Archives of Virology 131, 89-99). Amplification conditions were modified such that reactions contained 8 pmoles of each primer, 60mM Tris-HCl (pH9.0), 15mM NH3S04, 1.5mM MgCl2, 200sum dNTPs. Each reaction was partitioned by addition of a wax bead (GEM 100, Perkin Elmer) according to manufacturer's instructions. Heat inactivated rabbit swab specimen (10p1) was added and amplification consisted of an initial 1 min denaturation step at 950C followed by 35 cycles of 1 min at 950C, 1 min at 570C, 1 min at 720C. The final elongation step at 720C was extended to 5 mins.

A positive result was seen in samples from 3/5 rabbits by ethidium bromide staining of the agarose gel.

All 5 samples were positive following oligomer hybridisation which increased the sensitivity to under 10 molecules DNA.

C. Detection of amplified B virus DNA by oligomer hybridisation Amplification products were electrophoresed through 3.5% agarose and transferred to Hybond-N plus membranes (Amersham) by Southern blotting. An oligonucleotide probe of 188bp (Slomka et al, 1993 Archives of Virology 131, 89-99) was 3'end-labelled with digoxygenin by terminal transferase using a commercially available kit (BCL).

Hybridisation was carried out at 520C for 16 hours. Membranes were washed twice for 5 minutes at room temperature in 2XSSC, 0.1% SDS, followed by two washes for 15 minutes in 0.lXSSC, 0.1% SDS at 500C.

Detection of hybrids was performed using alkaline phosphatase conjugated anti-digoxygenin, diluted to 1:10,000.

D. "Hot-startW nested PCR for detection of B virus in rabbit ganglia From the 10 rabbits which had remained healthy at 31 days post B virus challenge, 4 were autopsied and the dorsal root ganglia adjacent to the inoculation site removed. Reconstruction experiments where dilutions of 9.6 kbp Bam HI fragment "g" were mixed with 250ng of total E6 cell (uninfected) DNA were amplified by the modified nested PCR. A detection limit of 40ng (approximately 10 molecules) was noted by ethidium bromide staining.

Primers which span a 512bp region of B virus DNA were used to initially amplify total DNA specimens extracted from rabbit ganglia (Slomka et al, 1993). Conditions were modified by the addition of a wax bead as before to partition the reaction. Extracted ganglion DNA (250ng) was added as a 10p1 volume. The reaction contained 40 pmoles of each primer, 60mM Tris-HCl (pH10.0), 15mM NH3SO, 1.5mM MgCl2, 200ZM dNTPs. Amplification consisted of an initial 1 min denaturation step at 950C followed by 30 cycles of 1 min at 950C, 1 min at 520C, 1 min at 720C with a final elongation step at 720C for 5 mins. An aliquot of 1.25 > 1 of product was added to a "hot start" PCR reaction mix and amplified for 30 cycles to produce the 188bp amplicon.

Duplicate nested amplification of 250ng ganglion DNA failed to reveal the B virus-specific amplicon (188bp) by ethidium bromide staining.

Failure to detect this amplicon by oligomer hybridisation as described in section C above, following Southern blotting confirmed this observation. In these 4 protected rabbits, absence of B virus DNA in the ganglia suggested protection against latent B virus infection.

Claims (34)

Claims

1. A prophylactic or therapeutic vaccine comprising a peptide having the amino acid sequence shown in Figure 1, or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered, or a vector which includes a nucleotide sequence which encodes said peptide, and is able to express said sequence when administered to a mammal.

2. A vaccine according to claim 1 which comprises a vector which includes a nucleotide sequence which encodes said peptide, and is able to express said sequence when administered to a mammal.

3. A vaccine according to claim 2 wherein the vector comprises a plasmid DNA vector.

4. A vaccine according to claim 2 wherein the vector comprises a virus vector.

5. A vaccine according to claim 4 wherein the virus is selected from an attenuated virus

6. A vaccine according to claim 4 or claim 5 wherein the virus is selected from vaccinia, adenovirus, HSV, BCG or BCC.

7. A vaccine according to claim 6 which comprises an attenuated vaccinia virus.

8. A vaccine according to any one of the preceding claims which comprises a further peptide which is a different B virus glycoproteir or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered, or a vector which includes a nucleotide sequence which encodes said further peptide, and is able to express said sequence when administered to a mammal.

9. A vaccine according to claim 8 wherein said different B virus glycoprotein comprises glycoprotein B.

10. A vaccine according to claim 8 or claim 9 which comprises a vector which includes a nucleotide sequence which encodes said further peptide, and is able to express said sequence when administered to a mammal.

11. A vaccine according to claim 10 wherein the vector which comprises the nucleotide sequence which encodes said further peptide also comprises a nucleotide sequence which encodes a pep tide having the amino acid sequence shown in Figure 1, or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered.

12. A vaccine according to any one of the preceding claims which further comprises a cytokine or an active fragement or variant thereof, or a vector which comprises a nucleotide sequence which encodes a cytokine or an active fragment or variant thereof.

13. A vaccine according to claim 12 which comprises a vector which comprises a nucleotide sequence which encodes a cytokine or an active fragment or variant thereof.

14. A vaccine according to claim 13 wherein said vector further comprises a nucleotide sequence which encodes a peptide having the amino acid sequence shown in Figure 1, or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered, and/or a nucleotide sequence which encodes a further peptide as defined in claim 8.

15. A vaccine according to any one of claims 12 to 14 wherein the cytokine is an interleukin.

16. A vaccine according to claim 15 wherein the interleukin is selected from human IL-2 or human IL-6.

17. A recombinant expression vector which comprises a nucleotide sequence which encodes a peptide the amino acid sequence shown in Figure 1, or a fragment or variant thereof which is capable of producing a protective immunogenic response against B virus in a mammal to which it is administered.

18. A recombinant expression vector according to claim 17 wherein the nucleotide sequence comprises the nucleotide sequence of Figure 1.

19. A recombinant expression vector according to claim 17 or claim 18 which further comprises a nucleotide sequence which encodes a further peptide which is a different B virus glycoprotein or a fragment or variant thereof which is capable of producing an immunogenic response in a mammal to which it is administered.

20. A recombinant expression vector according to claim 19 wherein the said different B virus glycoprotein is B virus glycoprotein B.

21. A recombinant expression vector according to any one of claims 17 to 20 which further comprises a nucleotide sequence which encodes a cytokine or an active fragment or variant thereof.

22. A recombinant expression vector according to any one of claims 17 to 21 for use in the preparation of a vaccine.

23. A peptide comprising B virus glycoprotein D or a fragment of variant thereof which is able to for use in the preparation of a vaccine 23. A method for preparing a peptide as defined in claim 1 which method comprises including nucleotide sequence which encodes said peptide into a recombinant expression vector, transforming a host cell with said vector, and culturing said cell and recovering the peptide from the culture.

24. A method according to claim 23 wherein the cell is a prokaryotic cell.

25. A method according to claim 24 wherein the host cell is an E.

coli.

26. A transformed host cell, for use in the method according to any one of claims 23 to 25.

27. A pharmaceutical composition comprising a peptide as defined in claim 1 and a pharmaceutically acceptable carrier or excipient.

28. A pharmaceutical composition comprising a recombinant expression vector according to any one of claims 17 to 22 in combination with a pharmaceutically acceptable carrier or excipient.

29. A method for producing a protective immune response against B virus in a mammal, which method comprises administering to said mammal, a vaccine according to any one of claims 1 to 16.

30. A method according to claim 29 wherein the mammal is either a human or a monkey.

31. A multivalent vaccine comprising a vaccine according to any one of claims 1 to 16 and a further vaccine.

32. A multivalent vaccine according to claim 31 wherein said further vaccine comprises a vaccine against Lassa fever virus and/or filoviruses and B virus may have more widespread application.

33. An antibody which reacts specifically with a peptide as defined in claim 1.

34. A diagnostic kit comprising an antibody according to claim 33.