Viral vaccines

The application provides a pharmaceutical which comprises a mutant non-retroviral virus (particularly HSV-1 and/or HSV-2) whose genome is defective in respect of a gene essential for the production of infectious virus. The virus can infect normal cells and undergo replication and expression of viral antigen genes in those cells but cannot produce normal infectious virus. The pharmaceutical is for prophylactic or therapeutic use in generating an immune response in a subject infected therewith. Where the non-retroviral virus is a herpes simplex virus eg HSV-1 or HSV-2, the defect can be in the glycoprotein gH gene. Vaccines and therapeutic pharmaceuticals are provided especially for epithelial, oral, vaginal and nasal administration. Also provided is use of a mutant based on HSV-1 for the preparation of a pharmaceutical for prophylactic or therapeutic use in generating an immune response in a subject against type-2 HSV infection.

The present invention relates to viral vaccines. 
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. These two types of vaccine each have their own 
disadvantages. Since killed vaccines do not replicate in the host, they 
are usually administered by injection, and hence may generate an 
inappropriate kind of immune response. For example 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. Hence 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". In addition, 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. However, the presentation 
occurs via MHC Class II molecules and leads to stimulation of T helper 
cell. In turn, the T helper cells help B cells to produce specific 
antibody against the antigen. 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 arid 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. 
To date, live attenuated viruses leave been made by deleting an unessential 
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). However, live attenuated viruses' often retain residual 
pathogenicity which can have a deleterious effect on the host. In 
addition, unless 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, as well as being used as vaccines in their own 
right, 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. Typically, members of the pox virus family 
eg. vaccinia virus, are used as vaccine vectors. When a virus, is used as 
a vaccine vector, it is important that it causes no pathogenic effects. In 
other words it may need to be attenuated in the same way that a simple 
virus vaccine is attenuated. The same disadvantages as those described 
above, therefore apply in this case. 
It has been found possible to delete an essential gene from a viral genome 
whilst also providing a so-called `complementing` cell which provides the 
virus with the product of the deleted gene. This has been achieved for 
certain viruses, for example adenoviruses, herpesviruses and retroviruses. 
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 (Harrison, Graham & Williams, Virology 77, 
319-329, 1977). Although the virus grew well on this cell line (the 
`complementing cell line`) and produced standard viral particles, it could 
not grow at all on normal human cells. Cells expressing the 
T-antigen-encoding region of the SV40 virus genome (a papovavirus) have 
also been shown capable of supporting the replication of viruses 
specifically deleted in this region (Gluzman, Cell, 23, 182-195, 1981). 
For herpes simplex virus, cell lines expressing the gB glycoprotein (Cai 
et al, J. Virol. 62, 714-721, 1987) the gD glycoprotein (Ligas and 
Johnson, J. Virol. 62, 1486, 1988) and the Immediate Early protein ICP4 
(Deluca et al., J. Virol., 56, 558, 1985) have been produced, and these 
have been shown capable of supporting the replication of viruses with 
specifically inactivated copies of the corresponding genes. 
WO92/05263 published on 2 Apr. 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. In preferred 
embodiments, 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. Thus 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. Hence, although 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. Thus, 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. 
However, preferably 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. There is thus the additional opportunity to stimulate 
the cellular arm of the host immune system. 
In particular, it is to be mentioned that 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. 
The applicants call the above described mutant viruses DISC viruses 
(standing for defective infectious single cycle) and the basic concept is 
illustrated In FIG. 1. The present application goes on from the work 
disclosed in WO92/05263. 
The present application makes the disclosures summarised below. 
(1) In a study using the mouse ear model the results reported in WO92/05263 
were confirmed. Intra-epithelial vaccination of mice with DISC HSV-1 led 
to complete protection against replication of the challenge virus wild 
type (w.t.) HSV-1. Little effective protection was provided by equivalent 
doses of inactivated HSV-1. DISC HSV-1 also protected against the 
establishment of latent infection in the cervical ganglia. 
(2) Also in the mouse ear model it is shown that no significant differences 
in antibody titres were observed between animals vaccinated with DISC 
HSV-1 and an equivalent amount of inactivated HSV-1. 
(3) Also in the mouse ear model it is shown that at low vaccination doses, 
inactivated HSV-1 failed to established a delayed-type hypersensitivity 
(DTH) response, whilst equivalent doses of DISC HSV-1 established a DTH 
response. At high doses, both DISc: HSV-1 and inactivated HSV-1 induced 
similar DTH responses. 
(4) Also in a mouse study it was shown that in contrast to vaccination with 
inactivated HSV-1, vaccination with DISC HSV-1 induced HSV-1 specific 
cytotoxic T cell activity. 
(5) The in vivo mouse ear model was used to study long term prophylactic 
effect of DISC HSV-1. Two vaccinations of DISC HSV-1 was found to provide 
hotter long term protection against challenge with w.t. HSV-1 than two 
vaccinations of inactivated DISC HSV-1. 
(6) The in vivo mouse ear model was used to investigate the prophylactic 
effect of DISC HSV-2 against HSV-2 infection. Intra-epithelial vaccination 
of mice with DISC HSV-2 provided better protection against replication of 
the challenge virus w.t. HSV-2 than inactivated DISC HSV-2. 
(7) The in vivo guinea-pig vaginal model was used to study the prophylactic 
effect of DISC HSV-1 against HSV-2 Infection. It was shown that 
intra-epithelial or intra-vaginal vaccination with DISC HSV-1 provided a 
high degree of protection against the primary symptoms of HSV-2 infection. 
Immunisation with DISC HSV-1 or inactivated virus retarded growth of 
challenge virus w.t. HSV-2 in the vagina. Further intra-vaginal 
vaccination with DISC HSV-1 lessened the number of recurrent HSV-2 lesions 
in a 100 day follow-up period. Intra-epithelial vaccination with DISC 
HSV-1 and inactivated virus also resulted in reduced recurrent lesions, 
but compared to intra-vaginal vaccination with DISC HSV-1, the reduction 
was less. 
(8) Oral and intranasal vaccination of guinea-pigs with DISC HSV-1 led to 
protection against acute disease symptoms following challenge with w.t. 
HSV-2. The intranasal route appeared to be more effective than the oral 
route. 
The per vaginum vaccination route in comparison to oral or intra-nasal 
vaccination resulted in significantly lower levels of recovered virus 
following challenge. 
(9) In guinea-pigs which had recovered fully from primary HSV-2 disease, 
the therapeutic administration of DISC HSV-1 either intra-vaginally or 
intra-epithelially resulted in an apparent reduction in the frequency of 
recurrent of disease symptoms compared with mock vaccinated animals. 
(10) In guinea-pigs which had recovered fully from primary HSV-2 disease, 
intra-vaginal therapeutic administration of DISC HSV-2 was more effective 
in reducing the frequency of recurrence of disease symptoms than treatment 
with DISC HSV-1. 
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 may 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-retroviral 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, Editors: A. Voller 
and H. Friedman, University Park Press, Baltimore, 1978 for further 
background details on vaccine preparation. 
Therapeutics comprising a mutant as herein provided can be formulated 
according to know methods to provide therapeutically useful compositions, 
whereby the mutant is combined in admixture with 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 Lo prepare 
therapeutically acceptable Compositions suitable for effective 
administration to the host. 
Typically vaccines are prepared as injectables, (traumatic or 
non-traumatic) either as liquid solutions or suspensions; solid forms 
suitable for solution in, or suspension in, liquid prior to injection may 
also be prepared. Preparations may also be encapsulated in liposomes. 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. In addition, if desired, 
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.

EXAMPLES 
Herpes Simplex Virus Deleted in Glycoprotein H (gH-HSV) 
Herpes simplex virus (HSV) 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 seeing 
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 (Dosai 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. 
All genetic manipulation procedures are carried out according to standard 
methods described in "Molecular Cloning", A Laboratory Manual, eds. 
Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. 
METHODOLOGY 
PREATION OFF A DISC HSV-1 
A. Generation of a Cell Line Expressing the HSV-1 gH gene 
This was carried out in accordance with the teaching of WO92/05263 
published on 2 Apr. 1992 and corresponding to U.S. Pat. No. 5,665,362 
issued Sep. 9, 1997, incorporated herein by reference and also using 
standard procedures in the art. 
B. Production of a DISC HSV Type 1 Virus Having an Interrupted gH Gene 
This was carried out in accordance with the teaching of WO92/05263 
published on 2 Apr. 1992 and corresponding to U.S. Pat. No. 5,665,362 
issued Sep. 9, 1997, incorporated herein by reference and also using 
standard procedures in the art. 
C. Relevant Publications 
(i) Forrester A. et al J. Virol. 1992; 66 p341-348 
(ii) Farrell H., et al. J. Virol. 1994: 68 p927-932 
PREATION OF A DISC HSV-2 
A. The HSV2 gH Gene 
(a) The Herpes Simplex type 2 (HSV2) gH gene is contained within two BamHI 
restriction fragments of the 25766 strain of HSV2. pTW49 is the BamH1 R 
fragment of HSV2 strain 25766 cloned into pBR322, pTW54 is the BamH1 S 
Fragment of HSV2 Strain 25766 cloned into pBR322. The construction of a 
single plasmid containing the complete gH gene is shown in FIG. 17. pTW49 
was digested with BamHI and Sall, and an 870 base pair (bp) fragment 
isolated from an agarose gel. Similarly pTW54 was digested with BamHI and 
Kpn1 and a 2620 bp fragment isolated from an agarose gel. The two 
fragments were ligated together with the plasmid pUC119 cut with Sall and 
Kpn1, resulting in the plasmid pIMMB24. 
(b) pIMMB24 was digested with Sall and Kpn1. In addition the plasmid was 
digested with Dra1 (which cuts in the vector sequences), to aid in 
isolation of the 3490 bp insert. The 3490 bp 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 
300-600 bp in length was ligated into M13mp11 cut with Smal (Amersham 
International UR). The ligated mixture was transformed into E. coli strain 
TG1, 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 .sup.35 
S dATP. 
In addition to sequencing in M13 using an oligonucleotide priming from 
within the M13 sequences, 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 HSV1 sequence. 
(c) The sequence (SEQ ID NO:1) of HSV2 strain 25766 in the region of the gH 
gene is shown in FIG. 18, along with a translation of the gH in single 
letter amino acid code (SEQ ID NO:2). FIG. 19 shows a comparison of the 
DNA sequence of HSV1 (SEQ ID NO:3) and HSV2 (SEQ ID NO:1) in this region. 
FIG. 20 shows a comparison of the deduced amino acid sequences of the HSV1 
(SEQ ID NO:4) and HSV2 (SEQ ID NO:2) gH proteins. At the DNA level the 
overall identity is 77%. At the protein level the overall identity is also 
77%, with a further 9.7% of amino acids being similar in properties. The 
degree of sequence similarity varies to some extent along the length of 
the gene, as can be seen from FIG. 21, which shows graphically the level 
of similarity. Even more marked than the variation along the gH gene is 
the difference in levels of identity between HSV1 and HSV2 at the DNA 
level between the coding and non-coding regions. As can be seen from FIG. 
19, the nucleotide sequence identity is higher within the coding sequence 
of the gH gene than it is in the intergenic regions. FIG. 21 shows this in 
a graphical form, with the positions of the TK, gH and UL21 genes marked. 
(d) The availability of nucleotide sequence data from around the HSV-2 gH 
gene enables further constructs to be made eg it allows the design of 
recombination vectors which enables precise deletion of the gene from the 
viral genome. Because of the differences between HSV1 and HSV2, 
particularly between the genes, may not have been possible from knowledge 
of the HSV1 sequence alone. 
Oligonucleotides MB57 (SEQ ID NO:5), MB58 (SEQ ID NO:6), MB59 (SEQ ID NO:7) 
and MB75 (SEQ ID NO:8) were designed to isolate and clone the regions of 
sequence flanking the HSV2 gH gene. As shown in FIG. 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: 
##STR1## 
The position of these oligonucleotides is also shown on FIG. 19. 
In accordance with the teachings made in PCT/GB91/01632 (WO 92/05263) and 
common general knowledge, 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. 
B Construction of a gH Defective Type 2 Herpes Simplex Virus (DISC HSV-2) 
Complementing Cell Lines 
It was found that cells expressing the HSV-1 gH gene (F6 cells, Forrester 
et al, Journal of Virology, 1992, 66, p. 341-348) can support the growth 
of an HSV-2 virus lacking the gH gene. However two new cell lines were 
made. 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. In the case of F6 cells and the 
gH-deleted virus in the Forrester paper, where there is overlap, wild-type 
gH-plus viruses occur by recombination at about 1 in 10.sup.6 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. 
Polymerase Chain Reaction (PER) of Flanking sequences 
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 TX 
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 oligonucleotide, cased art as follows: 
##STR2## 
Construction of Vectors 
The first stage recombination vector, pIMMB47+ 
The two PCR fragments made by oligos MB97 (SEQ ID NO:10)-MB96 (SEQ ID NO:9) 
and by oligos MB57 (SEQ ID NO:5)-MB58 (SEQ ID NO:6) 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 HindIII 
and Hpal. The MB57-MB58 fragment is digested with Hpal and EcoRI. These 
fragments are then ligated into the vector pUC1l9 which has been digested 
with HindIII and EcoRI. The resultant plasmid is called pIMMB45 (see FIG. 
24). 
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 a suitable plasmid carrying the 
promoter and gene using one or more 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. 
For example, 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 pCH110 (Pharmacia PL, Biochemicals) using BamHI and TthlllI, 
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 (SEQ ID NO:12) and by 
oligos MB57 (SEQ ID NO:5)-MB108 (SEQ ID NO:11) 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 HindIII 
and Hpal. The MB57-MB106 fragment is digested with Hpal and EcoRI. These 
fragments are then ligated into the vector pUC119 which has been digested 
with HindIII and EcoRI. The resultant plasmid is called pIIME46 (see FIG. 
26). The oligonucleotides used are as follows: 
##STR3## 
Construction of Recombinant Viruses 
a) First Stage. 
Virus DNA was made from strain HG52-D, which is a plaque-purified isolate 
of the HSV-2 strain HG52. Virus DNA (2.5 .mu.g) arid pIMMB47+ plasmid DNA 
(0.25 .mu.g) was transfected into CRl cells using the CaPO.sub.4 
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 
.mu.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 Voro 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 
CR1 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 (CR1) and non-complementing (Vero) cells. 
CR1 (gH+) Vero 
Virus dilutions 
10.sup.-4 
10.sup.-5 
10.sup.-6 
10.sup.-1 
10.sup.-2 
10.sup.-3 
10.sup.-4 
______________________________________ 
Number of 
&gt;350 174 22 0 0 0 0 
plaques &gt;350 169 19 0 0 0 0 
______________________________________ 
b) Second stage. 
DNA was made from this TK-minus DISC virus and a recombination was carried 
out as above with the plasmid pIMMB46. In this case TK-plus recombinants 
were selected, on a gH-expressing TK-minus BHK cell line, by growth in 
medium containing methotrexate, 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 BamHI 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 hand sizes after BamHI 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: 
______________________________________ 
Band size hybridising 
Virus (base pairs) 
______________________________________ 
HG52-D 3481 
TK-minus "first stage" DISC virus 
3140 
TK-plus "second stage" DISC virus 
4225 
______________________________________ 
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, and 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, Md. 20852 USA. The following are available from ATCC under the 
indicated accession numbers. 
______________________________________ 
HSV-1 strain F: 
ATCC accession no. VR-733 
HSV-1 strain MacIntyre: 
ATCC accession no. VR-539 
RSV-1 strain MP: 
ATCC accession no. VR-735 
HSV-2 strain G: 
ATCC accession no. VR-734 
HSV-2 strain MS: 
ATCC accession no. VR-540 
______________________________________ 
IN VIVO MOUSE STUDIES 
PROTECTION STUDIES 
The in vivo mouse ear model was used to study prophylactic effects. 
Equivalent doses of inactivated wild-type HSV-1 (Strain SC16 see Hill et 
al. J. Gen. Virol. 28, p341-353 (1975)) and DISC HSV-1 were compared for 
their effect on the replication of w.t. HSV-1, their ability to provide 
protection against w.t. HSV-1 challenge and to induce HSV-specific 
neutralising antibodies. 
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 .beta.-propiolactone (for further details see WO92/05263 
published on 2 Apr. 1992 and corresponding to U.S. Pat. No. 5,665,362 
issued Sep. 9, 1997, incorporated herein by reference). The mice were 
challenged with 2.times.10.sup.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 plaguing on BHK cells. (See 
FIG. 2.) 
It can be seen from FIG. 2 that vaccination with 5.times.10.sup.5 and 
5.times.10.sup.6 pfu DISC HSV-1 (pfu measured on complementing cell line 
for DISC viruses) led to complete protection against replication of the 
challenge virus, whilst mice vaccinated with inactivated virus still had 
live challenge virus present. 
A similar result was obtained when virus titres were assayed from the 
ganglia of vaccinated animals 5 days after challenge (data not shown). 
SEROLOGICAL RESPONSE TO DISC HSV-1 VACCINATION 
The role of antibody in protection conferred by the DISC HSV-1 vaccination 
was investigated. Both neutralising antibody titres and total antibody 
titres, as determined by ELISA, were measured. 
Groups of 6 mice were vaccinated with 5.times.10.sup.6 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. 
DELAYED-TYPE HYPERSENSITIVITY (DTH) RESPONSE to DISC HSV-1 VACCINATION 
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. 
Four doses (5.times.10.sup.3, 5.times.10.sup.4, 5.times.10.sup.5 and 
5.times.10.sup.6 pfu) of vaccine were used, and two weeks later the 
vaccinated animals were challenged in the opposite ear with 10.sup.6 pfu 
w.t. HSV-1 (strain SC16). The DTH response at the site of challenge was 
assessed by measurement of ear thickness at 24 and 48 hours post challenge 
and expressed as the difference between the challenged and unchallenged 
ears. (See FIG. 4.) 
It can be seen from FIG. 4 that at low vaccine doses (5.times.10.sup.3, 
5.times.10.sup.4 pfu), no DTH response was observed with killed DISC 
HSV-1, whilst a clear DTH response was demonstrated after DISC HSV-1 
vaccination. At high doses, (eg 5.times.10.sup.6 pfu), both the DISC HSV-1 
vaccine and killed DISC HSV-1 preparations induced similar DTH responses. 
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. 
DEMONSTRATION THAT DISC HSV TYPE 1 VIRUS IS CAPABLE OF GENERATING CYTOTOXIC 
T CELLS 
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: 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.times.10.sup.7 pfu of virus (DISC 
HSV-1; killed DISC HSV-1; MDK a thymidine 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 0.1 ml 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. 
Spleens were also removed from unimmunised 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.times.10.sup.7 cells/ml in 
effector medium and mitomycin C was added to a final concentration of 20 
.mu.g/ml. The feeder cells were incubated at 370.degree. 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 (MDK) 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.degree. C. the feeder cells 
were washed once with effector cell medium. 
Effector cells were resuspended to 5.times.10.sup.6 cells/ml, whilst feeder 
cells were resuspended to 2.5.times.10.sup.6 cells/ml. 500 .mu.l of 
effector cell suspension and 500 .mu.l feeder cell suspension were added 
to the wells of a 24 well plate. The plates were incubated in a humid 
atmosphere at 37.degree. C. (5% CO.sub.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 (5 
ml of medium per 2 plates). The cell suspension was layered onto 
lymphocyte separation medium and spun at 2500 rpm 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. 
Labelled target cells were prepared for the cytotoxicity assay. Uninfected 
syngeneic A202J target cells A20/2J cells were harvested from tissue 
culture flasks; 2.times.10.sup.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 10 pfu 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.degree. C. (in a waterbath) for 1 hour. The 
target cells were then washed three times (10 minutes, 1100 rpm) in target 
medium (DMEM supplemented with 10% FCS) and finally resuspended to the 
required cell concentration in target cell medium. 
Both uninfected and infected target cells were resuspended to 
1.times.10.sup.6 cells/ml and 1.times.10.sup.5 cells/ml and 100 .mu.l (ie 
to give 1.times.10.sup.5 targets/well and 1.times.10.sup.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.times.10.sup.6 
cells/ml in effector medium and two-fold dilutions were prepared. 100.mu.l 
of the effector cell suspensions were added to the wells containing the 
labelled target cells to give 8.times.10.sup.5 effector cells/well, 
4.times.10.sup.5 effector cells/well, 2.times.10.sup.5 effector cells/well 
and 1.times.10.sup.5 effector cells/well. Thus with 10.sup.5 target cells 
per well, effector to target ratios were: 8:1, 4:1, 2:1 and 1:1. With 
10.sup.4 target cells per well the 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 100 .mu.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 100.mu.l effector cell 
medium to wells containing target cells only. 
The plates were incubated at 37.degree. C. for four hours in a is humid 
atmosphere. After this time the plates were spun for four minutes at 1500 
rpm arid 100 .mu.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 
##EQU1## 
The results are shown in FIG. 5 and Table 1 
TABLE 1 
______________________________________ 
Inactivated 
E:T ratio 
DISC HSV-1 Virus MDK Unvaccinated 
______________________________________ 
8:1 53.9 1.5 48.3 ND 
4:1 49.6 0.0 42.2 0.0 
2:1 36.9 0.0 31.0 0.0 
1:1 23.9 0.0 21.9 0.0 
______________________________________ 
% HSV1 Specific Lysis 
(% lysis of HSVinfected cells minus % lysis of uninfected cells). 
DISC HSV-1 vaccination induced HSV-1 specific CTL activity comparable to 
that produced by infection with the fully replicative MDK virus. In 
contrast no 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 
4-5 week old BALB/c mice were divided into groups containing 6 animals 
each. The groups were vaccinated as follows: 
______________________________________ 
Group Vaccination 
______________________________________ 
PBS Mock immunisation with PBS 
1K 1 immunisation with inactivated DISC HSV-1 
2K 2 immunisations with inactivated DISC HSV-1 
1L 1 immunisation with (live) DISC HSV-1 
2L 2 immunisations with (live) DISC HSV-1 
1S 1 immunisation with w.t. HSV-1 (strain SC16) 
2S 2 immunisations with w.t. HSV-1 (strain SC16) 
______________________________________ 
All groups were immunised by scarification of the left ear pinna with 
5.times.10.sup.6 pfu on day 0 and blood samples taken on days 15, 27, 90, 
152 and 218. Groups PBS, 2K, 2L and 2S received additional immunisations 
of PBS or 5.times.10.sup.5 pfu on day 20. All groups were challenged with 
5.times.10.sup.5 w.t. HSV-1 (strain SC16) on day 223. The amount of virus 
present in the challenged ear (right) 5 days post challenge was assayed by 
plaquing on BHK cells. The results as depicted by FIG. 14 show that two 
vaccinations with DISC HSV-1 (group 2L) provides goods protection compared 
to inactivated DISC HSV-1 (group 2K), but that better protection was 
obtained with w.t. HSV-1 (strain SC16). The efficacy of vaccination with 
w.t. HSV-1 is of course, to be expected. However the use of normal live 
viruses as vaccines is generally undesirable. 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. 
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 
______________________________________ 
1 5 .times. 10.sup.2 pfu live DISC HSV-2 
2 5 .times. 10.sup.3 pfu live DISC HSV-2 
3 5 .times. 10.sup.4 pfu live DISC HSV-2 
4 5 .times. 10.sup.5 pfu live DISC HSV-2 
5 5 .times. 10.sup.2 pfu killed DISC HSV-2 
6 5 .times. 10.sup.3 pfu killed DISC HSV-2 
7 5 .times. 10.sup.4 pfu killed DISC HSV-2 
8 5 .times. 10.sup.5 pfu killed DISC HSV-2 
9 5 .times. 10.sup.4 pfu w.t. HSV-2 (strain HG52) 
10 5 .times. 10.sup.5 pfu w.t. HSV-2 (strain HG52) 
11 PBS 
______________________________________ 
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.times.10.sup.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 BKK cells (see FIG. 16). The results 
as depicted by the figure show that vaccination with DISC HSV-2 at doses 
of 5.times.10.sup.3, 5.times.10.sup.4 and 5.times.10.sup.5 pfu provides 
good protection against challenge with w.t. HSV-2 (strain HG52) compared 
to killed DISC HSV-2. However and as is to be expected, better protection 
was obtained with w.t. HSV-2 at doses of 5.times.10.sup.4 and 
5.times.10.sup.5 pfu, but the use of normal live wild type viruses as 
vaccines is undesirable. 
IN VIVO GUINEA PIG STUDIES 
As mentioned earlier, 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 immunogenicity 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. 
(1) Groups of 14 animals were immunised with two doses of the DISC HSV-1 
vaccine (2.times.10.sup.7 pfu, 3 weeks apart) either by non-traumatic 
introduction into the vagina (intra-vaginal route), or by scarification of 
the ear pinna (intra-epithelial route). 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 .beta.-propiolactone-inactivated w.t. HSV-1. 
Vaccinated animals were challenged 3 weeks later with 10.sup.5.2 pfu w.t. 
HSV-2 virus (strain MS) and monitored for the symptoms of primary and 
recurrent disease. 
(a) Following w.t. HSV-2 challenge, 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). 
The results show that intra-epithelial and intra-vaginal vaccination with 
the DISC HSV-1 both provided a high degree of protection against the 
primary symptoms of HSV-2 infection. Surprisingly, inactivated HSV-1 
administered by the intra-epithelial route also provided substantial 
protection, though apparently less than that afforded by the DISC virus 
vaccine. 
(b) Daily vaginal swabs were taken from all animals over a 12 day period 
post-challenge and virus titres determined by plaquing on Vero cells in 
order to monitor growth of the challenge virus in the vagina. The results 
as depicted in FIG. 8 shows that infection virus titres in mock-vaccinated 
animals rose to a maximum of 3.times.10.sup.4 at day 2 post challenge, and 
could be detected until day 10. By contrast, virus titres in the 
vaccinated animals declined steadily from day 1, and were undetectable by 
day 7. No significant different was observed between the groups immunised 
with the DISC HSV-1 or the inactivated virus preparation. 
(c) Following HSV-2 challenge, animals which had fully recovered from the 
acute phase of disease by 28 days were monitored daily for a further 100 
days for the recurrence of disease. Numbers of animals in each group were: 
DISC/Intra-vaginal -14; DISC/Intra-epithelial -12; 
Inactivated/Intra-epithelial -14; Mock/Intra-vaginal -12. Clinical lesions 
were scored as a direct numerical value, and erythema was scored on a 
scale of 1-5. The results are shown in FIGS. 9a and 9b. Points on the 
graphs represent the cumulative totals of mean values per day per animal. 
The results show that animals vaccinated with the DISC HSV-1 by the 
Intra-vaginal route showed approximately a 50% reduction in the number of 
recurrent HSV-2 lesions occurring over the 100 day follow-up period. 
Intra-epithelial vaccination with DISC HSV-1 and inactivated virus also 
resulted in a reduction of recurrent lesions, but to a lesser extent. 
(2) The following experiment was also designed to assess the immunogenicity 
of candidate DISC vaccines based on HSV-1 against genital HSV-2 infection. 
The experiment was designed to compare different vaccination routes (per 
vaginum, oral and nasal ie different mucosal surfaces) and different doses 
of either DISC HSV-1 or inactivated HSV-1 in the guinea pig. 
MATERIALS AND METHODS 
Virus. 
(i) DISC HSV-1 was propagated on Vero cells (F6) which had been transfected 
with the HSV-1 gH gene as described previously in w092/05263 published on 
2 Apr. 1992. Briefly, 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.degree. C. Virus titres were 
determined on F6 cells. 
(ii) DISC HSV-1 was inactivated by the addition of .beta.-propiolactone at 
a concentration of 0.05% for one hour at room temperature. Inactivation 
was checked by adding the virus to F6 cells. 
(iii) HSV-2 strain MS was propagated and titred on Vero cells in the same 
manner as DISC HSV-1 as described above. Animals 
Female Dunkin-Hartley guinea-pigs (300-350 g) were obtained from Davis 
Hall, Darley Oaks Farms, Newchurch, Nr. Burton-on-Trent. 
EXPERIMENTAL DESIGN 
Groups of 12 animals were immunised with two doses of 8.times.10.sup.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.sup.5.2 pfu HSV-2 
(strain MS) and the experiment blinded by randomisation of the cages by an 
independent worker. For a period of 11 days following challenge, animals 
were monitored for the symptoms of primary disease. Clinical observations 
were scored as the number of lesions present in the vaginal area and the 
presence of erythema (scored on a scale of 1-5). In addition, daily 
vaginal swabs were taken from all animals over a 12 day period post 
challenge and virus titres were determined by plaquing on Vero cells in 
order to monitor growth of the challenge virus in the vagina. Statistical 
methods 
Differences in group clinical scores were tested for significance using the 
Mann-Whitney U test. Values of p &lt;0.1 were considered significant. 
RESULTS 
Clinical disease profile. 
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. As 
compared to mock vaccinated animals, vaccination with DISC HSV-1 by the 
intravaginal route provided a high degree of protection from primary 
symptoms of infection. In contrast, 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. 
TABLE 2 
______________________________________ 
INCIDENCE OF PRIMARY DISEASE SYMPTOMS 
Disease 
Any disease 
Lesion Duration 
ongoing 
symptoms score &gt;5 of disease 
on day 11 
Immunisation 
(% of (% of (mean no. 
(% of 
with animals) animals) days) animals) 
______________________________________ 
mock 92 75 6.8 75 
DISC HSV-1 
33 17 4.5 8 
i.vag 
HSV-1 92 67 6.2 83 
inactivated 
i.vag 
DISC HSV-1 
33 0 2.3 0 
i.nas 
HSV-1 67 17 6.3 42 
inactivated 
i.nas 
DISC HSV-1 
90 20 4.1 20 
oral 
HSV-1 91 36 5.8 64 
inactivated 
oral 
______________________________________ 
Thus the following conclusions can be drawn from this experiment with the 
in vivo guinea pig model. 
A. Vaccination with DISC HSV-1 via the intravaginal and intranasal routes 
led to a high degree of protection from acute disease symptoms following a 
challenge with HSV-2. 
B. Intranasal administration of DISC HSV-1 gave the highest degree of 
protection when considering the number of days of severe disease (as 
defined by the presence of 6 or more lesions). 
C. 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 DISC HSV-1 vaccination via this 
route. 
D. A significant difference was observed between disease symptoms in 
animals vaccinated orally with DISC HSV-1 and mock-infected animals. 
However, this degree of protection was less than that observed in animals 
vaccinated with DISC HSV-1 via the intranasal or intravaginal route. 
E. Symptoms in animals vaccinated orally with inactivated DISC HSV-1 were 
not significantly different from those in the mock-infected group. 
F. The data on shed virus is interesting. Surprisingly the per vaginum 
vaccination route resulted in significantly lower levels of recovered 
virus following the challenge dose. This may be due to local antibody 
production. 
(3) The following experiment was designed to investigate HSV-2 induced 
recurrent disease following therapeutic vaccination. 
This was of interest as it has previously been shown that therapeutic 
administration of certain recombinant HSV-2 antigens, together with 
adjuvant, can decrease the frequency of subsequent recurrences. 
(Stanberry, L. R. et al. J. Inf. Dis. 1988; 157, p156-163; Stanberry, L. 
R. et al. J. Gen. Virol. 1989a; 70 p3177-3185; Ho, R. J. Y. et al, J. 
Virol. 1989; 63p 2951-2958). 
Accordingly 21 animals which had recovered fully from primary HSV-2 disease 
four weeks after challenge were randomised into three groups, and treated 
with live DISC HSV-1 intravaginally (10 animals), or intra-epithelially 
(11 animals). A group of 12 animals, which had previously acted as 
controls for prophylactic vaccination (see (2) above) and which had also 
recovered fully from primary disease were treated with an equivalent mock 
preparation (12 animals). The animals were given further identical 
treatments 24 and 48 days later. The frequency of recurrent disease was 
monitored from the day of first treatment for a further 100 days, and the 
cumulative results are shown in FIG. 13 and summarised in Table 3 below. 
TABLE 3 
__________________________________________________________________________ 
Effect of therapeutic vaccination on recurrent disease 
DISC HSV-1 
DISC HSV-1 
Mock Intra-epithelial 
Intra-vaginal 
Total 
% of Mock 
Total 
% of Mock 
Total 
% of Mock 
__________________________________________________________________________ 
1 Mean total disease/days 
9.41 
100 6.90 
73 7.32 
78 
per animal 
2 Mean total episodes per 
6.27 
100 4.67 
74 5.10 
81 
animal 
3 Disease incidence 
12/12 
100 9/11 
82 10/10 
100 
4 Severity per episode 
3.21 
100 3.00 
93 2.86 
89 
Mean duration of 
1.49 1.27 1.38 
episode (days) 
__________________________________________________________________________ 
1 Total number of days where disease was observed (either lesions or 
erythema) over the whole observation period (100 days from 1 month after 
challenge with HSV2) 
2 Total of days disease episodes over the whole observation period 
(episode length defined as period between two consecutive diseasefree day 
3 Proportion of animals showing any lesion or erythema score during whole 
observation period 
4 Total sum of erythema scores and lesion numbers over the whole 
observation period divided by number of episodes observed 
It can be seen that each of the groups treated with DISC HSV-1 appeared to 
experience a modest reduction (about 25%) in the overall number of 
disease/days and episodes especially over the 50 day period following 
second vaccination. 
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 riot 
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. 
Thus therapeutic administration of DISC HSV-1 virus either intra-vaginally 
or intra-epithelially resulted in an apparent reduction (20-25%) in the 
frequency of recurrence compared with mock-treated animals. 
(4) The following experiment was designed to investigate the therapeutic 
value of a DISC virus based on HSV-2. A DISC HSV-2 (strain HG 52) having a 
deletion of the gH gene was made as described earlier and in accordance 
with the general teaching of WO92/05263 published on 2 Apr. 1992 and 
corresponding to U.S. Pat. No. 5,665,362 issued Sep. 9, 1997, incorporated 
herein by reference and also using standard procedures in the art. The 
DISC version of the strain was grown in Vero cells transfected with the 
HSV-2 gH gene also in accordance with the teaching of WO92/05263. 
The experiment was a head to head comparison of DISC HSV-1 with DISC HSV-2 
in female 350-400 gms guinea-pigs. Guinea-pigs were divided into three 
groups. All guinea-pigs were infected with 10.sup.5.8 pfu HSV-2 strain MS. 
Four weeks were then allowed for the primary disease to have both 
developed and resolved and for recurrences to have started. The animals 
were then treated. A first group of 15 animals was treated intravaginally 
with a mock preparation of virus consisting of sonicated Vero cells. A 
second group of 13 animals was treated Intravaginally with 10.sup.7 pfu 
DISC HSV-1. A third group of 14 animals was treated intravaginally with 
10.sup.7 pfu DISC HSV-2. Treatment was repeated in 14 days. 
The results are shown in Table 4. 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. 
As shown by the results, it appears that 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. 
TABLE 4 
__________________________________________________________________________ 
Erythema scores Lesions scores 
Disease Days 
Group Total 
Per animal 
% of Mock 
Total 
Per animal 
% of Mock 
Total 
Per animal 
% of Mock 
__________________________________________________________________________ 
Days 1-13 
Mock 38 2.53 100 66 4.40 100 42 2.80 100 
DISC HSV-1 
34 2.62 103 48 3.69 84 34 2.62 93 
DISC HSV-2 
22 1.57 62 40 2.86 65 26 1.86 66 
Days 14-27 
Mock 13 0.87 100 23 1.53 100 17 1.13 100 
DISC HSV-1 
9 0.69 80 14 1.08 70 11 0.85 75 
DISC HSV-2 
2 0.14 16 3 0.21 14 3 0.21 19 
Days 1-27 
Mock 51 3.40 100 89 5.93 100 59 3.93 100 
DISC HSV-1 
43 3.31 97 62 4.77 80 45 3.46 88 
DISC HSV-2 
24 1.71 50 43 3.07 52 29 2.07 53 
__________________________________________________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 12 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3836 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CTGCAGCGCGGCGGGAGGTGGCGGGAGGACTGGGGCCGGCTGACGGGGGTCGCCGCGGCG60 
ACCCCGCGCCCCGACCCCGAGGACGGCGCGGGGTCTCTGCCCCGCATCGAGGACACGCTG120 
TTTGCCCTGTTCCGCGTTCCCGAGCTGCTGGCCCCCAACGGGGACTTGTACCACATTTTT180 
GCCTGGGTCTTGGACGTCTTGGCCGACCGCCTCCTTCCGATGCATCTATTTGTCCTGGAT240 
TACGATCAGTCGCCCGTCGGGTGTCGAGACGCCCTGTTGCGCCTCACCGCCGGGATGATC300 
CCAACCCGCGTCACAACCGCCGGGTCCATCGCCGAGATACGCGACCTGGCGCGCACGTTT360 
GCCCGCGAGGTGGGGGGAGTTTAGTTCAAACACGGAAGCCCGAACGGAAGGCCTCCCGGC420 
GATGACGGCAATAAAAGAACAGAATAAAAGGCATTGTTGTCGTGTGGTGTGTCCATAAGC480 
GCGGGGGTTCGGGGCCAGGGCTGGCACCGTATCAGCACCCCACCGAAAAACGGAGCGGGC540 
CGATCCGTCCTTGTTTTCGGTCTGGTACTCCCTTTGTGCTTTTACCCTCACCCCACCCCA600 
TCCTTTGGCCCGCGCTTACGGCAACAAAGGGCCTCCGATAGCCTCCGAGGTGCGGACGCT660 
CTTTGGGCCGTGGGTACGGACACCCCCCCATCTGCGGACTGGCAGCCGGGACGACGACC719 
ATGGGCCCCGGTCTGTGGGTGGTGATGGGGGTCCTGGTGGNCGTTGCC767 
MetGlyProGlyLeuTrpValValMetGlyValLeuValXaaValAla 
151015 
GGGGGCCATGACACGTACTGGACGGAGCAAATCGACCCGTGGTTTTTG815 
GlyGlyHisAspThrTyrTrpThrGluGlnIleAspProTrpPheLeu 
202530 
CACGGTCTGGGGTTGGCCCGCACGTACTGGCGCGACACAAACACCGGG863 
HisGlyLeuGlyLeuAlaArgThrTyrTrpArgAspThrAsnThrGly 
354045 
CGTCTGTGGTTGCCCAACACCCCCGACGACCAGCGACCCCCAGCGCGG911 
ArgLeuTrpLeuProAsnThrProAspAspGlnArgProProAlaArg 
505560 
ACGCTTGGCGCCCCCGGGCAACTCAACCTGACTACGGCATCCGTGCCC959 
ThrLeuGlyAlaProGlyGlnLeuAsnLeuThrThrAlaSerValPro 
65707580 
ATGCTTCGGTGGTACGCCGAGCGCTTTTGTTTCGTGTTGGTCACCACG1007 
MetLeuArgTrpTyrAlaGluArgPheCysPheValLeuValThrThr 
859095 
GCCGAGTTTCCTCGGGACCCCGGGCAGCTGCTTTACATCCCAAAGACC1055 
AlaGluPheProArgAspProGlyGlnLeuLeuTyrIleProLysThr 
100105110 
TATCTGCTCGGCCGGCCTCGGAACGCGAGCCTGCCCGAGCTCCCCGAG1103 
TyrLeuLeuGlyArgProArgAsnAlaSerLeuProGluLeuProGlu 
115120125 
GCGGGGCCCACGTCCCGTCCCCCCGCCGAGGTGACCCAGCTCAAGGGA1151 
AlaGlyProThrSerArgProProAlaGluValThrGlnLeuLysGly 
130135140 
CTGCTGCACAACCCCGGCGCCTCCGCGATGTTGCGGTCCCGGGCCTGG1199 
LeuLeuHisAsnProGlyAlaSerAlaMetLeuArgSerArgAlaTrp 
145150155160 
GTAACATTCGCGGCCGCGCCGGACCGCGAGGGGCTTACGTTNCCGCGG1247 
ValThrPheAlaAlaAlaProAspArgGluGlyLeuThrXaaProArg 
165170175 
GGAGACGACGGGGCGACCGAGAGGCACCCGGACGGCCGACGCAACGCG1295 
GlyAspAspGlyAlaThrGluArgHisProAspGlyArgArgAsnAla 
180185190 
NCCCCGGGGCCGCCCGCGGGGGCGCCGAGGCATCCGACGACGAACCTG1343 
XaaProGlyProProAlaGlyAlaProArgHisProThrThrAsnLeu 
195200205 
AGCATCGCGCATCTGCACAACGCGTCCGTGANCCTGCTGGCCGCCAGG1391 
SerIleAlaHisLeuHisAsnAlaSerValXaaLeuLeuAlaAlaArg 
210215220 
GGCCTGCTACGGACTCCGGGTCGGTACGTGTACCTCTCCCCGTCGGCC1439 
GlyLeuLeuArgThrProGlyArgTyrValTyrLeuSerProSerAla 
225230235240 
TCGACGTGGCCCGTGGGCGTCTGGACGACGGGCGGGCTGGCGTTCGGG1487 
SerThrTrpProValGlyValTrpThrThrGlyGlyLeuAlaPheGly 
245250255 
TGCGACGCCGCGCTCGTGCGCGCGCGATACGGGAAGGGCTTCATGGGG1535 
CysAspAlaAlaLeuValArgAlaArgTyrGlyLysGlyPheMetGly 
260265270 
CTCGTGATATCGATGCGGGACAGCCCTCCGGCCGAGATCATAGTGGTG1583 
LeuValIleSerMetArgAspSerProProAlaGluIleIleValVal 
275280285 
CCTGCGGACAAGACCCTCGCTCGGGTCGGAAATCCGACCGACGAAAAC1631 
ProAlaAspLysThrLeuAlaArgValGlyAsnProThrAspGluAsn 
290295300 
GCCCCGCGTGCTCCCCGCGCTCCGGCCGGCCCCAGGTATCGCGTCTTT1679 
AlaProArgAlaProArgAlaProAlaGlyProArgTyrArgValPhe 
305310315320 
GTCCTGGGGGCCCCGACGCCCGCCGACAACGGCNTCGGCGCTGGACCC1727 
ValLeuGlyAlaProThrProAlaAspAsnGlyXaaGlyAlaGlyPro 
325330335 
CCTCGGCGGGTGGCCGGCTACCCCGAGGAGAGCACGAACTACGCCCAG1775 
ProArgArgValAlaGlyTyrProGluGluSerThrAsnTyrAlaGln 
340345350 
TATATGTCGCGGGCCTATGCGGAGTTTTTGGGGGAGGACCCGGGCTCC1823 
TyrMetSerArgAlaTyrAlaGluPheLeuGlyGluAspProGlySer 
355360365 
GGCACGGACGACGCGCGTCCGTCCCTGTTCTGGCGCCTCGCGGGGCTG1871 
GlyThrAspAspAlaArgProSerLeuPheTrpArgLeuAlaGlyLeu 
370375380 
CTCGCCTCGTCGGGGTTTGCGTTCGTCAACGCGGCCCACGCCCACGAC1919 
LeuAlaSerSerGlyPheAlaPheValAsnAlaAlaHisAlaHisAsp 
385390395400 
GCGATTCGCCTCTCCGACCTGCTGGGTTTTTTGGCCCACTCGCGCGTG1967 
AlaIleArgLeuSerAspLeuLeuGlyPheLeuAlaHisSerArgVal 
405410415 
CTGGCCGGCCTGGCCGCCCGGGGAGCAGCGGGCTGCGCGGCCGACTCG2015 
LeuAlaGlyLeuAlaAlaArgGlyAlaAlaGlyCysAlaAlaAspSer 
420425430 
GTGTTCCTGAACGTGTCCGTGTTGGACCCGGCGGCCCGTCTGCGGCTG2063 
ValPheLeuAsnValSerValLeuAspProAlaAlaArgLeuArgLeu 
435440445 
GAGGCGCGCCTCGGGCATCTGGTGGCCGCGATCCTCGAGCGAGAGCAG2111 
GluAlaArgLeuGlyHisLeuValAlaAlaIleLeuGluArgGluGln 
450455460 
AGCCTGGCGGCGCACGCGCTGGGCTATCAGCTGGCGTTCGTGTTGGAC2159 
SerLeuAlaAlaHisAlaLeuGlyTyrGlnLeuAlaPheValLeuAsp 
465470475480 
AGCCCCGCGGCCTATGGCGGGTTGGCCCCGAGCGCGGCCCGCCTGATC2207 
SerProAlaAlaTyrGlyGlyLeuAlaProSerAlaAlaArgLeuIle 
485490495 
GACGCCCTTGTTACCGCGCAGTTTCTCGGCGGCCGCGTAACCGCCCCG2255 
AspAlaLeuValThrAlaGlnPheLeuGlyGlyArgValThrAlaPro 
500505510 
ATGGTCCGCCGAGCGCTGTTTTACGCCACGGCCGTCCTCCGGGCGCCG2303 
MetValArgArgAlaLeuPheTyrAlaThrAlaValLeuArgAlaPro 
515520525 
TTCCTGGCGGGCGTGCCCTCGGCCGGGCAGCGGGAACGCCCGCGGGGC2351 
PheLeuAlaGlyValProSerAlaGlyGlnArgGluArgProArgGly 
530535540 
CTCCTCATAACCACGGCCCTGTGTACGTCCGACGTCGCCGCGGCGACC2399 
LeuLeuIleThrThrAlaLeuCysThrSerAspValAlaAlaAlaThr 
545550555560 
CACGCCGATCTCCGGGCCGCGCTACGCAGGACCGACCACCAGAAAAAC2447 
HisAlaAspLeuArgAlaAlaLeuArgArgThrAspHisGlnLysAsn 
565570575 
CTCTTCTGGCTCCCGGACCACTTTTCCCCATGCGCACGTTCCCTGCCG2495 
LeuPheTrpLeuProAspHisPheSerProCysAlaArgSerLeuPro 
580585590 
TTCGATCTCGCCGAGGGCGGGTTCATCCTGGACGCGCTGGCCATGGCC2543 
PheAspLeuAlaGluGlyGlyPheIleLeuAspAlaLeuAlaMetAla 
595600605 
ACCCGATCCGACATCCCGGCGGACGTCATGGCACAACAGACCCGCGGC2591 
ThrArgSerAspIleProAlaAspValMetAlaGlnGlnThrArgGly 
610615620 
GTGGCCTCCGCTCTCACGCNCTGGGCGACTCACAACGCCCTGATCCGC2639 
ValAlaSerAlaLeuThrXaaTrpAlaThrHisAsnAlaLeuIleArg 
625630635640 
GCCTTCGTCCCGGAGGCCACCCACCAGTGTAGCGGCCCGTCGCACAAC2687 
AlaPheValProGluAlaThrHisGlnCysSerGlyProSerHisAsn 
645650655 
GNGGAGCCCCGGATCCTCGTGCCCATCACCCACAACGCCAGCTACGTC2735 
XaaGluProArgIleLeuValProIleThrHisAsnAlaSerTyrVal 
660665670 
GTCACCCACTACCCCCCTTGCCCCCGCGGGATCGGATACAAGCTTACG2783 
ValThrHisTyrProProCysProArgGlyIleGlyTyrLysLeuThr 
675680685 
GGCGTTGACGTCCGCCGCCCGCTGTTTATCACCTATCTCACCGCCACC2831 
GlyValAspValArgArgProLeuPheIleThrTyrLeuThrAlaThr 
690695700 
TGCGAAGGGCACGCGCGGGAGATTGAGCCGCCGCGGCTGGTGCGCACC2879 
CysGluGlyHisAlaArgGluIleGluProProArgLeuValArgThr 
705710715720 
GAAAACCGGCGCGACCTCGGCCTCGTGGGGGCCGTGTTTCTGCGCTAC2927 
GluAsnArgArgAspLeuGlyLeuValGlyAlaValPheLeuArgTyr 
725730735 
ACCCCGGCCGGGGAGGTCATGTCGGTGCTGCTGGTGGACACGGATGCC2975 
ThrProAlaGlyGluValMetSerValLeuLeuValAspThrAspAla 
740745750 
ACCCAACAGCAGCTGGCCCAGGGGCCGGTGGCGGGCACCCCGAACGTG3023 
ThrGlnGlnGlnLeuAlaGlnGlyProValAlaGlyThrProAsnVal 
755760765 
TTTTCCAGCGACGTGCCGTCCGTGGCCCTGTTGTTGTTCCCCAACGGA3071 
PheSerSerAspValProSerValAlaLeuLeuLeuPheProAsnGly 
770775780 
ACTGTGATTCATCTGCTGGCCTTTGACACGCTGCCCATCGCCACCATC3119 
ThrValIleHisLeuLeuAlaPheAspThrLeuProIleAlaThrIle 
785790795800 
GCCCCCGGGTTTCTGGCCGCGTCCGCGCTGGGGGTCGTTATGATTACC3167 
AlaProGlyPheLeuAlaAlaSerAlaLeuGlyValValMetIleThr 
805810815 
GCGGCCCTGGCGGGCATCCTCAGGGTGGTCCGAACGTGCGTCCCATTT3215 
AlaAlaLeuAlaGlyIleLeuArgValValArgThrCysValProPhe 
820825830 
TTGTGGAGACGCGAATAAACGGGTGTGTGGACGCAGCGGCGTCCAGCCCAACCCA3270 
LeuTrpArgArgGlu 
835 
ACCGACTCCCTCCGTGTCCGCGGTCTGTTTGTTATTGTGTCCGCCGTGGCTCCGCTACCG3330 
CCTCTGTTCCTTTCCCTTCTCCATTCCTGTTTCCTTTCCTTCCCCCCCCCCCATAGTCCC3390 
CCGTATAGGCATACAACGGCATCCGTGGGTTAGAAAACGACTGCACTTTATTGGGATATC3450 
TCACACAGACTGGCCGTGCTGGGCGCGAGCCAGGCAAACGGTAAGCAGCGCGTCCAGGTA3510 
CCCGGCGGTTCGCGTGCGGCCAGCCGCCCCCGCCGGCCCGCGGTCAAACGCGGACATCCG3570 
GTCGACGTCCCCCACGGTCAGGACCAGGGACGTCACGCCCGTCAGGCGCNCGGTATGCGT3630 
GGCCGCGGCCAGGCGTCCGTGGCCGGCGTACAACACGCCCAGGAACGCGCCGAGGTACAT3690 
GACGTGCTCGGGCGAGACGGACCCCCCCGGGGTCAGGCGTTGCGAGTCCACAAAGCGCAG3750 
CAGGGCGGCGCTGTCGGCCCGCGACGTCGCTCCCCACCGGCACGTCCTTGGGCGGGAGGA3810 
GGTCGAACATGAGGAGCTGCTCGCGA3836 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 837 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGlyProGlyLeuTrpValValMetGlyValLeuValGlyValAla 
151015 
GlyGlyHisAspThrTyrTrpThrGluGlnIleAspProTrpPheLeu 
202530 
HisGlyLeuGlyLeuAlaArgThrTyrTrpArgAspThrAsnThrGly 
354045 
ArgLeuTrpLeuProAsnThrProAspAspGlnArgProProAlaArg 
505560 
ThrLeuGlyAlaProGlyGlnLeuAsnLeuThrThrAlaSerValPro 
65707580 
MetLeuArgTrpTyrAlaGluArgPheCysPheValLeuValThrThr 
859095 
AlaGluPheProArgAspProGlyGlnLeuLeuTyrIleProLysThr 
100105110 
TyrLeuLeuGlyArgProArgAsnAlaSerLeuProGluLeuProGlu 
115120125 
AlaGlyProThrSerArgProProAlaGluValThrGlnLeuLysGly 
130135140 
LeuLeuHisAsnProGlyAlaSerAlaMetLeuArgSerArgAlaTrp 
145150155160 
ValThrPheAlaAlaAlaProAspArgGluGlyLeuThrLeuProArg 
165170175 
GlyAspAspGlyAlaThrGluArgHisProAspGlyArgArgAsnAla 
180185190 
AlaProGlyProProAlaGlyAlaProArgHisProThrThrAsnLeu 
195200205 
SerIleAlaHisLeuHisAsnAlaSerValSerLeuLeuAlaAlaArg 
210215220 
GlyLeuLeuArgThrProGlyArgTyrValTyrLeuSerProSerAla 
225230235240 
SerThrTrpProValGlyValTrpThrThrGlyGlyLeuAlaPheGly 
245250255 
CysAspAlaAlaLeuValArgAlaArgTyrGlyLysGlyPheMetGly 
260265270 
LeuValIleSerMetArgAspSerProProAlaGluIleIleValVal 
275280285 
ProAlaAspLysThrLeuAlaArgValGlyAsnProThrAspGluAsn 
290295300 
AlaProArgAlaProArgAlaProAlaGlyProArgTyrArgValPhe 
305310315320 
ValLeuGlyAlaProThrProAlaAspAsnGlyValGlyAlaGlyPro 
325330335 
ProArgArgValAlaGlyTyrProGluGluSerThrAsnTyrAlaGln 
340345350 
TyrMetSerArgAlaTyrAlaGluPheLeuGlyGluAspProGlySer 
355360365 
GlyThrAspAspAlaArgProSerLeuPheTrpArgLeuAlaGlyLeu 
370375380 
LeuAlaSerSerGlyPheAlaPheValAsnAlaAlaHisAlaHisAsp 
385390395400 
AlaIleArgLeuSerAspLeuLeuGlyPheLeuAlaHisSerArgVal 
405410415 
LeuAlaGlyLeuAlaAlaArgGlyAlaAlaGlyCysAlaAlaAspSer 
420425430 
ValPheLeuAsnValSerValLeuAspProAlaAlaArgLeuArgLeu 
435440445 
GluAlaArgLeuGlyHisLeuValAlaAlaIleLeuGluArgGluGln 
450455460 
SerLeuAlaAlaHisAlaLeuGlyTyrGlnLeuAlaPheValLeuAsp 
465470475480 
SerProAlaAlaTyrGlyGlyLeuAlaProSerAlaAlaArgLeuIle 
485490495 
AspAlaLeuValThrAlaGlnPheLeuGlyGlyArgValThrAlaPro 
500505510 
MetValArgArgAlaLeuPheTyrAlaThrAlaValLeuArgAlaPro 
515520525 
PheLeuAlaGlyValProSerAlaGlyGlnArgGluArgProArgGly 
530535540 
LeuLeuIleThrThrAlaLeuCysThrSerAspValAlaAlaAlaThr 
545550555560 
HisAlaAspLeuArgAlaAlaLeuArgArgThrAspHisGlnLysAsn 
565570575 
LeuPheTrpLeuProAspHisPheSerProCysAlaArgSerLeuPro 
580585590 
PheAspLeuAlaGluGlyGlyPheIleLeuAspAlaLeuAlaMetAla 
595600605 
ThrArgSerAspIleProAlaAspValMetAlaGlnGlnThrArgGly 
610615620 
ValAlaSerAlaLeuThrArgTrpAlaThrHisAsnAlaLeuIleArg 
625630635640 
AlaPheValProGluAlaThrHisGlnCysSerGlyProSerHisAsn 
645650655 
GlyGluProArgIleLeuValProIleThrHisAsnAlaSerTyrVal 
660665670 
ValThrHisTyrProProCysProArgGlyIleGlyTyrLysLeuThr 
675680685 
GlyValAspValArgArgProLeuPheIleThrTyrLeuThrAlaThr 
690695700 
CysGluGlyHisAlaArgGluIleGluProProArgLeuValArgThr 
705710715720 
GluAsnArgArgAspLeuGlyLeuValGlyAlaValPheLeuArgTyr 
725730735 
ThrProAlaGlyGluValMetSerValLeuLeuValAspThrAspAla 
740745750 
ThrGlnGlnGlnLeuAlaGlnGlyProValAlaGlyThrProAsnVal 
755760765 
PheSerSerAspValProSerValAlaLeuLeuLeuPheProAsnGly 
770775780 
ThrValIleHisLeuLeuAlaPheAspThrLeuProIleAlaThrIle 
785790795800 
AlaProGlyPheLeuAlaAlaSerAlaLeuGlyValValMetIleThr 
805810815 
AlaAlaLeuAlaGlyIleLeuArgValValArgThrCysValProPhe 
820825830 
LeuTrpArgArgGlu 
835 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3762 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CTGCAGGGCGGCGGGTCGTGGCGGGAGGATTGGGGACAGCTTTCGGGGGCGGCCGTGCCG60 
CCCCAGGGTGCCGAGCCCCAGAGCAACGCGGGCCCACGACCCCATATCGGGGACACGTTA120 
TTTACCCTGTTTCGGGCCCCCGAGTTGCTGGCCCCCAACGGCGACCTGTATAACGTGTTT180 
GCCTGGGCTTTGGACGTCTTGGCCAAACGCCTCCGTCCCATGCATGTCTTTATCCTGGAT240 
TACGACCAATCGCCCGCCGGCTGCCGGGACGCCCTGCTGCAACTTACCTCCGGGATGGTC300 
CAGACCCACGTCACCACCCCAGGCTCCATACCGACGATCTGCGACCTGGCGCGCACGTTT360 
GCCCGGGAGATCCGGGAGCCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCG420 
CGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATA480 
AACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGG540 
ACCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCAACCCCCAAGTTCGGGTGAAGGCC600 
CAGGGCTCGCAGCCAACGTCGGGGCGGCAAGCCCTGCCATAGCCACGGGCCCCGTGGGTT660 
AGGGACGGGGTCCCCCATGGGGAATGGTTTATGGTTCGTGGGGGTTATTATTTTGGGCGT720 
TGCGTGGGGTCAGGTCCACGACTGGACTGAGCAGACAGACCCATGGTTTTTGGATGGCCT780 
GGGCATGGACCGCATGTACTGGCGCGACACGAACACCGGGCGTCTGTGGCTGCCAAACAC840 
CCCCGACCCCCAAAAACCACCGCGCGGATTTCTGGCGCCGCCGGACGAACTAAACCTGAC900 
TACGGCATCTCTGCCCCTTCTTCGCTGGTACGAGGAGCGCTTTTGTTTTGTATTGGTCAC960 
CACGGCCGAGTTTCCGCGGGACCCCGGCCAGCTGCTTTACATCCCGAAGACCTACCTGCT1020 
CGGCCGGCCCCCGAACGCGAGCCTGCCCGCCCCCACCACGGTCGAGCCGACCGCCCAGCC1080 
TCCCCCCTCGGTCGCCCCCCTTAAGGGTCTCTTGCACAATCCAGCCGCCTCCGTGTTGCT1140 
GCGTTCCCGGGCCTGGGTAACGTTTTCGGCCGTCCCTGACCCCGAGGCCCTGACGTTCCC1200 
GCGGGGAGACAACGTGGCGACGGCGAGCCACCCGAGCGGGCCGCGTGATACCCGCCCCCC1260 
CGACCGCCGGTTGGGGCCCGGCGGCACCCGACGACGGAGCTGGACATCACGCACCTGCAC1320 
AACGCGTCCACGACCTGGTTGGCCACCCGGGGCCTGTTGAGATCCCCAGGTAGGTACGTG1380 
TATTTCTCCCCGTCGGCCTCGACGTGGCCCGTGGGCATCTGGACGACGGGGGAGCTGGTG1440 
CTCGGGTGCGATGCCGGGGTGGTGCGCGCGCGCTACGGGCGGGAATTCATGGGGCTCGTG1500 
ATATCCATGCACGACAGCCCTCCGGTGGAAGTGATGGTGGTCCCCGCGGGCCAGACGCTA1560 
GATCGGGTCGGGGACCCCGCGGACGAAAACCCCCCGGGGGCTCTTCCCGGGCCCCCGGGC1620 
GGCCCCCGGTATCGGGTCTTTGTCCTAGGGTCCCTGACGCGGGCCGACAACGGCTCCGCG1680 
CTGGACGCCCTCCGCCGCGTGGGCGGCTACCCGGAGGAGGGCACGAACTACGCCCAGTTC1740 
CTGTCGCGGGCATACGCGGAGTTTTTCTCGGGGGACGCGGGCGCCGAGCAGGGCCCGCGC1800 
CCCCCTCTCTTCTGGCGCCTAACGGGGCTGCTCGCGACGTCGGGTTTTGCTTTCGTGAAC1860 
GCCGCCCACGCAAACGGCGCGGTCTGCCTCTCCGACCTGCTAGGCTTTTTGGCCCACTCG1920 
CGCGCGCTTGCCGGGTTGGCCGCCCGCGCGGCCGCGGGCTGTGCCGCGGATTCTGTGTTT1980 
TTTAATGTGTCAGTCTTGGATCCCACGGCCCGCCTGCAGCTAGAGGCTCGGCTCCAGCAC2040 
CTGGTGGCCGAGATTCTGGAGCGCGAACAGAGCTTGGCATTACACGCGCTGGGCTATCAG2100 
CTGGCCTTCGTGCTGGATAGCCCCTCGGCGTACGACGCAGTGGCGCCCAGCGCAGCCCAT2160 
CTCATCGACGCCCTGCTATGCCCGAGTTTCTAGGGGGCCGCGTGCTGACCACCCCGGTCG2220 
TCCACCGGGCGCTATTTTACGCCTCGGCTGTCCTCCGGCAGCCGTTCTTGGCTGGCGTCC2280 
CCTCGGCGGTGCAGCGGGAACGCGCCCGCCGGACCCTTCTGATAGCCTCGGCCCTGTGTA2340 
CGTCCGACGTCGCCGCAGCGACCAACGCCGACCTCCGGACCGCGCTGGCCCGGGCCGACC2400 
ACCAGAAAACCCTCTTTTGGCTTCCGGACCACTTTTCGCCATGCGCGGCCTCCCTGCGCT2460 
TTGATCTAGACGAGAGCGTGTTTATCCTGGACGCGCTGGCTCAAGCCACCCGATCCGAGA2520 
CCCCGGTCGAAGTCCTGGCCCAGCAGACCCACGGCCTCGCCTCGACCCTGACGCGTTGGG2580 
CACACTACAACGCCCTGATCCGCGCCTTCGTCCCTGAGGCCTCACATCGGTGCGGGGGGC2640 
AGTCTGCCAACGTCGAGCCACGGATCCTGGTACCCATCACCCACAACGCCAGCTACGTCG2700 
TCACCCACTCCCCTCTGCCCCGGGGGATCGGCTACAAGCTCACCGGCGTCGACGTCCGAC2760 
GCCCACTGTTCCTAACCTACCTCACCGCGACATGCGAAGGCTCCACCCGGGATATCGAGT2820 
CCAAGCGGCTGGTGCGCACCCAAAACCAGCGCGACCTGGGGCTCGTGGGGGCCGTGTTTA2880 
TGCGCTACACCCCGGCCGGGGAGGTCATGTCTGTGTTGCTGGTGGATACGGACAACACAC2940 
AGCAGCAAATCGCCGCCGGGCCGACGGAGGGCGCCCCAAGCGTGTTTTCGAGCGACGTGC3000 
CGTCCACGGCCTTGTTGCTATTTCCAAACGGAACCGTCATTCATTTGCTAGCCTTTGACA3060 
CGCAGCCCGTGGCCGCAATTGCGCCCGGGTTTCTGGCCGCCTCTGCGCTGGGCGTGGTTA3120 
TGATTACCGCCGCCCTGGCTGGCATCCTAAAGGTTCTCCGGACAAGTGTCCCGTTTTTTT3180 
GGAGACGCGAATAAAGTGGGCGTGGCTTCGGCCGTTTCTCCGCCCGACCGAATAAACTGT3240 
AACCGTGTCTGTGGTTTGTTTGTTCAGGCCCCGGTGGTGCCGCTCCCCCAGCCCCTCTTT3300 
GCTTTCCCTCCCCCCCCCCCGGAGAGGCGTCCATTGACACACAAGGGTGTAGTAGCGATA3360 
TACGTTTATTGGGGTCTTTTACACAGACTGTCCGTGTTGGGAGCGAGCGAGACGAACGGT3420 
AAGAAGCACATCCAGGTACCCGGCGGCCCGCGTGCGGCTGGCCGCGCCCGCCGCTCCGCG3480 
GTCAAACGCGGAAAGACGGTCCACGTCACCCACCGCTAGCACCAGGGAGGTCACCCCTGT3540 
CAGCCGCGCGGTGTGCGTGGCTGCGGACATGCGCCCGCGGCCAGCGTACAGCACGCTCAG3600 
GAACGCACCAAGGTACGCGACGTGCTCGGGGGAGATCACCCCCCCGGGGACGGCGAGACG3660 
TTGCGATTCTATAAAGCGCAGCAGAGCGGTGCTGTCGGCCTGCACGTCGCTTCCCACCGG3720 
CACGTCCTTTGGGGGGAGAAGGTCGAACATGAGAGCTGCTCG3762 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 838 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetGlyAsnGlyLeuTrpPheValGlyValIleIleLeuGlyValAla 
151015 
TrpGlyGlnValHisAspTrpThrGluGlnThrAspProTrpPheLeu 
202530 
AspGlyLeuGlyMetAspArgMetTyrTrpArgAspThrAsnThrGly 
354045 
ArgLeuTrpLeuProAsnThrProAspProGlnLysProProArgGly 
505560 
PheLeuAlaProProAspGluLeuAsnLeuThrThrAlaSerLeuPro 
65707580 
LeuLeuArgTrpTyrGluGluArgPheCysPheValLeuValThrThr 
859095 
AlaGluPheProArgAspProGlyGlnLeuLeuTyrIleProLysThr 
100105110 
TyrLeuLeuGlyArgProProAsnAlaSerLeuProAlaProThrThr 
115120125 
ValGluProThrAlaGlnProProProSerValAlaProLeuLysGly 
130135140 
LeuLeuHisAsnProAlaAlaSerValLeuLeuArgSerArgAlaTrp 
145150155160 
ValThrPheSerAlaValProAspProGluAlaLeuThrPheProArg 
165170175 
GlyAspAsnValAlaThrAlaSerHisProSerGlyProArgAspThr 
180185190 
ProProProArgProProValGlyAlaArgArgHisProThrThrGlu 
195200205 
LeuAspIleThrHisLeuHisAsnAlaSerThrThrTrpLeuAlaThr 
210215220 
ArgGlyLeuLeuArgSerProGlyArgTyrValTyrPheSerProSer 
225230235240 
AlaSerThrTrpProValGlyIleTrpThrThrGlyGluLeuValLeu 
245250255 
GlyCysAspAlaAlaLeuValArgAlaArgTyrGlyArgGluPheMet 
260265270 
GlyLeuValIleSerMetHisAspSerProProValGluValMetVal 
275280285 
ValProAlaGlyGlnThrLeuAspArgValGlyAspProAlaAspGlu 
290295300 
AsnProProGlyAlaLeuProGlyProProGlyGlyProArgTyrArg 
305310315320 
ValPheValLeuGlySerLeuThrArgAlaAspAsnGlySerAlaLeu 
325330335 
AspAlaLeuArgArgValGlyGlyTyrProGluGluGlyThrAsnTyr 
340345350 
AlaGlnPheLeuSerArgAlaTyrAlaGluPhePheSerGlyAspAla 
355360365 
GlyAlaGluGlnGlyProArgProProLeuPheTrpArgLeuThrGly 
370375380 
LeuLeuAlaThrSerGlyPheAlaPheValAsnAlaAlaHisAlaAsn 
385390395400 
GlyAlaValCysLeuSerAspLeuLeuGlyPheLeuAlaHisSerArg 
405410415 
AlaLeuAlaGlyLeuAlaAlaArgGlyAlaAlaGlyCysAlaAlaAsp 
420425430 
SerValPhePheAsnValSerValLeuAspProThrAlaArgLeuGln 
435440445 
LeuGluAlaArgLeuGlnHisLeuValAlaGluIleLeuGluArgGlu 
450455460 
GlnSerLeuAlaLeuHisAlaLeuGlyTyrGlnLeuAlaPheValLeu 
465470475480 
AspSerProSerAlaTyrAspAlaValAlaProSerAlaAlaHisLeu 
485490495 
IleAspAlaLeuTyrAlaGluPheLeuGlyGlyArgValLeuThrThr 
500505510 
ProValValHisArgAlaLeuPheTyrAlaSerAlaValLeuArgGln 
515520525 
ProPheLeuAlaGlyValProSerAlaValGlnArgGluArgAlaArg 
530535540 
ArgSerLeuLeuIleAlaSerAlaLeuCysThrSerAspValAlaAla 
545550555560 
AlaThrAsnAlaAspLeuArgThrAlaLeuAlaArgAlaAspHisGln 
565570575 
LysThrLeuPheTrpLeuProAspHisPheSerProCysAlaAlaSer 
580585590 
LeuArgPheAspLeuAspGluSerValPheIleLeuAspAlaLeuAla 
595600605 
GlnAlaThrArgSerGluThrProValGluValLeuAlaGlnGlnThr 
610615620 
HisGlyLeuAlaSerThrLeuThrArgTrpAlaHisTyrAsnAlaLeu 
625630635640 
IleArgAlaPheValProGluAlaSerHisArgCysGlyGlyGlnSer 
645650655 
AlaAsnValGluProArgIleLeuValProIleThrHisAsnAlaSer 
660665670 
TyrValValThrHisSerProLeuProArgGlyIleGlyTyrLysLeu 
675680685 
ThrGlyValAspValArgArgProLeuPheLeuThrTyrLeuThrAla 
690695700 
ThrCysGluGlySerThrArgAspIleGluSerLysArgLeuValArg 
705710715720 
ThrGlnAsnGlnArgAspLeuGlyLeuValGlyAlaValPheMetArg 
725730735 
TyrThrProAlaGlyGluValMetSerValLeuLeuValAspThrAsp 
740745750 
AsnThrGlnGlnGlnIleAlaAlaGlyProThrGluGlyAlaProSer 
755760765 
ValPheSerSerAspValProSerThrAlaLeuLeuLeuPheProAsn 
770775780 
GlyThrValIleHisLeuLeuAlaPheAspThrGlnProValAlaAla 
785790795800 
IleAlaProGlyPheLeuAlaAlaSerAlaLeuGlyValValMetIle 
805810815 
ThrAlaAlaLeuAlaGlyIleLeuLysValLeuArgThrSerValPro 
820825830 
PhePheTrpArgArgGlu 
835 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TCAGTTAACGCCTCTGTTCCTTTCCCTTC29 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TCAGAATTCGAGCAGCTCCTCATGTTCGAC30 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TCAAAGCTTCTGCAGCGCGGCGGGAGGTGG30 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TCAGTTAACCGTCGTCCCGGCTGCCAGTC29 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TCAGTTAACGGACAGCATGGCCAGGTCAAG30 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TCGAAGCTTCAGGGAGTGGCGCAGC25 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TCAGAATTCGTTCCGGGAGCAGGCGTGGA29 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
TCAGTTAACTGCACTAGTTTTAATTAATACGTATGCCGTCCGTCCCGGCTGCCAGTC57 
__________________________________________________________________________