JP2003235588A - Vaccine for use in therapeutic treatment of hsv - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a vaccine effective for inhibiting recurrent herpes simplex virus-induced diseases by utilizing a polynucleotide encoding an isolated glycoprotein. <P>SOLUTION: A recombinant herpes simplex virus (HSV) type 1 glycoprotein (gB1) polypeptide which is produced in host cells and is matured peptide, wherein a signal peptide naturally binding thereto is deficient, or a short C-terminal fragment immunologically equivalent to the HSV gB1 polypeptide, is provided. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION Herpesviruses include herpes simplex viruses containing two closely related variants designated type 1 (HSV-1) and type 2 (HSV-2). These types of viruses show strong cross-reactivity, but can be distinguished by neutralization titration.
HSV-1 and HSV-2 are responsible for various human diseases such as skin infections, genital herpes, viral encephalitis and the like.

Herpes simplex virus is a double-stranded DNA virus, which has a genome of about 150 to 160 kbp in an icosahedral nuclear capsid encapsulated in the membrane. This membrane contains a number of virus-specific glycoproteins, the most common of which are gB, gC, gD, and gE. Where gB and gD are type 1 and 2
Shows cross-reactivity with molds.

Providing a safe and effective vaccine in humans against both HSV-1 and HSV-2, and in the event of an infection, providing a cure for the disease is both medically and scientifically. This is a very interesting problem.

One promising attempt has been to use isolated glycoproteins for prophylaxis. This glycoprotein has been shown to confer protection when injected into mice and then infected with live virus. However, the use of herpes simplex glycoproteins has hitherto mainly depended on viral culture and isolation of membrane proteins. In the commercial production of glycoproteins, handling dangerous pathogens,
Problems related to maintenance of viruses in cultured cells, isolation of glycoproteins that do not contain the viral genome or parts thereof, have prevented the use of glycoproteins as vaccines. Therefore, it is desirable to provide a vaccine using glycoproteins produced by methods other than culturing the virus and separating the membrane proteins.

It is also very useful to develop methods for the therapeutic treatment of herpes infections, that is to reduce the disease or prevent recurrence of the disease after the individual has been infected with the virus. Get interested. Viral infections are usually refractory to treatment with antibiotics, so other methods that do not have significant side effects are of great interest. There are several drugs (especially Acyclovir) for the treatment of recurrent herpes disease.
Although r)) was effective, it is not desirable due to the need for continuous administration over a long period of time to prevent recurrence, resulting in drug resistant mutants in the course of treatment.

[0006]

BACKGROUND OF THE INVENTION Everle and Mou (J. of
Infectious Diseases (198
3) 148: 436-444) report the relative titers of antibodies to the polypeptide antigens unique to HSV-1 in human serum. Marsden et al. (J.
of Virology (1978) 28: 624.
-642) is 117 kilodalton (k) due to intratype recombination between HSV-1 and HSV-2.
It has been reported that the gene corresponding to the glycoprotein of d) is located within 0.35 to 0.40 map units on the HSV genetic map. Ruyechan et al. (Ibid. (197
9) 29: 677-679) report that the position of the glycoprotein B gene lies between 0.30 and 0.42 map units. Skare and Summers V
irology (1977) 76: 581-59.
5) is EcoRI, Xba on HSV-1 DNA
I, HindIII endonuclease cleavage sites are reported. Roizman (Ann. Rev. G
enetics (1979) 13: 25-57)
Report the organization of the HSV genome. DeLucc
a et al. (Virology (1982) 122: 41.
1) is gB1 between 0.345 and 0.368 map units
It has localized several phenotypic variants that are believed to have mutations within the structural gene.

A subunit vaccine extracted from chicken embryo cells infected with HSV-1 or HSV-2
U.S. Pat. Nos. 4,317,811 and 4,374,4
127. Also, Hilfenhau
s et al. (Development. Biol. Standard.
d (1982) 52: 321-331) describes the preparation of a subunit vaccine from a particular HSV-1 species (BW3). Roizm
an et al. (Ibid. (1982) 52: 287-304).
Describes the preparation of non-infectious HSV-1 × HSV-2 recombinants and deletion mutants that have been shown to have efficacy in immunized mice. Wats
on et al. (Science (1982) 218: 381.
-384) not only express the cloned fragment by injection into the nucleus of frog oocytes but also clone the HSV-1 gD gene and E. coli (E. col).
i) describes low level expression of the gene. They also show the nucleotide sequence of the gD gene. Weis et al. (Nature (1983) 30.
2: 72-74) is the g in E. coli.
High level expression of D is reported. This polypeptide induces neutralizing antibodies in rabbits. Berm
an et al. (Science (1983) 222: 524.
-527) report the expression of glycoprotein D in cultured mammalian cells. Lasky et al. (Bio
technology (June 1984) 527-
532) reported the use of this glycoprotein D for immunization of mice. Cohen et al. (J. Viro
1 (1984) 49: 102-108) report on the position and chemical synthesis of specific antigenic determinants of gD contained within residues 8-23 of the mature protein.

[0008] The use of a membrane protein preparation from cells infected with HSV as a "treatment" as a post-infection vaccine for humans is described by Dundarov, S. et al. Et al (Do
v. Biol. Standard (1987) 5
7: 351-357) and Skinner, G .;
R. B. Et al. (Id. (1982) 52: 333-3.
4).

[0009]

SUMMARY OF THE INVENTION Vaccines and compositions for use in the treatment of herpes simplex virus types 1 and 2 and methods for their production are provided. These therapies use a combination of virus-specific polypeptides produced by recombinant DNA technology. In particular, HSV gB and gD were produced in a modified mammalian host and used in combination. These polypeptides can be used to treat herpes simplex virus infections in animals, including humans.

Accordingly, one aspect of the present invention is a vaccine used for the therapeutic treatment of herpes simplex virus (HSV) infections. The vaccine comprises a. Immunologically active herpes simplex virus glycoprotein B
(GB) polypeptide; and; b. Immunologically active herpes simplex virus glycoprotein D
(GD) polypeptide.

Here, the polypeptide is prepared by expressing a recombinant DNA construct in a mammalian cell.
HSV when administered to an individual after primary infection with
Is present in an amount effective to prevent recurrence of HSV-induced disease in the individual infected with S.

Another aspect of the invention is a vaccine used to treat herpes simplex virus (HSV) infection.
The vaccine comprises a. Immunologically active herpes simplex virus glycoprotein B
(GB) polypeptide; or b. Immunologically active herpes simplex virus glycoprotein D
(GD) polypeptide.

Here, the polypeptide is obtained by expressing a recombinant DNA construct in a mammalian cell, and the polypeptide is the HSV
HSV when administered to an individual after primary infection with
Is present in an amount effective to prevent recurrence of HSV-induced disease in the individual infected with S.

[0014]

According to the present invention, herpes simplex virus (HSV) glycoprotein type B1 (gB
Recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide produced in a host cell containing the isolated nucleotide sequence according to FIGS. Equivalent fragments, or mixtures thereof, are provided.

According to the present invention, a recombinant produced in a host cell containing the isolated nucleotide sequence set forth in Figures 4 to 7 which encodes herpes simplex virus (HSV) glycoprotein type B2 (gB2). Herpes simplex virus (HSV) glycoprotein B (gB) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof are provided.

According to the invention, herpes simplex virus (HSV) glycoprotein B (gB) and its precursors,
Alternatively, recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof produced in host cells containing nucleotide sequences encoding their analogs. Recombinant herpes simplex virus comprising a nucleotide sequence encoding i) the amino acid sequence set forth in Figures 4-7, or ii) an immunological equivalent of the amino acid sequence ( HSV) glycoprotein B (gB) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof are provided.

In one embodiment, the amino acid sequence of the immunological equivalent differs from the amino acid sequence by less than 5%.

In one embodiment, the nucleotide sequence is the plasmid pHS112 (ATCC No.
39650), or nucleotide sequences that differ from them only in the degeneracy of the codons.

In one embodiment said immunologically equivalent fragment has at least 15 amino acids.

In one embodiment said immunologically equivalent fragment has at least 30 amino acids.

In one embodiment, said polypeptide, immunologically equivalent fragment thereof, or a mixture thereof is a fusion protein.

According to the invention, said herpes simplex virus (HSV) glycoprotein B (gB) polypeptide and / or herpes simplex virus (HSV) glycoprotein D (gD), an immunologically equivalent fragment thereof, or Recurrent herpes simplex virus (HS) containing mixtures thereof
V) A vaccine effective to prevent induced diseases is provided.

In one embodiment, the herpes simplex virus (HSV) glycoprotein D (gD) is herpes simplex virus (HSV) glycoprotein type D1 (gD).
1) and / or herpes simplex virus (HSV) glycoprotein type D2 (gD2) polypeptides.

[0024] In one embodiment, the immunologically equivalent fragment of said herpes simplex virus (HSV) glycoprotein D (gD) is the mature herpes simplex virus (HSV).
8 to 23 amino acids of the glycoprotein D (gD) sequence,
And / or contains at least 15 amino acid sequences of the 253 to 283 amino acid sequences.

In one embodiment, the herpes simplex virus (HSV) glycoprotein B (gB) and / or
Or herpes simplex virus (HSV) glycoprotein D
(GD) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof prevent the recurrent HSV-induced disease when the vaccine is administered to an individual during the acute phase of herpes simplex virus (HSV) infection. Present in an amount effective to do so.

[0026] In one embodiment, the vaccine contains a pharmaceutically acceptable carrier and / or an adjuvant.

In one embodiment, the adjuvant is a liposome-forming component such as synthetic phosphatidylcholine and phosphatidylserine; CFA;
IFA; Squalene / Arlacel; Squalene and T
ween 80; and a compound selected from the group consisting of aluminum hydroxide, wherein the adjuvant is N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2 (1'-2'- It further optionally contains a muramyl dipeptide derivative such as dipalmitoyl-sn-glycero-3-hydroxyphosphorylyloxy-ethylamine (MTP-PE).

[0028] In one embodiment, the adjuvant is present in a low oil formulation.

[0029] In one embodiment, the individual infected with herpes simplex virus (HSV) is a mammal.

According to the present invention, it contains a polypeptide capable of binding both anti-herpes simplex virus (HSV) glycoprotein B (gB) antibody and anti-herpes simplex virus (HSV) glycoprotein D (gD) antibody. A composition for detecting both anti-herpes simplex virus (HSV) glycoprotein B (gB) antibody and anti-herpes simplex virus (HSV) glycoprotein D (gD) antibody, wherein the antibody is herpes simplex virus (HSV) Glycoprotein B
Provided is a composition produced by the (gB) polypeptide and the HSV gD polypeptide, or a mixture thereof.

According to the present invention, there is provided a polynucleotide encoding the herpes simplex virus (HSV) glycoprotein B (gB).

According to the present invention, there is provided a DNA construct containing the following nucleotide sequences: (a) an isolation having a nucleotide acid sequence encoding the herpes simplex virus (HSV) glycoprotein B (gB) described above. Nucleotide sequence: (b) a control sequence capable of binding to the nucleotide sequence to drive expression in a host cell.

According to the present invention, a host cell transformed with the above DNA construct is provided.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION (Vaccine) The vaccine of the present invention comprises both type 1 and type 2 recombinant HSV glycoprotein B.
And D are used. The mature (full-length) gB and gD proteins are fragments, precursors, immunologically equivalent (ie, conferring protection against infection) to these mature proteins.
And analogs as well. As used in the claims, the terms "glycoprotein B polypeptide" and "glycoprotein D polypeptide" are intended to include such fragments, precursors, and analogs. Recombinant gB and gD polypeptides are produced in eukaryotic cells, preferably yeast or mammalian cells, most preferably mammalian cells. The length of the fragment is at least about 15 amino acids, preferably at least about 30 amino acids. The vaccine may contain either a mixture of type 1 polypeptides, a mixture of type 2 polypeptides, or a mixture of both type 1 and type 2 polypeptides, or individual polypeptides.

The mixture of gB and gD polypeptides, although used alone, is usually in association with a physiologically and pharmacologically acceptable vehicle, typically water, saline, phosphate buffer, sugar and the like. Used. In addition, the polypeptide mixture can be used with physiologically acceptable adjuvants such as aluminum hydroxide, muramyl dipeptide derivatives and the like. Various adjuvants are effective, as demonstrated in Example 6.3. The choice of adjuvant depends, at least in part, on the stability of the vaccine containing the adjuvant, the route of administration, the efficacy of the adjuvant on the vaccinated individual, and in the case of humans, the adjuvant may be the Food and Drug Administration (FDA). Depends on whether the product is approved for use in humans. Vaccines are delivered by liposomes and / or with immunomodulators such as interleukins 1 and 2. The vaccine may be administered by any convenient parenteral route such as intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal. It may be advantageous to administer the vaccine in divided doses, which may be administered by the same or different routes. The vaccine is administered after a primary infection with herpes simplex virus.

The efficacy of post-infection administration is shown in Example 6.1. The effect of the vaccine on increasing the immune response in the host after viral infection is shown in Example 6.2. The viral surface glycoprotein of herpes simplex virus has been shown to be an antigen recognized by antibody-mediated virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Norrill B. Et al
ct. Immun. (1980) 28: 38-4
4. Patients who frequently relapse with HSV infection often have high levels of both neutralizing antibodies and ADCC. Co
rey, L.A. And Spear, P .; G. , En
g. N. , J. Med. 314: 686-69
1, 1986. For such patients, the predominant inducer of HSV-specific antibodies has been shown to be viral glycoproteins. Everle, R.M. , Mou,
S. W. , And Zia, J .; A. , J. Ge
nVirol. 65: 1839-1843, 198.
4. Therefore, it was not expected that a recombinant subunit vaccine composed of only two viral glycoproteins and containing no other viral antigen would be clinically used for the treatment of recurrent genital herpes.

Glycoproteins B and D are used without modification. However, with smaller related polypeptides (eg, fragments, etc.), and with a molecular weight of about 5,
Less than 000 Daltons (eg 1,500-5,
000 Daltons), modification is required to induce the desired immune response. The smaller hapten must be attached to a suitable immunogenic carrier, such as tetanus toxoid.

Short DNA fragments encoding gB or gD polypeptides can also be ligated to genes that express proteins from other pathogenic organisms or viruses. In this way, the resulting fusion protein can immunize against more than one disease.

Recombinant g used per dose
The total amount of B and gD polypeptides will usually be from about 0.1 μg to 2 mg / kg of host body weight, more usually from about 0.5 μg to 1 mg, and especially from about 0.5 μg to 10 mg.
μg. The ratio of gB to gD in the vaccine is
Usually about 0.1: 1 to 10: 1, more usually about 0.1.
5: 1 to 10: 1, and preferably about 0.5: 1 to
It is 5: 1. Single administration is repeated at intervals of daily to weekly, usually at intervals of 2 to 4 weeks, usually not more than about 2 to 10 times. However, as shown in Example 6.4, the time of administration after primary infection with the virus affects the recurrence rate of the disease.
Thus, depending on the species and / or individual being treated,
The most effective dosing time needs to be determined. The most effective time can be determined by rudimentary symptomology, which monitors the disease state, or by basic tests using antibody titers, for example. Furthermore, the data of Example 6.4 show that vaccination significantly reduces the recurrence of the disease in the acute phase of the disease, ie when the individual exhibits HSV-induced lesions in the body. (Recombinant Glycoprotein B) Preparation of recombinant gB polypeptide is described in International Publication N, published October 24, 1985.
o. International application No. WO 85/04587. PCT / U
This is described in detail in S85 / 00587. The materials and methods used to produce recombinant gB polypeptides are briefly described below.

4 to 7 in Examples show the nucleotide sequence of gB1 Patton strain and the amino acid sequence encoded by the nucleotide sequence. The substantial homology between gB1 and gB2 is also shown in FIGS. 4-7. There can be many variations in the nucleotide sequence. Various fragments with independent functions can be used. These fragments can bind proteins other than mature gB. further,
Various codons can be modified to encode the same amino acid, but show more efficient expression according to the nature of the host. For example, these codons can be modified according to the frequency of occurrence of particular codons in one or more proteins or proteins. These proteins are, for example, glycolytic proteins and can be used in specific hosts (eg yeast).
Occupies a high proportion in the total protein. In some cases, one or more codons may be modified to encode different amino acids by replacing one amino acid with another. In this case, the effect of the alteration is not detrimental to the immunogenicity of the protein or other biological factor of interest. In some cases it may be desirable to add amino acids to the N-terminus or C-terminus. Addition of such amino acids gives favorable results. This means that mature gB1
Alternatively, it can be easily achieved by adding a codon to the 5'end or 3'end of the sequence encoding the precursor. Furthermore, although the amino acid sequence of gB2 differs in number by 20% from the amino acid sequence of gB1, other strains of HSV-1 or HSV-2 show the same or similar gB glycoprotein as gB1 Patton strain or gB2333 strain, respectively. have. These gB glycoproteins usually contain less than 5%, more commonly less than 2%, and often less than 0.5% amino acids in the gB1 Patton strain or gB2. It differs from the amino acid sequence of strain 333.

The sequence of gB1, especially the gB1 Patton strain,
It is divided into four domains starting from the N-terminal of the protein: the first hydrophobic region extending from the 1st amino acid to about the 30th amino acid, about 72 from the first hydrophobic region
A region extending to the 6th amino acid, where the polarity can change; a second hydrophobic region extending from the region where the polarity can change to about the 795th amino acid; and extending to the C-terminus of the 904th amino acid, Second that can change polarity
Area.

Since gB is a membrane glycoprotein, by analogy with other glycoproteins, the first hydrophobic region is considered to be a signal leader sequence that controls secretion and / or membrane arrangement. Thus, the first sequence, which can change polarity, is outside the membrane and serves as a recognition sequence, to the extent that gB acts as a receptor for another protein or as an immunogen in a vaccine. The second hydrophobic sequence is a transmembrane integrator sequence (t
transmembrane integrators
equipment (often called "anchor"
r) ”). The second amino acid sequence, which can change polarity, is thought to be present in the cytoplasm and serves to alter one or more cytoplasmic processes to the extent that the receptor is outside the transmembrane integrator sequence. It can be achieved.

The polynucleotide sequence encoding the precursor of gB or a functional fragment thereof may be prepared by inserting the polynucleotide sequence into an appropriate expression vector and introducing the resulting expression construct into a compatible host. Can be cloned and expressed by. The coding fragment is less than about 0.1 map units, usually less than about 0.05 map units; where 1.0 map unit is the size of the entire HSV genome. The expression vector is extrachromosomal or integrated into the genome of the host cell,
It can be a low or high level multicopy vector. The expression vector then either secretes or excretes the polypeptide of interest or keeps the polypeptide of interest in the cytoplasm or in the membrane. Numerous expression vectors have been published in the literature, and in general,
It is useful for use in eukaryotic hosts. Eukaryotic hosts include yeast (eg, S. cerevisiae (S. cer)
evisiae)) and a wide range of permanently proliferating mammalian cells (eg, mouse cells, monkey cells, hamster cells (eg, 3T3, Vero, Chinese hamster ovary cells (CHO), or primary cell lines). If secretion is required, either a natural or non-natural secretory leader sequence can be used, depending on the host. Signals associated with the secretory leader sequence of Escherichia coli, or both, may be in tandem.

To obtain the polynucleotide sequence encoding gB1 Patton, the position of the gB1 coding sequence was mapped on the EcoRI restriction enzyme fragment F. Three subfragments of the F fragment were isolated and subcloned into pBR322 (Figure 2). The DNA fragments of these subclones were then used as probes for Northern blots of poly A ⁺ mRNA isolated from cells infected with HSV-1. The fragment that hybridized to the mRNA of the size predicted for gB was presumed to be within the gB coding region. The transfer direction of gB is also D
It was revealed by determining which strand of the NA probe hybridized to the mRNA. To confirm the identity of the gB sequence, a DNA fragment was used against Hybrid-Select HSV-1 mRNA. This mR
NA was then translated in vitro and the resulting protein was analyzed for gB using a gB-specific antibody.

Fragments encoding gB1 are handled today by a variety of methods including restriction enzyme mapping and nucleotide sequence determination to establish restriction enzyme cleavage sites and open reading frame regions for expression. Then DNA
The sequence is limited to providing the sequence encoding the complete gB precursor or a fragment thereof. These sequences are then inserted into a suitable expression vector with the transcriptional signals in the proper positions and the appropriate translational signals. This can be accomplished by filling the overhangs and making blunt end ligations with adapters and the like.

It is of particular interest to introduce a gene in tandem with respect to a gene capable of amplification. Useful genes include the dihydrofolate reductase (dhfr) gene (where the dhfr gene and flanking regions are repeated), which can be amplified by using methotrexate; and metallothionein, which can be amplified with heavy metals such as copper. Is included. Expression constructs can be introduced into a suitable host by any convenient means, including transformation, transfection, calcium phosphate precipitation, and the like. The host cell is then stressed with an appropriate biotoxin to a level that it chooses to amplify a particular gene. These cells are then cultured and grown until they effectively produce the desired polypeptide.

Following the procedure described above, HSV-2 333
The polynucleotide sequence encoding gB2 from the strain, both precursor and mature, has been isolated, cloned, and engineered to provide the construct. As a result, it can be expressed in one or more hosts. Considering the usefulness of the fragments encoding gB1 Patton, these fragments were cloned into the DNA of a specific HSV-2 restriction fragment clone.
It is used as either a probe for the localization of gB2 encoding a portion or for the isolation of gB2 mRNA from infected host cells. Conveniently, multiple probes encoding different regions of the gB1 gene are used. Either positive DNA fragments or abundant mRNAs of approximately the appropriate size to hybridize to this probe are selected. The mRNA can then be reverse transcribed to give the cDNA and / or used for hybridization with fragments of the HSV-2 genome to confirm its function encoding gB2. If desired, more than one cloned fragment containing a portion of the gB2 structural gene may be engineered and ligated to provide complete coding and flanking regions, as appropriate. This coding region is then introduced into an expression vector. (Recombinant glycoprotein D) Recombinant gD1 was prepared by the method of 198
International publication No. published on October 24, 2015 WO85
/ 04587 International Application No. PCT / US85 / 00
587. The materials and methods used to produce recombinant gD polypeptides are briefly described below. A detailed description of the preparation of recombinant gD2 is given in the examples below.

Polypeptides that immunologically cross-react with naturally occurring glycoprotein D are produced by recombinant DNA methodology in eukaryotic hosts such as yeast and mammalian cells (eg CHO cells). Production in eukaryotes offers advantages in relation to eukaryotic hosts, such as post-translational modification and / or secretion. The gD polypeptide is produced from a relatively short synthetic DNA fragment encoding at least about 9 amino acids, providing a hapten effective to induce a gD-specific immune response.

The gD DNA fragment may be of natural or synthetic origin. The native gD gene of HSV-1 is a short internal repeat (I
It is located between the Rs) sequence and the short termination repeat (TRs) sequence at its 3'end. Encoding the mature protein means that a fragment of approximately 1.6 kbp can be found located on the 2.9 kbp SacI restriction fragment of the genome. The entire coding region of the mature protein is HindI of the 2.9 kbp SacI fragment.
It is located in the II-NruI fragment. The naturally occurring gD gene may or may not be modified. The region of the gene can be deleted as desired, or
And / or can be combined with other DNA fragments. gD
The DNA fragment can be inserted and expressed in an expression vector using materials and procedures similar to those described above for expression of gB DNA. The preparation, cloning, and expression of specific fragments of the naturally occurring gD gene are detailed in the Examples below.

The following examples are given by way of illustration and not limitation. In the Examples below, Section 1 describes the general procedure used to produce recombinant proteins and Section 2 describes recombinant gB.
1 for the preparation; Section 3 for the preparation of recombinant gB2; Section 4 for the preparation of recombinant gD1; Section 5 for the preparation of recombinant gD2; Section 6 for the mixture of gB and gD polypeptides. The research of the used vaccine is described.

[0051]

EXAMPLES (1. Materials and Methods) Live preserved strains of HSV-1 Patton strain and HSV-2 333 strain were D
r. Richard Hyman (Hershey M
medical Center, Hershey, P
Ennsylvania). These viruses are known as Dr. Evelyn Linnette (Vi
ro Labs, Emeryville, Cali
fornia) or American Type T
It can be grown in Vero cells obtained from issue Culture Laboratory. This growth is done according to standard procedures. Plasmid pACYC18
4 (Chang and Cohen, J. Bacter
Ec of iology (1978) 134: 1141)
HSV-1 Patton Ec cloned into the oRI site
oRI DNA fragment (Kudler et al., Virolog
y (1983) 124: 86-99) was prepared by Dr. It may be obtained from Hyman, or may be independently prepared by a simple method. Two HSV-2 333 clones,
That is, pBR322 (Sutcliffe, Nucl
eic Acids Research (1978)
5: 2731) H inserted at the HindIII site
The indIII fragments H and L were also isolated from Dr. Hyman
You can get more.

The dhfr-deficient CHO cell line was transformed into Dr. Y.
W. Kan (University of Cali
fornia, San Francisco). This cell line was originally derived from Urlaub and Cha.
sin (Proc. Natl. Acad. Sc
i. USA (1980) 77: 4216-422.
0). Under non-selective conditions, these cells contained 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 15
Ham's F-12 supplemented with 0 μg / ml L-proline
Medium (obtained from Gibco, cat. No. 176)
Were grown in. Selection medium was 10% dialyzed fetal bovine serum plus penicillin, streptomycin, and 15
It was a DEM supplemented with 0 μg / ml L-proline. Concentrated M for selection of methotrexate (MTX)
TX stock medium was prepared from MTX obtained from Lederle and added to the above DME selective medium immediately before use. (1.1 Cloning) All DNA manipulations were done according to standard procedures. Maniatis et al., Mo
regular Cloning, CSH (198
See 2). Restriction enzymes, T4 DNA ligase, Escherichia coli DNA polymerase I Klenow fragment, and other biological reagents are Bethesda.
Purchased from Research Laboratories, or other indicated commercial suppliers, and used according to manufacturer's instructions. Double-stranded DNA was separated on a 1% agarose gel and isolated by electroelution. (1.2 RNA isolation, Northern blot analysis, and hybrid selective translation) Total RNA was 1 per cell
HSV-1 or HSV with a multiplicity of 0 viruses
It was prepared from Vero cells 6 hours after infection with -2. The cell monolayer was washed, incubated with extraction buffer, and Pachl et al. (Cell (1983) 33:33.
5-344) and processed.
Poly A ⁺ RNA is 500 mM NaCl, 10 mM
Tris HCl (pH 7.5), 1 mM EDT
A, 2 mg total RNA was passed through a 3 ml column of oligo dT cellulose (obtained from Collaborative Research) in 0.1% SDS, then 100 mM NaCl, 10 mM TrisHC
1 (pH 7.5), 1 mM EDTA, 0.1% S
Wash the column with DS, and 10 mM Tris H
Cl (pH 7.5), 1 mM EDTA, 0.1%
Prepared by eluting the poly A ⁺ fraction with SDS.

To perform Northern blot analysis, poly A ⁺ RNA was loaded with glyoxal (McMaster).
Et al., Proc. Natl. Acad. Sci.
USA (1977) 74: 4835-4838), and fractionated by electrophoresis on a 1% agarose gel.
Nitrocellulose paper (Thomas, ibid. (1
980) 77: 5201-5205), and
Hybridized with ³² P-labeled probe.

Details of the method used for the hybrid selective translation method have been previously described (Pachl et al., Ce.
11 (1983) 33: 335-344). DNA
The filter is a 3.5 kb Xho- that encodes gB.
It was prepared using either 3 μg of the Kpn fragment or 2 μg of the 3.0 kb SstI-SstI fragment encoding HSV-1 gD. The filter was incubated with 40 μg of poly A ⁺ RNA from cells infected with HSV-1, bound RNA was eluted and reticulocyte cell-free system (Pachl et al., J. Virol. (19).
83) 45: 133-139). The translation product was 12.5% SDS polyacrylamide gel (La
emmli, Nature (1970) 227: 6.
89). (1.3 DNA transfection) COS 7 cells (Gluzman, Cell (1981) 23:
175-182) or dhfr-deficient CHO cells (Ur
transformation of Laub and Chasin (1980) supra), except that the carrier DNA was omitted.
van der Eb and Graham (Metho
ds in Enz. (1980) 65: 826-8.
39) as described in Parker and Stark (J.
of Virol. (1979) 31: 306-
369). Calcium phosphate precipitation of plasmid DNA was 250 mM C
plasmid DNA in NaCl _2, and concentrated to 2 times the equivalent volume and this HEPES- buffered saline (2 × HB
S) was added drop by drop and mixed (1
× HBS is 0.14 M NaCl, 5 mM KCl,
0.7 mM Na ₂ HPO ₄ , 2.8 mM glucose, 10 mM HEPES (pH 7.0)). After incubating at room temperature for about 20 minutes, 1 ml of calcium phosphate-DNA suspension (containing 15 μg of DNA)
Was added to the medium of cells grown on 10 cm plates to reach 50% confluency. After 6-8 hours, the medium containing DNA was removed and the cells were washed with 15% glycerol-1 ×.
Incubated with HBS for 4 minutes. The cells were then grown in non-selective medium (F12) for 2 days, after which they were split or subcultured in selective medium. After 10 days, dhfr-positive cell colonies appeared, and after 14 days, these colonies were separated by transferring the colony cells from the plate with a Pasteur pipette. Separated cells were transferred to multiwell plates for growth. (1.4 In vivo labeling and immunoprecipitation of cells) ³⁵ S
-To label with methionine, cells were grown to confluency in 3.5 cm plates and washed with PBS (0.14 M NaCl).
2.7 mM KCl, 15.3 mM Na ₂ HPO ₄ )
Washed once with methionine and then in 0.5 ml of labeled medium DEM (Dulbecco's modified Eagle medium from Gibco, cat. No. 188G), 1% dialyzed fetal bovine serum and 400 μCi / ³⁵ S of ml
-Methionine (> 1000 μCi / mmol) was added to each plate. These cells were incubated at 37 ° C for an appropriate time. At the end of the sign period,
The medium was removed and the monolayer was washed once with PBS.
"Cold" methionine chase
The labeling medium was replaced with DME containing 2.5 mM methionine as the nicotine). To perform the immunoprecipitation, the cells were treated with 0.1 ml of cell solution buffer (20 ml).
mM Tris-HCl (pH 8), 100 mM Na
Cl, 1 mM EDTA, 0.5% nonidet P40,
0.5% sodium deoxycholate, bovine serum albumin, 0.1% SDS, 1.0 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 1% aprotenin, Sigma Chemical Compa
(obtained from ny). Cell lysates were scraped into tubes, vortexed briefly and then left at 4 ° C for 5-10 minutes. Cell debris was removed by centrifugation and the cleared lysate was -70 ° C.
Stored in.

To perform immunoprecipitation, cell lysate 0.1
ml was precleared by incubation with normal serum for 30 minutes at 4 ° C., then 50 μl of a 20% protein A sepharose (PAS) solution (in lysis buffer).
Was added and the incubation was continued at 4 ° C. for 30 minutes with gentle shaking. PAS was removed by centrifugation at 14,000 xg for 1 minute and 5 μl of HSV-1
Polyclonal antibody (obtained from DAKO) or gB-specific monoclonal antibody F3AB (Universit)
y of South Alabama Dr. Joh
n Oakes). When using the F3AB antibody, 0.1% SDS was omitted from the lysis buffer.
After 30 minutes at 4 ° C., 75 μl of PAS was added and incubated as above. The PAS-immune complex was collected by centrifugation, washed 3 times with lysis buffer lacking BSA and protease inhibitors, and washed with 0.12M Tris HC.
It was washed once with 1 (pH 7.0). The immunoprecipitated protein was boiled in SDS sample buffer to give PA.
It was dissociated from S and then analyzed on a 12% polyacrylamide gel. To immunoprecipitate the labeled protein from the cell culture medium, the medium was first clarified by centrifugation and then 1/10 volume of 10X lysis buffer was added and the protein was precipitated as described above. (1.5 Immunofluorescence) To analyze the expression of gB or gD in COS cells or CHO clones, cells grown in the wells of the slides were washed 3 times with PBS,
The cells were fixed with 100% methanol at −20 ° C. for 10 minutes, followed by further washing with PBS three times and once with PBS supplemented with 5% goat serum (GS) once. The fixed cells were then treated with the primary antibody (PBS-5% GS 1/10).
Incubated with HSV-1 or HSV-2 polyclonal antibody diluted to 0) for 30 minutes at 37 ° C. The cells were then washed 3 times with PBS-5% GS,
Secondary antibody (FI diluted 1/10 with PBS-5% GS
3 with TC-conjugated goat anti-rabbit IgG (Capel)
Incubated at 7 ° C for 30 minutes. PBS-5% G
After washing 4 times with S, slides are treated with 50% glycerol-
It was mounted with a cover piece using 100 mM Tris (pH 8.0) and observed with a Leitz optical microscope equipped with a fluorescence optical system. Immunofluorescence of live cells was performed using PBS-5% G
It was performed as described above, except that the first wash with S was followed immediately by incubation with the primary antibody. Live cells were fixed with PBS containing 5% formaldehyde before mounting with cover strips. Fluorescently stained cells were obtained from Kodak's Ektachrome film (Koda
c Ektachrome film; ASA400)
I took a picture with. (1.6 ELISA analysis) The indirect enzyme-linked immunosorbent assay (E) using the purified recombinant gB preparation as a standard substance was used to measure the gB protein concentration in the medium in which CHO cells were cultured.
LISA). 50 μl of F3AB antibody diluted 1: 1000 in PBS was incubated for 1 hour at room temperature to give 96-well polyvinyl chloride plates (Dynatech Laboratori).
es, Inc. ). Excess antibody was removed by washing 3 times with PBS-5% GS. 50 μl of media sample or gB protein standard diluted in PBS-1% GS was added to the wells and incubated at room temperature for 1 hour. Then, plate this plate
Wash 3 times with BS + 1% GS, then 1: 1 with the same buffer.
A third incubation was performed for 1 hour with 50 μl of rabbit anti-HSV-1 polyclonal antibody (obtained from DAKO) diluted to 1000. Excess secondary antibody was removed by washing 3 times with PBS + 1% GS. Finally, goat anti-rabbit antibody conjugated with horseradish peroxidase (B) diluted 1: 500 in PBS-1% GS.
50 μl of Oehringer Mannheim) was added to each well and incubated for 1 hour, then the wells were washed once with PBS + 1% GS followed by P
After washing 8 times with BS, 2,2 'at a concentration of 1 mg / ml
-Azido-di [3-ethylbenz-thioazoline sulfonate] (Boehringer Mannheim)
0.1 M citric acid (pH 4.0), 0.003% H
_The color was developed with 50 μl of the solution contained in ₂ O ₂ . The color reaction was stopped after 5 minutes by adding 50 μl of 10% SDS and the absorbance was read at 414 nm on a microtiter plate reader.

The concentration of the gD protein was such that the recombinant gD and F3AB purified as standards were replaced by gD-specific monoclonal antibody 8D2 (Rector et al., Inf.
ect. and Immun. (1982) 3
8: 168-174) was used. (2. Glycoprotein) (2.1 Isolation, cloning and characterization of gB1 gene) In order to isolate a gene corresponding to glycoprotein gB1, HSV-1 Patton strain (Patton; Ska).
re and Summers, Virology (19
77) The DNA fragment spanning the coordinates 0.345-0.40 of the EcoRIF restriction fragment map of 76: 581-595) was subcloned into the plasmid pBR322. These fragments are the EcoRI of plasmid pACY184.
Prepared from the appropriate restriction enzyme digests of the region and prepared in TAE buffer (0.04M Tris-acetate, 0.002M EDT.
Separated by electrophoresis on a 1% agarose gel using A) and electroeluted. The isolated fragment was ligated into pBR322 which had been previously cleaved with the appropriate restriction enzymes and treated with alkaline phosphatase. A restriction enzyme digestion map of the entire HSV-1 genome is shown in FIG. 1, and a more detailed map of the subcloned region is shown in FIG. Referring to FIG. 1, a conventional map is shown in the first two rows (Ro
izman, 1979). The dotted line shows the L-S connection. An EcoRI restriction enzyme cleavage map of the prototype isomer sequence is shown in line 3 with the EcoRI fragment F shown by enclosing in a hatched box (S
kare and Summers, 1977; Roiz
man, 1979). The HindIII restriction enzyme digestion map of HSV-2 is surrounded by a hatched frame Hi.
Shown in line 4 with ndIII fragment H (Roizma
n, 1979). 1 map unit is HSV-1 DNA
Corresponding to 98.9 megadaltons or 148.9 kbp of DNA and HSV-2 DNA corresponding to 105.6 megadaltons or 160.5 kbp.

Referring to FIG. 2, the restriction enzyme cleavage sites shown in detailed map line (I) are as follows:
E, EcoRI; B, BamHI; S, SalI;
P, PstI; X, XhoI (from DeLucca et al., 1983); N, NdeI; Xn, Xmn
I; V, EcoRV. The BstEII site mapped by DeLucca et al. At 0.355 is deleted in this strain and a new PstI site is present at 0.357. Line II shows three plasmid subclones containing the gB1 coding region. These are 0.3
PHS 106 ranging from 45 BamHI site to 0.360 SalI site; pHS107 ranging from 0.36 SalI site to 0.388 SalI site;
And B in the range of 0.345 to 0.40 map units
amHI fragment. Line III is gB1 mRNA
3 shows the three probes used to map H .; line IV shows the fragments used for hybrid selection; and line V shows these probes used to locate the gB2 gene (see below). See). The additional restriction enzyme cleavage sites used to generate these fragments are Nc, NcoI; K, Kp.
nI; and A, AluI.

To determine the location of the gB1 coding region in the EcoRI fragment, Northern blotting of poly A ⁺ mRNA isolated from HSV-1 infected Vero cells was mapped to detailed maps isolated from pHS106 and pHS107. The indicated DNA fragment was used as a probe. 0.5 kb Pst isolated from pHS106
When the I-SalI fragment was used as a probe for HSV-1, the major species detected was a 3 kb mRNA.
Met. When the same blotting was carried out using a 0.49 kb NcoI fragment located approximately 1 kb upstream of the PstI-SalI fragment as a probe, hybridization with a 3 kb mRNA presumed to be gB1 mRNA was also detected. This means that the sequence encoding gB1 is PstI-
It is suggested that it extends at least 1 kb upstream of the SalI fragment. 3 kb mRNA is PstI-S
It is not extended beyond the first XhoI site downstream from the alI fragment. Because, 0.5kb XhoI-Xh
This is because the oI fragment does not hybridize with this mRNA. The direction of transcription of the gB1 transcription unit is from right to left (3 '←
5 '), which means PstI-SalI and N
Only the strand in the 5'-3 'direction of the coI-NcoI fragment is 3
Proved by hybridizing to kb gB1 mRNA.

Hybrid selective translation was performed by hybridizing HSV-1 poly A ⁺ mRNA with a 3.2 kb KpnI-XhoI fragment (including the region designated as the gB1 coding region). Bound mRNA
Was eluted and translated in vitro, a 100 kd protein of the same size as gB1 derived from HSV-1 infected Vero cells was detected. Confirmation of the identity of this 100 kd protein was performed by immunoprecipitation with a gB1-specific monoclonal antibody. Hybrid selection with the KpnI-XhoI fragment also detected several other proteins, presumably a result of non-specific hybridization of the mRNA due to the high G + C content of the DNA. When the same RNA was selected using a 3.0 kb SstI-SstI DNA fragment encoding the HSV-1 glycoprotein gD, a similar protein pattern was observed, but the 100 kd gB protein was not detected. This result suggests that gB is specific for the KpnI-XhoI fragment.

FIG. 3 shows BamHI in 0.345 map units.
A restriction enzyme cleavage map of a 3.95 kb DNA fragment from the restriction enzyme cleavage site to the XhoI site of 0.373 map units is shown. The gB1 open reading frame is shown boxed and the direction of transcription is from right to left as shown. The actual code area is from map unit 0.348 to 0.367. The DNA sequence from the BamHI site to the 3640 non-unique AluI site is shown, the AluI site being indicated by (A). The restriction enzyme cleavage sites shown are B, BamHI; B
1, BalI; Bs, BstEII; K, Kp
nI; Nc, NcoI; P, PstI; Pv,
PvuII; S, SalI; Sc, SacI;
X, XhoI; including Xm and Xma3. The restriction enzyme cleavage site from the AluI site to the terminal XhoI site shown on the right is not shown. Possible glycosylation sites of the produced gB1 protein and the hydrophobic anchor and signal region (boxed) are shown.

DN from the BamHI site to the non-unique AluI site at nucleotide position 3,640.
The A sequence was determined using the Sanger M13 dideoxynucleotide synthesis method. Both DNA strands of the coding region were sequenced. The complete DNA sequence is assembled from the aligned restriction enzyme digests, thus reading the sequence throughout the diagnostic strip. Figures 4 to 7 show DN of gB1.
The A sequence is shown (line 3); the predicted amino acid sequence for gB1 is shown below the DNA sequence (line 4).

The amino acid sequence and DNA sequence corresponding to gB1 shown in FIGS. 4 to 7 are shown in International Patent Publication No. WO85.
1 of / 04587 (published on October 24, 1985)
It should be noted that it is different from the sequence first shown in. There is an error in the DNA sequence in Table 1 and a nucleotide (G) is added to the 607th position of the sequence. This nucleotide has been deleted in Figures 4-7 and the figure shows the correct DNA sequence. The amino acid sequences in Table 1 above were deduced from the incorrect DNA sequences shown therein; the sequences shown in Table 1 above because the reading frame was altered by the nucleotides added above. Is not correct. Figures 4-7 show the amino acid sequences based on the correct DNA sequence; the amino acid sequences of Figures 4-7 have been confirmed by amino acid sequencing of the N-terminal region of gB1. Changes in this deduced amino acid sequence also result in corrections for the predicted positions of hydrophobic and hydrophilic regions, and glycosylation sites within the gB1 molecule. The deduced amino acid sequence based on the correct sequence is shown below the sequence.

22 bp oligonucleotide (residue 47
3-494), primer extension using gB1 mR
It was shown that the 5'end of NA is located at residue 188.
The CAT and TATA transcriptional regulatory signals are residues 55-
It is estimated to be 62 and 125-131. Residue 4
There is an open reading frame of 2712 nucleotides starting at the ATG at 38-440 and ending at the TGA stop codon. Two putative polyadenylation signals are found at residues 3166-3173 and 34.
It is located in the non-coding region on the 3'side of 09-3416.

The amino acid sequences described are characteristic of membrane proteins. There is a very hydrophobic region ranging from the amino acid residues 726 to 795 near the carboxy terminus,
The 69 amino acids can be membrane bound. The first 30 amino acids at the N-terminus are predominantly hydrophobic. This hydrophobic amino acid domain precedes a highly concentrated region of charged or hydrophilic amino acids. The N-terminal hydrophilic sequence serves as a secretory leader or signal sequence, followed by processing signals for cleavage and removal of the secretory leader. The hydrophobic region near the C-terminus can act as a transmembrane integration sequence for binding proteins to cell membranes.

Sequence data are asn-X-thr / ser sequences in the hydrophilic ectodomain (see also Figure 3).
It also suggests that there are nine potential N-linked glycosylation sites as defined by If the first 30 amino acids are removed by processing and each potential N-linked glycosylation site is utilized to add an average of 2 kd of carbohydrate per site, the molecular weight of the mature protein is approximately 123 kd. Becomes (2.2 Expression of gB1 in mammalian cells) C which lacks dhfr in order to express gB1 in mammalian cells
Plasmids pHS112 and pHS114 in HO cells
Was introduced and transformed. This transformation was performed using the calcium precipitation method as described in Materials and Methods. E. coli transformed with plasmids pHS112 and pHS114. E. coli strain HB101 has been deposited with the ATCC and has accession numbers 39650 and 39, respectively.
It is entrusted with 651. Construction of these strains is described in International Patent Publication No. WO 85/04587 (supra). Transformed cells were selected with selection medium lacking thymidine, purine and glycine. Cells
It was isolated by removing with a Pasteur pipette and grown in plates with multiple wells. A large number of clones were isolated, which were shown to produce gB by immunofluorescence and radioimmunoprecipitation using HSV-1 polyclonal antibody or a monoclonal antibody specific for gB. 3 cell clones pHS112-1, p
HS112-9 and pHS112-23 were isolated,
The clone synthesized an intact form of the gB protein in the cell. The gB produced in these cells is believed to be glycosylated. Because, after pulse labeling for 1 hour, followed by chase for 5 hours, a larger molecular weight form could be detected compared to unchaseed cells, and about 10% of gB was secreted into the medium. .
5 cell clones expressing incomplete gB (pHS1
14-5, pHS114-6, pHS114-7, pH
S114-11 and pHS114-12) were also analyzed and shown to also secrete some gB into the medium. PHS114-7, one of these cell lines
Was selected for further amplification with MTX. Clones are initially 0.01, 0.05, 0.10 and 0.3
Selected with μM MTX. Three clones that synthesize high levels of gB as detected by immunofluorescence analysis have
Isolated from those selected with MMTX. By radioimmunoprecipitation these clones pHS114-0.3
μM-6, 23 and 25 are ³⁵ S-methionine 1
Clone pHS not amplified during time labeling
It synthesizes 2-3 times more gB than 114-7. Pulse chase experiments show that at least 8% of gB synthesized in these clones during the 1 hour pulse is secreted extracellularly at 5 hours.

Expression was also carried out using the expression vector pHS137. A map of this vector is shown in FIG. Plasmid pHS137 encodes an incomplete gB1 protein whose length after cleavage of the signal sequence is 690 amino acids.

PHS137 was constructed by digesting pHS108 (described in Section 2.1) with XhoI and BamHI, followed by isolation of the resulting 3.5kd fragment. The ends of this fragment were made blunt with Klenow.
The blunt-ended XhoI-BamHI fragment was used for PV
DNA partially digested with UII and migrating as a 2098 bp band on gel was isolated from the partially digested product. The isolated XhoI-PVUII band was previously labeled with SmaI.
DNA obtained by ligating to pSV7d which had been digested with
To E. It was used to transform E. coli. The resulting bacterial clones were screened for plasmids with the gB1 insert inserted in the proper orientation.

To obtain the expression, pHS137 was introduced into dhfr-deficient CHO cells together with the plasmid pADdhfr and transformed. The resulting clone produced and secreted gB1. One such clone is p
HS137-7-B-50 was 1-3 × 10 4 in 24 hours in a T75 culture flask containing 10 ml complete medium.
6.91 +/− 1.53 μg / ml gB per ⁷ cells
Produced one protein. (3. Glycoprotein B2) gB2 gene isolation, characterization, and cloning is described in International Patent Publication No. WO85 /
No. 04857 (supra). (3.1 Expression of gB2 in mammalian cells) HSV-2
Expression of glycoprotein gB is performed in COS cells (transient expression) by transformation with pHS210 alone or cotransformation with pHS210.
This second plasmid contains dhfr.

The plasmid pHS210 was constructed as follows; the entire gB2 gene was 3.8 kb NruI-.
It was subcloned into pBR322 as a BamHI fragment to generate pHS208. See FIG. PstI site at the 5'end of the gene (100 for NruI site
The bp right (downstream) was changed to the HindIII site by in vitro mutation with M13. Then 1.9 kb
The HindIII to PvuII fragment of pSV
1 / dhfr was inserted into pSV1 obtained by digestion with HindIII and BglII. See Figure 10; pSV1 / dhfr is described in PCT International Patent Publication No. WO 85/04587. For this cloning step, pHS208 was cut with PvuII to repair the ends to blunt ends. The molecule was then cleaved with HindIII and 1.9 kb HindII.
The I- (PvuII) fragment was isolated by gel electrophoresis. Similarly, pSV1 / dhfr was cut with BglII,
It was repaired to blunt ends, cut with HindIII, and subjected to 4.85 kb HindIII- (BglI by gel electrophoresis.
I) The vector fragment was isolated. These two fragments (1.
9 kb and 4.85 kb) are expressed in the expression vector p
Ligated to make HS210 (FIG. 10).

The plasmid pHS210 was directly transformed into COS.
Used to transform cells. Expression was determined by using the gB-specific monoclonal antibody F3AB and the commercially available polyclonal antibody H for the primary antibody screening.
Detected by immunofluorescence analysis, also using SV-2 antibody (DAKO). Secretion of gB2 into the medium was detected by gB2-specific ELISA analysis. For this purpose the plates were coated with a monoclonal antibody. A sample of cell culture medium was added to the coated plate and then bound gB2 was added to rabbit anti-HSV-2 polyclonal antibody (DAKO) followed by horseradish conjugated goat anti-rabbit I.
It was detected using gG.

Plasmid p for transformation of CHO cells
HS210 was used in a cotransformation protocol with a second plasmid containing dhfr as a selectable marker (Figure 10). Approximately 100 dhfr ⁺ clones were isolated by subsequent transformation and growth in selective medium and were analyzed by ELISA (EL plates were F
3AB-specific monoclonal antibody coated)
Was used to screen for gB2 synthesis and secretion. Clone pHS with the highest level of gB secretion
210 # 3-1 was further selected for gB2 polypeptide characteristics. gB2 protein is [ ³⁵ S]-
It was detected by labeling with methionine, followed by radioimmunoprecipitation. 79 kd and 8 after 1 hour pulse
Two diffuse bands corresponding to the 4 kd polypeptide were detected intracellularly. These proteins were 68,991 daltons larger than the size predicted for the 637 residue incomplete gene product, suggesting that the proteins represent a partially glycosylated precursor. 5
No gB2 was detected in the cells after time chase. Then, the 89 kd polypeptide was detected in the medium. Mature sized, fully glycosylated gB secreted into the medium of clone pHS210 # 3-1
2 is 100 kd gB secreted by pHS4-6
It was somewhat smaller than 1. This is because the coding sequence corresponding to 94 amino acids contained in the gB1 plasmid is p
This is because it has been removed from the HS210. (4. Glycoprotein D1) (4.1 Construction of mammalian expression vector of gD1) pBR
A library of HSV-1 EcoRI fragments of the Patton strain cloned into the EcoRI site of Dr.
Richard Hyman, Hershey Me
digital Center, Hershey, PA
It was made by. The gD1 gene is 2.9 kb Sac within the EcoRI fragment of clone H of this library.
It is completely contained in the I fragment. Clone H containing the 15 kb EcoRI insert was obtained from Dr. Obtained from Hyman. This 2.9 kb fragment was purified by gel electrophoresis and then Hin
Completely digested with dIII and NcoI. 74 bp of 5 '
60 encoding untranslated sequence and amino terminal 20 amino acids
The 5'end of the gD gene consisting of bp was isolated from gel as a 134 bp fragment. The 3'end of the gD gene is
pHYS119 (International Publication No. WO85 / 04587
(See above)) was digested with NcoI and SalI and the 873 bp fragment was isolated. These two fragments (5 ′ end and 3 ′ end) were ligated with the plasmid pUC12 which had been digested with HindIII and SalI in advance. The pUC12 vector contains P
harmasia and P-L Biochemica
is commercially available from Is and the resulting plasmid is pHS
I named it 131. Hind the plasmid pHS131
It was digested with III, the 5'-overhang of 4 base pairs was filled in with Klenow polymerase, and then digested with SalI. gD
The 1007 bp fragment containing the gene was gel isolated and ligated into plasmid pSV7d which had been previously cut with SmaI and SalI. This plasmid pSV7d is described below. The obtained expression vector is pHS1
Name it 32. The induction is outlined in FIG. This plasmid contains a gD containing the signal sequence of 25 amino acids of the total 399 amino acids of the complete protein.
Encodes 315 amino acids of one protein. This protein is cleaved at the carboxyl terminus and lacks 84 amino acids containing a hydrophobic membrane anchor domain and cytoplasmic domain, and the resulting protein is secreted into the medium.

The plasmid pSV7d was constructed as follows: 4 containing the SV40 origin of replication and the early promoter
The 00 bp BamHI / HindIII fragment was added to SVg
tI (Mulligan, R. et al., J. Mol.
Cell Biol. (1981) 1: 854-8.
It was excised from 64) and purified. 240 bp SV40BclI / Ba containing the poly A addition site of SV40
The mHI fragment was cloned into pSV2 / dhfr (Subraman
i, J. et al. Mol. Cell Biol. (198
1) It was cut out from 1: 854-864) and purified. Link these fragments below:

[0073]

[Chemical 1] Was fused by. This linker contains 5 restriction sites with stop codons in all 3 open reading frames. The resulting 670 bp fragment (SV40 origin of replication, SV40 early promoter, polylinker with stop codon,
And the polyadenylation site of SV40).
A 5 kb deleted pBR322 derivative (Lusky and Botchan, Cell (1984) 36: 3.
91), which was cloned into the BamHI site of pML,
pSV6 was obtained. EcoRI in the pML sequence of pSV6
And EcoRV sites with EcoRI and EcoRV
About 200b at each end after removal by decomposition with
Treatment with Bal31 nuclease to remove p and finally religation gave pSV7a. Bal31 excision causes SV about 200 bp away from EcoRV site
One BamHI restriction site flanking the 40 region was removed. PSV7a was digested with NruI to remove a second BamHI site adjacent to the SV40 region. This enzyme cleaves the pML sequence upstream of the origin of replication. This was recyclized by blunt end ligation to give pSV7b.

PSV7c and pSV7d refer to consecutive polylinker substitutions. First, set pSV7b to St
It was digested with uI and XbaI. The following linker was then ligated into the vector to give pSV7c:

[0075]

[Chemical 2] Then pSV7c was digested with BglII and XbaI and ligated with the following linker to give pSV7d:

[0076]

[Chemical 3] (Expression of 4.2 gD1 in mammalian cells) Plasmid p
HS132 gD1 expression has been shown in many experiments.
First, using the method described above and commercially available for detection HSV
Specific immunofluorescence was observed in COS7 cells after infection with -1 rabbit serum (DAKO). Second,
A stable CHO cell line secreting gD1 has been established. Expression levels were analyzed by ELISA and confirmed by radioimmunoprecipitation of pulse labeled, chased cell lysates and medium. Third, gD1 was purified from the medium of roller bottle cultures of CHO cell line D64 by a series of steps of ammonium sulfate precipitation, immunoaffinity chromatography, and ultrafiltration. For affinity chromatography, the one obtained by binding the gD monoclonal antibody 8D2 described in Rector et al. (1982) (supra) to cyanogen bromide-activated Sepharose 4B was used. (5. Glycoprotein D2) (5.1 Construction of mammalian expression vector of gD2) HSV
-2 HindIII fragment of the strain 333 was obtained from the literature (Kud
Ler et al., Virology (1983) 124:
86-99). Richa
It was cloned into pBR322 by rd Hyman. The gene for glycoprotein gD2 is Ruyechan
Et al., J. Virol. (1970) 29: 677-.
697 maps to a short unique region of the virus between 0.90 and 0.945 map units. HindII
The region occupied by the IL fragment is described by Roizman, B .; , A
nn. Rev. Genet (1979) 13: 2.
It is shown in the genomic map of 5-27. The DNA sequence of the gD2 gene is Watson, Gene (198
3) 26: 307-312.

Hind cloned in pBR322
The IIIL fragment was labeled with Dr. Obtained from Richard Hyman and the restriction map shown in Figure 12A was determined. gD
The second gene is 2.9 kb SacI which encodes gD1.
2.4 kbX was obtained by Southern blotting the restriction enzyme digestion product of the HindIIIL fragment using the fragment as a probe.
It was found to be present on the hoI fragment. XhoI
The map of the fragment and the position of the gD2 gene are shown in Fig. 12B. This 2.4 kb XhoI fragment was cloned into a pBR322 derivative vector containing an XhoI site, resulting in plasmid pHS204. Three different gD2 expression vectors, plasmids pHS211, pHS212, and pHS213 were constructed as follows and a schematic diagram is shown in FIG. The plasmid pHS211 contains a signal sequence g
It encodes the first 305 amino acids of D2. For its construction, pHS204 was cut with SmaI and BamHI and two restriction fragments were isolated from the gel: 250 containing the 5'end of the gene containing 82 bp of 5'untranslated sequence.
bp SmaI fragment and 3'containing the inside of the gene
The flanking 746 bp SmaI-BamHI fragment. Mammalian cell expression vector pSV7d (described in Section 4.2)
It was cut with coRI, the 5'4 bp overhang was made blunt with Klenow polymerase, then cut with BamHI. The two fragments of pHS204 were ligated to the digested pSV7d and selected from the bacterial transformants with the SmaI fragment in the correct orientation, the vector pHS2
I got 11.

352 amino acids and pHS of gD2
The plasmid pHS212, which encodes an additional 47 residues over the gene present in 211, was transformed into pHS204
Was digested with HaeII and blunted with Klenow polymerase and digested with BamHI. A 141 bp fragment from (HaeII) (brackets indicate end filled) to BamHI was gel isolated. Plasmid pHS211 was transformed into E. coli
It was introduced into strain GM272 to prepare plasmid DNA, which was then digested with BclI, blunted with Klenow polymerase, and digested with BamHI. The large vector fragment (about 3.4 kb) was gel isolated and isolated with 141 bp (Ha
eII) -BamHI fragment and ligated to plasmid pHS
212 was obtained. The fusion of the gD2 sequence and the plasmid vector sequence at the 3'end of the gene adds 27 codons of nonsense DNA to the 3'end of the gD2 gene. To remove these nonsense sequences, the plasmid pHS213 was digested with SalI of pHS211 by partial digestion, gel isolation of the single-cut plasmid, followed by blunting with Klenow polymerase and BamHI.
It was constructed by disassembling with. pHS204 (HaeI
The 141 bp fragment of BamHI from I) was linearized into p
Ligation into HS211 gave plasmid pHS213. (5.2 Expression of gD2 in Mammalian Cells) The expression of gD2 in mammalian cells is expressed by pHS211 and pHS21.
Transient expression was first analyzed by transfecting COS7 cells with 2 and pHS213. Expression of gD2 was determined by both immunofluorescence and capture ELISA analysis of the medium in which COS7 was cultured, using a rabbit anti-HSV-2 antibody for immunofluorescence and for the capture antibody in the ELISA. , GD type common antibody 8D2 (Rector et al. (1982) supra).

Next, the plasmid p containing Addhfr was added.
Transfected with HS211 or pHS213,
Select for dhfr acquisition and E for gD2 expression
Permanent C by screening with RISA analysis
The HO cell line was established. Description of Ad-dhfr: Construction of a plasmid containing the dhfr gene by fusing a major aerovirus promoter derived from adenovirus-2 (Ad-MLP, map units 16-17.3) to mouse dhfr cDNA at the 5'end. did. Intron for SV40 small t antigen and SV4
DNA encoding 0 initial region polyadenylation site
In Southern and Berg, J .; Mo
l. Appl. Genet. (1982) 1:
It was obtained from pSV2-neo described in 327-341 and fused to the 3'end of the dhfr cDNA. These three fragments were subcloned into pBR322 to obtain the plasmid Ad-dhfr. This plasmid is K
Auhman and Sharp, Molec. an
d Cell Biol. , (1982) 2: 130.
Functionally similar to the dhfr plasmid described in 4-1319. (6. Therapeutic Treatment Using gB-gD Vaccine) (6.1 Effect of Recombinant HSV Glycoprotein Vaccine Administered After Primary Infection on Recurrent Herpes Disease in Guinea Pigs) On the first day, 5 × 10 ⁵ pfu HSV-2 MS strain was inoculated. The animals were treated on days 1-10 by adding acyclovir (5 mg / ml) to the drinking water. Acyclovir reduces the symptoms of primary infections and thus reduces the incidence of secondary bacterial infections and genital wounds. The use of acyclovir during primary infection has been shown to have no effect on the course of the disease after cessation of treatment for guinea pigs (Bernstei).
N et al., Virology, (1986) 67: 160.
1). After recovery from the primary infection, these animals were treated with HSV.
-2 total glycoprotein preparation (gP2) and immunized with a mixture of recombinant gB1 and gD1 (HSV-1 gB + g
Immunized with D) or not treated. The treatment groups are shown below. Group Treatment Dose Adjuvant Route N I None None None None None 11 II HSV-1 gB + gD 25 μg + 25 μg Freund's foot pad 11 III gP2 50 μg Freund's foot pad 11 IV Control adjuvant only None Freund's foot pad injected with 9 foot posterior footpad injection The animals were immunized on day 21 and then on day 42. Both recombinant proteins gB1 and gD1 were produced in mammalian cells as described above. The results are reported in Table 1 and Figure 14. These results showed that the pattern of recurrent herpes disease was the same for groups I and IV, therefore these groups were pooled for analysis (control, n = 20).

The results shown in Table 1 and FIG. 14 are:
It has been shown that vaccination with recombinant glycoproteins has a significant impact on the frequency of disease recurrence. In addition, the gB + gD combination is superior to the mixture of natural glycoproteins.

The recurrence rate of a disease, measured by the number of disease days occurring within a specific time period, is an assessment that considers both the frequency and duration of recurrent episodes. FIG. 15A shows the infection rate of recurrent herpes disease expressed as the average number of days per week in which herpes disease was observed. The immunized group includes both gBgD and gD-2 vaccinated animals. As shown in FIG. 15A, the disease recurrence rate (days of disease per week) decreased in all groups as the evaluation period moved away from the first infection, but the reduction rate was in vaccinated animals. It was great. The difference in relapsing herpes disease infection rates between control and immunized animals is shown in Figure 15B. As is clear from FIG. 15B, the effect of the immunization with glycoprotein on the recurrence rate of the disease is as follows from FIG. 14, and it is inferred from FIG. Seemed to be established.

[0082]

[Table 1] (6.2 Effect of Recombinant HSV Glycoprotein Vaccine Administered After Primary Infection on Host Immune Response) The effect of post-infection glycoprotein administration on host immune response is the anti-HSV antibody produced by infected animals. Before infection,
It was determined by measuring after immunization with the HSV glycoprotein vaccine.

As described in section 6.1, H
SV-2 ms strain inoculated and treated with acyclovir,
Then treated with HSV glycoprotein vaccine. Serum was collected from these animals on days 41 and 95. Anti-HSV antibodies in serum are essentially as described by Pachl, C. et al. Et al, J
of Virology (1987) 61: 315.
-325, the method described in Section 1.6, measured by ELISA. The capture antigens include
HSV-1 glycoprotein mixture (gP-1), HSV-1
Glycoprotein D (gD-1) or HSV-2 glycoprotein D (gD-2) was contained.

HSV glycoprotein vaccine administration is anti-HSV
The effect on antibody titer is shown in Table 2, where the data are expressed as geometric means. No antibody was detected in serum collected before HSV inoculation. As seen in Table 2, in untreated control animals the titer of anti-HSV antibody was 9th.
The 41st day was larger than the 5th day. In contrast, animals treated with glycoproteins generally received 95th
It had been showing a high titer until the first day. Vaccination with HSV glycoprotein also significantly increased anti-HSV antibody titers compared to untreated controls (P <.05). Furthermore, treatment with the gP-2 mixture resulted in an antibody titer of 1.4
Although there was an ~ 7-fold increase, treatment with the recombinant HSV-1gBgD vaccine resulted in a 9-31-fold increase in titer compared to control values. Thus, administering an HSV glycoprotein to an animal, particularly a recombinant HSV glycoprotein gBgD, increases the host's immune response and, as shown in Section 6.1, recurrent HSV disease. Reduce the frequency and symptoms of.

[0085]

[Table 2] Effect of Adjuvants on Immune Response Induced by HSV Glycoprotein Vaccine Containing 6.3 gD1 Several adjuvants were tested to investigate their effect on immunotherapeutic treatment with HSV glycoprotein vaccine. The adjuvants tested were complete Freund's adjuvant (CFA), aluminum hydroxide, and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn. -Glycero-3-hydroxyphosphoryloxy) -ethylamine (called MTP-PE).

The effect of adjuvants was investigated by measuring the amount of anti-gD1 antibody after administration of vaccines containing gD1, which also contains various adjuvants. A vaccine containing only gD1 serves as a model for a vaccine containing gBgD. This is because the effect of adjuvants does not appear to be specific to the type of HSV glycoprotein in the vaccine.

In the following studies, gD1 was added to pHS13
Synthesized from 2 and isolated as described in Section 4.2. Female guinea pigs were immunized with a mixture consisting of 35 μg of gD1 and various adjuvant compositions by footpad administration three times at 3 week intervals. One week after the second immunization and 1, 5, 9 and 1 of the third immunization
After 3 weeks, the animals were bled and the anti-gD titers were
Determined by ELISA as described in Section 1.6.

The following results suggest that MTP-PE is the most promising of the tested adjuvants. This is because MTP-PE constitutively produces high anti-gD1 titers in experimental animals. Although titers were not retained as long as CFA, these levels were comparable to those seen for CFA. (6.3.1 Comparison of CFA and aluminum hydroxide)
Animals were immunized with a vaccine containing gD1 and either CFA or aluminum hydroxide. The effect of adjuvant on anti-gD titers is shown in Table 3. In Table 3, the data are represented by geometric mean. As seen in Table 5, the most effective adjuvant was CFA. The longest lasting and highest antibody titers were found in the group immunized with the CFA vaccine. The effect of other adjuvants is shown as a percentage compared to the titer obtained with CFA. 1
The lowest anti-gD1 titers were obtained by using 0% aluminum hydroxide suspension as an adjuvant.

[0089]

[Table 3] (6.3.2 CFA, nor-MDP, and MTP
-Comparison of PE) Animals were treated with gD1, CFA, nor-M
Immunizations were with vaccines containing DP and either MTP-PE. MTP-PE was encapsulated in liposomes and this latter adjuvant was co-administered with exogenous gD1 and incorporated into liposomes.
It was administered together with 1. Liposomes consist of synthetic phosphatidylcholine, phosphatidylserine and MTP-PE.
(Or MTP-PE and gD1) in suspension medium (sterile Ca ⁺⁺ and Mg ⁺⁺ salt-free isotonic Dulbecco's buffer, pH 7.2) at a ratio of 175: 75: 1 and vortexed. It was prepared by As seen in Table 4, immunization with a vaccine containing CFA yielded the highest mean anti-gD1 titers. nor
-The titers obtained with MDP ranged from 44 to 74% of the mean titers obtained with the CFA immunized group. MT
The mean titers obtained with P-PE and exogenous gD1 are n
somewhat lower than that obtained with or-MDP, CF
It was in the range of 32 to 72% of that obtained in A. M
GD1 encapsulated with TP-PE and liposomes
The very low titers obtained with can be due to the very low levels of gD1 in encapsulated form. The dose of encapsulated gD1 was only about 7% of the dose of exogenous gD1. This low dose was due to the very low efficiency of incorporation of gD1 into liposomes. This very low uptake can be caused by the size of the antigen. Other formulations of liposomes were able to uptake the antigen with better efficiency.

[0090]

[Table 4] (6.3.3 Comparison of different formulations containing MTP-PE) Animals were prepared with vaccine formulated with MTP-PE in a high oil delivery system (squalene / arracel) and MTP-PE in a low oil delivery system. Immunized with gD1 containing vaccine formulated in. Low oiliness MTP-
The PE formulation contained 4% squalene and 0.008% Tween 80. The gD1 antibody titers obtained with these adjuvant formulations are shown in Table 5.

As can be seen in Table 5, the MTP-PE formulation was effective as an adjuvant even when used as the sole ingredient in the low oil-surfactant formulation. [It was also an effective alternative to the CWS component in RIBI, and its effect increased over time when compared to RIBI as well. At the third blood draw, the titer obtained with MTP-RIBI was twice that obtained with RIBI. ] After the first blood collection, MTP-
The PE low oil formulation showed higher potency than the high oil delivery system (squalene / arracel).

[0092]

[Table 5] (6.4 Therapeutic Study: Impact of Adjuvants in Vaccines Effective in Preventing Recurrent Herpes Disease in Guinea Pigs,
Administration site and timing) gBgD vaccine is SD
It contains 25 μg each of recombinant gB1 and gD1 purified to approximately 70-80% unity as judged by S-PAGE. The recombinant gB1 protein is pHS1
13-9-10-21 and pHS137-7-B-5
It was a 50:50 mixture of gB1 prepared from the 0 cell line. These cell lines are based on the vector pHS11.
3 and pHS137 respectively. The preparation of pHS113 and pHS137 is described in Section 2.2. gB protein is described in Pachl et al., J of Virology, 6
1: 315-325 (1987), which is described in Section 2.2. GD1 was prepared as described in Section 4.2 except that 8D2 was used in place of C4D2 during purification by affinity chromatography.

In this example, the effects of two adjuvants, nor-MDP and CFA, are compared. Adjuvant nor-MAP was emulsified with 50% squalene / arracel and antigen and used at 50 μg / dose.

This study also compares two routes of administration. That is, the intra-footpad administration is compared with the intramuscular or subcutaneous administration. Finally, we compare various times of administration in the prevention of recurrent herpes disease in guinea pigs. The experimental design is shown in Table 6.

[0095]

[Table 6] For female Hartley guinea pigs weighing 350-400 g,
5.7 / log ₁₀ pfu of HSV-2 M on day 1
The S strain was inoculated in the vagina. Animals were confirmed to be infected by collecting HSV from vaginal swab samples collected 24 hours after vaginal inoculation. The medical course of primary infection was reviewed by Stanbury et al., J INF Dis.
(1987) 155: 914,
The lesions on the genital skin were followed and quantified. After recovery from the primary infection, animals were randomized into treatment groups as shown in Table 10. Animals were examined daily for signs of recurrence of the disease from day 11 to day 100 after the acute disease had resolved. Disease day is defined as the day on which recurrent disease was observed, severe recurrence was the day on which more than one blisters were observed, and episodes were days with no disease followed by new disease. Indicates that

The results obtained from the analysis of the animals between the 22nd and the 76th day are shown in Table 7. The data in Table 9 suggests that nor-MDP is an effective adjuvant for IM injection. This is reflected in a lower overall illness, a lower rate of severe recurrence, and a reduction in the total number of herpetic episodes. Furthermore, the vaccine containing nor-MDP and the administered IM are considered to be as effective as the vaccine containing CFA and administered in the footpad.

[0097]

[Table 7] Local reactions caused by injection of vaccines containing various adjuvants were also monitored. The results are shown in Table 8. The incidence of local erythema and cirrhosis at the injection site is
The same was true for vaccines containing nor-MDP.
Furthermore, based on local reaction productivity, nor-MDP
Is considered to have potential use in vaccines.

[0098]

[Table 8] The results in Table 7 also show that as the interval between the start of immunotherapy and the onset of acute illness decreases, the relative efficacy of treatment increases. 8, after the first infection
Of the animals vaccinated with the gBgD vaccine containing CFA after 15 or 21 days, the animals vaccinated the shortest after vaginal inoculation had the shortest duration of disease compared to untreated controls. , Had the lowest rate of severe recurrence, and had the lowest total episodes. Infection 1
After 5 days the values obtained for the vaccinated animals are
Higher than that obtained for the group vaccinated after 8 days, and higher for the group vaccinated after 21 days than for the group vaccinated after 15 days. This effect is also shown in FIG. FIG. 16 shows a graph of the number of recurrences on days following vaginal inoculation for the first vaccinated animals 8, 15 or 21 days after HSV-2 inoculation.

The data in FIG. 16 was used to calculate the percent reduction in relapsed disease (see Section 6.1 for a description of the significance of disease recurrence rate),
This data is shown in Table 9. In Table 9, it can be observed that the first vaccination on day 8 in the earliest period (ie 14-50 days) has the greatest reduction in the percentage of recurrent disease. However, the most effective protection was obtained from 51-92 days with the first vaccination 15 days after vaginal inoculation of HSV. The weakest protective effect was obtained when the first vaccination was given 21 days after the first contact with HSV-2.

It should be noted that in guinea pigs, the acute phase of the disease occurs in the period 14-21 days after infection with the virus. In this acute period,
Diseases induced by HSV are found in the body of animals. Thus, the data in Figure 16 show that the most effective protection at days 51-92 was obtained by administration of the vaccine during the acute phase of infection.

[0101]

[Table 9] According to the present invention, a vaccine effective for therapeutic treatment of herpes simplex virus type 1 and type 2 when administered after infection with herpes simplex virus is provided.

While the above invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications may be made within the scope of the appended claims. Is clear.

[0103]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to produce a vaccine effective for preventing recurrent herpes simplex virus-induced diseases using the isolated polynucleotide encoding a glycoprotein.

[Sequence list] SEQUENCE LISTING <110> Chiron Corporation <120> VACCINE FOR USE IN THE THERAPEUTIC TREATMENT OF HSV <130> 10-222018 Chiron <140> JP P1998-222018 <141> 1987-10-20 <150> US921,213 <151> 1986-10-20 <160> 6 <170> PatentIn Ver. 2.0 <210> 1 <211> 3472 <212> DNA <213> herpes simplex virus 7 <220> <221> CDS <222> (309) .. (3023) <400> 1 tcgcgagctc attatcgcca ccacactctt tgcgtcggtc taccggtgcg gggagcttga 60 gttgcgccgc cccgactgca gccgcccgac ctccgaaggt ctgtaccgct acccgccggg 120 cgtgtacctc acgtacaact ccgactgtcc gctggtggcc atcgtcgaga gcggccccga 180 cggctgcatc ggaccccgct cggtcgtggt ttacgaccga gacgtttttt ccatcctcta 240 ctcggtcctg cagcacctcg cccccagact agcgggcggc gggagcgacg cgcccccgta 300 ggcccgcc atg cgc ggg ggg ggc ttg att tgc gcg ctg gtc gtg ggg gcg 350 Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala 1 5 10 ctg gtg gcc gcg gtg gcg tcg gcg gcc ccg gcg gcc ccg gcg gcc ccc 398 Leu Val Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro 15 20 25 30 cgc gcc tcg ggc ggc gtg gcc gcg acc gtc gcg gcg aac ggg ggt ccc 446 Arg Ala Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro 35 40 45 gcc tcc cgg ccg ccc ccc gtc ccg agc ccc gcg acc acc aag gcc cgg 494 Ala Ser Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg 50 55 60 aag cgg aaa acc aaa aag ccg ccc aag cgg ccc gag gcg acc ccg ccc 542 Lys Arg Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro 65 70 75 ccc gac gcc aac gcg acc gtc gcc gcc ggc cac gcc acg ctg cgc gcg 590 Pro Asp Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala 80 85 90 cac ctg cgg gaa atc aag gtc gag aac gcc gat gcc cag ttt tac gtg 638 His Leu Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val 95 100 105 110 tgc ccg ccc ccg acg ggc gcc acg gtg gtg cag ttt gag cag ccg cgc 686 Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg 115 120 125 cgc tgc ccg acg cgc ccg gag ggg cag aac tac acg gag ggc atc gcg 734 Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala 130 135 140 gtg gtc ttc aag gag aac atc gcc ccg tac aaa ttc aag gcc acc atg 782 Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met 145 150 155 tac tac aaa gac gtg acc gtg tcg cag gtg tgg ttc ggc cac cgc tac 830 Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr 160 165 170 tcc cag ttt atg ggg ata ttc gag gac cgc gcc ccc gtt ccc ttc gag 878 Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu 175 180 185 190 gag gtg atc gac aag att aac gcc aag ggg gtc tgc cgc tcc acg gcc 926 Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala 195 200 205 aag tac gtg cgg aac aac atg gag acc acc gcg ttt cac cgg gac gac 974 Lys Tyr Val Arg Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp 210 215 220 cac gag acc gac atg gag ctc aag ccg gcg aag gtc gcc acg cgc acg 1022 His Glu Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr 225 230 235 agc cgg ggg tgg cac acc acc gac ctc aag tac aac ccc tcg cgg gtg 1070 Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val 240 245 250 gag gcg ttc cat cgg tac ggc acg acg gtc aac tgc atc gtc gag gag 1118 Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu 255 260 265 270 gtg gac gcg cgg tcg gtg tac ccg tac gat gag ttt gtg ttg gcg acg 1166 Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr 275 280 285 ggc gac ttt gtg tac atg tcc ccg ttt tac ggc tac cgg gag ggg tcg 1214 Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser 290 295 300 cac acc gag cac acc agc tac gcc gcc gac cgc ttc aag cag gtc gac 1262 His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp 305 310 315 ggc ttc tac gcg cgc gac ctc acc acg aag gcc cgg gcc acg tcg ccg 1310 Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro 320 325 330 acg acc cgc aac ttg ctg acg acc ccc aag ttt acc gtg gcc tgg gac 1358 Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp 335 340 345 350 tgg gtg ccg aag cga ccg gcg gtc tgc acc atg acc aag tgg cag gag 1406 Trp Val Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu 355 360 365 gtg gac gag atg ctc cgc gcc gag tac ggc ggc tcc ttc cgc ttc tcc 1454 Val Asp Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser 370 375 380 tcc gac gcc atc tcg acc acc ttc acc acc aac ctg acc cag tac tcg 1502 Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln Tyr Ser 385 390 395 ctc tcg cgc gtc gac ctg ggc gac tgc att ggc cgg gat gcc cgc gag 1550 Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu 400 405 410 gcc atc gac cgc atg ttt gcg cgc aag tac aac gcc acg cac atc aag 1598 Ala Ile Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys 415 420 425 430 gtg ggc cag ccg cag tac tac ctg gcc acg ggg ggc ttc ctc atc gcg 1646 Val Gly Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala 435 440 445 tac cag ccc ctc ctc agc aac acg ctc gcc gag ctg tac gtg cgg gag 1694 Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu 450 455 460 tac atg cgg gag cag gac cgc aag ccc cgg aat gcc acg ccc gcg cca 1742 Tyr Met Arg Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro 465 470 475 ctg cgg gag gcg ccc agc gcc aac gcg tcc gtg gag cgc atc aag acc 1790 Leu Arg Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr 480 485 490 acc tcc tcg atc gag ttc gcc cgg ctg cag ttt acg tat aac cac ata 1838 Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile 495 500 505 510 cag cgc cac gtg aac gac atg ctg ggg cgc atc gcc gtc gcg tgg tgc 1886 Gln Arg His Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys 515 520 525 gag ctg cag aac cac gag ctg act ctc tgg aac gag gcc cgc aag ctc 1934 Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu 530 535 540 aac ccc aac gcc atc gcc tcc gcc acc gtc ggc cgg cgg gtg agc gcg 1982 Asn Pro Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala 545 550 555 cgc atg ctc gga gac gtc atg gcc gtc tcc acg tgc gtg ccc gtc gcc 2030 Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala 560 565 570 ccg gac aac gtg atc gtg cag aac tcg atg cgc gtc agc tcg cgg ccg 2078 Pro Asp Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro 575 580 585 590 ggg acg tgc tac agc cgc ccc ctg gtc agc ttt cgg tac gaa gac cag 2126 Gly Thr Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln 595 600 605 ggc ccg ctg atc gag ggg cag ctg ggc gag aac aac gag ctg cgc ctc 2174 Gly Pro Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu 610 615 620 acc cgc gac gcg ctc gag ccg tgc acc gtg ggc cac cgg cgc tac ttc 2222 Thr Arg Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe 625 630 635 atc ttc ggc ggg ggc tac gtg tac ttc gag gag tac gcg tac tct cac 2270 Ile Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His 640 645 650 cag ctg agt cgc gcc gac gtc acc acc gtc agc acc ttc atc gac ctg 2318 Gln Leu Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu 655 660 665 670 aac atc acc atg ctg gag gac cac gag ttt gtg ccc ctg gag gtc tac 2366 Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr 675 680 685 acg cgc cac gag atc aag gac agc ggc ctg ctg gac tac acg gag gtc 2414 Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val 690 695 700 cag cgc cgc aac cag ctg cac gac ctg cgc ttt gcc gac atc gac acg 2462 Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr 705 710 715 gtc atc cgc gcc gac gcc aac gcc gcc atg ttc gcg ggg ctg tgc gcg 2510 Val Ile Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala 720 725 730 ttc ttc gag ggg atg ggg gac ttg ggg cgc gcg gtc ggc aag gta gtc 2558 Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val 735 740 745 750 atg gga gta gtg ggg ggc gtg gtg tcg gcc gtc tcg ggc gtg tcc tcc 2606 Met Gly Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser 755 760 765 ttt atg tcc aac ccc ttc ggg gcg ctt gcc gtg ggg ctg ctg gtc ctg 2654 Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu 770 775 780 gcc ggc ctg gtc gcg gcc ttc ttc gcc ttc cgc tac gtc ctg caa ctg 2702 Ala Gly Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu 785 790 795 caa cgc aat ccc atg aag gcc ctg tat ccg ctc acc acc aag gaa ctc 2750 Gln Arg Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu 800 805 810 aag act tcc gac ccc ggg ggc gtg ggc ggg gag ggg gag gaa ggc gcg 2798 Lys Thr Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala 815 820 825 830 gag ggg ggc ggg ttt gac gag gcc aag ttg gcc gag gcc cga gaa atg 2846 Glu Gly Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met 835 840 845 atc cga tat atg gct ttg gtg tcg gcc atg gag cgc acg gaa cac aag 2894 Ile Arg Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys 850 855 860 gcc aga aag aag ggc acg agc gcc ctg ctc agc tcc aag gtc acc aac 2942 Ala Arg Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn 865 870 875 atg gtt ctg cgc aag cgc aac aaa gcc agg tac tct ccg ctc cac aac 2990 Met Val Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn 880 885 890 gag gac gag gcc gga gac gaa gac gag ctc taa gggaggggag gggagctggg 3043 Glu Asp Glu Ala Gly Asp Glu Asp Glu Leu 895 900 905 cttgtgtata aataaaaaga caccgatgtt caaaaataca catgacttct ggtattgttt 3103 tgccttggtt tttatttggg gggggggcgt gtgactagaa aaacaaatgc agacatgtgc 3163 taacgggaaa accaacccca aaccaacccc aaaccaaccc cgtctccctg cgaccggtcg 3223 ctttccacac cccctccccg tggtagtctt ccgggccttc cgtcgcgtgt gggggccatc 3283 ggttcggctc ctagcccccc ccccctcacc cctccgacct aatttttgtg tcattcggcc 3343 cactttcccc cccactccac cccccccctc tcaaacaaaa acacaagcac acgaagtggg 3403 tatacttttg tccggttgtt tgtttattta aaatatatga aaacacac cccccccaag 3463 tccggatcc 3472 <210> 2 <211> 2712 <212> DNA <213> herpes simplex virus 7 <220> <221> CDS <222> (1) .. (2712) <400> 2 atg cgc ggg ggg ggc ttg att tgc gcg ctg gtc gtg ggg gcg ctg gtg 48 Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val 1 5 10 15 gcc gcg gtg gcg tcg gcg gcc ccg gcg gcc ccg gcg gcc ccc cgc gcc 96 Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala 20 25 30 tcg ggc ggc gtg gcc gcg acc gtc gcg gcg aac ggg ggt ccc gcc tcc 144 Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser 35 40 45 cgg ccg ccc ccc gtc ccg agc ccc gcg acc acc aag gcc cgg aag cgg 192 Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg 50 55 60 aaa acc aaa aag ccg ccc aag cgg ccc gag gcg acc ccg ccc ccc gac 240 Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp 65 70 75 80 gcc aac gcg acc gtc gcc gcc ggc cac gcc acg ctg cgc gcg cac ctg 288 Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu 85 90 95 cgg gaa atc aag gtc gag aac gcc gat gcc cag ttt tac gtg tgc ccg 336 Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro 100 105 110 ccc ccg acg ggc gcc acg gtg gtg cag ttt gag cag ccg cgc cgc tgc 384 Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys 115 120 125 ccg acg cgc ccg gag ggg cag aac tac acg gag ggc atc gcg gtg gtc 432 Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val 130 135 140 ttc aag gag aac atc gcc ccg tac aaa ttc aag gcc acc atg tac tac 480 Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr 145 150 155 160 aaa gac gtg acc gtg tcg cag gtg tgg ttc ggc cac cgc tac tcc cag 528 Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln 165 170 175 ttt atg ggg ata ttc gag gac cgc gcc ccc gtt ccc ttc gag gag gtg 576 Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu Glu Val 180 185 190 atc gac aag att aac gcc aag ggg gtc tgc cgc tcc acg gcc aag tac 624 Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr 195 200 205 gtg cgg aac aac atg gag acc acc gcg ttt cac cgg gac gac cac gag 672 Val Arg Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu 210 215 220 acc gac atg gag ctc aag ccg gcg aag gtc gcc acg cgc acg agc cgg 720 Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg 225 230 235 240 ggg tgg cac acc acc gac ctc aag tac aac ccc tcg cgg gtg gag gcg 768 Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala 245 250 255 ttc cat cgg tac ggc acg acg gtc aac tgc atc gtc gag gag gtg gac 816 Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp 260 265 270 gcg cgg tcg gtg tac ccg tac gat gag ttt gtg ttg gcg acg ggc gac 864 Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp 275 280 285 ttt gtg tac atg tcc ccg ttt tac ggc tac cgg gag ggg tcg cac acc 912 Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr 290 295 300 gag cac acc agc tac gcc gcc gac cgc ttc aag cag gtc gac ggc ttc 960 Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe 305 310 315 320 tac gcg cgc gac ctc acc acg aag gcc cgg gcc acg tcg ccg acg acc 1008 Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr 325 330 335 cgc aac ttg ctg acg acc ccc aag ttt acc gtg gcc tgg gac tgg gtg 1056 Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val 340 345 350 ccg aag cga ccg gcg gtc tgc acc atg acc aag tgg cag gag gtg gac 1104 Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp 355 360 365 gag atg ctc cgc gcc gag tac ggc ggc tcc ttc cgc ttc tcc tcc gac 1152 Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp 370 375 380 gcc atc tcg acc acc ttc acc acc aac ctg acc cag tac tcg ctc tcg 1200 Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln Tyr Ser Leu Ser 385 390 395 400 cgc gtc gac ctg ggc gac tgc att ggc cgg gat gcc cgc gag gcc atc 1248 Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile 405 410 415 gac cgc atg ttt gcg cgc aag tac aac gcc acg cac atc aag gtg ggc 1296 Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys Val Gly 420 425 430 cag ccg cag tac tac ctg gcc acg ggg ggc ttc ctc atc gcg tac cag 1344 Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln 435 440 445 ccc ctc ctc agc aac acg ctc gcc gag ctg tac gtg cgg gag tac atg 1392 Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met 450 455 460 cgg gag cag gac cgc aag ccc cgg aat gcc acg ccc gcg cca ctg cgg 1440 Arg Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg 465 470 475 480 gag gcg ccc agc gcc aac gcg tcc gtg gag cgc atc aag acc acc tcc 1488 Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser 485 490 495 tcg atc gag ttc gcc cgg ctg cag ttt acg tat aac cac ata cag cgc 1536 Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg 500 505 510 cac gtg aac gac atg ctg ggg cgc atc gcc gtc gcg tgg tgc gag ctg 1584 His Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu 515 520 525 cag aac cac gag ctg act ctc tgg aac gag gcc cgc aag ctc aac ccc 1632 Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro 530 535 540 aac gcc atc gcc tcc gcc acc gtc ggc cgg cgg gtg agc gcg cgc atg 1680 Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met 545 550 555 560 ctc gga gac gtc atg gcc gtc tcc acg tgc gtg ccc gtc gcc ccg gac 1728 Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp 565 570 575 aac gtg atc gtg cag aac tcg atg cgc gtc agc tcg cgg ccg ggg acg 1776 Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr 580 585 590 tgc tac agc cgc ccc ctg gtc agc ttt cgg tac gaa gac cag ggc ccg 1824 Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro 595 600 605 ctg atc gag ggg cag ctg ggc gag aac aac gag ctg cgc ctc acc cgc 1872 Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg 610 615 620 gac gcg ctc gag ccg tgc acc gtg ggc cac cgg cgc tac ttc atc ttc 1920 Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Ile Phe 625 630 635 640 ggc ggg ggc tac gtg tac ttc gag gag tac gcg tac tct cac cag ctg 1968 Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu 645 650 655 agt cgc gcc gac gtc acc acc gtc agc acc ttc atc gac ctg aac atc 2016 Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile 660 665 670 acc atg ctg gag gac cac gag ttt gtg ccc ctg gag gtc tac acg cgc 2064 Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg 675 680 685 cac gag atc aag gac agc ggc ctg ctg gac tac acg gag gtc cag cgc 2112 His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg 690 695 700 cgc aac cag ctg cac gac ctg cgc ttt gcc gac atc gac acg gtc atc 2160 Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile 705 710 715 720 cgc gcc gac gcc aac gcc gcc atg ttc gcg ggg ctg tgc gcg ttc ttc 2208 Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe 725 730 735 gag ggg atg ggg gac ttg ggg cgc gcg gtc ggc aag gta gtc atg gga 2256 Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly 740 745 750 gta gtg ggg ggc gtg gtg tcg gcc gtc tcg ggc gtg tcc tcc ttt atg 2304 Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met 755 760 765 tcc aac ccc ttc ggg gcg ctt gcc gtg ggg ctg ctg gtc ctg gcc ggc 2352 Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly 770 775 780 ctg gtc gcg gcc ttc ttc gcc ttc cgc tac gtc ctg caa ctg caa cgc 2400 Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg 785 790 795 800 aat ccc atg aag gcc ctg tat ccg ctc acc acc aag gaa ctc aag act 2448 Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr 805 810 815 tcc gac ccc ggg ggc gtg ggc ggg gag ggg gag gaa ggc gcg gag ggg 2496 Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly 820 825 830 ggc ggg ttt gac gag gcc aag ttg gcc gag gcc cga gaa atg atc cga 2544 Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845 tat atg gct ttg gtg tcg gcc atg gag cgc acg gaa cac aag gcc aga 2592 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg 850 855 860 aag aag ggc acg agc gcc ctg ctc agc tcc aag gtc acc aac atg gtt 2640 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn Met Val 865 870 875 880 ctg cgc aag cgc aac aaa gcc agg tac tct ccg ctc cac aac gag gac 2688 Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp 885 890 895 gag gcc gga gac gaa gac gag ctc 2712 Glu Ala Gly Asp Glu Asp Glu Leu 900 <210> 3 <211> 904 <212> PRT <213> herpes simplex virus 7 <400> 3 Met Arg Gly Gly Gly Leu Ile Cys Ala Leu Val Val Gly Ala Leu Val 1 5 10 15 Ala Ala Val Ala Ser Ala Ala Pro Ala Ala Pro Ala Ala Pro Arg Ala 20 25 30 Ser Gly Gly Val Ala Ala Thr Val Ala Ala Asn Gly Gly Pro Ala Ser 35 40 45 Arg Pro Pro Pro Val Pro Ser Pro Ala Thr Thr Lys Ala Arg Lys Arg 50 55 60 Lys Thr Lys Lys Pro Pro Lys Arg Pro Glu Ala Thr Pro Pro Pro Asp 65 70 75 80 Ala Asn Ala Thr Val Ala Ala Gly His Ala Thr Leu Arg Ala His Leu 85 90 95 Arg Glu Ile Lys Val Glu Asn Ala Asp Ala Gln Phe Tyr Val Cys Pro 100 105 110 Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu Gln Pro Arg Arg Cys 115 120 125 Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu Gly Ile Ala Val Val 130 135 140 Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys Ala Thr Met Tyr Tyr 145 150 155 160 Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly His Arg Tyr Ser Gln 165 170 175 Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val Pro Phe Glu Glu Val 180 185 190 Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg Ser Thr Ala Lys Tyr 195 200 205 Val Arg Asn Asn Met Glu Thr Thr Ala Phe His Arg Asp Asp His Glu 210 215 220 Thr Asp Met Glu Leu Lys Pro Ala Lys Val Ala Thr Arg Thr Ser Arg 225 230 235 240 Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro Ser Arg Val Glu Ala 245 250 255 Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile Val Glu Glu Val Asp 260 265 270 Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val Leu Ala Thr Gly Asp 275 280 285 Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg Glu Gly Ser His Thr 290 295 300 Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys Gln Val Asp Gly Phe 305 310 315 320 Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala Thr Ser Pro Thr Thr 325 330 335 Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val Ala Trp Asp Trp Val 340 345 350 Pro Lys Arg Pro Ala Val Cys Thr Met Thr Lys Trp Gln Glu Val Asp 355 360 365 Glu Met Leu Arg Ala Glu Tyr Gly Gly Ser Phe Arg Phe Ser Ser Asp 370 375 380 Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr Gln Tyr Ser Leu Ser 385 390 395 400 Arg Val Asp Leu Gly Asp Cys Ile Gly Arg Asp Ala Arg Glu Ala Ile 405 410 415 Asp Arg Met Phe Ala Arg Lys Tyr Asn Ala Thr His Ile Lys Val Gly 420 425 430 Gln Pro Gln Tyr Tyr Leu Ala Thr Gly Gly Phe Leu Ile Ala Tyr Gln 435 440 445 Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr Val Arg Glu Tyr Met 450 455 460 Arg Glu Gln Asp Arg Lys Pro Arg Asn Ala Thr Pro Ala Pro Leu Arg 465 470 475 480 Glu Ala Pro Ser Ala Asn Ala Ser Val Glu Arg Ile Lys Thr Thr Ser 485 490 495 Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His Ile Gln Arg 500 505 510 His Val Asn Asp Met Leu Gly Arg Ile Ala Val Ala Trp Cys Glu Leu 515 520 525 Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys Leu Asn Pro 530 535 540 Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser Ala Arg Met 545 550 555 560 Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val Ala Pro Asp 565 570 575 Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro Gly Thr 580 585 590 Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp Gln Gly Pro 595 600 605 Leu Ile Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg Leu Thr Arg 610 615 620 Asp Ala Leu Glu Pro Cys Thr Val Gly His Arg Arg Tyr Phe Ile Phe 625 630 635 640 Gly Gly Gly Tyr Val Tyr Phe Glu Glu Tyr Ala Tyr Ser His Gln Leu 645 650 655 Ser Arg Ala Asp Val Thr Thr Val Ser Thr Phe Ile Asp Leu Asn Ile 660 665 670 Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val Tyr Thr Arg 675 680 685 His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu Val Gln Arg 690 695 700 Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp Thr Val Ile 705 710 715 720 Arg Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Cys Ala Phe Phe 725 730 735 Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val Val Met Gly 740 745 750 Val Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser Ser Phe Met 755 760 765 Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly 770 775 780 Leu Val Ala Ala Phe Phe Ala Phe Arg Tyr Val Leu Gln Leu Gln Arg 785 790 795 800 Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu Leu Lys Thr 805 810 815 Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala Glu Gly 820 825 830 Gly Gly Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Arg 850 855 860 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ser Lys Val Thr Asn Met Val 865 870 875 880 Leu Arg Lys Arg Asn Lys Ala Arg Tyr Ser Pro Leu His Asn Glu Asp 885 890 895 Glu Ala Gly Asp Glu Asp Glu Leu 900 <210> 4 <211> 3472 <212> DNA <213> herpes simplex virus 7 <220> <221> CDS <222> (309) .. (3023) <400> 4 tcgcgagctg attatcgcca ccacactctt tgcctcggtc taccggtgcg gggagctcga 60 gttgcgcngc ccggactgca gccgcccgac ctccgaaggt cgttaccgtt acccgcccgg 120 cgtatatctc acgtacgact ccgactgtcc gctggtggcc atcgtcgaga gcgcccccga 180 cggctgtatc ggcccccggt cggtcgtggt ctacgaccga gacgttttct cgatcctcta 240 ctcggtcctc cagcacctcg cccccaggct acctgacggg gggcacgacg ggcccccgta 300 gtcccgcc atg cgc cag ggc gcc ccc gcg cgg ggg tgc cgg tgg ttc gtc 350 Met Arg Gln Gly Ala Pro Ala Arg Gly Cys Arg Trp Phe Val 1 5 10 gta tgg gcg ctc ttg ggg ttg acg ctg ggg gtc ctg gtg gcg tcg gcg 398 Val Trp Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala 15 20 25 30 gct ccg agt tcc ccc ggc acg cct ggg gtc gcg gcc gcg acc cag gcg 446 Ala Pro Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala 35 40 45 gcg aac ggg ggc cct gcc act ccg gcg ccg ccc gcc ctt ggc gcc gcc 494 Ala Asn Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala 50 55 60 cca acg ggg gac ccg aaa ccg aag aag aac aaa aaa ccg aaa aac cca 542 Pro Thr Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro 65 70 75 acg ccg cca cgc ccc gcc ggc gac aac gcg acc gtc gcc gcg ggc cac 590 Thr Pro Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His 80 85 90 gcc acc ctg cgc gag cac ctg cgg gac atc aag gcg gag aac acc gat 638 Ala Thr Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp 95 100 105 110 gca aac ttt tac gtg tgc cca ccc ccc acg ggc gcc acg gtg gtg cag 686 Ala Asn Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln 115 120 125 ttc gag cag ccg cgc cgc tgc ccg acc cgg ccc gag ggt cag aac tac 734 Phe Glu Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr 130 135 140 acg gag ggc atc gcg gtg gtc ttc aag gag aac atc gcc ccg tac aag 782 Thr Glu Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys 145 150 155 ttc aag gcc acc atg tac tac aaa gac gtc acc gtt tcg cag gtg tgg 830 Phe Lys Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp 160 165 170 ttc ggc cac cgc tac tcc cag ttt atg ggg atc ttt gag gac cgc gcc 878 Phe Gly His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala 175 180 185 190 ccc gtc ccc ttc gag gag gtg atc gac aag atc aac gcc aag ggg gtc 926 Pro Val Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val 195 200 205 tgt cgg tcc acg gcc aag tac gtg cgc aac aac ctg gag acc acc gcg 974 Cys Arg Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala 210 215 220 ttt cac cgg gac gac cac gag acc gac atg gag ctg aaa ccg gcc aac 1022 Phe His Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn 225 230 235 gcc gcg acc cgc acg agc cgg ggc tgg cac acc acc gac ctc aag tac 1070 Ala Ala Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr 240 245 250 aac ccc tcg cgg gtg gag gcg ttc cac cgg tac ggg acg acg gta aac 1118 Asn Pro Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn 255 260 265 270 tgc atc gtc gag gag gtg gac gcg cgc tcg gtg tac ccg tac gac gag 1166 Cys Ile Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu 275 280 285 ttt gtg ctg gcg act ggc gac ttt gtg tac atg tcc ccg ttt tac ggc 1214 Phe Val Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly 290 295 300 tac cgg gag ggg tcg cac acc gaa cac acc agc tac gcc gcc gac cgc 1262 Tyr Arg Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg 305 310 315 ttc aag cag gtc gac ggc ttc tac gcg cgc gac ctc acc acc aag gcc 1310 Phe Lys Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala 320 325 330 cgg gcc acg gcg ccg acc acc cgg aac ctg ctc acg acc ccc aag ttc 1358 Arg Ala Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe 335 340 345 350 acc gtg gcc tgg gac tgg gtg cca aag cgc ccg tcg gtc tgc acc atg 1406 Thr Val Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met 355 360 365 acc aag tgg cag gag gtg gac gag atg ctg cgc tcc gag tac ggc ggc 1454 Thr Lys Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly 370 375 380 tcc ttc cga ttc tcc tcc gac gcc ata tcc acc acc ttc acc acc aac 1502 Ser Phe Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn 385 390 395 ctg acc gag tac ccg ctc tcg cgc gtt gac ctg ggg gac tgc atc ggc 1550 Leu Thr Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly 400 405 410 aag gac gcc cgc gac gcc atg gac cgc atc ttc gcc cgc agg tac aac 1598 Lys Asp Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn 415 420 425 430 gcg acg cac atc aag gtg ggc cag ccg cag tac tac ctg gcc aat ggg 1646 Ala Thr His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly 435 440 445 ggc ttt ctg atc gcg tac cag ccc ctt ctc agc aac acg ctc gcg gag 1694 Gly Phe Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu 450 455 460 ctg tac gtg cgg gaa cac ctc cga gag cag agc cgc aag ccc cca aac 1742 Leu Tyr Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn 465 470 475 ccc acg ccc ccg ccg ccc ggg gcc agc gcc aac gcg tcc gtg gag cgc 1790 Pro Thr Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg 480 485 490 atc aag acc acc tcc tcc atc gag ttc gcc cgg ctg cag ttt acg tac 1838 Ile Lys Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr 495 500 505 510 aac cac ata cag cgc cat gtc aac gat atg ttg ggc cgc gtt gcc atc 1886 Asn His Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile 515 520 525 gcg tgg tgc gag ctg cag aat cac gag ctg acc ctg tgg aac gag gcc 1934 Ala Trp Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala 530 535 540 cgc aag ctg aac ccc aac gcc atc gcc tcg gcc acc gtg ggc cgg cgg 1982 Arg Lys Leu Asn Pro Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg 545 550 555 gtg agc gcg cgg atg ctc ggc gac gtg atg gcc gtc tcc acg tgc gtg 2030 Val Ser Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val 560 565 570 ccg gtc gcc gcg gac aac gtg atc gtc caa aac tcg atg cgc atc agc 2078 Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser 575 580 585 590 tcg cgg ccc ggg gcc tgc tac agc cgc ccc ctg gtc agc ttt cgg tac 2126 Ser Arg Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr 595 600 605 gaa gac cag ggc ccg ttg gtc gag ggg cag ctg ggg gag aac aac gag 2174 Glu Asp Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu 610 615 620 ctg cgg ctg acg cgc gat gcg atc gag ccg tgc acc gtg gga cac cgg 2222 Leu Arg Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg 625 630 635 cgc tac ttc acc ttc ggt ggg ggc tac gtg tac ttc gag gag tca gcg 2270 Arg Tyr Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala 640 645 650 tac tcc cac cag ctg agc cgc gcc gac atc acc acc gtc agc acc ttc 2318 Tyr Ser His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe 655 660 665 670 atc gac ctc aac atc acc atg ctg gag gat cac gag ttt gtc ccc ctg 2366 Ile Asp Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu 675 680 685 gag gtg tac acc cgc cac gag atc aag gac agc ggc ctg ctg gac tac 2414 Glu Val Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr 690 695 700 acg gag gtc cag cgc cgc aac cag ctg cac gac ctg cgc ttc gcc gac 2462 Thr Glu Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp 705 710 715 atc gac acg gtc atc cac gcc gac gcc aac gcc gcc atg ttc gcg ggc 2510 Ile Asp Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly 720 725 730 ctg ggt gcg ttt ttc gag ggg atg ggc gac ctg ggg cgc gcg gtc ggc 2558 Leu Gly Ala Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly 735 740 745 750 aag gtg gtg atg ggc atc gtg ggc ggc gtg gta tcg gcc gtg tcg ggc 2606 Lys Val Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly 755 760 765 gtg tcc tcc ttc atg tcc aac ccc ttt ggg gcg ctg gcc gtg ggt ctg 2654 Val Ser Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu 770 775 780 ttg gtc ctg gcc ggc ctg gcg gcg gcc ttc ttc gcc ttt cgc tac gtc 2702 Leu Val Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val 785 790 795 atg cgg ctg cag agc aac ccc atg aag gcc ctg tac ccg cta acc acc 2750 Met Arg Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr 800 805 810 aag gag ctc aag aac ccc acc aac ccg gac gcg tcc ggg gag ggc gag 2798 Lys Glu Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu 815 820 825 830 gag ggc ggc gac ttt gac gag gcc aag cta gcc gag gcc cgg gag atg 2846 Glu Gly Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met 835 840 845 ata cgg tac atg gcc ctg gtg tct gcc atg gag cgc acg gaa cac aag 2894 Ile Arg Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys 850 855 860 gcc aag aag aag ggc acg agc gcg ctg ctc agc gcc aag gtc acc gac 2942 Ala Lys Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp 865 870 875 atg gtc atg cgc aag cgc cgc aac acc aac tac acc caa gtt ccc aac 2990 Met Val Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn 880 885 890 aaa gac ggt gac gcc gac gag gac gac ctg tga cggggggttt gttgtaaata 3043 Lys Asp Gly Asp Ala Asp Glu Asp Asp Leu 895 900 905 aaaaccacgg gtgttaaacc gcatgtgcat cttttggtgt gtttgtttgg tacgcctttt 3103 gtgtgtgtgg gaagaaagaa aagggaacac ataaactccc ccgggtgtcc gcggcctgtt 3163 tcctctttcc tttcccgtga caaaacggac ccccttggtc agtgccgatt cccccccacg 3223 ccttcctcca cgtcgaaggc ttttgcattg taaagctacc cgcctacccg cgcctcccaa 3283 taaaaaaaaa gaacatacac caatgggtct tatttggtat tacctggttt atttaaaaag 3343 atatacagta agacatccca tggtaccaaa gaccggggcg aatcagcggg cccccatcat 3403 ctgagagacg aacaaatcgg cggcgcgggc cgtgtcaacg tccacgtgtg ctgcgctgct 3463 ggcgttgac 3472 <210> 5 <211> 2712 <212> DNA <213> herpes simplex virus 7 <220> <221> CDS <222> (1) .. (2712) <400> 5 atg cgc cag ggc gcc ccc gcg cgg ggg tgc cgg tgg ttc gtc gta tgg 48 Met Arg Gln Gly Ala Pro Ala Arg Gly Cys Arg Trp Phe Val Val Trp 1 5 10 15 gcg ctc ttg ggg ttg acg ctg ggg gtc ctg gtg gcg tcg gcg gct ccg 96 Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro 20 25 30 agt tcc ccc ggc acg cct ggg gtc gcg gcc gcg acc cag gcg gcg aac 144 Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn 35 40 45 ggg ggc cct gcc act ccg gcg ccg ccc gcc ctt ggc gcc gcc cca acg 192 Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr 50 55 60 ggg gac ccg aaa ccg aag aag aac aaa aaa ccg aaa aac cca acg ccg 240 Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro 65 70 75 80 cca cgc ccc gcc ggc gac aac gcg acc gtc gcc gcg ggc cac gcc acc 288 Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr 85 90 95 ctg cgc gag cac ctg cgg gac atc aag gcg gag aac acc gat gca aac 336 Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn 100 105 110 ttt tac gtg tgc cca ccc ccc acg ggc gcc acg gtg gtg cag ttc gag 384 Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu 115 120 125 cag ccg cgc cgc tgc ccg acc cgg ccc gag ggt cag aac tac acg gag 432 Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu 130 135 140 ggc atc gcg gtg gtc ttc aag gag aac atc gcc ccg tac aag ttc aag 480 Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys 145 150 155 160 gcc acc atg tac tac aaa gac gtc acc gtt tcg cag gtg tgg ttc ggc 528 Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly 165 170 175 cac cgc tac tcc cag ttt atg ggg atc ttt gag gac cgc gcc ccc gtc 576 His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val 180 185 190 ccc ttc gag gag gtg atc gac aag atc aac gcc aag ggg gtc tgt cgg 624 Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg 195 200 205 tcc acg gcc aag tac gtg cgc aac aac ctg gag acc acc gcg ttt cac 672 Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His 210 215 220 cgg gac gac cac gag acc gac atg gag ctg aaa ccg gcc aac gcc gcg 720 Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala 225 230 235 240 acc cgc acg agc cgg ggc tgg cac acc acc gac ctc aag tac aac ccc 768 Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro 245 250 255 tcg cgg gtg gag gcg ttc cac cgg tac ggg acg acg gta aac tgc atc 816 Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile 260 265 270 gtc gag gag gtg gac gcg cgc tcg gtg tac ccg tac gac gag ttt gtg 864 Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val 275 280 285 ctg gcg act ggc gac ttt gtg tac atg tcc ccg ttt tac ggc tac cgg 912 Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg 290 295 300 gag ggg tcg cac acc gaa cac acc agc tac gcc gcc gac cgc ttc aag 960 Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys 305 310 315 320 cag gtc gac ggc ttc tac gcg cgc gac ctc acc acc aag gcc cgg gcc 1008 Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala 325 330 335 acg gcg ccg acc acc cgg aac ctg ctc acg acc ccc aag ttc acc gtg 1056 Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val 340 345 350 gcc tgg gac tgg gtg cca aag cgc ccg tcg gtc tgc acc atg acc aag 1104 Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys 355 360 365 tgg cag gag gtg gac gag atg ctg cgc tcc gag tac ggc ggc tcc ttc 1152 Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe 370 375 380 cga ttc tcc tcc gac gcc ata tcc acc acc ttc acc acc aac ctg acc 1200 Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr 385 390 395 400 gag tac ccg ctc tcg cgc gtt gac ctg ggg gac tgc atc ggc aag gac 1248 Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp 405 410 415 gcc cgc gac gcc atg gac cgc atc ttc gcc cgc agg tac aac gcg acg 1296 Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr 420 425 430 cac atc aag gtg ggc cag ccg cag tac tac ctg gcc aat ggg ggc ttt 1344 His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe 435 440 445 ctg atc gcg tac cag ccc ctt ctc agc aac acg ctc gcg gag ctg tac 1392 Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr 450 455 460 gtg cgg gaa cac ctc cga gag cag agc cgc aag ccc cca aac ccc acg 1440 Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr 465 470 475 480 ccc ccg ccg ccc ggg gcc agc gcc aac gcg tcc gtg gag cgc atc aag 1488 Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys 485 490 495 acc acc tcc tcc atc gag ttc gcc cgg ctg cag ttt acg tac aac cac 1536 Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His 500 505 510 ata cag cgc cat gtc aac gat atg ttg ggc cgc gtt gcc atc gcg tgg 1584 Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp 515 520 525 tgc gag ctg cag aat cac gag ctg acc ctg tgg aac gag gcc cgc aag 1632 Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys 530 535 540 ctg aac ccc aac gcc atc gcc tcg gcc acc gtg ggc cgg cgg gtg agc 1680 Leu Asn Pro Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser 545 550 555 560 gcg cgg atg ctc ggc gac gtg atg gcc gtc tcc acg tgc gtg ccg gtc 1728 Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val 565 570 575 gcc gcg gac aac gtg atc gtc caa aac tcg atg cgc atc agc tcg cgg 1776 Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg 580 585 590 ccc ggg gcc tgc tac agc cgc ccc ctg gtc agc ttt cgg tac gaa gac 1824 Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp 595 600 605 cag ggc ccg ttg gtc gag ggg cag ctg ggg gag aac aac gag ctg cgg 1872 Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg 610 615 620 ctg acg cgc gat gcg atc gag ccg tgc acc gtg gga cac cgg cgc tac 1920 Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr 625 630 635 640 ttc acc ttc ggt ggg ggc tac gtg tac ttc gag gag tca gcg tac tcc 1968 Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser 645 650 655 cac cag ctg agc cgc gcc gac atc acc acc gtc agc acc ttc atc gac 2016 His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp 660 665 670 ctc aac atc acc atg ctg gag gat cac gag ttt gtc ccc ctg gag gtg 2064 Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val 675 680 685 tac acc cgc cac gag atc aag gac agc ggc ctg ctg gac tac acg gag 2112 Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu 690 695 700 gtc cag cgc cgc aac cag ctg cac gac ctg cgc ttc gcc gac atc gac 2160 Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp 705 710 715 720 acg gtc atc cac gcc gac gcc aac gcc gcc atg ttc gcg ggc ctg ggt 2208 Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly 725 730 735 gcg ttt ttc gag ggg atg ggc gac ctg ggg cgc gcg gtc ggc aag gtg 2256 Ala Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val 740 745 750 gtg atg ggc atc gtg ggc ggc gtg gta tcg gcc gtg tcg ggc gtg tcc 2304 Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser 755 760 765 tcc ttc atg tcc aac ccc ttt ggg gcg ctg gcc gtg ggt ctg ttg gtc 2352 Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val 770 775 780 ctg gcc ggc ctg gcg gcg gcc ttc ttc gcc ttt cgc tac gtc atg cgg 2400 Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg 785 790 795 800 ctg cag agc aac ccc atg aag gcc ctg tac ccg cta acc acc aag gag 2448 Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu 805 810 815 ctc aag aac ccc acc aac ccg gac gcg tcc ggg gag ggc gag gag ggc 2496 Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly 820 825 830 ggc gac ttt gac gag gcc aag cta gcc gag gcc cgg gag atg ata cgg 2544 Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845 tac atg gcc ctg gtg tct gcc atg gag cgc acg gaa cac aag gcc aag 2592 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Lys 850 855 860 aag aag ggc acg agc gcg ctg ctc agc gcc aag gtc acc gac atg gtc 2640 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp Met Val 865 870 875 880 atg cgc aag cgc cgc aac acc aac tac acc caa gtt ccc aac aaa gac 2688 Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp 885 890 895 ggt gac gcc gac gag gac gac ctg 2712 Gly Asp Ala Asp Glu Asp Asp Leu 900 <210> 6 <211> 904 <212> PRT <213> herpes simplex virus 7 <400> 6 Met Arg Gln Gly Ala Pro Ala Arg Gly Cys Arg Trp Phe Val Val Trp 1 5 10 15 Ala Leu Leu Gly Leu Thr Leu Gly Val Leu Val Ala Ser Ala Ala Pro 20 25 30 Ser Ser Pro Gly Thr Pro Gly Val Ala Ala Ala Thr Gln Ala Ala Asn 35 40 45 Gly Gly Pro Ala Thr Pro Ala Pro Pro Ala Leu Gly Ala Ala Pro Thr 50 55 60 Gly Asp Pro Lys Pro Lys Lys Asn Lys Lys Pro Lys Asn Pro Thr Pro 65 70 75 80 Pro Arg Pro Ala Gly Asp Asn Ala Thr Val Ala Ala Gly His Ala Thr 85 90 95 Leu Arg Glu His Leu Arg Asp Ile Lys Ala Glu Asn Thr Asp Ala Asn 100 105 110 Phe Tyr Val Cys Pro Pro Pro Thr Gly Ala Thr Val Val Gln Phe Glu 115 120 125 Gln Pro Arg Arg Cys Pro Thr Arg Pro Glu Gly Gln Asn Tyr Thr Glu 130 135 140 Gly Ile Ala Val Val Phe Lys Glu Asn Ile Ala Pro Tyr Lys Phe Lys 145 150 155 160 Ala Thr Met Tyr Tyr Lys Asp Val Thr Val Ser Gln Val Trp Phe Gly 165 170 175 His Arg Tyr Ser Gln Phe Met Gly Ile Phe Glu Asp Arg Ala Pro Val 180 185 190 Pro Phe Glu Glu Val Ile Asp Lys Ile Asn Ala Lys Gly Val Cys Arg 195 200 205 Ser Thr Ala Lys Tyr Val Arg Asn Asn Leu Glu Thr Thr Ala Phe His 210 215 220 Arg Asp Asp His Glu Thr Asp Met Glu Leu Lys Pro Ala Asn Ala Ala 225 230 235 240 Thr Arg Thr Ser Arg Gly Trp His Thr Thr Asp Leu Lys Tyr Asn Pro 245 250 255 Ser Arg Val Glu Ala Phe His Arg Tyr Gly Thr Thr Val Asn Cys Ile 260 265 270 Val Glu Glu Val Asp Ala Arg Ser Val Tyr Pro Tyr Asp Glu Phe Val 275 280 285 Leu Ala Thr Gly Asp Phe Val Tyr Met Ser Pro Phe Tyr Gly Tyr Arg 290 295 300 Glu Gly Ser His Thr Glu His Thr Ser Tyr Ala Ala Asp Arg Phe Lys 305 310 315 320 Gln Val Asp Gly Phe Tyr Ala Arg Asp Leu Thr Thr Lys Ala Arg Ala 325 330 335 Thr Ala Pro Thr Thr Arg Asn Leu Leu Thr Thr Pro Lys Phe Thr Val 340 345 350 Ala Trp Asp Trp Val Pro Lys Arg Pro Ser Val Cys Thr Met Thr Lys 355 360 365 Trp Gln Glu Val Asp Glu Met Leu Arg Ser Glu Tyr Gly Gly Ser Phe 370 375 380 Arg Phe Ser Ser Asp Ala Ile Ser Thr Thr Phe Thr Thr Asn Leu Thr 385 390 395 400 Glu Tyr Pro Leu Ser Arg Val Asp Leu Gly Asp Cys Ile Gly Lys Asp 405 410 415 Ala Arg Asp Ala Met Asp Arg Ile Phe Ala Arg Arg Tyr Asn Ala Thr 420 425 430 His Ile Lys Val Gly Gln Pro Gln Tyr Tyr Leu Ala Asn Gly Gly Phe 435 440 445 Leu Ile Ala Tyr Gln Pro Leu Leu Ser Asn Thr Leu Ala Glu Leu Tyr 450 455 460 Val Arg Glu His Leu Arg Glu Gln Ser Arg Lys Pro Pro Asn Pro Thr 465 470 475 480 Pro Pro Pro Pro Gly Ala Ser Ala Asn Ala Ser Val Glu Arg Ile Lys 485 490 495 Thr Thr Ser Ser Ile Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asn His 500 505 510 Ile Gln Arg His Val Asn Asp Met Leu Gly Arg Val Ala Ile Ala Trp 515 520 525 Cys Glu Leu Gln Asn His Glu Leu Thr Leu Trp Asn Glu Ala Arg Lys 530 535 540 Leu Asn Pro Asn Ala Ile Ala Ser Ala Thr Val Gly Arg Arg Val Ser 545 550 555 560 Ala Arg Met Leu Gly Asp Val Met Ala Val Ser Thr Cys Val Pro Val 565 570 575 Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser Ser Arg 580 585 590 Pro Gly Ala Cys Tyr Ser Arg Pro Leu Val Ser Phe Arg Tyr Glu Asp 595 600 605 Gln Gly Pro Leu Val Glu Gly Gln Leu Gly Glu Asn Asn Glu Leu Arg 610 615 620 Leu Thr Arg Asp Ala Ile Glu Pro Cys Thr Val Gly His Arg Arg Tyr 625 630 635 640 Phe Thr Phe Gly Gly Gly Tyr Val Tyr Phe Glu Glu Ser Ala Tyr Ser 645 650 655 His Gln Leu Ser Arg Ala Asp Ile Thr Thr Val Ser Thr Phe Ile Asp 660 665 670 Leu Asn Ile Thr Met Leu Glu Asp His Glu Phe Val Pro Leu Glu Val 675 680 685 Tyr Thr Arg His Glu Ile Lys Asp Ser Gly Leu Leu Asp Tyr Thr Glu 690 695 700 Val Gln Arg Arg Asn Gln Leu His Asp Leu Arg Phe Ala Asp Ile Asp 705 710 715 720 Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly 725 730 735 Ala Phe Phe Glu Gly Met Gly Asp Leu Gly Arg Ala Val Gly Lys Val 740 745 750 Val Met Gly Ile Val Gly Gly Val Val Ser Ala Val Ser Gly Val Ser 755 760 765 Ser Phe Met Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val 770 775 780 Leu Ala Gly Leu Ala Ala Ala Phe Phe Ala Phe Arg Tyr Val Met Arg 785 790 795 800 Leu Gln Ser Asn Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Glu 805 810 815 Leu Lys Asn Pro Thr Asn Pro Asp Ala Ser Gly Glu Gly Glu Glu Gly 820 825 830 Gly Asp Phe Asp Glu Ala Lys Leu Ala Glu Ala Arg Glu Met Ile Arg 835 840 845 Tyr Met Ala Leu Val Ser Ala Met Glu Arg Thr Glu His Lys Ala Lys 850 855 860 Lys Lys Gly Thr Ser Ala Leu Leu Ser Ala Lys Val Thr Asp Met Val 865 870 875 880 Met Arg Lys Arg Arg Asn Thr Asn Tyr Thr Gln Val Pro Asn Lys Asp 885 890 895 Gly Asp Ala Asp Glu Asp Asp Leu 900

[Brief description of drawings]

1 depicts a physical map of HSV-1 and HSV-2, an Eco RI cleavage map for the placement of the prototype isomer, and a HindIII restriction enzyme cleavage map of HSV-2.

FIG. 2 shows a restriction map of the gB1 coding region in the HSV-1 map.

FIG. 3 is a restriction enzyme cleavage map of the gB1 coding region.

FIG. 4 represents the DNA amino acid sequences of gB1 and gB2.

FIG. 5 is a continuation of FIG.

FIG. 6 is a continuation of FIG.

FIG. 7 is a continuation of FIG.

FIG. 8 is a physical map of HSV-2, showing the gB2 coding region.

FIG. 9 is a map showing some important features of plasmid pHS137.

FIG. 10 is a restriction enzyme digestion map of gB2.

FIG. 11. pHS, a mammalian expression vector for gD1.
13 is a flowchart regarding the construction of 132.

FIG. 12 is a physical map of HSV-2, showing the gD2 coding region.

FIG. 13 is a flow chart for the construction of mammalian vectors for gD2.

FIG. 14: Recombinant gB-gD performed after primary infection
Fig. 7 shows the effect of vaccination with sucrose on recurrent herpes disease.

FIG. 15A shows the effect of immunization with herpesvirus glycoproteins on recurrent herpes infection rates. B
Represents the difference in weekly recurrence rates between control and sensitized guinea pigs.

FIG. 16 is a graph showing the effect of administration time of the gBgD vaccine on recurrence of herpes disease.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 19/00 C12P 21/02 C C12P 21/02 C12N 15/00 ZNAA (72) Inventor Carol Pakul USA California 94611 Auckland, Somerset Road 321 (72) Inventor Pablo Dee. tea. Valenzuela United States California 94117 San Francisco, Upper Terrace 455 F-term (reference) 4B024 AA01 BA32 CA02 HA01 4B064 AG32 CA19 CC24 DA01 4C085 AA03 AA38 BA78 BB12 CC08 CC21 DD62 EE01 EE03 EE06 FF02 FF12 4H045 AA11 AA31 CA30 FA03 BA30 FA30 BA30 DA03

Claims (49)

[Claims] 1. A recombinant herpes simplex virus (HSV) type 1 glycoprotein B (gB1) produced in a host cell.
A polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 and being a mature peptide, ie said H
An HSV gB1 polypeptide lacking a signal peptide that naturally binds to an SV gB1 polypeptide, or a C-terminal truncated fragment thereof immunologically equivalent to said HSV gB1 polypeptide. 2. A recombinant herpes simplex virus (HSV) type 2 glycoprotein B (gB2) produced in a host cell.
A polypeptide which has the amino acid sequence set forth in SEQ ID NO: 3 and is a mature peptide, ie said H
An HSV gB2 polypeptide lacking a signal peptide that naturally binds to an SV gB2 polypeptide, or a C-terminal truncated fragment thereof immunologically equivalent to said HSV gB2 polypeptide. 3. A recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide produced in a host cell, or an analog or C-terminal truncated fragment thereof immunologically equivalent to the HSV gB polypeptide. Wherein the HSVgB polypeptide is SEQ ID NO: 3 or 6
A mature peptide, that is, lacking a signal peptide that naturally binds to the HSV gB polypeptide, the analog being a substitution of one or more amino acids in the amino acid sequence,
An HSV glycoprotein B polypeptide, analog or C-terminal truncated fragment thereof having additions and / or deletions. 4. A recombinant HSVgB polypeptide or analog or C-terminal truncated fragment thereof according to claim 3, wherein the amino acid sequence of said polypeptide, analog or fragment differs from said amino acid sequence by a number less than 5%. 5. The nucleotide sequence encoding the amino acid sequence is plasmid pHS112 (ATCC No. 39650).
Available from) or a nucleotide sequence which differs from it in nucleotide degeneracy only in codon degeneracy.
A gB polypeptide or analog or C-terminal truncated fragment thereof. 6. The recombinant HSV according to claim 1, wherein the HSV gB polypeptide or its analog or C-terminal truncated fragment is a fusion protein.
A gB polypeptide or analog or C-terminal truncated fragment thereof. 7. The severity of recurrent herpes simplex virus (HSV) infection comprising the herpes simplex virus (HSV) type 1 glycoprotein B (gB1) polypeptide or the C-terminal truncated fragment of claim 1, and Vaccine effective in reducing the rate. 8. The severity of recurrent herpes simplex virus (HSV) infection comprising the herpes simplex virus (HSV) type 2 glycoprotein B (gB2) polypeptide or the C-terminal truncated fragment of claim 2, and Vaccine effective in reducing the rate. 9. Recurrent herpes simplex virus (HSV)
A vaccine effective in reducing the severity and / or rate of infection, comprising the HSVgB polypeptide of claim 3 or an analog or C-terminal truncated fragment thereof. 10. The vaccine of claim 9, wherein the amino acid sequence of the analog differs from the amino acid sequence by a number less than 5%. 11. The nucleotide sequence encoding the amino acid sequence is plasmid pHS112 (ATCC No. 3965).
Vaccine according to any one of claims 7 to 10, selected from the nucleotide sequences in (available from 0) or nucleotide sequences that differ from it only in the degeneracy of codons. 12. The vaccine according to any one of claims 7 to 11, wherein the vaccine contains a pharmaceutically acceptable carrier and / or an adjuvant. 13. A component capable of forming liposomes such as synthetic phosphatidylcholine and phosphatidylserine; CFA; IFA; squalene /
Consisting essentially of a compound selected from the group consisting of Arlacel; squalene and Tween 80; and aluminum hydroxide, wherein the adjuvant is N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl-L-alanine-2
(1'-2'-dipalmitoyl-sn-glycero-3-
Hydroxyphosphorylyloxy-ethylamine (MTP
Vaccine according to claim 12, optionally containing a muramyl dipeptide derivative such as -PE). 14. The vaccine of claim 13, wherein the adjuvant is present in a low oil formulation. 15. The vaccine according to claim 9, wherein the individual infected with HSV is a mammal. 16. An effective method for reducing the severity and / or rate of recurrent herpes simplex virus (HSV) infection, comprising herpes simplex virus (HSV) type 1 glycoprotein B (gB1) polypeptide or fragment. A method for producing a vaccine, comprising the HSV according to claim 1.
A method comprising adding a gB1 polypeptide or a C-terminal truncated fragment in an amount effective to reduce the severity and / or rate of recurrent HSV infection. 17. An effective method for reducing the severity and / or rate of recurrent herpes simplex virus (HSV) infection, comprising herpes simplex virus (HSV) type 2 glycoprotein B (gB2) polypeptide or fragment. A method for producing a vaccine, comprising the HSV according to claim 2.
A method comprising adding a gB2 polypeptide or a C-terminal truncated fragment in an amount effective to reduce the severity and / or rate of recurrent HSV infection. 18. A recurrent herpes simplex virus (HSV) comprising a herpes simplex virus (HSV) glycoprotein B (gB) polypeptide or analog or fragment.
A method for producing a vaccine effective in reducing the severity and / or rate of infection, which method comprises recurring HSV gB polypeptide or analog or C-terminal truncated fragment of claim 3 in a recurrent manner. A method comprising adding in an amount effective to reduce the severity and / or rate of HSV infection. 19. The amino acid sequence of the analog differs from the amino acid sequence by less than 5%.
The method described in. 20. The nucleotide sequence encoding the amino acid sequence is plasmid pHS112 (ATCC No. 3965).
(Available from 0) or a nucleotide sequence that differs from it in only the degeneracy of the codons. 21. The method of any of claims 16-20, wherein the vaccine comprises a pharmaceutically acceptable carrier and / or an adjuvant. 22. Components in which the adjuvant is capable of forming liposomes such as synthetic phosphatidylcholines and phosphatidylserines; CFA; IFA; squalene /
Consisting essentially of a compound selected from the group consisting of Arlacel; squalene and Tween 80; and aluminum hydroxide, wherein the adjuvant is N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl-L-alanine-2
(1'-2'-dipalmitoyl-sn-glycero-3-
Hydroxyphosphorylyloxy-ethylamine (MTP
22. The method of claim 21, optionally containing a muramyl dipeptide derivative such as -PE). 23. The method of claim 22, wherein the adjuvant is present in a low oil formulation. 24. The method according to claim 21, wherein the individual infected with HSV is a mammal. 25. Recombinant herpes simplex virus (HSV) produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 5, which encodes herpes simplex virus (HSV) glycoprotein type B1 (gB1). ) A glycoprotein B (gB) polypeptide, or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region. 26. Recombinant herpes simplex virus (HSV) produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 2, which encodes herpes simplex virus (HSV) glycoprotein type B2 (gB2). ) A glycoprotein B (gB) polypeptide, or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region. 27. Recombinant herpes simplex virus (HS) produced in a host cell containing nucleotide sequences encoding herpes simplex virus (HSV) glycoprotein B (gB) and its precursors, or analogs thereof.
V) Glycoprotein B (gB) polypeptide or recombinant HSV g containing a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region
A recombinant HSV gB polypeptide, which is a B polypeptide, wherein the nucleotide sequence encoding the HSV gB contains a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or 6. 28. The nucleotide sequence selected from the nucleotide sequences in plasmid pHS112 (available from ATCC No. 39650) or nucleotide sequences which differ from them only in degeneracy of codons.
7. The recombinant herpes simplex virus (H
SV) Glycoprotein B (gB) polypeptide or recombinant HSV containing deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region
gB polypeptide. 29. The recombinant HSV gB polypeptide, or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region, is a fusion protein. 7. A recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide according to any of the above, or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region. 30. Relapsing comprising a herpes simplex virus (HSV) glycoprotein B (gB) polypeptide or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region. Herpes simplex virus (HSV)
A vaccine effective in reducing the severity and / or rate of infection. 31. The herpes simplex virus (HSV)
31. The glycoprotein B (gB) polypeptide is produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 5 which encodes herpes simplex virus (HSV) glycoprotein type B1 (gB1). Vaccine according to. 32. The herpes simplex virus (HSV)
31. The glycoprotein B (gB) polypeptide is produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 2 encoding herpes simplex virus (HSV) glycoprotein type B2 (gB2). Vaccine according to. 33. The herpes simplex virus (HSV)
31. The glycoprotein B (gB) polypeptide is produced in a host cell containing a nucleotide sequence encoding herpes simplex virus (HSV) glycoprotein B (gB) and its precursors, or analogs thereof.
7. The vaccine of claim 7, wherein the nucleotide sequence encoding HSVgB contains a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or 6. 34. The nucleotide sequence selected from the nucleotide sequences in plasmid pHS112 (available from ATCC No. 39650) or nucleotide sequences which differ from them only in degeneracy of codons.
The vaccine according to any one of 3 above. 35. The herpes simplex virus (HSV)
Glycoprotein B (gB) or cytoplasmic domain and / or
Alternatively, a recombinant HSV gB polypeptide comprising a deletion of all or a portion of the transmembrane anchor region may give rise to the recurrent HSV when the vaccine is administered to an individual during the acute phase of herpes simplex virus (HSV) infection.
The vaccine according to any of claims 30 to 34, which is present in an amount effective to reduce the severity and / or rate of infection. 36. The vaccine according to any one of claims 30 to 35, wherein the vaccine contains a pharmaceutically acceptable carrier and / or an adjuvant. 37. Components in which the adjuvant is capable of forming liposomes such as synthetic phosphatidylcholines and phosphatidylserines; CFA; IFA; squalene /
Aralcel; squalene and Tween 80; and consisting essentially of a compound selected from the group consisting of aluminum hydroxide, wherein the adjuvant further comprises N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl-L-alanine-2
(1'-2'-dipalmitoyl-sn-glycero-3-
Hydroxyphosphorylyloxy-ethylamine (MTP
A vaccine according to claim 36, optionally containing a muramyl dipeptide derivative such as -PE). 38. The vaccine of claim 37, wherein the adjuvant is present in a low oil formulation. 39. The vaccine according to any of claims 36 to 38, wherein the individual infected with herpes simplex virus (HSV) is a mammal. 40. Relapsing comprising a herpes simplex virus (HSV) glycoprotein B (gB) polypeptide or a recombinant HSV gB polypeptide comprising a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region. Herpes simplex virus (HSV)
What is claimed is: 1. A method for producing a vaccine effective to reduce the severity and / or rate of infection, said method comprising:
Recombinant HSV gB polypeptides containing deletions of all or part of the polypeptide or cytoplasmic domain and / or transmembrane anchor region are labeled with recurrent H
A method comprising adding in an amount effective to reduce the severity and / or rate of SV infection. 41. The herpes simplex virus (HSV)
41. The glycoprotein B (gB) polypeptide is produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 5 encoding herpes simplex virus (HSV) glycoprotein type B1 (gB1). The method described in. 42. The herpes simplex virus (HSV)
41. The glycoprotein B (gB) polypeptide is produced in a host cell containing the isolated nucleotide sequence set forth in SEQ ID NO: 2 encoding herpes simplex virus (HSV) glycoprotein type B2 (gB2). The method described in. 43. The herpes simplex virus (HSV)
41. The glycoprotein B (gB) polypeptide is produced in a host cell containing a nucleotide sequence encoding herpes simplex virus (HSV) glycoprotein B (gB) and its precursors, or analogs thereof.
The method according to claim 7, wherein the nucleotide sequence encoding HSV gB contains a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or 6. 44. The nucleotide sequence selected from nucleotide sequences in plasmid pHS112 (available from ATCC No. 39650) or nucleotide sequences which differ from them only in degeneracy of codons.
The method according to any one of 3 above. 45. The herpes simplex virus (HSV)
A recombinant HSV gB polypeptide comprising a glycoprotein B (gB) polypeptide or a deletion of all or part of the cytoplasmic domain and / or transmembrane anchor region confers the vaccine on the acute phase of herpes simplex virus (HSV) infection. 45. The method of any of claims 40-44, which is present in an amount effective to reduce the severity and / or rate of recurrent HSV infection when administered to an individual. 46. The method of any of claims 40-45, wherein the vaccine contains a pharmaceutically acceptable carrier and / or an adjuvant. 47. Components in which the adjuvant is capable of forming liposomes such as synthetic phosphatidylcholines and phosphatidylserines; CFA; IFA; squalene /
Aralcel; squalene and Tween 80; and consisting essentially of a compound selected from the group consisting of aluminum hydroxide, wherein the adjuvant further comprises N-acetylmuramyl-L-
Alanyl-D-isoglutaminyl-L-alanine-2
(1'-2'-dipalmitoyl-sn-glycero-3-
Hydroxyphosphorylyloxy-ethylamine (MTP
47. The method of claim 46, optionally containing a muramyl dipeptide derivative such as -PE). 48. The method of claim 47, wherein the adjuvant is in a low oil formulation. 49. The method according to any of claims 45 to 48, wherein the individual infected with herpes simplex virus (HSV) is a mammal.