JP2002167398A - Vaccine for treatment of hsv - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polypeptide effective for detecting the infection with a herpes simplex virus(HSV), a vaccine effective for preventing a disease induced by a recurrent herpes simplex virus(HSV), and a method for producing them. SOLUTION: This vaccine is composed of a fragment which is immunologically equal (or, provide a protection against infection) to a recombinant herpes simplex virus(HSV) glycoprotein (gB) yielded in a host cell coding a herpes simplex virus(HSV) glycoprotein (gB) and containing an isolated nucleotide sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Herpesviruses include herpes simplex viruses containing two closely related variants, designated as 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 a variety of human diseases, such as skin infections, genital herpes, viral encephalitis, and the like.

[0002] Herpes simplex virus is a double-stranded DNA virus having a genome of about 150 to 160 kbp in an icosahedral nuclear capsid encapsulated in a membrane. This membrane contains a number of virus-specific glycoproteins, of which gB, gC, gD, and gE are the most common. Here, gB and gD show a cross-reaction between type 1 and type 2.

[0003] Providing safe and effective vaccines against humans against both HSV-1 and HSV-2 and, if an infection occurs, providing a cure for the disease has been medically and scientifically justified. 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, utilization of the herpes simplex glycoprotein has so far largely relied on virus culture and membrane protein isolation. In the commercial production of glycoproteins, the handling of dangerous pathogens,
Problems with maintaining the virus in cultured cells, isolating glycoproteins that do not contain the viral genome or portions thereof, etc., have prevented the use of glycoproteins as vaccines. Therefore, it is desirable to provide vaccines using glycoproteins produced by methods other than culturing the virus and isolating membrane proteins.

[0005] It is also very much to develop a method for the therapeutic treatment of herpes infections, ie, a treatment that reduces the disease or prevents the recurrence of the disease after the individual has been infected with the virus. Interested. Since viral infections are usually resistant to treatment with antibiotics, other methods that do not have significant side effects are of great interest. Although some drugs (especially Acyclovir) have been effective in treating recurrent herpes disease, they are not desirable because of the need for long-term continuous administration to prevent relapse. In the course of treatment, drug-resistant mutants are generated.

[0006]

2. Description of the Related Art Eberle and Mou (J. of Infectious Di
seases (1983) 148: 436-444) shows that H
The relative titers of antibodies to polypeptide antigens specific to SV-1 are reported. Marsden et al. (J. of Virology (197
8) 28: 624-642) shows that the gene corresponding to the 117-kilodalton (kd) glycoprotein is 0.35-0.4 on the HSV genetic map due to intra-type recombination between HSV-1 and HSV-2.
It reports that it is located within 0 map units. Ruyechan
(Id. (1979) 29: 677-679) report that the location of the glycoprotein B gene lies between 0.30 and 0.42 map units. Skare and Summers Virology (1977) 76: 5
81-595) report the endonuclease cleavage sites of EcoRI, XbaI and HindIII on HSV-1 DNA. Roizman
(Ann. Rev. Genetics (1979) 13: 25-57) report the organization of the HSV genome. DeLucca et al. (Virology (198
2) 122: 411) has determined the location of several phenotypic variants that are thought to have mutations in the gB1 structural gene between 0.345 and 0.368 map units.

[0007] A subunit vaccine extracted from chicken embryo cells infected with HSV-1 or HSV-2 is disclosed in US Pat.
Nos. 4,317,811 and 4,374,127. In addition, Hilfenhaus et al. (Develop. Biol. Standard (1982) 5
2: 321-331), which describes the preparation of a subunit vaccine from a specific HSV-1 species (BW3). (Ibid. (1982) 52: 287-304) describe a non-infectious HSV-1 × HSV-2 recombinant and deletion mutant that has been shown to be effective in immunized mice. The preparation is described. Watson et al. (Science (1982) 21
8: 381-384) show not only the expression of cloned fragments by injection into the nucleus of frog oocytes, but also the cloning of the HSV-1gD gene and the low level expression of this gene in E. coli. Is described. They also show the nucleotide sequence of the gD gene. Weis et al. (Natu
re (1983) 302: 72-74) report the high level expression of gD in Escherichia coli. This polypeptide induces neutralizing antibodies in rabbits. Berm
(Science (1983) 222: 524-527) report the expression of glycoprotein D in cultured mammalian cells. Lasky et al. (Biotechnology (June 1984) 527-532)
Report the use of this glycoprotein D for immunization of mice. Cohen et al. (J. Virol (1984) 49: 102-10).
8) reports the location and chemical synthesis of specific antigenic determinants of gD, contained within residues 8-23 of the mature protein.

[0008] The use of membrane protein preparations from HSV-infected cells as a "treatment" as a human post-infection vaccine has been described by Dundarov, S. et al. (Dov. Biol. Standa
rd (1987) 57: 351-357) and Skinner, GRB et al. (ibid. (1982) 52: 333-34).

[0009]

SUMMARY OF THE INVENTION The present invention provides vaccines and compositions for treating herpes simplex virus types 1 and 2, and methods for producing them. 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 infection in animals, including humans.

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

[0011] The polypeptide is prepared by expressing a recombinant DNA construct in mammalian cells, and the polypeptide is administered to an individual after the vaccine has been primarily infected with HSV. In some cases, it is present in an amount effective to prevent relapse of HSV-induced disease in the individual infected with HSV.

Another aspect of the present invention is a vaccine for use in treating herpes simplex virus (HSV) infection. The vaccine comprises: a. Immunologically active herpes simplex virus glycoprotein B
(GB) a polypeptide; or b. Immunologically active herpes simplex virus glycoprotein D
(GD) containing a polypeptide.

Here, the polypeptide is obtained by expressing a recombinant DNA construct in a mammalian cell, and the polypeptide is administered to an individual after the vaccine has been primarily infected with HSV. If so, it is present in an amount effective to prevent relapse of the HSV-induced disease in the individual infected with HSV.

[0014]

According to the present invention, there is provided a herpes simplex virus (HSV) glycoprotein B1 (gB
Recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide produced in a host cell containing the isolated nucleotide sequence of Figures 4-7 encoding 1), its immunological properties Equivalent fragments , or mixtures thereof, are provided.

According to the present invention, there is provided a recombinant herpes simplex virus (HSV) glycoprotein type B2 (gB2) produced in a host cell containing the isolated nucleotide sequence described in FIGS. Provided is a herpes simplex virus (HSV) glycoprotein B (gB) polypeptide, an immunologically equivalent fragment thereof, or a mixture thereof.

According to the present invention, there is provided a herpes simplex virus (HSV) glycoprotein B (gB) and a precursor thereof,
Alternatively, a recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide, an immunologically equivalent fragment thereof, or a mixture thereof, produced in a host cell containing a nucleotide sequence encoding an analog thereof. A recombinant herpes simplex virus comprising a nucleotide sequence encoding: i) the amino acid sequence set forth in FIGS. 4-7, or ii) an immunological equivalent of the amino acid sequence. HSV) glycoprotein B (gB) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof.

According to the present 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), including mixtures thereof
V) Vaccines are provided that are effective in preventing induced disease.

According to the present invention, there is provided a polypeptide capable of binding to both an anti-herpes simplex virus (HSV) glycoprotein B (gB) antibody and an 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 comprises herpes simplex virus (HSV) Glycoprotein B
(GB) Compositions are provided that are produced by the polypeptide and the HSV gD polypeptide, or a mixture thereof.

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

According to the present invention, there is provided a DNA construct containing the following nucleotide sequence: (a) Isolation having a nucleotide acid sequence encoding the aforementioned herpes simplex virus (HSV) glycoprotein B (gB). (B) a control sequence capable of binding to the nucleotide sequence for promoting expression in a host cell.

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

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION (Vaccine) The vaccine of the present invention uses both type 1 and type 2 recombinant HSV glycoproteins B and D. Mature (full-length) gB and gD proteins are used as well as fragments, precursors, and analogs that are immunologically equivalent (ie, confer protection against infection) to these mature proteins. As used in the claims, the terms "glycoprotein B polypeptide" and "glycoprotein D polypeptide" refer to such fragments,
It is intended to include 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 a mixture of type 1 polypeptides, a mixture of type 2 polypeptides, or a mixture of both type 1 and type 2 polypeptides, or any of the individual polypeptides.

The mixture of gB and gD polypeptides can be used alone, but usually together with a physiologically and pharmacologically acceptable medium, generally water, saline, phosphate buffer, sugar and the like. Used. Further, the polypeptide mixture can be used with a physiologically acceptable adjuvant (eg, aluminum hydroxide, muramyl dipeptide derivatives, etc.). Various adjuvants are effective, as shown 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 species to be vaccinated, and in the case of humans, the adjuvant may be Depending on whether or not it has been approved for use in humans. The vaccine is delivered by liposomes and / or with an immunomodulator such as interleukins 1 and 2. Vaccines may be administered by any convenient parenteral route (eg, intravenous, intraarterial, subcutaneous, intradermal,
(Intramuscular or intraperitoneal). It is advantageous to divide and administer the vaccines administered by the same or different routes. The vaccine is administered after a primary infection with the 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). Norrild B. et al., Infect. Immun. (1980)
28: 38-44. Patients who recur frequently with HSV infection often have high levels of both neutralizing antibodies and ADCC. Core
y, L. and Spear, PG, Eng. N., J. Med. 314: 686-
691, 1986. For such patients, a potent inducer of HSV-specific antibodies has been shown to be viral glycoproteins. Eberle, R., Mou, SW, and Zaia, JA, J.
Gen Virol. 65: 1839-1843, 1984. Therefore, there was no expectation that a recombinant subunit vaccine composed of only two viral glycoproteins and containing other viral antigens would be clinically used for the treatment of recurrent genital herpes.

Glycoproteins B and D are used without modification. However, when using smaller related polypeptides (eg, fragments, etc.), and when the molecular weight is about 5,000
If it is less than 0 daltons (eg, 1,500-5,000 daltons), modifications are required to induce the desired immune response. Smaller haptens must be conjugated to a suitable immunogenic carrier (eg, tetanus toxoid, etc.).

A short coding for a gB or gD polypeptide
DNA fragments can also be ligated to genes that express proteins from other pathogenic organisms or viruses. In this way, the resulting fusion protein may confer immunity to more than one disease.

Recombinant gB used per dose
And the total amount of gD polypeptide is usually 1 kg of host weight.
About 0.1 μg to 2 mg, more usually about 0.5 μg to 1
mg, and especially about 0.5 μg to 10 μg. G in vaccine
The ratio of gB to D is usually about 0.1: 1 to 10: 1, more usually about 0.5: 1 to 10: 1, and preferably about 0.5.
5: 1 to 5: 1. A single dose may be repeated at daily to weekly intervals, usually at intervals of two to four weeks, usually no more than about two to ten 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. Therefore, 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 basic tests using, for example, disease symptomatology or antibody titers to monitor disease status. In addition, the data in Example 6.4 show that vaccination significantly reduces disease recurrence during the acute phase of the disease, ie, when the individual exhibits HSV-induced pathology in the body. (Recombinant glycoprotein B) Preparation of recombinant gB polypeptide is described in International Publication No. WO85 / 0458 published October 24, 1985.
7 International Application No. PCT / US85 / 00587. Materials and methods used to produce recombinant gB polypeptides are briefly described below.

FIGS. 4 to 7 in the examples show the nucleotide sequence of the gB1 Patton strain and the amino acid sequence encoded by the nucleotide sequence. FIGS. 4-7 also show substantial homology between gB1 and gB2. There can be many variations in the nucleotide sequence. Various fragments having independent functions can be used. These fragments can bind to proteins other than mature gB. In addition, various codons can be modified to encode the same amino acid, but exhibit more efficient expression depending on the nature of the host.
For example, these codons can be modified according to the frequency of occurrence of one or more proteins, or a particular codon in a group of proteins. These proteins are, for example, glycolytic proteins and occupy a high ratio in all proteins of a specific host (for example, yeast). In some cases, 1
One or more codons can be modified to encode a different amino acid by replacing one amino acid with another. In this case, the effects of the alteration are not deleterious to the immunogenicity of the protein or other biological factors of interest. In some cases, it is desirable to add an amino acid at the N-terminus or C-terminus. The addition of such amino acids produces favorable results. This can be easily achieved by adding a codon to the 5 'or 3' end of the sequence encoding mature gB1 or its precursor. Further gB
The amino acid sequence of 2 differs from the amino acid sequence of gB1 by 20%, but the other strains of HSV-1 or HSV-2 are identical or similar to gB1 Patton or gB2333, respectively.
Has gB glycoprotein. These gB glycoproteins usually contain less than 5%, more usually less than 2%, and often less than 0.5% of amino acids in gB1 Patton or gB2333 strains. Amino acid sequence.

The sequence of gB1, especially the gB1 Patton strain, is divided into four domains starting from the N-terminus of the protein:
A first hydrophobic region extending from the first amino acid to about the 30th amino acid, a region extending from the first hydrophobic region to the about 726th amino acid, and having a variable polarity; And a second hydrophobic region extending from the C-terminus of the 904th amino acid to the amino acid at about the 795th amino acid;

Since gB is a membrane glycoprotein, the first hydrophobic region is considered to be a signal leader sequence that controls secretion and / or membrane arrangement, by analogy with other glycoproteins. Thus, the first sequence of variable 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
Transmembrane integrator array (transme
mbrane integrator sequence) (often "anchor
(called "anchor"). The second amino acid sequence, whose polarity can be changed, 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. Can play.

A polynucleotide sequence encoding a precursor of gB or a functional fragment thereof can be obtained by inserting the polynucleotide sequence into an appropriate expression vector and introducing the resulting expression construct into a suitable host. Can be cloned and expressed. The code fragment is
Less than about 0.1 map unit, usually less than about 0.05 map unit; where 1.0 map unit is the size of the entire HSV genome. The expression vector can be a low or high level multicopy vector that is extrachromosomal or integrated into the genome of the host cell. And
Expression vectors secrete or excrete the polypeptide of interest, or retain the polypeptide of interest in the cytoplasm or membrane. Numerous expression vectors have been published in the literature and are generally useful for use in eukaryotic hosts. Eukaryotic hosts include yeast (eg, S. cerevisiae) and a wide range of permanently growing 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 natural or unnatural secretion leader sequences can be used, depending on the host. The processing signal that cleaves the secretory leader sequence can be a natural signal, or a signal associated with a non-natural secretory leader sequence, or both in tandem.

In order to obtain a polynucleotide sequence encoding gB1 Patton, gB1
The location of the coding sequence is shown on the map. Three subfragments of the F fragment were isolated and subcloned into pBR322 (FIG. 2). Next, the DNA fragments of these subclones were HSV-1
Was used as a probe for Northern blots of polyA ⁺ mRNA isolated from infected cells. The fragment that hybridized to the mRNA of the expected size for gB
Presumed to be within the gB coding region. The direction of transcription of gB was also revealed by determining which strand of the DNA probe hybridized to the mRNA. To confirm gB sequence identity, DNA fragments were used for hybrid-select HSV-1 mRNA. This mRNA was then translated in vitro and the resulting protein was analyzed for gB using a gB-specific antibody.

The fragment encoding gB1 is nowadays handled in various ways, including restriction enzyme mapping and nucleotide sequence determination, to establish restriction enzyme cleavage sites and open reading frame regions for expression. The DNA sequence is then limited to providing the sequence encoding the complete gB precursor or a fragment thereof. These sequences are then:
It is inserted into a suitable expression vector having a transcription signal at a proper position and a proper translation signal. this is,
This can be achieved by filling overhangs and performing blunt end ligation using an adapter or the like.

It is of particular interest to introduce the gene in tandem with respect to the gene that has the ability to amplify. Useful genes include the dihydrofolate reductase (dhfr) gene, which can be amplified by using methotrexate (where the dhfr gene and adjacent regions are repeated); and metallothionein, which can be amplified by heavy metals such as copper. Is included. The expression construct product can be introduced into a suitable host by any useful means, including transformation, transfection, calcium phosphate precipitation, and the like. The host cell is then stressed with a suitable biotoxin to a level that selects to amplify the particular gene. These cells are then cultured and expanded until they effectively produce the desired polypeptide.

According to the procedure described above, g from HSV-2 333 strain
The polynucleotide sequence encoding B2, both precursor and mature, is isolated, cloned, and manipulated to give a construct. As a result, it can be expressed in one or more hosts. Given the usefulness of the fragments encoding gB1 Patton, these fragments were identified as gB2 encoding the DNA portion to the specific HSV-2 restriction fragment clone.
GB2 mRNA from infected host cells
Used as any probe for the isolation of For convenience, multiple probes encoding different regions of the gB1 gene are used. Either a positive DNA fragment or abundant mRNA having approximately the appropriate size to hybridize to this probe is selected. The mRNA can then be reverse transcribed to give a cDNA and / or used for hybridization with a fragment 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 can be manipulated and ligated to provide the entire coding region and flanking regions, as appropriate. This coding region is then introduced into an expression vector. (Recombinant glycoprotein D) Preparation of recombinant gD1 was
International Application No. WO85 / 04587 published on March 24
o. Detailed in PCT / US85 / 00587. 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 can be obtained from recombinant DNA in eukaryotic hosts such as yeast and mammalian cells (eg, CHO cells).
Produced by the methodology of Production in eukaryotes confers advantages on eukaryotic hosts, such as, for example, post-translational modification and / or secretion. gD polypeptides are produced from relatively short synthetic DNA fragments encoding at least about 9 amino acids, and provide an effective hapten to induce a gD-specific immune response.

[0037] The gD DNA fragment can be of natural or synthetic origin. The native gD gene of HSV-1 has short internal repeat (IRs) sequences on the viral genome,
It is located between its 3 'terminating short termination repeat (TRs) sequences. Encoding a mature protein means that an approximately 1.6 kbp fragment is found located on the 2.9 kbp SacI restriction enzyme fragment of the genome. The entire coding region of the mature protein is the Hin of the 2.9 kbp SacI fragment.
Located in the dIII-NruI fragment. Naturally occurring gD
Genes may or may not be modified. Regions of the gene can be deleted as desired and / or combined with other DNA fragments. The gD DNA fragment can be inserted into an expression vector and expressed using materials and procedures similar to those described above for the expression of gB DNA. The preparation, cloning, and expression of specific fragments of the naturally occurring gD gene are described in detail in the Examples below.

The following examples are given by way of illustration and not limitation. In the following examples, Section 1 describes the general procedure used to produce recombinant protein, Section 2 describes the recombinant gB1
Section 3 describes the preparation of recombinant gB2; Section 4 describes the preparation of recombinant gD1; Section 5 describes the preparation of recombinant gD2; Section 6 uses a polypeptide mixture of gB and gD. This section describes the research on vaccines.

[0039]

EXAMPLES (1. Materials and Methods) Living stocks of HSV-1 Patton and HSV-2 333 strains were obtained from Dr. Richard Hyman.
(Hershey Medical Center, Hershey, Pennsylvania)
Obtained from. These viruses are Dr. Evelyn Linnet
te (Viro Labs, Emeryville, California) or Ameri
Can be grown in Vero cells obtained from Tissue Culture Laboratory. This propagation is performed according to standard procedures. Plasmid pACYC184 (Chang and Cohen,
J. Bacteriology (1978) 134: 1141) HSV-1 Patton EcoRI DNA fragment cloned into the EcoRI site (Kudler
The library of Virology (1983) 124: 86-99)
It can be obtained from Dr. Hyman or can be prepared independently by a simple method. Two HSV-2 333 clones, pBR322 (Sutcl
Hind by iffe, Nucleic Acids Research (1978) 5: 2731)
The HindIII fragments H and L inserted at the III site also
It can be obtained from r.Hyman.

The dhfr-deficient CHO cell line was obtained from Dr. YWKan (Unive
rsity of California, San Francisco). This cell line was originally derived from Urlaub and Chasin (Proc. Natl.
Acad. Sci. USA (1980) 77: 4216-4220). Under non-selective conditions, these cells were derived from Ham's F- supplemented with 10% fetal calf serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 150 μg / ml L-proline.
The cells were grown in 12 media (obtained from Gibco, cat. No. 176).
The selection medium was a DEM supplemented with 10% dialyzed fetal calf serum and penicillin, streptomycin, and 150 μg / ml L-proline. For methotrexate (MTX) selection, the concentrated MTX storage medium was MTX obtained from Lederle.
And added to the above DME selective medium immediately before use. (1.1 Cloning) All DNA manipulations were performed according to standard procedures. Maniatis et al., Molecular Cloning,
See CSH (1982). Restriction enzymes, T4 DNA ligase, Escherichia coli DNA polymerase I Klenow fragment, and other biological reagents are available from Bethesda Research La
Boratories or other indicated commercial suppliers were used and used according to the manufacturer's instructions. Double-stranded DN
A 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 prepared from Vero cells 6 hours after infection with HSV-1 or HSV-2 at a multiplicity of 10 viruses per cell. did. The cell monolayer is washed, incubated with extraction buffer, and added to Pachl et al. (Cell (1
983) 33: 335-344). Poly A ⁺ RNA is 500 mM NaCl, 10 mM Tris HCl (pH
7.5) Pass 2 mg of total RNA through a 3 ml column of oligo dT cellulose (obtained from Collaborative Research) in 1 mM EDTA, 0.1% SDS, then 100 mM NaCl, 10 mM Tr
Wash the column with isHCl (pH 7.5), 1 mM EDTA, 0.1% SDS, and 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 0.1% SDS.
Prepared by eluting the poly A ⁺ fraction with DS.

To perform a Northern blot analysis,
A ⁺ RNA is purified using glyoxal (McMaster et al., Proc. Natl.
Acad. Sci. USA (1977) 74: 4835-4838)
Separation was performed by electrophoresis on a 1% agarose gel, and nitrocellulose paper (Thomas, ibid. (1980) 77: 5201-520)
5) and hybridized with a ³² P-labeled probe.

The details of the method used for the hybrid selection translation method have been described previously (Pachl et al., Cell (198).
3) 33: 335-344). DNA filter encodes gB
It was prepared using either 3 μg of the 3.5 kb Xho-Kpn fragment or 2 μg of the 3.0 kb SstI-SstI fragment encoding HSV-1 gD. This filter was incubated with 40 μg of poly A ⁺ RNA from HSV-1 infected cells, bound RN
A was eluted and reticulocyte cell-free system (Pachl et al., J. Virol.
(1983) 45: 133-139). The translation product is 12.5
% SDS polyacrylamide gel (Laemmli, Nature (197
0) 227: 689). (1.3 DNA transfection) COS 7 cells (Gluzma
n, Cell (1981) 23: 175-182) or dhfr-deficient CHO cells (Urlaub and Chasin (1980) supra) were transformed using van der Eb and G, except that carrier DNA was omitted.
The procedure of raham (Methods in Enz. (1980) 65: 826-839) was followed by Parker and Stark (J. of Virol. (1979) 31: 306).
-369) using the modified procedure. Calcium phosphate precipitation of plasmid DNA was prepared by adding drop-wise HEPES-buffered saline (2 × HBS), which was twice as concentrated, to the plasmid DNA in 250 mM CaCl ₂ and mixing. (1 × HBS is 0.14 M NaCl, 5 mM KC
l, 0.7 mM Na ₂ HPO ₄ , 2.8 mM glucose, 10 mM HEPES (pH
7.0)). After incubation for about 20 minutes at room temperature, 1 ml of the calcium phosphate-DNA suspension (containing 15 μg of DNA) was added to the medium of the cells that had grown on a 10 cm plate until the confluence reached 50%. After 6-8 hours, the medium containing the DNA was removed and the cells were replaced with 15% glycerol-1 × HBS.
For 4 minutes. The cells were then grown in non-selective medium (F12) for 2 days, after which the cells were split, ie, subcultured, into selective medium. Ten days later, dhfr-positive cell colonies appeared, and 14 days later, these colonies were separated by transferring the colony cells from the plate with a Pasteur pipette. Dissociated cells were transferred to multiwell plates for growth. 1.4 In Vivo Labeling and Immunoprecipitation of Cells For labeling with ³⁵ S-methionine, cells were grown to confluence on 3.5 cm plates and PBS (0.14 M NaCl 2.7 mM KCl, 15.3 mM Na ₂ H).
PO ₄ ), then washed with 0.5 ml of labeled medium DEM (Dulbecco's modified Eagle's medium from Gibco, cat. No. 188G) depleted of methionine, 1% dialyzed fetal bovine serum, 400 μCi / ml of ³⁵ S-methionine (> 1000 μCi / mmo
l) was added to each plate. These cells
Incubate at 7 ° C. for appropriate time. At the end of the labeling period, the medium was removed and the monolayer was washed once with PBS. "Cold" methionine chase
As a chase, the labeling medium was replaced with DME containing 2.5 mM methionine. To perform immunoprecipitation, cells were
l of cell solution buffer (20 mM Tris-HCl (pH 8), 100 mM NaC
l, 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, obtained from Sigma Chemical Company). The cell lysate is scraped into a tube, vortexed briefly and then
It was left at 5 ° C for 5-10 minutes. Cell debris was removed by centrifugation and clarified lysates were stored at -70 ° C.

For performing immunoprecipitation, 0.1 ml of cell lysate
Are pre-cleared by incubating with normal serum for 30 minutes at 4 ° C., then adding 50 μl of a 20% solution of protein A sepharose (PAS) in lysis buffer,
For 30 minutes with gentle shaking. PAS was removed by centrifugation at 14,000 × g for 1 minute and 5 μl of HSV-1 polyclonal antibody (obtained from DAKO) or gB-specific monoclonal antibody F3AB (Univer
sity of South Alabama, obtained from Dr. John Oakes). When using F3AB antibody, 0.1% from lysis buffer
SDS omitted. After 30 minutes at 4 ° C., add 75 μl of PAS,
Incubate as described above. The PAS-immune complex was collected by centrifugation, washed three times with lysis buffer lacking BSA and protease inhibitors and once with 0.12 M Tris HCl (pH 7.0). The immunoprecipitated protein is dissociated from the PAS by boiling in SDS sample buffer, then
Analysis was performed on a 2% 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 10-fold lysis buffer was added, causing the protein to precipitate as described above. (1.5 Immunofluorescence) gB in COS cells or CHO clones
Alternatively, to analyze gD expression, cells grown in the wells of the slides were washed three times with PBS, fixed with 100% methanol for 10 minutes at −20 ° C., followed by an additional 3 times with PBS.
Washed once and washed once with PBS supplemented with 5% goat serum (GS). Next, the fixed cells were removed from the primary antibody (PBS-5%
(HSV-1 or HSV-2 polyclonal antibody diluted 1/100 in GS) at 37 ° C. for 30 minutes. Next, the cells were washed three times with PBS-5% GS, and the secondary antibody (PB
FITC-conjugated goat anti-rabbit I diluted 1/10 with S-5% GS
Incubated with gG (Capel) at 37 ° C. for 30 minutes. After washing 4 times with PBS-5% GS, the slide was mounted with a cover piece using 50% glycerol-100 mM Tris (pH 8.0) and observed with a rights optical microscope equipped with a fluorescence optical system. Immunofluorescence of live cells was performed as described above, except that the cells were incubated with the primary antibody immediately following an initial wash with PBS-5% GS.
Live cells were fixed with PBS containing 5% formaldehyde before mounting with cover strips. Fluorescently stained cells were from Kodak Ektachro film.
me film; ASA400). (1.6 ELISA analysis) The concentration of gB protein in the medium in which the CHO cells were cultured was measured by an indirect enzyme-linked immunosorbent assay (ELISA) using a recombinant gB preparation purified as a standard substance. A 96-well polyvinyl chloride plate (Dynatech Laboratories, Id.) Was incubated for 1 hour at room temperature with 50 μl of F3AB antibody diluted 1: 1000 in PBS.
nc.). Remove excess antibody from PBS-5% GS
And removed by washing three times. 50 μl of media sample or gB protein standard diluted in PBS-1% GS was added to the wells and incubated for 1 hour at room temperature. The plate was then washed three times with PBS + 1% GS, then 50 μl of rabbit anti-HSV-1 diluted 1: 1000 in the same buffer.
A third incubation with a polyclonal antibody (obtained from DAKO) was performed for 1 hour. Excess secondary antibody was removed by washing three times with PBS + 1% GS.
Finally, a horseradish peroxidase-conjugated goat anti-rabbit antibody (Boehringer Mann) diluted 1: 500 in PBS-1% GS.
nheim) was added to each well and incubated for 1 hour, then the wells were washed once with PBS + 1% GS, followed by 8 washes with PBS, followed by 2,2 'at a concentration of 1 mg / ml.
-Azido-di [3-ethylbenz-thioazoline sulfonate] (Boehringer Mannheim) with 0.1 M citric acid (pH
4.0), color was developed with 50 μl of a solution contained in 0.003% H 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 determined by using the gD-specific monoclonal antibody 8D2 (Rector et al., Infect. And Immun. (1982) instead of the purified recombinant gD and F3AB as standard substances.
38: 168-174), except that it 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; Skare and Summers, Vis.)
rology (1977) 76: 581-595), a DNA fragment spanning the coordinates 0.345-0.40 of the EcoRIF restriction fragment map into plasmid pBR32
Subcloned into 2. These fragments are
It was prepared from an appropriate restriction enzyme digest of the EcoRI region of ACY184, separated by electrophoresis on a 1% agarose gel using TAE buffer (0.04M Tris-acetic acid, 0.002M EDTA) and electroeluted. The isolated fragment was excised with the appropriate restriction enzymes and ligated to pBR322 which had been treated with alkaline phosphatase. A restriction map of the entire genome of HSV-1 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 (Roizman, 1979).
The dotted line indicates the LS connection. The EcoRI restriction map of the sequence of the prototype isomer is shown on line 3 together with the EcoRI fragment F indicated by hatching (Skare and Summers, 1977; Roizman, 197).
9). The HindIII restriction map of HSV-2 is shown in line 4 with the HindIII fragment H boxed (Roizman, 1979). One map unit is 98.9% of DNA of HSV-1.
Megadalton or 148.9 kbp, which corresponds to 10% of the HSV-2 DNA
It corresponds to 5.6 megadaltons or 160.5 kbp.

Referring to FIG. 2, the restriction sites shown in the detailed map line (I) are as follows:
E, EcoRI; B, BamHI; S, SalI; P, PstI; X, XhoI (DeL
ucca et al., 1983); N, NdeI; Xn, XmnI; V, EcoRV.
The BstEII site mapped by DeLucca et al. At 0.355 is deleted in this strain, with a new PstI site at 0.357. Line II shows a subclone of the three plasmids containing the gB1 coding region. These are 0.345 B
pHS106 ranging from the amHI site to the SalI site at 0.360;
PHS ranging from 0.36 SalI site to 0.388 SalI site
107; and BamHI ranging from 0.345 to 0.40 map units
It is a fragment. Line III shows the three probes used to map gB1 mRNA; line IV shows the fragments used for hybrid selection; and line V shows those used to determine the location of the gB2 gene. (See below). Additional restriction sites used to generate these fragments are Nc, NcoI; K, KpnI; and A, AluI.

To determine the location of the gB1 coding region in the EcoRI fragment, Northern blotting of polyA ⁺ mRNA isolated from Vero cells infected with HSV-1 was performed on detailed maps isolated from pHS106 and pHS107. The indicated DNA fragment was used as a probe. 0.5k isolated from pHS106
b When the PstI-SalI fragment was used as a probe for HSV-1, the major species detected was 3 kb mRNA. 0.49 kb Nc located about 1 kb upstream of the PstI-SalI fragment
When similar blotting was carried out using the oI fragment as a probe, hybridization with a putative 3 kb mRNA of gB1 mRNA was also detected. This suggests that the sequence encoding gB1 has been extended at least 1 kb upstream of the PstI-SalI fragment. The 3 kb mRNA is not extended beyond the first XhoI site downstream from the PstI-SalI fragment. This is because the 0.5 kb XhoI-XhoI fragment does not hybridize to this mRNA. The direction of transcription of the gB1 transcription unit is from right to left (3 '← 5'), indicating that PstI
Only the 5'-3 'strand of the -SalI and NcoI-NcoI fragments is demonstrated by hybridizing to 3 kb gB1 mRNA.

Hybrid selective translation was performed using HSV-1 polyA ⁺ mR
NA was performed by hybridizing with a 3.2 kb KpnI-XhoI fragment (including the region indicated as the gB1 coding region). When the bound mRNA was eluted and translated in vitro, a 100 kd protein of the same size as gB1 from HSV-1-infected Vero cells was detected. The identity of this 100 kd protein was confirmed by immunoprecipitation with a gB1-specific monoclonal antibody. Hybrid selection with the KpnI-XhoI fragment also detected some other proteins, probably as a result of nonspecific hybridization of mRNA that occurred due to the high G + C content of DNA. 3.0 kb SstI encoding HSV-1 glycoprotein gD
When the same RNA was selected using the -SstI DNA fragment, a similar protein pattern was observed, but the 100 kd gB protein was not detected. This result indicates that gB is KpnI-XhoI.
Suggests that it is specific for the fragment.

FIG. 3 shows a 3.95 kb fragment from the BamHI restriction site at 0.345 map units to the XhoI site at 0.373 map units.
1 shows a restriction map of a DNA fragment. The gB1 open reading frame is boxed and the direction of transcription is from right to left as shown. The actual code area is from map units 0.348 to 0.367. BamH
DN from the I site to the 3,640th unique AluI site
The A sequence is shown and the AluI site is shown in (A). The restriction sites shown are B, BamHI; B1, BalI; Bs, BstE.
II; K, KpnI; Nc, NcoI; P, PstI; Pv, PvuII; S, Sal
I; Sc, SacI; X, XhoI; Xm, Xma3. Restriction enzyme cleavage sites from the AluI site on the right side to the terminal XhoI site are not shown. Potential glycosylation sites of the produced gB1 protein and hydrophobic anchor and signal regions (filled boxes) are indicated.

3,640 nucleotide residues from the BamHI site
The DNA sequence up to the second and only AluI site was
Was determined using the M13 dideoxynucleotide synthesis method. Both DNA strands of the coding region were sequenced. The complete DNA sequence is assembled from the aligned restriction fragments, and the sequence is thus read through the entire diagnostic strip. FIG.
7 shows the DNA sequence of gB1 (line 3); the predicted amino acid sequence for gB1 is shown below the DNA sequence (line 4).
line).

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

A 22 bp oligonucleotide (residues 473-49)
Primer extension using 4) indicated that the 5 'end of gB1 mRNA was located at residue 188. CAT and TATA transcriptional regulatory signals are predicted to be residues 55-62 and 125-131. There is an open reading frame of 2712 nucleotides starting from the ATG at residues 438-440 and ending at the TGA stop codon. Two putative polyadenylation signals are located in the non-coding region 3 'to residues 3166-3173 and 3409-3416.

The amino acid sequences described are characteristic of a membrane protein. Amino acid residues 726 to 795 near the carboxy terminus
There are up to two very hydrophobic regions, with 69 amino acids capable of binding to the membrane. The first 30 at the N-terminus
The amino acids are mainly hydrophobic. This hydrophobic amino acid domain precedes a region of high concentration of charged or hydrophilic amino acids. The N-terminal hydrophilic sequence serves as a secretory leader or signal sequence, followed by a processing signal for cleavage and removal of the secretory leader. The hydrophobic region near the C-terminus can serve as a transmembrane integration sequence for binding proteins to cell membranes.

The sequence data is in the hydrophilic ectodomain
It also suggests that there are nine potential N-linked glycosylation sites as defined by the asn-X-thr / ser sequence (see also FIG. 3). 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 about 123 kd Becomes (2.2 Expression of gB1 in Mammalian Cells) To express gB1 in mammalian cells, CHO cells lacking dhfr were transformed by introducing plasmids pHS112 and pHS114.
This transformation was performed using the calcium precipitation method as described in Materials and Methods. Plasmid pHS112 and
The E. coli HB101 strain transformed with pHS114 has been deposited with the ATCC and has been deposited under accession numbers 39650 and 39951, respectively. The construction of these strains is described in International Patent Publication No. WO85 / 085.
No. 4587 (supra). Transformed cells were selected using a selective medium lacking thymidine, purine and glycine. Cells were isolated by removal with a Pasteur pipette and grown on plates with multiple wells. A large number of clones have been isolated,
It was shown to produce gB by immunofluorescence and radioimmunoprecipitation using an HSV-1 polyclonal antibody or a monoclonal antibody specific for gB. 3 cell clones pH
S112-1, pHS112-9 and pHS112-23 were isolated and the clone synthesized an intracellular form of the complete gB protein.
GB produced in these cells is considered to be glycosylated. Because, after pulse labeling for 1 hour and then chasing for 5 hours, a larger molecular weight form can be detected compared to non-chaseed cells, about 10%
Of gB was secreted into the medium. Five cell clones expressing incomplete gB (pHS114-5, pHS114-6, pHS1
14-7, pHS114-11 and pHS114-12) were also analyzed and also showed to secrete some gB into the medium. One of these cell lines, pHS114-7, was selected for further amplification with MTX. Clones are initially 0.01, 0.05,
Selected at 0.10 and 0.3 μM MTX. Three clones that synthesize high levels of gB as detected by immunofluorescence analysis are 0.
Isolated from selection at 3 μM MTX. By radioimmunoprecipitation, these clones, pHS114-0.3 μM-6, 23 and 25, had 2-3 fold more unlabeled clone pHS114-7 while labeling with ³⁵ S-methionine for 1 hour. Synthesize gB. Pulse chase experiments indicate that at least 8% of the gB synthesized in these clones during the 1 hour pulse is secreted extracellularly in 5 hours.

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

PHS137 is obtained by converting pHS108 (described in Chapter 2.1) to X
It was constructed by digestion with hoI and BamHI, followed by isolation of the resulting 3.5 kd fragment. The ends of this fragment were blunt-ended with Klenow. This blunt-ended XhoI
The BamHI fragment was partially digested with PVUII, and the DNA that migrated as a 2098 bp band on the gel was isolated from the partially digested product.
The isolated XhoI-PVUII band was ligated into pSV7d, which had been digested with SmaI, and the resulting DNA was used to transform E. coli. The resulting bacterial clone was cloned into gB1
Screening was performed for plasmids in which the insert was inserted in the appropriate orientation.

To obtain expression, pHS137 was converted to plasmid pA
Dhfr-deficient CHO cells were introduced together with Ddhfr for transformation. The resulting clone produced and secreted gB1. One such clone, pHS137-7-B-50, was placed in a T75 culture flask containing 10 ml of complete medium at 1-3 × for 24 hours.
The 10 ⁷ 6.91 +/- 1.53μg / ml gB1 protein per cell was produced. (3. Glycoprotein B2) gB2 gene isolation, characterization,
And cloning are described in International Patent Publication No. WO85 / 04857 (supra). (3.1 Expression of gB2 in mammalian cells) HSV-2 glycoprotein g
Expression of B was transformed with pHS210 alone or pHS21.
Co-transformation with COS is performed in COS cells (temporary expression). This second plasmid is dhfr
including.

The plasmid pHS210 was constructed as follows; the entire gB2 gene was constructed as a 3.8 kb NruI-BamHI fragment.
It was subcloned into BR322 to produce pHS208. FIG.
checking ... The PstI site at the 5 'end of the gene (100 bp right (downstream) of the NruI site) was changed to a HindIII site by in vitro mutation at M13. Then, 1.9 kb HindIII to P
The fragment up to vuII was inserted into pSV1 obtained by digesting pSV1 / dhfr with HindIII and BglII. See FIG. 10; pSV1 / dhfr is described in PCT International Patent Publication No. WO85 / 045.
No. 87. For this cloning step, pHS208 was cut with PvuII and the ends were repaired to blunt ends. This molecule was then cut with HindIII and 1.9 kb HindI
The II- (PvuII) fragment was isolated by gel electrophoresis. Similarly, pSV1 / dhfr was cut with BglII, repaired to blunt ends, Hind
Cleavage with III and gel electrophoresis 4.85 kb HindIII- (Bgl
II) The vector fragment was isolated. These two fragments (1.9 kb
And 4.85 kb) with the expression vector pHS210 (FIG. 1).
0).

[0058] Plasmid pHS210 was used to directly transform COS cells. Expression was determined using a gB-specific monoclonal antibody, F3AB, and a commercially available polyclonal antibody, HSV-2 antibody (DAK), for primary antibody screening.
O) was also detected using immunofluorescence analysis. Secretion of gB2 into the medium was detected by gB2-specific ELISA analysis. For this purpose, the plates were coated with a monoclonal antibody. Cell culture media samples were added to the coated plates and bound gB2 was detected using a rabbit anti-HSV-2 polyclonal antibody (DAKO) followed by horseradish-conjugated goat anti-rabbit IgG.

Plasmid pHS21 for CHO cell transformation
0 is d as a selectable marker in the co-transformation protocol
used with a second plasmid containing hfr (FIG. 1).
0). Subsequent transformation and growth in selective medium resulted in the isolation of approximately 100 dhfr ⁺ clones and synthesis and secretion of gB2 using ELISA analysis (ELISA plates coated with F3AB specific monoclonal antibodies). Was screened for The clone pHS210 # 3-1 with the highest level of gB secretion was further selected for gB2 polypeptide characteristics. The gB2 protein was detected by labeling with [ ³⁵ S] -methionine followed by radioimmunoprecipitation. After a one hour pulse, two diffuse bands corresponding to the 79 kd and 84 kd polypeptides were detected in the cells. These proteins were 68,991 daltons larger than the size predicted for the incomplete gene product of 637 residues, suggesting that the proteins corresponded to partially glycosylated precursors. After chasing for 5 hours, no gB2 was detected in the cells. Then, a polypeptide of 89 kd was detected in the medium. Clone pHS210 #
The mature, fully glycosylated gB2 secreted into the medium of 3-1 is 100 kd secreted by pHS4-6.
Was somewhat smaller than gB1. This means that the coding sequence corresponding to 94 amino acids contained in the gB1 plasmid
This is because it has been removed from S210. (4. Glycoprotein D1) (Construction of 4.1 gD1 mammalian expression vector) EcoR of pBR322
A library of EcoRI fragments of the HSV-1 of Patton strain cloned into the I site was obtained from Dr. Richard Hyman, Hershey Medi.
Cal Center, Hershey, PA. The gD1 gene is located in the EcoRI fragment of clone H of this library.
Completely contained in the 2.9 kb SacI fragment. 15kb EcoRI
Clone H containing the insert was obtained from Dr. Hyman. This 2.9k
The b fragment was purified by gel electrophoresis and then completely digested with HindIII and NcoI. The 5 'end of the gD gene consisting of a 74 bp 5' untranslated sequence and a 60 bp
Isolated from the gel as a 134 bp fragment. The 3 'end of the gD gene was obtained by digesting pHYS119 (see International Publication No. WO85 / 04587, supra) with NcoI and SalI and isolating a 873 bp fragment. These two fragments (5 'end and 3' end) were ligated to plasmid pUC12 which had been digested with HindIII and SalI in advance. The pUC12 vector is Ph
Commercially available from armasia and PL Biochemicals,
The resulting plasmid was named pHS131. Plasmid pH
S131 was digested with HindIII, the 5 '4 bp overhang was filled in with Klenow polymerase, and then digested with SalI. g
The 1007 bp fragment containing the D gene was gel isolated and ligated into plasmid pSV7d which had been cut with SmaI and SalI. This plasmid pSV7d is described below. The resulting expression vector is named pHS132. The guidance is schematically shown in FIG.

This plasmid contains the total protein
Encodes 315 amino acids of the gD1 protein, including the signal sequence of 25 of the 399 amino acids. This protein is truncated at the carboxyl terminus and lacks 84 amino acids including a hydrophobic membrane anchor domain and a cytoplasmic domain, and the resulting protein is secreted into the medium.

The plasmid pSV7d was constructed as follows: 400 bp B containing the SV40 origin of replication and the early promoter.
The amHI / HindIII fragment was converted to SVgtI (Mulligan, R. et al., J. M.
ol. Cell Biol. (1981) 1: 854-864) and purified. 240 bp SV4 containing the polyA addition site of SV40
The 0BclI / BamHI fragment was ligated with pSV2 / dhfr (Subramani et al., J. M.
ol. Cell Biol. (1981) 1: 854-864) and purified. Link these fragments below:

[0062]

Embedded image And fused. This linker contains five restriction sites with stop codons in all three open reading frames. The resulting 670 bp fragment (containing the SV40 origin of replication, the SV40 early promoter, the polylinker with a stop codon, and the SV40 polyadenylation site) was deleted by about 1.5 kb in pBR32
2 derivatives (Lusky and Botchan, Cell (1984) 36: 391)
Was cloned into the BamHI site of pML to obtain pSV6. p
The EcoRI and EcoRV sites in the pML sequence of SV6 were removed by digestion with EcoRI and EcoRV, followed by treatment with Bal31 nuclease to remove approximately 200 bp at each end, and finally religation to give pSV7a. Was. By Bal31 resection,
One adjacent to the SV40 region about 200 bp away from the EcoRV site
The BamHI restriction site was removed. The second adjacent to the SV40 region
PSV7a was digested with NruI to remove the BamHI site. This enzyme cleaves pML sequences upstream of the origin of replication.
This was recyclized by blunt end ligation to yield pSV7b.

PSV7c and pSV7d refer to contiguous polylinker substitutions. First, pSV7b was digested with StuI and XbaI. Next, the following linkers were ligated into the vector to yield pSV7c:

[0064]

Embedded image Subsequently, pSV7c was digested with BglII and XbaI and ligated with the following linkers to give pSV7d:

[0065]

Embedded image (Expression in mammalian cells of 4.2 gD1) Plasmid pHS132
gD1 expression has been demonstrated in a number of experiments. First, a commercially available anti-HSV-1 rabbit serum (DAK
After infection with O), specific immunofluorescence was observed in COS7 cells. Second, a stable CHO cell line secreting gD1 was established. Expression levels were analyzed by ELISA and confirmed by radioimmunoprecipitation of pulse-labeled and chased cell lysates with media. Third, gD1 is CHO cell line D64
Was purified from a series of roller bottle cultures by a series of steps: ammonium sulfate precipitation, immunoaffinity chromatography, and ultrafiltration. Affinity chromatography includes gD described in Rector et al. (1982) (supra).
A monoclonal antibody 8D2 bound to cyanogen bromide-activated Sepharose 4B was used. (5. Glycoprotein D2) (Construction of a mammalian expression vector of 5.1 gD2) The HindIII fragment of HSV-2 333 strain was described in the literature (Kudler et al., Virology (1983) 1
24: 86-99), as shown by Dr. Richard Hyman
Was cloned into pBR322. The gene for glycoprotein gD2 has been mapped to a short specific region of the virus between 0.90 and 0.945 map units by Ruyechan et al., J. Virol. (1970) 29: 677-697. The region occupied by the HindIIIL fragment is shown in the genome map of Roizman, B., Ann. Rev. Genet (1979) 13: 25-27. gD2 gene DNA sequence is W
atson, Gene (1983) 26: 307-312.

The HindIIIL fragment cloned into pBR322 was obtained from Dr. Richard Hyman and the restriction map shown in FIG. 12A was determined. The gD2 gene encodes gD1 2.
Using a 9 kb SacI fragment as a probe, the restriction enzyme digest of the HindIIIL fragment was subjected to Southern blot to obtain a 2.4 kb XhoI fragment.
It was found to be on the I fragment. The map of the XhoI fragment and the location of the gD2 gene are shown in FIG. This 2.
The 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 is shown in FIG. Plasmid pHS211 encodes the first 305 amino acids of gD2 including the signal sequence. PHS20
4 was cut with SmaI and BamHI and two restriction fragments were isolated from the gel: 5 ′ of the gene containing 82 bp of 5 ′ untranslated sequence.
A 250 bp SmaI fragment containing the terminus and a 3 ′ flanking 746 bp SmaI-BamHI fragment containing the interior of the gene. Cutting the mammalian cell expression vector pSV7d (described in section 4.2) with EcoRI,
The 5 '4 bp overhang was blunt-ended with Klenow polymerase and then cut with BamHI. The two fragments of pHS204 were ligated into the digested pSV7d and Sma from the bacterial transformants.
The one in which the I fragment was in the correct orientation was selected to obtain the vector pHS211.

Plasmid pHS212, encoding 352 amino acids of gD2 and 47 additional residues beyond the gene present in pHS211 was digested with pHS204 with HaeII, and blunt-ended with Klenow polymerase, and Constructed by digestion with BamHI. A 141 bp fragment from (HaeII) (parentheses indicate filled ends) to BamHI was gel isolated. Plasmid pHS211 was introduced into E. coli GM272 strain, plasmid DNA was prepared, which was then digested with BclI, blunted with Klenow polymerase, and
Decomposed with I. The large vector fragment (about 3.4 kb) was gel isolated and ligated to the 141 bp (HaeII) -BamHI fragment to obtain plasmid pHS212. By fusion of the gD2 sequence and the plasmid vector sequence at the 3 'end of the gene, the gD2 gene
27 codons of nonsense DNA are added to the 3 'end. To remove these nonsense sequences, the plasmid pH
S213 was constructed by partial digestion of pHS211 with SalI, gel isolation of the single-cut plasmid, followed by blunting with Klenow polymerase and digestion with BamHI. pH
A 141 bp fragment of BamHI from (HaeII) of S204 was linearized.
Ligation to pHS211 yielded plasmid pHS213. (5.2 Expression of gD2 in mammalian cells) The expression of gD2 in mammalian cells was determined by pHOS21, pHS212 and pHS213.
Seven cells were first analyzed for transient expression by transfection. 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. And gD-type common antibody 8D2 (Rector et al. (1982) supra).

Next, a permanent CHO cell line was established by transfecting with the plasmids pHS211 or pHS213 with Addhfr, selecting for dhfr acquisition and screening by g ELISA for expression of gD2. Description of Ad-dhfr: Plasmids carrying the dhfr gene were replaced with a major aerobic promoter from Adenovirus-2 (Ad-MLP,
Map unit 16-17.3) was fused to the mouse dhfr cDNA at the 5 'end. DNA encoding the intron for the SV40 small t antigen and the SV40 early region polyadenylation site was obtained from Southern and Berg, J. Mol. Appl. Genet.
(1982) 1: 327-341 and obtained from pSV2-neo and fused to the 3 'end of dhfr cDNA. These three fragments were subcloned into pBR322 to obtain the plasmid Ad-dhfr. This plasmid was obtained from Kauhman and Sharp, Molec.
and Cell Biol., (1982) 2: 1304-1319.
Functionally similar to the dhfr plasmid. (6. Therapeutic treatment with gB-gD vaccine) (6.1 Effect of recombinant HSV glycoprotein vaccine administered after primary infection on recurrent herpes disease in guinea pigs)
In the vagina of a female Hartley guinea pig, 5 × 10 on the first day
⁵ pfu HSV-2 MS strain was inoculated. The animals were primed by adding acyclovir (5 mg / ml) to drinking water.
Treated on day 1010. Acyclovir reduces the symptoms of primary infection, 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 (Bernstein et al., Virology, (1986) 67: 1601). After recovery from the primary infection, the animals were immunized with an HSV-2 total glycoprotein preparation (gP2), and the recombinant gB1 and gD1
(HSV-1 gB + gD) or no treatment. The treatment groups are shown below. Group Treatment Dose Adjuvant route NI None None None None 11 II HSV-1 gB + gD 25 μg + 25 μg Freund footpad 11 III gP2 50 μg Freund footpad 11 IV Control adjuvant only None Freund footpad 9 By injecting vaccine into rear footpad Animals were immunized on day 21 and further 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 FIG.

These results indicate that the pattern of recurrent herpes disease was the same for groups I and IV, and these groups were pooled for analysis (control, n = 20).

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

The recurrence rate of a disease, as measured by the number of days of disease occurring within a specified period of time, is an assessment that takes into account both the frequency and duration of recurrent episodes. FIG. 15A shows the prevalence of recurrent herpes disease, expressed as the average number of days per week when herpes disease was observed. The immunized group includes both animals vaccinated with gBgD and gD-2. As shown in FIG.
The rate of disease recurrence (days of disease per week) decreased in all groups as the evaluation period moved away from the initial infection, but the rate of reduction was greater in vaccinated animals. The difference in the rate of recurrent herpes disease infection between control and immunized animals is shown in FIG. 15B. As is evident from FIG. 15B, the effect of glycoprotein immunization on the recurrence rate of the disease, as inferred from FIG. 14, was greater following the first immunization rather than after the second administration. It seemed to be established.

[0072]

[Table 1] (6.2 Effect of recombinant HSV glycoprotein vaccine administered after primary infection on host immune response) The effect of glycoprotein administration after infection on host immune response is based on the anti-HSV antibodies produced by infected animals. By measuring before infection and after immunization with the HSV glycoprotein vaccine,
It has been determined.

As described in section 6.1, these animals were given HSV-2
Inoculated with the ms strain, treated with acyclovir, and treated with the HSV glycoprotein vaccine. Serum from these animals
Collected on days 41 and 95. Anti-HSV antibodies in serum are essentially defined by Pachl, C. et al., J of Virology (1987) 61: 315-325.
As described in Section 1.6, the method described in section 1.6, E
Measured by LISA. Capture antigens included HSV-1 glycoprotein mixture (gP-1), HSV-1 glycoprotein D (gD-1), or HSV-2 glycoprotein D (gD-2).

The effect of HSV glycoprotein vaccine administration on anti-HSV antibody titers is shown in Table 2, where data are presented as geometric means. No antibodies were detected in serum collected before HSV inoculation. As seen in Table 2, untreated control animals had higher titers of anti-HSV antibody on day 41 than on day 95. In contrast, animals treated with glycoproteins generally showed much higher titers by day 95. Vaccination with HSV glycoprotein also significantly increased anti-HSV antibody titers compared to untreated controls (P <.05). In addition, treatment with the gP-2 mixture increased antibody titers by a factor of 1.4-7, while recombinant HSV-1 g
Treatment with the BgD vaccine resulted in titers of 9-31 compared to control values.
Doubled. Thus, administering an HSV glycoprotein to an animal, especially administering a recombinant HSV glycoprotein gBgD, increases the immune response of the host and, as shown in Section 6.1, the frequency of recurrent HSV disease. And reduce symptoms.

[0075]

[Table 2] Effect of Adjuvant on Immune Response Induced by HSV Glycoprotein Vaccine Containing 6.3 gD1 Several adjuvants were tested to determine 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 (MTP-
Called PE).

The effect of the adjuvant was determined by measuring the amount of anti-gD1 antibody after administration of a vaccine 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 the adjuvant does not seem to be specific to the type of HSV glycoprotein in the vaccine.

In the following studies, gD1 was synthesized from pHS132 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 three times at three-week intervals via the footpad. One week after the second immunization, and 1, 5, 9 and 13 weeks after the third immunization, the animals were bled and the anti-gD titers were determined as described in Section 1.6.
Determined by LISA.

The results shown below suggest that the most promising adjuvant tested is MTP-PE. This is because MTP-PE constantly produces high anti-gD1 titers in experimental animals. Although the titers were not held as long as CFA, these levels were comparable to those seen for CFA. (6.3.1 Comparison of CFA and aluminum hydroxide)
Immunization was performed with a vaccine containing D1 and either CFA or aluminum hydroxide. Table 3 shows the effect of the adjuvant on the anti-gD titer. In Table 3, the data is represented by a geometric mean. As seen in Table 5, the most effective adjuvant was CFA. The longest and highest antibody titers were seen in the group immunized with the CFA vaccine. The effect of other adjuvants is shown as a percentage compared to the titers obtained with CFA. The lowest anti-gD1 titers were obtained by using a 10% aluminum hydroxide suspension as adjuvant.

[0079]

[Table 3] (6.3.2 Comparison of CFA, nor-MDP, and MTP-PE) Animals were immunized with a vaccine containing gD1 and one of CFA, nor-MDP, and MTP-PE. MTP-PE was encapsulated in liposomes and this latter adjuvant was administered with exogenous gD1 and with gD1 incorporated into liposomes. Liposomes include synthetic phosphatidylcholine, phosphatidylserine and MTP-
PE (or MTP-PE and gD1) was added to the suspension medium (sterile
Ca ⁺⁺ and Mg ⁺⁺ salt-free isotonic Dulbecco's buffer, pH
It was prepared by adding 175: 75: 1 in 7.2) and stirring by vortex. As seen in Table 4, immunization with the vaccine containing CFA resulted in the highest anti-gD
One average titer was obtained. The titers obtained with nor-MDP ranged from 44 to 74% of the average titers obtained in the group immunized with CFA. The average titers obtained with MTP-PE and exogenous gD1 are:
It was somewhat lower than that obtained with nor-MDP, ranging from 32 to 72% of that obtained with CFA. The very low titers obtained with gTPl encapsulated in MTP-PE and liposomes may be due to very low levels of gDl 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 allowed for more efficient incorporation of antigen.

[0080]

[Table 4] (6.3.3 Comparison of different formulations containing MTP-PE) Animals were treated with MTP- in a highly oily delivery system (squalene / arlacell).
Immunization was carried out with gD1 containing a vaccine formulated with PE and a vaccine formulated with MTP-PE in a low oil delivery system. The less oily MTP-PE formulation contained 4% squalene and 0.008% Tween80. Table 5 shows the gD1 antibody titers obtained with these adjuvant formulations.

As seen in Table 5, the MTP-PE formulation was:
It was effective as an adjuvant even when used as the only component in a low oil-surfactant formulation. [It was also an effective replacement for the CWS component in RIBI, and its effect increased over time compared to RIBI. At the third blood draw, the titer obtained with MTP-RIBI was twice that obtained with RIBI. ] 1
After the second blood draw, the MTP-PE low oil formulation showed higher titers than the high oil delivery system (squalene / arlacell).

[0082]

[Table 5] (6.4 Therapeutic study: Effects of adjuvants in vaccines effective in preventing recurrent herpes disease in guinea pigs, site of administration, and timing of administration) The gBgD vaccine has a unity of about 70-80% as judged by SDS-PAGE. Contains 25 μg of each of recombinant gB1 and gD1 purified to 25 g. Recombinant gB
One protein was a 50:50 mixture of gB1 prepared from pHS113-9-10-21 and pHS137-7-B-50 cell lines.
These cell lines are CHO cell lines that carry the vectors pHS113 and pHS137, respectively. pHS113 and p
The preparation of HS137 is described in section 2.2. The gB protein is described in Pachl et al., J of Virology, 61: 315-325 (198
Purified as described in 7), which is described in section 2.2. 8D2 instead of C4D2 during purification by affinity chromatography
G, as described in Section 4.2, except that
D1 was prepared.

This example compares the effects of two adjuvants, nor-MDP and CFA. The adjuvant nor-MAP was emulsified with 50% squalene / arlacell and antigen and used at 50 μg / dose.

This study also compares the two routes of administration. That is, a comparison is made between intra-footpad administration and intramuscular or subcutaneous administration. Finally, different times of administration in the prevention of recurrent herpes disease in guinea pigs are compared. The experimental design is shown in Table 6.

[0085]

[Table 6] Female Hartley guinea pigs weighing 350-400 g were inoculated intravaginally on day 1 with 5.7 / log ₁₀ pfu of HSV-2 MS strain. The animals were confirmed to be infected by recovering HSV from vaginal swabs collected 24 hours after vaginal inoculation. Stanberry et al., J Infec Dis
(1987) 155: 914, followed and quantified by genital skin lesions. After recovery from primary infection, animals were randomized into treatment groups as shown in Table 10. Animals were examined daily for signs of disease recurrence from day 11 to day 100 after the acute illness had resolved. The disease day is defined as the day on which recurrent disease was observed, a severe relapse is the day on which more than one blister was observed, and the episode is a new disease on the day after no disease To do so.

The results obtained from the analysis of the animals between days 22 and 76 are shown in Table 7. The data in Table 9 suggest that nor-MDP is an effective adjuvant for IM injection. This is reflected in lower overall disease days, lower rates of severe relapses, and a reduction in the total number of herpetic episodes. Furthermore, no
Vaccines containing r-MDP and administered IM appear to be as effective as vaccines containing CFA and administered in the footpad.

[0087]

[Table 7] Local reactions resulting from injection of vaccines containing various adjuvants were also monitored. Table 8 shows the results. The incidence of local erythema and cirrhosis at the injection site,
The same was true for vaccines containing nor-MDP. Furthermore, nor-MDP could be used in vaccines based on the local reactivity.

[0088]

[Table 8] The results in Table 7 also show that as the interval between the onset of immunotherapy and the onset of acute illness decreases, the relative efficacy of the treatment increases. After the first infection, 8,
Of the animals vaccinated with the gBgD vaccine containing CFA 15 or 21 days later, those vaccinated the shortest time after vaginal inoculation have the shortest duration of disease compared to untreated controls The rate of severe relapses was the lowest, and all episodes were the least. At 15 days post infection, the values obtained for the vaccinated animals were higher than those obtained for the vaccinated group at 8 days and 21
The group inoculated 15 days later was higher than the group inoculated 15 days later. This effect is also shown in FIG. FIG.
Shown is a graph of the number of recurrences daily after vaginal inoculation of the first vaccinated animal at 8, 15, or 21 days after V-2 inoculation.

The data in FIG. 16 was used to calculate the percentage reduction in relapsed disease (see Section 6.1 for an explanation of the significance of disease recurrence). It is shown in Table 9. In Table 9, it can be observed that the first vaccination on day 8 in the earliest period (i.e., 14-50 days) has the greatest reduction in the percentage of relapsed disease. But,
The first vaccination, 15 days after the vaginal inoculation of HSV, provided the most effective protection from days 51-92. The weakest protective effect was obtained when the first vaccination was performed 21 days after the initial exposure to HSV-2.

It should be noted that in guinea pigs, the acute phase of the disease occurs between 14 and 21 days after infection with the virus. During this acute period, HSV
The disease induced by is found in the animal's body.
Thus, the data in FIG. 16 shows that the most effective protection at days 51-92 was obtained by administration of the vaccine during the acute period of infection.

[0091]

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

While the above invention has been described in some detail by way of illustration and example to provide a clearer understanding of its contents, certain changes and modifications may be made within the scope of the appended claims. Is clear.

[0093]

Industrial Applicability According to the present invention, it has become possible to produce a vaccine which is effective for preventing recurrent herpes simplex virus-induced disease using the isolated polynucleotide encoding the glycoprotein.

[0094]

[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 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Val 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 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 L ys 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 gt 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 ttcag 8 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 255 260 265 270 270 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 tgg ggc tac 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 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 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 g gc 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 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 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 gg ac 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 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 aa c 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 cgag gcc cagag 1 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 atg gtg cag cgc gtc agc tcg cgg ccg 2078 Pro Asp Asn Val Ile Val Gln Asn Ser Met Arg Val Ser Ser Arg Pro 575 580 585 585 590 ggg acg tgc tac agc cgc ccc ctg gtc agc ttt cgg tac gaa gac cag 2126 Gly Thr Cys 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 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 Val Gly His Arg Arg Tyr Phe 625 630 635 atc ttc ggc ggg ggc tac gc tg gac tg gc tg gac tg gc 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 gc 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 gacg acg 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 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 gg ggt g 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 Ast 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 Leu 790 G 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 ggc gcg 2798 Lys Thr Ser Asp Pro Gly Gly Val Gly Gly Glu Gly Glu Glu Gly Ala 815 820 825 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 ys 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 ag 860860 gcc 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 ccccct 990 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 gt aca tca cata c ata 900 g 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 aaacacacac 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 gcggcgg 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 Aln 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 gggcg 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 Iag 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 Glu210 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 g tt 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 ggc 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 340 345 350 350 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 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 cgac ag acg 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 ttc atc gcg tac cag 1344 Gln Pro Gln Tyr Tyr Ayr 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 460460 cgg 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 465 470 475 475 480 gag gcg ccc agc gcc aac gcg tcc gtg gag accg atc 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 ctgag 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 As le 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 ggt gtg 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 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 630 630 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 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 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 gcgccccccttc 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 Vals 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 gt 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 Pc Acac Lacac Acahe Acahe Lac 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 805 810 815 815 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 gga gag 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 Get 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 865 870 875 875 880 ctg cgc aag c gc 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 Gap 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 Alu 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 As n 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 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 Ph e 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 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 Pr o 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 705 710 715 715 720 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 Gl u 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 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 P ro 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 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 ttg tac atg tcc tcc 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 accg 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 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 c tg 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 M 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 gg 440 440 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 ag ag ag 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 cg c 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 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 Gly Ar 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 gtc gac gac gac ag aac tcg atg cgc atc agc 2078 Pro Val Ala Ala Asp Asn Val Ile Val Gln Asn Ser Met Arg Ile Ser 575 580 585 585 590 tcg cgg ccc ggg gcc tgc tac agc cgc ccc ctg gtc agc ttt cgg tac 2126 Ser Arg Pro G 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 gg ggc tac gtg 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 675 680 680 685 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 ttgcc gcttccc 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 Thror 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 g ag gt g gt g gt g gt g gt g 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 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 The Ala Phe Arg 790 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 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 L ys 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 ag 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 caac gcc c990 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 gt gttacacc gg gg cc 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> CD <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 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 Threl Pro 65 70 75 80 cca cgc ccc gcc ggc gac aac gcg acc gtc gcc 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 caac 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 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 His210 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 280c 285 g 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 tt 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 340 345 350 350 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 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 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 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 Tgt 450 455 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 465 470 475 475 480 ccc ccg ccg ccc ggg gcc agc gcc aac gcg tccg gggg aag 1488 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 515 520 525 525 tgc gag ctg gag atg 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 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 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 cgcgc gc 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 Gly Pro Leu Val 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 640 630 635 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 ag acc c 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 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 Thr 208 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 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 gtt g 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 Agla A400 G 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 805 810 815 815 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 Glu Gly 820 825 830 ggc gac ttt gac gag gcc aag cta gcc gag gcc ggg 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 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 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 20 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 Ply 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 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 530 535 540 540 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 Ser 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 705 710 715 720 720 Thr Val Ile His Ala Asp Ala Asn Ala Ala Met Phe Ala Gly Leu Gly 725 730 730 735 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 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 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 the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Physical maps of HSV-1 and HSV-2, Eco RI cleavage maps for the placement of prototype isomers, and HS.
1 shows a HindIII restriction enzyme cleavage map of V-2.

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

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

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

FIG. 5 is a continuation of FIG. 4;

FIG. 6 is a continuation of FIG. 5;

FIG. 7 is a continuation of FIG. 6;

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 map of gB2.

FIG. 11 is a flowchart relating to the construction of pHS132, a mammalian expression vector for gD1.

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

FIG. 13 is a flowchart relating to construction of a mammalian vector for gD2.

FIG. 14 shows the effect of vaccination with recombinant gB-gD following primary infection on recurrent herpes disease.

FIG. 15A shows the effect of immunization with herpesvirus glycoprotein on relapse 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 gBgD vaccine on recurrence of herpes disease.

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Carroll Pacl United States of America 94611 Auckland, Somerset Road 321 (72) Inventor Pablo, De. tea. Valenzuela USA California 94117 San Francisco, Upper Terrace 455 F-term (Reference) 4B024 AA01 BA32 CA04 DA02 EA04 GA11 HA01 4C085 AA03 BA78 CC08 DD23 DD43 EE03 GG02 GG03 GG04 GG05 GG06 4H045 AA11 BA10 CA03 DA86 EA31 FA74

Claims (1)

[Claims] 1. A recombinant herpes simplex virus (HSV) glycoprotein B (gB) polyproduced in a host cell containing an isolated nucleotide sequence encoding a herpes simplex virus (HSV) glycoprotein (gB). An immunologically equivalent fragment of the peptide.