JP2004242679A - Vaccine used for curative treatment of hsv - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polypeptide useful for detecting herpes simplex virus (HSV) infection as well as a vaccine useful for inhibiting a recurrent HSV-induced disease, and a production method thereof. <P>SOLUTION: An immumologically equivalent fragment of a polypeptide being a recombinant HSV glycoprotein B (gB) is produced in a host cell containing an isolated nucleotide sequence encoding HSV gB. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  Herpesviruses include herpes simplex viruses containing two closely related variants, termed 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.

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

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

  One promising attempt has been to use isolated glycoproteins for prophylaxis. This glycoprotein has been shown to confer protection when injected into mice and then infected with live virus. However, the 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 problems associated with handling dangerous pathogens, maintaining the virus in cultured cells, and isolating glycoproteins that do not contain the viral genome or parts thereof have led to the use of glycoproteins as vaccines. Has been hindered. Therefore, it is desirable to provide a vaccine using glycoproteins produced by methods other than culturing the virus and isolating the membrane proteins.

  It is also of great interest to develop methods for therapeutically treating herpes infections, i.e., treatments that reduce the disease or prevent the disease from recurring after the individual has been infected with the virus. It is. 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.

  Eberle and Mou (J. of Infectious Diseases (1983) 148: 436-444) report the relative titers of antibodies to HSV-1 specific polypeptide antigens in human serum. Marsden et al. (J. of Virology (1978) 28: 624-642) reported that the gene corresponding to the 117-kilodalton (kd) glycoprotein was HSV due to intra-type recombination between HSV-1 and HSV-2. Are located within 0.35-0.40 map units on the genetic map. (Id. (1979) 29: 677-679) report that the location of the glycoprotein B gene is between 0.30 and 0.42 map units. Skare and Summers Virology (1977) 76: 581-595) report EcoRI, XbaI, HindIII endonuclease cleavage sites on HSV-1 DNA. Roizman (Ann. Rev. Genetics (1979) 13: 25-57) reports the organization of the HSV genome. (Virology (1982) 122: 411) have 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.

  Subunit vaccines extracted from chicken embryo cells infected with HSV-1 or HSV-2 are described in US Pat. Nos. 4,317,811 and 4,374,127. See also Hilfenhaus et al. (Develop. Biol. Standard (1982) 52: 321-331) which describes the preparation of subunit vaccines from specific HSV-1 species (BW3). (Id. (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) 218: 381-384) describe not only the expression of cloned fragments by injection into the nucleus of frog oocytes, but also the cloning of the HSV-1 gD gene and in E. coli. Describes the low level expression of the gene. They also show the nucleotide sequence of the gD gene. Weis et al. (Nature (1983) 302: 72-74) report on the high level expression of gD in Escherichia coli. This polypeptide induces neutralizing antibodies in rabbits. Berman et al. (Science (1983) 222: 524-527) report on 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: 102-108) report the location and chemical synthesis of specific antigenic determinants of gD within residues 8-23 of the mature protein.

  For the use of "membrane protein preparations from HSV infected cells as" therapeutic "as a post-infection vaccine in humans, see Dundarov, S. et al. (Dov. Biol. Standard (1987) 57: 351-357). And Skinner, GRB et al. (Ibid. (1982) 52: 333-34).

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

Accordingly, one aspect of the invention is a vaccine for use in the therapeutic treatment of herpes simplex virus (HSV) infection. The vaccine is
a. An immunologically active herpes simplex virus glycoprotein B (gB) polypeptide;
b. It contains an immunologically active herpes simplex virus glycoprotein D (gD) polypeptide.

  Here, the polypeptide is prepared by expressing a recombinant DNA construct in a mammalian cell, and the polypeptide is used when the vaccine is administered to an individual after primary infection with HSV. Is present in an amount effective to prevent recurrence 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 is
a. An immunologically active herpes simplex virus glycoprotein B (gB) polypeptide; or b. It contains an immunologically active herpes simplex virus glycoprotein D (gD) polypeptide.

  Here, the polypeptide is obtained 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. Is present in an amount effective to prevent recurrence of HSV-induced disease in the individual infected with HSV.

  According to the present invention, a recombinant herpes simplex virus produced in a host cell containing the isolated nucleotide sequence according to FIGS. 4 to 7 encoding herpes simplex virus (HSV) glycoprotein type B1 (gB1). (HSV) glycoprotein B (gB) polypeptides, immunologically equivalent fragments thereof, or mixtures thereof are provided.

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

According to the present invention, a recombinant herpes simplex virus (HSV) produced in a host cell containing a nucleotide sequence encoding herpes simplex virus (HSV) glycoprotein B (gB) and its precursors, or analogs thereof, is provided. A) a glycoprotein B (gB) polypeptide, an immunologically equivalent fragment thereof, or a mixture thereof:
The nucleotide sequence is
i) the amino acid sequence according to FIGS.
ii) an immunological equivalent of the amino acid sequence,
A recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide, an immunologically equivalent fragment thereof, or a mixture thereof, comprising a nucleotide sequence encoding

  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 a mixture thereof A vaccine is provided that is effective in preventing recurrent herpes simplex virus (HSV) -induced disease, including:

  According to the present invention, an anti-herpes simplex virus containing 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 a virus (HSV) glycoprotein B (gB) antibody and an anti-herpes simplex virus (HSV) glycoprotein D (gD) antibody, wherein the antibody comprises the 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) an isolated nucleotide sequence having a nucleotide acid sequence encoding herpes simplex virus (HSV) glycoprotein B (gB) as described above:
(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.

  ADVANTAGE OF THE INVENTION By this invention, it became possible to manufacture the vaccine effective in preventing recurrent herpes simplex virus-induced disease using the isolated polynucleotide encoding a glycoprotein.

(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" are intended to include such fragments, precursors, and analogs. Recombinant gB and gD polypeptides are produced in eukaryotic cells, preferably yeast or mammalian cells, most preferably mammalian cells. The length of the fragment is at least about 15 amino acids, preferably at least about 30 amino acids. A 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 gB and gD polypeptide mixtures can be used alone, but are usually used with a physiologically and pharmacologically acceptable medium, generally water, saline, phosphate buffer, sugar and the like. In addition, the polypeptide mixtures can be used with physiologically acceptable adjuvants such as aluminum hydroxide, muramyl dipeptide derivatives, and the like. Various adjuvants are effective, as shown in Example 6.3. The choice of adjuvant will depend, 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 humans, the adjuvant will 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. The vaccine 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 neutralization of the virus and antibody-dependent cellular cytotoxicity (ADCC). NorrildB. 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. Corey, L. and Spear, P.G., 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, S.W., and Zaia, J.A., 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 less than about 5,000 daltons (eg, 1,500-5,000 daltons), modifications are required to induce the desired immune response. Is done. Smaller haptens must be conjugated to a suitable immunogenic carrier (eg, tetanus toxoid, etc.).

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

The total amount of recombinant gB and gD polypeptides used per dose will usually be about 0.1 μg to 2 mg, more usually about 0.5 μg to 1 mg, and especially about 0.5 μg / kg of host body weight. 1010 μg. The ratio of gB to gD in the vaccine is usually about 0.1: 1 to 10: 1, more usually about 0.5: 1 to 10: 1, and preferably about 0.5: 1 to 5: 1. A single administration 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. Furthermore, the data in Example 6.4 show that vaccination significantly reduces disease recurrence during the acute phase of the disease, ie, when the individual shows HSV-induced pathology to the body.
(Recombinant glycoprotein B)
The preparation of a recombinant gB polypeptide is described in detail in International Application No. WO85 / 04587, International Application No. PCT / US85 / 00587, published October 24, 1985. 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 particular codons in a group of proteins. These proteins are, for example, glycolytic proteins and account for a high proportion of all proteins of a specific host (for example, yeast). In some cases, 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. Furthermore, the amino acid sequence of gB2 differs from the amino acid sequence of gB1 by 20%, whereas the other strains of HSV-1 or HSV-2 have the same or similar gB glycoprotein as the gB1 Patton strain or gB2333 strain, respectively. have. 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. It differs from the 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 of variable polarity that extends to about amino acid 726; a second hydrophobic region that extends from the region of variable polarity to about amino acid 795; and extends to the C-terminus of amino acid 904 , A second region where the polarity can change.

  Since gB is a membrane glycoprotein, by analogy with other glycoproteins, the first hydrophobic region is considered to be a signal leader sequence that controls secretion and / or membrane arrangement. Thus, the first sequence 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 may serve as a transmembrane integrator sequence (often called an "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 cloned by inserting the polynucleotide sequence into an appropriate expression vector and introducing the resulting expression construct into a compatible host. And can be expressed. The encoding fragment is less than about 0.1 map units, usually less than about 0.05 map units; where 1.0 map units 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. The expression vector then secretes or excretes the polypeptide of interest, or retains 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 secretory leader sequences can be used, depending on the host. The processing signal that cleaves the leader sequence can be a natural signal, or a signal associated with a non-natural secretory leader sequence, or both in tandem.

To obtain the polynucleotide sequence encoding gB1 Patton, the location of the gB1 coding sequence was mapped on EcoRI restriction fragment F. Three subfragments of the F fragment were isolated and subcloned into pBR322 (FIG. 2). The DNA fragments of these subclones were then used as probes to Northern blots of poly A ⁺ mRNA isolated from cells infected with HSV-1. A fragment that hybridized to mRNA of the expected size for gB was 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 gB1-encoding fragment is now handled in a variety of ways, including restriction mapping and nucleotide sequence determination, confirming 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 inserted into a suitable expression vector with the transcriptional and translational signals at the appropriate locations. This can be accomplished by filling overhangs and performing blunt end ligation using adapters and the like.

  It is of particular interest to introduce the gene in tandem with respect to the gene having the amplification ability. 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 the appropriate biotoxin to a level that will select for amplification of the particular gene. These cells are then cultured and expanded until they effectively produce the desired polypeptide.

In accordance with the procedures described above, the polynucleotide sequence encoding gB2 from HSV-2333 strain is isolated, cloned, and engineered to give a construct, both precursor and mature. As a result, it can be expressed in one or more hosts. In view of the utility of the fragments encoding gB1 Patton, these fragments are either probes for positioning gB2 encoding the DNA portion to specific HSV-2 restriction fragment clones or for isolating gB2 mRNA from infected host cells. Also used as For convenience, multiple probes encoding different regions of the gB1 gene are used. Either a positive DNA fragment or abundant mRNA having a size approximately appropriate for hybridizing 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)
The preparation of recombinant gD1 is described in detail in International Application No. WO85 / 04587, International Application No. PCT / US85 / 00587, published October 24, 1985. Materials and methods used to produce recombinant gD polypeptides are briefly described below. A detailed description of the preparation of recombinant gD2 is given in the examples below.

  Polypeptides that immunologically cross-react with naturally occurring glycoprotein D are produced by recombinant DNA methodology, for example, in eukaryotic hosts such as yeast and mammalian cells (eg, CHO cells). 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, providing an effective hapten to induce a gD-specific immune response.

  The gD DNA fragment can be of natural or synthetic origin. The native gD gene of HSV-1 is located on the viral genome between a short internal repeat (IRs) sequence and a short terminal repeat (TRs) sequence at its 3 'end. 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 located in the HindIII-NruI fragment of the 2.9 kbp SacI fragment. The naturally occurring gD gene may or may not be modified. Regions of the gene can be deleted as desired and / or ligated to other DNA fragments. The gDDNA 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 the recombinant protein, Section 2 describes the preparation of recombinant gB1; Section 3 describes the preparation of recombinant gB2; Section describes the preparation of recombinant gD1; Section 5 describes the preparation of recombinant gD2; Section 6 describes the study of vaccines using a mixture of gB and gD polypeptides.

(1. Material and method)
Live stocks of HSV-1 Patton and HSV-2 333 strains were obtained from Dr. Richard Hyman (Hershey Medical Center, Hershey, Pennsylvania). These viruses can be propagated in Vero cells obtained from Dr. Evelyn Linnette (Viro Labs, Emeryville, California) or American Type Tissue Culture Laboratory. This propagation is performed according to standard procedures. The library of the HSV-1 Patton EcoRI DNA fragment (Kudler et al., Virology (1983) 124: 86-99) cloned into the EcoRI site of plasmid pACYC184 (Chang and Cohen, J. Bacteriology (1978) 134: 1141) , Dr. Hyman, or can be prepared independently by a convenient method. Two HSV-2 333 clones, HindIII fragments H and L inserted into the HindIII site of pBR322 (Sutcliffe, Nucleic Acids Research (1978) 5: 2731) can also be obtained from Dr. Hyman.

The dhfr-deficient CHO cell line was obtained from Dr. YWKan (University of California, San Francisco). This cell line was originally described by Urlaub and Chasin (Proc. Natl. Acad. Sci. USA (1980) 77: 4216-4220). Under non-selective conditions, these cells were grown in Ham's F-12 medium (obtained from Gibco, cat. No.) Supplemented with 10% fetal calf serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 150 μg / ml L-proline. .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, a concentrated MTX stock medium was prepared from MTX obtained from Lederle and added to the above DME selection medium immediately before use.
(1.1 Cloning)
All DNA manipulations were performed according to standard procedures. Maniatis et al., Molecular
See Cloning, CSH (1982). Restriction enzymes, T4 DNA ligase, Escherichia coli DNA polymerase I Klenow fragment, and other biological reagents were purchased from Bethesda Research Laboratories or other indicated commercial suppliers and used according to the manufacturer's instructions. Double-stranded DNA was separated on a 1% agarose gel and isolated by electroelution.
(1.2 RNA isolation, Northern blot analysis, and hybrid selective translation)
Total RNA was prepared from Vero cells 6 hours after infection with HSV-1 or HSV-2 at a multiplicity of 10 viruses per cell. Cell monolayers were washed, incubated with extraction buffer, and processed as described by Pachl et al. (Cell (1983) 33: 335-344). Poly A ⁺ RNA was passed through a 3 ml column of oligo dT cellulose (obtained from Collaborative Research) in 500 mM NaCl, 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 0.1% SDS, passing 2 mg of total RNA, 100mM NaCl, 10mM Tris
Prepared by washing the column with HCl (pH 7.5), 1 mM EDTA, 0.1% SDS, and eluting the poly A ⁺ fraction with 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 0.1% SDS.

To perform Northern blot analysis, poly A ⁺ RNA was denatured with glyoxal (McMaster et al., Proc. Natl. Acad. Sci. USA (1977) 74: 4835-4838) and fractionated by electrophoresis on a 1% agarose gel. And transferred to nitrocellulose paper (Thomas, supra (1980) 77: 5201-5205) and hybridized with a ³² P-labeled probe.

Details of the method used for the hybrid selection translation method have been described previously (Pachl et al., Cell (1983) 33: 335-344). DNA filters were prepared using either 3 μg of a 3.5 kb Xho-Kpn fragment encoding gB or 2 μg of a 3.0 kb SstI-SstI fragment encoding HSV-1 gD. The filters were incubated with 40 μg of poly A ⁺ RNA from cells infected with HSV-1, eluted bound RNA and purified with a reticulocyte cell-free system (Pachl et al., J. Virol. (1983) 45: 133-139). Translated. Translation products were analyzed on a 12.5% SDS polyacrylamide gel (Laemmli, Nature (1970) 227: 689).
(1.3 DNA transfection)
Transformation of COS 7 cells (Gluzman, Cell (1981) 23: 175-182) or dhfr-deficient CHO cells (Urlaub and Chasin (1980) supra) were performed except for omitting carrier DNA, except for vander Eb and Graham ( Methods in Enz. (1980) 65: 826-839) was performed using a procedure modified by Parker and Stark (J. of Virol. (1979) 31: 306-369). 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 KCl, 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 cells that had grown on a 10 cm plate to 50% confluency. After 6-8 hours, the medium containing DNA was removed and cells were incubated with 15% glycerol-1 × HBS for 4 minutes. The cells were then grown on 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, washed once with PBS (0.14 M NaCl 2.7 mM KCl, 15.3 mM Na ₂ HPO ₄ ), and then methionine was removed. In 0.5 ml of labeling medium DEM (Dulbecco's modified Eagle's medium from Gibco, cat. No. 188G), 1% dialyzed fetal calf serum and 400 μCi / ml ³⁵ S-methionine (> 1000 μCi / mmol). Was added to each plate. These cells were incubated at 37 ° C. for an appropriate time. At the end of the labeling period, the medium was removed and the monolayer was washed once with PBS. As a "cold" methionine chase, the labeling medium was replaced with DME containing 2.5 mM methionine. To perform immunoprecipitation, cells were washed with 0.1 ml of cell solution buffer (20 mM Tris-HCl (pH 8), 100 mM NaCl, 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). Cell lysates were scraped into tubes, vortexed briefly and then left at 4 ° C. for 5-10 minutes. Cell debris was removed by centrifugation and clarified lysates were stored at -70 ° C.

For immunoprecipitation, 0.1 ml of cell lysate is precleared by incubating with normal serum for 30 minutes at 4 ° C., followed by addition of 50 μl of a 20% solution of protein A sepharose (PAS) in lysis buffer. Incubation was continued at 4 ° C. 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 (obtained from Dr. John Oakes of University of South Alabama). added. When using the F3AB antibody, 0.1% SDS was omitted from the lysis buffer. After 30 minutes at 4 ° C., 75 μl of PAS was added and incubated as 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). Immunoprecipitated proteins were dissociated from PAS by boiling in SDS sample buffer and then analyzed on a 12% polyacrylamide gel. To immunoprecipitate the labeled protein from the cell culture medium, the medium was first clarified by centrifugation and then 1/10 volume of 10-fold lysis buffer was added, causing the protein to precipitate as described above.
(1.5 immunofluorescence)
To analyze gB or gD expression in COS cells or CHO clones, cells grown in slide wells were washed three times with PBS, fixed with 100% methanol at -20 ° C for 10 minutes, followed by The plate was further washed three times with PBS, and once with PBS supplemented with 5% goat serum (GS). The fixed cells were then incubated with a primary antibody (HSV-1 or HSV-2 polyclonal antibody diluted 1/100 in PBS-5% GS) at 37 ° C. for 30 minutes. The cells were then washed three times with PBS-5% GS and incubated at 37 ° C. with a secondary antibody (FITC-conjugated goat anti-rabbit IgG (Capel) diluted 1/10 in PBS-5% GS) at 37 ° C. After washing 4 times with PBS-5% GS, slides were mounted with a cover piece using 50% glycerol-100 mM Tris (pH 8.0) and observed with a rights optical microscope equipped with fluorescence optics. 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. Previously, live cells were fixed in PBS containing 5% formaldehyde, and fluorescently stained cells were photographed using Kodac Ektachrome film (ASA400).
(1.6 ELISA analysis)
The gB protein concentration in the medium in which the CHO cells were cultured was measured by indirect enzyme-linked immunosorbent assay (ELISA) using a purified recombinant gB preparation as a standard. 50 μl of F3AB antibody diluted 1: 1000 in PBS was adsorbed to the wells of a 96-well polyvinyl chloride plate (DynatechLaboratories, Inc.) by incubating at room temperature for 1 hour. Excess antibody was removed by washing three times with PBS-5% GS. 50 μl of media sample or gB protein standard diluted in PBS-1% GS was added to the wells and incubated for 1 hour at room temperature. The plate was then washed three times with PBS + 1% GS, followed by a third incubation with 50 μl rabbit anti-HSV-1 polyclonal antibody (obtained from DAKO) diluted 1: 1000 with the same buffer. Excess secondary antibody was removed by washing three times with PBS + 1% GS. Finally, 50 μl of horseradish peroxidase-conjugated goat anti-rabbit antibody (Boehringer Mannheim) diluted 1: 500 in PBS-1% GS was added to each well and incubated for 1 hour, then the wells were washed once with PBS + 1% GS. After washing, followed by 8 washes with PBS, 2,2'-azido-di [3-ethylbenz-thioazoline sulfonate] (Boehringer Mannheim) at a concentration of 1 mg / ml was added to 0.1 M citric acid (pH 4.0). And 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 gD protein was determined using the gD-specific monoclonal antibody 8D2 (Rector et al., Infect. and Immun. (1982) 38: 168-174) in place of purified recombinant gD and F3AB as standards. Was measured in the same manner.
(2. sugar protein)
(2.1 Isolation, cloning and characterization of gB1 gene)
In order to isolate the gene corresponding to glycoprotein gB1, DNA spanning the coordinates 0.345-0.40 of the EcoRIF restriction fragment map of the HSV-1 Patton strain (Patton; Skare and Summers, Virology (1977) 76: 581-595). The fragment was subcloned into plasmid pBR322. These fragments were prepared from appropriate restriction digests of the EcoRI region of plasmid pACY184 and separated by electrophoresis on a 1% agarose gel using TAE buffer (0.04M Tris-acetic acid, 0.002M EDTA). 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 HSV-1 genome is shown in FIG. 1, and a more detailed map of the subcloned region is shown in FIG. Referring to FIG. 1, a conventional map is shown in the first two rows (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 with EcoRI fragment F indicated by hatching (Skare and Summers, 1977; Roizman, 1979). The HindIII restriction enzyme cut map of HSV-2 is shown on line 4 with the HindIII fragment H boxed (Roizman, 1979). One map unit corresponds to 98.9 megadaltons or 148.9 kbp of HSV-1 DNA and 105.6 megadaltons or 160.5 kbp of HSV-2 DNA.

  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 (DeLucca 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 subclones of the three plasmids containing the gB1 coding region. These are pHS106 ranging from a BamHI site of 0.345 to a SalI site of 0.360; pHS107 ranging from a SalI site of 0.36 to a SalI site of 0.388; and a BamHI fragment ranging from 0.345 to 0.40 map units. Line III shows the three probes used to map gB1 mRNA; line IV shows the fragments used for hybrid selection; and line V shows these used to determine the location of the gB2 gene. (See below). Additional restriction enzyme cleavage 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 HSV-1 infected Vero cells was shown on detailed maps isolated from pHS106 and pHS107. DNA fragments were used as probes. When a 0.5 kb PstI-SalI fragment isolated from pHS106 was used as a probe for HSV-1, the major species detected was 3 kb mRNA. When a similar blotting was carried out using a 0.49 kb NcoI fragment located about 1 kb upstream of the PstI-SalI 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 ′), which means that only the 5′-3 ′ strand of the PstI-SalI and NcoI-NcoI fragments hybridizes to 3 kbgB1 mRNA. Is proved by

Hybrid selective translation was performed by hybridizing HSV-1 poly A ⁺ mRNA with a 3.2 kb KpnI-XhoI fragment (including the region designated as gB1 coding region). When the bound mRNA was eluted and translated in vitro, a 100 kd protein similar in size to gB1 from HSV-1 infected Vero cells was detected. The identification of this 100 kd protein was confirmed by immunoprecipitation with a gB1-specific monoclonal antibody. Hybrid selection using the KpnI-XhoI fragment also detected some other proteins, probably as a result of non-specific hybridization of mRNA that occurred due to the high G + C content of DNA. When the same RNA was selected using a 3.0 kb SstI-SstI DNA fragment encoding HSV-1 glycoprotein gD, a similar protein pattern was observed, but no 100 kd gB protein was detected. This result suggests that gB is specific for the KpnI-XhoI fragment.

FIG. 3 shows a restriction map of a 3.95 kb DNA fragment from the BamHI restriction site of 0.345 map units to the XhoI site of 0.373 map units. 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. The DNA sequence from the BamHI site to the 3,640th non-unique AluI site is shown, with the AluI site shown in (A). The restriction sites shown are B, BamHI; B1, BalI;
Bs, BstEII; K, KpnI; Nc, NcoI; P, PstI; Pv, PvuII; S, SalI; Sc, SacI; X, XhoI; Restriction enzyme cleavage sites from the AluI site on the right to the terminal XhoI site are not shown. The potential glycosylation sites of the produced gB1 protein and the hydrophobic anchor and signal regions (filled box) are indicated.

  The DNA sequence from the BamHI site to the non-unique AluI site at nucleotide position 3640 was determined using Sanger's 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. Figures 4-7 show the DNA sequence of gBl (line 3); the predicted amino acid sequence for gBl is shown below the DNA sequence (line 4).

  The amino acid sequence and the DNA sequence corresponding to gB1 shown in FIGS. 4 to 7 are different from the sequence first shown in Table 1 of International Patent Publication No. WO85 / 04587 (published on October 24, 1985). It should be noted that The DNA sequence in Table 1 contains an error, and a nucleotide (G) is added at position 607 of the sequence. This nucleotide has been deleted in FIGS. 4 to 7 and this figure shows the correct 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, resulting in the sequences shown in Table 1 above. Is not correct. FIGS. 4-7 show the amino acid sequences based on the correct DNA sequence; the amino acid sequences of FIGS. 4-7 have been confirmed by amino acid sequencing of the N-terminal region of gB1. 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.

  Primer extension with a 22 bp oligonucleotide (residues 473-494) 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 sequence described is characteristic of a membrane protein. There is a highly hydrophobic region ranging from amino acid residues 726 to 795 near the carboxy terminus, where 69 amino acids can bind to the membrane. The first 30 amino acids at the N-terminus 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. A hydrophobic region near the C-terminus can act as a transmembrane integration sequence for binding proteins to cell membranes.

The sequence data shows that there are nine possible N-linked glycosylation sites as defined by the asn-X-thr / ser sequence (see also FIG. 3) in the hydrophilic ectodomain. It also suggests. 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 It becomes.
(2.2 Expression of gB1 in mammalian cells)
In order to express gB1 in mammalian cells, CHO cells lacking dhfr were transformed with plasmids pHS112 and pHS114. This transformation was performed using the calcium precipitation method as described in Materials and Methods. The E. coli HB101 strain transformed with plasmids pHS112 and pHS114 has been deposited with the ATCC and has been deposited under accession numbers 39650 and 39651, respectively. The construction of these strains is described in International Patent Publication No. WO85 / 04587 (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 number of clones have been isolated and shown to produce gB by immunofluorescence and radioimmunoprecipitation using HSV-1 polyclonal antibodies or monoclonal antibodies specific for gB. Three cell clones, pHS112-1, pHS112-9, and pHS112-23 were isolated, and the clones synthesized intact, complete gB protein in the cell. GB produced in these cells is considered to be glycosylated. Because, after pulse labeling for 1 hour, followed by 5 hours of chasing, a larger molecular weight form could be detected compared to unchaseed cells, and about 10% of gB was secreted into the medium. . Five cell clones expressing incomplete gB (pHS114-5, pHS114-6, pHS114-7, pHS114-11 and pHS114-12) were also analyzed, again showing that they secrete some gB into the medium. Was done. One of these cell lines, pHS114-7, was selected for further amplification with MTX. Clones were initially selected at 0.01, 0.05, 0.10 and 0.3 μMMTX. Three clones that synthesized high levels of gB as detected by immunofluorescence were isolated from those selected with 0.3 μM MTX. By radioimmunoprecipitation, these clones, pHS114-0.3 μM-6, 23, and 25, were 2-3 times more abundant than non-amplified clone, pHS114-7, during 1 hour of labeling with ³⁵ S-methionine. Synthesize gB. Pulse chase experiments indicate that at least 8% of the gB synthesized in these clones during the 1 hour pulse is extracellularly secreted 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 was constructed by digesting pHS108 (described in section 2.1) with XhoI 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-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 clones were screened for a plasmid with the gB1 insert inserted in the appropriate orientation.

To obtain expression, pHS137 was introduced into dhfr-deficient CHO cells together with plasmid pADdhfr and transformed. 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 6.91 +/- 1.53 µg / ml gB1 per 1-3 x 10 ⁷ cells in 24 hours. Produced protein.
(3. sugar protein B2)
The isolation, characterization, and cloning of the gB2 gene is described in International Patent Publication No. WO85 / 04857, supra.
(3.1 Expression of gB2 in mammalian cells)
Expression of HSV-2 glycoprotein gB is performed in COS cells (transient expression) by transformation with pHS210 alone or by co-transformation with pHS210. This second plasmid contains dhfr.

  Plasmid pHS210 was constructed as follows; the entire gB2 gene was subcloned into pBR322 as a 3.8 kb NruI-BamHI fragment to create pHS208. See FIG. 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. Next, a 1.9 kb fragment from HindIII to PvuII was inserted into pSV1 obtained by digesting pSV1 / dhfr with HindIII and BglII. See FIG. 10; pSV1 / dhfr is described in PCT International Publication No. WO 85/04587. 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 a 1.9 kb HindIII- (PvuII) fragment was isolated by gel electrophoresis. Similarly, pSV1 / dhfr was digested with BglII, repaired to blunt ends, digested with HindIII, and a 4.85 kb HindIII- (BglII) vector fragment was isolated by gel electrophoresis. These two fragments (1.9 kb and 4.85 kb) were ligated to create an expression vector, pHS210 (FIG. 10).

  Plasmid pHS210 was used to directly transform COS cells. Expression was detected by immunofluorescence analysis using a gB-specific monoclonal antibody, F3AB, and a commercially available polyclonal antibody, HSV-2 antibody (DAKO), for primary antibody screening. 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 medium samples were added to the coated plates and bound gB2 was then detected using rabbit anti-HSV-2 polyclonal antibody (DAKO) followed by horseradish-conjugated goat anti-rabbit IgG.

Plasmid pHS210 for CHO cell transformation was used in a co-transformation protocol with a second plasmid containing dhfr as a selectable marker (FIG. 10). Subsequent transformation and growth in selective media 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 expected 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. The mature size, fully glycosylated gB2 secreted into the medium of clone pHS210 # 3-1 was somewhat smaller than the 100 kd gB1 secreted by pHS4-6. This is because the coding sequence corresponding to 94 amino acids contained in the gB1 plasmid was removed from pHS210.
(4. Glycoprotein D1)
(Construction of 4.1 gD1 mammalian expression vector)
A library of EcoRI fragments of the HSV-1 of Patton strain cloned into the EcoRI site of pBR322 was created by Dr. Richard Hyman, Hershey Medical Center, Hershey, PA. The gD1 gene is completely contained in the 2.9 kb SacI fragment within the EcoRI fragment of clone H of this library. Clone H containing the 15 kb EcoRI insert was obtained from Dr. Hyman. This 2.9 kb fragment was purified by gel electrophoresis and then completely digested with HindIII and NcoI. The 5 'end of the gD gene, consisting of 74 bp of 5' untranslated sequence and 60 bp encoding the amino terminal 20 amino acids, was 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 commercially available from Pharmacia and P-L Biochemicals, and the resulting plasmid was named pHS131. Plasmid pHS131 was digested with HindIII, the 5 '4 bp overhang was filled in with Klenow polymerase, and then digested with SalI. The 1007 bp fragment containing the gD 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 encodes 315 amino acids of the gD1 protein, including a signal sequence of 25 amino acids out of a total of 399 amino acids of the complete protein. 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.

  Plasmid pSV7d was constructed as follows: A 400 bp BamHI / HindIII fragment containing the SV40 origin of replication and the early promoter was replaced with SVgtI (Mulligan, R. et al., J. Mol. Cell Biol. (1981) 1: 854-864. ) And purified. A 240 bp SV40 BclI / BamHI fragment containing the polyA addition site of SV40 was excised from pSV2 / dhfr (Subramani et al., J. Mol. Cell Biol. (1981) 1: 854-864) and purified. Link these fragments below:

And fused. This linker contains five restriction sites with stop codons in all three open reading frames. The resulting 670 bp fragment (including the SV40 origin of replication, the SV40 early promoter, the polylinker having a stop codon, and the SV40 polyadenylation site) was deleted by a pBR322 derivative (Lusky and Botchan, Cell (1984). ) 36: 391), which was cloned into the BamHI site of pML to obtain pSV6. The EcoRI and EcoRV sites in the pML sequence of pSV6 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. Bal31 excision removed one BamHI restriction site adjacent to the SV40 region about 200 bp away from the EcoRV site. PSV7a was digested with NruI to remove a second BamHI site adjacent to the SV40 region. This enzyme cleaves pML sequences upstream of the origin of replication. This was recyclized by blunt end ligation to yield pSV7b.

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

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

(Expression of 4.2 gD1 in mammalian cells)
The gD1 expression of plasmid pHS132 has been demonstrated in a number of experiments. First, specific immunofluorescence was observed in COS7 cells after infection using the method described above and using commercially available HSV-1 rabbit serum (DAKO) for detection. 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 was purified from roller bottle culture medium of CHO cell line D64 by a series of steps: ammonium sulfate precipitation, immunoaffinity chromatography, and ultrafiltration. The affinity chromatography used was one in which the gD monoclonal antibody 8D2 described in Rector et al. (1982) (supra) was bound to cyanogen bromide-activated Sepharose 4B.
(5. Sugar protein D2)
(Construction of mammalian expression vector of 5.1 gD2)
A HindIII fragment of HSV-2 strain 333 was cloned into pBR322 by Dr. RichardHyman as shown in the literature (Kudler et al., Virology (1983) 124: 86-99). The gene for the glycoprotein gD2 has been mapped by Ruyechan et al., J. Virol. (1970) 29: 677-697 to a short specific region of the virus between 0.90 and 0.945 map units. The region occupied by the HindIIIL fragment is shown in the genome map of Roizman, B., Ann. Rev. Genet (1979) 13: 25-27. The DNA sequence of the gD2 gene has been reported by Watson, 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 was found to be present on the 2.4 kb XhoI fragment by Southern blotting the digestion product of the HindIIIL fragment with the 2.9 kb SacI fragment encoding gD1 as a probe. The map of the XhoI fragment and the location of the gD2 gene are shown in FIG. This 2.4 kb XhoI fragment was cloned into a pBR322 derivative vector containing an XhoI site to obtain 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. For its construction, pHS204 was cut with SmaI and BamHI, and two restriction fragments were isolated from the gel: a 250 bp SmaI fragment containing the 5 ′ end of the gene, containing 82 bp of 5 ′ untranslated sequence, and the interior of the gene. And a 746 bp SmaI-BamHI fragment flanking 3 ′. The mammalian cell expression vector pSV7d (described in Section 4.2) was cut 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 to the digested pSV7d, and those having the SmaI fragment in the correct orientation were selected from bacterial transformants to obtain the vector pHS211.

Plasmid pHS212, encoding 352 amino acids of gD2 and an additional 47 residues beyond the gene present in pHS211 was digested with HaeII of pHS204, and blunt-ended with Klenow polymerase and BamHI. Constructed by decomposition. 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 digested with BamHI. The large vector fragment (about 3.4 kb) was gel isolated and ligated to the 141 bp (HaeII) -BamHI fragment to obtain plasmid pHS212. The fusion of the gD2 sequence and the plasmid vector sequence at the 3 ′ end of the gene adds 27 codons of nonsense DNA to the 3 ′ end of the gD2 gene. To remove these nonsense sequences, plasmid pHS213 was constructed by partial digestion of pHS211 with SalI, gel isolation of the truncated plasmid, followed by blunting with Klenow polymerase and digestion with BamHI. . A 141 bp fragment of BamHI from (HaeII) of pHS204 was ligated to linearized pHS211 to obtain plasmid pHS213.
(5.2 Expression of gD2 in mammalian cells)
GD2 expression in mammalian cells was first analyzed for transient expression by transfecting COS7 cells with pHS211, pHS212 and pHS213. Expression of gD2 was determined by both immunofluorescence and capture ELISA analysis of the medium in which COS7 was cultured, using rabbit anti-HSV-2 antibody for immunofluorescence, and for capture antibody in ELISA. And 8D2 (Rector et al. (1982) supra) which is a gD-type common antibody.

Next, a permanent CHO cell line was established by transfecting with plasmids pHS211 or pHS213 with Addhfr, selecting for dhfr acquisition and screening by g ELISA for expression of gD2.
Ad-dhfr description:
A plasmid carrying the dhfr gene was constructed by fusing a major aerobic promoter from Adenovirus-2 (Ad-MLP, map unit 16-17.3) 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 pSV2-neo described in Southern and Berg, J. Mol. Appl. Genet. (1982) 1: 327-341. 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 used in Kauhman and Sharp, Molec. And Cell Biol., (1982)
2: functionally similar to the dhfr plasmid described in 1304-1319.
(6. Therapeutic treatment with gB-gD vaccine)
(6.1 Effect of recombinant HSV glycoprotein vaccine given after primary infection on recurrent herpes disease in guinea pigs)
Female Hartley guinea pigs were inoculated intravaginally on day 1 with 5 × 10 ⁵ pfu HSV-2 MS strain. The animals were treated on days 1-10 by adding acyclovir (5 mg / ml) to drinking water. 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 whole glycoprotein preparation (gP2) and immunized with a mixture of recombinant gB1 and gD1 (HSV-1 gB + gD) or untreated. The treatment groups are shown below.
Group Treatment Dose Adjuvant Route N
I None None None None 11
II HSV-1 gB + gD 25μg + 25μg Freund Footpad 11
III gP2 50μg Freund Footpad 11
IV Control adjuvant only None Freund Footpad 9
Animals were immunized on day 21 and further day 42 by injecting the vaccine into the hind footpad. 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, therefore these groups were pooled for analysis (control, n = 20).

  The results, shown in Table 1 and FIG. 14, indicate that vaccination with recombinant glycoprotein has a significant effect on the frequency of disease recurrence. In addition, the gB + gD combination is superior to the natural glycoprotein mixture.

  The rate of disease recurrence, measured by the number of days of disease occurring within a particular time period, is an assessment that considers 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 gBgD and gD-2 vaccinated animals. As shown in FIG. 15A, the rate of disease recurrence (days of disease per week) decreased in all groups as the evaluation period was further away from the initial infection, but the rate of reduction was lower in the vaccinated animals. Then it was big. 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 immunization. It seemed to be established.

(6.2 Effect of recombinant HSV glycoprotein vaccine administered after primary infection on host immune response)
The effect of post-infection glycoprotein administration on the host immune response is determined by measuring anti-HSV antibodies produced by infected animals before infection and after immunization with an HSV glycoprotein vaccine. Was.

  The animals were inoculated with the HSV-2 ms strain, treated with acyclovir, and treated with the HSV glycoprotein vaccine as described in section 6.1. Serum was collected from these animals on days 41 and 95. Anti-HSV antibodies in serum were measured by ELISA, essentially as described in Section 1.6, as described in Pachl, C. et al., Jof Virology (1987) 61: 315-325. 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 can be seen in Table 2, in untreated control animals, the titer of anti-HSV antibody was higher 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 the antibody titer by 1.4-7 fold, whereas treatment with the recombinant HSV-1gBgD vaccine increased the titer by 9 to 31 fold compared to control values. 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.

(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 vaccines. 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 (termed MTP-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 appear 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 35 μg of a mixture of gD1 and various adjuvant compositions administered via the footpad three times at three week intervals. One week after the second immunization, and 1, 5, 9 and 13 weeks after the third immunization, animals were bled and anti-gD titers were determined by ELISA as described in section 1.6. .

The results shown below suggest that MTP-PE is the most promising of the adjuvants tested. This is because MTP-PE constantly produces high anti-gD1 titers in experimental animals. The titers were not held as long as CFA, but these levels were comparable to those seen for CFA.
(6.3.1 Comparison between CFA and aluminum hydroxide)
Animals were immunized with a vaccine containing gD1 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.

(6.3.2 Comparison of CFA, nor-MDP, and MTP-PE)
Animals were immunized with a vaccine containing gD1 and any 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. The liposomes are made up of synthetic phosphatidylcholine, phosphatidylserine and MTP-PE (or MTP-PE and gD1) in suspension medium (sterile Ca ⁺⁺ and Mg ⁺⁺ salt-free, isotonic Dulbecco's buffer, pH 7.2). Was prepared by adding 175: 75: 1 to the mixture and stirring by vortexing. As seen in Table 4, immunization with the vaccine containing CFA resulted in the highest anti-gD1 mean titers. The titers obtained with nor-MDP ranged from 44 to 74% of the average titers obtained in the group immunized with CFA. The mean titers obtained with MTP-PE and exogenous gD1 were somewhat lower than those obtained with nor-MDP, ranging from 32 to 72% of those 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. According to other formulations of liposomes, the antigen could be taken up with even higher efficiency.

(6.3.3 Comparison of different formulations containing MTP-PE)
Animals were immunized with gD1 containing a vaccine formulated with MTP-PE in a high oil delivery system (squalene / arlacell) 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 can be seen in Table 5, the MTP-PE formulation was effective as an adjuvant, even when used as the only component in the 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. After the first blood draw, the MTP-PE low-oil formulation had higher titers than the high-oil delivery system (squalene / arlacell).

(6.4 Therapeutic study: Effect of adjuvant on vaccine effective for prevention of recurrent herpes disease in guinea pigs, administration site, and administration timing)
The gBgD vaccine contains 25 μg each of recombinant gB1 and gD1 purified to approximately 70-80% unity as judged by SDS-PAGE. The recombinant gB1 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. The preparation of pHS113 and pHS137 is described in Section 2.2. The gB protein was purified as described in Pachl et al., Jof Virology, 61: 315-325 (1987), which is described in Section 2.2. GD1 was prepared as described in section 4.2 except that 8D2 was used instead of C4D2 during purification by affinity chromatography.

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

  The 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.

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. The medical course of primary infection was followed and quantified by genital skin lesions as described in Stanberry et al., J Infec Dis (1987) 155: 914. After recovery from primary infection, animals were randomized to 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. Sick days are defined as the days when recurrent disease is observed, severe relapses are the days when more than one blister is observed, and episodes are days after the absence of lesions when new disease occurs 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. In addition, vaccines containing nor-MDP and administered IM appear to be as effective as vaccines containing CFA and administered in the footpad.

  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 was similar for vaccines containing nor-MDP. Furthermore, based on local reactivity, nor-MDP could be used in vaccines.

  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. 8, 15, or 21 days after the initial infection, the animals vaccinated with the gBgD vaccine containing CFA in the shortest time after vaginal inoculation were compared to untreated controls. The shortest duration of the disease, the lowest rate of severe relapse, and the lowest number of episodes. At 15 days post infection, the values obtained for the vaccinated animals were higher than those obtained for the group vaccinated 8 days later, and the group vaccinated 21 days later was higher than the group vaccinated 15 days later. This effect is also shown in FIG. FIG. 16 shows a graph of the number of recurrences daily after vaginal inoculation of the first vaccinated animal 8, 15, or 21 days after HSV-2 inoculation.

  The data in FIG. 16 was used to calculate the percentage reduction in disease that had recurred (see Section 6.1 for an explanation of the significance of the rate of disease recurrence). It is shown. 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. However, the first vaccination, 15 days after inoculation of the vagina with 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-induced disease 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.

  According to the present invention, there is provided a vaccine which is 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 in order to provide a clearer understanding of its contents, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. is there.

1 shows the physical maps of HSV-1 and HSV-2, the EcoRI cleavage map for the configuration of the prototype isomers, and the HindIII restriction enzyme cleavage map of HSV-2. 1 shows a restriction map of the gB1 coding region in the HSV-1 map. 3 is a restriction map of the gB1 coding region. 1 shows the DNA amino acid sequences of gB1 and gB2. FIG. 4 is a continuation of FIG. 4. It is a continuation of FIG. It is a continuation of FIG. 2 is a physical map of HSV-2, showing the gB2 coding region. 1 is a map showing some important features of plasmid pHS137. It is a restriction map of gB2. It is a flowchart regarding construction of pHS132 which is a mammalian expression vector of gD1. 2 is a physical map of HSV-2, showing the gD2 coding region. It is a flowchart regarding construction of a mammalian vector for gD2. FIG. 3 shows the effect of vaccination with recombinant gB-gD performed after primary infection on recurrent herpes disease. A shows the effect of immunization with herpesvirus glycoprotein on the rate of recurrent herpes infection. B represents the difference in weekly recurrence rate between control and sensitized guinea pigs. Fig. 2 is a graph showing the effect of administration time of gBgD vaccine on recurrence of herpes disease.

Claims (1)

Immunological analysis of recombinant herpes simplex virus (HSV) glycoprotein B (gB) polypeptide produced in a host cell containing the isolated nucleotide sequence encoding herpes simplex virus (HSV) glycoprotein (gB) Fragment equivalent to

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