JPH08266289A - Simple herpesvirus protein and vaccine containing it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the new subject protein comprising a nonglycosylated polypeptide of gD glycoprotein of herpes simplex virus(HSV) types-1 and -2 having a specific amino acid sequence, and useful for a vaccine or the like for protection against HSV infection.

SOLUTION: This new herpes simplex virus(HSV) protein comprising a nonglycosylated polypeptide of a gD glycoprotein of herpes simplex virus type-1 (HSV-1) having an amino acid sequence containing the amino acid sequence of formula I, a partial sequence of the HSV-1 polypeptide having at least one immunologically antigen-determining group, a nonglycosylated polypeptide of the gD glycoprotein of herpes simplex virus type-2 (HSV-2) of formula II, a partial sequence of the HSV-2 polypeptide having at least one immunologically antigen-determining group of the gD glycoprotein, and usable for an immunogen for producing vaccine for the protection of HSV infection and antibody for neutralizing both of the HSV-1 and the HSV-2.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

1. FIELD OF THE INVENTION The present invention relates to a protein related to herpes simplex virus (HSV) glycoprotein, a method for producing the same and a vaccine containing the same, and a novel DNA sequence, a plasmid and a microorganism producing the protein (eukaryote and Both methods and compositions for making and using prokaryotes).

The present invention relates to viral DNA, plasmid D
Utilizing recombinant DNA technology to insert a DNA sequence encoding glycoprotein D (gD) or a portion thereof into a DNA vector such as NA or bacteriophage DNA, whereby the vector is a bacterial host. Alternatively, it is capable of replicating the gD gene and inducing its expression in other single cell lines. When the obtained recombinant DNA molecule is introduced into a host cell, gD or a part thereof or a mutant molecule can be produced by the host cell. The protein produced was then isolated and purified, and HSV type 1 (HSV-
1) and modified for use as an immunogen for vaccines against both type 2 (HSV-2) infections.

[0003]

[Prior art]

2. BACKGROUND OF THE INVENTION 2.1. Recombinant DNA Technology and Gene Expression Recombinant DNA technology inserts a specific DNA sequence into a DNA vector to produce a recombinant DNA molecule capable of replicating in a host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vector. That is, the inserted DNA sequence and the DNA vector are derived from organisms having no exchange of genetic information in nature, or the inserted DNA sequence is wholly or partially synthesized. Recently, recombinant DNA
Several general methods have been developed that allow the construction of molecules. For example, US Pat. No. 4,237,224 (Co
(Hen & Boyer) describe making such recombinant plasmids using a method known as restriction enzymes and ligation (ligation). These recombinant plasmids are then introduced into the unicellular organism by transformation and replicated. U.S. Pat. No. 4,23
Because of the general availability of the technology described in 7,224, this patent is incorporated herein by reference.

Other methods of introducing recombinant DNA molecules into unicellular organisms are described by Collins and Hohn in US Pat. No. 4,304,863, which is also incorporated herein by reference. This method utilizes a packaging / transduction system that uses bacteriophage vectors. Regardless of the method used for construction, the recombinant DNA molecule should be compatible with the host cell, ie it should be capable of autonomous replication in the host cell. The recombinant DNA molecule should also have a marker function that allows for selection of host cells transformed with the recombinant DNA molecule. Furthermore, a foreign gene will be properly expressed in transformed cells and their progeny if all of the appropriate replication, transcription and translation signals are correctly placed on the plasmid.

As is characteristic of all viruses that infect eukaryotic cells, herpes simplex virus requires a eukaryotic host cell line in which the viral genes are expressed in order to replicate its genome. , Its offspring arise. Signaling and regulatory elements for DNA replication in eukaryotic cells, gene expression and viral assembly differ from those of prokaryotic cells. This becomes very important when trying to express a gene that is naturally expressed only in eukaryotic cells in prokaryotic host cells.

These different gene signals and processing processes are associated with many levels of gene expression (eg,
DNA transcription and messenger RNA translation). Transcription of DNA relies on the presence of a promoter in the DNA sequence that directs the binding of RNA polymerase, thereby promoting transcription. The DNA sequence of eukaryotic promoters differs from that of prokaryotic promoters.
Furthermore, eukaryotic promoters and associated genetic signals are either unrecognized or non-functional in prokaryotic systems.

Similarly, the messenger RN in prokaryotes
Translation of A (mRNA) relies on the presence of appropriate prokaryotic signals distinct from eukaryotes. M in prokaryotes
Efficient translation of RNA requires a ribosome binding site on the mRNA called the Shine Dalgarno (SD) sequence. This sequence is a short nucleotide sequence of mRNA, which is the initiation codon (AU) encoding the amino-terminal methionine of the protein.
It is located in front of G). SD sequence is 16S rRNA
MRNA that is complementary to the 3'end of (ribosomal RNA), possibly forming a double helix with rRNA and allowing correct positioning of the ribosome.
Promotes binding to the ribosome. For a discussion on maximizing gene expression, see Roberts
and Lauer, Methods in Enzym
Seeology 68: 473 (1979).

Many factors complicate the expression of eukaryotic genes in prokaryotes, even after the proper signals have been inserted and properly positioned. A clear understanding of the nature and mechanism of action of these factors is currently lacking. One of these factors is Escherichia coli (Esche
Richia coli) and other bacteria are the presence of active proteolytic systems. This proteolytic system appears to selectively destroy "abnormal" or foreign proteins such as eukaryotic proteins. Thus, it would be versatile to develop means to protect eukaryotic proteins expressed in bacteria from hydrolysis. One strategy is to link the eukaryotic sequence to the prokaryotic gene in the inphase (ie, the correct reading frame), thereby linking the fusion protein product (the prokaryotic amino acid sequence with the foreign or eukaryotic amino acid sequence). Protein) that is a hybrid) is to be constructed.

The construction of hybrid genes was the method used in the molecular cloning of genes encoding many eukaryotic proteins such as somatostatin, rat proinsulin, growth hormone and egg albumin-like proteins. Furthermore, in the case of ovalbumin and β-globin, a prokaryotic promoter was linked to such a fusion gene sequence (Guarente).
Et al., Cell 20: 543 (1980)). Guar
The ente et al. system involves inserting the lac promoter containing the SD sequence at different distances in front of the fusion gene ATG. Molecular cloning and expression of several eukaryotic genes has been achieved, but this has not previously been done for the gD gene. Also, the expression of foreign or eukaryotic genes in prokaryotic host cells is not in the state of the art that can be routinely done.

2.2. There are three basic approaches to protection and treatment of vaccine virus infections: That is, (1) a vaccine that elicits an active immune response, (2) a chemotherapeutic agent that suppresses viral replication,
And (3) a drug that induces the synthesis of interferon. All three methods can be used to prevent infection, but are more effective when applied early in infection. Vaccination, or active immunization, is usually ineffective after the onset of infection.

Vaccines are generally prepared by detoxifying the virus without destroying its immunological properties. Traditionally, this is to inactivate the infectivity of the virus for inactivated vaccines (ie, "kill" the virus by treatment with various chemical agents such as formaldehyde) or live vaccines (attenuated vaccines). Non-toxic or attenuated (modified) for
Achieved by selecting the virus. Attenuation can be caused by adaptation of the virus to unusual conditions (different animal hosts and cell cultures) or by passage of a large viral inoculum with no virulence or no symptoms at all. Obtained by selecting mutants that show few signs. The few vaccines still used in the veterinary field consist of completely virulent infectious virus injected at sites where viral growth is of little concern. This method is considered too dangerous for human use.

Attenuated viruses used in live vaccines are generally good immunogens. This is because the attenuated virus grows in the host and elicits long lasting immunity. Attenuated vaccines induce humoral antibodies against all viral antigens (both surface and internal antigens of viral particles). However, many problems have been raised with the use of live vaccines, such as inadequate attenuation of the virus, genetic instability of the virus, contamination with foreign viruses in the cell culture used to grow the vaccine virus, And finally, vaccine instability (eg heat instability).

Inactivated vaccine ("dead" that does not grow)
Alternatively, killed virus does not grow in the body of the immunized animal, usually resulting in only antibodies to surface components. However, the main drawback with inactivated vaccines is that they produce enough virus to supply the required amount of relevant antigens. Other drawbacks associated with the use of inactivated vaccines are that additional immunity is often required due to the short immunity achieved and weakened immunogenicity as antigenic sites are altered by chemical treatment of the inactivation. , Or induce antibodies that are less effective at neutralizing viral infections, and that the vaccine often does not induce satisfactory levels of IgA.

The subunit vaccines contain only the necessary and appropriate immunogenic substances, such as the capsid protein of the icosahedral virus without envelope or the peplomer of the virus with envelope (spike of glycoprotein). Subunit vaccines can be made by isolating the relevant subunits from the highly purified viral fraction or by synthesizing the relevant polypeptides. The main advantages of subunit vaccines are:
Elimination of viral-derived genetic material as well as elimination of host or donor-derived interfering material. However, at present, the production of subunit vaccines using these methods is too expensive for widely used commercial applications. Recombinant DNA technology has provided much for the production of subunit vaccines, in which the molecular cloning and expression in host cells of the viral gene encoding the relevant immunogenic portion of the virus or its protein has been demonstrated in subunit vaccines. A sufficient amount of immunogen can be provided to be used.

Vaccines are often administered as an emulsion containing various adjuvants. Adjuvants help to obtain sustained high levels of immunity at lower doses with less antigen than when the immunogen alone was administered. The mechanism of action of adjuvants is complex and not fully understood. However, it may include phagocytosis of the reticuloendothelial system, stimulation of other activities, delayed release and degradation of antigen. Examples of adjuvants include Freund's adjuvant (complete or incomplete), adjuvant 65 (including falling oil, mannide monooleate and aluminum monostearate), and minerals such as aluminum hydroxide, aluminum phosphate or alum. There is a gel. Freund's adjuvant is no longer used in vaccines for humans and food animals. Because it contains non-metabolizable mineral oils and is a potential carcinogen. However, mineral gels are widely used in commercial veterinary vaccines.

2.3. Herpesviruses Herpesviruses (Herpetobiridae: Herper
toviridae) is a large DNA virus,
It is famous for establishing a potential infection that can persist for the life of the host. After the primary infection, which does not appear in the table, the virus
It remains quiescent until reactivated by any of several suspected stimuli, such as irradiation or immunosuppression.

The active state, ie the acute form of infection, is characterized by a productive or lytic infection. That is, the virus replicates, hijacks the host cell machinery, produces infectious mature viral particles, and causes cell death. However, in the latent state, the virus is lurking in the ganglia of the host. There is little evidence that infectious virus is produced in the latent state.

Most primary infections occur in childhood and are not superficial. Infection can result from virus inoculation of the mucous membranes or by virus entering the skin through epidermal rupture. Thus, infectious virus can be transmitted by close contact. Once acquired, HSV is retained throughout the body for life and the patient is asymptomatic or has, for example, cold sores (a disorder on the lips commonly referred to as "febrosis" or "cold sores"). , Gingivostomatitis (vesicles and gums covered with vesicles that rupture into ulcers), pharyngitis, tonsillitis, keratoconjunctivitis (keratitis or inflammation of the cornea of the eye, progresses to a dendritic ulcer, and finally (Causes scarring of the cornea, resulting in blindness)
And vulnerable to recurrent attacks leading to many clinical signs such as mucocutaneous diseases including genital herpes. In rare cases, H
SV infections can cause encephalitis, herpes zoster, traumatic herpes, hepatitis and neonatal herpes.

During active outbreak, the infectious virus can be isolated from the lesion site or from surrounding body fluids, such as saliva in the case of lip sores. These lesions are
It tends to occur in the same part of the body of a particular individual and is caused by many stimuli such as menstruation, overexposure to sunlight or cold wind, pituitary or adrenal hormones, allergic reactions, or classically fever. During these outbreaks, the patient is releasing the virus and can transmit the infectious virus to others through contact.

During the latency of infection, researchers have demonstrated that the virus is retained at sites other than where lesions subsequently occur. During this stationary phase, the virus is localized in neurons of the sensory ganglion (in the case of facial lesions,
It usually involves the trigeminal ganglion) and cannot be isolated from normal lesions. Most children recover rapidly from the primary infection, but at times can defeat newborns affected by the disseminated neonatal herpes infection characterized by hepatitis. The most severe infections are acquired from an infected attendant, and more commonly from an infected mother, at birth when the baby encounters genital herpes in the birth canal. Vulvar vaginitis and penile infections in women and children were previously thought to be caused by HSV-2, but recent results have shown that HSV-1
Or that HSV-2 may be the cause,
Furthermore, herpes venereal infections in women are associated with cervical cancer.

Herpes infections are common in today's society. Currently, there is no cure for HSV infection and recent treatments have used compounds that inhibit DNA synthesis. Of course, these compounds are viral DNA
It is non-selective in that it inhibits not only synthesis but also DNA synthesis in dividing cells. Furthermore, these compounds are effective only during active infections, and until now no latent effect has been demonstrated.

Herpesviruses are eukaryotic viruses and contain a linear double-stranded DNA genome ranging in size from 80 × 10 ^{6 to} 150 × 10 ⁶ Daltons. Each viral particle has an icosahedral nucleocapsid and a membrane envelope formed by separation from the lipid bilayer of the host cell during maturation and budding. The viral envelope is a lipid bilayer that contains many virus-specific proteins that are partly located in the membrane but project outward from the membrane envelope. During primary infection, the membrane envelope is required for viral particles to efficiently enter the cell. Antibodies that specifically bind to viral proteins outside the lipid bilayer are capable of neutralizing viral infection. The most important viral proteins for virus entry into cells are glycoproteins (protein molecules to which sugar molecules are bound). Herpes simplex virus type-1 (HSV-1) and type-2 (HS
V-2) makes at least four different glycoproteins that are antigenically distinct from each other. HSV-1 and HSV
Some of these proteins from -2 have a common epitope (ie, antigenic site or antibody binding site). An example of such a protein is 49,000-5
It is a glycoprotein of 8,000 daltons and is called gD. The multivalent antiserum against HSV-1 gD is HSV-
Ability to neutralize both 1 and HSV-2 infections
(Norrild, 1979, Current Top
ics in MiCrobiol. and Immu
nol. 19:67).

[0023]

[Problems to be Solved by the Invention]

3. SUMMARY OF THE INVENTION Methods and compositions are provided for cloning and expression of HSV glycoprotein genes in unicellular host organisms. In addition, by culturing these novel unicellular organisms, H
Methods for producing SV gene products and methods for purifying the gene products are described. Recombination D described here
The gD-related protein produced by NA technology is
It can be formulated for use as an immunogen in a vaccine to protect against SV-1 and HSV-2 infections.

The HSV-1 gD gene (isolated from the HSV-1 Patton strain) was identified in the viral genome by intertype recombination analysis and mRNA mapping. Once the gene was localized on a particular segment of DNA, its nucleotide sequence was determined and the amino acid sequence of the gD protein was deduced.

The HSV-2 gD gene (HSV-2 G
(Isolated from the strain) is gD in the HSV-1 genome.
By analogy with the localization of A., was also identified in the viral genome by hybridization analysis. The isolated gD gene or gene fragment (type 1 or type 2) was then inserted into a plasmid vector to create a recombinant plasmid that served as a biologically functional replication unit. These recombinant plasmids were constructed to facilitate replication and expression of the gD gene upon transformation of compatible host cells. Furthermore, these plasmids provide a one-step identification of transformed microorganisms that actively express the gD gene. Finally, methods for isolating the expressed gene product and for use in formulating vaccines are described.

[0026]

[Means for Solving the Problems]

4. DESCRIPTION OF THE DRAWINGS The invention will be more fully understood by reference to the following detailed description of the invention, examples of specific embodiments of the invention and the accompanying drawings. FIG. 1a shows the HSV-1 genome, and shows the position of a short unique region (Us) in the HSV-1 gD gene (hereinafter referred to as the gD-1 gene).

FIG. 1b shows the lambda gtWES :: EcoR.
Figure 3 shows a restriction map of the I-H HSV- 1 EcoRI-H fragment insert. Only the restriction sites relevant to the discussion are shown. The restriction enzyme recognition sequences are indicated by the following abbreviations: Bam
HI (Ba4 to Ba8); BglII (Bg2); Bs
tEII (Bs); EcoRI (RI); HindII
I (H3); KpnI (Kp); PvuII (Pv);
SacI (Sc); SalI (Sa); and XhoI
(Xh).

FIG. 1c shows the lambda gtW into pBR322.
ES: EcoRI-H BamHI-8 to BamHI
Construction of pRWF6 with insertion of fragments up to -7. FIG. 1d shows HSV-1 Sa of pSC30-4.
3 shows a restriction map of the cI DNA fragment insert. Bars (enlarged area) indicate the localization and location of the gD-1 mRNA coding sequence.

FIG. 2 shows the sequencing strategy and restriction map of the gD-1 gene sequence. Coding regions are indicated by bars (enlarged regions) and non-coding regions are indicated by solid lines (thin lines). The restriction endonuclease cleavage sites used for fragment isolation, labeling and secondary digestion are indicated. Horizontal arrows indicate the regions of the sequenced DNA, those above the restriction map indicate that the non-coding strand sequence was determined, and those below the restriction map indicate that the template strand sequence was determined. Show.

FIG. 3 shows the nucleotide sequence of the gD-1 gene and the deduced amino acid sequence of the gD-1 protein. The relevant restriction sites are indicated and gD-1
The numbered vertical arrows at the amino-coding end of the gD-to pJS413 in the following recombinant plasmids:
1 shows the ligation site at the amino code end: pEH51, p
EH60, pEH62, pEH66, pEH71, pE
H73 and pEH73 (which include the rest of the gD-1 sequence through its native TAG); and pEH4-
2, pEH82, pEH3-25 and pEH90-N
Series (these encode the Cro / gD-1 / β-galactosidase fusion protein). The numbered vertical arrows at the carboxy coding end of gD-1 are:
(1) The following recombinant plasmids: pEH4-2, pEH8
2. β-galactosidase gene (z gene) of pJS413 in pEH3-25; or (2) g to the z gene of pHK414 in the pEH90-N series of plasmids.
The connection site at the D-1 carboxy code end is shown.

FIG. 4 (not drawn to scale) shows
Part of gD-1 gene and expression vector pJ of E. coli
3 shows the construction of recombinant plasmid pEH25 derived from S413. Recombinant plasmid pEH25 results in the production of HSV-1 gD-related protein encoded by about 87% of the gD-1 coding sequence linked to the "leader" sequence (cro) derived from the expression vector.

FIG. 5 shows Cro / gD-1 in pEH25.
The DNA sequence and deduced amino acid sequence of the junction are shown. FIG. 6 shows inhibition of immunoprecipitation of the pEH25gD product by adding a competing antigen present in the lysate of HSV infected Hela cells. FIG. 7 (not drawn to scale) shows the construction of the gE-1 expression plasmid pEH4-2 derived from pEH25, which
The 3'terminal 205 nucleotides (69 amino acids) of the gD-1 coding sequence were deleted and replaced with approximately 3,000 nucleotides encoding the E. coli β-galactosidase protein. This recombinant plasmid is gD-1
A "sandwich" protein (ie, fusion protein) in which E. coli-related polypeptides (ie, Cro and β-galactosidase) are fused to each end of a particular protein
Bring about the production of.

FIG. 8 (not drawn to scale) shows
Many gD-1 expression plasmids pEH50 + x (each containing a variable portion at the amino coding end of the gD gene)
A method for manufacturing the is shown. FIG. 9 (not drawn to scale) shows gD-1 fused to β-galactosidase.
6 shows a method of reconstructing the amino coding end of the gD-1 gene in pEH4-2 so that the protein will be expressed by host cells transformed with pEH82.

FIG. 10 (not drawn to scale)
Is a gD derived from pEH3-25 and pHK414
1 shows the construction of expression plasmid pEH90-10am. Recombinant plasmid pEH90-10am is β in E. coli transformants containing amber suppressor tRNA.
-Allows production of both gD-1 fused or unfused to galactosidase.

FIG. 11a represents the HSV-2 genome and also shows the position of the BglII fragment within the short unique region (Us) where the HSV-2 gD gene (hereinafter referred to as the gD-2 gene) is located. B that defines the L fragment
The glII restriction site is designated Bg. FIG. 11b shows p
3 shows a restriction map of the BglII L fragment insert of HV1. Only relevant restriction endonuclease recognition sites are shown. Restriction enzyme recognition sites are indicated by the following abbreviations: Bam
HI (Ba); BglII (Bg); ClaI (C);
HindIII (H3); PvuII (Pv); Sal
I (Sa); SacI (Sc); SphI (Sp); and XhoI (Xh). The bar (enlarged area) is gD
-2 shows the localization and location of the mRNA coding sequence.

FIG. 11c shows HSV-2 Xh of pHV2.
3 shows a restriction map of the oI DNA fragment insert. FIG. 12 shows the nucleotide sequence of gD-2 gene and gD
2 shows the deduced amino acid sequence of the -2 protein. The relevant restriction sites are indicated and the numbered vertical arrows at the amino coding ends of the gD-2 gene indicate the recombinant plasmid pHV5 (including the entire carboxy coding end of gD-2) and pJS413 in pHV6. To gD-2
The ligation site at the amino code end is shown. The BamHI site is gD-2 to the z gene of pHK414 in pHV6.
It is the ligation site at the end of the carboxy code.

FIG. 13 shows a comparison of the deduced amino acid sequences of gD-1 and gD-2. Amino acid residues are represented by a single letter code. Amino acid residues homologous in gD-1 and gD-2 are indicated by a dash in the gD-2 sequence. g
The D-2 sequence is one amino acid residue shorter than the gD-1 sequence. Amino acids that are "deleted" in gD-2 are marked with an asterisk (*).

FIG. 14 (not drawn to scale)
Shows the construction of a recombinant plasmid pHV5 derived from part of the gD-2 gene and pJS413. Recombinant plasmid pHV5 results in the production of an HSV-2gD-related protein encoded by about 90% of the gD-2 coding sequence linked to a "leader" sequence (cro) derived from the expression vector.

FIG. 15 shows the gD- derived from pHV5.
2 shows the construction of the expression plasmid pHV6, in which the 3'terminal 259 nucleotides (86 amino acids) of the gD-2 coding sequence have been deleted, and the plasmid derived from the expression plasmid pHK414 encoding the β-galactosidase protein of E. coli It is replaced with 3,000 additional nucleotides. This recombinant plasmid pHV6 has gD-2
A "sandwich" protein (ie, fusion protein) in which an Escherichia coli-related polypeptide (ie, Cro and β-galactosidase) is fused to each end of a specific protein
Bring about the production of.

5. DESCRIPTION OF THE INVENTION The present invention relates to the use of recombinant DNA technology to produce polypeptides related to HSV proteins that can be used as immunogens in vaccine formulations.
More specifically, the production of gD-related proteins is described.
Multivalent antisera against HSV-1gD are HSV-1 and HS
Since it has the ability to neutralize both V-2, pure gD
The protein or antigenically important portion thereof is HSV-
1 and HSV-2 could be used in a subunit vaccine that could efficiently protect the recipient from primary infections.

Recombinant plasmids constructed as described herein are stable gD resistant to degradation in host cells.
It results in host cell production of proteins that are related polypeptides. Such plasmids allow large-scale production of gD-related proteins or fusion proteins containing the immunological antigenic determinants of gD. However, the DNA compositions described herein are not limited to the production of gD-related proteins and can be used to produce polypeptides related to HSV proteins. It will be readily appreciated that a wide variety of immunogens and vaccine formulations can be produced.

The method of the present invention can be divided into the following steps for purposes of illustration. (1) identification and isolation of the HSV glycoprotein gene or gene fragment,
(2) Insertion of the gene or gene fragment into a cloning expression vector for producing a recombinant DNA molecule used for transformation of a unicellular organism, (3) Transformed single cell capable of replicating and expressing the gene Measurement of the immunopotency of a gene product by assessing its ability to elicit organism identification and growth, (4) identification and purification of the gene product of interest, and (5) neutralizing antibody production. For clarity purposes, all methods are discussed with respect to the gD gene.
However, the same techniques can be used similarly to similarly produce polypeptides related to HSV glycoproteins.

5.1. Identification of HSV glycoprotein gene
And the isolated HSV glycoprotein (gD) can be obtained from strains of HSV type 1 or type 2. Isolation of the gD gene (or a portion thereof) is carried out by first isolating HSV DNA, producing an HSV DNA fragment,
And identifying a fragment containing the glycoprotein gene sequence. Prior to identification, the HSV DNA fragment is usually ligated into a cloning vector, which vector is used to transform host cells. This allows the production of multiple copy number HSV DNA fragments, which is sufficient for analysis and identification operations.

The HSV DNA may be (1) directly isolated from the virus purified from a virus-infected eukaryotic cell, or (2) a bacterium containing a part of the viral genome containing the gD gene. It can be obtained from a phage or a plasmid. HSV D
To make an NA fragment, the DNA is cleaved with a restriction enzyme at a specific position, the DNA is fragmented using DNase in the presence of manganese, or
A is physically crushed, for example by sonication. The linear DNA fragments can then be separated by size by standard techniques including, but not limited to, agarose or polyacrylamide gel electrophoresis, column chromatography.

Any restriction enzyme or combination of restriction enzymes can be used to produce an HSV DNA fragment containing the gD sequence, provided that the enzyme does not destroy the immunocompetence of the gD gene product. For example, the antigenic site of a protein can consist of about 7-14 amino acids. Thus, a gD-sized protein can have many different antigenic sites, probably considering thousands of antigenic sites, given overlapping sequences, secondary structure, and processing processes such as acetylation, glycosylation, and phosphorylation. It seems that there is. Therefore, many partial gD gene sequences could encode antigenic sites. As a result, a large number of restriction enzyme combinations can be used to generate DNA fragments which, when inserted into an appropriate vector, give a gD-specific amino acid sequence with a different antigenic determinant to a unicellular organism. Could be produced in.

Transformation of host cells with these recombinant DNA molecules containing HSV DNA fragments allows the production of high copy numbers of viral DNA, which is then analyzed to identify fragments containing the gD gene sequence. Can be done. H to cloning vector
Insertion of the SV DNA restriction fragment is readily accomplished when the cloning vector has complementary cohesive ends. However, if the complementary restriction sites used to fragment the HSV DNA are not present in the cloning vector, both ends of the HSV DNA or cloning vector can be modified. Such modifications include digesting single stranded DNA ends to blunt ends, or repairing single stranded ends to allow blunt end ligation. In addition, a desired end can be prepared by linking a nucleotide sequence (linker) to the DNA end, and the linked linker can include a chemically synthesized specific oligonucleotide encoding a restriction enzyme recognition sequence. According to another method, the cleaved vector and the HSV DNA fragment may be modified by homopolymer tailing (Morrow, 1979,
Methods in Enzymology 68: 3
checking).

The identification of a particular DNA fragment containing the gD gene can be done in a number of ways. First, the viral DNA fragment was sequenced (Maxam
& Gilbert, 1980, Methods in
Enzymology 65: 499), and it is then possible to identify fragments containing the gD gene sequence by comparing the deduced amino acid sequence with the amino acid sequence of the gD protein. Second, the fragment containing the gD gene can be identified by mRNA selection. HSV
The DNA fragment is used to isolate complementary mRNA by hybridization. The mRNA is identified by immunoprecipitation analysis of the in vitro translation product of the isolated mRNA and thus the complementary HSV DNA fragment containing the gD gene sequence. Finally, gD-specific mRNA can be selected by adsorbing polysomes isolated from HSV infected cells to immobilized monoclonal antibodies against gD. Radiolabeled gD mRNA or cDNA can be synthesized using the selected mRNA (from the adsorbed polysome) as a template. Then, using radiolabeled mRNA or cDNA as a probe, gD
HSV DNA fragments containing sequences can be identified (Southern, 1975, j. Mol.
Biol. 98: 503; Alwine et al., 1979,
Methods in Enzymology 68: 2
20).

Other methods for isolating the gD gene include, but are not limited to, chemically synthesizing the gene sequence itself (provided that the sequence is known), or gD gene It involves producing a cDNA (complementary DNA) for the encoding mRNA. To achieve this, gD-specific mRNA is isolated from virus-infected cells. The mRNA isolated by standard techniques is then converted to a cDNA copy and inserted directly into a cloning vector.

Isolated gene, cDNA or synthetic DN
Transforming a host cell with a recombinant DNA molecule containing an A sequence allows the production of high copy number genes. Therefore, the transformant is propagated, the recombinant DNA molecule is isolated from the transformant, and the isolated recombinant DNA is isolated.
The gene can be obtained in a microgram amount by recovering the inserted gene from. Microgram quantities of the gD gene can also be obtained from the gD gene source, eg, herpes simplex virus in infected animal cells, or bacteria or phage containing the viral gene in a recombinant plasmid. Viral or plasmid DNA is isolated, fragmented, and size-fractionated by the same methods used to localize the gene.

5.2. The HSV glycoprotein gene clone
Insertion into a Growning Expression Vector Once isolated, the gD gene or gene fragment is inserted into a suitable expression vector, ie, a vector containing the elements necessary for transcription and translation of the inserted gene (Roberts & Lauer). , 197
9, Methods in Enzymology, 6
8: 473). gD gene (or part of the gene)
In order to efficiently express Escherichia coli, a promoter must be present in the expression vector. RNA polymerase normally binds to a promoter to initiate transcription of a gene or group of linked genes and regulatory elements (called operons). Promoters vary in their "strength," or ability to promote transcription. For molecular cloning, it is desirable to use strong promoters to obtain high levels of transcription and thus high levels of gene expression. Depending on the host cell system used, one of many suitable promoters can be used. For example, when cloning into an E. coli host cell system, a promoter isolated from Taiyobacterium, its bacteriophage or plasmid can be used (eg: la
c promoter). In addition, the E. coli promoter produced by recombinant DNA or other synthetic DNA techniques may be used to drive transcription of the inserted gene. More particularly, P _R and P _L promoters of E. coli phage lambda dominates the high levels of transcription of adjacent DNA segments. Furthermore, the recA promoter from E. coli leads to high levels of gene transcription of flanking fragments.

Specific initiation signals are also required for efficient gene transcription and translation in prokaryotes and eukaryotes. These transcription and translation initiation signals vary in "strength" as measured by the amount of gene-specific messenger RNA and protein synthesized.
The DNA expression vector containing the promoter may further contain any combination of various "strong" transcription and / or translation initiation signals. Thus, the SD-ATG combination that can be utilized by the host cell ribosome is used. Such combinations include S from the cro gene or N gene of coliphage lambda or from the E. coli tryptophan E, D, C, B or A gene.
There are, but are not limited to, D-ATG combinations. In addition, SD-ATG combinations made by recombinant DNA or other synthetic techniques can also be used.

To link the promoter and other regulatory elements to a particular site within the vector, the methods described above for inserting the DNA fragment into the vector can be used. Thus, the gD gene (or part of it) is
It will be linked at a specific position to the promoter and regulatory elements of the expression vector so that the gD gene sequence is in the correct translational reading frame (ie, in phase) with respect to the vector ATG sequence.
Alternatively, gD ATG or synthetic ATG may be used. The resulting recombinant DNA molecule is then introduced into a suitable host cell by transformation (depending on the vector / host cell system), transduction or transfection. Transformants are selected on the basis of expression of the appropriate genetic marker normally present in the vector, eg pBR3
22 resistance to ampicillin or tetracycline, or thymidine kinase activity in eukaryotic host systems. Expression of such a marker protein is an indicator that the recombinant DNA molecule is intact and replicating. Expression vectors (which usually include a marker function) include the following vectors or derivatives thereof: SV40 and adenovirus vectors, yeast vectors, bacteriophage vectors (eg lambda g).
t-WES-Lambda B, Charon 28, Char
on 4A, Lambda gt-1-Lambda Bc, Lambda gt
-1-Lambda B, M13mp7, M13mp8, M13
mp9) or a plasmid DNA vector (eg p
BR322, pAC105, pVA51, pACY17
7, pKH47, pACYC184, pUB110, p
MB9, pBR325, ColE1, pSC101, p
BR313, pML21, RSF2124, pCR1 or RP4). The expression vector used in the examples of the present invention is described in US Patent Application No. 449,187 (December 1, 1982).
(3rd application), the teachings of which are incorporated herein by reference.

Further, as the host cell strain, one which suppresses the action of the promoter is selected except when it is specifically induced. In this way, 95% or more of the vector promoter effectiveness can be suppressed in non-induced cells. Some operons require the addition of specific inducers for efficient transcription and translation of inserted DNA,
For example, the lac operon is lactose or IPTG
(Ie, isopropylthio-β-D-galactoside). Various other operons, such as trp, pro, etc., are under different control. The trp operon is induced when tryptophan is not present in the growth medium, and the lambda P _L promoter is a temperature-sensitive lambda repressor protein (eg CI85).
Induced by an increase in temperature in the host cell containing 7). In this way, the expression of the genetically engineered gD protein can be controlled. This is important if the protein product of the cloned gene is lethal to the host cell. In such cases, the foreign gene is replicated but not expressed during growth of the transformant. After the cells reach the appropriate density in the growth medium, the promoter can be induced for protein production.

A plasmid constructed as described above (ie, AT which terminates with the native gD translation termination signal and is provided by the vector, gD gene or synthetic sequence).
G starting with G, a plasmid which induces the expression of gD-related proteins)
D-related proteins are hereinafter referred to as unfused gD proteins or unfused gD-related proteins.

5.3. Production of Fusion Proteins In order to maximize the level of gene expression by a particular transformant, it is desirable to link the gene of interest to genes encoding other proteins such as host cell proteins. Additional advantages are obtained if the host cell protein naturally contains a detectable function. Expression of the linked genes results in a fusion protein product (hereinafter referred to as gD fusion protein), which can be identified based on its large molecular weight and detectable function. For example, production of the gD / β-galactosidase fusion protein offers several advantages for cloning and expression of gD in E. coli hosts. First, Miller
Method (Experiments in Molecu
lar Genetics, Cold SpringH
arbor Press, New York, N.A. Y.
1972, pp. 47-55 and 352-355), it is possible to estimate the level of vector-induced protein production (hence expression) using a colorimetric assay specific for β-galactosidase activity. Second, production of the fusion protein simplifies identification and isolation of the protein product. The unfused gD protein produced by the E. coli transformant is gD / β-
It is smaller than the galactosidase fusion protein and therefore co-migrates with some other host proteins analyzed by SDS-polyacrylamide gel electrophoresis. However, the fusion protein produced can be easily detected and identified by SDS-polyacrylamide electrophoresis (SDS PAGE) due to its unique large molecular weight.

The present invention is not limited to the production of β-galactosidase fusion proteins. Any gene of eukaryotic or prokaryotic origin can be read in the same reading frame as the gD gene (or HSV protein gene).
phase) to obtain similar advantages as the β-galactosidase fusion protein product. Examples are galactokinase; trpD, E or leaders; pili (pilus) genes; and eukaryotic genes such as thymidine kinase, β-globin, SV-.
Examples include, but are not limited to, 40T-antigen or Rous sarcoma virus transforming gene.

In order to construct the gene encoding the fusion protein, the two genes must be joined within their coding sequence so that the translation reading frame is maintained and not interrupted by the termination signal.
Also, as explained above, when the host cell is a strain that suppresses the action of the promoter, the fusion protein is produced only when it responds to induction.

5.4. Identification of the gene product Once transformants containing the correct DNA construct are identified, analysis of the immunoreactivity and antigenicity of the recombinant DNA gD gene product is required. The gD gene product will differ from the native gD glycoprotein if the host cell is not capable of glycosylating the gD gene product in the same pattern as naturally occurring HSV-gD. Therefore, immunological analysis is of particular importance for the gD gene product. This is because the ultimate goal is to use the gD-related protein thus produced in a vaccine formulation. The immunoreactivity analysis is most easily carried out using antisera directed against the gD glycoprotein of HSV-infected cells, but the antigenicity is the antiserum potency of the test animals in response to immunization with the gD gene product. Titer and the ability of the antiserum to neutralize HSV infection can be assessed by measuring.

A variety of antisera can be utilized in analyzing immunoreactivity, including multivalent antibody preparations directed against whole virus or especially gD.
Higher specificity is obtained by using a monoclonal antibody that recognizes only one antigenic site on the gD protein molecule. There are many monoclonal antibodies against gD, some of which are g of HSV-1 and HSV-2.
It not only specifically immunoprecipitates the D gene product, but also neutralizes the infectivity of both viruses.

Therefore, the identification of the proteins mentioned in the present invention is based on two requirements. First, gD-related proteins should only be produced in response to promoter induction. Second, the gD-related protein should be immunoreactive with various polyclonal antibodies against HSV or with monoclonal antibodies against gD, which protein may be from the entire gene sequence, from a portion of the gene sequence, or from fusion proteins. Whether it arises from two or more gene sequences linked so as to bring about production,
Should be immunoreactive. This reactivity is separated by standard immunological techniques, such as immunoprecipitation, immunodiffusion, radioimmunocompetition, immunoelectrophoresis, or polyacrylamide gel electrophoresis, and then transferred to nitrocellulose. It can be detected by immunological detection.

5.5. Purification of gene product gD gene (or any HSV glycoprotein gene)
Cells containing H. coli are grown in large volumes, and the protein produced after induction of the promoter is isolated from such cells and, if the protein is secreted, from its culture medium. Proteins can be isolated and purified by standard chromatography with ion exchange, affinity or sizing resins, by centrifugation, or by any other standard technique for protein purification.

Since certain fusion proteins form aggregates when overproduced in cells or when produced in solution, the method of isolating aggregate-forming proteins is a fusion produced by the present invention. It is particularly useful for protein isolation.
These fusion proteins can then be used in vaccine formulations. Purification of the fusion protein (hereinafter referred to as aggregate purification) involves the extraction, separation and / or purification of aggregate-forming proteins from a protein mixture such as cell culture lysate by cell disruption. In addition, the aggregates are solubilized by adding a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a mixed solvent in which each solvent has one of these properties), after which aggregate-forming proteins are removed. It can be diluted with an appropriate buffer and sedimented. Suitable protein solvents include, but are not limited to, urea (about 4-8M), formamide (at least about 80%, volume / volume basis) and guanidine hydrochloride (about 4-8M). Solvents that can solubilize aggregate-forming proteins, such as SDS (sodium dodecyl sulfate), 70% formic acid, are unsuitable for use in this procedure. This is because the subsequent precipitation of the solubilized aggregate-forming protein was found to be inadequate. Although guanidine hydrochloride and other similar reagents are denaturants, experiments have shown that this denaturation is not irreversible and refolding occurs upon removal (eg by dialysis) or dilution of the denaturant, resulting in immunological Target and /
Or it has been found to allow the reformation of biologically active proteins.

One embodiment of the fusion protein isolation technique is outlined as follows (hereinafter referred to as the non-denaturing aggregate purification method). The cell pellet is quickly frozen with dry ice / methanol, weighed, and 3-4 g of cells in at least 25 ml of buffer solution [eg 50 m
M Tris-HCl (Trishydroxymethylaminomethane hydrochloride), pH 8.0, 2 mM EDTA (ethylenediaminetetraacetic acid) and 200 mM NaCl]. That is, the cells are about 100-
Suspend at an approximate concentration of 200 g cells / L. About 160g
A concentration of / L or less is suitable. Lysozyme is added to this suspension at a final concentration of about 130 μg / ml and the resulting mixture is left at 4 ° C. for 20 minutes with occasional shaking. Nonidet P40 (NP-40, She, a nonionic surfactant used to solubilize membranes.
ll registered trademark, polyoxyethylene (9) p-ter
t-octylphenol) is added to a final concentration of about 0.1%, after which the solution is mixed. Next, the suspension is Poly
tron grinder (Brinkman Instrument
nts, Westbury, N.N. Y. ) Or an equivalent device for about 1 minute.

The suspension is centrifuged at 8,000 × g for 30 minutes and the pellet washed with a wash buffer [eg 2 m.
M Tris-HCl (pH 7.2), 1 mM EDTA,
150 mM NaCl and 2% Triton-X100
(Polyoxyethylene (9-10) p-tert-octylphenol, nonionic surfactant)] and milled with a Polytron mill. The steps of centrifugation, washing and grinding can be repeated to further wash the pellet and remove as much cell debris as possible.

The aggregate pellets can be further treated by adding a denaturing agent as follows (hereinafter referred to as a modified aggregate purification method). The suspension is centrifuged, the supernatant is completely removed, and the pellet is resuspended in approximately 1/5 volume of 6M guanidine hydrochloric acid (in distilled water). For example, 25
3 g cells washed with ml buffer should be resuspended in 5 ml 6M guanidine hydrochloride solution at this stage. Resuspending the pellet at this stage is difficult and sonication or homogenization may be necessary to obtain a homogeneous solution. The solution is left at 22 ° C. for 20 minutes and then centrifuged at 8000 × g for 30 minutes to remove debris, at which point a supernatant containing fusion protein aggregates is obtained.

The fusion protein is precipitated from the guanidine hydrochloride supernatant by the addition of about 4 volumes of aqueous buffer. Almost any aqueous buffer can be used at this stage, but with a small amount of nonionic surfactant [eg:
It may be helpful to add 0.5% NP-40 or Triton X-100]. The suspension is left at 4 ° C. for 30 minutes and then centrifuged at 8,000 × g for 10 minutes. The supernatant is discarded and the pellet (containing the fusion protein precipitate) is resuspended in a suitable volume of a suitable buffer, eg phosphate buffered saline (PBS). Short-term sonication or homogenization will help obtain a homogeneous suspension or slurry (since aggregate-forming proteins are insoluble in water).

5.6. Production of Unfused gD Proteins It may be desirable to use fusion proteins in vaccine formulations. However, as explained previously, transformants producing the non-fusion gD protein produce it in lesser amounts than those producing the gD fusion protein, which means that the gene of the non-fusion protein is Even when the sequence is under the control of an inducible promoter. Furthermore, the unfused gD protein produced by the bacterial transformants may be less stable than the gD fusion protein.

In another embodiment of the present invention, host cell transformants can be engineered to produce large amounts of both fused and unfused gD-related proteins that can be coaggregated and readily purified. . According to this embodiment, g
The recombinant plasmid encoding the D fusion protein is g
At the juncture of the D gene sequence and the host cell protein gene sequence (eg, β-galactosidase z gene sequence), the nonsense codon sequence, ie, amber.
A chain terminator such as r) (TAG), ochre (TAA) or opal (TGA) is modified to be located between the two gene sequences. This chain terminator must be placed in phase with the translation reading frame of both the gD sequence and the host cell gene sequence. Such a modification cleaves the plasmid at a restriction site located between the two gene sequences and then ligates a DNA linker sequence encoding a chain terminator such as amber, ocher or opal to the cleavage site of the plasmid. , The chain terminator is placed in phase with the translational reading frame of both gene sequences.

Introduction of these amber, ocher or opal modified plasmids into host cells containing the appropriate tRNA suppressor results in the synthesis of both unfused gD-related proteins as well as gD fusion proteins. If amber, ocher or opal modified plasmids are used to transform non-suppressor cell lines, predominantly non-fused gD-related proteins will be produced.

There are at least two ways to introduce the amber, ochre or opal modified plasmid into the background of suppressor cells. That is,
(1) Transformant (ie, host cell transformed with amber, ocher or opal modified plasmid)
Alternatively, a lysogenic transducing phage with the appropriate suppressor tRNA gene (eg, φ80pSU3 carries the amber mutant supF suppressor) can be infected. Alternatively, (2) using amber, ocher or opal modified plasmid, amber,
Cell lines containing the ocher or opal suppressor tRNA can be transformed. Examples of such strains include LE392 (amber mutant supE and s
There are but not limited to upF suppressors), YMC (including supF) and C600 (supE). Various amber suppressor tRNs in E. coli
A gene includes supB, supC, supD, s
upE, supF, supG, supL, supM, s
There are, but are not limited to, upN, supO, supP, supU, supV. Various ocher suppressor tRNA genes in E. coli include supB, supC,
supG, supL, supM, supN, supO,
supV, but not limited to these. Various opal suppressor tRNA genes in E. coli include but are not limited to supK (Backmann and Lo).
w, 1980, Microbiological Re
views 44 (1): 1-56).

Host cells containing the appropriate suppressor tRNA gene transformed with amber, ocher or opal modified plasmids can produce gD-related proteins as both fusion and non-fusion proteins. The ratio of gD to unfused gD protein depends on the degree of inhibition in the host cell). Both proteins immunoreact with antisera to HSV. When using the non-denaturing aggregate purification method described in Section 5.5, where the method is a non-denaturing co- aggregate purification method, both unfused gD and gD fusion protein are co-purified. After solubilization, unfused gD can be separated from gD fusion protein based on size or charge. Consequently, large amounts of unfused gD-related proteins produced by host cell transformants can be easily purified and formulated for use as vaccines.

5.7. Vaccine production The object of the present invention is to produce HSV-
1 and / or to produce HSV glycoprotein-related polypeptides, such as gD-related proteins, which can be used as immunogens in vaccines to protect against HSV-2 infection. If the produced gD-related protein immunoreacts with a specific HSV-1 and / or HSV-2 neutralizing antibody, the gD-related protein produces an immune response capable of neutralizing the related virus in vivo. You will be expected to withdraw. Vaccines made from genetically engineered immunogens should be safer than conventional vaccines made from attenuated viruses. This is because the risk of infection of the recipient is completely gone. Also, the genetically engineered gD product can be used to make antibodies useful in passive immunotherapy.

Although the gD / β-galactosidase fusion protein product itself may be useful in vaccine formulations,
It may be necessary first to remove the β-galactosidase moiety.
Also, the rearrangement of the amino coding end of the gD gene may be important for the immunogenicity of the protein. Since the amino terminus may contain additional important antigenic sites. The amino coding end of the gD gene is gD
DNA sequence encoding the amino terminus of
It can be reconstituted by linking it to the appropriate site within the gD coding region of the molecule. Since the recombinant DNA molecule retains all necessary expression control elements, full-length (or nearly full-length) gD-related proteins are produced by cells transformed with the DNA molecule containing the reconstituted gene. .

Finally, genetically engineered gD-
Related proteins can be isolated and purified from host cells using standard protein isolation techniques or by the aggregate purification methods described herein. The final purified product is then diluted to the appropriate concentration, formulated with the appropriate vaccine adjuvant, and packaged for use. Suitable adjuvants include surface-active substances such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecyl ammonium bromide,
N, N-dioctadecyl-N'-N-bis (2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid, carbohydrate Paul; peptides such as, but not limited to, muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and alum. Finally, the protein product can be retained in liposomes for vaccine formulations,
It may form a complex with a polysaccharide or another polymer.

The genetically engineered D described herein
NA molecules offer great flexibility in vaccine production.
For example, gD- produced by a transformant containing a recombinant DNA molecule containing in tandem any portion of the gD gene sequence or multiple copies of the gD gene (or a portion thereof).
Vaccines can be formulated with related proteins. The gD gene sequence (or part thereof) may be linked to genes encoding other immunogens and the fusion protein product used in the manufacture of multivalent vaccines. Furthermore,
The gD gene sequence (or part thereof) could be linked to other HSV glycoprotein sequences (or part thereof) in any combination. Finally, the gD sequence may be rearranged to enhance the immunogenicity of the vaccine. For example, the gene sequence is altered such that the protein product presents a particular epitope to the immune system (eg, the normally unexposed antigenic site of gD is presented to the immune system). Alternatively, the region of the gD gene sequence that encodes the immunosuppressive portion of the protein can be deleted.

[0076]

BEST MODE FOR CARRYING OUT THE INVENTION

6. Example: HSV-1gD According to the method of the present invention, a DNA fragment of the Us portion of the HSV-1 genome (see FIG. 1a) was added to the vector pBR3.
The gD-1 gene was localized and identified by insertion into 22 to create recombinant plasmids, each containing a different fragment of the HSV-1 genome. gD-
Plasmids containing one gene were identified by the following three techniques (not necessarily in the order shown). That is, (1) DNA sequencing, (2) gD-1 specific mR
NA hybridization experiment, and (3) in vitro of mRNA that hybridizes to recombinant plasmid
tro translation.

Once the plasmid containing the gD-1 gene was identified, the gene was characterized by DNA sequencing and by locating the ends of the gD-1 gene in the recombinant plasmid. A DNA fragment containing the gD-1 gene lacking the first 156 nucleotides of the coding sequence was isolated from the identified plasmid. This DNA fragment was ligated into the DNA vector pJS413 to create pEH25 used for cloning and expression of the gene in E. coli. The gene product isolated from the induced transformants contained a monoclonal antibody against gD-1 and HSV.
GD- by immunoprecipitation with a multivalent antibody against -1
1 was identified as a unique protein.

Finally, the gD-1 gene fragment was
PEH2 by restriction endonuclease cleavage at a specific site
5 from which the termination sequence (TAG) of the gD-1 coding sequence was deleted. Parts of the gD-1 gene and plasmid promoters and regulatory elements (eg S
This DNA fragment containing D-ATG) was ligated into the vector containing the β-galactosidase coding sequence. The resulting plasmid pEH4-2 was croSD-
The fusion protein was expressed in induced E. coli transformants containing the gD-1 coding sequence flanked by the lac promoter of the ATG-bearing plasmid and the β-galactosidase gene. The fusion protein was then isolated from host cell lysates and formulated for use as an immunogen. Also, the amino coding end of the gD-1 gene was reconstituted and the resulting gD-1 protein was formulated for injection into animals and preliminary clinical trials. Details of each construction process are described in the following sections.

The gD-1 related proteins and fusion proteins produced here are not glycosylated and are therefore different from the naturally occurring HSV-1 gD glycoprotein. However, gD-1 related proteins
It has immunoreactivity with antibodies against gD of SV-1 and HSV-2. 6.1. General Procedures Used for Plasmid Construction The following sections describe general procedures and materials used for DNA isolation, enzymatic reactions and ligation reactions.

6.1.1. Plasmid DNA Isolation Host Cell Bacteria Escherichia
E. coli) (Gautier & Bon
Ewald, 1980, Molec. Gen. Gene
t. 178: 375), and large quantities (micrograms) of plasmid DNA were isolated from cultures of E. coli transformants grown in M-9 broth (Miller, E.
xperiments in Molecular G
enetics, Cold Spring Harbo
r Press, New York, N .; Y. , 197
2, p. 431). The plasmid was prepared according to the method of Guerry et al. (1973, J. Bacteriol. 116: 10.
It was isolated from cells in late logarithmic growth phase using a modified method of 63).

6.1.2. Conditions for restriction enzyme digestion The restriction enzymes used in the present invention, unless otherwise indicated,
New England, Beverly, Massachusetts
Biolads, Inc. It was obtained from.
Enzyme units are defined as the amount required to digest 1.0 μg lambda DNA for 15 minutes at 37 ° C. in 50 μl total reaction mixture.

Complete digestion with all restriction enzymes was performed under the following conditions. 1 μg of each DNA was incubated with 0.5 units of enzyme for 60 minutes at 37 ° C. in 20 μl of buffer. Partial digestion was performed by changing the conditions used for complete digestion as follows. 1 μg of D each
NA was incubated with 0.1 units of enzyme for 15 minutes at 37 ° C. 0.1% sodium dodecyl sulfate (SD
The reaction was stopped by the addition of S). Thus, DN
The reaction conditions were adjusted so that an average of 1 cleavage per A molecule.

6.1.3. The buffer used for restriction enzyme buffer SacI, SacII or SmaI digestion was 6.6 mM Tris-HCl (pH 7.4),
6.6 mM MgCl ₂ and 6.6 mM β-ME
(Β-mercaptoethanol).
BglII, BstEII, EcoRI, HindII
The buffer used for I, NruI, PstI or PvuII digestion was 60 mM NaCl, 6.6 mM Tris-.
It consisted of HCl (pH 7.4), 6.6 mM MgCl ₂ and 6.6 mM β-mercaptoethanol (β-ME).

The buffer used for BamHI, SalI or XbaI digestion was 150 mM Tris-HCl (pH
7.4), 6.6 mM MgCl ₂ and 6.6 mM
It consisted of β-ME. It should be noted that if more than one restriction endonuclease reaction is performed on the DNA, the reaction in the low salt buffer will precede the reaction in the high salt buffer. The reactions may be performed simultaneously if the two enzymes require the same buffer.

6.1.4. The modified Bal 31 nuclease of DNA is a multifunctional enzyme containing highly specific single-stranded endodeoxyribonuclease activity and forward exonuclease activity.
Both 3'- and 5'-ends of A (dsDNA) are cleaved simultaneously. Nuclease Bal 31 (New
England Biolabs, Inc. , Beve
rly, Ma. ) Is one unit of denatured calf thymus DNA (6
50 μg / ml) to 1.0 μg of acid-soluble nucleotide is defined as the amount required to release 1 minute at 30 ° C. The reaction buffer used for Bal 31 digestion was 20 mM Tris HCl (pH 8.0), 600 mM.
NaCl, 12m MgCl ₂ , 12mM CaC
It consisted of ₁₂ , 1.0 mM EDTA and DNA. Incubation was performed at 30 ° C. Ba
l31 digestion was performed by adding 30 μg of DNA to 0.5 unit of Ba
This was done by incubating with l31 for 1, 2, 4, 6, 8 and 10 minutes. The reaction was stopped by the addition of EDTA to 50 mM or heat inactivation of Bal 31 (eg, 65 ° C. for 10 minutes).

S1 nuclease is RNA or denatured DN
A (that is, single-stranded DNA) is decomposed into mononucleotides, but double-stranded DNA and DNA / RNA hybrids are not decomposed (under appropriate conditions). S1 nuclease (B
oehringer Mannheim, Indian
apolis, Ind. 1) is defined as the amount of enzyme required to acid solubilize 1 μg of denatured calf thymus DNA at 37 ° C. for 30 minutes. The reaction buffer used for S1 nuclease was 30 mM sodium acetate (pH 4.6), 250 mM NaCl, 1 mM Zn.
It consisted of SO ₄ and 5% glycerol.
S1 digestion uses 2000 units of enzyme with 0.1 μg of DNA
And 20 μg of RNA by incubation at 45 ° C. for 30 minutes.

Exonuclease VII (Exo VI
I) degrades single-stranded DNA (ssDNA). Ex
The mechanism of action of oVII appears to be that of a forward exonuclease. Exo VII (Bethesda
Research Laboratories, Roc
kville, Md. 1 unit of) is a linear denatured [ ³ H] -SV40 DNA as a substrate at 37 ° C. for 30
Defined as the amount of enzyme that yields 1 nmol of nucleotide monomer in minutes. The reaction buffer used for Exo VII was 10 mM Tris HCl (pH 7.
9), 100 mM NaCl, 10 mM β-ME and 8 mM EDTA. Exo V
II digestion was performed with 0.1 μg D in a 250 μl reaction volume.
1 at 45 ° C with 4 units of Exo VII per NA
I went on time.

6.1.5. DNA Polymerase Reaction DNA polymerase catalyzes the stepwise elongation of DNA strands. Chain growth is in the 5'to 3'direction (ie, the addition of nucleotides occurs at the 3'end) and the sequence of the polymer depends on the sequence of the template. This is because the incoming nucleotide must be complementary to its template, ie it must form the correct base pair with the template strand. Known DNA polymerases are unable to initiate strand synthesis. Therefore, DNA synthesis requires a primer with a free 3'-hydroxy end that anneals to the template strand. Strand extension is 3'of the primer to the 5'-phosphate of the incoming nucleotide.
-Proceeds with nucleophilic attack of the hydroxy terminus. The new strand is base paired and antiparallel to the template strand. As a result of the DNA polymerase reaction, the single-stranded template strand is "repaired" or converted into a double-stranded DNA molecule.

The second important feature of DNA polymerase is "proof-readin.
g) ”function. The 3'-5 'exonuclease activity associated with DNA polymerase I acts at the non-base paired ends, and in both single-stranded and double-stranded DNA.
Is decomposed in the 3'to 5'direction to produce a mononucleotide. Thus, erroneously added nucleotides are removed before polymerization continues, further increasing the fidelity of the enzyme. In addition, DNA polymerase I is a double-stranded DNA
It also has a 5'-3 'exonuclease activity specific for (produces 5'-mononucleotides and oligonucleotides), which can excise mismatched regions of DNA.

Klenow's method (Klenow et al., 1
971, Eur. J. Biochem. 22: 371)
Hydrolysis treatment of DNA polymerase I with 2 gives two fragments, a large one and a small one.
The large fragment, the Klenow fragment (76,000 daltons), has polymerase activity and 3'-
It retains 5'exonuclease activity, while the smaller fragment retains 5'-3 'exonuclease activity. 1 unit of DNA Polymerase I or DNA Polymerase I-Large Fragment (New England
Biolabs, Inc. , Beverly, M
a. ) Is defined as the amount that converts 10 nmoles of deoxyribonucleotides to the acid insoluble form at 37 ° C. for 30 minutes. The assay conditions for both enzymes are the same, but DN
The reaction mixture for A polymerase I was 6.6 mM M
gCl ₂ , 1.0 mM β-ME, 2 mM dAT copolymer, 33 μM dATP, 33 μM ³ H-dTT
67 mM KPO in a buffer consisting of P and enzyme
₄ (pH 7.5), while the Klenow fragment reaction mixture contained 40 mM KPO ₄ (pH 7.5).
Was included.

In the present application, when DNA polymerase large (Klenow) fragments were used to repair single-stranded DNA or sticky ends resulting from restriction enzyme cleavage, the following procedure was used. Restriction enzyme reaction at 68 ℃ for 10
It was stopped by heating for a minute. Each deoxynucleoside triphosphate was added to the same reaction mixture at a final concentration of 50 μM and 10-100 units of DNA polymerase Kleno per microgram of DNA in the reaction mixture.
w fragment was added. In other words, excess DNA polymerase Klenow fragment was used to ensure complete repair of the single stranded ends.

6.1.6. Gel purification of DNA fragments
After digestion with restriction enzymes or nucleases, DNA fragments of various sizes were treated with agarose or polyacrylamide slab gel at low voltage (about 2 volt / cm for agarose gel and 10 volt / cm for polyacrylamide gel). Gel electrophoresis was performed to separate, stain with ethidium bromide, and visualized under UV light (Southern, 1979, Me.
methods in Enzymology 68:15
2).

In order to recover the specific DNA fragment from the gel, the appropriate band was cut out from the gel and the DNA was electroeluted into a dialysis tube. Next, the DNA is DEA
Isolated on E-cellulose or ethanol precipitated and resuspended in an appropriate buffer (Smi.
th, 1980, Methods in Enzymo
65: 371). 6.1.7. Ligation of DNA All ligation reactions were performed using T4 DNA ligase (New England Biolabs, In.
c. Beverly, Ma. ). One unit of T4 DNA ligase is 20 μl ligase buffer and 0.12 μM (300 μg / ml) 5′-DNA end concentration at the bacteriophage lambda DNA Hin.
Defined as the amount required to produce 50% ligation of the dIII fragment in 16 minutes at 16 ° C.

DNA ligation was performed using 60 mM Tris-HCl.
(PH 7.8) was carried out in a ligase buffer consisting of 10 mM MgCl ₂ , 20 mM dithiothreitol (DTT), 1.0 mM ATP and a DNA concentration ranging from 15 to 50 μg / ml. The ligation reaction was incubated for 4-24 hours at room temperature with about 300 units of T4 DNA ligase per 10 μl reaction volume.

6.2. localization of the gD-1 gene and
Analysis of the protein identified by the isolated HSV-1 × HSV-2 recombinant showed that the gD-1 gene was present between 0.9 and 0.945 genomic map units within the Us region of DNA. (Ruyechan et al., 19
79, J. Virol. 29: 667). See Figures Ia and Ib. The Us region was fragmented with a restriction enzyme, and these fragments were inserted into a cloning vector to prepare various recombinant plasmids. Each plasmid contained a specific portion of the Us region. These recombinant plasmids were then analyzed to locate the gD-1 gene within the Us region. The map position of gD-1 in HSV-1 was recently reported by Lee et al. (1982, J. Virol.
43:41).

6.2.1. Identification of Us region of HSV-1
Recombinant DNA plasmid vector pBR322 containing part and HSV-1 (Patton strain)
Several recombinant plasmids were made using various fragments of the Us region of E. coli. One of these plasmids, pRWF6, was found to contain the entire gD-1 gene. pRWF6 is described below.

HSV-1 of recombinant plasmid pRWF6
The insert (Fig. 1c) is lambda gtWES: EcoRI-H.
Obtained from the Us region of the clone (Umene & E
nguist, 1981, Gene 13: 251).
Lambda gtWES: EcoRI-H clone was EcmR
Digested completely with I. Lambda gtWES: EcoRI-
EcoRI-H fragment of H clone, about 15-1
6 kb (kilobase) contains the entire Us region of HSV-1.

Plasmids pBR322 and HSV-1
EcoRI-H fragment (isolated above)
Were each digested to completion with BamHI. The obtained pBR
322 4.4 kb fragment and HSV-1 6.4.
The kb fragment was annealed and ligated in a 1: 1 ratio to give pRWF6. 6.2.2. Position of the mRNA coding sequence unique to gD-1
The viral DNA insert was subcloned back into pBR322 for localization and selection of the gD-1 specific coding sequence within confirmation pRWF6. A denatured viral DNA restriction fragment of subclone pSC30-4 (FIG. 1d and description below) was immobilized on nitrocellulose and mRNA was extracted from cytosolic RNA extracts of HSV-1 infected cells (by complementary base pair hybridization). Used for isolation. Two mRNA species (3.0 kb and 1.7 kb) hybridized with pSC30-4. In vitro of these mRNAs
o 3.0 kb or 1.7 kb mRNA by translation
Was found to encode the gD-1 protein. The details of this procedure are described below and shown in FIG.

The plasmid pRWF6 was isolated from the E. coli transformant and digested with the restriction enzyme SacI. This resulted in 3 fragments. That is, all pBR
322 vector and a 6.2 kb fragment containing part of the HSV-1 DNA sequence, 2.9 kb HSV-1.
DNA fragment, and 1.7 kb HSV-1D
NA fragment.

To subclone the 2.9 kb HSV-1 DNA fragment (see FIG. 1d), p
BR322 is PvuII (pBR322 is linearized)
Cleaved with SacI linker (New E
ngland Biolabs, Inc. , Bever
ly, Ma. ) Was used to ligate with T4 DNA ligase. Therefore, the unique Pvu of pBR322
The II site was converted to a unique SacI site [Sac
I (Pvu II ^(-) )]. After cleaving the modified pBR322 vector with the SacI enzyme, this vector was ligated with the 2.9 kb HSV-1 SacI DNA fragment to give pSC30-4. E. coli was transformed with this recombinant plasmid. The transformants are screened and the microlysate technique (mini-lys) is used.
ate technique; Clewell & H
elinski, 1970, Biochem. 9:44
28) was selected by restriction mapping.

Subclone pSC30-4 was transformed into mRNA
Used for hybridization selection as follows (Ricciardo et al., 1979, Proc. Nat.
l. Acad. Sci. , U. S. A. 76: 492
7). The plasmid pSC30-4 was isolated from the E. coli transformant, and 200 μg of pSC30-4 DNA was added to Sac
The HSV-1 DNA insert was excised by digesting with I.
Cleaved plasmid with chloroform / phenol (1: 1)
Extracted with and ethanol precipitated. 2m of sediment
DNA was denatured by resuspending in 1M 0.3M NaOH and incubating at room temperature for 10 minutes. This suspension is then treated with 4.4 ml distilled water, 0.2 ml 3M HCl,
0.2 ml of 1 M Tris-HCl (pH 7.5) and 3 ml of 20 × SSC (SSC is 0.15 M NaC
1, 0.015M sodium citrate). The pH, measured by indicator paper, was between 6-8. The denatured and neutralized DNA was diluted with distilled water and then pre-soaked in 6 × SSC with a 25 mm nitrocellulose filter (Schleicher and
Schuell, Keene, N .; H. ) Through vacuum filtration. After vacuum filtration, the nitrocellulose filter was washed with 6 × SSC, dried and calcined at 80 ° C. for 2 hours. A half nitrocellulose filter was used in the hybridization method. This is briefly described below.

Nitrocellulose containing viral DNA was cut into small pieces and incubated with total cytoplasmic RNA. The RNA was HSV-infected V prepared as follows.
It was isolated from a lysate of ero cells. Ve
Ro cells were infected with 10 plaque forming units (pfu) / cell and 50 μg to block DNA replication
/ Ml cytarabine (β-cytosine arabinoside) was added and incubated in Dulbecco's medium at 37 ° C for 7 hours. Cells are treated with 0.15M NaCl, 1.5mM MgCl
Dissolved with 0.65% NP-40 in ₂ and 0.01 M Tris-HCl (pH 7.9). Nuclei were sedimented by centrifugation at 3000 xg for 2 minutes, and the supernatant was diluted to 1 mM.
Adjusted to final concentrations of EDTA (pH 7.9) and 0.5% SDS. This solution was extracted twice with chloroform / phenol (1: 1) and once with chloroform. Ethanol-precipitated cytosolic RNA (one confluent roller bottle of Vero cells contained approximately 1.5 mg RN).
Resulting in A) and partially dried. RNA precipitate (about 0.4-0.8 mg of RNA) was added to 400 μl of hybridization buffer (50% formamide, 0.4 M NaCl, 40 mM PIPES, pH).
6.4, 1 mM EDTA and 1% SDS) and added to chopped nitrocellulose filters. Hybridization was performed in a shaking water bath at 55 ° C. for 3 hours.

Next, the filter piece is placed in 0.5% S at 60 ° C.
Wash 10 times with SSC containing DS, followed by SS at 60 ° C
Washed twice with 1 ml of 2 mM EDTA in C. Finally, the filter pieces were washed with 2 mM EDTA, pH 8.0 for 5 minutes at 60 ° C. The solution was removed and the filter pieces were thoroughly dried with a cotton swab. The hybridized mRNA was eluted from the nitrocellulose strip using formamide and heat as follows. 120 μl
95% formamide / 10 mM PIPES (pH
6.5) was added to the filter and incubated for 5 minutes at 65 ° C. The eluate was transferred to a microcentrifuge tube and kept on ice. A second 120 μl elution buffer was added to the nitrocellulose pieces and incubated again at 65 ° C. for 5 minutes. This eluate was transferred to a second microcentrifuge tube and kept on ice. A 720 μl aliquot of sterile distilled water was added to the filter and then stirred. About 360 μl was transferred to each microcentrifuge tube containing the eluate. RNA eluted in microcentrifuge tubes
To this was added 20 μl of 5M NaCl, 5 μg of a suitable carrier (eg rabbit liver tRNA) and 1 ml absolute ethanol. Incubate the mixture at -70 ° C for 1 hour or centrifuge the tube in dry ice / E.
RNA was precipitated by immersion in a slurry of tOH for 20 minutes. Precipitated RNA was pelleted in a microcentrifuge (12,000 xg for 10 minutes) and one tube of sediment was added to 1 ml of buffer (0.5 M NaCl, 10 min).
mM Tris-HCl (pH 7.9) and 0.5% S
RNA was lysed by resuspension in DS). The lysed RNA was added to a homotypic tube to combine aliquots and to lyse all precipitated RNA. Polyadenylated RNA
[Poly (A) RNA] was added to oligo (dT) cellulose (Bethesda Research Laboratory).
ories, Inc. Rockville, Md. )
Was isolated from the lysed RNA by chromatography using. Poly (A) RNA was used as an elution buffer at 10
mM Tris HCl (pH 7.9) and 0.1% SDS
Was used to elute from oligo (dT) cellulose and then poly (A) RNA was ethanol precipitated as described above.

Two types of mRNA hybridized with pSC30-4 DNA, namely 3.0 kb and 1.7
A kb mRNA was isolated. Rabbit reticulocyte cell free system (including ³⁵ S-methionine)
Was translated in vitro using Pelha (Pelha
m & Jackson, 1976, Eur. J. Bi
ochem. 67: 247). The in vitro translation extract was immunoprecipitated with the monoclonal antibody 4S directed against gD-1 (donated by M. Zweig of the National Institutes of Health), and S as described previously.
Prepared for DS-PAGE. Electrophoretic analysis of the immunoprecipitated protein products of the cell-free translation system demonstrated that these selected mRNAs specified a 50,000 dalton gD-1 specific protein. According to the size of the gD-1 protein, the smallest mRNA coding sequence would be approximately 1.6 kb. Therefore, considering the mRNA leader sequence and poly (A) tail, the larger (3.0 kb) mRNA was gD-
It was predicted to encode one polypeptide.

6.2.3. Characterization of gD-1 mRNA
Mapping the position of the 3.0 kb mRNA sequence contained in the constant pSC30-4, and further characterizing the mRNA, was performed by S1 mapping, that is, by using the single-strand specific nuclease S1 and exonuclease VII. By mapping the region of the DNA probe that hybridizes to the sequence (Berk & Shar
p, 1978, Proc. Natl. Acad. Sc
i. , U. S. A. 75: 1274), and gD-
By sequencing the DNA of one gene coding region (Maxam & Gilbert, 1980, Me.
methods in Enzymology, 65:49.
9) Implemented.

By the S1 mapping technique, both 3.0 kb and 1.7 kb mRNA species are unspliced (ie, contain no intervening sequences or introns), and they have a common 5'end and a common 3'end. 'It turned out to have an end. 3.0 kb mRNA
A 1068 nucleotide DNA sequence containing the coding region of the 5'end of was determined and the reading frame of translation was deduced by locating the start codon (ATG) closest to the 5'end.

The principle of the S1 mapping technique is to form a duplex between the RNA and a radiolabeled single-stranded DNA (ssDNA) probe (eg obtained from pSC30-4). If the RNA is a spliced mature mRNA, the intron will form an ssDNA loop. The enzyme nuclease S1 is RNA
Digests all ssDNA regions of radiolabeled DNA that are not protected by double-stranded formation with. Exonuclease VII, on the other hand, will digest ssDNA only at the ends and thus will not digest the ssDNA loop. By comparing the size of DNA that is not sensitive to these nucleases (by hybridization with RNA),
Detection of spliced mRNA transcripts is possible.

Radiolabeled DNA used in the present invention to locate the 5'end of a 3.0 kb mRNA.
At its BstEII 5'end before cleavage with BamHI (using a polynucleotide kinase enzyme,
³² P by the method of Maxam & Gilbert)
3.8 kb BamHI-8 / of pRWF6 labeled with
It was the BstEII fragment. 3.0 kb mr
Radiolabel D used to locate the 3'end of NA
NA has its HindIII before it is cut with SalI.
At the 3'end (Klenow of DNA polymerase
Maxam & Gilber with fragments
³² P-labeled pSC30-4 4.7 kb HindIII / SalI fragment. Cytoplasmic mRNA was isolated from HSV-1 infected Vero cells grown for 7 hours in the presence of cytarabine as described above. The S1 mapping technique used is Berk &
A modification of the Sharp method (Watson et al., 198).
1, J. Virol. 37: 431). gD-
1 5'end of mRNA is HindI of pSC30-4
It was mapped near the II site, while the 3'end was mapped approximately 2.8 kb downstream (Fig. 1d).

Finally, HSV-1 D of pSC30-4
NA was sequenced using the method of Maxam and Gilbert. Figure 2 shows the sequencing strategy for the HSV-1 gD gene coding region. Both coding and non-coding strands were sequenced. FIG. 3 shows the resulting DNA sequence of the HSV-1gD gene. This DNA sequence was found to contain an open reading frame of 394 codons extending from the ATG at position 241. This site is presumed to be the initiator of the gD-1 gene, and this putative gD-1 gene is used as the expression vector pJS41.
It was found to be so by cloning into 3 (see Section 6.3).

6.3. Cloning of the gD-1 gene and
The expressed gD-1 coding sequence was placed under the control of the lac promoter. For this reason, a part of the putative gD-1 gene (hereinafter gD-
1 gene) was ligated to the DNA cloning expression vector pJS413 to prepare pEH25 (see FIG. 4). The partial gD-1 gene was inserted so that the protein coding sequence was in the correct open reading frame with respect to the starting ATG of the vector. As a result, translation of the transcribed mRNA is initiated at the vector's initiation sequence (ATG), through the gD-1 gene (which lacks its own initiation ATG and the first 156 nucleotides of the gD-1 coding sequence), Go to the -1 natural termination signal.

The pEH25 plasmid containing the gD-1 gene was used to transform an E. coli host strain,
Transcription of DNA from the lac operon is repressed unless the promoter is specifically induced. Primary transformants were assayed for drug resistance (vector carrying the ampicillin resistance gene) and resistant clones were further analyzed. The structure of the resulting recombinant plasmid pEH25 was confirmed by restriction analysis and DNA sequencing. pEH
Induction of 25 transformants produced a polypeptide of 46,000 daltons, which was immunoprecipitated with either antiserum to HSV or a monoclonal antibody to gD. These procedures are detailed in the following sections.

6.3.1. Expression vector pJS413 Expression vector pJS413 (FIG. 4) was expressed as amp ^r (β-
Lactamase) gene, lac promoter (Pla
c), lac and cro ribosome binding sites (SD
^LacZ and SD ^cro are SD in all drawings
^Z and SD ^CRO ), cro 69
A chain initiation ATG with nucleotides and a modified β-galactosidase gene (lac i-z gene, hereafter z
-Denoted as a gene). p
Between the cro initiation ATG and the z-gene of JS413 (ie BglII, SmaI or Bam of pJS413).
Insertion of the gene in the correct reading frame (within the HI cloning site) allows expression of the fusion protein by transformed cells. However, in this example, the z-gene was deleted and the partial gD-1 gene was ligated in phase with the cro initiation ATG and cro nucleotides of pJS413.

PJS4 for inserting the gD-1 gene
PJS413 was added to Sm in order to produce 13 (see FIG. 4).
aI (resulting in blunt ends) and SacI (SacI
Resulting in 3'sticky ends). Promoter, S
4.7 including D sequence, initiation ATG and partial cro sequence
The kb fragment was isolated by gel electrophoresis. A 1.8 kb fragment containing the 5'end of the z-gene was deleted.

6.3.2. pJS41 of the gD-1 gene
Insertion into 3 After mapping the gD-1 gene within pSC30-4 (Section 6.2.3), 2.2 containing the last 1026 bp (base pairs) at the carboxy-coding end of the gD-1 gene.
The kb DNA fragment was selectively isolated from pSC30-4 by digestion with PvuII (which produces blunt ends) and SacI (which produces SacI 3 ′ sticky ends) (FIG. 4).

2.2 kb PvuII / SacIpSC
30-4 fragment and 4.7 kb SmaI / S
The acIpJS413 fragment was T4 at a 1: 1 ratio.
Ligation was performed using DNA ligase (Fig. 4). Escherichia coli NF1829 strain was transformed with the obtained recombinant plasmid. E. coli NF1829 share, F'-la with the lacI ^Q mutation for lac repressor overproduction
K-12 MC1000 derivative carrying c episome. The lacz-gene encoding β-galactosidase present on the F′-lac episome is Tn5.
(Transposon) is inactivated by insertion.
Therefore, in the NF1829 strain, the lac promoter must be induced in order to obtain the expression of the gene inserted in the pJS413 plasmid.

6.3.3. Form expressing gD-1 gene
Identification of the transformant The plasmid isolated from the ampicillin-resistant E. coli transformant was further subjected to restriction enzyme mapping to obtain pJS4.
D at the junction between the 13 vector and the gD-1 gene insert
Analyzed by NA sequencing. Plasmid pEH2
5 (FIG. 4) has the correct nucleotide sequence along the Cro-gD-1 junction (shown in FIG. 5) and gD-
Its ability to express one related polypeptide was investigated. l
Since the ac promoter is transcriptionally inactive in NF1829 (due to overproduction of the lac repressor),
gD-1 protein is 1 mM IPTG or 1-10
It was only detectable after inducing the promoter with either mM lactose.

The clones transformed with pEH25 were tested for IPTG-specific induction of gD-1 related proteins and showed that 23 amino acids of the cro protein (encoded within pJS413) and 342 amino acids of the gD-1 protein (ie , The first 52 of gD-1
The production of a protein of 46,000 daltons consisting of amino acids disappeared) was confirmed. This protein is a rabbit antiserum against HSV-1 (DAKO C
chemicals, Inc. , Hicksville,
N. Y. ) And a monoclonal antibody specific for gD of HSV-1 (1S, 4S, 55S and 57S).
(Showalter et al., 1981, Infectio
n and Immunity, 34: 684).

Monoclonal antibodies 1S, 4S, 55S and 57S recognize many different sites on the gD-1 molecule (Eisenberg et al., 1982, J. Viro.
l. 41: 478). Of note is the ability of monoclonal antibody 4S to neutralize the infectivity of HSV-1 and HSV-2 and to immunoprecipitate the gD protein produced by both viruses. The protein produced by pEH25 was immunoprecipitated by the 4S antibody, which indicates that pEH2
5 gD-1 related proteins are HSV-1 and HSV-2
It was demonstrated to express an antigenic determinant shared by both gD proteins of E. coli. Furthermore, gE of pEH25
-1-related protein is a 1S that neutralizes only HSV-1
It was also immunoprecipitated with a monoclonal antibody. Also,
pEH25gD-1 related proteins are associated with HSV-1 and H
55S and 5 that do not neutralize any infectivity of SV-2
Immunoprecipitated with 7S monoclonal antibody. Each immunoprecipitate was analyzed by SPS-PAGE. Details of all procedures are described below.

All pEH25 transformants were taken in aliquots of an overnight culture at 37 ° C. in L broth, M
Diluted 20-fold with -9 broth and grown by shaking for 90 minutes at 37 ° C (Miller, Ex.
periments in Molecular Gen
etics, Cold Spring Harbor
Rless, New York, N.M. Y. , 197
2). Assays of these cultures for expression of gD-1 protein showed 1 mM IPTG and 25 μCi.
/ Ml of ³⁵ S-methionine was added to the culture (control labeled with ³⁵ S-methionine but no induction). After 60 minutes at 37 ° C., the culture was pelleted by centrifugation. Each pellet of cells is treated with an equal volume of lysis buffer IP to lyse the cells and release the cell contents.
-3 (20 mM Tris-HCl (pH 8.1), 100
mM NaCl, 1 mM EDTA, 1% NP-40,
Resuspended in 1% deoxycholic acid and 0.1% SPS), immediately frozen in liquid nitrogen and sonicated. Centrifuge the cell lysate at 5,000 xg for 5 minutes at 4 ° C,
The supernatant was divided into aliquots. Control serum (non-immune or pre-immune serum) or test antiserum (multivalent antiserum against HSV-1 or monoclonal antiserum against gD)
Was added to each aliquot and then incubated at 4 ° C. for 60 minutes. (The amount of antiserum added is determined by calibrating the antiserum titer by testing serial dilutions of the antiserum with known amounts of antigen.) The immune complex was washed with pansorbin ( Pansorb
in) (Staphylococcus aureus
Protein A, Calbiochem-Behring
Corp. , LaJolla, Cal. ) Is added,
Harvested by centrifugation (unless otherwise noted, all centrifugation in this procedure was performed at 5,000 xg at 4 ° C for 5 minutes). IP-2 pelleted immunoprecipitate
Resuspend in IP3 (same as IP-3 but with 20 mg / ml bovine serum albumin BSA added to eliminate non-specific adsorption), wash twice, and wash with IP-3
P-1 [20 mM Tris HCl (pH 8.1), 10
0 mM NaCl, 1 mM EDTA and 1% NP-4
0]. Transfer the suspension to a new tube, centrifuge it and pellet the SDS-polyacrylamide gel sample buffer (Laemml).
i, 1970, Nature 227: 680). After heating at 95 ° C. for 3 minutes, the sample was centrifuged for 2 minutes at 12,000 × g in a microcentrifuge to remove insoluble components. The supernatant was removed and loaded on an SDS polyacrylamide gel (10%). After electrophoresis, proteins were stained with Coomassie blue dye, treated with sodium salicylate and dried for fluorography (Chamberrain, 1979, An.
al. Biochem. 98: 132). The results of SDS polyacrylamide gel electrophoresis (SDS-PAGE) showed that the 46,000 dalton gD-1 related protein produced from pEH25 after induction showed that the whole rabbit antiserum against HSV-1 and monoclonal antibody 1S, 4.
It clearly showed that it was immunoprecipitated by S, 55S and 57S.

Finally, a competition experiment was performed to examine the effect of lysates of HSV-1-infected Hela cells on the immunoprecipitation of induced gD-1-related proteins expressed from E. coli NF1829 transformed with pEH25. (See Figure 6). Control (non-infected) and HSV-1 infected Hel
Serial dilutions were prepared from lysates of a cells (infection was performed as previously described for Vero cells). A 5 μl aliquot of a 100-fold dilution of monoclonal 55S ascites (gD-1 specific monoclonal antibody) was added to He
La cell lysate was added to each aliquot of serial dilutions. Induced p after incubation for 30 minutes at 4 ° C
Equal amounts of ³⁵ S-methionine labeled protein from lysates of EH25 transformants were added to Hela cell lysates and incubated for an additional 60 minutes at 4 ° C. Immune complexes were recovered by immunoprecipitation and SD was used as described above.
Analyzed by S-PAGE and fluorography. The specifically immunoprecipitated protein band was excised from the gel and radioactivity was measured by scintillation counting. Figure 6 shows the results of these experiments. The radioactivity of each sample represents the immunoprecipitated labeled protein product of pEH25 and was plotted as a% of control. Circles are serially diluted controls (uninfected)
Represents Hela cell lysate. Squares are serially diluted H
SV-1 infected Hela cell lysate is shown. This clearly indicates that the HSV-1 protein competes with the radiolabeled protein from pEH25 for the formation of immune complexes with the 55S monoclonal antibody.

6.4. Cro / gD-1 / β-galacto
PEH4-2 leading to the production of sidase fusion protein
The gD-1 gene was isolated from pEH25 and its termination signal was deleted. For this, the DNA fragment containing the gD-1 gene sequence was cleaved at a restriction site beyond the gD-1 termination sequence TAG. Then, TAG was digested with Bal 31 of the DNA nuclease by forward digestion of the ends.
Excluded. This gD-1 gene fragment was then inserted into pJS413 so as to encode the fusion protein: Cro / gD-1 / β-galactosidase.
This procedure is described below and is shown in FIG.

Plasmid pEH25 was digested to completion with NruI and progressively digested with Bal 31 nuclease. Next, the obtained DNAs of various lengths were digested with the restriction enzyme Ps.
Cleavage with tI generated a wide range of fragments, many lacking the gD-1 stop codon, but most gD
-1 gene sequence was carried. Appropriate DNA fragments (1.5-1.9 kb) were separated by gel electrophoresis and eluted as previously described. Vector pJS4
13 was digested to completion with PstI and SmaI and the appropriate 5.5 kb DNA fragment was isolated (FIG. 7).
The pEH25 fragment and the pJS413 fragment were ligated in a 1: 1 ratio and used to transform E. coli NF1829. Ampicillin resistant colonies were examined for fusion protein production by assaying β-galactosidase activity on the indicated agar plates (Mil.
ler, Experiments in Molec
lar Genetics, Cold Spring
Harbor Press, New York, N.M.
Y. , 1972). Positive colonies were tested for the presence of the Cro / gD-1 / β-galactosidase fusion protein by SDS-PAGE analysis of total lysates of IPTG-induced transformants. Cro / gD-1 /
From these clones expressing the β-galactosidase fusion protein, a high level producer of the 160,000 dalton fusion protein was isolated. The plasmid contained in this clone was designated as pEH4-2 (Fig. 7). pE
The fusion protein produced by the E. coli clone transformed with H4-2 was found to be inducible by IPTG and yet cross immunoreactive with both HSV-1 and HSV-2. The pEH4-2 plasmid was isolated and analyzed by restriction mapping and DNA sequencing. pEH4-2 does not contain the BamHI site of pJS413 (see Figure 8).

Another E. coli isolate transformed with the plasmid designated pEH3-25 produced lesser amounts of the Cro / gD-1 / β-galactosidase fusion protein than the pEH4-2 transformants. The pEH3-25 transformant will be described later in the text (see Section 6.5). 6.4.1. pEH4-2 fusion protein is anti-HSV-
A fusion protein produced from E. coli transformed with pEH4-2 that immunoreacted with 1 serum specifically interacted with a rabbit antiserum against HSV-1 (data not shown). pEH4
-2 and pEH25 IPTG-induced proteins to S
Separated by DS-PAGE and transferred to nitrocellulose (ie protein "blot" was performed).
Lysates of uninduced pEH4-2 and pEH25 transformants were treated by the same procedure as controls. next,
Nitrocellulose was treated with rabbit antiserum to HSV-1 followed by ¹²⁵ I-labelled goat antiserum to rabbit immunoglobulin as a probe (Towb.
in et al., 1979, Proc. Natl. Acad. S
ci. , U. S. A. 76: 4350).

An autoradiogram of the protein blot shows that the 160,000 dalton pEH4-2 derived fusion protein immunoreacts with a rabbit antiserum directed against HSV-1 and 46,000 dalton pEH25.
It was demonstrated that the derived gD-1 related protein immunoreacts with a rabbit antiserum against HSV-1. 6.4.2. Anti-blood against pEH4-2 fusion protein
Qing immunoprecipitates HSV-1 and HSV-2 proteins
The antiserum against the pEH4-2 fusion protein is HSV
It was found to immunoreact with -1gD and HSV-2gD, thus demonstrating that the pEH4-2 fusion protein contains gD-specific antigenic determinants. This is pE
SDS-PAGE analysis of HSV-1 and HSV-2 proteins immunoprecipitated with antisera to H4-2 fusion protein. See discussion below.

Rabbit antiserum to the pEH4-2 fusion protein was prepared as follows. E. coli clones transformed with pEH4-2 were grown to mid-log phase, 1
Induction was performed with mM IPTG. 4 hours after induction,
Bacteria were pelleted by centrifugation and SDS-PAG
E Lysis with sample buffer and SDS-for separation
Placed on a polyacrylamide gel. After electrophoresis, the outer lane was stained with Coomassie blue dye to visualize the proteins. The 160,000 Dalton fusion protein band was then excised from the gel. Gel sections were soaked in liquid nitrogen, ground and resuspended in PBS. An equal volume of Freund's complete adjuvant was added. After mixing well, this solution was mixed with two New Zealand rabbits (01
8 and 019) subcutaneously. 25 to 5 for each rabbit
0 μg of protein was injected. After 28 days, rabbits were boosted with the same amount of fusion protein suspended in incomplete Freund's adjuvant. Two or more booster immunizations were performed every 10 days. 55 days after the first injection, blood was collected from the ear, serum was collected, and subjected to immunoprecipitation analysis.

Immunoprecipitation was performed as follows. Confluent cultures were infected with HSV at 10 pfu / cell (10 plaque forming units / cell) and post infection with ³⁵ S-methionine.
Labeled for 6 hours. [GBK (Georgia bovine kidney) cells were infected with HSV-1, while HeLa cells were infected with HSV-1.
-2 was infected. Vero cells were infected with either HSV-1 or HSV-2. ] The lysate of HSV-infected cells labeled with ³⁵ S-methionine was added to 10 μm.
Incubated with 1 pre-immune rabbit serum, rabbit antiserum to pEH4-2 fusion protein (eg, 018 or 019 antiserum), or 1 μl of monoclonal antibody 4S. Immune complexes were recovered with pansorbin as described in Section 6.3.3, immunoprecipitated radiolabeled proteins were separated by SDS-PAGE and subjected to fluorography. SDS-PA
The results of GE were as follows: (1) The 52,000 dalton gD protein produced from GBK cells or Vero cells infected with HSV-1 was treated with antiserum against the fusion protein produced from pEH4-2 transformants; 4 more
Immunoreactivity with S monoclonal antibody, (2)
The 50,000 dalton gD protein produced from HSV-2 infected HeLa or Vero cells was shown to be immunoreactive with antisera to the pEH4-2 fusion protein and the 4S monoclonal antibody. HSV-2 when compared to HSV-1 derived gD
The apparent low molecular weight of gD from which is consistent with published observations (Ruyechan et al., 1979, J. Viro.
l. 29: 677). The result of fluorography is H
G produced by SV-1 or HSV-2 infected cells
The D protein was shown to be immunoreactive with antisera to the fusion protein produced from pEH4-2 transformants.

64.3. Antiserum against pEH4-2
Infected with HSV-1 and HSV-2 in vitro
Rabbit antiserum against the pEH4-2 fusion protein that neutralizes
It was also analyzed for its ability to neutralize the infectivity of the virus. Virus neutralization was assayed by the reduction in plaque number of infected cells in tissue culture (Showalter).
Et al., 1981, Infection and Immu.
(Nity 34: 684). For this, 50-100p
Pre-treatment of fu HSV-1 or HSV-2 with preimmune serum (control), immune antiserum (018 or 019) or dilutions of monoclonal antibody 4S in the presence or absence of active serum complement (C ′). Incubated. Vero cells were infected with a preparation of HSV-1 or HSV-2. After 3-4 days, HSV plaques were counted to determine the effect of antisera on the reduction of plaque numbers.
The results shown in Table 1 show that the antiserum obtained from each rabbit was in
It has been shown to be capable of neutralizing HSV-1 and HSV-2 in vitro. Neutralization was also demonstrated in the absence of active complement, but complement significantly increased neutralizing activity. Neutralization was much more effective against HSV-1 than HSV-2.

[0128]

[Table 1] Serum 018 and 019 from ¹ rabbit in virus on Vero cell monolayers in the presence of 5% heat inactivated complement (30 min at 56 ° C, -C ') or active guinea pig complement (+ C'). Tested for sum. The number is the number of plaques is 5
Antibody titers as the reciprocal of the 0% reduced serum dilution are shown. ² In these tests were used GBK cells instead of Vero cells. The displayed results are HSV-1 and HSV.
PEH4-as a subunit vaccine against C-2
The availability of two fusion proteins is shown.

6.4.4. gD-1 remains in pEH4-2
Using the gene reconstructed plasmids pJS413 and pRWF6, g
The D-1 gene was reconstructed (Fig. 8). Thus, pRW
F6 was digested with HindIII and Bal31 to generate randomly sized blunt end fragments. After digesting these fragments with SacI, 2.2-
The 2.4 kb blunt ended / SacI fragment was isolated. These fragments have different lengths of gD
It included the amino-coding end of the -1 gene (see Figure 8).

The gD-1 fragment was subcloned into pJS413. For this purpose, pJS413 is added to Sm
aI (resulting in blunt ends) and SacI (SacI
Resulting in 3'sticky ends). 4.7 kb S
The maI / SacI pJS413 fragment was blunted / SacI pRWF6 fragment (2.2-
2.4 kb) and ligated at a ratio of 1: 1 to E. coli NF18
Used to transform 29 strains. This resulted in a population of clones transformed with a plasmid containing the gD-1 gene randomly "deleted" at the amino coding end (Fig. 8). pEH50 + x (where x is 1 to
A total of 24 plasmids (meaning 24) (about 7 kb each) were analyzed by mapping restriction enzyme recognition sites. Those containing a PvuII site within the gD-1 gene (19 out of 24 transformants) were further analyzed to identify clones containing the longest amino-coded end of gD-1.

In the complete gD-1 gene sequence, gD
The distance between the starting ATG and the internal PvuII site is 156 base pairs. Therefore, each of the 19 pEH50 + x plasmids was digested with BglII and PvuII.
The resulting fragments were separated by gel electrophoresis and the Bg
The size of the II / PvuII fragment was examined. 1
Fifteen pEH50 + x plasmids with a BglII / PvuII fragment smaller than 60 base pairs were identified. That is, the end point of Bal31 digestion shown in FIG. 8 is gD-
1 was somewhere between the starting ATG and PvuII sites.
By analyzing these 15 sequences, the ligation site to the pJS413 plasmid was accurately determined. 7 plasmids, pE
H51, pEH60, pEH62, pEH66, pEH
71, pEH73 and pEH74 are the translation reading frame of croATG of pJS413 and inph.
It contained an asse-linked gD-1 sequence (see Figure 3, where these ligation sites are indicated by the corresponding pEH numbers and vertical arrows). In fact, pEH51 is g
D-1 encodes all but the first 6 amino acids at the amino terminus.

Transformants of pEH51, pEH60, pEH62 and pEH71 were labeled with ³⁵ S-methionine with or without IPTG induction and cell lysates were treated with monoclonal antibody 55S. Immunoprecipitates were analyzed by SDS-PAGE as described above. pE
Transformants containing H51, pEH60, pEH62 or pEH71 each produced an inducible protein of 46,000 daltons that was precipitated by monoclonal 55S (data not shown).

Finally, the recombinant plasmid encoding the fusion protein was constructed in pEH4-2 in order to reconstruct the amino coding end of the gD-1 gene sequence present in pEH4-2.
It was constructed using the amino-coded end of H51 (see Figure 9). Plasmid pEH4-2 was added to PstI and Sac
After digestion with II, a 6.2 kb fragment containing the partial gD-1 / β-galactosidase gene was isolated. Plasmid pEH51 was digested to completion with PstI and SacI digested.
Partially digested with I. 1.25 kb P of pEH51
The stI / SacII fragment was added to pEH4-2 6.
Ligation with the 2 kb PstI / SacII fragment at a 1: 1 ratio created pEH82. Transformants were identified by screening for β-galactosidase activity as described above. The E. coli transformant carrying the pEH51 plasmid was named E. coli WW51 strain, and pEH8
The transformant containing 2 plasmids is designated as Escherichia coli WW82 strain.

We have shown that the HSV gD-1 fusion protein can be produced in large quantities in E. coli. The fusion protein is probably very stable due to the composition of the fusion protein itself, or due to sequestration of the fusion protein in dense, insoluble inclusion bodies. We have shown that the fusion protein can be extracted from E. coli and used to immunize animals. The resulting antiserum is able to neutralize both HSV-1 and HSV-2 plaque formation in vitro.

6.5. Cro / gD-1 and Cro /
Produces gD-1 / β-galactosidase fusion protein
Generation of pEH90-10amLE392 Containing both fused and unfused gD in a suitable host cell
A recombinant plasmid pEH90-10am capable of producing -1-related proteins was constructed (see Figure 10).

The pEH90-10am plasmid contains Cr
It was derived from a series of recombinant plasmids pEH-90-N, which was derived from pEH3-25 encoding the o / gD-1 / β-galactosidase fusion protein (see Section 6.4) (FIG. 3). And FIG. 10). pEH
The -90-N series is matched in the translation reading frames of the cro, gD-1 and z-genes (in pha
is similar to pEH4-2 (section 6.4), but the pEH-90-N sequence has an additional unique restriction endonuclease recognition sequence at the junction of the gD-1 and z-genes. Is included.

The recombinant plasmid designated pEH-90-10, isolated from one transformant of the pEH-90-N series, was further modified as follows. (1)
pEH-90-10 is cleaved at the junction of the gD-1 gene sequence and the z-gene sequence, (2) then pEH-90-
10 were ligated in the presence of a DNA linker sequence containing the amber chain termination sequence TAG. The resulting recombinant plasmid pEH-90-10am contained the gD-1 gene and z-
It contains the amber chain termination sequence TAG which matches both translational reading frames of the gene.

PEH90-10am was transformed into E. coli NF182.
When used to transform 9, ampicillin-resistant transformants produced no detectable fusion protein. However, when pEH-90-10am transformants were infected with the lysogenic transducing phage φ80 SuIII containing the amber suppressor tRNA gene, the induced transformants were Cro / gD-1 / β-galactosidase fusion protein. Produced. This lysogen is named pEH90-10am SuIII.

The pEH90-10am plasmid was also used to transform E. coli LE392 with two amber suppressor mutations (SupE and SupF). The pEH90-10amLE 392 transformant produced the Cro / gD-1 / β-galactosidase fusion protein under both inducing and non-inducing conditions (LE392 cells lead to overproduction of the lac repressor NF1.
No 829 lacI ^Q mutations). pEH90-1
SDS-PAGE analysis of the protein produced by the 0amLE392 transformant confirmed that both fusion protein (approximately 160,000 daltons) and unfused gD-1 related protein (approximately 38,000 daltons) were produced. It was Both proteins are rabbit anti-HS
Immunoreacts with V-1 serum. pEH90-10amLE
The 392 transformants appear to produce both proteins in approximately equimolar amounts, and the two proteins co-purify when isolated by the non-denaturing co-aggregate purification method described in Section 5.5. The details of this procedure are described in the following sections.

6.5.1. Generation of pEH90-N Series As described previously in Section 6.4, pEH3-25 transformants produce the Cro / gD-1 / β-galactosidase fusion protein, but the yield is pEH4-2. Less than convertible. The pEH3-25 recombinant plasmid contains pEH4-2 except that it has the transmembrane sequence of gD-1.
(See FIG. 3). The transmembrane sequence may be involved in inefficient expression of the fusion protein by host cell transformants.

The expression vector pHK414 (FIG. 10) is p
It is a JS413 derivative. In fact, pHK414 is pJS
It contains all the elements of 413, but differs in having its unique cloning site across the cro-z junction. p
The cloning sites of HK414 are BglII, Hin
dIII, SmaI and BamHI. The z-gene is not in reading frame with croATG and therefore the intact plasmid does not control β-galactosidase production. However, the appropriate length (3n +
When the DNA fragment of 2) is inserted into any of these cloning sites of the plasmid, the reading frame of the z-gene is readjusted to croATG, and the inserted DNA sequence is croATG or z-gene. The β-galactosidase fusion protein will be produced by the host cell transformant, provided that it does not contain the termination signal present in the inphase (eg, TGA, TAA or TAG).

The pEH90-N series was constructed as follows (see FIG. 10). First, pEH3-25 was digested with BamHI, and then the cleaved plasmid was digested with Bal31 to remove the gD-1 transmembrane sequence at the carboxy-coding end.
Processed in. After Bal31 digestion, pEH3-25 was cleaved with PstI to remove the PstI sticky end, the amino coding end of the amp ^r gene, the lac promoter and translation regulatory elements, all or part of the transmembrane sequence gD-. 1 gene, and blunt ends (BamH
A population of 1.7-1.9 kb DNA fragments characterized by I ^(-) ) was isolated by agarose gel electrophoresis.

The expression plasmid pEK414 was prepared by
Cleavage with glII and the BglII sticky ends were repaired with the Klenow fragment of DNA polymerase.
Next, the linear blunt-ended pHK414 was digested with PstI to give blunt ends (BglII ^(-1) ), z gene, a
the carboxy coding end of the mp ^r gene and PstI
A 5.5 kb DNA fragment characterized by sticky ends was isolated by agarose gel electrophoresis.

1.7 to 1.9 kb P of pEH3-25
stI / BamHI ^(-1) DNA fragment and pH
5.5 kb BglII ^(-1) / PstI of K414
DNA fragments in a 1: 1 molar ratio to T4 DNA
Ligation was used to ligate. The resulting population of plasmids, designated pEH90-N, was used to transform E. coli NF1829 grown on the indicated agar plates.

The following recombinant plasmids, derived from 10 of the 24 transformants that produced the fusion protein, were passed through the gD-1 / z-gene junction (see FIG. 3).
Analyzed by NA sequencing. That is, pEH90-
2, pEH90-3, pEH90-4, pEH90-
5, pEH90-6, pEH90-7, pEH90-
9, pEH90-10, pEH90-11 and pEH
It was 90-12. All but pEH90-12 contain deletions of all or part of the transmembrane sequence. pEH90-
4 and pEH90-5 contain almost half of the transmembrane sequence. With the exception of pEH90-12, these plasmids show high levels of Cr produced by pEH4-2.
This led to the production of o / gD-1 / β-galactosidase fusion protein. Plasmid pEH90-10 was selected for the next step in this procedure (the gD-1 fusion protein produced by the pEH90-10 transformants was
The same as that produced by the pEH4-2 transformant. However, the last four amino acids of gD-1 produced by the pEH4-2 transformant were pEH90-10.
It is missing from the fusion protein produced by the transformants).

6.5.2. Construction of pEH90-10am The following procedure was used to introduce an amber chain termination sequence between the gD-1 sequence and the z-gene of the pEH90-10 plasmid (see Figure 10). pEH90-10 for Hi
cleaved with ndIII and then with BamHI to give gD
The plasmid was cleaved at its unique restriction endonuclease site located at the junction between the -1 and z-genes (see below).

[0147]

Embedded image

7.3 kb HindIII / BamHI
The cleaved plasmid was isolated by agarose gel electrophoresis and HindIII (using T4 DNA ligase).
/ BglII sticky end and next DN characterized by internal XbaI site and amber sequence (TAG)
A linker was connected.

[0149]

[Chemical 6]

Each complementary strand of the DNA linker is
Et al., 1981, Nucleic Acids Res9
(12): 2807-2817 and was synthesized by the solid phase method. After ligating the linker to the digested plasmid, the resulting recombinant plasmid was digested with XbaI and the linear 7.3 kb DNA fragment was isolated by agarose gel electrophoresis and ligated with T4 DNA ligase for recircularization. (This is in fact indicated by the presence of a single XbaI site on the plasmid, gD-
The pEH90-10 plasmid was selected which contains a linker sequence linked between the HindIII and BamHI sites at the 1 / z junction). As a result of the ligation, the amber sequence of the DNA linker was in translation reading frame with the gD-1 and z-genes (see below).

[0151]

[Chemical 7]

The resulting plasmid was pEH90-10a.
It was named m. 6.5.3. The pEH90-10am transformant pEH90-10am was used to transform E. coli NF1829. Ampicillin-resistant transformants did not synthesize detectable levels of fusion protein when induced with IPTG (no red colonies on MacConkey-directed agar plates). The transformants were then infected with the lysogenic transducing phage φ80 SuIII carrying the amber suppressor tRNA gene. Lysogenic transformants induced with IPTG formed red colonies on MacConkey-indicated agar plates, indicating production of the fusion protein. These lysogens were transformed into pEH90-10a
It was named mSuIII.

Furthermore, the pEH90-10am plasmid contains two amber suppressors (supE and su).
It has a pF) using E. coli LE392 not excessively synthesized lac repressor for no lacI ^Q mutation NF1829 to transform. These transformants, named pEH90-10amLE392, produced fusion proteins whether induced by IPTG.

6.5.4. pEH90-10amLE3
Analysis of proteins produced by 92 transformants Fusion proteins (approximately 160,000 daltons) and 3
A protein of 8,000 daltons, p in almost equimolar amounts
Produced from EH90-10amL E392 transformants. Each protein was found to immunoreact with a rabbit antiserum against HSV-1. For this, cellular proteins were isolated by the mini-aggregate method (described below) and
Separation by DS-PAGE, transfer to nitrocellulose, followed by treatment with rabbit antiserum against HSV-1, and against rabbit immunoglobulin as a probe.
Treated with ¹²⁵ I-labeled goat antiserum (Towbin
Et al., 1979, Proc. Natl. Acad. Sc
i. , U. S. A. 76: 4350).

The following mini-aggregate method was employed in isolating host cell aggregates for screening analysis. (1) Fresh L containing 100 μg / ml ampicillin
Overlapping culture tubes containing 5 ml of uria broth were inoculated with 200 μl of cells obtained from an overnight culture and grown at 37 ° C. for 90 minutes. One for each duplicate
Induction was by the addition of mM IPTG (final concentration). The inoculum was then grown for another 5 hours at 37 ° C with agitation.

(2) The cells contained in a 3 ml aliquot of the culture were mixed with a microcentrifuge (12,000 × g) to give 2 cells.
Centrifuge for minutes to pellet. (Caution: The total volume of the microcentrifuge tube is 1.5 ml. Therefore, the 1.5 ml aliquot was first pelleted and the supernatant was removed. Add another 1.5 ml aliquot to the same microcentrifuge tube (3) The cell pellet was suspended in 50 μl of 50 mM Tris-HCl, pH 8 containing 25% sucrose, and the suspension was frozen by placing the centrifuge tube in a dry ice / ethanol bath. .

(4) The frozen suspension was thawed, 50 μl of 10 mg / ml lysozyme in 0.25 M Tris-HCl, pH 8 was added and the suspension was incubated on ice for 10-15 minutes. (5) 400 after incubation for 10 to 15 minutes
μl TET buffer (100 mM Tris-HCl
pH 8, 50 mM EDTA, 2% Triton X-10
0; Triton X-100 was added nonionic surfactant polyoxyethylene (9,10) p-tert-octylphenol), the suspension was gently mixed and incubated on ice for 5 minutes. Then 500 μl of 2
X RIPA (2 x RIPA is 20 mM Tris-HCl,
pH 7.4, 300 mM NaCl, 2% sodium deoxycholate, 2% NP-40, 0.2% SPS) was added and the suspension was gently mixed and incubated on ice for 5 minutes.

(6) Next, the cells in the suspension were sonicated with a microprobe for 10 seconds to suspend the suspension.
20,000 rpm (4
(7,800 xg) and clarified by centrifugation for 30 minutes. (7) The supernatant was decanted, and the pellet containing the coaggregate protein was resuspended in 50 μl of distilled water, to which 50 μl was added.
1 × 2 × sample buffer (2 × sample buffer is 10% SDS, 40 mM Tris-HCl, pH 6.
0.8% of 2% β-ME and bromophenol blue.
02 volume saturated solution) was added and mixed well. The mixture was boiled for 5 minutes and a 25 μl aliquot was applied to a 10% SDS-polyacrylamide gel for electrophoresis.

After separating the proteins by SDS-PAGE, they were transferred to nitrocellulose (ie:
A protein "blot" was performed). The nitrocellulose is then treated with a rabbit antiserum against HSV-1
Then against rabbit immunoglobulin as a probe
Treated with ¹²⁵ I-labeled goat antiserum (Towbin,
1979, supra). The autoradiogram of the protein blot clearly showed that the two bands immunoreacted with the anti-HSV-1 antibody. A 160,000 Dalton fusion protein (Cro / gD-1 / β-galactosidase) and a 38,000 Dalton gD-.
1-related protein (Cro / gD-1) or unfused g
D protein (not fused to β-galactosidase)
Is. Thus, when using the coaggregate isolation method to isolate proteins, the non-fusion protein is co-purified with the fusion protein. And non-fused gD (Cro
/ GD-1) and gD fusion protein (Cro / gD-1
/ Β-galactosidase) both immunoreact with anti-HSV sera. 7. Example: HSV-2gD The genomic map of HSV-1 was compared to that of HSV-2. The position of the gD coding sequence within the HSV-2 genome is
It was determined by analogy with the position and size of gD within the HSV-1 genome. BglII in the Us region of HSV-2
The L fragment was inserted into pBR322 to create recombinant plasmid pHV1. gD contained in pHV1
The position of the −2 coding sequence was determined by performing hybridization using a part of gD-1 DNA obtained from pSC30-4 as a hybridization probe. pHV1 containing the gD-2 coding sequence
The portion of was then subcloned into pBR322 to generate recombinant plasmid pHV2. DNA sequence of gD-2 gene within pHV2 was determined and
The -2 sequence was compared to that of gD-1. gD-2 D
The NA sequence is one codon shorter than the gD-1 DNA sequence, but there is considerable homology between these two sequences.

To put the gD-2 gene under the control of the lac promoter, the first 12 of the protein coding sequences were placed.
Part of the gD-2 gene sequence lacking 0 nucleotides
Isolated from HV2. This DNA fragment was
It was ligated to a small DNA fragment encoding the c promoter and translational regulatory elements. The resulting recombinant plasmid pHV5 produced gD-2-related proteins from host cell transformants.

Finally, the gD-2 gene fragment was digested with pHV by restriction endonuclease cleavage at a specific site.
5 from which the termination sequence (TAG) of the gD-2 coding sequence was deleted. This DNA fragment contains part of the gD-2 gene and the promoter and regulatory elements of the expression plasmid and was ligated into the vector pHK414 containing the β-galactosidase coding sequence. The obtained recombinant plasmid pHV6 was croSD
The fusion protein was expressed in induced E. coli transformants containing the gD-2 coding sequence flanked by the plasmid lac promoter with -ATG and the β-galactosidase gene. The pHV6 fusion protein was isolated from host cell lysates and formulated for use as an immunogen. Details of each construction process are described in the following sections.

7.1. General procedure used for plasmid construction
Unless this order otherwise stated, DNA isolation, the general method described in Section 6 and its segments for enzymatic reactions Oyohi ligation were utilized in the cloning of gD-2. 7.1.1. The buffer used for the restriction enzyme buffer SmaI digestion was 6.6 mM Tris-.
Consists of HCl (pH 7.4), 6.6 mM MgCl ₂ and 6.6 mM β-ME.

The buffer used for BglII, ClaI, EcoRI or PstI digestion was 60 mM NaC.
1, 6.6 mM Tris-HCl (pH 7.4), 6.6
Consists of mM MgCl ₂ and 6.6 mM β-ME.
The buffer used for BamHI, SalI or XhoI digestion was 150 mM NaCl, 6.6 mM Tris-HC.
1 (pH 7.4), 6.6 mM MgCl ₂ and 6.
Consists of 6 mM β-ME.

Two or more restriction endonuclease reactions are D
It should be noted that when performed on NA, the reaction in low salt buffer is performed before the reaction in high salt buffer. If the two enzymes require the same buffer, the reactions may be performed simultaneously. 7.2. Localization and isolation of the gD-2 gene The HSV-1 and HSV-2 genomic maps were compared as previously described to determine the approximate location and size of gD within the HSV-2 genome. The gD-2 gene is 8.
Us contained within a 5 kb BglII L fragment
Mapped between 0.9 to 0.945 genomic map units within the region. BglII L fragment into HSV
-2 DNA, excised from pBR3 for further analysis
Inserted in 22.

7.2.1. pHV containing the gD-2 gene
Construction of 1 HSV-2 (G strain) genomic DNA was digested with BglII and a DNA fragment of about 8.5 kb containing the gD-2 gene was isolated by agarose gel electrophoresis. The plasmid pBR322 was completely digested with BamHI and the resulting 4.4 kb pBR322 DNA and HSV-2 were obtained.
Of the 8.5 kb BglII L DNA fragment of BglII (BamHI cohesive ends and BglII
PHV1 was obtained by ligation at a 1: 1 ratio (the sticky ends are complementary) (FIG. 11b).

7.2.2. gD-2 specific mRNA coding
The localization of the gD-2 mRNA coding sequence was obtained from pSC-30-4 as a hybridization probe.
-1 Hybridization using a part of DNA (Southern, 1975, J. Mol. Bio
l. 98: 503) for localization in pHV1 (for protocols used for Southern transfer, see Maniatis et al., 1982, Molecular).
r Cloning, A Laboratory Ma
numeric, Cold Spring Harbor L
laboratory, pp. 382-389).

Thus, pHV1 was cleaved with XhoI,
The obtained DNA fragments were separated by agarose gel electrophoresis. Next, the DNA fragment in the gel was denatured in alkali, neutralized, and transferred to a nitrocellulose filter. After that, D attached to the filter
The NA fragment was hybridized to the ³² P-labeled gD-1 probe.

The ³² P-labeled gD-1 probe was prepared as follows. pSC30-4 (Fig. 1d and Fig. 4) was Pv
cleaved with uII, and the first 52 codons of gD-1 (15
A 500 bp DNA fragment containing 6 nucleotides) was isolated by polyacrylamide gel electrophoresis. gD-1 DNA was radioactively labeled by nick translation (BRL Kit, Bethesda R).
essearch Laboratories, In
c. , Gaithersburg, MD). Nick translation converts double-stranded DNA into high radioactivity i
A preferred method for n vitro labeling. This method utilizes two of several activities of E. coli DNA polymerase I. That is, it has a 5'-3 'exonuclease activity and a 5'-3' polymerase activity. In nick translation, this enzyme binds to the nick on one strand of double-stranded DNA. The 5'-3 'exonuclease activity then hydrolyzes the nucleotides of the nicked strand prior to the advancing polymerase. Thus, the nick is moved (translated) along the chain. There is no net synthesis as the rate of hydrolysis is equal to the rate of polymerization. However, during the course of this exchange reaction, the radioactive deoxynucleoside triphosphates present in the reaction mixture are incorporated into the DNA (Kelly et al., 1
970, J.I. Biol. Chem. 245: 39). The ³² P-labeled gD-1 double-stranded DNA was then denatured for use as a single-stranded probe (Maniatis).
Et al., Molecular Cloning, A Lab
oratory Manual, ColdSpring
Harbor Laboratories, pp. 1
09-112).

By autoradiography, pHV1
Of the approximately 2.3 kb XhoI / XhoI DNA fragment was demonstrated to be complementary to the radioactive gD-1 probe and thus contained the gD-2 coding sequence. The XhoI / XhoI DNA fragment of pHV1 was then subcloned into pBR322 to further characterize the gD-2 coding sequence. To that end, pHV1 was digested with XhoI and a 2.3 kb DNA fragment containing the gD-2 coding sequence was digested with SalI.
Anneal to linear pBR322 that had been previously cleaved with (the SalI generated sticky ends are complementary to the XhoI generated sticky ends) and ligate at a 1: 1 ratio to create pHV2 ( FIG. 11c).

7.2.3. gD-2 mRNA coding sequence
Row Characterization The HSV-DNA of pHV2 is described by Maxam and Gilber.
Method of t (1980, Methods in Enzy
Sequencing was carried out using the chemistry, 65: 499). FIG. 12 shows the obtained DN of the HSV-2 gD gene.
The A sequence is shown. This DNA sequence has 393 codons (gD
It contained an open reading frame (1 codon less than the -1 DNA coding sequence) and extended from the ATG at position 267. Since this site is presumed to be the initiator of the gD-2 gene, this putative gD-2
Gene sequences are expressed in expression vectors pJS413 and pHK4
This was confirmed by cloning into 13 (see below).

The predicted amino acid sequence of the gD-2 protein was compared with the predicted amino acid sequence of the gD-1 protein (FIG. 13). The two sequences are about 82% homologous, and there appears to be a non-homologous region in the next three regions. A leader sequence, a transmembrane region, and a putative anchor region. gD-2 protein is gD-1
One amino acid residue shorter than a protein.

7.3. Cloning of gD-2 gene
In order to place the expressed gD-2 gene under the control of the lac promoter, a portion of the putative gene sequence lacking the first 120 nucleotides of the amino coding end (5 'end of the gene) was
Isolated from HV2. The pHV2 DNA fragment was added to the lac promoter, translational regulatory element, croATG.
And small pJS4 containing 69 nucleotides of cro
It was ligated to 13 DNA fragments (about 200 bp). The resulting recombinant plasmid pHV5 (Fig. 14)
It contains all but the first 40 codons of the gD-2 sequence. In fact, pHV5 actually encodes all but 15 amino acids of the mature gD-2 protein. Usually, the first 25 amino acid residues of gD-2 (signal sequence) are cleaved when the native gD-2 protein is processed. The construction of pHV5 is described below.

7.3.1. Construction of pHV5 The recombinant plasmid pHV2 was digested with ClaI (Fig. 14).
), The resulting cohesive ends were labeled with Kle of DNA polymerase.
It was repaired with the now fragment. Next, the blunt-ended linear pHV2 was cut with EcoRI. 5.4 kb ClaI ^(-) / EcoR containing all but 120 nucleotides of the amp ^r gene and gD-2 coding sequence.
The I DNA fragment was isolated by agarose gel electrophoresis.

Expression vector pJS413 (6.3.1)
Section) was cut with SmaI and EcoRI. lac
200 bp encoding 69 nucleotides of promoter, SD ^z , SD ^cro , ^cro ATG and cro
The (0.2 kb) fragment was isolated by polyacrylamide gel electrophoresis. 5.4 kb ClaI
⁽⁻⁾ / EcoRI pHV2 DNA fragment and 0.2 kb EcoRI / SmaI pJS413D
The NA fragments were ligated with T4 DNA ligase in a 1: 1 molar ratio. Obtained recombinant plasmid p
HV5 was used for transformation of Escherichia coli NF1829 strain.

7.3.2. Form expressing gD-2 gene
Identification of Transformants Plasmids isolated from ampicillin-resistant E. coli transformants were analyzed by restriction endonuclease mapping to further examine their ability to express gD-2 related polypeptides. In NF1829, (l
Since the lac promoter is transcriptionally inactive (due to overproduction of the ac repressor), gD-2 related proteins would only be detectable after inducing the promoter with either 1 mM IPTG or 1-10 mM lactose.

A clone transformed with a recombinant plasmid designated pHV5 was found to produce an inducible gD-2 related protein. The inducible protein was found to be immunoprecipitated by a rabbit antiserum against HSV-2. 7.4. Cro / gD-2 / β-galactosidase fusion
Generation of pHV6 to Produce Protein To make a fusion protein, the gD-2 sequence was linked to a bacterial host gene sequence so that the translational reading frame was not interrupted by a termination signal. To that end, the pHV5 gD-2 coding sequence was truncated so that the gD-2 termination signal (TAG) was deleted. The pHV5 DNA fragment containing the lac promoter, translational regulatory elements and the Cro / gD-2 sequence was ligated to the pHK414 DNA fragment containing the z-gene.
The resulting recombinant plasmid pHV6 (Fig. 15) was Cro
/ GD-2 / β-galactosidase fusion protein.

7.4.1. Construction of pHV6 Plasmid pHV5 was digested with PstI and BamHI (see Figure 15). part of the amino-coding end of amp ^r gene, lac promoter, SD ^z SD ^cro , c
1.7 encoding roATG and cro / gD-2
The 5 kb PstI / BamHI pHV5 DNA fragment was isolated by agarose gel electrophoresis.

Expression vector pHK414 was added to PstI and Bst.
Digested with amHI. 5.54 kb encoding part of the carboxy coding end of the z and amp ^r genes
BamHI / PstI pHK414 DNA fragment of was isolated by agarose gel electrophoresis.
1.75 kb PstI / BamHI pHV5 DN
A fragment and 5.54 kb BamHI / PstI
The pHK414 DNA fragment was annealed, ligated at a 1: 1 molar ratio, and E. coli NF1.
Used to transform 829. Ampicillin resistant colonies were tested for fusion protein productivity by assaying β-galactosidase activity on the indicated agar plates. Positive colonies were Cro / gD by SDS-PAGE analysis of total lysates of transformants induced by IPTG.
The presence of -2 / β-galactosidase fusion protein was tested. A high level producer of the 160,000 dalton fusion protein was isolated and the plasmid obtained from this transformant was designated pHV6.

The fusion protein produced from the E. coli clone transformed with pHV6 is inducible by IPTG and is also HSV-1 and HSV-2.
Were found to be cross-immunoreactive with both. The fusion protein produced contains 265 amino acid residues of the gD-2 protein. 7.4.2. Antiserum against pHV6 fusion protein
Immunoprecipitation analysis The pHV6 fusion protein was purified using the denaturation protocol described in Section 6.4.2 and purified from 3 rabbits (R15
9, R160, R161) was used as follows to produce antiserum. Escherichia coli clones transformed with pHV6 were grown to mid-log phase and treated with 1 mM IPTG.
Induced by. Four hours after induction, the bacteria were pelleted by centrifugation, lysed with SDS-PAGE sample buffer and placed on a preparative SDS-polyacrylamide gel. After electrophoresis, the outer lane was stained with Coomassie blue dye to visualize the proteins. The 160,000 Dalton fusion protein band was then excised from the gel. Gel slices were dipped in liquid nitrogen, ground and then suspended in PBS. An equal volume of Freund's complete adjuvant was then added. After thorough mixing, this solution was subcutaneously injected into 3 New Zealand rabbits (R159, R160, R161). Each rabbit was injected with 100-200 μg of protein. After 28 days, rabbits were boosted with the same amount of fusion protein suspended in incomplete Freund's adjuvant. Animals received a total of 5 boosts every 10 days. Sera collected 48 days after the first injection (ie after two boosts) were used for immunoprecipitation analysis.

Immunoprecipitation was performed as follows. Confluently grown cells were infected with HSV at 10 pfu / cell (Vero cells were infected with HSV-1 while Hela was infected).
The cells were infected with HSV-2). Cells were labeled with ³⁵ S-methionine for 16 hours post infection. ³⁵ S-methionine-labeled HSV-1 infected Vero cells or HSV-2
Lysates of infected Hela cells were mixed with 10 μl of preimmune rabbit serum or rabbit antiserum against pHV6 fusion protein (ie, R159, R160 or R161 antiserum).
Incubated with 1. Immune complexes were recovered using pansorbin as described in Section 6.3.3 and the resulting radiolabeled immunoprecipitated protein was used for SDS-P.
Separated by AGE and subjected to fluorography. SDS
-PAGE results showed that the 50,000 dalton gD protein produced by HSV-2-infected Hela cells immunoreacted with antisera against the fusion protein produced from the transformants. R159 and R161
Antisera are specific for gD-2, while R160
The antiserum immunoprecipitates both gD-1 (52,000 dalton gD protein produced by HSV-1 infected Vero cells) and gD-2. Therefore, R16
0 is “type-common”. It should be noted that 018 (Section 6.4.2) directed against the gD-1 fusion protein encoded by pEH4-2 is also type-common.

74.3. Herpes simplex virus in
The rabbit antiserum described above against the neutralizing pHV6 fusion protein in vitro was used to examine its ability to neutralize HSV infection in vitro. To that end, virus neutralization experiments using the plaque assay were performed as described above. Each dish (35 mm) of confluent cells was tested with control (preimmune serum) or test antiserum dilutions (the following antisera: 018,
R159, R160, R161) were infected with 50 pfu of HSV that had been pre-incubated (Vero
Cells were infected with HSV-1, while Hela cells were infected with H
SV-2 infected). After 3 days, cells were fixed, stained and plaques were counted. Table 2 shows the results of two such experiments. Neutralization is expressed as the serum dilution required to reduce plaque numbers by 50%. From Table 2, p
Antiserum against HV6 fusion protein (R159, R1
60, R161) can neutralize HSV-2 infection in vitro, while antiserum to the pEH4-2 fusion protein (018) appears to be able to neutralize HSV-1 infection in vitro. 018 and R160 are type-common (based on immunoprecipitation data in Section 7.4.2), but 018 (anti-gD-1 fusion protein)
Is more effective at neutralizing HSV-1 in vitro, and R160 (anti-gD-2 fusion protein)
It is more effective at neutralizing HSV-2 infections in vitro.

[0182]

[Table 2]

^One number represents the antibody titer as the reciprocal of the serum dilution that reduced the plaque number by 50%. The assay was performed in the presence of serum complement. 7.4.4. Antiserum against pHV6 fusion protein
Immunofluorescence analysis An immunofluorescence assay was performed as follows. Confluent cells were infected with herpesvirus at 0.1 pfu / cell for 20 hours at 37 ° C. (Vero cells were infected with HSV-1 while Hela cells were infected with HSV-2). P cells
It was washed with BS and then fixed with acetone: methanol (1: 1) for 90 seconds at room temperature. The fixative was removed and the cells were allowed to air dry. Next, each rabbit antiserum diluted with PBS 1:20 (018, R159, R160 and R160).
A 20 μl aliquot of 161) was added to the cells on the plate as a separate drop at the marked position. 37 ° C, 3
After 0 minutes of incubation, cells were washed with PBS and air dried. Then a 20 μl aliquot of fluorescein-conjugated porcine anti-rabbit serum was added to the marked position. After incubation at 37 ° C. for 30 minutes, the cells were washed, naturally dried, fixed with glycerol, and observed under an ultraviolet ray using an ultraviolet microscope. The presence of fluorescence indicates that the rabbit antiserum is immunoreactive with HSV infected cells. The results are shown in Table 3.

[0184]

[Table 3]

¹ antiserum was added at a 1:20 dilution. ² ++ is the strongest cell staining ++ is a moderate cell staining + is a positive but weak staining 8. Example: Vaccination 8.1. Protection from in vivo HSV-2 infection with gD-1 fusion protein in vaccine formulation One group of 10 Balb C mice was injected intraperitoneally (IP) with the following preparations: Group 1 (non-vaccinated control) received saline, group 2 received 3 μg of native HSV-1 gD protein in complete Freund's adjuvant, and group 3 pEH4-2 fusion protein in saline (5. (Isolated by the modified aggregate purification method described in Section 5) 30 μ
I received g. All groups received 4 IP injections, each injection was given at 2 week intervals, and boosts contained 1 μg gD (group 2) or 10 μg fusion protein.

Mice were bled retroorbitally after the fourth injection and in vitro (complement absence was performed using the plaque assay described previously in Section 6.4.3 and Section 7.4.2). Sera were tested for herpes virus neutralization. One week after the fourth inoculation, 10L
Mice were challenged with D ₅₀ HSV-2 strain 186. HSV-2 was suspended in 0.5 ml and injected intraperitoneally. Survival of challenged mice indicates protection.
The results are shown in Table 4.

[0187]

[Table 4]

8.2. GD-2 fusion tag in vaccine formulation
From in vivo HSV-2 infection using protein
The defense 1 group of 10 mice of 4 groups of mice, the following preparations were vaccinated. Group 1 (control) received pNB9-1 bovine growth hormone / β-galactosidase fusion protein, group 2 pEH4-2 Cro / gD-1 / β.
-Receiving the galactosidase fusion protein (section 6.4), group 3 was pEH82-reconstructed Cro / gD-
Receives the 1 / β-galactosidase fusion protein (see Section 6.5), and Group 4 receives pHV6Cro
/ GD-2 / β-galactosidase fusion protein (see Section 7.4).

The fusion protein disrupts the induced transformants with lysozyme / detergent, followed by pelleting of aggregates (non-denaturing aggregate purification method as described in Section 5.5). By isolation from various E. coli transformants. The immunization schedule was as follows: 0.2 ml for Balb C mice (6-7 weeks old)
Non-denatured fusion protein 150-200 in saline
μg was inoculated intraperitoneally. Mice were reinoculated (boosted) intraperitoneally with 75-100 μg fusion protein every 2 weeks for 3 or more times (4 times in total). After the fourth injection,
Mice were bled retroorbitally and sera for herpesvirus neutralization in vitro (in the absence of complement) using the plaque assay described previously in Sections 6.4.3 and 7.4.2. Was tested.

One week after the fourth inoculation, 1
Mice were challenged with 7 LD ₅₀ of HSV-2 strain 186. HSV-2 was suspended in 0.5 ml and injected intraperitoneally. Survival of challenged mice indicates protection. The results are shown in Table 5.

[0191]

[Table 5]

¹ Fusion protein was inoculated with IP in saline. Sera were tested for HSV-1 or HSV-2 neutralization in the absence of ² complement. Numbers represent antibody titers expressed as the reciprocal of the serum dilution with 50% plaque reduction. 8.8. Comparison of Vaccine Formulations Mice were immunized with a mixture of fusion protein preparations produced from pEH4-2 and pEH90-10amLE392 transformants and different adjuvants described below. The obtained antisera were evaluated by their ability to immunoprecipitate proteins from HSV-1 infected cells in culture.

PEH4-2 and pEH90-10amLEE39 by the non-denaturing technique described in Section 8.2.
The fusion protein was isolated from the two transformants. Aggregates were resuspended by sonication (forming a slurry) and solubilized by treatment in hot alkali as follows. 0.5 MNa
OH was added to the pellet of aggregates at a final concentration of 50 mM. Aggregates were solubilized by heating at 65 ° C for 15 minutes. The solution is then cooled and Tris-HCl (pH
7.5) was added (final concentration: 100 mM Tris-
HCl).

The fusion protein preparation (slurry or hot alkaline preparation) was mixed with the following adjuvants before inoculation. (1) L121 (Hunter et al., 1981,
J. of Immunol. 127 (3): 1244-
1250; BASF Wyandotte Corpo
relation, Wyandotte, Mich. ) Pluronic polyol (administered 2.5 mg per animal), or (2) aluminum hydroxide gel (Rehe
is Chemical Company, Berke
ley Heights, N.M. J. ) 0.2% final concentration.

These preparations were used to immunize mice (CD-1 strain) according to the following schedule. Each mouse received a primary injection of 100 μg of fusion protein,
After 22 days, 50 μg of fusion protein was reinjected. The fusion protein was suspended in a total volume of 0.2 ml and injected intramuscularly in the thigh. Seven days after the last injection, blood was collected from the retroorbital of the mouse.

The collected sera were analyzed by immunoprecipitation as follows. ³⁵ S-methionine labeled HSV-1 infection V
Lysates of ero cells were treated with 5 μl of mouse serum (anti-pE
H4-2 or anti-pEH90-10amLE392),
Incubate with preimmune serum (negative control) or monoclonal 4S (positive control) for 2 hours at 4 ° C., 5 μ
Incubated with 1 rabbit anti-mouse serum (Dako for 30 minutes at 4 ° C.). Immune complexes were recovered with pansorbin as described in Section 6.3.3 and immunoprecipitated radiolabeled proteins were separated by SDS-PAGE and subjected to fluorography. From the results of fluorography, the alkali-solubilized fusion proteins (pEH4-2 and pEH90-1) with L121 adjuvant were added.
All mice immunized with 0 amLE392) were found to have a strong immune response. Alkali-solubilized pEH4 administered with aluminum hydroxide
A weaker immune response was observed with the -2 fusion protein. Other mice did not appear to develop an immune response as judged by this test method. 9. It should be understood that the sizes of all base pairs given to the deposited nucleotides of the microorganism are approximate and are used for illustration. Moreover, it will be apparent that many modifications and variations of the present invention set forth above can be made without departing from its spirit and scope. The particular embodiments described are merely illustrative and the invention is defined only by the claims.

The following E. coli strains carrying the indicated plasmids are American Type Culture Collection (Am
erican Type Culture Colle
action: ATCC) (Rockville, M
d. ) Has been deposited with the following deposit numbers. The next E. coli strain carrying the indicated plasmid is the Agricultural Research Culture Collection (Agr
icultural Research Culture
e Collection: NRRL) (Peori
a, Ill. ) Has been deposited with the following deposit number. The present invention is not limited in scope by the deposited microorganism.
This is because the deposited embodiments are illustrative of some aspects of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are also included in the scope of the claims.

[Brief description of drawings]

1 shows (a) HSV-1 genome, (b) HSV-1 EcoRI of lambda gtWES :: EcoRI-H.
-Shows a restriction map of the -H fragment insert, (c) pR
3 shows the construction of WF6 and (d) H of pSC30-4
FIG. 6 shows a restriction map of SV-1 SacI DNA fragment insert.

FIG. 2 shows the sequencing strategy and restriction map of the gD-1 gene sequence.

FIG. 3: Nucleotide sequence of gD-1 gene and gD
It is the figure which showed the deduced amino acid sequence of -1 protein.

FIG. 4 shows a recombinant plasmid pEH2 derived from a part of the gD-1 gene and the E. coli expression vector pJS413.
It is a figure showing the construction of No. 5.

FIG. 5. DN of Cro / gD-1 junction in pEH25
It is the figure which showed A sequence and deduced amino acid sequence.

FIG. 6 shows inhibition of immunoprecipitation of pEH25gD product by adding a competing antigen present in lysates of HSV infected Hela cells.

FIG. 7 shows the construction of gD-1 expression plasmid pEH4-2 derived from pEH25.

FIG. 8: Many gD-1 expression plasmids pEH50 + x
It is a figure showing the method of manufacturing.

FIG. 9 shows a method for reconstructing the amino coding end of the gD-1 gene in pEH4-2.

FIG. 10 shows the construction of the gD-1 expression plasmid pEH90-10am derived from pEH3-25 and pHK414.

FIG. 11 (a) shows the HSV-2 genome and (b) pH.
FIG. 6 shows a restriction map of the BglII L fragment insert of V1 and (c) HSV-2 XhoI of pHV2.
It is the figure which showed the restriction map of a DNA fragment insert.

FIG. 12: Nucleotide sequence of the gD-2 gene and g
It is the figure which showed the deduced amino acid sequence of D-2 protein.

FIG. 13 is a diagram showing a comparison of deduced amino acid sequences of gD-1 and gD-2.

FIG. 14 shows the construction of a recombinant plasmid pHV5 derived from a part of the gD-2 gene and pJS413.

FIG. 15 shows the construction of pHV5-derived gD-2 expression plasmid pHV6.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // C12N 1/21 8828-4B C12N 1/21 (C12N 15/09 ZNA C12R 1:92) ( (C12P 21/02 C12R 1:91) (72) Inventor John Haynes Weiss United States 01246 Bricklin Pond Avenue 33 Massachusetts 33 Apartment Bee 606 (72) Inventor Lynn William Enquist United States 55331 Minnesota Exelcia Zambra Drive 5691

Claims (6)

[Claims] 1. The following amino acid sequence: Herpes simplex virus type 1 (HSV-1)
A non-glycosylated polypeptide of the gD glycoprotein; a partial sequence of an HSV-1 polypeptide having at least one immunological antigenic determinant of the gD glycoprotein; the following amino acid sequence: Herpes simplex virus type 2 (HSV-2)
A non-glycosylated polypeptide of the gD glycoprotein; and a partial sequence of an HSV-2 polypeptide having at least one immunological antigenic determinant of the gD glycoprotein;
A non-glycosylated polypeptide of the herpes simplex virus gD glycoprotein selected from the group consisting of: 2. E. coli having ATCC deposit number 39,159, E. coli having ATCC deposit number 39,160, E. coli having NRRL deposit number B-15449,
E. coli with NRRL deposit number B-15450, NR
E. coli having RL accession number B-15451, and N
A non-glycosylated polypeptide of herpes simplex virus gD glycoprotein produced by E. coli selected from the group consisting of E. coli having RRL accession number B-15471. 3. A vaccine preparation for protection against herpes simplex virus infection, which comprises the non-glycosylated polypeptide according to claim 1 or 2 or a partial sequence thereof. 4. A method for producing a non-glycosylated polypeptide of the herpes simplex virus gD glycoprotein of claim 1, which comprises: a) the following DNA sequence: A DNA sequence encoding a polypeptide having the amino acid sequence of the gD glycoprotein of HSV-1 consisting of
A partial sequence of HSV-1 DNA encoding a polypeptide having at least one immunological determinant of a glycoprotein; the following DNA sequence: A DNA sequence encoding a polypeptide having the amino acid sequence of the gD glycoprotein of HSV-2 consisting of: and HSV-2DN encoding a polypeptide having at least one immunological determinant of the gD glycoprotein.
A subsequence of A; culturing a unicellular organism that comprises a DNA sequence selected from the group consisting of: A) and having the ability to express said DNA sequence or subsequence to produce said polypeptide, and b) from said culture Isolating the peptide. 5. The method according to claim 4, wherein the unicellular organism carries a recombinant vector containing the DNA sequence or a partial sequence thereof. 6. The unicellular organism is ATCC Deposit No. 3
E. coli having 9,159, ATCC Deposit No. 39,1
E. coli having 60, NRRL accession number B-15449
6. An E. coli strain selected from the group consisting of E. coli having NRRL accession number B-15450, E. coli having NRRL accession number B-15451, and E. coli having NRRL accession number B-15471. the method of.