WO1989010965A2

WO1989010965A2 - Glycoprotein h of herpes viruses

Info

Publication number: WO1989010965A2
Application number: PCT/US1989/001563
Authority: WO
Inventors: Erik A. Petrovskis; Leonard E. Post
Original assignee: The Upjohn Company
Priority date: 1988-05-03
Filing date: 1989-04-18
Publication date: 1989-11-16
Also published as: WO1989010965A3; AU3579089A

Abstract

The present invention provides recombinant DNA molecules comprising a sequence encoding pseudorabies virus (PRV) or infectious bovine rhinotracheitis virus (IBRV) glycoprotein gH, host cells transformed by said recombinant DNA molecule sequences, and the gH polypeptides. The present invention also provides subunit vaccines for PRV and IBRV and methods for detecting PRV and IBRV infections and methods for protecting animals against PRV AND IBRV infections.

Description

GLYCOPROTEIN H OF HERPESVIRUSES FIELD OF INVENTION

This invention relates to DNA sequences encoding glycoprotein H of herpesviruses, specifically, pseudorabies virus glycoprotein H (PRV gH) and infectious bovine rhinotracheitis virus glycoprotein H (IBRV gH) and polypeptides related thereto are disclosed. These DNA sequences are useful for screening animals to determine if they are infected with PRV or IBRV and also for expressing the glycoproteins encoded thereby which are useful for diagnostic and vaccination purposes.

BACKGROUND OF THE INVENTION

Pseudorabies virus (PRV) is a disease which infects many species of animals worldwide. PRV infections are variously called infectious Bulbar paralysis, Aujeszky's disease, and mad itch. Infections are known in important domestic animals such as swine, cattle, dogs, cats, sheep, rats and mink. The host range is very broad and includes most mammals and, experimentally at least, many kinds of birds (for a detailed list of hosts, see D.P. Gustafson, "Pseudorabies", in Diseases of Swine, 5th ed., A.D. Leman et al., eds., (1981)). For most infected animals the disease is fatal. Adult swine and possibly rats, however, are not killed by the disease and are therefore carriers.

Populations of swine are particularly susceptible to PRV.

Although the adult swine rarely show symptoms or die from the disease, piglets become acutely ill when infected and death usually ensues in 24 to 48 hours, often without specific clinical signs (T.C. Jones and R.D. Hunt, Veterinary Pathology, 5th ed., Lea &

Febiger (1983)).

PRV vaccines have been produced by a variety of techniques and vaccination in endemic areas of Europe has been practiced for more than 15 years. Losses have been reduced by vaccination, but vaccination has maintained the virus in the environment. No vaccine has been produced that will prevent infection. Vaccinated animals that are exposed to virulent virus survive the infection and then shed more virulent virus. Vaccinated animals may, therefore, harbor a latent infection that can flare up again. (See, D.P. Gustafson, supra).

Live attenuated and inactivated vaccines for PRV are available commercially in the United States and have been approved by the USDA (See, C.E. Aronson, ed., Veterinary Pharmaceuticals & Biologicals, (1983)).

Because adult swine are carriers of PRV, many states have instituted screening programs to detect infected animals. DNA/DNA hybridization can be used to diagnose actively infected animals utilizing the DNA sequence of the instant invention. Some of the PRV glycoproteins of the present invention are also useful in producing diagnostics for PRV infections and also to produce vaccines against PRV.

PRV is a herpesvirus. The herpesviruses generally are among the most complex of animal viruses. Their genomes encode at least 50 virus specific proteins and contain upwards of 150,000 nucleotides.

Among the most immunologically reactive proteins of herpesviruses are the glycoproteins found, among other places, in virion membranes and the membranes of infected cells. The literature on PRV glycoproteins refers to at least four viral glycoproteins (T. Ben-Porat and

A.S. Kaplan, Virology, 41, pp. 265-73 (1970); A.S. Kaplan and

T. Ben-Porat, Proc. Natl. Acad. Sci. USA, 66, pp. 799-806 (1970)). IBRV also is a herpesvirus. The spread of IBRV in cattle is a serious problem. It is associated with respiratory, reproductive, enteric, occular, central nervous system, neonatal, mammary, and dermal infections of cattle. Infectious bovine rhinotracheitis (IBR) is found worldwide and is characterized by a sudden onset of hyperthermia, anorexia, and depression.

IBRV causes abortion, stillbirths, conjunctivitis, vulvovaginitis, alimentary tract disease, infertility and meningoencephalitis. However, IBR is known mainly as a respiratory tract disease characterized by tracheitis and rhinitis. Fever is another sequela to IBRV infection.

IBRV infects species other than cattle including swine, goats, and mink.

Currently killed virus vaccines, modified live-virus vaccines and subunit vaccines against IBRV are in use. INFORMATION DISCLOSURE

M.W. Wathen and L.K. Wathen, J. Virol., 51, pp. 57-62 (1984) refer to a PRV containing a mutation in a viral glycoprotein (gp50) and a method for selecting the mutant utilizing neutralizing monoclo nal antibody directed against gp50. Wathen and Wathen also indicate that a monoclonal antibody directed against gp50 is a strong neutralizer of PRV, with or without the aid of complement, and that polyvalent immune serum is highly reactive against gp50, therefore concluding that gp50 may be one of the important PRV immunogens. On the other hand, it has been reported that monoclonal antibodies that react with the 98,000 MW envelope glycoprotein neutralize PRV infectivity but that monoclonal antibodies directed against some of the other membrane glycoproteins have very little neutralizing activity (H. Hampl , et al., J. Virol., 52, pp. 583-90 (1984); and T. Ben-Porat and A.S. Kaplan, "Molecular Biology of Pseudorabies Virus", in B. Roizman ed., The Herpesviruses, 3, pp. 105-73 (1984)).

L.M.K. Wathen, et al., Virus Research, 4, pp. 19-29 (1985) refer to the production and characterization of monoclonal antibodies directed against PRV glycoproteins identified as gp50 and gp83 (now known to be gill) and their use for passively immunizing mice against PRV infection.

A.K. Robbins, et al., in Herpesvirus, pp. 551-61 (1984), refer to the construction of a library of E. coli plasmids containing PRV DNA. They also refer to the identification of a PRV gene that encodes glycoproteins of 74,000 and 92,000 MW (now known to be gill). They do not refer to the glycoproteins of the instant invention.

A.K. Robbins, et al., European patent application No. 85400704.4 (publication No. 0 162 738) refers to the isolation, cloning and expression of PRV glycoproteins identified as gll and gill. They do not refer to the PRV glycoproteins of the instant invention.

T.C. Mettenleiter, et al., J. Virol., 53, pp. 52-57 (1985), refer to the mapping of the coding region of glycoprotein gA (which they equate with gl) to the BamHI 7 fragment of PRV DNA. They also state that the BamHI 7 fragment codes for at least three other viral proteins of 65K, 60K, and 40K MW. They do not disclose or suggest the DNA sequences encoding the glycoproteins of the instant invention or the production of such polypeptides by recombinant DNA methods. B. Lomniczi, et al., J. Virol., 49, pp. 970-79 (1984), refer to PRV vaccine strains that have deletions in the unique short sequence between 0.855 and 0.882 map units. This is in the vicinity of the gl gene. T.C. Mettenleiter, et al., J. Virol., 56, pp. 307-11 (1985), demonstrated that three commercial PRV vaccine strains lack glyccpro- tein gl. We have also found recently that the Bartha vaccine strain contains a deletion for most of the gp63 gene.

T.J. Rea et al., J. Virol., 54, pp. 21-29 (1985), refers to the mapping and the sequencing of the gene for the PRV glycoprotein that accumulates in the medium of infected cells (gX) .

European published patent application 0 133 200 refers to a diagnostic antigenic factor to be used together with certain lectin- - bound PRV glycoprotein subunit vaccines to distinguish carriers and noncarriers of PRV.

U. Gompels and A. Minson, Virology, 153, pp. 230-47 (1986) refer to the location and sequence of glycoprotein H (gH) of Herpes simplex Type 1 (HSV). They also show that antibodies to HSV gH neutralize HSV and prevent cell to cell spread of HSV. P.M. Keller, et al, Virology, 157, pp. 526-33 (1987) refer to the location and sequence of glycoprotein H of Varicella-Zoster Virus. They also show that antibodies to Varicella gH neutralize the virus . Neither of these documents refers to PRV or IBRV or PRV or IBRV gH and neither of them teaches the use of use of the respective gH antigens as subunit vaccines to prevent viral infection.

T. Heineman, et al., J. Virol., 62, pp. 1101-07 (1988) and D.E. Oba and L.M. Hutt-Fletcher, J. Virol., 62, pp. 1108-14 (1988), refer to the Epstein-Barr Virus glycoprotein H, including identification of the gene. M.P. Cranage, et al., J. Virol., 62, pp. 1416-22 (1988) refer to the Identification expression of human Cytomegalovirus glycoprotein H.

A portion of the PRV gH gene is disclosed in U.S patent- 4,514,497 as unidentified sequence downstream from the thymidine kinase gene (see Figure 5). However, this sequence contains errors that make the predicted amino acid sequence incorrect and including making it look like something other than a glycoprotein (i.e., there is no N-terminal hydrophoblc signal sequence).

United States patent No. 4,291,019 refers to a vaccine for IBR prepared by non-ionic detergent extraction of IBRV-infected cells. This vaccine comprises a number of unidentified IBR proteins referred to as "viral envelope protein" . One of the advantages of the instant invention is that it provides essentially pure PRV and IBRV gH which is free from other PRV and IBRV polypeptides and including con taminating virus. This has obvious advantages in production of the vaccines and diagnostics and for their registration with regulatory agencies.

SUMMARY OF INVENTION The present invention provides recombinant DNA molecules comprising DNA sequences encoding polypeptides displaying PRV or IBRV glycoprotein H-like antigenicity.

More particularly, the present invention provides host cells transformed with recombinant DNA molecules comprising the DNA sequences set forth in Charts A and B and fragments thereof.

The present invention also provides essentially pure PRV gH and IBRV gH. Also provided are polypeptides expressed by hosts transformed with recombinant DNA molecules comprising DNA sequences of the formulas set forth in Charts A and B, and immunologically functional equivalents and immunogenic fragments and derivatives of the polypeptides.

More particularly, the present invention provides polypeptides having the formulas set forth in Charts A and B, immunogenic fragments thereof and immunologically functional equivalents thereof. The present invention also provides recombinant DNA molecules comprising the DNA sequences encoding PRV or IBRV glycoprotein gH or immunogenic fragments thereof operatively linked to an expression control sequence.

The present invention also provides vaccines comprising gH-like polypeptides and methods of protecting animals from PRV and IBRV infection by vaccinating them with these polypeptides. DETAILED DESCRIPTION OF INVENTION

The gene encoding PRV gH maps to the BamHI 4, 11 and 15 fragments and one additional very small BamHI fragment of the PRV DNA. These DNA fragments can be obtained by preparing DNA from any isolate of PRV, for example, PRV Aujeszky (ATCC VR-135). The isolation is described in more detail in the Examples that follow.

To produce large amounts of PRV gH gene, E. coli HB101 containing plasmids comprising the PRV gH encoding DNA can be grown up in L-broth by well known procedures. Typically the culture is grown to an optical density of 0.6 after which chloramphenicol is added and the culture is left to shake overnight. The culture is then lysed by. e.g., using high salt SDS and the supernatant is subjected to a cesium chloride/ethidium bromide equilibrium density gradient centrifugation to yield the plasmids.

The gene encoding IBRV gH maps to the HindIII A fragment of IBRV

DNA. Such IBRV DNA can be obtained by preparing DNA from any IBRV isolate, for example, IBRV Colorado-1 (ATCC VR-864). The exact cleavages required to extract the gH gene are set forth in more detail in the Examples.

The availability of these gene sequences permits direct manipulation of the genes and gene sequences which allows modifications of the regulation of expression and/or the structure of the protein encoded by the genes or fragments thereof. Knowledge of these gene sequences also allows one to clone the gene, or fragment thereof, from any strain of PRV or IBRV using the known sequence as a hybridization probe, and to express the entire protein or fragment thereof by recombinant techniques generally known in the art.

Knowledge of these gene sequences enabled us to deduce the amino acid sequence of the corresponding polypeptides (Charts A and B) . As a result, fragments of these polypeptides having PRV or IBRV immunogenicity can be produced by standard methods of protein synthesis or recombinant DNA techniques. As used herein, immunogenicity and antigenicity are used interchangeably to refer to the ability to stimulate any type of adaptive immune response, i.e., antigen and antigenicity are not limited in meaning to substances that stimulate the production of antibodies. The primary structure (sequence) of the genes coding for PRV and IBRV gH also are set forth in Charts A and B.

All restriction endonucleases referred to herein are commercially available and their use is well known in the art. Directions for use generally are provided by commercial suppliers of the restriction enzymes.

The excised gene or fragments thereof can be ligated to various cloning vehicles or vectors for use in transforming a host cell. The vectors preferably contain DNA sequences to initiate, control and terminate transcription and translation (which together comprise expression) of the PRV and IBRV glycoprotein genes and are, therefore, operatively linked thereto. These "expression control sequences" are preferably compatible with the host cell to be transformed. When the host cell is a higher animal cell, e.g., a mammalian cell, the naturally occurring expression control sequences of the glycoprotein genes can be employed alone or together with heterologous expression control sequences. Heterologous sequences may also be employed alone. The vectors additionally preferably contain a marker gene (e.g., antibiotic resistance) to provide a phenotypic trait for selection of transformed host cells. Additionally a replicating vector will contain a replicon.

Typical vectors are plasmids, phages, and viruses that infect animal cells. In essence, one can use any DNA sequence that is capable of transforming a host cell (R.L. Rodriquez and D.T. Denhardt, eds., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths (1988)).

The terminology "host cell" as used herein means a cell capable of being transformed with the DNA sequence coding for a polypeptide displaying PRV or IBRV gH-like antigenicity. The term "transformed" as used herein, includes the case of a host cell infected with a recombinant virus, e.g., a baculovirus. Preferably, the host cell is capable of expressing the PRV or IBRV polypeptide or fragments thereof. The host cell can be procaryotic or eucaryotic. Illustrative procaryotic cells are bacteria such as E. coli, B. subtilis, Pseudomonas, and B. stearothermophilus. Illustrative eucaryotic cells are yeast or higher animal cells such as cells of insect, plant or mammalian origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. Mammalian cell lines include, for example, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7 or MDCK cell lines. Insect cell lines include the Sf9 line of Spodoptera frugiperda (ATCC CRL1711). A summary of some available eucaryotic plasmids, host cells and methods which are useful for employing them for cloning and expressing PRV or IBRV glycoproteins can be found in K. Esser, et al., Plasmids of Eukaryotes (Fundamentals and Applications), Springer-Verlag (1986) which is incorporated herein by reference.

As indicated above, the vector, e.g., a plasmid, which is used to transform the host cell preferably contains compatible expression control sequences for expression of the PRV or IBRV gH gene or fragments thereof. The expression control sequences are, therefore, operatively linked to the gene or fragment. When the host cells are bacteria, illustrative useful expression control sequences include the trp promoter and operator (Goeddel, et al., Nucl. Acids Res., 8, 4057 (1980)); the lac promoter and operator (Chang, et al., Nature, 275, 615 (1978)); the outer membrane protein promoter (EMBO J., 1, 771-775 (1982)); the bacteriophage λ promoters and operators (Nucl. Acids Res., 11, 4677-4688 (1983)); the α-amylase (B. subtilis) promoter and operator, termination sequences and other expression enhancement and control sequences compatible with the selected host cell. When the host cell is yeast, illustrative useful expression control sequences include, e.g., α-mating factor. For insect cells the polyhedrin promoter of baculoviruses can be used (Mol. Cell. Biol., 3, pp. 2156-65 (1983)). When the host cell is of insect or mammalian origin illustrative useful expression control sequences include, e.g., the SV-40 promoter (Science, 222, 524-527 (1983)) or, e.g., the metallothionein promoter (Nature, 296, 39-42 (1982)) or a heat shock promoter (Voellmy, et al., Proc. Natl. Acad. Sci. USA, 82, pp. 4949-53 (1985)). As noted above, when the host cell is mammalian one may use the expression control sequences for the PRV or IBRV glycoprotein gene but preferably in combination with heterologous expression control sequences.

The plasmid or replicating or integrating DNA material containing the expression control sequences is cleaved using restriction enzymes, adjusted in size as necessary or desirable, and ligated with the PRV or IBRV glycoprotein H gene or fragments thereof by means well known in the art. When yeast or higher animal host cells are employed, polyadenylation or terminator sequences from known yeast or mammalian genes may be incorporated into the vector. For example, the bovine growth hormone polyadenylation sequence may be used as set forth in European publication number 0 093 619 and incorporated herein by reference. Additionally gene sequences to control replication of the host cell may be incorporated Into the vector.

The host cells are competent or rendered competent for transformation by various means. When bacterial cells are the host cells they can be rendered competent by treatment with salts, typically a calcium salt, as generally described by Cohen, PNAS, 69, 2110 (1972). A yeast host cell generally is rendered competent by removal of its cell wall or by other means such as ionic treatment (J. Bacteriol., 153, 163-168 (1983)). There are several well-known methods of introducing DNA into aniraal cells including, e.g., calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation treatment of the recipient cells with liposomes containing the DNA, and microinjection of the DNA directly into the cells.

The transformed cells are grown up by means well known in the art (Molecular Cloning, Maniatis, T., et al., Cold Spring Harbor Laboratory, (1982); Biochemical Methods In Cell Culture And Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc., (1977); Methods In Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982)) and the expressed PRV or IBRV gH or fragment thereof is harvested from the cell medium in those systems where the protein is excreted from the host cell, or from the cell suspension after disruption of the host cell system by, e.g., mechanical or enzymatic means which are well known in the art.

As noted above, the amino acid sequences of the PRV and IBRV gH's as deduced from the gene structures are set forth in Charts A and B. Polypeptides displaying PRV or IBRV gH antigenicity include the sequences set forth in Charts A and B and any portions of the polypeptide sequences which are capable of eliciting an immune response in an animal, e.g., a mammal, which has been injected with the polypeptide sequence and also immunogenically functional analogs of the polypeptides.

As indicated hereinabove the entire gene coding for the PRV or IBRV glycoprotein can be employed in constructing the vectors and transforming the host cells to express the PRV or IBRV glycoprotein, or fragments of the gene coding for the PRV or IBRV glycoprotein can be employed, whereby the resulting host cell will express polypeptides displaying PRV or IBRV antigenicity. Any fragment of the PRV or IBRV glycoprotein gene can be employed which results in the expression of a polypeptide which is an immunogenic fragment of the PRV or IBRV glycoprotein or an analog thereof. As is well known in the art, the degeneracy of the genetic code permits easy substitution of base pairs to produce functionally equivalent genes and fragments thereof encoding polypeptides displaying PRV or IBRV gH antigenicity. These functional equivalents also are included within the scope of the invention.

Charts C and D are set forth to illustrate the constructions of the Examples. Certain conventions are used to illustrate plasmids and DNA fragments as follows:

(1) The single line figures represent both circular and linear double-stranded DNA. (2) Asterisks (*) indicate that the molecule represented is circular. Lack of an asterisk indicates the molecule is linear.

(3) Endonuclease restriction sites of interest are indicated above the line.

(4) Genes are indicated below the line. (5) Distances between genes and restriction sites are not to scale. The figures show the relative positions only unless indicated otherwise.

Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail, for example, in Molecular Cloning, T. Maniatis, et al., Cold Spring Harbor Laboratory, (1982) and B. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984), which are incorporated herein by reference. EXAMPLE 1 Isolation, Characterization and Expression of PRV gH The PRV gH gene is first constructed in a three step procedure. PRV genomic DNA prepared as described by Rea, et al, J. Virol., 54, pp. 21-29 (1985) is digested with BamHI. The 3.1 kb fragment, designated BamHI 11 (Feldman, et al, Virology, 97, pp. 316-27 (1979)) is isolated by agarose gel electrophoresis and ligated to pBR322, previously also digested with BamHI, to yield plasmid pTK11 (Chart C). A 0.6 kb SalI-Kpnl fragment containing the 5' end of the gH gene is isolated from pTK11 and ligated to pUC19 (purchased from Pharmacia, Inc., Piscataway, N.J.), previously digested with SalI and Kpnl, to produce plasmid pTKHSKS. pTKllSKS is then digested with EcoRI and Hindlll and the 0.6 kb fragment is isolated by gel electrophoresis. This fragment is digested with HphI, and the ends are made blunt with T4 DNA polymerase and ligated to BamHI linkers. The fragment so produced is digested with BamHI and KpnI and the 0.3 kb fragment produced thereby is ligated to pUC19, previously digested with BamHI and Kpnl, to produce a plasmid designated pBgH. Next, PRV genomic DNA (as above) is digested with BglII and Kpnl, and an 8.0 kb fragment is isolated. The 8.0 kb fragment and the 0.3 kb BamHI-KpnI fragment from pBgH are ligated to pUC19, previously digested with BamHI, to yield pUCgH-14 (Chart C).

Plasmids pTKll and pUCgH-14 are used to generate end-labelled fragments for DNA sequencing of the gH gene by the method of Maxam and Gilbert, (Methods Enzymol., 65, 499-560 (1980)). The DNA sequence for gH so determined is set forth in Chart A. This DNA may be employed to detect animals actively infected with PRV. For example, a nasal or throat swab is taken, and then by standard DNA/DNA hybridization techniques the presence or absence of PRV is determined. To express the PRV gH gene using a baculovirus vector and host, plasmid pUCgH-14 is digested with BamHI and the 2.0 kb fragment containing most of the gH gene is isolated. This BamHI fragment is cloned into a BamHI site downstream from the polyhedrin promoter in plasmid pAC373 (Mol. Cell. Biol., 5, pp. 2860-65 (1985)) to produce plasmid pAcBgH (Chart C). Plasmid pUCgH-14 is also digested with Hpal and a Kpnl linker is ligated on. After digestion with Kpnl, the 3.8 kb Kpnl fragment is isolated and ligated to pAcBgH, previously digested with Kpnl , to yield plasmid pAcgH (Chart C). Plasmid pAcgH is co-transfected with DNA from baculovirus Autographa californica (AcNPV) into Sf9 cells, and a recombinant virus designated AcNPVgH is isolated by methods set forth in Mol. Cell. Biol., 5, pp. 2860-65 (1985). This recombinant virus produces a glycosylated form of gH of about 70,000 MW upon infecting Sf9 cells. The baculovirus-related technology used here is set forth in detail in U.S. patent application S.N. 068,211, filed 30 June 1987, which is incorporated herein by reference. EXAMPLE 2 Isolation, Characterization and Expression of IBRV gH

IBRV genomic DNA is prepared by growing IBRV (IBR Colorado, ATCC VR-864) virus on MDBK cells (ATCC CCL22), extracting the cytoplasm followed by sodium iodide density gradient ultracentr.ifugation as described for PRV DNA in Rea, et al, J. Virol., 54, 21-29 (1985). IBRV genomic DNA is digested with HindIII and ligated to pACYC184 (ATCC #37033) previously digested with Hindlll. The plasmid so produced which contains the Hindlll A fragment of IBRV DNA is designated pHA. pHA is digested with SalI. The resulting 2.6 kb, 1.6 kb and 0.9 kb fragments are isolated and ligated to pUC19, previously digested with SalI, to yield three plasmids designated pHAS4, pHAS5 and pHAS6 (Chart D). These plasmids are used to generate end-labelled fragments for DNA sequencing by the method of Maxam and Gilbert (supra). The DNA sequence for gH is set forth in Chart B. This DNA may be employed to detect animals actively infected with IBRV as with the PRV DNA above. To reconstruct an IBRV gH gene for expression, plasmid pHA is digested with BamHI which cuts 45 bases upstream from the gH initiation codon, and StuI, to generate a 1.9 kb fragment. The downstream piece of the gH gene is generated by digesting pHASS with StuI and SalI and by isolating the 0.9 kb fragment. These fragments are ligated to pUC19 previously digested with BamHI and SalI to produce plasmid pUCIBRVgH. Plasmid pUCIBRVgH is digested with HindIII, the ends are filled with T4 DNA polymerase, BamHI linkers are added followed by a BamHI digestion, and finally a 2.8 kb BamHI fragment is isolated by agarose gel electrophoresis. This fragment is cloned into the BamHI site of pAc373 in the orientation such that the gH gene is transcribed by the polyhedrin promoter. The IBRV gH gene is then introduced and expressed in the baculovirus genome as described by Summers and Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures , Texas Agricultural Experiment Station (1987) which is incorporated herein by reference.

In another aspect of the instant invention a derivative of PRV or IBRV gH is produced by removing the DNA coding for the C-terminal end of gH and insertion of a termination codon. The resulting polypeptide has a deletion for the amino acid sequence necessary to anchor gH into the cell membrane. When expressed in mammalian cells this gH derivative is secreted into the medium. Purification of this gH derivative from the medium for use as a subunit vaccine is easier than fractionation of whole cells. Removal of the anchor sequence to convert a membrane protein into a secreted protein was first demonstrated for the influenza hemagglutinin gene (M.-J. Gething and J. Sambrook, Nature, 300, pp. 598-603 (1982)).

A vaccine prepared utilizing a glycoprotein of the Instant invention, or an immunogenic fragment thereof, can consist of fixed host cells, a host cell extract, or a partially or completely purified PRV or IBRV glycoprotein preparation from the host cells, or produced by chemical synthesis. The PRV or IBRV glycoprotein immunogen prepared in accordance with the present invention is preferably free of PRV or IBRV virus. Thus, the vaccine immunogen of the invention is composed substantially entirely of the desired immunogenic PRV or IBRV polypeptide and/or other PRV or IBRV polypeptides displaying PRV or IBRV antigenicity.

The immunogen can be prepared in vaccine dose form by well-known procedures. The vaccine can be administered intramuscularly, subcutaneously or intranasally. For parenteral administration, such as intramuscular injection, the immunogen may be combined with a suitable carrier, for example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunomodulating agents including aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, Quil A, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Another suitable adjuvant is Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Michigan).

The proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts. For example, aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (Al₂O₃ basis). On a per dose basis, the concentration of the immunogen can range from about 1.0 μg to about 100 mg per animal. A preferable range is from about 100 μg to about 3.0 mg per animal. A suitable dose size is about 1-10 ml, preferably about 1.0 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 1 ml containing 1.0 mg of immunogen in admixture with 0.5% aluminum hydroxide. Comparable dose forms can also be prepared for parenteral administration to, for example, baby pigs, but the amount of immunogen per dose will be smaller, for example, about 0.25 to about 1.0 mg per dose. For vaccination of sows or cows against PRV or IBRV infection respectively, for example, a two dose regimen can be used. The first dose can be given from about several months to about 5 to 7 weeks prior to birthing. The second dose of the vaccine then should be administered some weeks after the first dose, for example, about 2 to 4 weeks later, and vaccine can then be administered up to, but prior to, birthing. Alternatively, the vaccine can be administered as a single 2 ml dose, for example, at about 5 to 7 weeks prior to birthing. However, a 2 dose regimen is considered preferable for the most effective immunization of the baby animals. Semi-annual revaccination is recommended for breeding animals. Males may be revaccinated at any time. Also, females can be revaccinated before breeding. Piglets born to unvaccinated sows may be vaccinated at about 3-10 days, again at 4-6 months and yearly or preferably semi-annually thereafter. Cattle may be vaccinated before shipping.

The vaccine may also be combined with other vaccines for other diseases to produce polyvalent vaccines. It may also be combined with other medicaments, for example, antibiotics. A pharmaceutically effective amount of the vaccine can be employed with a pharmaceutically acceptable carrier or diluent to vaccinate animals such as swine, cattle, sheep, goats, and other mammals.

Other vaccines may be prepared according to methods well known to those skilled in the art as set forth, for example, in I. Tizard, An Introduction to Veterinary Immunology, 2nd ed. (1982), which is incorporated herein by reference.

Claims

1. A recombinant DNA molecule comprising a DNA sequence coding for a polypeptide displaying pseudorabies virus (PRV) or infectious bovine rhinotracheitis (IBRV) glycoprotein gH immunogenicity, said DNA sequence being operatively linked to an expression control sequence.

derivatives thereof encoding polypeptides displaying PRV or IBRV glycoprotein H antigenicity.

3. A host cell transformed with a recombinant DNA molecule of claim 1.

4. A host cell of claim 3 which is of bacterial, fungal, plant, or animal origin.

5. A host cell of claim 4 which is an insect cell infected with a baculovirus.

6. Essentially pure IBRV gH or PRV gH.

7. An essentially pure polypeptide according to claim 6 as follows :

fragments and derivatives thereof displaying infectious bovine rhinotracheitis virus antigenicity.

8. A vaccine comprising a polypeptide displaying herpesvirus gH antigenicity .

9. A vaccine according to claim 8 , wherein the polypeptide isPRV gH or IBRV gH.

10. A method of protecting an animal susceptible to herpes virus infection from said infection, comprising administering a vaccine of claim 8 to the animal.

11. The method according to claim 10, wherein said animal is a swine and said polypeptide is PRV gH.

12. The method according to claim 10, wherein said animal is a bovine and said polypeptide is IBRV gH.

13. A method for producing a polypeptide displaying PRV or IBRV gH antigenicity, comprising:

(a) preparing a recombinant DNA molecule, said molecule comprising a DNA sequence coding for a polypeptide displaying PRV or IBRV gH antigenicity, said DNA sequence having operatively linked thereto an expression control sequence; (b) transforming an appropriate host cell with said recombinant DNA molecule;

(c) culturing said host cell;

(d) and collecting said polypeptide.

14. A method according to claim 13, wherein the DNA sequence is

15. A method according to claim 13, wherein the host cell is selected from the group consisting of bacteria, fungi, plant cells and animal cells.

1.6. A method according to claia 13, wherein the host cell is an insect cell infected with a baculovirus.