VIRUS PROTEINS HAVING REDUCED O-LINKED GLYCOSYLATION

Abstract: The present invention provides viral glycoproteins having a lower amount of O-linked glycosylation than the naturally occurring glycoprotein. These glycoproteins are produced in insect host cells infected with a recombinant DNA molecule comprising a baculovirus vector and the DNA sequence encoding said glycoprotein. The present invention also provides subunit vaccines for pseudorabies virus.

Description: VIRUS PROTEINS HAVING REDUCED O-LINKED GLYCOSYLATION This application is a continuation-in-part of PCT International Application No. PCT/US86/01761, which is incorporated herein by reference. FIELD OF INVENTION This invention relates to- viral glycoproteins having reduced 0- linked glycosylation. The invention also relates to methods for producing such glycoproteins. More specifically, the invention relates to a pseudorabies virus glycoprotein having reduced 0-linked glycosylation and methods for producing . said glycoprotein with baculovirus vectors in insect cell hosts. These glycoproteins having reduced 0-linked glycosylation are useful for protecting animals against infection by the viruses they are derived from. 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, "Pseudo¬ rabies", 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 vaccina- tion 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)). 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)). INFORMATION DISCLOSURE European patent publication number 0 127 839 and European patent publication number 0 155 476 refer to methods for producing recom¬ binant baculovirus expression vectors for expression of selected genes together with such vectors. They do not disclose the use of such vectors to produce viral glycoproteins having less 0-linked glycosylation than the naturally occurring proteins for use in vaccines. Both of these documents are incorporated herein by reference. S. Alexander and J.H. Elder, "Carbohydrate Dramatically Influ¬ ences Immune Reactivity of Antisera to Viral Glycoprotein Antigens", Science, 226, pp. 1328-30 (1984) demonstrated that viral glyco¬ proteins with N-linked carbohydrate removed by endoglycosidase F had varying antigenic effects. Heteroantisera prepared against virus were virtually unreactive toward the viral glycoproteins after N- linked carbohydrate removal. The reactivity of monoclonal antibodies prepared to glycosylated antigen in most cases improved, while in a smaller number of cases it stayed the same, or decreased in reactivity when reacted with the antigen without N-linked carbo- hydrate. Most antibodies to synthetic peptide sequences also improved in reactivity after N- inked carbohydrate removal. D.W. Miller, et al, "An Insect Baculovirus Host-Vector System for High-Level Expression of Foreign Genes", Genetic Engineering, 8, pp. 277-98 (1986) refer to a baculovirus expression system for expressing heterologous genes. They also provide a review of the use of baculovirus expression systems. During this review, they discuss post-translational modifications in baculovirus systems, including glycosylation, as follows: "Very little is known about the nature of protein glycosylation by insect cells [citations omitted] ....The ability of these cells to carry out N-linked glycosylation appears to end at the addition of the high mannose complex [citation omitted] . 0- linked glycosylation has been examined only indirectly [citation omitted] . "A protein expressed in insect cells will be rich in mannose and lacking sialic acid. At this point, the ramifica¬ tions of this are not clear and are likely to be of more or less concern depending on the end use of the product. When analyzed by SDS-acrylamide gel electrophoresis, proteins expressed in insect cells are frequently smaller than their native counter¬ parts. We attribute this to less complex glycosylation and perhaps substantial hydrolysis of the high mannose complex unit [citation omitted] . This has not been directly determined for heterologous gene products of the baculovirus expression system. "With regard to the possible in vivo physiological effects of this glycosylation difference, much work needs to be done. We have no clear-cut case where the glycosylation difference has altered biological activity of a protein but do consider that this will inevitably be observed." T.D. Butters, et al, "Steps in the Biosynthesis of Mosquito Cell Membrane Glycoproteins and the Effects of Tunicamycin" , Bioch. Bioph. Acta, 640, pp. 672-86 (1981) set forth evidence indicating that mosquito cells synthesize N-glycosidically-linked carbohydrate chains and O-glycosidically-linked chains which are attached to glyco¬ proteins. G.E. Smith, et al, "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector", Mol. Cell. Biol., 3, pp. 2156-65 (1983) and S. Maeda, et al., "Production of Human α-Interferon in Silkworm Using a Baculovirus Vector," Nature, 315, pp. 592-94 (1985) refer to the production of β- and a- interferon in Spodoptera frugiperda. cells and silkworm using the Autographs californica and Bonibyx mori nuclear polyhedris virus (AcNPV and BmNPV) as expression vectors, respectively. Smith, et al., indicate that the ^-interferon produced thereby is glycosylated. International patent application PCT/US85/02319 relates to N- 5 linked carbohydrate-free viral polypeptides, and methods for produc¬ ing them by reacting the glycoprotein with an endoglycosidase enzyme, which can be used to induce antibody production in a host animal. 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 (gρ50) Iff- and a method for selecting, the mupant " utilizing neutralizing monoclo¬ nal antibody directed against gp50. Wathen and Wathen also indicate that a monoclonal antibody directed against gp50 is a strong neutral- izer of PRV, with or without the aid of complement, and that polyval¬ ent immune serum is highly reactive against gp50, therefore conclud- 15 ing 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 20 activity (H. Ha pl, 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 25 directed against PRV glycoproteins identified as gp50 and gp83 and their use for passively immunizing mice against PRV infection. A.K. Robbins, et al. , "Localization of a Pseudorabies Virus Glycoprotein Gene Using an E. coli Expression Plasmid Library", in Herpesvirus, pp. 551-61 (1984), refer to the construction of a 30 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. They do not refer to the glycoproteins of the instant invention. A.K. Robbins, et al., European patent application No. 85400704.4 35 (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 having reduced 0-linked glyco¬ sylation of the instant invention. T.C. Mettenleiter, et al., "Mapping of the Structural Gene of Pseudorabies Virus Glycoprotein A and Identification of Two Non- -Glycosylated Precursor Polypeptides", 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 of PRV codes for at least three other viral proteins of 65K, 60K, and 40K MW. They do not disclose or suggest a viral glycoprotein having reduced 0-linked glycosylation of the instant invention or the production of similar polypeptides in insect cells with baculovirus vectors. B. Lomniczi, et al., "Deletions in the Genomes of Pseudorabies Virus Vaccine Strains and Existence of Four Isomers of the Genomes", 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. Metten¬ leiter, et al. , "Pseudorabies Virus Avirulent Strains Fail to Express a Major Glycoprotein", J. Virol., 56, pp. 307-11 (1985), demonstrated that three commercial PRV vaccine strains lack glycoprotein 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) . Included among the flanking sequences of the gX gene shown therein is a small portion of the gp50 sequence, specifically beginning at base 1682 of Figure 6 therein. 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. G. E. Smith, et al. , "Modification and Secretion of Human Interleukin 2 Produced in Insect Cells by a Baculovirus Expression Vector," Proc. Natl. Acad. Sci. U.S.A., 82, pp. 8404-08 (1985) refer to production of IL-2 by inserting its cDNA into the genome of Autographa califomica nuclear polyhedrosis virus adjacent to the polyhedrin promoter and then using this recombinant virus to infect an insect cell host. They indicated that there was no evidence that the recombinant IL-2 produced was glycosylated. They do not disclose or suggest producing viral glycoproteins having a lower amount of 0- linked glycosylation than their naturally occurring homologs for use as vaccines. SUMMARY OF THE INVENTION The present invention relates to viral glycoproteins having a lower amount of 0-linked glycosylation than their naturally occurring glycoprotein, more particularly wherein said glycoprotein is a pseudorabies virus glycoprotein, and even more particularly pseudo¬ rabies virus gp50. The glycoproteins having a lower amount of glycosylation than the natural glycoproteins are produced by an insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein. More specifically, the insect cell host referred to above is selected from the group consisting of Spodoptera frugiperda, Bomhyx mori , Trichoplusia ni , and other lepidopteran cell lines. Intact worms can also be used as hosts for example, for AcNPV vectors, cabbage loopers or fall armyworms. See Maeda, et al. , supra. More specifically, the glycoprotein having a lower amount of glycosylation than the natural glycoprotein is produced by a Spodop¬ tera frugiperda insect cell host infected with a baculovirus vector comprising a DNA sequence encoding said glycoprotein, more specifi¬ cally, gp50. Also provided is a method for producing a glycoprotein having such reduced glycosylation, comprising: (a) preparing a recombinant DNA molecule, said molecule com¬ prising a DNA sequence coding for a glycoprotein, and a baculovirus vector; (b) infecting an insect host cell with said recombinant DNA molecule; (c) culturing said infected insect host cell; (d) and collecting said glycoprotein. More specifically in the method, the glycoprotein is a pseudo¬ rabies virus glycoprotein, more specifically, gp50. Also, more specifically, the insect host cell used in the method set forth above is Spodoptera frugiperda. DETAILED DESCRIPTION OF THE INVENTION The existence and location of the gene encoding glycoprotein gp50 of PRV was demonstrated by M.W. Wathen and L.M. Wathen, supra. The glycoprotein encoded by the gene was defined as a glycoprot¬ ein that reacted with a particular monoclonal antibody. This glycoprotein did not correspond to any of the previously known PRV glycoproteins. Wathen and Wathen mapped a mutation resistant to the monoclonal antibody, which, based on experience with herpes simplex virus (e.g., T.C. Holland et al., J. Virol., 52, pp.566-74 (1984)), indicated that they had mapped the location of the structural gene for gp50. Wathen and Wathen mapped the gp50 gene to the smaller Sall/BamHI fragment from within the BamHI 7 fragment of PRV. Rea et al, supra, have mapped the PRV glycoprotein gX gene to the same region. Many of the details related to production of PRV glycoprotein gp50 and its use as a subunit vaccine for the prevention of PRV infection are set forth in PCT patent application PCT US/01761 and in E.A. Petrovskis, et al, "DNA Sequence of the Gene for Pseudorabies Virus gp50, a Glycoprotein without N-Linked Glycosylation", Virology, 59, pp. 216-23 (1986), which are incorporated herein by reference. We cloned the gp50 gene under control of the polyhedrin promoter in the baculovirus Autographa califomica nuclear polyhedrosis virus (AcNPV) . Two forms of the gene were inserted into AcNPV: (1) the intact gene encoding the complete gp50 amino acid sequence to produce a recombinant virus designated AcNPV-gp50, and (2) the gene with the base pairs encoding the anchor sequence of gp50 deleted to produce a recombinant virus designated AcNPV-gp50T. AcNPV-gp50-infected cells produced a cellular form of gp50 and AcNPV-gp50T-infected cells produced a truncated form of gρ50 which is secreted into the medium. Both of these forms of gp50 produced in insect cells had a sig¬ nificantly lower molecular weight than the same proteins produced in mammalian cells because of reduced 0-linked glycosylation. The gp50 produced in insect cells is, however, labeled with • ' ■ ^'C-glucosamine. Since the gp50 sequence contains no N-linked glycosylation sites (E.A. Petrovskis,et al, supra), this is presumed to be 0-linked glycosylation in insect cells although at a substantially reduced level as compared to that produced in mammalian cells and as compared to the native glycoprotein. As a result, we refer to the glyco¬ protein of the instant invention as "having a lower amount of 0- 1inked glycosylation than its corresponding naturally occurring viral glycoprotein." It should be clear to those skilled in the art that this does not include such glycoproteins produced in prokaryotic hosts, e.g., E. coli , which would produce glycoproteins having no 0- linked glycosylation. In contrast the polypeptides of the instant invention have a decreased amount of 0-linked glycosylation as compared to the corresponding native polypeptide, but 0-linked carbohydrates are not entirely absent. Insect cells infected with AcNPV- p50 were used to immunize mice. After a series of three immunizations with 10° infected cells, most of the mice survived a challenge with virulent PRV. - As mentioned above, the gene encoding gp50, mapped to the BamHI 7 fragment of the PRV DNA. The BamHI 7 fragment from PRV can be derived from plasmid pPRXhl (also known as pUC1129) and fragments convenient for DNA sequence analysis can be derived by standard subcloning procedures. Plasmid pUC1129 is available from E. coli HB101, NRRL B-15772. This culture is available from the permanent collection of the Northern Regional Research Center Fermentation Laboratory (NRRL), U.S. Department of Agriculture, in Peoria, Illi¬ nois, U.S.A. E. coli HB101 containing pUC1129 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 a ino acid sequence of PRV glycoprotein gp50 is set forth in Chart A of PCT/US86/01761. - Glycoproteins having a smaller amount of 0-linked glycosylation than the naturally occurring glycoproteins produced by the method of the instant invention and displaying PRV glycoprotein antigenicity, include the sequence set forth in Chart A and any portion of the polypeptide sequence which is 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. The entire gene coding for the PRV glycoprotein can be employed in constructing the vectors and infecting the insect host cells to express the PRV glycoprotein having reduced 0-lihked glycosylation, or fragments of the gene coding for the PRV glycoprotein can be em¬ ployed, whereby the resulting insect host cell will express polypep¬ tides displaying PRV antigenicity. Any fragment of the PRV glycopro¬ tein gene can be employed which results in the expression of a polypeptide which is an immunogenic fragment of the PRV 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 polypep¬ tides displaying PRV glycoprotein antigenicity. These functional equivalents also are included within the scope of the invention. Also included are PRV glycoproteins having non-essential amino acid substitutions, which are also well-known in the art, and deletions, which do not affect the PRV antigenicity of the polypeptides. Charts A and B 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 Molecu- lar 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. The procedures specifically related to baculoviruses are set forth in M.D. Summers and G.E. Smith, A Manual for Baculovirus Vectors and Insect Cell Culture Procedures, published by the College of Agricul¬ ture, Texas Agricultural Experiment Station, Texas Agricultural Extension Service, College Station, Texas (1986) which is incor¬ porated herein by reference. While the specific Examples herein relate primarily to PRV, it is clear to those skilled in the art that other viruses and other polypeptides could be used in a similar fashion, for example, respiratory syncitial virus glycoprotein G and herpes simplex virus glycoprotein C. Example 1 Construction of Plasmid pAcgp50-4 In this example we set forth the construction of a plasmid with the gp50 gene downstream from the polyhedrin promoter, and flanked by baculovirus DNA. The starting material containing the gp50 gene was plasmid pD50 which is disclosed in Chart I of patent application PCT/US86/01761 and textual materials related thereto. Referring now to Chart A, pD50 was cut with EcoRI and the ends made blunt by treatment with T4 DNA polymerase. BamHI linkers were then ligated on and the fragment so produced was treated with BamHI. The fragment, containing the gp50 gene (about 1300 base pairs), was isolated by agarose gel electrophoresis. Plasmid pAc373 (7.1 kb) is a baculovirus expression vector having a unique BamHI site immediately downstream from the polyhedron promoter of AcNPV. The plasmid is available from Professor Max Summers of the Department of Entomology, Texas A&M University, College Station, Texas 77843 and is fully described in Mol. and Cell. Biol., 3, pp. 2156-65 (1983) and in European patent publication number 0 127 839. It can also be obtained from the Agricultural Research Culture Collection (NRRL) where it has been assigned accession number NRRL B-15778. pAc373 was digested with BamHI, and the ends were dephosphory- lated with bacterial alkaline phosphatase. The BamHI fragment containing the gp50 gene from above was ligated into the linearized pAc373 to produce plasmid pAcgp50-4. Recombinants having the gp50 gene in the proper orientation with relation to the polyhedron promoter were determined by digestion with Sail which produced a 975 base pair fragment for clones with the correct orientation. Example 2 Construction of Plasmid pAcgp50T-5 We constructed this plasmid to put the gene encoding the truncated secreted form of gp50 (see PCT/US86/01761) into the pAc373 vector. Referring now to Chart B, plasmid pD50T was constructed exactly -li¬ the same as plasmid pDIE50T was constructed in PCT/US86/01761, chart L and materials related thereto, except that pD50 was the starting plasmid instead of ρDIE50. When pD50 is digested with Sail and EcoRI a 4.2 kb Sall/Eco RI fragment is produced instead of the 5.0 kb fragment for pDIE50. The only difference between the resulting plasmids is that the cytomegalovirus (CMV) promoter is not in plasmid pD50T and a BamHI site remains. By following the techniques set forth above in Example 1 for pD50, pD50T was manipulated to introduce the gene for the truncated gp5° into the pAc373 vector to produce pAcgp50-T-5. The same 975 bp Sail fragment was diagnostic for the correct orientation of the sequence encoding the truncated gp50. Example 3 Construction of Recombinant Baculoviruses The methods for construction of baculoviruses have been de- scribed in detail in publications by Dr. Max Summers (e.g., Mol. and Cell Biol., 3, pp. 2156-65 (1983)) and particularly in the manual referred to above (M.D. Summers and G.E. Smith, supra) which are incorporated herein by reference. The plasmids produced above in Examples 1 and 2 were co-trans- fected along with baculovirus AcNPV DNA into Spodoptera frugiperda Sf9 cells (available from the American Type Culture Collection, Rockville Maryland as deposit number ATCC CRL 1711) . Recombinant viruses containing the gp50 genes were enriched by limited dilution of approximately 10^ plaque forming units per well in microtiter wells, followed by hybridization with -^^p-iabeled gρ50 DNA to detect wells where recombinant viruses were growing. Recombinant viruses were isolated by plating out dilute virus and picking individual plaques that lacked occluded virus. The viruses were plaque-purified four times to be sure that occlusion positive virus was eliminated before growing up virus stocks. Recombinant viruses were isolated from transfections with both pAcgp50-4 and pAcgp50T-5 and were designated AcNPV-gp50 and AcNPV- gp50T respectively. Example 4 Detection of gp50 Expression from Recombinant Baculo- viruses Sf9 cells are infected with one of the recombinant viruses produced in Example 3 at a multiplicity of infection of at least 1.0 plaque forming unit per cell. At 24-48 hours post infection the cells are suspended in Grace's medium, gently centrifuged out, and resuspended in Grace's medium lacking me hionine but to which 100 microcuries per ml of --S-methionine is added. Samples are harvested and gp50 expression is analyzed by immunoprecipita ion as described in E.A. Petrovskis, et al, J. Virol., 59, pp. 216-23 (1986). AcNPVgpSO-infected cells produced three forms of gp50, having molecular weights of 44, 46 and 47 kd. AcNPVgp50T-infected cells produced a form of gp50 having a molecular weight of about 41 kd. Two forms of gp50 having molecular weights of 43 and 44 kd were found in the medium of these latter infected cells. The lower molecular weight polypeptides correspond to the expected molecular weight of the protein backbone of gp50 molecules with little or no 0-linked glycosylatio . Similar experiments with -^C-glucosamine indicated that some of these forms of gp50 could be labeled by carbohydrate precursors. Although not wishing to bound by theory, it is likely that insect cells can add 0-linked carbohydrate to glycoproteins, but to a much lesser extent than mammalian cells do and to a much lesser degree than is found for the native glycoproteins, but more than prokaryotic cells. Example 5 Use of gp50 Produced in Baculovirus as a Vaccine Table 1 sets forth the protection of mice from challenge by virulent PRV by immunization with gp50 produced in Sf9 cells or tissue plasminogen activator (tPA) produced in Sf9 cells as a control (AcNPVgp50 or AcNPV-tPA respectively) . Mice were immunized at 28 days, 18 days and 7 days prior to challenge. Table 1 Immunizing Number of % Agent/Adjuvant Cells Survival 5 gp50/CFA b 1 x 10 5 88 (7/8) tPA/CFA 1 x 10 6 25 (2/8) gp50/none 1 x 10 6 100 (8/8) gp50/none 1 x 10 5 75 (6/8) gp50/none 1 x 10 4 38 (3/8) unvaccinated 38 (3/8) a Challenged with 30 LD50 of PRV Rice strain by footpad route. "Complete Freund's adjuvant. As can be seen from Table 1, cells expressing the gp50 antigen were effective in protecting mice from infection with PRV as compared to both an unvaccinated control and cells expressing a non-PRV antigen, tPA. It also appears that such protection decreases with a decreasing amount of gp50. A vaccine prepared utilizing a gp50 protein 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 glycoprotein preparation from the host cells. The gp50 immunogen prepared in accordance with the present invention is preferably free of PRV virus. Thus, the vaccine immunogen of the invention is composed substantially entirely of the desired im¬ munogenic PRV gp50 polypeptide and/or other PRV polypeptides display¬ ing PRV antigenicity. The immunogen can be prepared in vaccine dose form by well-known procedures. The vaccine can be administered intramuscularly, subcu- taneously 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 emul¬ sions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebac- terium parvu (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such adjuvants are available commer¬ cially 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, Mich¬ igan) . 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 (AI2O3 basis). On a per dose basis, the concentration of the immunogen can range from about 1.0 μg to about 100 mg per animal. In pigs, a preferable range is from about 100 μg to about 3.0 mg per pig. 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 5 forms can also be prepared for parenteral administration to 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, a two dose regimen can be used. The first dose can be given from about several months to about 5 to 7 Iff weeks- prior to farrowing. 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, farrowing. Alternatively, the vaccine can be administered as a single 2 ml dose, for example, at about 5 to 7 weeks prior to 15 farrowing. However, a 2 dose regimen is considered preferable for the most effective immunization of the baby pigs. Semi-annual revaccination is recommended for breeding animals. Boars may be revaccinated at any time. Also, sows can be revaccinated before breeding. Piglets born to unvaccinated sows may be vaccinated at 20 about 3-10 days, again at 4-6 months and yearly or preferably semi-annually thereafter. The vaccine may also be combined with other vaccines for other diseases to produce multivalent vaccines. It may also be combined with other medicaments, for example, antibiotics. A pharmaceutically 25 effective amount of the vaccine can be employed with a pharmaceuti¬ cally 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, 30 An Introduction to Veterinary Immunology, 2nd ed. (1982) , which is incorporated herein by reference. CHART A. Construction of plasmid pAcgp50-4 (a) Plasmid pD50 is cut with EcoRI to produce fragment 1. BamHI Hindlll PvuII EcoRI PvuII * I I I I I * dhfr SV40 Amp R 50505050505050 Ori Fragment 1 EcoRI BamHI EcoRI 505050505050 | | dhfr Amρ R (b) Fragment 1 is blunt-ended, BamHI linkers are added and then digested with BamHI to produce fragment 2. BamHI BamHI 505050505050505050505050 (c) Plasmid pAc373 is digested with BamHI and BAP to produce fragment 3. pAc373 BamHI * I * I I P poly Fragment 3 BamHI BamHI poly P (d) Fragments 2 and 3 are ligated to produce plasmid pAcgp50-4. BamHI BamHI * I μ 505050505050 L_| * P poly dhfr -> Dihydrofolate reductase gene SV40 Ori - * • SV40 promotor and origin of replication Amp* ** = Ampicillin resistance gene P — polyhedrin promoter poly - polyhedrin gene CHART B. Construction of plasmid pAcgp50T-5 (a) Plasmid pAcgp50T-5 is produced from plasmid pD50T by the same manipulations as were done in Chart B, but starting with pD50T. pD50T EcoRI BamHI * 50T50T50T I I dhfr Amp R pAcgp50T-5 BamHI BamHI * I 50T50T50T I P poly 50T = Transacted form of gp50 gene

Claims: CLAIMS 1. A viral glycoprotein having a lower amount of 0-linked glycosy¬ lation than its corresponding naturally occurring viral glycoprotein, produced by an insect cell host infected with a recombinant DNA molecule comprising a baculovirus vector and a DNA sequence encoding said glycoprotein. 2. A glycoprotein according to claim 1, wherein the glycoprotein is a pseudorabies virus glycoprotein. A glycoprotein according to claim 2, pseudorabies virus gp50. 4. A glycoprotein according to claim 1, wherein the insect cell host is selected from the group consisting of Spodoptera frugiperda , Bombyx mori and Trichoplusia ni . 5. A glycoprotein according to claim 1, wherein said glycoprotein is pseudorabies virus gp50 and said insect cell host is Spodoptera frugiperda . 6. A method for producing a viral glycoprotein having reduced 0- linked glycosylation as compared to its naturally occurring glycosyl- ated form comprising: (a) preparing a recombinant DNA molecule, said molecule com- prising a DNA sequence coding for a glycoprotein and a baculovirus vector; (b) infecting an insect host cell with said recombinant DNA molecule; (c) culturing said infected insect host cell; (d) and collecting said glycoprotein. 7. A method according to claim 6 wherein said glycoprotein is pseudorabies virus gp50. 8. A method according to claim 6 wherein said insect host cell is Spodoptera frugiperda . 9. A method according to claim 6 wherein said glycoprotein is pseudorabies virus gp50 and said insect host cell is Spodoptera frugiperda.. 10. A pseudorabies virus gp50 polypeptide having a lower amount of O-linked glycosylation than its corresponding naturally occurring form. 11. A vaccine comprising the glycoprotein according to claim 10.

Inventors: POST LEONARD E, THOMSEN DARRELL R, POST, LEONARD, E., THOMSEN, DARRELL R.
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1988
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N15, C12P21, C12R1