VIRUS VACCINE

Abstract: Virus vaccine, comprising a properly incapacitated virus lacking an antigen of the wild-type virus which is useful for serologically distinguishing between vaccinated and infected animals, methods for distinguishing between vaccinated and infected animals and multivalent vaccines.

Description: VIRUS VACCINE FIELD OF INVENTION This invention relates to a serologically identifiable vi vaccine. The vaccine of the present invention allows one to disti guish between animals infected with a virulent wild-type virus, those which have been vaccinated, by utilizing a serologically disti virus for the vaccine. BACKGROUND OF THE INVENTION Pseudorabies virus (PRV) i3 a disease which infects many spec of animals worldwide. PRV infections are variously called infecti Bulbar paralysis, Aujeszky's disease, and mad itch. Infections known in important domestic animals such as swine, cattle, dogs, ca sheep, rats and mink. The host range is very broad and includes m mammals and, experimentally at least, many kinds of birds (for detailed list of hosts, see D.P. Gustafson, "P3eudora ies ,, , in Disea of Swine, 5th ed., A.D. eman et al., eds., (1981)). For moat infec animals the disease is fatal. Adult swine and possibly rats, howev are not killed by the disease and are therefore carriers for disease. Populations of swine are particularly susceptible to P Although the adult swine rarely show symptoms or die from the disea piglets become acutely ill when infected and death usually ensues in to 48 hours often without specific clinical signs (T.C. Jones R.D. Hunt, Veterinary Pathology, 5th ed., Lea & Febiger (1983)). PRV vaccines have been produced by a variety of techniques vaccination in endemic areas of Europe has been practiced for more t 15 years. Losses have been reduced by vaccination, but vaccination maintained the virus in the environment. No vaccine has been produ that will prevent infection. Vaccinated animals that are exposed virulent virus survive the infection and then shed more virulent vir Vaccinated animals may therefore harbor a latent infection that flare up again. (See, D.P. Gustafson, supra). Live attenuated and inactivated vaccines for PRV are availa commercially in the United States and have been approved by the U (see, C.E. Aronson, ed., Veterinary Pharmaceuticals 4 Biologica ( 1983 ) ). Because adult swine are carriers of PRV, many states have ins tuted screening programs to detect infected animals. A problem ariβe in distinguishing between those animals carrying virulent PRV and thos which have been vaccinated. The antigenic profile of the virulen viruses and the viruses used in vaccines are the same and therefore i may be impossible to distinguish between infected and vaccinate animals. As a result, regulations concerning movement of seropositi swine would apply to both vaccinated swine and to swine that have be previously infected with PRV (C.E. Aronson, supra.). PRV is a herpesvirus. The herpes viruses generally are among t most complex of animal viruses. Their genomes encode at least 50 vir specific proteins and contain upwards of 150,000 nucleotides. Amo the most immunologically reactive proteins of herpesviruses are t glycoproteins found, among other places, in virion membranes and t membranes of infected cells. The literature on PRV glycoprotei refers to at least four viral glycoproteins (T. Ben-Porat an 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)). Several herpesviruses reportedly secrete glycoproteins into t medium of infected cells. Herpes simplex virus (HSV) releases glyc protein C and several truncated forms of glycoprotein D into the medi (B. Norrild and B.F. Vestergaard, Intervirology , 11, pp. 104-10 (1979) R.E. Randall, et al . , J. Gen. Virol., 48, pp. 297"310 (1980)). arek' disease virus releases a considerable amount of the virion glycoprote A into the medium (D. Van Zaane, et al . , Virology, 121, pp. 116- (1982)); and herpes saimiri virus also releases a virion glycoprotei in the medium (R.E. Randall and R. . Honess, J.Gen. Virol., 51 pp.445-49 (1980)). PRV releases a glycoprotein into the medium whi reportedly is not incorporated into the viral particles (T. Ben-Por and A.S. Kaplan, Virology, 41, pp.265-73 (1970); T.J. Rea, et al . J. Virol., 54, pp. 21-29 (1985)). The PRV protein which is secreted into the medium has be referred to as 3a (T. Ben-Porat and A. S. Kaplan, supra) , and i3 al referred to as glycoprotein X (gX) (T. J. Rea, et al., supra.). gX h the following characteristics when isolated from PRV-infected cells: (1) it is the predominant protein in the culture medium of P infected animal cell3 in culture; (2) it is a glycoprotein; (3) it has a molecular weight of about 95 kd on SDS polyacr amide gels; (4) It is a sulfated protein; (5 ) it is soluble in about \ % perchloric acid; and ( 6) it is immunogenic in standard laboratory mice . . The instant invention overcomes the problems referred to abo for example in screening swine for PRV infection, by providing a strain which is immunologically distinct from the wild-type virus , t allowing one to distinguish between vaccinated and infected anim without the need for sacrificing the tested animals . These antigenic differences may be a result of deletion of one more detectable antigenic polypeptides from the vaccine virus. A result of these genetic changes , it is possible to immunologica distinguish between infected and vaccinated animals on the basis their serological profiles without the need for sacrificing the tes animals . INFORMATION DISCLOSURE M. . Wathen and L .K . Wathen , J. Virol . , 51 , pp .57-62 ( 1984) ref to a PRV containing a mutation in a viral glycoprotein (gp50 ) an method for sel ecting the mutant utilizing neutralizing monoclo antibody directed against gp50. Wathen and Wathen do not describe use of this virus aa a vaccine . Further , animals immuni zed wi th t virus would be serologically indistinguishable from infected animals T .C . Holland , et al . , J. Virol . , 15 , pp .672-82 ( 1983 ) refers antigenic variants of HSV selected with glycoprotein-specif ic mo clonal antibodies . Included among the variants selected are two w fail to express HSV glycoprotein gC. Holland , et al . also do not t or suggest the use of these variants for vaccines . European patent publication 0 1 33 200 refers to a diagnos ant i genic factor to be used together wi th certain lectin-bound glycoprotein subunit vaccines to distinguish carriers and noncarr of PRV. European patent publication 0 074 808 refers to specif ic sequence insertions , deletions and substitutions in eukaryotic cell viral genomes that are stably effected through the use of select DNA sequences comprising a herpesvirus thymidine kinaae gene. A the genomes listed as susceptible to mani pulation are PRV . Ano -it- related publication also sets forth similar methods (L. E. Post an B. Roizman, "A Generalized Technique for Deletion of Specific Genes i Large Genomes: α Gene 22 of Herpes simplex Virus 1 Is Not Essentia for Growth," Cell, 25, pp. 227-32 (1981 )). The methods set forth i these documents are employed in producing the PRV of the presen invention, infra. A.J.M. Berns and A.L.J. Gielkens, European Publication No. 0 14 458 refers to deletion mutants of PRV. The deletions are not within gene encoding a secreted glycoprotein. Furthermore Berns neithe suggests or describes the use of such mutants to distinguish serologic ally between a vaccine and wild- ype virus. A.L.J. Gielkens, et al., "Genome Differences Among Field Isolate and Vaccine Strains of Pseudorabies Virus", J. Gen. Virol., 66 pp. 69-82 (1985) refers to comparing the genomes of different fiel isolates and modified live virus vaccine strains of pseudorabies vir (PRV) by BamHI restriction mapping. They reported observing two typ of variations, (1) additions and/ or deletions of nucleotide sequence to fragments derived from the TR S and IR S regions of the PRV genom and (2) loss or gain of BamHI cleavage sites within the Uτ_ region the genome. They speculate that analysis of viral DNA with restricti endonucleases may provide a method to distinguish PRV field strains. We have determined that one of the PRV vaccines now commercial available contains a deletion for the gene encoding glycoprotein I have Mettenleiter, et al., J. Virol., 56, pp. 307-11 (1985). We ha also shown that another commercial strain (Bartha) lacks gp63- The vaccines may be useful in certain of the embodiments of the insta invention as described, infra. B. Lomniczi, et al., "Deletions in the Genomes of Pseudorabi Virus Vaccine Strains and Existence of Four Isomera of the Genomes J. Virol., 49, pp. 970-79 (1984) refers to characterization of t commercial vaccine strains of PRV (from Bartha and Norden) showing th they have deletions in the unique short sequence of the PRV geno between 0.855 and 0.882 map units. This area is within the BamHI fragment of PRV. Nowhere do either of these documents describe suggest a PRV lacking a secreted glycoprotein, a vaccine comprisi such a mutant or a method of distinguishing between a vaccinated a infected animal by using such a PRV mutant. United States patent 4, 51 4, 497 refers to PRV tk~ deletions . does not refer to PRV having deletions which allows one to serologic lly distinguish between animals infected with a virulent wild-ty virus and those which are vaccinated . SUMMARY OF THE INVENTION A3 used herein, the expressions "properly incapacitated virus and variants of that expression , and "avirulent" , refer to both kill and attenuated viruses . As used herein, "secreted glycoprotein" referβ to a glycoprote that accumulates in the medium of infected cells in culture . The present invention relates to a vaccine comprising a proper incapacitated virus lacking at least one detectable antigen of t wild- type virus whi ch allowβ the serological distinction betwe vaccinated and infected animals . More particularly, the present invention relates to a pseudorabi virus lacking a serologically detectable polypeptide of the wild-ty PRV . More particularly, the present invention relates to a pseudorabi virus lacking a secreted glycoprotein of the wild-type PRV . More particularly, the present invention relates to a pseudorabi virus lacking glycoprotein X . The present invention also provides methods for distinguishi between vaccinated and infected animals and a mul ti valent vacci comprising the above-described viruses and vaccines . DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vaccine which allows one serologically distinguish between vaccinated and i nfected anima wi thout the need for sacrif icing the tested animals . The vacci comprises a virus having a deletion for an antigenic poly pepti d particularly a secreted polypeptide , and more particularly a secre glycoprotein . We produced such a PRV by utilizing recombinant DNA techni ques . Starting with a readily available plasmid (pPRXh l , also known PUC1 129) containing the gX gene that we wished to delete, together w another publicly available plasmid (pACYCl 84) , we constructed a plas (pPRXK4) carrying the gX gene subcloned for convenient mani pulati We then removed the gX promoter from pPRXK4 and cloned it into anot publicly available plasmid, pUC9, to construct plasmid pPGX1. Next, we removed the HSV thymidine kinase (tk) gene from plasmid pRB103 and inserted it into a site in pPGX1 so that it was fused to the gX promoter to produce plasmid pGXTK2. We then removed the BamHI 7 fragment of PRV containing the C-terminal coding region of the gX gene from pPRXhl and inserted it downstream from the tk gene in pGXTK2 to form a plasmid (pGXTK3) in which the HSV tk gene is flanked by the PRV gX promoter and the C-terminal coding region for the gX gene. We next co^-transfected rabbit skin cells with a tk- gX+ PRV and pGXTK3 (tk + SX~) to produce a tk+ gX- PRV by the method of L.E. Post and B. Roizman, infra. Finally, we converted the tk + gX~ PRV to a tlc'gX' * PRV to attenuate the virus for use as a vaccine. We have also demonstrated the efficacy of such a tlό-gX- PRV as a vaccine against pseudorabies disease. Charts A^-K are set forth to illustrate the constructions of the present invention. 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 are indicated above the line. (4) Genes are indicated below the line. (5) Distances between genes and restriction sites are not to scale. The drawings show their relative positions only. The methods used in the plasmid constructions are standard recombinant DNA procedures, well known to those skilled in the art. These methods are described in, for example, T. Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982) and B. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984), which are incorporated herein by reference. Many of the specific methods employed herein are set forth i L. E. Post and B. Roizman, "A Generalized Technique for Deletion of Specific Genes in Large Genomes: α Gene 22 of Herpes simplex Virus 1 Is Not Essential for Growth," Cell, 25, pp. 227-32 (1981), which is incorporated herein by reference. In particular, the methods for cc transfection and selection procedures are found therein. EXAMPLE 1 1. Construction of pPRXK4 Referring now to Chart A, we describe the construction of plasmid for subcloning the complete gX gene. Plasmid pPRXhl (also known as pUC1129 and available as depo No. B-15772 from the Northern Regional Research Laboratory, U.S. partment of Agriculture, Peoria, Illinois) which contains the gX g and gX promoter from PRV, is digested with restriction endonuclea Xhol and Kpnl. The third largest of the four fragments produ (fragment 1, about 2.6 kb) is isolated by polyacryl amide gel elect phoresis. Fragment 1 is blunt-ended with T4 DNA polymerase and Ec linkers are added. Vector pACYCl84 (available as deposit No. 37033 from the Ameri Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryl 20852) is digested with EcoRI and treated with bacterial alkal phosphatase (BAP) to yield fragment 2. EcoRI cuts the Cm* * gene. Fragments 1 and 2 are then ligated to produce plasmid pPRX This plasmid contains the complete gX gene, including the likely promoter (see Rea, et al., supra). 2. Construction of pPGX1 Referring now to Chart B, we describe the subcloning of the promoter. The nucleotide sequence recognized by restriction endonucle Mstl (TGCGCA) is located in the DNA sequence putatively encoding 5 '-untranslated region of the gX mRNA (Rea, et al . , supra). pPRXK4 digested with Mstl and the second largest fragment (fragment 3 . ab 2.1 kb) is isolated. Fragment 3 is then cut with EcoRI, and smaller piece (fragment 4, about 400 bp) is isolated. Plasmid p (available from Pharmacia P/L, Inc., Piscataway, NJ, USA) is diges with EcoRI and Smal, and the larger fragment (fragment 5, about 2.6 is isolated. Fragments 4 and 5 are then ligated at the EcoRI sites by a Mstl/Smal fusion to produce pPGX1. This plasmid contains the promoter with a BamHI cleavage site immediately downstream from it. 3. Construction of pGXTK2 Referring now to Chart C, we describe the construction o plasmid in which the gX promoter is fused with the HSV tk gene. Plasmid pPGX1 from above is digested with BamHI and treated w BAP to yield fragment 6. Plasmid pRB103 contains the BamHI Q fragme from HSV-1 strain F (L.E. Post, et al., Proc. Natl. Acad. Sci. USA, 7 pp. 4201-05 (1980)). (Alternatively, plasmid pHSV106, which is comme cially available from Bethesda Research Laboratories, Gaithersburg, M USA, al30 contains the BamHI Q fragment and can be used in thi construction.) pRB103 is digested with BamHI plus Bglll and the seco largest fragment (fragment 7, about 2.9 kb) is isolated. This fragme contains the HSV tk gene without its promoter. Ligation of t digested pPGX1 (fragment 6) with the fragment containing the tk ge (fragment 7) at the BamHI sites and by a Bglll/BamHI fusion gives t plasmids containing the tk fragment in opposite orientations. T plasmid with the tk gene immediately downstream from the gX promoter selected by examination of the BamHI plus EcoRI digestion patterns a is called pGXTK2. 4. Construction of pGXTK3 Referring now to Chart D, we describe a plasmid comprising the H tk gene and PRV sequences flanking it. pPRXhl (see 1, above) is digested with BamHI and fragment 8 (abo 6.9 kb) is isolated. (This fragment is known in the literature BamHI 7 (see Rea, et al . , supra.)). pGXTK2 is digested with BamHI a treated with BAP to produce fragment 9. Fragment 3 is ligated into t BamHI site of pGXTK2 (fragment 9). The resulting plasmid with fragme 8 in the same orientation as the gX promoter is called pGXTK3. Th plasmid has the tk gene immediately downstream from the gX promoter a replacing the DNA coding for the N-terminal amino acids of gX. 5. Co- trans feet ion Referring now to Chart E, pGXTK3 is cut with Clal. The D fragment so produced which contains the C-terminal region of (effectively gX~) and the entire HSV tk gene fused to the gX promot is used to co-transfect rabbit skin cells together with DNA from a t gX + mutant of PRV (which we call PRV HR) which is selected by growth PRV in the presence of iododeoxyuridine according to the method Tatarov, Zentralblatt Veterinarmedizin, 15, pp. 847-53 (1968). The tk + gX~ recombinant viruses (which may be used for vacci after proper incapacitation) are selected by growth in tk" human 1 cells (J.P. Weir, et al., Proc. Natl. Acad. Sci. USA, 79, pp. 1210- (1982); Panicali and Paoletti, Proc. Natl. Acad. Sci. USA, 7 pp. 4927-31 (1982); Ca pione-Piccardo , et al . , J. Virol., 3 pp. 281-87 (1982); K.L. Pof f enberger , et al., Proc. Natl. Acad. Sc USA, 80, pp. 2690-94 (1983); M.F. Stinski , et al., J. Virol., 5 pp. 431-41 (1985)) in HAT medium (L. E. Post and B. Roizman, supr We called the virus so produced PRVΔgXI or DT-A. DT-A is tk + and fully capable of killing mice. Viruses selected for growth in HAT (e.g., DT-A) are analyzed synthesis of gX by labeling viral proteins with 35s-methionine 1 ^C-glucosamine, followed by immunopreci itation with anti-gX ser No gX is detected. Proteins from cells infected with the tk + gX~ virus are a analyzed by western blots with anti-gX serum and no gX is detected cells infected with the mutant virus. It is also possible to remove the entire gX gene. For example, digesting fragment 8 with Narl, one produces a fragment having entire gX gene deleted (see Chart D). This fragment can then employed in place of fragment 8 to produce a gX" PRV entirely lack the gX gene. It has been known for some time that tk" PRV are avirulent make good vaccines. Therefore, to properly incapacitate DT-A to mak tk" virus useful as a vaccine one could mutagenize and select for PRV by the metho^ of Tatarov (see, e.g., G. Tatarov, "Apathoge Mutant of the Aujeszky Virus Induced by 5-1 odo-2-Deoxyuri dine (IUDR Zentralblatt Veterinarmedizin, 15, pp. 847-53 (1968); G. Tatarov, al., "Investigation of Harmlessness and Immunogenicity of the MK Str of Aujeszky's Virus", Vet. Nauki, 6, pp. 49-54 (1969); G. Tatar "Results of Use of Live Vaccine MK-25 Against Aujeszky's Disease", V Sbirka, 7, pp. 10-12 (1974); G. Tatarov, "Bulgarian MK-25 Vacc Against Aujeszky's Disease", Cah. Med. Vet., 43, pp. 3^7-52 (1974); Tatarov, et al., "Development of an Avirulent Mutant of Aujeszk Disease Virus Under the Influence of 5-Bromo-Desoxyuri ine" , Veterin Science, 18, pp. 3-12 (1981); V. Khristova, et al . , "Thymidine Kin Activity of Virulent and Vaccinal Strains of Aujezsky's Disease Viru Veterinary Science, 22, pp. 15-22 (1985)); or by similar techniq (W.C. Topp, Virology, 113, pp. 408-11 (1981); S. Kit, et a Exp. Cell Res., 31, pp. 97-312 (1963); S. Kit et al., Virology, 1 pp.381-89 (1983)). PRVΔgXI which contains the HSV tk gene can also be converted to tk" PRV by a recombinational event to remove the HSV tk gene by co transfecting rabbit skin cells with this tk + gX~ PRV and a plasmi carrying a gX deletion, and selecting for tk~ recorabinants with ara according to the method of L. E. Post and B. Roizman, supra, a follows: Referring now to Chart F, plasmid pPGX 1 was digested with BamH and treated with BAP as set forth in Chart C, step (a) to produc fragment 6 comprising the gX promoter. Plasmid pPRXhl is digested wit BamHI to produce fragment 8 comprising the C-terminal coding region o gX as set forth above and in Chart D, step (a). This fragment is als known as the PRV BamHI 7 fragment. Fragments 6 and 8 are then ligate together, transformed into E. coli, and the clones are screened for th desired orientation of the BamHI 7 fragment by digestion with EcoR plus PyuII. Proper orientation is indicated by the generation of 2. kb fragment by the digestion. The plasmid so produced is calle PΔGXB7. Plasmid pΔGXB7 is co-transf ected with viral DNA from PRVΔgXI usin the calcium phosphate transfection technique (Mocarski , et al., Cell 22:243 (1980)), into rabbit skin cells. The viruses that result fro this transfection are plated onto vero cells in the presence of 10 μg/ml araT as described by Mocarski, et al., supra. Individual plaque are picked and analyzed for the desired tk" recombinant virus b restriction enzyme digestion of DNA as described in Mocarski, et al. supra. Thiβ virus is designated PRVΔGXTK" or DT-B (Chart G). Thes tk~ PRV are useful attenuated viruses for vaccines as set forth i Example 2 below. To differentiate between infected and vaccinated animals usi these viruses, one could employ, for example, an ELISA assay. Genera ly, gX protein, produced, for example, in E. coli by recombinant DN techniques (Rea, et al . , supra.), is added to the wells of suitabl plastic plates and allowed sufficient time to absorb to the plasti (overnight, 20-25° C). The plates are washed and a blocking age (e.g.,BSA) is added to neutralize any unreacted sites on the plasti surface. A wash follows and then the pig serum is added to the well After about 1 hour incubation at 20-25° C, the wells are washed and protein A-horseradish peroxidase conjugate is added to each well for 1 hour incubation at 20-25° C. Another wash follows and the enzy substrate (o-phenylenediamine) is added to the wells and the reacti is terminated with acid. Absorbance is measured at 492 nmeters quantitate the amount of gX antibody present in the serum. Lack of antibody indicates that an animal is not infected. By testing f other antigens one can establish whether or not a given animal vaccinated. Although PRVΔGXTK" is an effective vaccine, the PRV tk" mutati selected by growth in iododeoxyuridine could be a point mutation. it were a point mutation it could revert to tk + and possirbly virulent. Therefore, a tk" PRV having a deletion is preferabl United States patent 4,51 ,497 refers to PRV tk" deletions. The mark rescue procedure used therein to make tk" PRV was first used to ma tk " deletions in herpes simplex virus (J.R. Smiley, Nature, 285:333- (1980) and L.E. Post, et al . , Cell, 24:555-65 (1981)). Referring now to Chart H, the PRV tk" gene has been mapped to t BamHI 11 fragment of PRV (T. Ben-Porat, et al., Virology, 127:194-2 (1983)). The BamHI 11 fragment was isolated by agarose gel electr phoresis of BamHI-digested DNA from PRV Rice strain and cloned in pBR322 to produce plasmid pTKI 1. pTK11 has a single Xhol cleavage si within the tk gene. pTK11 was cut with Xhol and digested for vario amounts of time with exonuclease Bal31, recircularized with ligase a transformed into E. coli DH1 (Maniatis, supra., p. 505). This produc a collection of plasmids with various deletions in the tk gene. Two of these plasmidβ were selected, purified by CsCl densi gradient centrif ugation and sequenced by the method of Maxam Gilbert, Methods in Enzymology, 65(part1), pp. 497-559 (1980). Plas pΔtk-3 had a deletion of 329 base pairs from within the tk gene plasmid pΔtk-4 had a deletion of 275 base pairs. Before the tk deletions could be crossed into the gX" viruses was first necessary to make the viruses tk + so the tk" deleti recorabinants could be selected. PRVΔGXTK" DNA was co-transfected w plasmid pTK11 into rabbit skin cells. The resulting recombin viruses were isolated and grown in 143 cells in HAT medium to selec tk + virus which we called PRVΔgXtk + PRV. The DNA from this virus then co-transfected with pΔtk-4 DNA into rabbit skin cells. resulting viruses were plated on vero cells in the presence of araT select for tk" recombinants . The resulting viruses were screened f the proper recombinant by preparation of DNA and restriction enzy analysis. These recombinants had incorporated the tk deletion. One o these viruses , which we called P RVΔgXΔtk or DT-C , contained th expected tk deletion and a gX deletion. We determined that the LD50 o DT-C is greater than 107 plaque forming uni ts in mice . For compariso the LD50 of PRVΔgXPRVTK + was 3 pfu. Although the particular embodiment described herein is directed t PRV and utilizes recombinant DNA techniques , it will be clear to tho skill ed in the art that similar vaccines containing deletions f secreted glycoproteins can be produced by other techniques and utili ing other viruses , such as Marek' s disease virus , and infectious bovi rhinotracheitis virus . Although the di sclosure herein relates primarily to PRV , t techniques employed herein may be used to produce serologi call distinct herpesviruses in any situation where it is advantageous distinguish between vaccinated and infected animals such as is the ca with the PRV . Included, for example are Marek' s disease virus a infectious bovine rhinotrachei tis virus . Whil e we placed the HSV tk gene under the control of the promoter , the DNA fragment containing tk could be inserted so that is under control of another PRV promoter , or any other promoter whi could function for expression of the tk gene . For example , other H promoters , for example , the HSV ICP4 promoter , that can be expressed PRV-infected cells could also be employed . For example , one cou insert the PRV tk gene into the gX gene , or insert any other tk ge under the control of a promoter that can be expressed in PRV-infect cells . While we used the tk gene as the marker gene insert , any mark gene could be inserted into the gX gene to inactivate it as long as t marker would allow selection of viruses carrying that marker from tho that lack it . For example , antibiotic resistance genes could be us as the marker genes such as resistance to G418 or hygroraycin. It is also possible to insert genes other than a selectable mark into the gX gene . To construct such a virus , one could place t foreign gene into a plasmid such as pGXTK3 , with the foreign ge replacing the tk sequences . This plasmid could then be co-transfect with DNA from the tk + gX~ virus illustrated in Chart E. Selection wi araT will give recombinant viruses with the foreign gene inserted a expressed. Such a gene, for example, could encode an antigen another type of virus, for example, transmissible gastroenteritis vir or porcine parvovirus, or a of a bacteria such as E. coli, which wou then raise an immunogenic response against that other virus. In th embodiment of the invention one would therefore have produced a " ult valent" vaccine comprising antigens for two or more types of viruses other pathogens. As an example of the foregoing, we inserted the tissue plasminog activator (tPA) gene into the gX gene as follows: PPSA18 is a plasmid which contains the entire coding region f tPA, with a unique BamHI cleavage site in the 5 '-untranslated region the coding region. It is constructed as set forth in copending U. patent application SN 758,517, filed July 26, 1985. Referring now Chart I, pPSAIδ is cut with Ball and BamHI linkers are added to prod fragment 11. Fragment 11 is then digested with BamHI to produce a 1. kb fragment (fragment 12) containing the entire coding region of tP Plasmid pPGX1 (Chart B) is then cut with BamHI to produce fragment Fragments 12 and 6 are then ligated to produce plasmid pGXTPA. proper orientation of the tPA cDNA with relation to the gX promoter determined by digestion with EcoRI which produces a 1.1 kb fragm when the orientation is proper. Referring now to Chart J, plasmid pGXTPA is digested with Hind to produce fragment 13. The BamHI 7 fragment of PRV DNA is isolated set forth above (fragment 8), and Hindlll linkers are added to prod fragment 14. Fragments 13 and 14 are then ligated together to prod pGXTPA-B7. The proper orientation of the BamHI 7 fragment inser into this plasmid is determined by digestion with EcoRI and Sphl. 5.8 kb fragment is produced thereby when the orientation is proper. Plasmid pGXTPA-B7 is then co-transfected with viral DNA f PRVΔGX1 into rabbit skin cells. The resulting viruses are plated vero cells in the presence of araT to select tk" recombinants. called one of the viruses so selected PRV-GXTPA. When this vi infects cells, the cells produce and secrete tPA as detected imraunoprecipitation with anti-tPA antiserum. tPA activity of 20 ng was detected by a fibrin solubilization assay (Unkeless, et al., Exp. Med., 137, pp.85-111 (1973); Luskutoff, et al., Proc. Natl. Acad Sci. USA, 74, 3903-07 (1977)). Example 2 In this example we set forth the protection of mice and swine fro PRV- induced mortality by vaccination with a thymidine kinase-deficien glycoprotein X deletion mutant (tk-gX-) of PRV. 1. Animals Female CF-1 mice, 5-6 weeks old, were obtained from the Charle River Co. Eighteen crossbred pigs, 5-6 weeks old and of mixed sex, wer obtained from J. Gilmore Enterprises. Pigs were randomly allotted int three rooms, six pigs per room. Pigs and mice were given non-medicated food and water ad libitum. 2. Viruses and Cells PRV deletion mutants DT-A (HSVtk + gX") and DT-B (tk"gX~) wer constructed from the parental PRV (HR strain; .tk~gX + ) as set fort above. DT-B does not synthesize thymidine kinase and it does no synthesize gX due to deletion of the gX gene. The virulent PRV (Ric strain) was used as the challenge virus in all protection studies Viruses were propagated in either Vero cells or porcine kidney-15 (P 15) cells. 3. Microneutralization Assay The microneutralization assay was done as described by T.C Holland et al., J. Virol., 45, pp. 672-82 (1983). Mouse sera wer incubated with virus and 10% rabbit complement for 3 hr at 37°C whil pig sera were incubated with virus in the absence of complement for hr at 37°C. The neutralization titer was expressed as the reciproc of the highest dilution of serum which protected greater than 50$ the cells from cytopathic effects. 4. Enzyme-linked Immunosorbent Assay (ELISA) The antigen solution was prepared by diluting gX fusion prote (p60-11, produced as set forth in copending U.S. patent application 853,087, filed April 17, 1986) to 20 μg/ml in Voller's buffer (50 NaHC0 3> 0.03$ aN3, adjusted to pH 9.6 with NaOH). This was filter through a 0.45 micron sterilizing filter (Sartorius SM 165 55 K). needed, the filtered solution was further diluted with Voller's buff before use. 100 μl of p60-11 protein in Voller's buffer (concentration abo 2μg/ml) was added to each well of a 96 well plate (Costar 3590 EIA Adsorption occurred during an overnight, room-temperature incubatio The wells were washed three times with 300 μl of Dulbecco's PBS (8 g NaCl, 0.2 g/1 KC1, 0.2 g/1 KH P04, and 1.14 g/1 a2HP0 ; resultant was 7.3-7.4). Unreacted sites on the plastic surface were neutraliz during a 2 hour 37° incubation with 3% BSA in Dulbecco's PBS (200 per well). A single wash of each well with 300 μl of Dulbecco's P followed. Then the adsorbed antigen was reacted with antibodies in 1 μl of diluted serum (obtained from pigs exposed to PRV) and incubat overnight at 4°. Unreacted antibodies were removed by three wash with Dulbecco's PBS (300 Ml/well). Then 100 Ml of Protein A-hors radish peroxidase conjugate (diluted 1/800 for mouse sera and 1/15,0 for pig sera; diluted in 50 mM Tris, 0.05$ Tween-20, 1$ BSA, 0.0 N-tf.3, pH 8.0) was added to each well for a 2 hour, 37° incubatio Again, the wells were washed three times with Dulbecco's PBS (3 Ml/well). One hundred μl of substrate solution was added to ea well. This solution was prepared by adding 10 mg of o-phenylenediami (previously dissolved in 0.5 ml CH3OH) and 25 μl of 30$ (w/v) H2O2 49.5 ml buffer (17 mM Citric acid, 65 mM phosphate and 0.01$ merthi late adjusted to pH 6.3 with NaOH). The enzyme reaction continued f 10 minutes at room- temperature before 100 μl of 4.5 M H2SO4 was add to each well. Absorbance of the chromophore was measured at 4 nmeters using a Titertek Multiskan. 5. Viral Isolation from Nasal Swabs Nasal swabs collected from pigs were each placed in one ml Eagles Basal Medium (BME; M.A. Bioproducts) supplemented with 3$ fet bovine serum (FBS) and antibiotics. Swabs were stored at -70° unt they were assayed for the presence of virus. For the virus isolati assay, the nasal swabs in BME were thawed and the individual swabs w discarded. Samples (0.1 ml) were inoculated in duplicate onto porci kidney- 15 (PK-15; ATCC CCL33) cell monolayers and incubated for 1 hr 37° to allow virus adsorption. An overlay of medium-199 (Flow Labo tories) supplemented with 4$ FBS, antibiotics, and 1$ agar was pla on the cell cultures. After 3 days the cell monolayers were stai with neutral red and the plaques were enumerated. 6. Experimental Design for the Mouse Study The virulence of four PRV strains was evaluated in mi ce b determining the 50$ lethal dose (LD50) of each strain . The four P strains were: 1 ) the wild-type (Rice strain) , 2) DT-A, 3) DT-B, and HR. Each virus strain was administered to either f ive or six groups mice ( 1 0 mice/ group ) at various doses . Mice were inoculated with 50 of the respective viruses by the footpad route (Day 0) . The LD determinations were made 1 4 days (Day 1 4) after administration of t viruses and sera were obtained from the surviving mice of groups giv the highest dose of the respective viruses. The LD50 for each vir strain was calculated by the Spearman-Karber method. Surviving mice from the LD50 virulence study were challeng intraperitoneally at day 14 with PRV Rice strain at approximately 1 times the LD50 (determined for the intraperitoneal route ) for PRV Ri strain (10 LD50 =« 5. 4 x 102 pfu/mouse) . The study was concluded 1 days after challenge (Day ' 28) . 7. Experimental Design for the Swine Study Pigs were injected intramuscularly on Day 0 with 2 ml of DT strain (about 1 .5 x 107 pfu/pig) , 2 ml of Norden live vaccine (PR-Va as recommended by the manufacturer , or saline. All pigs were cha lenged on Day 20 with about 40 LD50 of virulent P RV (Rice strain) ( LD50 = 1 . 1 x 105 pfu/pig) . The study was terminated on Day 34. Serum samples were obtained and each pig was weighed on Days 1 20, and 34. Nasal swabs were taken daily on Days 19 through 3 1 *. 8. Criteria for Efficacy Evaluation Efficacy of PRV DT-B strain (tk~gX~ ) as a vaccine was evaluated the basi3 of protection against PRV-induced mortality and the abili to serologically distinguish between PRV DT-B-treated swine and swi exposed to field-type virus . Table 1 shows the relative virulence of the four PRV strains mice in terms of the respective LD50 determinations . DT-B and HR ha greatly reduced virulence compared to the wild-type strain and DT- being at least 1000-fold less virulent . Mice that survived the administration of the various PRV strai were subsequently challenged on day 1 4 by the intraperitoneal rou with Yirulent wild-type PRV at a dose of 10 times the LD50 ( 5. 4 x 1 pfu/mouse ) . As shown in Table 2, mice given a dose of 1 x 1 θ3 pfu greater of DT-B and mice given a dose of 2. 3 x 103 pfu or greater of were protected from virulent wild-type PRV challenge. In additio sera taken prior to challenge from mice given DT-B contained a hi titer of neutralizing antibodies (neutrali zation titer = 1024) whi the reaction with gX antigenic determinants , based on the ELISA dat was relatively insignificant (ELISA absorbance = 0.05) . Sera tak pr ior to chall enge from mi ce given HR contained a high titer neutralizing anti bodies (neutrali zation ti t er == 2048 ) while t reaction with gX antigenic determinants was substantial (ELISA absor ance = 0.60) . To evaluate the protective eff icacy and diagnostic impl ications PRV DT-B 3train , swine were inoculated with DT-B, Norden live vacci ( PR-Vac) or ealine by the intramuscular route on Day 0 and challeng intranasally with wild-type Rice strain on day 1 4. The results sho in Table 3 demonstrate that DT-B strain is comparable to Norden li vaccine (PR-Vac ) in elici ting a signif i cant ne utr al i zing anti bo response and affording protection from virulent Rice strain challeng The data obtained from an ELISA using the gX fusion protein sugge that swine i mmunized wi th DT-B did not have antibodies against whereas sera taken from convalescent pigs that survived exposure to t wild-type Rice strain did. In this study sera taken from swine giv Norden live vaccine (PR-Vac ) did not react with gX antigenic determ nants to a statistically signif icant extent (P > 0.05) , however , bas on previous studies , swine given Norden live vaccine (PR-Vac) may fo ant ibodies against gX because the Norden strain possesses the ge encoding gX. Therefore , sera taken from swine vaccinated with Nord l ive vaccine (PR-Vac) may vary in terms of reactivity with gX antigen determinants . Swine treated with DT-B and Norden l ive vaccine sporadi cally s virus after challenge whereas saline-treated pigs shed virus conti ously until death in most cases . No virus was detected in the nas secretions of one pig treated with DT-B , and one pig treated wi Norden live vaccine (PR-Vac ) . These results suggest that vaccinat pigs do not shed virus as readily as non-vaccinated pigs . The average weight on Day 20 (prior to challenge ) of pigs trea with DT-B was not significantly different ( P > 0.05 ) from the aver weight of sal ine- treated control pigs , indicating that vaccination w DT-B did not adversely affect the growth of swine. These results indicate that DT-B protects swine from virulent P challenge and allows the serological differentiation between vaccinat swine and convalescent swine previously exposed to virulent PRV. DT did not reduce the growth of treated swine. In addition, the virulen study in mice demonstrated that DT-B is less virulent than tk + P strains in a species other than swine. Table 1 Mice LD50 Virus Strain Genetic Characteristics LD50 Rice strain PRVtk + gX + 40 pfu mouse DT-A HSVtk + gX~ 34 pfu/mouse DT-B tk"gX" 9.4x10^ pfu/mouse HR tk"gX + >2.3x1θ6 pfu/mouse Table 2 Protection of mice vaccinated with attenuated PRV strains Genetic Mortality ELISA Virus Charac¬ DθS€ After Neutraliza¬ Absorb- Strain teristics (Pfu 'mouse) Challenge tion Titer anceb DT-B tk-gX" 1.0 X 105 0/6 1024 0.05 1.0 X 104 0/10 1.0 X 103 0/10 1.0 X 102 4/10 1.0 X 101 7/10 1.0 X 100 9/10 HR tk"gX + 2.3 X 1θ6 0/10 2048 0.60 2.3 X 105 0/10 2.3 X 10^ 0/10 2.3 X 103 0/10 2.3 X 102 2/10 Control 10/10 0.00 a Neutralization titer = reciprocal of the highest dilution of ser taken from survivors (prior to challenge with PRV Rice strain) th protected greater than 50$ of cells from cytopathic effects. b Absorbance values represent ELISA reactions obtained using a 1/ dilution of serum. Sera were the same as used for the neutralizati assay. Table 3 Protection of swine vaccinated with attenuated PRV DT-B Geometric Mean Arithmetic Mean Preparation Mortality Titerb of ELISA Absorbanc DT-B 0/6 91 0.072 ± 0.01 6e PR-Vac 0/6 36 0. 1 30 ± O. Oδi e Saline 6/6 <4c 0.093 ± 0.024 Rice — — 0.942 ± 0. 228f a Five convalescent pigs that survived exposure to Rice strain were bl in a previous study . They are included here to provide gX reacti sera as a control used in the ELISA. ^Geometri c mean titer is the geometric mean of the neutralizati titers obtained for each of the six pigs in each group prior challenge . Each neutralization titer is the reciprocal of the highe serum dilution that protected >50$ of the cells from cytopath effects . c No detectable antibody . ^Arithmetic mean of ELISA absorbances is the arithmetic mean of t ELISA absorbance values ( 1 /40 dilution of sera) obtained for sera tak from each of the 6 pigs/group prior to challenge , except the last val (see note a above) . e Not signi icantly different (P > 0.05 ; two-tailed Student ' s t tes from saline-treated pigs . Signif icantly different (P < 0.05 ; two-tailed Student ' s t test ) fr saline-treated pigs . Example 3 Following essentially the same procedures as set forth in Exam 2, we have also done similar experiments for the DT-C strain of P DT-C has a deletion for the PRV tk gene and for that reason Is preferred embodiment of the present invention. The results of th experiments are set forth in tables 4-8. Tables 4 and 8 show reduced virulence of DT-C in swine, sheep, and calves. Tables 5 and show the protective ability of DT-C. Table 7 shows the dose titrati for DT-C in swine. Table 4 Mice LD50 Virus Strain Genetic Characteristics LD50 Rice strain PRVtk + gX + 9 pfu mouse DT-C tk"gX- >1.0x107 pfu/mouse HR tk"gX + >1.5x1θ7 pfu/mouse Table 5 Protection of mice vaccinated with attenuated PRV strains Genetic Mortality ELISA Virus Charac¬ Dose After Neutraliza¬ Absor Strain teristics (pfu/mouse) Challenge tion Titera ancet> DT-C tk"gX" 1.0 x 107 0/8 5120 0.05 1.0 x 1θ6 0/8 1.0 x 105 0/8 1.0 x 10 0/8 1.0 x 103 0/8 1.0 x 102 2/7 1.0 x 101 7/8 HR tk " gX + 1.5 x 107 0/6 2560 0.97 1.5 x 1θ6 0/7 1.5 x 105 0/8 1.5 x 10 1 * 0/8 1.5 x 103 0/8 1.5 x 102 3/8 1.5 x 101 5/8 Control 7/8 <20 0.00 a Neutralization titer =» reciprocal of the highest dilution of ser taken from survivors (prior to challenge with PRV Rice strain) t protected greater than 50$ of cells from cytopathic effects. b Absorbance values represent ELISA reactions obtained using a 1 dilution of serum. Sera were the 3ame as used for the neutralizati assay. Table 6 Protection of swine vaccinated with attenuated PRV DT-C Geometric Mean Arithmetic Mean Preparation* Mortality Titerb of ELISA Absorbance 0 DT-C 0/6 26 0.072<* 0.388© PR-Vac 0/6 1 6 0. 105 e 0.645 e BME 3/6 <8 0.059 0. 250e a Pigs receiving DT-C, PR-Vac , and BME had weight gains from 8. 1 t 27. 1 , 6.5 to 23.0, and 6.0 to 1 1 .4 kg respectively for survivors fr day 0 to day 35 of the test . b Geometric mean of the neutralization ti ters obtained for each of t six pigs in each group prior to challenge. Each neutralization tit is the reciprocal of the highest serum dilution that protected >50$ the cells from cytopathic effects . arithmetic mean of the ELISA absorbance values ( 1 /40 dilution of ser obtained for sera taken from each of the 6 pigs/group prior to chal enge . Means with different superscripts were signif icantly, differe (P < 0.05) from the day 20, BME (Eagle' s basal medium) control valu First No. is pre-challenge , second No . is p>ost-challenge . Table 7 Dose Titration of DT-C in Swine Dose Mortal ity* Geometric Mea (pfu/pig) (Rice challenge ) Titer 1 x 107 0/6 23 1 x 1θ6 0/6 25 1 x 105 0/6 18 1 x 10* 0/6 18 1 x 103 0/6 18 1 x 10 0/6 20 control 5/6 <4 a Swine were administered 2 ml of each preparation at the indicat dosage by intramuscular injection on day 0 and were challenged on d 21 with about 80 LD50 ( 2.2 x 105 pfu/pig) of PRV Rice strain. b See note b, Table 6. Table 8 Virulence of DT-C for Sheep and Calves Animal Inoculum Route* Dead/ Positive (pfu animal ) tested Nasal swabs sheep 4 x 107 intranasal 1/3° 0/3 4 x 107 intramuscular 0/3 0/3 calves 4 x 107 intranasal 0/3 0/3 4 x 107 intramuscular 0/3 0/3 a The virus suspension was administered in a total volume of 2 ml by t intranasal ( 1 ml per nostril ) or intramuscular routes. Animals we observed for symptoms of Aujeskey' s disease for 21 days post-admin stration. b Swabs of the nasal mucosa were taken from each animal at terminati of the experiment and these were tested for PRV as described above . c 0ne sheep died on day 1 4 exhibi ting no symptoms of Auj eskey' s disea or viral shedding. The probable cause of death in this animal w coccidiosis . This disease was diagnosed in other sheep in both group In another aspect of the present invention , to ensure that a g tk + virulent PRV does not result from a theoretically possible recom ination between the gX~tk~ PRV of the present invention and a wild-ty PRV in the field, the gX~tk"PRV is further engineered. By followi the general methods set forth above a deletion i s made in the PRV gp gene at i ts ori ginal locus and a copy of the gp50 gene is inserted in close linkage , or inserted within , the tk gene . To delete the origin gp50 gene , one can employ the PvuII /BamHI fragment of fragment 8 (s Chart D ) to perform a marker rescue experiment as set forth above . T product virus will be gX~tk" and have a copy of the gp50 gene close linked to ,- or within what remains of the tk gene sequence. Since gp is essential to the virus viabil ity , any gX~ virus resulting from recombination would also be gp50" and nonviable . Therefore , it will impossible to separate the gX" and tk" deletions by recombination the f ield to produce a virulent gX~ PRV . In more detail , referring now to Chart K , pΔTK-4 from above digested with Sphl and BamHI to produce a fragment containing the deletion , and the fragment is then isolated . pUC1 9 (available fr Pharmacia/PL) is digested with BamHI and Sphl and the larger fragme so produced is isolated. These two fragments are then ligated produce plasmid pUCΔTK4-V which contains a single Sail site adjacent the tk deletion. pUCΔTK4-V is then cut with Sail and the ends ma blunt with T4 DNA polymer ase to produce fragment A. Fragment 8 from above (BamHI 7 ) is digested with Ndel , which cu between the gX and gp50 genes , and Stul , which cuts within the gp gene, to produce a Ndel/Stul fragment containing the gp50 gene (fra ment B, about 1 .8 kb) . This i s f illed in with T4 DNA polymerase a then fragments A and B are ligated to produce plasmid pΔTK4gp50-8. Plasmid pΔTK4gp50-8 is cut with Hindlll , and then co-transfect with PRV DNA into rabbit skin cells . The resulting virus are grown a selective medium containing araT to isolate tk" recombinants havi the structure shown in Chart K (c) . We called this virus PRVΔTKgp50. PRVΔTKgp50 is then co-transfected with p>GXTK3 from above , and t viruses are selected on 1 43 cells in HAT medium. This virus , call PRVΔTKgp50tk + (HSV) , is gX" . Next , the PvuII/BamHI fragment containing the gp63 and gl gen made by digesting BamHI 7 with these enzymes is subcloned into t PvuII/BamHI fragment of pBR322 to produce plasmid pPR28-1 (see copen ing U. S . patent application SN 844 , 1 1 3 , f iled March 26, 1986) . Plasm pPR28-1 is cut with PvuII , BamHI linkers are added , and the fragment produced is digested with BamHI to convert the 5 kb PvuI I /Bam fragment into a BamHI fragment . This BamHI fragment is then clon into the BamHI site of pPGX1 (produced above) . The resulting plasm is co-transfected with PRVΔTKgp50tk + (HSV) . The resulting viruses a selected for the tk" phenotype by growth on araT . The selected virus have the gp50 gene inserted into the remaining portion of the tk ge as well as deleted from its normal locus . They are also gX" . Ther fore , any recombination wi th a f ield virus that gives a tk + gX~ vir would produce a virus that lacks a gp50 gene . S ince gp50 is essential gene, such a virus would be non-viable . Although our example relates to PRV , i t should be clear to tho skilled in the " art the same technique is useful to produce simil recombination-proof vaccine viruses in other her pesviruses . general , the steps include 1 ) insertion of an essential gene adjace to a mutation conferring avirulence , and 2) deletion of that essenti gene from its normal locus, which is linked to the deleted gene for secreted protein. Even more generally, moving essential genes fr their normal positions will reduce the probability of recombinati with wild- type viruses. Insertion of a selectable marker is n absolutely required to construct a PRV lacking gX. For example, plasmid containing a deletion in the gX coding region can be made deleting the base pair BamHI fragment from within the gX gene. Th plasmid is then be co-transfected with PRV DNA followed by screeni the viruses derived from that transfection for either lack of t deleted piece of DNA by nucleic acid hybridization, or by screening f lack of gX by an antibody screen (Holland, et al . , J.Virol., 4 pp. 649-52 (1983)). The polypeptide (e.g., gX") deletion viruses employed in t vaccines of the present invention can also be produced by oth techniques of inducing mutations followed by screening for virus lacking the polypeptide (e.g., gX) or any other technique which is us to produce a virus that has a deletion which renders the vaccine vir serologically distinct from the wild-type virus. For example, althou one could not select gX" PRV (anti-gX antibodies do not neutralize PR one can use the method of T.C. Holland, et al . , supra., to select f gl or gill (Wathen, J. Virol., 58, 173"78 (1986)) deletions in P which are useful in the vaccines of the present invention. While attenuation by inactivating or deleting the tk ge (Tatarov, supra.; Post and Roizman, supra.) is the preferred metho the gX" viruses of the instant invention may be subjected to conve tional chemical or physical inactivation procedures whereby the vir is rendered nonvirulent but still retains its antigenic propertie These inactivated vaccines may be formulated with a suitable adjuvan e.g., alum. For a general description of various vaccine preparati techniques see J.I. Duffy, Vaccine Preparation Techniques, Noyes Da Corporation (1980), and G.W. Warr, "Preparation of Antigens a Principles of Immunization", in J.J. Marchalonis and G.W. Warr, eds Antibody As A Tool - The Applications Of Immunochemi3try , pp. 21-5 John Wiley & Sons (1982). The virulent PRV virus may be propagated in animal tissue cultur until the virus is rendered nonpathogenlc, i.e., avirulent. PRV can propagated in a wide variety of tissue culture systems including, f example, chick embryo, duck embryo, porcine kidney, porcine teste embryonic bovine kidney, feline kidney, canine kidney and monk kidney; and also in established cell lines, such as, for example, Mad Darby bovine kidney (MDBK), and Madin Darby canine kidney (MDCK). Attenuation of PRV may be accomplished by standard serial passag including terminal dilution passage techniques wherein a sufficie number of passages in a susceptible tissue culture is employed unt the virus is rendered nonpathogenic without loss of immunogenicity. The passage time intervals should be such as to sufficiently all the virus to replicate between passages, and incubation temperatur are preferably from about 30-38°C. The optimum passage time depends the particular virus, culture system, and temperature employed. The final vaccine product should contain an amount of avirule PRV sufficient to stimulate an- immune response in disease-susceptib animals and still be nonpathogenic. The recommended titer to be admi istered to the susceptible animal is about 103-105 plaque-formi units, preferably about 10* plaque-forming units. The viral preparations of this invention may be diluted -with wat to adjust their potency, and they may have added to them stabilizer such as dextrose and lactose, or other nontoxic substances. The vir preparation may also be desiccated, e.g., by freeze drying, for stora purposes or for subsequent formulation into liquid vaccines. The vaccines may be administered to animals- by various route including intramuscular, intravenous, subcutaneous, intratracheal a intranasal. The preferred route of administration is intramuscular. For vaccination of sows a two dose regimen can be used. The fir dose can be given from about several months to about 5 to 7 weeks pri to farrowing. The second dose of the vaccine then should be admini tered several weeks after the first dose, for example, about 2 to weeks later and vaccine can then be administered up to, but prior t farrowing. Alternatively, the vaccine can be administered as a sing 2 ml dose, for example, at about 5 to 7 weeks prior to farrowin However, a 2 dose regimen Is considered preferable for the mo effective immunization of the baby pigs. Semi-annual revaccination recommended for breeding animals. Boars may be revaccinated at time. Also, sows can be revaccinated before breeding. Piglets born The vaccine may also be combined with other vaccines for oth diseases to produce a multivalent vaccine which may also be admini tered by any of the foregoing routes. It may also be combined wi other medicaments , for example, antibiotics . A vaccine prepared according to the pr esent invention wi stimulate an immune response in an animal susceptible to the disea without producing the clinical symptoms caused by the virulent virus any signif icant degree. A pharmaceuti cally effective amount of t vaccine can be employed with a pharmaceutically acceptable carrier diluent to vaccinate animals such as swine , cattle, sheep , goats , a other mammals . CHART A. Construction of pPRXK4 (a) pPRXhl is digested with Xhol and Kpnl to yield fragment (2.6 kb). Xhol Kpnl Kpnl Xhol * I I I I * p gX"* xxxxxxx < 2.6kb -» • x hoI Kpnl J L p gX — ÷ xxxxxx fragment 1 (b ) Fragment 1 is blunt-ended with T4 DNA polymerase and Eco linkers are added. EcoRI EcoRI J L p gX— ■ * xxxxxxxxx (c ) PACYC184 is digested with EcoRI and treated with BAP to yi fragment 2. EcoRI EcoRI J 1 I I Cmr Tetr * (d) Fragments 1 and 2 are ligated to produce pPRXK4. Mstl EcoRI Mstl EcoRI Mstl * _J j J J J * p gX " —- xxxxx X = glycoprotein X gene Cmi * = chloramphenicol resistance gene Tetr - tetracycline resistance gene p gχ » gX promoter CHART B. Construction of pPGX1 (a) pPRXK4 is digested with Mstl to yield fragment 3 (2.1 kb). Mstl EcoRI Mstl J J L |— p x (b) Fragment 3 is cut with EcoRI to produce fragment 4 (400 bp) EcoRI Mstl j L I— p gχ (c) pUC9 is digested with EcoRI and Smal to yield fragment 5 (2 kb). EcoRI ' BamHI Smal 1 J L (d) Fragments 4 and 5 are l igated to yield pPGX1 . EcoRI BamHI p gχ CHART C. Construction of pGXTK2 (a) pPGX1 is digested with BamHI and treated with BAP to yie fragment 6. BamHI EcoRI BamHI *g χ (b) pRB103 is digested with BamHI and Bglll to produce fragment (2.9 kb). Bglll BamHI BamHI * I I I * [ ■ + ttttttttt 'tk * ■ 2.9 kb- • Bglll BamHI j L ttttttttt Fragment 7 (c ) Fragments 6 and 7 are l igated to produce pGXTK2. EcoRI BamHI * J 1 * I ÷ tfctttttt PgX p tk = thymidine kinase promoter t = thymidine kinase gene CHART D. Construction of pGXTK3 (a) pPRXhl is digested with BamHI to produce fragment 8 (6.9 kb) BamHI Narl PvuII BamHI J J J L XXXXXXXX 5050505050 6363636363 gX-Ct (b) pGXTK2 is digested with BamHI and treated with BAP to yiel fragment 9. BamHI EcoRI BamHI J J L I ■ » • ttttttt p gχ (c) Fragments 8 and 9 are ligated to produce pGXTK3. EcoRI ' ■ BamHI BamHI I f ttttttt |XXXXXX gX-Ct = C-terminal coding region of gX gene 50 = glycoprotein 50 gene 63 = glycoprotein 63 gene I = glycoprotein I gene CHART E. Co-transfection with pGXTK3 and a tk" PRV . (a) Co-transfection of pGXTK3 and a tk"gX-PRV produces the t gX" product PRVΔgXI (or DT-A) . BamHI BamHI BamHI BamHI J I I J . I •>Ittttttt |xxxxx P g χ gX-Ct CHART F. Construction of pΔGXB7 (a) pPGXI is digested with BamHI and treated with BAP to yiel fragment 6. BamHI EcoRI BamHI p sx (b) pPRXhl is digested with BamHI to produce fragment 8 (6.9 kb BamHI Narl PvuII BamHI J J J L XXXXXXXX 5050505050 6363636363 g -Ct (c) Fragments 6 and 8 are ligated to produce plasmid pΔGXB7. EcoRI BamHI • BamH * I I L I -t xxxxxxx 5050505063636363 PgX gX-Ct CHART G. Construction of PRVΔGXTK" by recombination (a) Co-transfection of PRVΔGX1 and pΔGXB7 and recombinati produces- PRVΔGXTK" . BamHI BamHI /Bglll BamHI BamHI * I I I I * [ + 1 ttttttt |XXXXX p sx gX-Ct EcoRI BamHI BamH __ j →- XXXXXXX PgX gX-Ct = region of crossover (scale of right hand crossover region is extremely distorted ) CHART H. Construction of tk deletion plasmlds (a) BamHI 11 is cloned into pBR322 to produce plasmid pTK11. BamHI Xhol BamHI * I I I tktktktktktktktk (b) pTK11 is digested with Xhol to produce fragment 10. Xhol BamHI BamHI Xhol J I I L tktktktktktk tktktktktktktktktktktk (c) Fragment 1 0 is digested with Bal31 and then recircularized produce plasmids , e .g . , pΔtk-3 and pΔtk-4 , having varying leng deletions in the tk gene . BamHI BamHI * I I * tktktktkddddddtktktktktk tk = thymidine kinase gene d = deletion in the thymidine kinase gene CHART I. Construction of pGXTPA (a) Plasmid pPSAIδ is cut with Ball and BamHI linkers are adde to produce fragment 11. pPSA18: BamHI Ball * | I * tptptptptptptptp Fragment 11: BamHI LLLL tptptptptptptptptptp LLLL (b) Fragment 11 is digested with BamHI to produce fragment 1 (1.95 kb). BamHI BamHI tptptptptptptptptptptptp (c) Plasmid pPGXI (Chart B) is cut with BamHI to produce fragmen 6. BamHI Hindlll EcoRI BamHI p gχ (d) Fragments 6 and 12 are ligated to " produce plasmid pGXTPA. EcoRI BamHI BamHI Hindlll * I I I 1 j ■ * tptptptptptptptp p gx tp = tissue plasminogen activator gene L = BamHI linkers CHART J. Construction of plasmid pGXTPA-B7. (a) Plasmid pGXTPA is digested with Hindlll to produce fragme 13. Hindlll EcoRI BamHI BamHI Hindl J I I I j + tptptptp p gX (b) Hindlll linkers are added to fragment 8 (Chart D ) to produ fragment 1 . BamHI BamHI HLHLHL XXXXXXXXXXXXXXX HLHLHL gX-Ct (c ) Fragments 1 3 and 14 are then ligated together to produ pGXTPA -B7. EcoRI BamHI BamHI Hindlll Hindlll * I I 1 1 l_ I + tptptptp XXXXXXXXXXXXXXXXXX p gX HL = Hindlll linkers CHART K. Producution of recombination-proof viruses . (a) pΔTK-4 BamHI Sail Sphl Sail BamHI * I I 1 1 1 tktktktkddddddtktktktk (b) BamHI 7 BamHI Ndel PvuII Stul BamHI J J J J L XXXXXXXX 5050505050 6363636363 gX-Ct (c) PRVΔTKgp50 BamHI Sall/Ndel Sall/Stul Sphl » _l I J L tktktktk 50505050505050505050 tktktktk

Claims: CLAIMS 1. A herpesvirus vaccine comprising a properly incapacitated viru having a deletion for a gene encoding a secreted glycoprotein whic allows the serological distinction between the vaccine virus and it wild-type progenitor virus. 2. A vaccine according to claim 1, wherein the virus is selected fro the group consisting of pseudorabies virus, Marek's disease virus, an infectious bovine rhinotracheitis virus. 3. A vaccine according to claim 1 , wherein the virus is pseudorabie virus. 4. A vaccine according to claim 1, wherein the virus is pseudorabie virus and wherein the secreted glycoprotein is glycoprotein X. 5. A herpesvirus having a deletion for at least one gene encoding secreted glycoprotein. 6. A herpesvirus according to claim 5, selected from the grou consisting of pseudorabies virus, Marek's disease virus, and infectiou bovine rhinotracheitis virus. 7. A herpesvirus according to claim 5, wherein the herpesvirus i pseudorabies virus. 8. A herpesvirus according to claim 7, wherein the secrete glycoprotein is glycoprotein X. 9. The herpesvirus according to claim 8, wherein the virus produces n functional thymidine kinase. 10. A method for distinguishing between animals infected with a viru and animals vaccinated against said virus, comprising vaccinatin animals susceptible to the virus with a vaccine comprising said viru having a deletion for at least one gene encoding an antigen of th wild-type progenitor virus, and then serologically distinguishin between such vaccinated animals and those infected with virulent virus without sacrificing the animal. 11. A method according to claim 10, wherein the virus is a herpesvirus. 12. A method according to claim 10, wherein the virus is selected from the group consisting ' of pseudorabies virus, Marek's disease virus, and infectious bovine rhinotracheitis virus. 13. A method according to claim 10, wherein the virus is pseudorabies virus. 14. A method according to claim 10, wherein the antigen is' a glycoprotein. 15. A method according to claim 10, wherein the antigen is a secreted glycoprotein. 16. A method according to claim 10, wherein the virus is pseudorabies virus and the glycoprotein is glycoprotein X. 17. A multivalent vaccine comprising- a properly incapacitated virus having inserted within a gene encoding an antigen, a DNA sequence encoding a heterologous polypeptide capable of raising an immune response. 18. A vaccine according to claim 17, wherein the virus is a herpesvirus and the antigen is a secreted glycoprotein. 19. A vaccine according to claim 17, wherein the virus is selected from the group consisting of pseudorabies virus, Marek's disease virus, and infectious rhinotracheitis virus. 20. A vaccine according to claim 18, wherein the herpesvirus is pseudorabies virus, and wherein the secreted glycoprotein is glycoprotein X. 21 . A vaccine according to claim 20 , wherein the polypeptide capable of raising an immune response is selected from the group consisting of a polypeptide from transmissible gastroenteri tis virus or porcine parvovirus .

Inventors: POST LEONARD E, THOMSEN DARRELL R, POST, LEONARD, E., THOMSEN, DARRELL, R.
Applicants: UPJOHN CO, THE UPJOHN COMPANY
Application year: 1986
Main classification: C12N15
IPC codes: A61K39, C07K14, C12N7, C12N15, G01N33