Deletion mutant of a herpesvirus and vaccine containing said virus

Abstract

The invention relates to live deletion mutants of a herpesvirus, especially of Pseudorabies virus. The genomes of the mutants differ from the genomes of the parent strain by the presence of one or more deletions selected from a deletion in the inverted repeats, a deletion in the unique sequence, and a deletion in the thymidine kinase gene. The new deletion mutants are avirulent, do not induce latent infections, are stable and possess good immunogenic properties. The invention also relates to vaccines containing the new mutant strains.

We claim: 
     
       1. A live deletion mutant of a pseudorabies virus .[.derived from a.]. .Iadd.obtained by introducing, by means of recombinant DNA techniques, one or more deletions into the genome of a virulent .Iaddend.parental strain having a parental genome of about 160,000 nucleotide pairs, said parental genome comprising (a) two inverted repeats, each of which contains cleavage sites for the restriction endonucleases BamHI and KpnI, BamHI digestion of the parental genome yielding two pairs of restriction fragments derived entirely from the sequence of the inverted repeats, the larger fragment of each pair being designated as BamHI fragments 5; and KpnI digestion of the genome yielding two fragments, designated KpnI fragment E and .[.kpnI.]. .Iadd.KpnI .Iaddend.fragment H, each of said KpnI fragments containing the distal end of one of the inverted repeats;   (b) a unique sequence located between the inverted repeats, said unique sequence comprising a BamHI restriction fragment of about 7 kilobase pairs in length designated BamHI fragment 7, said BamHI fragment 7 containing a cleavage site for the restriction endonuclease SalI, wherein cleavage at the SalI site produces two unequal fragments from BamHI fragment 7, one fragment of about five kilobase pairs in length and the other of about two kilobase pairs in length; and   (c) a gene for thymidine kinase, said deletion mutant .[.being characterized by one or more deletions, said deletions being located in (i) a portion of an inverted repeat which is common to BamHI fragment 5 and either KpnI fragment E or KpnI fragment H;   (ii).]. .Iadd.having a deletion in .Iaddend.the about five kilobase pair SalI fragment of BamHI fragment 7, .[.or   (iii) both.]. .Iadd.such that the mutant is less virulent that the parental strain.Iaddend.. .[.2. Virus according to claim 1, characterized in that     
     
     
        deletion (i) comprises about 100 nucleotides..]. 3. Live Pseudorabies virus deletion mutants according to claim 1, which are derived from NIA-3 
     
     
        strain of PRV. 4. Deletion mutant 2.4N3A according to claim 3 as deposited at the Phabagen Collection at Utrecht, Netherlands, deposit number PC PV2. 
     
     
        . Deletion mutant 2.8N3A according to claim 3 as deposited at the Phabagen 
     
     
        Collection at Utrecht, Netherlands, deposit number PC PV3. 6. Vaccine containing a live virus mutant according to one of claims 1, .[.2.]. 
     
     
        .Iadd.or .Iaddend.3-5. 7. A live deletion mutant according to claim 1, 
     
     
        including a further deletion in the thymidine kinase gene. 8. A live deletion mutant according to claim 7, wherein the deletion in the 
     
     
        thymidine kinase gene is from 100 to 500 nucleotides in length. .Iadd.9. Virus according to claim 1, having a further deletion in a portion of an inverted repeat which is common to BamHI fragment 5 and either KpnI fragment E or KpnI fragment H. .Iaddend. .Iadd.10. Virus according to claim 9, including a further deletion in the thymidine kinase gene. .Iaddend.