Pseudorabies virus mutants, vaccines containing same, methods for the production of same and methods for the use of same

Claim 2

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of a deletion, an insertion or both a deletion and an insertion in the g92 gene.

Claim 3

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus fails to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene.

Claim 4

The pseudorabies virus as claimed in claim 1, wherein said deletion is about 10 to 1500 bp in size.

Claim 5

The pseudorabies virus as claimed in claim 3, wherein said deletion is about 75 to 200 bp in size.

Claim 6

The pseudorabies virus as claimed in claim 1, wherein said insertion is about 8 to 5000 bp in size.

Claim 7

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus also fails to produce any functional TK as a result of a mutation in the tk gene.

Claim 8

The pseudorabies virus as claimed in claim 6, wherein said mutation in the tk gene is a deletion mutation.

Claim 9

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus also fails to produce any glycoprotein gI as a result of a mutation in the gI gene.

Claim 10

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus is also temperature-resistant.

Claim 11

The pseudorabies virus as claimed in claim 1, wherein said virus has the identifying characteristics of PRV(dlg92/dltk) (ATCC No. VR-2116).

Claim 12

The pseudorabies virus as claimed in claim 1, wherein said pseudorabies virus is lyophilized.

Claim 13

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequence thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene and selecting for pseudorabies virus recombinants which produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 14

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequence thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene and selecting for pseudorabies virus recombinants which produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 15

The pseudorabies virus as claimed in claim 12, wherein said insertion is about 8 to 5000 bp in size.

Claim 16

The pseudorabies virus as claimed in claim 12, wherein the infectious DNA of step (3) is derived from a pseudorabies virus mutant which fails to produce any functional TK such that the resulting mutants of step (3) fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene and fail to produce any functional TK as a result of a mutation in the tk gene.

Claim 17

The pseudorabies virus as claimed in claim 14, wherein said pseudorabies virus mutant which fails to produce any functional TK, fails to produce such as a result of deletion in the tk gene.

Claim 18

The pseudorabies virus as claimed in claim 15, wherein said PRV mutant is pseudorabies virus (BUK-dl 3).

Claim 19

The pseudorabies virus as claimed in claim 12, wherein said pseudorabies virus mutant also fails to produce any gI glycoprotein as a result of a mutation in the gI gene.

Claim 20

The pseudorabies virus as claimed in claim 12, wherein the infectious DNA of step (3) is derived from temperature-resistant pseudorabies virus such that the resulting mutants of step (3) are temperature-resistant pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 21

The pseudorabies virus as claimed in claim 12, additionally comprising step (4): (4) Propagating the resulting pseudorabies virus of step (3) at a non-permissive temperature for a temperature-sensitive virus so as to select for and produce temperature-resistant pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 22

The pseudorabies virus as claimed in claim 12, wherein said cloning vector is selected from the group consisting of pBR322, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10 and pMAR420.

Claim 23

The pseudorabies virus as claimed in claim 20, wherein said cloning vector is pBR322.

Claim 24

The pseudorabies virus as claimed in claim 12, wherein the resulting hybrid plasmid of step (2) is pPRVTK/Stu12.

Claim 25

The pseudorabies virus as claimed in claim 12, wherein said selectable gene is selected from the group consisting of a tk gene, the transposon Tn5 gene and the E. coli lacZ gene.

Claim 26

The pseudorabies virus as claimed in claim 23, wherein said selectable gene is a tk gene.

Claim 27

The pseudorabies virus as claimed in claim 24, wherein said tk gene is selected from the group consisting of the HSV-1 tk gene, the HSV-2 tk gene, the marmoset herpesvirus tk gene, the chicken tk gene, the human tk gene and the pseudorabies virus tk gene.

Claim 28

The pseudorabies virus as claimed in claim 25, wherein said tk gene is the pseudorabies virus tk gene.

Claim 29

The pseudorabies virus as claimed in claim 12, wherein said pseudorabies virus is lyophilized.

Claim 30

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequences thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene, and selecting for pseudorabies virus which produce the product of the selectable gene; (4) Deleting DNA sequences from the hybrid plasmid of step (1) such that less than substantially all of the g92 gene is present, while retaining pseudorabies virus DNA sequences adjacent to each side of the deletion; (5) Co-transfecting in pseudorabies virus host cells the resulting hybrid plasmid of step (4) with infectious DNA from the selected pseudorabies virus of step (3), and selecting for PRV which do not produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene.

Claim 31

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequences thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene, and selecting for pseudorabies virus which produce the product of the selectable gene; (4) Deleting DNA sequences from the hybrid plasmid of step (1) such that less than substantially all of the g92 gene is present, while retaining pseudorabies virus DNA sequences adjacent to each side of the deletion; (5) Co-transfecting in pseudorabies virus host cells the resulting hybrid plasmid of step (4) with infectious DNA from the selected pseudorabies virus of step (3), and selecting for PRV which do not produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene.

Claim 32

The pseudorabies virus as claimed in claim 28, wherein the deletion is about 10 to 1500 bp in size.

Claim 33

The pseudorabies virus as claimed in claim 29, wherein the deletion is about 74 to 200 bp in size.

Claim 34

The pseudorabies virus as claimed in claim 28, wherein a foreign DNA sequence is inserted in place of the deleted g92 gene sequence in step (4) such that no antigenic g92 polypeptides are produced and such that pseudorabies virus DNA sequences adjacent to each side of the deleted g92 gene sequences are retained, so that the resulting pseudorabies virus mutants of step (5) fail to produce any antigenic g92 polypeptides as a result of a combined deletion and insertion in the g92 gene.

Claim 35

The pseudorabies virus as claimed in claim 31, wherein the foreign DNA sequence is about 8 to 5000 bp in size.

Claim 36

The pseudorabies virus as claimed in claim 28, wherein the infectious DNA of step (3) is derived from a pseudorabies virus mutant which fails to produce any functional TK such that the resulting pseudorabies virus mutants of step (5) fail to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene and fail to produce any functional TK as a result of a mutation in the tk gene.

Claim 37

The pseudorabies virus as claimed in claim 33, wherein said pseudorabies virus mutant which fails to produce any functional TK, fails to produce such as a result of deletion in the tk gene.

Claim 38

The pseudorabies virus as claimed in claim 34, wherein said PRV mutant is pseudorabies virus (BUK-dl 3).

Claim 39

The pseudorabies virus as claimed in claim 28, wherein said pseudorabies virus mutant also fails to produce any gI glycoprotein as a result of a mutation in the gI gene.

Claim 40

The pseudorabies virus as claimed in claim 28, wherein the infectious DNA of step (3) is derived from a temperature-resistant pseudorabies virus such that the resulting pseudorabies virus mutants of step (5) are temperature-resistant pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene.

Claim 41

The pseudorabies virus as claimed in claim 28, additionally comprising step (6): (6) Propagating the resulting pseudorabies virus of step (5) at a non-permissive temperature for a temperature-sensitive virus so as to select for and produce a temperature-resistant pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of a deletion in the g92 gene.

Claim 42

The pseudorabies virus as claimed in claim 28, wherein said cloning vector is selected from the group consisting of pBR322, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10 and pMAR420.

Claim 43

The pseudorabies virus as claimed in claim 39, wherein said cloning vector is pBR322.

Claim 44

The pseudorabies virus as claimed in claim 28, wherein the resulting hybrid plasmid of step (2) is pPRVTK/Stu12.

Claim 45

The pseudorabies virus as claimed in claim 28, wherein the resulting hybrid plasmid of step (4) is pBUK:gCdlSal.

Claim 46

The pseudorabies virus as claimed in claim 28, wherein said selectable gene is selected from the group consisting of a tk gene, the transposon Tn5 gene and the E. coli lacZ gene.

Claim 47

The pseudorabies virus as claimed in claim 43, wherein said selectable gene is a tk gene.

Claim 48

The pseudorabies virus as claimed in claim 44, wherein said tk gene is selected from the group consisting of the HSV-1 tk gene, the HSV-2 tk gene, the marmoset herpesvirus tk gene, the chicken tk gene, the human tk gene and the pseudorabies virus tk gene.

Claim 49

The pseudorabies virus as claimed in claim 45, wherein said tk gene is the pseudorabies virus tk gene.

Claim 50

The pseudorabies virus as claimed in claim 28, wherein said pseudorabies virus is lyophilized.

Claim 51

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequences thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene, and selecting for pseudorabies virus which produce the product of the selectable gene; (4) Inserting a foreign DNA sequence into the plasmid of step (1) such that no antigenic g92 polypeptides are produced and such that pseudorabies virus DNA sequences adjacent to each side of the insertion are retained; (5) Co-transfecting in pseudorabies virus host cells the resulting hybrid plasmid of step (4) with infectious DNA from the selected pseudorabies virus of step (3), and selecting for pseudorabies virus which do not produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 52

A pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene produced by the process comprising: (1) Constructing a hybrid plasmid comprising a cloning vector and a DNA fragment of pseudorabies virus containing substantially all of the pseudorabies virus g92 gene and flanking sequences thereof; (2) Inserting a DNA fragment which encodes a functional selectable gene into the pseudorabies virus g92 gene of the resulting hybrid plasmid of step (1) such that the functional selectable gene is flanked by DNA sequences of the pseudorabies virus g92 gene; (3) Co-transfecting into pseudorabies virus host cells the hybrid plasmid of step (2) with infectious DNA from a pseudorabies virus which fails to produce the product of the selectable gene, and selecting for pseudorabies virus which produce the product of the selectable gene; (4) Inserting a foreign DNA sequence into the plasmid of step (1) such that no antigenic g92 polypeptides are produced and such that pseudorabies virus DNA sequences adjacent to each side of the insertion are retained; (5) Co-transfecting in pseudorabies virus host cells the resulting hybrid plasmid of step (4) with infectious DNA from the selected pseudorabies virus of step (3), and selecting for pseudorabies virus which do not produce the product of the selectable gene so as to produce pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene.

Claim 53

The pseudorabies virus as claimed in claim 48, wherein said insertion is about 8 to 5000 bp in size.

Claim 54

The pseudorabies virus as claimed in claim 48, wherein the infectious DNA of step (3) is derived from a pseudorabies virus mutant which fails to produce any functional TK such that the resulting mutants of step (5) fail to produce any antigenic g92 polypeptides as a result of an insertion in the g92 gene and fail to produce any functional TK as a result of a mutation in the tk gene.

Claim 55

The pseudorabies virus as claimed in claim 50, wherein said pseudorabies virus mutant which fails to produce any functional TK, fails to produce such as a result of deletion in the tk gene.

Claim 56

The pseudorabies virus as claimed in claim 51, wherein said pseudorabies virus mutant is PRV(BUK-dl 3).

Claim 57

The pseudorabies virus as claimed in claim 48, wherein said pseudorabies virus mutant also fails to produce any gI as a result of a mutation in the gI gene.

Claim 58

The pseudorabies virus as claimed in claim 48, wherein the infectious DNA of step (3) is derived from temperature-resistant pseudorabies virus such that the resulting PRV mutants of step (5) are temperature-resistant pseudorabies virus mutants which fail to produce any antigenic g92 polypeptides as a result of an insertion mutation in the g92 gene.

Claim 59

The pseudorabies virus as claimed in claim 48, additionally comprising step (6): (6) Propagating the resulting pseudorabies virus of step (5) at a non-permissive temperature for a temperature-sensitive virus so as to select for and produce a temperature-resistant pseudorabies virus which fails to produce any antigenic g92 polypeptides as a result of an insertion mutation in the g92 gene.

Claim 60

The pseudorabies virus as claimed in claim 48, wherein said cloning vector is selected from the group consisting of pBR322, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10 and pMAR420.

Claim 61

The pseudorabies virus as claimed in claim 56, wherein said cloning vector is pBR322.

Claim 62

The pseudorabies virus as claimed in claim 48, wherein the resulting hybrid of step (2) plasmid is pPRVTK/Stu12.

Claim 63

The pseudorabies virus as claimed in claim 48, wherein said selectable gene is selected from the group consisting of a tk gene, the transposon Tn5 gene and the E. coli lacZ gene.

Claim 64

The pseudorabies virus as claimed in claim 59, wherein said selectable gene is a tk gene.

Claim 65

The pseudorabies virus as claimed in claim 60, wherein said tk gene is selected from the group consisting of the HSV-1 tk gene, the HSV-2 tk gene, the marmoset herpesvirus tk gene, the chicken tk gene, the human tk gene and the pseudorabies virus tk gene.

Claim 66

The pseudorabies virus as claimed in claim 61, wherein said tk gene is the pseudorabies virus tk gene.

Claim 67

The pseudorabies virus as claimed in claim 48, wherein said pseudorabies virus is lyophilized.