Live attenuated vaccines based on cp45 HPIV-3 strain and method to ensure attenuation in such vaccines

• US 6,110,457 A
• Filed: 12/09/1997
• Issued: 08/29/2000
• Est. Priority Date: 12/08/1995
• Status: Expired due to Term

• Current Assignee: St. Louis University of St. Louis
• Original Assignee: St. Louis University
• Inventors: Ray, Ranjit, Belshe, Robert B.
• Primary Examiner(s): REYNOLDS, DEBORAH J
• Application Number:
  US08/987,439
• Time in Patent Office: 994 Days
• Field of Search: 435/235.1, 435/320.1, 435/4, 435/5, 435/6, 435/325, 435/455, 435/440, 424/93.1, 424/93.2, 514/44
• US Class Current: 424/93.2
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/155 Paramyxoviridae, e.g. parainfluenza virus
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2760/18622 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/18634 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The present invention is based upon correlation of two attenuating lesions of the cp45 strain to specific genetic defects in the viral genome of cp45. Specifically, it is now understood that a significant level of attenuation of cp45 giving rise to its temperature-sensitive and cold-adapted phenotypes is directly associated with mutation of the large, or L, gene of cp45 relative to the corresponding gene in the wild-type JS strain. Moreover, it is further understood that a second attenuating lesion exits independent of the temperature-sensitive lesion, and is directly associated with mutation of the hemagglutinin-neuraminidase gene, or HN gene, of cp45 relative to the corresponding gene in the wild-type HPIV-3 (JS) strain. The correlation of these two attenuating lesions of cp45 to specific genes enables several practical applications. It is now possible to create vaccines directed at other wild-type HPIV-3 viruses and, additionally, vaccines directed at target viruses other than HPIV-3 using genetic engineering techniques. For example, the mutated L and/or HN genes of cp45 can be incorporated into the viral genome of a target virus. Alternatively, the genes of the target virus which encode its surface antigens can be incorporated into the viral genome of cp45. Moreover, it is possible to determine whether an HPIV-3 strain or a hybrid virus strain made by the methods disclosed herein is attenuated by confirming the presence or absence of mutations in its L and/or HN genes.

Claims

1. An enveloped, negative-sense, single-stranded RNA hybrid virus, the hybrid virus having a genome comprising, operatively linked for expression, (i) a nucleic acid sequence which encodes at least one surface antigen of an enveloped target virus of the family Paramyxoviridae, the surface antigen being different from surface antigens of cp45 and (ii) a nucleic acid sequence which encodes a variant HPIV-3 large protein, L, the variant L protein having at least one variation in amino acid sequence relative to the L protein of wild-type HPIV-3 (JS), the variation being a substitution selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1, and Ile for Thr at residue 1558 of SEQ ID NO. 1, the variant L protein having polymerase activity.

2. A live vaccine suitable for use against an enveloped target virus of the family Paramyxoviridae, the vaccine comprising the hybrid virus of claim 1 and a pharmaceutically appropriate carrier.

3. The hybrid virus of claim 1 wherein the variant L protein has an amino acid sequence having at least a 90% sequence identity with the amino acid sequence of the wild type HPIV-3 (JS) L protein, and the variant L protein has an RNA-polymerase activity which is at least about 10 times less than the polymerase activity normally associated with the target virus at a temperature of about 39°

C.

4. The hybrid virus of claim 3 wherein the hybrid virus has a genome further comprising (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of cp45;

(ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of cp45;

(iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of cp45;

(iv) a nucleic acid sequence which encodes the matrix protein, M, of cp45; and

(v) wherein the variant large protein, L, has at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Thr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

5. A live vaccine suitable for use against an enveloped target virus of the family Paramyxoviridae, the vaccine comprising the hybrid virus of claim 3 and a pharmaceutically appropriate carrier.

6. The vaccine of claim 5 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

7. The vaccine of claim 5 wherein the amino acid sequence of the variant L protein has at least a 99.5% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

8. The vaccine of claim 5 wherein the variant L protein has an amino acid sequence having a substitution in one, two or three amino acids relative to the sequence of the L protein of wild-type HPIV-3 (JS), the substitution being selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1, and Ile for Thr at residue 1558 of SEQ ID NO. 1.

9. The vaccine of claim 5 wherein the variant L protein has an amino acid sequence having at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

10. The vaccine of claim 5 wherein the variant L protein is the cp45 L protein.

11. The vaccine of claim 5 wherein the target virus is selected from the group consisting of HPIV-1, HPIV-2, RSV, and Morbillivirus viruses.

12. The vaccine of claim 5 wherein the target virus is selected from the group consisting of HPIV-1, HPIV-2 and RSV.

13. The vaccine of claim 5 wherein the variant L protein is an RNA-dependent RNA polymerase having an activity which is at least about 10 times less than the activity of the wild-type HPIV-3 (JS) polymerase.

14. The vaccine of claim 5 wherein the genome of the virus further comprises:

(i) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of a HPIV-3 virus and (ii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of a HPIV-3 virus.

15. The vaccine of claim 5 wherein the genome of the virus further comprises:

(i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of a HPIV-3 virus;

(ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of a HPIV-3 virus;

(iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of a HPIV-3 virus; and

(iv) a nucleic acid sequence which encodes the matrix protein, M, of a HPIV-3 virus.

16. The vaccine of claim 15 wherein the 3'"'"' leader region of the HPIV-3 genome is the 3'"'"' leader region of the cp45 genome.

17. The vaccine of claim 15 wherein the HPIV-3 phosphoprotein is the phosphoprotein of cp45.

18. The vaccine of claim 15 wherein the HPIV-3 matrix protein is the matrix protein of cp45.

19. The vaccine of claim 15 wherein the 3'"'"' leader region of the HPIV-3 genome is the 3'"'"' leader region of the cp45 genome and the HPIV-3 NP, P(+C), and M proteins are cp45 NP, P(+C) and M proteins.

20. The vaccine of claim 5 wherein the genome of the hybrid virus further comprises (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of cp45;

(ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of cp45;

(iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of cp45;

(iv) a nucleic acid sequence which encodes the matrix protein, M, of cp45;

(v) wherein the surface antigen encoding nucleic acid sequence encodes a surface antigen of a target virus selected from the group consisting of HPIV-1, HPIV-2 and RSV; and

(vi) wherein the L protein encoding nucleic acid sequence encodes a variant large protein, L, having RNA-polymerase activity which is at least ten times less than the polymerase activity normally associated with the target virus at a temperature of about 39°

C., having at least a 99.8% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and having at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

21. The enveloped, negative-sense, single-stranded RNA hybrid virus of claim 1, wherein the genome comprises, operatively linked for expression, a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of a wild-type HPIV-3 target virus or which encodes at least one protein selected from the group consisting of the matrix protein, M, of said target virus, and the fusion protein, F, of said target virus and the hemagglutinin-neuraminidase protein, HN, of the target virus.

22. A live vaccine suitable for use against a HPIV-3 target virus, the vaccine comprising the hybrid virus of claim 21 and a pharmaceutically appropriate carrier.

23. The vaccine of claim 22 wherein the amino acid sequence of the variant L protein has at least a 90% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

24. The vaccine of claim 22 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

25. The vaccine of claim 22 wherein the amino acid sequence of the variant L protein has at least a 99.5% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

26. The vaccine of claim 22 wherein the amino acid sequence of the variant L protein has a substitution in one, two or three amino acids relative to the sequence of the L protein of wild-type HPIV-3 (JS), the substitution being selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1 and Ile for Thr at residue 1558 of SEQ ID NO. 1.

27. The vaccine of claim 22 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and has at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

28. The vaccine of claim 22 wherein the variant L protein is the L protein of the HPIV-3 strain designated as cp45.

29. The vaccine of claim 27 wherein the viral genome comprises a nucleic acid sequence which encodes a variant hemagglutinin-neuraminidase protein, HN, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the HN protein of wild-type HPIV-3 (JS) virus, which has at least one variation in amino acid sequence relative to the HN protein of the target virus and which has at least one variation in amino acid sequence relative to the HN protein of the HPIV-3 (JS) virus, the variation relative to the HN protein of HPIV-3 (JS) being at residue 384 of SEQ ID NO. 3, the variant HN protein having neuraminidase activity which is less than the neuraminidase activity normally associated with the HN protein of the target virus.

30. The vaccine of claim 29 wherein the amino acid sequence of the variant HN protein has a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) HN protein.

31. The vaccine of claim 29 wherein the amino acid sequence of the variant HN protein has a 99.5% sequence identity with the amino acid sequence of the wild-type PIV-3 (JS) HN protein.

32. The vaccine of claim 29 wherein the variant HN protein has at least one substitution in amino acid sequence relative to the wild-type HPIV-3 (JS) HN protein, the substitution being Val for Ala at residue 384 of SEQ ID NO. 3.

33. The vaccine of claim 29 wherein the viral genome comprises a nucleic acid sequence which encodes the hemagglutinin-neuraminidase protein, HN, of the HPIV-3 strain designated as cp45.

34. The vaccine of claim 29 wherein the viral genome comprises a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of the HPIV-3 strain designated as cp45 or which encodes a protein selected from the group consisting of the nucleocapsid protein, NP, of cp45, the phosphoprotein, P(+C), of cp45, the matrix protein, M, of cp45, and the fusion protein, F, of cp45.

35. The vaccine of claim 29 wherein the viral genome comprises a nucleic acid sequence which encodes the hemagglutinin-neuraminidase protein, HN, of the HPIV-3 target virus.

36. The vaccine of claim 29 wherein the viral genome comprises (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of cp45, (ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of cp45, (iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of cp45, (iv) a nucleic acid sequence which encodes the matrix protein, M, of cp45, (v) a nucleic acid sequence which encodes the fusion protein, F, of cp45, (vi) a nucleic acid sequence which encodes the hemagglutinin-neuraminidase protein, HN, of the wild-type HPIV-3 target virus, and (vii) a nucleic acid sequence which encodes the L protein of cp45.

37. The vaccine of claim 29 wherein the hemagglutinin-neuraminidase protein encoded by the viral genome is the hemagglutinin-neuraminidase protein, HN, of wild-type HPIV-3 (JS).

38. The vaccine of claim 29 wherein the viral genome comprises (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of the target virus, (ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of the target virus, (iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of the target virus, (iv) a nucleic acid sequence which encodes the matrix protein, M, of the target virus, (v) a nucleic acid sequence which encodes the fusion protein, F, of the target virus, (vi) a nucleic acid sequence which encodes the hemagglutinin-neuraminidase protein, HN, of the target virus, and (vii) a nucleic acid sequence which encodes the variant L protein.

40. A host cell transfected with the plasmid vector set forth in claim 34, wherein said host cell is cultured or isolated.

39. A plasmid vector comprising a positive or negative sense genome which includes, operatively linked for expression, (i) a nucleic acid sequence which encodes the surface antigens of a target virus of the family Paramyxoviridae, the surface antigens being different from surface antigens of the HPIV-3 strain designated as cp45, and (ii) a nucleic acid sequence which encodes a variant large protein, L, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and which has at least one variation in amino acid sequence relative to the L protein of wild-type HPIV-3 (JS), the variation being a substitution selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1, and Ile for Thr at residue 1558 of SEQ ID NO. 1, the variant L protein having an RNA-polymerase activity which is less than the polymerase activity normally associated with the target virus at a temperature of about 39°

C.

41. A method for producing an enveloped, negative-sense, single-stranded RNA hybrid virus suitable for use as a live vaccine, the method comprising:

preparing the chimeric plasmid vector of claim 39,

transfecting a host cell with the chimeric plasmid vector,

cotransfecting the host cell with plasmid vectors that express wild-type HPIV-3 NP, P and L proteins;

incubating the transfected host cell to produce a hybrid virus; and

isolating the hybrid virus in a pharmaceutically acceptable medium.

42. The method as set forth in claim 41 wherein the chimeric plasmid vector is prepared by

preparing a cDNA clone of the genome of the HPIV-3 strain designated as cp45, the genome comprising a nucleic acid sequence which encodes the HN protein of cp45 and the L protein of cp45,

incorporating the cDNA clone of the cp45 genome into a plasmid vector,

preparing a cDNA clone of or obtaining DNA of the genome of a target virus, the genome comprising a nucleic acid sequence which encodes the surface antigens of the target virus, and

replacing the region of the plasmid vector'"'"'s genome which encodes the HN protein of cp45 with the nucleic acid sequence which encodes the surface antigens of the target virus.

43. The method as set forth in claim 41 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and has at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

44. The method as set forth in claim 41 wherein the genome further includes (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of cp45, (ii) a nucleic acid sequence which encodes the nucleocapsid protein, NP, of cp45, (iii) a nucleic acid sequence which encodes the phosphoprotein, P(+C), of cp45, (iv) a nucleic acid sequence which encodes the matrix protein, M, of cp45.

66. A non-human host cell transfected with the plasmid vector set forth in claim 39, wherein said host cell is contained within an organism.

45. An enveloped, negative-sense, single-stranded RNA hybrid virus, the hybrid virus having a genome comprising, operatively linked for expression, a nucleic acid sequence which encodes (i) at least one surface antigen of a target virus of the family Paramyxoviridae, said target virus being one which depends upon a protein exhibiting neuraminidase activity to complete its replication cycle, the surface antigen being different from surface antigens of the HPIV-3 strain designated as cp45;

and (ii) one protein which exhibits neuraminidase activity, said protein comprising a portion of the cp45 HN protein, the encoded portion having a neuraminidase activity and including an amino acid sequence which is the same as the amino acid sequence from residue 160 to residue 385 of SEQ. ID. NO. 3.

46. A live vaccine suitable for use against a target virus of the family Paramyxoviridae, the vaccine comprising the hybrid virus of claim 45 and a pharmaceutically appropriate carrier.

47. The vaccine as set forth in claim 46 wherein the viral genome further comprises a nucleic acid sequence which encodes a variant large protein, L, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and which has at least one variation in amino acid sequence relative to the L protein of wild-type HPIV-3 (JS), the variation being a substitution being selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1 and Ile for Thr at residue 1558 of SEQ ID NO. 1, the variant L protein having an RNA-polymerase activity which is less than the polymerase activity normally associated with the target virus at a temperature of about 39°

C.

48. The vaccine as set forth in claim 47 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein.

49. The vaccine as set forth in claim 47 wherein the amino acid sequence of the variant L protein has at least a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and has at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Leu at residue 992 of SEQ ID NO. 1.

50. The vaccine as set forth in claim 46 wherein the viral genome further comprises a nucleic acid sequence which encodes the large protein, L, of cp45.

51. An enveloped, negative-sense, single-stranded RNA hybrid virus having a genome comprising, operatively linked for expression, (i) a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of a wild-type HPIV-3 target virus or which encodes at least one protein selected from the group consisting of the matrix protein, M, of the target virus, the fusion protein, F, of the target virus and the large protein, L, of the target virus, and (ii) a nucleic acid sequence which encodes a variant hemagglutinin-neuraminidase protein, HN, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the HN protein of wild-type HPIV-3 (JS) virus, which has at least one variation in amino acid sequence relative to the HN protein of the target virus and which has at least one variation in amino acid sequence relative to the HN protein of the HPIV-3 (JS) virus, the variation relative to the HN protein of HPIV-3 (JS) being at residue 384 of SEQ ID NO. 3, the variant HN protein having neuraminidase activity which is less than the neuraminidase activity normally associated with the HN protein of the target virus.

52. A live vaccine suitable for use against a HPIV-3 target virus, the vaccine comprising the hybrid virus of claim 51 and a pharmaceutically appropriate carrier.

53. The vaccine of claim 52 wherein the amino acid sequence of the variant HN protein has a 99% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) HN protein.

54. The vaccine of claim 52 wherein the amino acid sequence of the variant HN protein has a 99.5% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) HN protein.

55. The vaccine of claim 52 wherein the variant HN protein has at least one substitution in amino acid sequence relative to the wild-type HPIV-3 (JS) HN protein, the substitution being Val for Ala at residue 384 of SEQ ID NO. 3.

56. The vaccine of claim 52 wherein the variant HN protein is the HN protein of the HPIV-3 strain designated as cp45.

57. The vaccine of claim 52 wherein the selection from group (i) is not the large protein, L, of the target virus, and wherein the viral genome further comprises a nucleic acid sequence which encodes a variant large protein, L, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and which has at least one substitution in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitution being selected from the group consisting of His for Tyr at residue 942 of SEQ ID NO. 1, Phe for Leu at residue 992 of SEQ ID NO. 1 and Ile for Thr at residue 1558 of SEQ ID NO. 1, the variant L protein having an RNA-polymerase activity which is less than the RNA-polymerase activity normally associated with the L protein of the target virus at a temperature of about 39°

C.

58. The vaccine of claim 52 wherein the viral genome comprises a nucleic acid sequence which encodes a variant large protein, L, having an amino acid sequence which has at least a 90% sequence identity with the amino acid sequence of the wild-type HPIV-3 (JS) L protein and which has at least two substitutions in amino acid sequence relative to the wild-type HPIV-3 (JS) L protein, the substitutions being His for Tyr at residue 942 of SEQ ID NO. 1 and Phe for Ile at residue 992 of SEQ ID NO. 1, the variant L protein having an RNA-polymerase activity which is less than the RNA-polymerase activity normally associated with the L protein of the target virus at a temperature of about 39°

C.

59. The vaccine of claim 52 wherein the viral genome comprises a nucleic acid sequence which encodes the large protein, L, of cp45.

60. The vaccine of claim 52 wherein the viral genome comprises a nucleic acid sequence which is the same as the nucleic acid sequence of the 3'"'"' leader region of cp45 or which encodes a protein selected from the group consisting of the nucleocapsid protein, NP, of cp45, the phosphoprotein, P(+C), of cp45, the matrix protein, M, of cp45, and the fusion protein, F, of cp45.

61. A method for determining whether a HPIV-3 virus or a cp45-hybrid virus is attenuated, the method comprising confirming the presence of at least one variation in the genome of the virus relative to the genome of wild-type HPIV-3, the variation being selected from the group consisting of those which would cause a variation in the amino acid corresponding to position 942, 992, or 1558 of SEQ ID NO. 1 or position 384 of SEQ ID NO. 3.

62. The method of claim 61 wherein the presence of at least one variation in the gene encoding the L protein of the virus is confirmed by performing a complementation assay, wherein the virus is complemented in a host cell with L protein of the wild-type HPIV-3 virus.

63. A method for determining whether a HPIV-3 or a cp45-hybrid virus has a temperature sensitive phenotype, the method comprising

obtaining a sample of a HPIV-3 virus or a cp45-hybrid virus,

performing a first plaque assay at a non-permissive temperature on the virus as a control assay;

transfecting a mammalian host cell with a plasmid vector that expresses L protein of wild-type HPIV-3 (JS);

infecting the host cell with the virus;

incubating to yield a complemented virus;

performing a second plaque assay at a non-permissive temperature on the complemented viral sample; and

comparing the second plaque assay to the control assay to confirm the presence of the temperature sensitive phenotype.

64. The method of claim 63 further comprising cotransfecting the host cell with a plasmid vector that expresses P protein of wild-type HPIV-3 (JS).

65. The method of claim 63 further comprising cotransfecting the host cell with a plasmid vector that expresses NP protein of wild-type HPIV-3 (JS).