Identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp4 5) candidate vaccine was shown previously to be safe, immunogenic, and phen otypically stable in seronegative human infants. Previous findings indicate d that each of the three amino acid substitutions in the L polymerase prote in of cp45 independently confers the temperature-sensitive (ts) and attenua tion (aft) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other prot eins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting se quences (the leader region and the transcription gene start [GSI signal of the N gene), and their contribution to these phenotypes was undefined. To f urther characterize the genetic basis for the ts, ca, and an phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutati ons mentioned above, and found that it was essentially indistinguishable fr om the biologically derived cp45 on the basis of plaque size, level of temp erature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruse s containing the cp45 mutations in individual proteins as well as several c ombinations of mutations. Analysis of these recombinant viruses revealed th at multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and an phenotypes, In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the fs phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ea phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3' N region, the L protein, and the C-M-F-HN region. The an phenotype is a comp osite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C an d F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.