Replacement of the ectodomains of the hemagglutinin-neuraminidase and fusion glycoproteins of recombinant parainfluenza virus type 3 (PIV3) with their counterparts from PIV2 yields attenuated PIV2 vaccine candidates

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) va ccine strain for use in infants and young children, using reverse genetic t echniques that previously were used to rapidly produce a live attenuated PI V1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, w as generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955-2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503-51 0, 1999). However, using the same strategy, we failed to recover recombinan t chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when c himeric HN and F open reading frames (ORFs) rather than complete PIV;I F an d HN ORFs were used to construct the full-length cDNA, The recovered viruse s, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane dom ain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail do main, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was requ ired for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly a ttenuated for replication in the respiratory tracts of hamsters, African gr een monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified a n attenuation phenotype in vivo, Despite this attenuation, these viruses we re highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further a ttenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additiv e with that of the chimerization, and thus inclusion of all or some of the cp45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.