Characterization of a Live, Attenuated Human Parainfluenza Type 3 Virus Candidate Vaccine Strain

Characterization of a temperature-sensitive and live, attenuated human parainfluenza type 3 virus strain (cp45)grownatapermissivetemperature(32C however, shifting to a permissive temperature allowed virus growth. Although at a nonpermissive temperature virus polypeptide synthesis was significantly reduced, the hemagglutinin-neuraminidase and fusion glycoproteins were transported to cell surfaces and retained their characteristic biologic activities. Studies on mRNA synthesis from the P protein gene suggested a poor transcriptional activity of the cp45 virus at a nonpermissive temperature. Results from this study indicate that the temperature sensitivity of cp45 virus is related to altered transcriptional activity and a marked reduction in virus polypeptide synthesis. Human parainfluenza viruses (HPIVs), members of the Paramyxoviridae family, are responsible for serious lower respiratory tract infections in infants (4, 37). HPIV type 3 (HPIV-3) often infects young infants less than 6 months of age and causes bronchiolitis, pneumonia, or croup. It is second only to respiratory syncytial virus as a cause of lower respiratorytractinfectioninneonatesandyounginfants.TheHPIV-3 genome contains a single-stranded negative-sense RNA of approximately15,000nucleotides(27,33)andencodesatleastsix structural proteins (39-nucleocapsid protein [NP]-P[1C]-matrix protein [M]-fusion protein [F]-hemagglutinin-neuraminidase protein [HN]-L-59) (30, 31). The genome RNA and its full-length complement, the antigenome RNA, are found both in the virion and in the infected cell as an RNase-resistant nucleocapsid because of the tight association of the major NP with the RNA (12). Two other viral proteins, P and L, are associated less tightly with the nucleocapsid and function as subunits of the RNA-dependent RNA polymerase. Three additional viral proteins, HN, F, and M, are associated with the lipid envelope of the virion. Both HN and F glycoproteins are responsible for initiation and progress of the infection process (11,15)andhavebeenshowntoinduceneutralizingantibodies and correlate with protection from infection (16, 24). On the other hand, HN, F, NP, and M proteins have been shown to induce cytotoxic T cells (1, 20). HPIV-3-mediated immunoregulation of human T lymphocytes has been suggested to play an important role in the failure of the virus to induce lifelong immunity (29). Cold-adapted and temperature-sensitive (ts) HPIV-3 mutants were generated by serial passages at a suboptimal temperature (2). Cold-adapted viruses were also temperature sensitive and attenuated for growth in the lungs and nasal turbinates of experimental animals. The reduction of virus replication was directly related to the cold passage level of the virus (3, 6, 9). cp45 virus was also evaluated in seronegative chimpanzees and found to be highly attenuated in both the upper and lower respiratory tracts and showed resistance to challenge infection (13). cp45 virus is currently under evaluation in humans as a candidate vaccine against HPIV-3 infection. In this study, we determined the basis of temperature sensitivity of the cp45 candidate vaccine strain. Our results suggest that the transcriptional activity of the vaccine strain is temperaturesensitiveandsignificantlyreducestheoverallprotein synthesis and replication of the virus at the nonpermissive temperature.