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We evaluated a combination respiratory syncytial virus (RSV) and parainfluenza 3 virus (PIV3) live, attenuated intranasal vaccine for safety, viral replication, and immunogenicity in doubly seronegative children 6-18 months old. RSV cpts-248/404 and PIV3-cp45 vaccines were combined in a dose of 10(5) plaque-forming units of each per 0.5-mL dose and compared with monovalent vaccines or placebo. The virus shedding pattern of RSV was not different between monovalent RSV cpts-248/404 vaccine and combination vaccine. Modest reductions in the shedding of PIV3-cp45 vaccine virus were found after the administration of RSV cpts-248/404 and PIV3-cp45 vaccine, relative to monovalent PIV3 vaccine; 16 (76%) of 21 children given combination vaccine shed PIV3-cp45 versus 11 (92%) of 12 of those given monovalent PIV3 vaccine. Both vaccines were immunogenic, and antibody responses were similar between the monovalent groups and the combination group. Combined RSV/PV3 vaccine is feasible for simultaneous administration, and further studies are warranted.

Parainfluenza type 3 virus (PIV-3) infections cause lower respiratory tract illness in children throughout the world. A licensed PIV-3 vaccine is not yet available.A live attenuated cold-adapted (ca) and temperature-sensitive (ts) PIV-3 vaccine, designated cp-45, was evaluated sequentially in open label studies in 20 adults and in placebo-controlled, double blind studies in 24 PIV-3-seropositive children, 52 PIV-3-seronegative infants and children and 49 infants 1 to 2 months old. A single dose of this intranasal vaccine was evaluated in adults [106 plaque-forming units (pfu)] and seropositive children, and 104 and 105 pfu were evaluated in seronegative children. In the infant study, two 104 pfu doses of vaccine were administered at 1- or 3-month intervals. Safety, infectivity, immunogenicity and phenotypic stability of the vaccine were evaluated in all cohorts.The cp-45 vaccine was well-tolerated in all age groups and infected 94% of vaccinated seronegative children and 94% of vaccinated infants. Although immunization with the first dose of cp-45 diminished the replication of a second dose in all infants, those immunized after 3 months shed vaccine virus more frequently than those immunized after 1 month (62% vs. 24%, respectively). Antibody responses to PIV-3 were readily detected in seronegative children with a variety of assays; however, the IgA response to the viral hemagglutinin-neuraminidase was the best measure of immunogenicity in young infants. Of 109 vaccine virus specimens recovered from nasal washes, 98 were ts and 11 were temperature-sensitive intermediate (tsi) viruses, with pinpoint plaques visible at 40 degrees C. tsi viruses appeared transiently at the time of peak viral replication, represented a very small proportion of the total virus shed and were not associated with changes in clinical status. ca revertants were not detected.The cp-45 vaccine is appropriately attenuated and immunogenic in infants as young as 1 month of age. Further development of this vaccine is warranted.

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 ( cp 45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp 45 independently confers the temperature-sensitive ( ts ) and attenuation ( att ) phenotypes but not the cold-adaptation ( ca ) phenotype (29). cp 45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis -acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts , ca , and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp 45 virus that contained all 15 cp 45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp 45 on the basis of plaque size, level of temperature 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 viruses containing the cp 45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp 45 mutations distributed throughout the genome contribute to the ts , ca , and att 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 ts phenotype. A recombinant virus containing all the cp 45 mutations except those in L was more ts than cp 45, illustrating the complex nature of this phenotype. The ca phenotype of cp 45 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 att phenotype is a composite 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 and F proteins. The presence of multiple ts and non- ts attenuating mutations in cp 45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

Studies were initiated to define the genetic basis of the temperature-sensitive ( ts ), cold adaptation ( ca ), and attenuation ( att ) phenotypes of the human parainfluenza virus type 3 (PIV3) cp 45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp 45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580–584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43–52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts , att , and ca phenotypes of cp 45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts , att , and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 10 5 -fold reduction in plaque formation in cell culture at 40°C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp 45, indicating that these mutations are the major contributors to the ts phenotype of cp 45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp 45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp 45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp 45 virus is a major attenuating component of this candidate vaccine.

A phase 2 evaluation of live attenuated parainfluenza type 3 (PIV3)-cold passage mutant 45 (cp45) vaccine was conducted in 380 children 6-18 months old; 226 children (59%) were seronegative for PIV3. Of the 226 seronegative children, 114 received PIV3-cp45 vaccine, and 112 received placebo. No significant difference in the occurrence of adverse events (i.e., runny nose, cough, or temperature > or =38 degrees C) was noted during the 14 days after vaccination. There was no difference between groups in the occurrence of acute otitis media or serous otitis media. Paired serum samples were available for 109 of the seronegative vaccine recipients and for 110 of the seronegative placebo recipients; 84% of seronegative vaccine recipients developed a > or =4-fold increase in antibody titers. The geometric mean antibody titer after vaccination was 1 : 25 in the vaccine group and

A recombinant live-attenuated chimeric human parainfluenza virus type 1 (HPIV1) candidate vaccine was previously generated by replacing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein open reading frames (ORFs) of the HPIV3 candidate vaccine, rHPIV3 cp 45, with those of wild-type HPIV1. Previously, this recombinant chimeric virus, designated rHPIV3–1 cp 45, exhibited a greater level of the temperature sensitivity of replication in vitro and a greater level of attenuation of replication in the respiratory tract of immunized hamsters when compared to its HPIV3 cp 45 parent virus. In the present study, rHPIV3–1 cp 45 was evaluated for its level of attenuation and efficacy in African green monkeys ( Cercopithecus aethiops ), a primate in which both HPIV1 and HPIV3 wild-type viruses replicate efficiently. The rHPIV3–1 cp 45 candidate vaccine was as restricted in replication in the upper and lower respiratory tract as its thoroughly characterized rHPIV3 cp 45 parent indicating that the attenuating mutations present in the rHPIV3 cp 45 backbone specified an appropriate level of attenuation of rHPIV3–1 cp 45 for primates. The level to which rHPIV3–1 cp 45 replicated in the respiratory tract of African green monkeys was also sufficient to induce a strong immune response to HPIV1 and provided protection against challenge with wild-type HPIV1. These results provide a basis for further evaluation of this HPIV1 candidate vaccine in humans.

Cold-adapted, temperature-sensitive (ts), attenuated strains of respiratory syncytial virus have been developed from a B subgroup clinical isolate for potential use as vaccine candidates. The replication of two B subgroup ts mutant viruses (2B33F and 2B20L) at the permissive and nonpermissive temperatures have been compared with that of the parental 2B virus to establish differences that may account for their ts and/or attenuated phenotypes. We have shown that the ts restriction at 39 degrees C in the replication of the two mutant viruses in tissue culture occurs at a step after virus adsorption but before or including initiation of virus-specific mRNA transcription. At the permissive temperature of 32 degrees C a 12- to 24-h delay in the accumulation of mRNA for both mutant viruses in comparison to that of the parental 2B virus was exhibited. This effect was mirrored by equivalent delays in viral protein synthesis and production of infectious virus. By 36 h postinfection both mutants had produced levels of viral mRNA, protein, and infectious virus that were similar to those of the parent virus at 32 degrees C. ts+ revertant viruses derived from both mutants have also reverted in their viral mRNA, protein, and infectious virus production kinetics at 32 degrees C to rates more like those exhibited by the parental 2B virus. This suggests a positive correlation between the ts step in the replication of the mutant viruses and the initial delay in mRNA production that occurs at the permissive temperature.