Human parainfluenza virus type 1 (HPIV1) is a significant cause of severe respiratory tract disease in infants and young children. HPIV1 is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the subfamily Paramyxovirinae within the Paramyxoviridae family, which also includes the HPIV2 and HPIV3 serotypes. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV1 genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 20% of all pediatric hospitalizations due to respiratory disease. A licensed vaccine is currently not available for any of the HPIVs. HPIV2 vaccine development Reverse genetics was used previously to generate attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and to enhance their genetic stability. An attenuating mutation at nucleotide 15 (15TC ) in the 3 genomic promoter was identified and was found to be present in the previously characterized mutants. We evaluated the properties of this promoter mutation alone and in various combinations with the L polymerase mutations. Amino acid substitutions at L protein positions 460 (460A or 460P) or 948 (948L), or deletion of amino acids 1724 and 1725 (1724), each conferred a temperature sensitivity (ts) phenotype whereas the 15TC mutation did not. The 460A and 948L mutations each contributed to restricted replication in the lower respiratory tract of African green monkeys, but the 1724 mutation increased attenuation only in certain combinations with other mutations. We constructed two highly attenuated viruses, rV94(15C)460A948L and rV94(15C)948L1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys and, therefore, appear to be suitable for evaluation in humans. Studies have been initiated to generate HPIV2 backbones in with the P and V genes have been separated in two cistrons to permit introductions of mutations in V that do not affect P. HPIV1 vaccine development Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the PC gene, namely CR84GHNT553A (two point mutations used together as a set) and C170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and L1710-11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). The rHPIV1 mutant bearing the novel L1710-11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G170HNT553ALY942A and rHPIV1-CR84G170HNT553AL1710-11 vaccine candidates were highly ts, with shut-off temperatures of 38C and 35C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G170HNT553AL1710-11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. The rHPIV1-CR84G170HNT553ALY942A and rHPIV1-CR84G170HNT553AL1710-11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans. Studies have been initiated to generate HPIV1 backbones in with the P and C genes have been separated in two cistrons to permit introductions of mutations in C that do not affect P. HPIV1 mutations in C and the interferon response. Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion () mutations in the PC gene (r-C10-15HNT553A, r-CR84G, r-CF170S and r-C170), which have previously been evaluated as HPIV1 vaccine candidates, were evaluated for their effect on the type I interferon (IFN) response in vitro. HPIV1 wt infection inhibited the IFN response by inhibiting IFN regulatory factor-3 (IRF-3) activation and IFN production in A549 cells and IFN signaling in Vero cells. In contrast, r-CR84G, r-CF170S and r-C170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-C170 did not inhibit IFN signaling. Thus, HPIV1 antagonizes the IFN response at both the level of induction and signaling, and antagonism at both levels was disrupted by mutations in the PC gene. Since CF170S affects C and not P, the anti-IFN function can be attributed to the C proteins. These data, in the context of previous in vivo studies, suggest that the loss of antagonism of the IFN response at both the level of induction and signaling, observed with the PC mutants, r-CF170S and r-C170, was necessary for significant attenuation in African green monkeys (AGMs). Plasmid only system for recovery of PIV vaccine candidates A method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids was developed. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.