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.? The development of a reverse genetics system for HPIV1 provides the capability to generate live attenuated recombinant HPIV1 (rHPIV1) vaccine candidates by the introduction of one or more temperature (ts) and non-ts attenuating (att) mutations into wild type HPIV1. Since respiratory viruses with mutations in proteins with anti-interferon activities are attenuated in vivo, the C accessory proteins are prime targets for inactivation by mutation. In addition, mutations in L have been identified that attenuate respiratory viruses for rodents or primates. Attenuating mutations identified in the L genes of RSV and HPIV3 and the C genes of MPIV1 and HPIV3 were previously transferred to the homologous loci of HPIV1 identified by sequence alignments to generate live attenuated HPIV1 vaccine candidates. Specifically, amino acid substitutions introduced individually at position 170 in the C protein of HPIV1 and at positions 456, 942, 992 and 1558 in L attenuated HPIV1 for replication in the respiratory tract of hamsters. The mutation at position 170 in C specified a non-ts att phenotype whereas those in L specified either a ts or non-ts att phenotype. The combination of L gene mutations rendered viruses more ts and more attenuated in hamsters than either mutation alone. The codons at positions 942 and 992 were systematically mutated to achieve enhanced phenotypic stability and increased attenuation. At position 942, the original rL-Y942H virus was mutated to generate rL-Y942A, a virus that possessed a similar level of temperature sensitivity and attenuation as rL-Y942H but that would require three nucleotide substitutions in the Y942A codon to generate a codon that specified a wild type phenotype. The rL-Y942A mutant was confirmed to exhibit increased genetic and phenotypic stability over that of rL-Y942H. Similarly, a 2-nucleotide substitution at position 992 (Leu to Cys) was found to specify the highest level of temperature sensitivity and attenuation among recombinants with a change at codon 992. ? The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C??/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, CR84G/HNT553A, and a separate amino acid substitution, CF170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation (att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: i) creating a two-amino acid deletion and substitution at the site of the CF170S mutation, yielding C??170; ii) introducing a 6 amino acid deletion in the N-terminal region of C, C??10-15; and iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The C??10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The C??170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the CR84G/HNT553A mutation set and the C??170 deletion mutation yielded a virus, rCR84G/??170HNT553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus was modified to contain the stabilized codon at 942, and this virus will be manufactured by Charles River Laboratories and evaluated clinically as a live intranasal HPIV1 vaccine.? Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (??) mutations in the P/C gene (r-C??10-15HNT553A, r-CR84G, r-CF170S and r-C??170), 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-C??170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-C??170 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 P/C 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 P/C mutants, r-CF170S and r-C??170, was necessary for significant attenuation in African green monkeys (AGMs).? Recombinant human parainfluenza virus type 2 (rHPIV2) vaccine candidates were created using reverse genetics by importing known attenuating mutations in the L polymerase protein from heterologous paramyxoviruses into the homologous sites of the HPIV2 L protein. Four recombinants (rF460L, rY948H, rL1566I, and rS1724I) were recovered and three were attenuated for replication in hamsters. The genetic stability of the imported mutations at three of the four sites was enhanced by use of alternative codons or by deletion of a pair of amino acids. rHPIV2s bearing these modified mutations exhibited enhanced attenuation. A live attenuated HPIV2 vaccine candidate has been identified.
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