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 ts and non-ts 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. These previous observations were extended in several respects. First, rHPIV1 vaccine candidates were generated to contain new combinations of mutations that included the stabilized codon at 942 and the partially-stabilized codon at 992 in L. Second, a pair of novel spontaneous mutations in C (R84G) and HN (T553A) was identified, characterized, and used to generate novel vaccine candidates. Third, new rHPIV1 combination vaccine candidates were evaluated in hamsters. Fourth, the rHPIV1 vaccine candidates were evaluated in African green monkeys (AGMs), whose anatomical and phylogenic relatedness to humans makes them suitable as the penultimate step prior to clinical trials. rHPIV1 vaccine candidates were identified that exhibited a spectrum in their level of attenuation, immunogenicity, and efficacy in hamsters or AGMs. Several of the rHPIV1 vaccine candidates appeared to have achieved an acceptable balance between attenuation and immunogenicity for AGMs and thus represent promising vaccine candidates for use in humans. In addition, progress has been made on the development of a live-attenuated HPIV2 vaccine. 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. The genetically stabilized mutations conferring a high level of attenuation will be useful in generating a live-attenuated virus vaccine for HPIV2.
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