The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (>6.0 log 10 ) in the upper respiratory tract of hamsters and to moderate titers (>3.6 log 10 ) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPVinfected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.

We evaluated the individual contributions of the three surface glycoproteins of human metapneumovirus (HMPV), namely the fusion F, attachment G, and small hydrophobic SH proteins, to the induction of serum HMPV-binding antibodies, serum HMPV-neutralizing antibodies, and protective immunity. Using reverse genetics, each HMPV protein was expressed individually from an added gene in recombinant human parainfluenza virus type 1 (rHPIV1) and used to infect hamsters once or twice by the intranasal route. The F protein was highly immunogenic and protective, whereas G and SH were only weakly or negligibly immunogenic and protective, respectively. Thus, in contrast to other paramyxoviruses, the HMPV attachment G protein is not a major neutralization or protective antigen. Also, although the SH protein of HMPV is a virion protein that is much larger than its counterparts in previously studied paramyxoviruses, it does not appear to be a significant neutralization or protective antigen.

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F H HN H ) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F H HN H replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F H HN H conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F H HN H as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.Bovine parainfluenza virus type 3 (BPIV3) is restricted in replication in the respiratory tract of rhesus monkeys, chimpanzees, and humans, and it is being evaluated as a vaccine against human PIV3 (HPIV3) (8,10,12,26,27). HPIV3 and BPIV3 are closely related enveloped, nonsegmented, negativestrand RNA viruses within the Respirovirus genus of the Paramyxoviridae family (2, 10). The two viruses are 25% related antigenically by cross-neutralization studies (8), and they share neutralization epitopes on their fusion (F) and hemagglutininneuraminidase (HN) surface glycoproteins (9, 30). HPIV3 and BPIV3 are essentially identical in genome organization (2). Both viruses encode nine proteins: the nucleoprotein (N), phosphoprotein (P), and large polymerase protein (L) are nucleocapsid-associated proteins; the C, D, and V accessory proteins are proteins of unknown function encoded by the P mRNA or by an edited version thereof; the M protein is an internal matrix protein; and the F and HN glycoproteins are protective antigens of the vi...

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. H uman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are enveloped, nonsegmented, negative-stranded RNA viruses of the family Paramyxoviridae. They are, respectively, the first and second leading viral causes of severe acute lower respiratory tract infections in infants and children worldwide. RSV alone is responsible for an estimated 34 million annual pediatric cases of lower respiratory tract illness worldwide, with Ͼ3.5 million hospitalizations and 66,000 to 199,000 pediatric deaths, which occur predominantly in the developing world (1). Licensed vaccines or effective antiviral drugs are not available for either RSV or HPIV3. Experimental inactivated (RSV and HPIV3) and subunit (RSV) vaccines have been linked to vaccine-induced enhanced disease in young children (inactivated RSV) and experimental animals (subunit RSV and inactivated HPIV3) (2-4). In contrast, disease enhancement is not observed with live attenuated RSV strains or vectors expressing RSV antigens (5-7), indicating that suitably attenuated candidates are safe for immunization of infants and young children.A chimeric recombinant bovine-human PIV3 (rB/HPIV3) ( Fig. 1) has been under development as a replication-competent intranasal pediatric vaccine vector. The PIV3 genome is a negative-sense RNA of 15.5 kb that contains six genes in the order 3=-N (nucleoprotein)-P (phosphoprotein)-M (matrix protein)-F (fusion glycoprotein)...

Recombinant human parainfluenza virus type 1 (HPIV1) and mutants containing point and deletion (Delta) mutations in the P/C gene (r-CDelta10-15HNT553A, r-CR84G, r-CF170S and r-CDelta170), 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-CDelta170 were defective for inhibition of IRF-3 activation and IFN production and r-CF170S and r-CDelta170 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. Because 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-CDelta170, was necessary for significant attenuation in African green monkeys (AGMs).

Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the "rule of six." To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.Human parainfluenza virus type 2 (HPIV2) is an enveloped single-stranded negative-sense RNA virus of the genus Rubulavirus in the family Paramyxoviridae. Other rubulaviruses include simian virus 41 (SV41), SV5, HPIV4 A and B, and mumps virus. Like HPIV3 and HPIV1, which are members of the genus Respiroviridae in the paramyxovirus family, HPIV2 is a major cause of acute respiratory tract disease in infants and young children (4). HPIV2 has a genomic organization that is similar to that of the other parainfluenza viruses (4, 24), encoding seven proteins from six mRNAs. The HPIV2 gene map, based on the encoded mRNAs, is 3Ј-N-P/V-M-F-HN-L. The nucleoprotein (N), phosphoprotein (P), and large polymerase (L) complexed with genomic RNA make up the nucleocapsid/ polymerase complex. The P protein is encoded by a version of the P/V mRNA that is cotranscriptionally edited to contain an insertion of two guanosine residues at the P gene editing site (28). The accessory V protein is encoded from an unedited version of the P/V mRNA. HPIV2 does not have genes encoding the accessory C and D proteins that are found in HPIV1 or HPIV3 and does not have a gene for the SH protein that is found in SV5 and mumps vir...

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C؊), a virus in which expression of the C proteins (C, C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C؊) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C؊) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C؊) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C؊) and rHPIV1-C F170S , a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C؊) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C؊), whereas only the anti-IFN activity is disabled in rHPIV1-C F170S . In African green monkeys (AGMs), rHPIV1-P(C؊) was considerably more attenuated than rHPIV1-C F170S , suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C؊) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.

The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.