Sonja R. Surman

The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness.

ABSTRACT 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 log10) in the upper respiratory tract of hamsters and to moderate titers (≥3.6 log10) 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. HMPV-infected 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.

Bovine Parainfluenza Virus Type 3 (BPIV3) Fusion and Hemagglutinin-Neuraminidase Glycoproteins Make an Important Contribution to the Restricted Replication of BPIV3 in Primates

ABSTRACT 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-FHHNH) 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-FHHNH 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-FHHNH 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-FHHNH as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Human Parainfluenza Virus Type 3 (PIV3) Expressing the Hemagglutinin Protein of Measles Virus Provides a Potential Method for Immunization against Measles Virus and PIV3 in Early Infancy

ABSTRACT Recombinant human parainfluenza virus type 3 (PIV3) was used as a vector to express the major protective antigen of measles virus, the hemagglutinin (HA) glycoprotein, in order to create a bivalent PIV3-measles virus that can be administered intranasally. The measles virus HA open reading frame (ORF) was inserted as an additional transcriptional unit into the N-P, P-M, or HA-neuraminidase (HN)-L gene junction of wild-type PIV3 or into the N-P or P-M gene junction of an attenuated derivative of PIV3, termed rcp45L. The recombinant PIV3 (rPIV3) viruses bearing the HA inserts replicated more slowly in vitro than their parental viruses but reached comparable peak titers of ≥107.5 50% tissue culture infective doses per ml. Each of the wild-type or cold-passaged 45L (cp45L) PIV3(HA) chimeric viruses replicated 5- to 10-fold less well than its respective parent virus in the upper respiratory tract of hamsters. Thus, insertion of the ∼2-kb ORF itself conferred attenuation, and this attenuation was additive to that conferred by thecp45L mutations. The attenuated cp45L PIV3(HA) recombinants induced a high level of resistance to replication of PIV3 challenge virus in hamsters and induced very high levels of measles virus neutralizing antibodies (>1:8,000) that are well in excess of those known to be protective in humans. rPIV3s expressing the HA gene in the N-P or P-M junction induced about 400-fold more measles virus-neutralizing antibody than did the rPIV3 with the HA gene in the HN-L junction, indicating that the N-P or P-M junction appears to be the preferred insertion site. Previous studies indicated that the PIV3cp45 virus, a more attenuated version of rcp45L, replicates efficiently in the respiratory tract of monkeys and is immunogenic and protective even when administered in the presence of very high titers of passively transferred PIV3 antibodies (A. P. Durbin, C. J. Cho, W. R. Elkins, L. S. Wyatt, B. Moss, and B. R. Murphy, J. Infect. Dis. 179:1345–1351, 1999). This suggests that this intranasally administered PIV3(HA) chimeric virus can be used to immunize infants with maternally acquired measles virus antibodies in whom the current parenterally administered live measles virus vaccine is ineffective.

Sequence analysis of the washington/1964 strain of human parainfluenza virus type 1 (HPIV1) and recovery and characterization of wild-type recombinant HPIV1 produced by reverse genetics

A complete consensus sequence was determined for the genomic RNA of human parainfluenza virus type 1 (HPIV1) strain Washington/20993/1964 (HPIV1 WASH/64), a clinical isolate that previously was shown to be virulent in adults. The sequence exhibited a high degree of relatedness to both Sendai virus, a PIV1 virus recovered from mice, and human PIV3 (HPIV3) with regard to cis-acting regulatory regions and protein-coding sequences. This consensus sequence was used to generate a full-length antigenomic cDNA and to recover a recombinant wild-type HPIV1 (rHPIV1). Interestingly, the rHPIV1 could be rescued from full-length antigenomic rHPIV1 cDNA using HPIV3 support plasmids, HPIV1 support plasmids, or a mixture thereof. The replication of rHPIV1 in vitro and in the respiratory tract of hamsters was similar to that of its biologically derived parent virus. The similar biological properties of rHPIV1 and HPIV1 WASH/64 in vitro and in vivo, together with the previous demonstration of the virulence of this specific isolate in humans, authenticates the rHPIV1 sequence as that of a wild-type virus. This rHPIV1 can now be used to study the biological properties of HPIV1 and as a substrate to introduce attenuating mutations for the generation of live-attenuated HPIV1 vaccine candidates.

The Genome Length of Human Parainfluenza Virus Type 2 Follows the Rule of Six, and Recombinant Viruses Recovered from Non-Polyhexameric-Length Antigenomic cDNAs Contain a Biased Distribution of Correcting Mutations

ABSTRACT 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.

Generation of a parainfluenza virus type 1 vaccine candidate by replacing the HN and F glycoproteins of the live-attenuated PIV3 cp45 vaccine virus with their PIV1 counterparts

Parainfluenza virus type 1 (PIV1) is a major cause of croup in infants and young children, and a vaccine is needed to prevent the serious disease caused by this virus. In the present study, a live attenuated PIV1 vaccine candidate was generated by modification of the extensively-studied PIV3 cold-passaged (cp) cp45 vaccine candidate using the techniques of reverse genetics. The HN and F glycoproteins of the PIV3 cp45 candidate vaccine virus were replaced with those of PIV1. This created a live attenuated PIV1 vaccine candidate, termed rPIV3-1 cp45, which contained the attenuated background of the PIV3 cp45 vaccine virus together with the HN and F protective antigens of PIV1. Three of the 15 mutations of cp45 lie within the HN and F genes, and those in the F gene are attenuating. Thus, some attenuation might be lost by the HN and F glycoprotein replacement. To address this issue we also constructed a derivative of PIV3 cp45, designated rPIV3 cp45 (F(wt)HN(wt)), that possessed wild type PIV3 HN and F glycoproteins but retained the 12 other cp45 mutations. rPIV3 cp45 (F(wt)HN(wt)) replicated in the respiratory tract of hamsters to a level three- to four-fold higher than rPIV3 cp45, indicating that loss of the two attenuating mutations in the cp45 F gene effected a slight reduction in the overall attenuation of cp45 for hamsters. However, the chimeric rPIV3-1 cp45 virus was about 5-fold more restricted in replication in hamsters than rPIV3 cp45 and about 15- to 20-fold more restricted than rPIV3 cp45 (F(wt)HN(wt)). This suggests that two components contribute to the attenuation of the new chimeric rPIV3-1 cp45 PIV1 vaccine candidate: one being the 12 cp45 mutations, which provide most of the observed attenuation, and the other resulting from the introduction of the heterologous PIV1 HN and F proteins into PIV3 (i.e., a chimerization effect). rPIV3-1 cp45 was observed to be immunogenic and protective against challenge with wild type PIV1 in hamsters. This virus shows sufficient promise that it should be evaluated further as a candidate live attenuated vaccine strain for preventing severe lower respiratory tract PIV1 disease in infants and young children.

Codon substitution mutations at two positions in the L polymerase protein of human parainfluenza virus type 1 yield viruses with a spectrum of attenuation in vivo and increased phenotypic stability in vitro.

ABSTRACT The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

Generation of Recombinant Human Parainfluenza Virus Type 1 Vaccine Candidates by Importation of Temperature-Sensitive and Attenuating Mutations from Heterologous Paramyxoviruses

ABSTRACT Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.

A Chimeric Human-Bovine Parainfluenza Virus Type 3 Expressing Measles Virus Hemagglutinin Is Attenuated for Replication but Is Still Immunogenic in Rhesus Monkeys

ABSTRACT The chimeric recombinant virus rHPIV3-NB, a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-NBHA, replicated like its attenuated rHPIV3-NB parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-NB in vitro or in vivo. Monkeys immunized with rHPIV3-NBHA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate

ABSTRACT 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. IMPORTANCE Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypofusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of prefusion and postfusion RSV F protein. Prefusion RSV F induced a larger quantity and higher quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation.