Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes

<p>Abstract</p> <p>Background</p> <p>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-<it>ts</it>) attenuating (<it>att</it>) mutations primarily in the P/C gene, namely C^R84GHN^T553A(two point mutations used together as a set) and C^Δ170(a short deletion mutation), and two <it>ts att </it>mutations in the L gene, namely L^Y942A(a point mutation), and L^Δ1710–11(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).</p> <p>Results</p> <p>The rHPIV1 mutant bearing the novel L^Δ1710–11mutation was highly <it>ts </it>and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aand rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11vaccine candidates were highly <it>ts</it>, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aprotected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11was 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.</p> <p>Conclusion</p> <p>The rHPIV1-C^R84G/Δ170HN^T553AL^Y942Aand rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.</p

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

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

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

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

Determinants of the Host Range Restriction of Replication of Bovine Parainfluenza Virus Type 3 in Rhesus Monkeys Are Polygenic

The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3

Sendai Virus, a Murine Parainfluenza Virus Type 1, Replicates to a Level Similar to Human PIV1 in the Upper and Lower Respiratory Tract of African Green Monkeys and Chimpanzees

AbstractHuman parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533–540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 104 and 106 and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans

Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes-2

<p><b>Copyright information:</b></p><p>Taken from "Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes"</p><p>http://www.virologyj.com/content/4/1/67</p><p>Virology Journal 2007;4():67-67.</p><p>Published online 2 Jul 2007</p><p>PMCID:PMC1939843.</p><p></p> challenge virus titers (logTCID/ml; x-axis) in the same groups (Table 4). A curve of best fit has been inserted (solid line) to demonstrate the association between these two data sets