Live human metapneumovirus vaccine candidates attenuated by temperature sensitivity mutations from human respiratory syncytial virus

ABSTRACT Human metapneumovirus (HMPV, genus Metapneumovirus ) and respiratory syncytial virus (HRSV, genus Orthopneumovirus ) are pediatric respiratory pathogens of the Pneumoviridae family. We previously developed live-intranasal HRSV vaccine candidates attenuated by genetically stabilized temperature sensitivity/attenuating ( ts/att ) mutations in the HRSV polymerase (L) ORF, including sites “ 1030s ” (L codon changes S1313 (AGC-to-TCA) /Y1321K (AAA) , named after clone number 1030), and “Δ1313/I1314L,” comprising a genetically stabilized deletion of L codon 1313. In pediatric vaccine studies, HRSV candidates with “ 1030s ” or Δ1313/I1314L were attenuated, immunogenic, and genetically stable. Given the similarities between HRSV and HMPV, we introduced corresponding modifications into the HMPV L ORF, resulting in HMPV with deletion of codon 1238 (Δ1238), corresponding to HRSV Δ1313/I1314L, and HMPV with C1238S (TCA) /Y1246K (AAA) substitutions together (C1238S/Y1246K, corresponding to HRSV “ 1030s ”), or individually (C1238S, Y1246K), serving as experimental controls. All versions replicated well in Vero cells at 32°C. At 37°C, replication of Δ1238, C1238S/Y1246K, and Y1246K was delayed, and peak titers were ~80-fold, 200-fold, and 25-fold lower than wild-type HMPV. As predicted, the C1238S control was not temperature-sensitive (shutoff temperature [ T SH ] > 40°C, similar to wild-type HMPV), while Y1246K and Δ1238 ( T SH = 39°C) and C1238S/Y1246K ( T SH = 38°C) had a ts phenotype. Thus, the addition of the C1238S (TCA) substitution increased the temperature sensitivity of Y1246K. The HMPV ts/att sites were genetically stable under temperature stress. In hamsters inoculated intranasally, C1238S/Y1246K and Δ1238 were restricted in replication, yet immunogenic and protective against HMPV challenge. Thus, inclusion in the HMPV L ORF of C1238S/Y1246K and Δ1238 mutations, corresponding to HRSV ts/att sites, provided genetically stable live-attenuated HMPV, suitable for clinical development. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of lower respiratory illness in young children, and there are no licensed vaccines. Similarly to immunization strategies against the related human respiratory syncytial virus (HRSV), live-attenuated HMPV vaccines for intranasal immunization would be most suitable for young children. Live-intranasal vaccines infect and induce immunity in the presence of residual maternal antibodies without priming for enhanced respiratory disease. Genetic stability of attenuating mutations represents a challenge in vaccine design. We generated live-attenuated HMPV vaccine candidates by introducing into corresponding sites of the HMPV polymerase gene temperature-sensitivity-inducing attenuating mutations that had been originally developed for HRSV, genetically stabilized, and proven to be free of reversions in HRSV vaccine studies. The resulting HMPV vaccine candidates are temperature-sensitive, attenuated, and genetically stable under temperature stress. In the hamster model, the new HMPV candidates were immunogenic and protective. These live-attenuated HMPV candidates will be advanced to pediatric vaccine studies.