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

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.

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.