Introducing point and deletion mutations into the P/C gene of human parainfluenza virus type 1 (HPIV1) by reverse genetics generates attenuated and efficacious vaccine candidates

Abstract

The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C′/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, C R84G/HN T553A, and a separate amino acid substitution, C F170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation ( att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: (i) creating a two-amino acid deletion and substitution at the site of the C F170S mutation, yielding C Δ170; (ii) introducing a six amino acid deletion in the N-terminal region of C, C Δ10–15; and (iii) combining these stable deletion mutations with the att C R84G/HN T553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The C Δ10–15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the C R84G/HN T553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The C Δ170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of C F170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the C R84G/HN T553A mutation set and the C Δ170 deletion mutation yielded a virus, rC R84G/Δ170HN T553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus will be evaluated clinically as a live intranasal HPIV1 vaccine, one that can be further attenuated as necessary by the introduction of additional stabilized att mutations previously developed in the L protein.