The two amino acid substitutions in the L protein of cpts530/1009, a live-attenuated respiratory syncytial virus candidate vaccine, are independent temperature-sensitive and attenuation mutations

Abstract

cpts530/1009 is a live-attenuated, temperature-sensitive ( ts) RSV vaccine candidate that was shown previously to be attenuated for seronegative humans. It was generated by two rounds of chemical mutagenesis: first, a partially attenuated, cold-passaged ( cp), non- ts RSV mutant ( cpRSV) was mutagenized to yield the ts derivative cpts530, and then cpts530 was mutagenized to yield cpts530/1009, which is more ts. Previous nucleotide (nt) sequence analysis of cpts530 showed that it has a single nt change compared to cpRSV that results in an amino acid substitution at residue 521 in the L protein. Reverse genetics confirmed that this mutation is responsible for the ts phenotype of cpts530. Here, determination of the complete 15,222-nt sequence of cpts530/1009 identified a single change compared to cpts530, namely a point mutation at nt 12002, which results in a methionine-to-valine substitution at amino acid 1169 in the L protein. The contribution of the 1009 mutation to the level of temperature sensitivity and attenuation exhibited by cpts530/1009 was evaluated by its introduction alone or with the 530 and cp mutations into the full-length cDNA clone of wild-type (wt) RSV. Subsequent analysis of infectious viruses recovered from the mutant cDNAs indicated that (i) the 1009 mutation indeed was a ts mutation and the level of temperature sensitivity specified by the 1009 mutation was less than that specified by the 530 mutation, (ii) the 530 and 1009 mutations each contributed to attenuation in the upper respiratory tract of mice and their effects were additive, (iii) viruses bearing the 1009 mutation were more attenuated in the lower respiratory tract of mice than viruses bearing the 530 mutation and (iv) the combination of the 530 and 1009 mutations in the cpRSV background resulted in the same level of temperature sensitivity and attenuation in mice as that observed for the biologically-derived cpts530/1009 mutant. These data show that the genetic basis of the attenuation and temperature sensitivity of the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e. the 5 cpRSV mutations, the 530 mutation and the 1009 mutation.