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

(cpts530/1009 is a live-attenuated, temperature-sensitive (ts) RsV vaccine candidate that was shown previously to be attenuated for seronegative human s. It was generated by two rounds of chemical mutagenesis: first, a partial ly attenuated, cold-passaged (cp). non-ts RSV mutant (cpRSV) was mutagenize d to yield the ts derivative cpts530, and then cpts530 was mutagenized to y ield cpts530/1009, which is more ts, Previous nucleotide (nt) sequence anal ysis of cpts530 showed that it has a single nt change compared to cpRSV tha t results in an amino acid substitution at residue 521 in the L protein. Re verse genetics confirmed that this mutation is responsible for the ts pheno type 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 cpts5 30/1009 was evaluated by its introduction alone or with the 530 cp mutation s into the full-length cDNA clone of wild-type (wt) RSV, Subsequent analysi s of infectious viruses recovered from the mutant cDNAs indicated that ii) the 1009 mutation indeed was a rs mutation and the level of temperature sen sitivity specified by the 1009 mutation was less than that specified by the 530 mutation, (ii) the 530 and 1009 mutations each contributed to attenuat ion 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) th e combination of the 530 and 1009 mutations in the cpRSV background resulte d in the same level of temperature sensitivity and attenuation in mice as t hat observed for the biologically-derived cpts530/1009 mutant. These data s how that the genetic basis of the attenuation and temperature sensitivity o f the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e. the 5 cpRSV mutations, t he 530 mutation and the 1009 mutation, (C) 1999 Elsevier Science Ltd. All r ights reserved.