Satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis

Abstract

A cold-passaged RSV mutant, designated cp-RSV, which acquired host range mutations during 52 passages at low temperature in bovine tissue culture, was completely attenuated for seropositive adults and children but retained the capacity to cause upper respiratory disease in seronegative infants. We sought to introduce additional attenuating mutations, such as temperature-sensitive ( ts) and small-plaque ( sp) mutations, into the cp-RSV mutant, which is a ts + virus, in order to generate a mutant which would be satisfactorily attenuated in seronegative infants and young children. Nine mutants of cp-RSV, which had acquired either the ts or small-plaque sp phenotype, were generated by chemical mutagenesis with 5-fluorouracil. The two ts mutants with the lowest in vitro shut-off temperature, namely the cpts-248 (38°C) and cpts-530 (39°C) mutants, were the most restricted of the nine cp-RSV mutant progeny tested for efficiency of replication in Balb/c mice. In seronegative chimpanzees, the cpts-248 mutant replicated fourfold less efficiently in the nasopharynx and caused significantly less rhinorrhoea than its cp-RSV parent. The cpts-248 mutant virus, like its cp-RSV parent, was 1000-fold restricted in replication in the trachea compared with wild-type RSV. Previously, another candidate RSV live attenuated vaccine strain, a mutant designated ts-1, exhibited some instability of its ts phenotype following replication in susceptible humans or chimpanzees. Hence, we sought cp-RSV ts progeny that exhibited a greater degree of stability of the ts phenotype than the prototype ts-1 mutant. The cpts-248 and cpts-530 progeny viruses exhibited a greater degree of stability of the ts phenotype in nude mice than the ts-1 virus, and in chimpanzees, the former mutant also exhibited a greater stability of its ts phenotype than ts-1. The cpts-248 mutant was immunogenic and induced a high level of resistance in chimpanzees to subsequent challenge with wild-type RSV. The cpts-248 mutant therefore exhibits a set of properties that make it a promising vaccine candidate. These desirable properties of cpts-248 suggest that the mutant should be tested in humans for its suitability in immunoprophylaxis.