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

A cold-passaged RSV mutant, designated cp-RSV, which acquired host ran ge mutations during 52 passages at low temperature in bovine tissue cu lture, was completely attenuated for seropositive adults and children but retained the capacity to cause upper respiratory disease in serone gative infants. We sought to introduce additional attenuating mutation s, 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 infan ts and young children. Nine mutants of cp-RSV which had acquired eithe r the ts or small-plaque sp phenotype, were generated by chemical muta genesis with 5-fluorouracil. The two ts mutants with the lowest in vit ro shut-off temperature, namely the cpts-248 (38 degrees C) and cpts-5 30 (39 degrees C) mutants, were the most restricted of the nine cp-RSV mutant progeny tested for efficiency of replication in Balb/c mice. I n seronegative chimpanzees, the cpts-248 mutant replicated fourfold le ss efficiently in the nasopharynx and caused significantly less rhinor rhoea than its cp-RSV parent. The cpts-248 mutant virus, like its cp-R SV parent, was 1000-fold restricted in replication in the trachea comp ared with wild-type RSV. Previously, another candidate RSV live attenu ated vaccine str ain, a mutant designated ts-l, exhibited some instabi lity of its ts phenotype following replication in susceptible humans o r chimpanzees. Hence, we sought cp-RSV ts progeny that exhibited a gre ater degree of stability of the ts phenotype than the prototype ts-1 m utant. The cpts-248 and cpts-530 progeny viruses exhibited a greater d egree of stability of the ts phenotype in nude mice than the ts-1 viru s, and in chimpanzees, the former mutant also exhibited a greater stab ility of its ts phenotype than ts-1. The cpts-248 mutant was immunogen ic 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 tes ted in humans for its suitability in immunoprophylaxis.