A FURTHER ATTENUATED DERIVATIVE OF A COLD-PASSAGED TEMPERATURE-SENSITIVE MUTANT OF HUMAN RESPIRATORY SYNCYTIAL VIRUS RETAINS IMMUNOGENICITY AND PROTECTIVE EFFICACY AGAINST WILD-TYPE CHALLENGE IN SERONEGATIVE CHIMPANZEES

A cold-passaged (cp), temperature-sensitive (ts) RSV mutant designated RSV cpts-248 (shut-off temperature 38 degrees C), which possesses host-range mutations acquired during 52 passages at low temperature in bovine tissue culture and a ts phenotype introduced by subsequent chemical mutagenesis, was found previously to be attenuated, immunogenic, and protective against wild-type challenge in seronegative chimpanzees. We sought to introduce additional attenuating mutations such as small-plaque (sp) and ts mutations into RSV cpts-248 by chemical mutagenesis with 5-fluorouracil with the intent of obtaining cpts-248 derivatives that are more attenuated in mice or chimpanzees and that are move genetically stable following replication in vivo. Ten mutants of RSV cpts-248 which had acquired a sp phenotype or a second ts mutation were generated by chemical mutagenesis. Five cpts-248 derivatives which had acquired mutations that specified a 36 degrees C shut-off temperature for plaque formation and one which had acquired only a sp phenotype were more restricted in replication in Balb/c mice than the cpts-248 parental strain. One mutant, designated RSV cpts-248/404 (shut-off temperature 36 degrees C), was 100 times more restricted in replication in the nasal turbinates of mice and 1000 times more restricted in the nasopharynx of seronegative chimpanzees than its cpts-248 parent. The cpts-248/404 mutant was completely restricted in replication in the lower respiratory tract of chimpanzees even following direct intratracheal administration. The ts phenotype of the cpts-248/404 mutant was stable during replication in vivo in mice and chimpanzees. Chimpanzees immunized with cpts-248/404 were fully protected against upper respiratory tract disease and lower respiratory tract virus replication upon subsequent challenge with wild-type virus. The cpts-248/404 virus and related mutants exhibit many desirable characteristics which make them promising vaccine candidates.