ISOLATION AND CHARACTERIZATION OF A HIGHLY ATTENUATED RESPIRATORY SYNCYTIAL VIRUS (RSV) VACCINE CANDIDATE BY MUTAGENESIS OF THE INCOMPLETELY ATTENUATED RSV A2 TS-1 NG-1 MUTANT VIRUS

Ts-1, a temperature sensitive (ts) mutant of RSV was previously derive d from RSV A2 virus by mutagenesis with 5-fluorouracil (5-FU). Ts-1 wa s attenuated for adult volunteers and seropositive children but retain ed a low level of virulence in seronegative infant vaccinees as indica ted by the occurrence of upper respiratory tract disease. Ts-1 NG-1, a more defective derivative of ts-1, has produced by mutagenesis of ts- 1 with nitrosoguanidine. However, ts-1 NG-1 still retained a low level of virulence for the upper respiratory tract and showed some genetic instability in chimpanzees. With renewed interest in the goal of devel oping a live, attenuated RSV vaccine, we have now attempted to further attenuate ts-1 NG-1 by mutagenesis with 5-FU and 5-azacytidine. Four mutants that are phenotypically different from the ts-1 NG-1 parental virus were identified. Each of the four mutants was more restricted in replication in BALB/c mice compared with the ts-1 NG-1 parental virus . One of the ts-1 NG-1 derivatives, termed A-20-4, which showed the lo west (35 degrees C) in vitro shutoff temperature and which was also co mpletely restricted in replication in BALB/c mice, was selected for fu rther evaluation in seronegative chimpanzees, A-20-4 did not cause rhi norrhea in chimpanzees but induced detectable titers of serum RSV neut ralizing antibodies in 2 of 4 chimpanzees. Apparent complete protectio n to subsequent challenge with, wild-type RSV was observed in each of the four chimpanzees previously immunized with A-20-4, The ts-1 NG-1 A -20-4 mutant thus represents a promising live attenuated RSV vaccine c andidate.