Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees

Abstract

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.

FIG. 1

Deletion of the SH gene to generate rA2ΔSH. The map of the negative-sense wt RSV genome is shown (not to scale), as is the sequence illustrating the deletion, which involved nucleotides 4205 to 4623 (arrows). To construct rA2ΔSH, plasmid D50, containing the left-hand end of the genome from the 3′ leader region to the end of the M2 gene, was digested with ScaI and PacI and the resulting fragment was replaced with a short double-stranded DNA constructed by hybridizing the two synthetic oligonucleotides ACTCAAATAAGTTAAT and TAACTTATTTGAGT. In the final construction, the deletion extended from the middle of the M GE signal to the middle of the SH GE signal. In the ΔSH genome, compared to its wt recombinant parent, the M GE signal sustained a single nucleotide change (AGTTAATAAAAAA to AGTTAATTAAAAA, with the change underlined) to become identical to that of the wt SH GE signal. The modified D50 plasmid was then used to assemble a complete antigenome by ligation with a cDNA containing the L gene and trailer region as described previously (5, 33, 34). IG, intergenic region; GS, gene start signal; GE, gene end signal.