Respiratory syncytial virus modified by deletions of the NS2 gene and amino acid S1313 of the L polymerase protein is a temperature-sensitive, live-attenuated vaccine candidate that is phenotypically stable at physiological temperature

Abstract

Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. In previous work to develop point mutations in RSV with improved genetic stability, we observed that an attenuating mutation at amino acid position 1321 in the L polymerase protein was subject to deattenuation by a spontaneous second-site compensatory mutation at position 1313 (C. Luongo, C. C. Winter, P. L. Collins, and U. J. Buchholz, J. Virol. 86:10792-10804, 2012). In the present study, we found that deletion of position 1313 (Δ1313), irrespective of the presence of an attenuating mutation at position 1321, provided a new attenuating mutation. RSV bearing Δ1313 replicated in cell culture as efficiently as wild-type virus at 32°C, was restricted for replication at 37°C, and was restricted 50-fold and 150-fold in the upper and lower respiratory tracts, respectively, of mice. We combined the Δ1313 deletion with the previously described, attenuating NS2 gene deletion (ΔNS2) to produce the recombinant live-attenuated RSV vaccine candidate ΔNS2/Δ1313. During in vitro stress tests involving serial passage at incrementally increasing temperatures, a second-site compensatory mutation was detected in close proximity of Δ1313, namely, I1314T. This site was genetically and phenotypically stabilized by an I1314L substitution. Combination of I1314L with ΔNS2/Δ1313 yielded a virus, ΔNS2/Δ1313/1314L, with genetic stability at physiological temperature. This stabilized vaccine candidate was moderately temperature sensitive and had a level of restriction in chimpanzees comparable to that of MEDI-559, a promising RSV vaccine candidate that presently is in clinical trials but lacks stabilized attenuating mutations. The level of attenuation and genetic stability identify ΔNS2/Δ1313/1314L as a promising candidate for evaluation in pediatric phase I studies.

Fig 1

Deletion of L gene codon 1313 and temperature stress test of the Δ1313 virus. (A) Deletion of codon 1313. Sequences of the relevant portions of the L gene and protein of wt RSV are shown on the top line, while the line below indicates those of a mutant RSV with deletion of codon 1313. Note that codon 1321 also is indicated: this was the location of the attenuating 1030 mutation for which an S1313C change was a compensatory second-site mutation. (B) An abbreviated temperature stress test was performed for the Δ1313 virus. The Δ1313 virus was passaged in 10 replicate, independent cultures twice at 37°C and twice at 38°C, for a total of four passages (solid lines). The T_SH of this virus is 37°C (Table 1). As a control, the Δ1313 virus was passaged in parallel in two replicate cultures at the permissive temperature of 32°C for four passages (dotted lines). Virus titers at different passage numbers are shown. The substantial decrease in titer for the independent parallel cultures of the Δ1313 mutant at restrictive temperatures (solid lines) compared to the permissive temperature of 32°C (dotted lines) indicates that there was a substantial restriction of growth in all cultures at the restrictive temperature. This indicates that the Δ1313 virus remained attenuated at restrictive temperatures, consistent with genetic and phenotypic stability.

Fig 2

Gene maps of five attenuated recombinant RSVs bearing the Δ1313 mutation in combination with various other mutations. Note that some of the mutations involved are derived from previously described attenuated mutants, such as the rA2cp248/404/1030ΔSH virus, that have been evaluated in clinical studies (9, 11, 23, 26, 28, 37). The mutation set noted as “cp” comprises the following 5 amino acid substitutions: V267I in the N protein, E218A and T523I in the F protein, and C319Y and H1690Y in the L protein (26). The 404 mutation involves a nucleotide substitution in the GS signal of the M2 gene (T to C, positive sense, at the ninth nucleotide position in the signal) (23). ΔSH refers to deletion of the SH gene (28) in this case involving nucleotides 4210 to 4628 and joining the last nucleotide of the M gene end signal to the first nucleotide of the SH-G intergenic region. ΔNS2 refers to deletion of the NS2 gene, involving deletion of nucleotides 577 to 1098 and joining the gene end signal of the NS1 gene to the NS2-N intergenic region (14). The cp/ΔSH/Δ1313 virus was constructed in the complete wt RSV backbone, whereas the other recombinants were constructed in the 6120 background that lacks part of the downstream noncoding region of the SH gene (see Materials and Methods).

Fig 3

Temperature stress test of the 1321K(AAA)/Δ1313 virus, which bears the ts attenuating mutations 1321K(AAA) and Δ1313 in the L protein. Virus was passaged in 10 replicate cultures of HEp-2 cells twice at 34°C, twice at 35°C, and twice at 36°C, for a total of six passages (solid lines). The T_SH for this virus is 36°C (Table 2). Two replicate cultures were passaged at the permissive temperature of 32°C for six passages (dotted lines). Virus titers of the mutants at different passage numbers are shown, as detailed above. The substantial decrease in titer for the independent parallel cultures of the mutant virus at the restrictive temperatures (solid lines) compared to the permissive temperature of 32°C (dotted lines) indicates that there was a substantial restriction of growth in all cultures at 35 to 36°C, consistent with genetic and phenotypic stability of the attenuated virus.

Fig 4

Temperature stress test of the ΔNS2/Δ1313 virus, which bears the ΔNS2 and Δ1313 mutations. (A) Virus was passaged in 10 replicate cultures of Vero cells twice at each of the following temperatures, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C, for a total of 12 passages (solid lines). The T_SH for this virus is 38°C (Table 2). Two replicate control cultures were passaged at the permissive temperature of 32°C for 12 passages (dotted lines). Virus titers of the mutants at different passage levels are shown, as detailed above. Efficient growth at and above the T_SH of 38°C is indicative of loss of the ts phenotype. (B) Identification of a putative compensatory mutation at position 1314 in the L gene. Sequence analysis of viral populations following the 10th passage showed that all of the populations from the restrictive passages contained a single-nucleotide substitution conferring a I1314T missense mutation in the L gene (substitutions are in bold and underlined).

Fig 5

Nucleotide and amino acid sequences of wt RSV and mutant RSVs illustrating substitutions and deletions that were introduced at amino acid sequence positions 1313, 1314, 1316, and 1320 of the L protein. The provided sequences show either the wt or corresponding mutant sequences for nucleotides 12428 to 12457 and amino acids 1311 to 1320 of the RSV L protein.

Fig 6

Gene map and temperature stress test of the ΔNS2/Δ1313/1314L virus, which bears ΔNS2, Δ1313, and I1314L(CTG) mutations. (A) Gene map of the ΔNS2/Δ1313/1314L virus. (B) Temperature stress test. Virus was passaged in 10 replicate cultures of Vero cells twice at each of the following temperatures, 36°C, 37°C, 38°C, 39°C, and 40°C, for a total of 10 passages (solid lines). Two replicate cultures were passaged at the permissive temperature of 32°C for the same number of passages (dotted lines). Virus titers of the mutants at different passage numbers are shown, as detailed above. This showed that virus in some cultures was severely restricted at 39 to 40°C, virus in other cultures replicated nearly as efficiently as in the control cultures, and virus in the remaining cultures replicated to an intermediate level.

Fig 7

RSV vaccine candidates induce serum RSV-neutralizing antibodies in chimpanzees. Seronegative juvenile chimpanzees were inoculated by the intranasal/intratracheal route with the indicated viruses in the experiment described in Tables 4 and 5. Sera were obtained at the indicated time points, and neutralizing antibody titers were determined by complement-enhanced 60% plaque reduction neutralizing assay. Neutralizing titers were calculated as described previously (38). Animal identification codes and vaccine virus are indicated on the right.