Replacement of the F and G proteins of respiratory syncytial virus (RSV) subgroup A with those of subgroup B generates chimeric live attenuated RSV subgroup B vaccine candidates

Abstract

Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

FIG. 1

Insertion of the F and G genes of RSV strain B1 into recombinant strain A2. (A) The G and F genes of RSV B1 (stippled rectangles) were amplified by PCR as a single cassette with oligonucleotide primers designed to create upstream PacI and downstream SphI restriction sites. Following an additional modification described below, this cassette was cloned into the naturally occurring PacI site and the previously introduced SphI site of the parental RSV A2 cDNA plasmid (6). The RSV genes are shown as open rectangles; the GS and GE transcription signals are shown as shaded and solid bars, respectively. (B) In order to stabilize the G gene end signal of the B1 cDNA, the sequence in this region was modified as shown (new): the third nucleotide in each of the last 11 codons of the G ORF was changed without altering the amino acid coding assignment; the termination codon of the G ORF was changed to an alternative termination codon; 4 nucleotides were introduced into the downstream nontranslated region of the G gene between the ORF and the gene end signal; the G gene end signal was made identical to that of the F gene of strain A2 by substituting 2 nucleotides and deleting a nucleotide in the A tract; and the G-F intergenic region was shortened by 47 nucleotides and an MfeI restriction site was introduced. The sequence is divided into triplets at coding nucleotides, with the amino acid assignment shown directly below each codon. Nucleotides which differ from the B1 wt sequence are underlined, and the introduced MfeI restriction site is shown in boldface italics.

FIG. 2

Kinetics of replication in the upper (A) and lower (B) respiratory tracts of chimpanzees inoculated intranasally and intratracheally with wt control viruses A2 and B1 and chimeric viruses rAB and rABcp248/404/1030. The number of animals in each group (n) is shown. The dose of virus administered intranasally and intratracheally to each group was 5.0 log₁₀ PFU/ml, except for wt RSV A2, which was administered at 4.0 log₁₀ PFU/ml (11). Nasopharyngeal-swab and tracheal-lavage samples were collected on the days shown and titered by plaque assay on HEp-2 cell monolayers. The limit of detection for this assay is 0.7 log₁₀ PFU/ml of sample. The data for wt RSV A2 and wt RSV B1 are from Crowe et al. (11, 12).