#40 An RSV live-attenuated vaccine candidate lacking G protein mucin domains is highly attenuated, immunogenic, and effective in RSV prevention in BALB/c mice

Abstract

Abstract Background RSV is a leading viral respiratory pathogen in infants. Live attenuated vaccines (LAV) show promise to reduce the disease burden of RSV disease. The RSV attachment protein (G protein) is heavily glycosylated with mucin domains that promote viral attachment. They may also aid in immune evasion through steric shielding of immunogenic epitopes. The objective of this study was to create and rescue RSV LAV candidates lacking the RSV G protein mucin domains with attenuated LAV replication and increased immunogenicity. Method LAV vaccine candidates “G155” and “G155-S” were generated from recombinant A2-line19F strain RSV by deletion of the G-protein mucin domains. G155-S additionally underwent deletion of the transmembrane domain, such that G155-S only expressed the secreted form of the G protein, while G155 expressed both transmembrane and secreted forms. Western blotting was used to characterize F and G protein expression in both vaccine candidates in comparison to wild-type (wt) A2-line19F. Enzyme-linked immunosorbent assays (ELISAs) were used to measure monoclonal antibody binding to LAV virus lysate. Viral growth kinetics were measured in HEp2 cells and in BALB/c mouse lungs by a fluorescent focus unit (FFU) assay. Immunogenicity was determined by measuring serum neutralizing antibody titers in BALB/c mice following a prime/boost regimen on days 0 and 28. Mice were challenged with wt RSV on day 60, and LAV efficacy was determined by measuring RSV lung viral loads by FFU assay and RT-PCR on day 4 post-RSV challenge. Results The vaccine candidates showed increased monoclonal antibody binding to surface F protein by ELISA, despite similar levels of F protein expression to wt, consistent with F epitope de-shielding. Both vaccine candidates were significantly attenuated in vitro and were undetectable in mouse lungs via FFU assay. By RT-PCR, LAV viral RNA was reduced 3-fold for G155-S, and 22-fold for G155 compared to wt RSV in mouse lungs. Both vaccine candidates elicited similar neutralizing antibody titers compared to wt A2-line19F on day 28 (G155 GMT 13, 95% CI 3, 53; G155-S GMT 10, 95% CI 2, 51; A2-line19F GMT 13, 95% CI 12, 15), and significant increases in titers were observed on day 59 (G155 GMT 440, 95% CI 5, 36057; G155-S 436, 95% CI 354, 537; A2-line19F 275, 95% CI 134, 567). Following RSV challenge, vaccinated mice showed no detectable lung virus by FFU assay and a reduction in lung viral RNA by RT-PCR of 560-fold for G155 and 604-fold for G155-S compared to RSV-challenged unvaccinated mice. Conclusion G155 and G155-S are highly attenuated RSV LAV candidates that protect against RSV challenge in mice and produce high levels of neutralizing antibodies comparable to wt RSV.