Mechanisms of resistance for human respiratory syncytial virus isolates against anti-fusion agents in tissue culture

PhD ThesisHuman respiratory syncytial viruses (hRSV) are a major cause of lower respiratory tract disease on primary infection of infants and children. There is no effective vaccine against the virus, but high risk infants can be protected by administration of palivizumab (PZ), a humanised anti-fusion glycoprotein. In previous studies, hRSV isolated from the naso-pharyngeal secretions of infected infants were found to be dominated by slow growing variants which were largely refractory to neutralization by PZ. On further passage in tissue culture, the slow growing variants were replaced by fast growing, neutralization susceptible variants. The aim of this study was to investigate the mechanism of neutralization phenotype shift between slow-growing neutralization resistant and fast-growing neutralization susceptible clones. Neutralization resistance was found to be cell-line dependent with cell lines varying in their permissiveness to antibody treated virus. Antibody resistant and susceptible viruses showed no differences in the amount of the membrane expressed F glycoprotein or post-translational processing of the F polypeptide. They were also equally susceptible to inhibition by clathrin endocytosis inhibitors (monodansylcadaverine and chlorpromazine) suggesting that endocytosis was required for entry of both. They were further tested for susceptibility to anti-fusion inhibitors. These included a peptide derived from the F glycoprotein heptad repeat 2 and two small molecular weight compouds BMS-433771 and BTA9881. It was found that all three compounds failed to inhibit slow growing PZ resistant virus clones in parallel with PZ, but efficiently blocked fast growing PZ susceptible virus clones. These studies suggest that differences in antibody susceptibility stem from differences in the mechanism of fusion for the two virus clones. The full genomes of virus clones with resistant and susceptible phenotypes were sequenced to identify mutations which correlate with the difference in antibody susceptibility. The two clones differed at four sites in the SH, G, F and L genes. These mutations were sought in a number of virus clones expressing a range of susceptibility to antibody neutralization. No single mutation was associated with the shift from neutralization resistant to susceptible phenotype in all clones. However, a mutation at nucleotide 6162 in the L gene was associated with the shift from resistance to susceptibility in one virus lineage.Faculty of Medicine, Thammasat Universit