VACCINE EFFICACY: CHARACTERIZATION OF THE PROTECTIVE IMMUNE RESPONSE TO RESPIRAT

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Respiratory syncytial virus is the most common cause of lower respiratory tract disease in young children worldwide. The F and G glycoproteins of this virus are known to be targets of a protective immune response; however, following natural infection, immunity is incomplete. Repeated infections with viruses of the homologous or heterologous subtypes occur despite high levels of neutralizing antibody. In addition, antibodies that neutralize extracellular virus may not be able to inhibit fusion, allowing cell-to-cell spread of virus. These findings suggest that B cell responses seen following infection may be deficient in antibodies that prevent virus- receptor interactions. In order to better understand the virus-cell interactions required for infection, studies were initiated to more completely define the receptor (s) used by RSV. The binding of viral proteins to cell surface glycosaminoclycans was evaluated using heparin agarose affinity chromatography. Studies using whole virions and infected cell lysates showed that both F and G glycoproteins bind to heparin. Strain RSV/B cp 52, which expresses F glycoprotein but lacks G and SH, was used to show that F-heparin binding was independent of G. In addition, cp52 infection of Vero cells was diminished in the presence of heparin, Bovine lung heparin and de-N sulfated heparin and this inhibition was dose-dependent. These results indicate that RSV-F-heparin sulfate interactions are important in the biology of the virus. In order to identify linear F sequences responsible for heparin binding, overlapping F peptides were evaluated for their ability to bind during heparin agarose affinity chromatography. These studies identified a linear sequence on RSV subgroup A F that bound to heparin. We plan to evaluate the ability of these peptides to bind to Vero, Hep-2 and A549 cells and assess the ability of the peptides to limit infection with strains such as A2 and cp52. Work in this laboratory has also shown that RSV F,G and SH are expressed in a complex on the surface of infected cells. Interestingly, the affinity of the F-G-SH complex for heparin is greater than the affinity of F or G glycoproteins alone. These results may explain, in part, the incomplete inhibition observed when heparin and heparin sulfate derivatives are used to inhibit RSV infection. In addition, working with colleagues in OBRR, we have determined that the dominant human neutralizing antibody response is of the IgG1 isotype. Human IgG1 reacts with whole virions and binds to F and G expressed on the surface of infected cells as well as purified RSV F and G. Studies are in progress to determine linear and conformational sites on RSV F and G recognized by human neutralizing antibody.