Respiratory syncytial virus is the most common cause of lower respiratory tract disease in young children worldwide. The F glycoprotein of this virus is one of the targets of a protective immune response; however, despite significant conservation in the F glycoprotein and the development of anti-F antibodies following natural infection, immunity is incomplete and repeated infections with viruses of the homologous or heterologous subtype 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 intratypic antigenic differences in the F glycoprotein might be able to convey an epidemiological advantage during the course of natural infection. In order to study intratypic antigenic variation and the role it plays in the development of a protective immune response, we have characterized clinical isolates that vary in fusion epitopes. Although these viruses bind to and are neutralized by F-antibodies, fusion of infected cells is not inhibited by the presence of antibody to the F glycoprotein. Sequence analysis of the envelope glycoproteins has identified several amino acids that may contribute to the ability of these viruses to cause fusion. Serial passage of the clinical isolates in VERO cells produces viruses that have gained or lost reactivity with the fusion inhibiting antibody. Sequence analysis of these passaged viruses should help to identify the specific amino acid changes that result in the ability to escape from fusion inhibition. Another interesting property of some clinical isolates is the ability to cause giant syncytia in tissue culture (LPV); in contrast, the laboratory strain, A2, produces small syncytia, (SPV). Replication of the viruses were equivalent and DI particles were not present. Studies to quantitate the level of F expression and density of fusion epitopes on infected cell membranes indicate that the level of expression was greatest for SPV. FACS analysis also demonstrated two peaks of F-Mab binding on SPV. In contrast, there was a single peak seen for the LPV. Western blot and IP studies have indicated that SPV-F is expressed as both uncleaved monomer as well as cleaved F1+F2 while the LPV expresses only cleaved F1+F2. Sequence analysis of LPV and SPV indicate that the region around the cleavage activation sites are identical. It appears that mutations outside of the cleavage activation site determine accessibility to cellular proteases and determine the efficiency of cleavage of RSV-F.
