Respiratory Syncytial virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and children. The immune mediators of resistance to infection with RSV are still under investigation. In previous work the Principal Investigator generated large panels of live attenuated RSV viruses and performed the preclinical testing of these viruses in rodents and chimpanzees. New data from the applicant s clinical trials with these vaccines suggest that an IgA response correlates with protection against reinfection even in the absence of an IgG or neutralizing antibody response in the nasopharyngeal secretions or serum. The investigator's observations led to the hypothesis that IgA is indeed the principal mediator of protection against RSV reinfection in neonates. The question is: how do IgA antibodies that do not exhibit neutralizing activity protect against RSV? The hypothesis to be tested is that polymeric IgA (pIgAs) antibodies mediate intracellular neutralization of RSV. IgA transcytosis mediated by the polymeric immunoglobulin receptor (pIgR) was thought to be simply a mechanism for delivering high concentrations of antibodies to repertory secretions. Recent studies with several viruses, including Sendai virus, influenza virus, murine rotavirus, and HIV-1 suggest that IgA antibodies may neutralize viruses inside infected cells as the antibodies traverse the cell. The Principal Investigator will test this hypothesis using polarized epithelial cell cultures that express human or mouse pIgR. The Principal Investigator has developed panels of murine hybridoma lines secreting anti-RSV antibodies to either surface or internal proteins. The panels contain matched pairs of hybridoma lines that are identical except they are of the IgG or IgA isotype. The Principal Investigator will determine if murine PIgA binds to RSV inside polarized epithelial cells expressing the mouse pIgR and neutralizes RSV inside the cell. The applicants also will demonstrate this mechanism of virus inhibition in a cell line expressing human pIgR using pIgA purified from secretions of RSV vaccinees. Next, they will test the hypothesis that the compartment in which IgA complexes with RSV surface proteins is the apical recycling endosome, a site in which 80% of the intracellular IgA normally resides. Finally, the investigators will develop novel recombinant bi- specific antibodies that bind to both RSV and human pIgR. The Principal Investigator proposes that such novel antibody fragments will mimic the ability of IgA to enter cells and neutralize RSV, but will be vastly more potent in vivo due to their smaller size and hence better ability to exit the intravascular space following parenteral administration.
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