Neutralización del virus respiratorio sincitial humano por agentes dirigidos frente a la proteína de fusión y detección de distintas formas de la proteína F ditinguibles por su reactividad con anticuerpos neutralizantes

Resumen

Human respiratory syncytial virus (HRSV), a member of the Pneumovirus of the Paramyxoviridae family, is the main cause of severe lower respiratory tract infections in very young children and is a pathogen of considerable importance in the elderly and in immunocompromised adults. Currently, there is no effective vaccine against the virus, although it is known that passive administration of neutralizing antibodies to individuals at high risk is an effective immunoprophylaxis. The HRSV fusion (F) protein is an attractive target for drug and vaccine development as it is essential for viral entry, is highly conserved, and is the major virus neutralization antigen. To approach a detailed analysis of neutralization of HRSV infectivity, a panel of monoclonal anti-F antibodies were tested for neutralization. No direct correlation was observed between antibody ELISA titer and neutralizing ability. Therefore, the neutralizing activity of anti F antibodies is likely related to the epitope specificity, and is somewhat independent of their affinity for the F protein. Bivalency is needed to inhibit viral infectivity by weakly neutralizing antibodies (i.e., mAb 2F). However, potent neutralizing antibodies (i.e., mAb 47F and 101F) exert their neutralizing activity even if monovalent. These antibodies bind to the F protein present in the viral membrane, prior to its activation, while F is presumably in its prefusion form. In contrast, low molecular weight compounds, like HRB F derived peptides and other organic compounds, have to be present during virus entry to inhibit HRSV virus infectivity, suggesting that a conformational change in the F protein in contact with the target membrane is required for these compounds to be effective. This work also shows that immunization of rabbits with a recombinant vaccinia virus expressing the full-length F protein of HRSV (VAC/FC) elicits neutralizing antibodies that recognize different conformations of the F protein, a situation similar to that occurred through natural infections in humans. Specific antibodies present in the anti-VAC/FC serum were separated by affinity chromatography with immobilized F soluble protein. These antibodies allowed us to identify two forms of the F protein which are found in HRSV-infected cells. The most abundant form appears to be the highly thermostable postfusion conformation. The other is less abundant and less thermostable, suggesting that it adopts the prefusion conformation. The results presented here help to explain the virus neutralizing ability of antibodies specific to the F protein of HRSV, as well as offer a foundation for future searches for neutralizing antibodies. Identification of new, highly potent neutralizing antibodies together with further investigation of the molecular basis of neutralization will help to improve prophylactic antibody treatment to prevent HRSV infection.