Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries.