The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries

Abstract

Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy-and light-chain germ-line gene usage, suggesting that a broad antibody repertoire was selected. The single-chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full-length human IgG 1 format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti-RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post-exposure prophylaxis.

Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus

“…Screening for in vitro functional activity identified a subset of MAbs with neutralizing and/or CF activity that varied over a 2-log-unit concentration range; however, the majority exhibited activity only at higher concentrations (see Table S1 in the supplemental material). These observations are in contrast to a previous antibody repertoire cloned against rabies virus, where 44% of the repertoire was focused on only one VH3 gene (22) and 25% had potent or very potent neutralizing activities. These observations could stem from differences in natural infection versus immunization.…”

“…Peripheral-blood samples were drawn 1, 2, and 3 months after recovery from acute illness, and total RNA, prepared from whole blood from each sample, was converted into cDNA using random hexamers. The library was constructed as previously described (22). PCR-amplified light chain (LC) genes were pooled and cloned into the phagemid vector PDV-C06 using SalI and NotI; PCR-amplified heavy-chain genes were then pooled and ligated into the LC library using SfiI and XhoI.…”

“…Selections were carried out as described previously (6,22). CT helper phage was used to rescue phage libraries (21).…”

“…The immune antibody phage display library was incubated with inactivated WNV or VLP or soluble E protein immobilized on immunotubes. A second round of selection was again carried out with the three antigens so that all nine possible selection combinations were performed, a strategy that generally results in a diverse panel of MAbs (22). A total of 480 monoclonal scFv phages were screened for binding activity to inactivated WNV preparations.…”

Antibody fragments, expressed by a fowl adenovirus vector, are able to neutralize infectious bursal disease virus

“…Anti-viral scFv have been used to neutralize a wide range of animal viruses in vivo including infectious bursal disease virus (IBDV) (Ignjatovic et al, 2006), severe acute respiratory syndrome virus (Sui et al, 2004), rabies virus (Muller et al, 1997;Ray et al, 2001;Kramer et al, 2005), canine parvovirus (Yuan & Parrish, 2000) and western equine encephalitis virus (Long et al, 2001). It is also possible to engineer proteins onto the C terminus of scFv such as the immunoglobulin Fc domain (scFv-Fc), to confer greater stability and prolonged in vivo persistence (Powers et al, 2001;Borsi et al, 2002;Ono et al, 2003).…”