Characterization of monoclonal antibodies targeting the RtxA1 toxin of Vibrio vulnificus

Vibrio vulnificus is a lethal pathogen that is responsible for most seafood-related deaths. The mortality rate for susceptible individuals is higher than 50% within 1-2 days after contact with the bacteria. Recent studies suggest that RtxA1, a secreted bacterial toxin, plays a key role in the cytotoxicity and pathogenesis of V. vulnificus. Despite the important role of RtxA1 in the virulence of V. vulnificus, few monoclonal antibodies (MAbs) against RtxA1 have been generated or characterized. In this study, we purified a recombinant RtxA1 protein with a mass of approximately 130 kDa that contains GD-rich repeats proposed to play an important role in host cell killing, and generated a panel of 10 new MAbs against the recombinant RtxA1 protein. By performing competition binding assays and expressing RtxA1 fragments via nested deletions of the gene, we spatially mapped our new MAbs to three fragments of the recombinant RtxA1 protein. Western blot analysis and immunostaining studies showed that the MAbs efficiently reacted with secreted and host cell-targeted RtxA1 from V. vulnificus. These data suggest that the RtxA1 MAbs might be effectively used as important biomaterials for manufacturing toxin-neutralizing therapeutic MAbs targeting the bacterial RtxA1 toxin and could be relevant for developing rapid and specific diagnostic tools against lethal V. vulnificus infections.