E-032. Probing the Mechanism of a Complement-Independent Bactericidal IgM against Relapsing Fever Borrelia

T. J. LaRocca, L. I. Katona, D. G. Thanassi, J. L. Benach;
Stony Brook Univ., Stony Brook, NY.

A single chain variable fragment (scFv) of CB515, a complement-independent bactericidal monoclonal IgM against a relapsing fever Borrelia, was constructed to investigate the region wherein the unique bactericidal function resides. Monomeric CB515 scFv (26 kDa) was capable of binding its antigen on whole organisms and by immunoblot. This binding was shown to be species and serotype-specific to the 19 kDa variable small protein (Vsp), recognized by its parent monoclonal IgM. A dose-dependent bactericidal effect of the CB515 scFv was detected by direct enumeration of spirochetes. Spirochetes incubated with the CB515 scFv prior to inoculation into mice grew into escape mutants, whereas spirochetes incubated with an irrelevant scFv developed as the original infecting serotype, demonstrating the bactericidal efficiency of the effect. As seen at the ultrastructural level, this bactericidal effect was due to disruption of the outer membrane (OM) and to severe membrane blebbing eventually progressing to lysis. These results indicate that the variable region of CB515 is responsible for this bactericidal activity and that the constant region of the antibody is dispensable. Additionally, disruption of the Borrelia OM was shown to require the presence of the divalent cations Ca2+ and Mg2+. Once the OM was disrupted, inner membrane damage and spirochete death was resultant of osmotic lysis dependent on the presence of pores, as shown through osmoprotection studies.