INVESTIGATION OF THE OUTER SURFACE PROTEIN, BBK19, AS A POTENTIAL COMPONENT OF A CHIMERIC ANTIGEN FOR DIAGNOSIS OF HUMAN LYME DISEASE

Lyme Disease (LD), caused by spirochetes of the genus Borreliella, is the most common tick-borne infection in North America and Europe. Borreliella plasmids have been shown to be essential for virulence, including B. burgdorferi B31 lp36. In this study, the lp36 encoded lipoprotein, BBK19, was characterized. Triton X-114 extraction and phase partitioning and size-exclusion chromatography revealed that BBK19 localizes to the outer membrane and is a monomer. A B. burgdorferi B31 gene deletion strain (B31-Δbbk19) displayed significantly attenuated chemotactic response and infectivity in mice via needle inoculation (n=7/group). ELISA analyses of experimentally infected mice (n=45) found BBK19 to be antigenic in murine infection. This work indicates that BBK19 may play a role in B. burgdorferi pathogenesis and may be a potential diagnostic antigen. The development of a single antigen-based diagnostic approach has been complicated by issues, including differential gene regulation, antigen diversity, and cross-reactivity. Therefore, this study developed a multi-protein chimeric antigen for diagnostic applications. The antigens DbpA, DbpB, BBK53, BBA73, and VlsE were identified as candidate diagnostic proteins based on their conservation and immunogenicity. Based on this work, two diagnostic chimeric proteins were designed with the most promising being HDFL4. Early (n=53) and late (n=16) stage human serum samples demonstrated comparable reactivity to HDFL4 compared to VlsE in early disease (45%) and increased reactivity in late disease (81% versus 63%, respectively). Serum from patients with cross-reactive diseases demonstrated no reactivity to HDFL4 (n=12). European human serum samples demonstrated reactivity to HDFL4 indicating its potential utility in a global market