Development of recombinant monoclonal antibodies targeting conserved VlsE epitopes in Lyme disease pathogens

VlsE (variable major protein-like sequence, expressed) is an outer surface protein of the Lyme disease pathogen (Borreliella species) and a key diagnostic biomarker of Lyme disease. However, the high sequence variability of VlsE poses a challenge to the development of consistent VlsE-based diagnostics and therapeutics. In addition, the standard diagnostic protocols detect immunoglobins elicited by the Lyme pathogen, not the presence of pathogen or its derived antigens. Here we describe the development of recombinant monoclonal antibodies (rMAbs) that bind specifically to conserved epitopes on VlsE. We first quantified amino-acid sequence variability encoded by the vls genes from thirteen B. burgdorferi genomes by evolutionary analyses. We showed broad inconsistencies of the sequence phylogeny with the genome phylogeny, indicating rapid gene duplications, losses, and recombination at the vls locus. To identify conserved epitopes, we synthesized peptides representing five long conserved invariant regions (IRs) on VlsE. We tested the antigenicity of these five IR peptides using sera from three mammalian host species including human patients, the natural reservoir white-footed mouse (Peromyscus leucopus), and VlsE-immunized New Zealand rabbits (Oryctolagus cuniculus). The IR4 and IR6 peptides emerged as the most antigenic and reacted strongly with both the human and rabbit sera, while all IR peptides reacted poorly with sera from natural hosts. Four rMAbs binding specifically to the IR4 and IR6 peptides were identified, cloned, and purified. Given their specific recognition of the conserved epitopes on VlsE, these IR-specific rMAbs are promising diagnostic and theragnostic agents for direct detection of Lyme disease pathogens regardless of strain heterogeneity.