Serum epitope repertoire analysis enables early detection of Lyme disease with improved sensitivity in an expandable multiplex format.

Widely employed diagnostic antibody serology for Lyme disease, known as standard two-tier testing (STTT), exhibits insufficient sensitivity in early Lyme disease yielding many thousands of false negative test results each year. Given this problem, we applied serum antibody repertoire analysis (SERA), or NGS-based serology, to discover IgG and IgM antibody epitope motifs capable of detecting Lyme disease specific antibodies with high sensitivity and specificity. Iterative motif discovery and bioinformatic analysis of epitope repertoires from subjects with Lyme disease (n = 264) and controls (n = 391) yielded a set of 28 epitope motifs representing 20 distinct IgG antibody epitopes, and set of 38 epitope motifs representing 21 distinct IgM epitopes which performed equivalently in a large validation cohort of STTT positive samples. In a second validation set from subjects with clinically-defined early Lyme disease (n=119) and controls (n = 257), the SERA Lyme IgG and IgM assay exhibited significantly improved sensitivity relative to STTT (77% vs. 62%, z-test, p = 0.013) and improved specificity (99% vs. 97%). Early Lyme disease subjects exhibited significantly fewer reactive epitopes (Mann-Whitney U-test, p < 0.0001), relative to subjects with Lyme arthritis. Thus, SERA Lyme IgG and M panels provided increased accuracy in early Lyme disease, in a readily expandable multiplex assay format.