4.2. Epitope Mapping and Peptide Sequences

Overlapping peptide libraries (ProImmune, Oxford, UK), consisting of 15-AA long peptides overlapping by 10-AA, were generated using sequences of p35 (BBH32) (accession# {"type":"entrez-protein","attrs":{"text":"WP_010256558.1","term_id":"497942402"}}WP_010256558.1) and ErpP (accession # {"type":"entrez-protein","attrs":{"text":"WP_010883886.1","term_id":"499186346"}}WP_010883886.1) from the B31 strain of Borrelia burgdorferi sensu stricto. ProImmune, Inc. performed the epitope mapping under contract using their proprietary ProArray Ultra technology, as previously described [19]. Sera from 8 patients with physician-diagnosed EM+ early Lyme disease that were seropositive by two-tier testing, with an IgG immunoblot sensitivity of 9-10, of 10 bands were used for the epitope mapping. Using the 17-mer B31 sequence previously described, mod-VlsE(275–291) was engineered. [17]. It includes substitutions at AA278 (D→ N), AA282 (A→ V), and AA290 (M→ V), which represent natural sequence variability found in Borrelia strains other than B31. It is similar in sequence to the IR6 epitope of VlsE, which has been shown to be sensitive as a first- or second-tier assay target [16,27]. FlaB(211–223) is a previously described immunodominant peptide of the Borrelia Flagella [42] contained within a region of the protein previously identified to have little cross-reactivity with flagella from other species [39,40]. Peptides: p35(101–115): DTGSERSIRYRRRVY, ErpP(51–65): KIEFSKFTVKIKNKD, FlaB(211–223): (C)VQEGVQQEGAQQP, and mod-VlsE(275–291): MKKNDQIVAAIALRGVA were synthesized by Lifetein (Hillsborough, NJ, USA). Dual-epitope peptides were synthesized containing three glycine (G) residues between two epitope sequences.