FIG 1.
Antigens and a representative immunization protocol used in this study. (A) Schematic of the recombinant monomeric and dimeric OspC proteins. OspC from strain B31 is given as an example. The signal peptide (amino acids [aa] 1 to 18) is replaced with an oligohistidine tag. Monomeric OspC also carries a C19G mutation. Both monomeric and dimeric variants carry a C→S mutation of the central cysteine, thereby preventing aggregation. Peptides B31loop5 and B31Cterm are indicated in orange and yellow, respectively. (B) Schematic overview of a representative immunization schedule. Mice were immunized up to 4 times using up to 100 μg of protein per injection with adjuvant (typically alum). Adjuvant only was used as a control. Sera were sampled before the initial immunization, ∼12 days after each immunization, on the day of challenge, and on the day of sacrifice. Challenge was done either by subcutaneous injection of in vitro-cultured B. burgdorferi (needle inoculation) or by applying B. burgdorferi B31-infected I. scapularis ticks onto each individually caged mouse for a period of 14 days. (C) SDS-PAGE of monomeric and dimeric recombinant OspC proteins, showing dimerization mediated by Cys19. Left panel, SDS-PAGE of M-OspCB31, D-OspCB31, M-OspC297, D-OspC297, M-OspCN40, and D-OspCN40 in the absence of dithiothreitol (DTT) reducing agent. Right panel, SDS-PAGE migration of recombinant B31 OspC proteins in the presence and absence of DTT. Each lane contains 1 μg of purified recombinant protein. M, molecular mass (in kilodaltons) ladder; M-OspCB31, monomeric OspC of strain B31; D-OspCB31, dimeric OspC of strain B31; M-OspC297, monomeric OspC of strain 297; D-OspC297, dimeric OspC of strain 297; M-OspCN40, monomeric OspC of strain N40; and D-OspCN40, dimeric OspC of strain N40.