Conformational Nature of the Borrelia burgdorferi B31 Outer Surface Protein C Protective Epitope

Serological profiles of individual mice immunized with recombinant OspC expressed by the pSCREEN (pET) vector system as assayed by immunoblotting. (A) Mouse groups 1 to 5 prior to challenge; (B) mouse groups 1 to 5 at 2 weeks following challenge, with seroconversion, indicative of infection, beginning in most mice. Mice were immunized with E. coli lysate containing (pET) pSCREEN vector only with no insert (group 1), heat-denatured recombinant OspC (group 2), guanidine-HCl-denatured recombinant OspC (group 3), nondenatured, insoluble recombinant OspC (group 4), or recombinant OspC, denatured with guanidine, with removal of guanidine following antigen purification on a Ni²⁺cation column. Sizes of antigen bands (in kilodaltons) are given on the right. (C) Serological profile of mouse group 4, at 4 weeks following challenge, with three of five mice seroconverting, indicative of infection. Sizes of antigen bands (in kilodaltons) are given on the right.

(A) Western blot demonstrating MAb B5 reactivity againstB. burgdorferi B31 low-passage-number cells (B31 low) and recombinant (Rec.) OspC proteins generated in the expression vectors pBluescript and pSCREEN. The recombinant proteins are larger than the B31 OspC due to the presence of fusion partners. Molecular masses (in kilodaltons) are given on the left. (B) Spot blot demonstrating MAb B5 reactivity against non-heat-denatured (“not boiled”) and heat-denatured (“boiled”) antigens. B31 high, B. burgdorferi B31 high-passage-number (>50 passages) cells, which produce small amounts of OspC; E. coli pSCREEN only, lysate of cells harboring the vector with no cloned insert. Recombinant (Rec.) OspC E. coli lysates are the same as in the Western blot in panel A.

Diagram showing truncated constructs of theospC gene coding sequence. The long solid arrow at the top represents the coding sequence minus the signal peptide, beginning at amino acid (aa) 33. The dotted line at the left represents the missing signal peptide. The BglII restriction site is indicated. Arrows indicate the relative positions of the primers used to amplify and clone the constructs shown below. Lines bounded by dots represent the regions encoded by the truncated constructs, whose designations appear at the right. 5′ Bgl II and 3′ Bgl II were clones made by digestion of the gene with BglII. HK-12 and BS-32 were made by unidirectional deletions. 117-590, F1-590, 117-R2, and F1-R1 were made by PCR with the primers shown. (+) and (−), reactivity and no reactivity, respectively, with MAb B5.