VraA (BBI16) protein of Borrelia burgdorferi is a surface-exposed antigen with a repetitive motif that confers partial protection against experimental Lyme borreliosis

Abstract

We have previously described the expression cloning of nine Borrelia burgdorferi antigens, using rabbit serum enriched for antibodies specific for infection-associated antigens, and determined that seven of these antigens were associated with infectious B. burgdorferi strain B31. One of these infection-associated antigens encoded a 451-amino-acid putative lipoprotein containing 21 consecutive and invariant 9-amino-acid repeat sequences near the amino terminus that we have designated VraA for virulent strain-associated repetitive antigen A. The vraA locus (designated BBI16 by The Institute for Genomic Research) maps to one of the 28-kb linear plasmids (designated lp28-4) that is not present in noninfectious strain B31 isolates. Subsequent PCR analysis of clonal isolates of B. burgdorferi B31 from infected mouse skin revealed a clone that lacked only lp28-4. Southern blot and Western blot analyses indicated that the lp28-4 and VraA proteins, respectively, were missing from this clone. We have also determined that VraA is a surface-exposed protein based on protease accessibility assays of intact whole cells. Furthermore, vraA expression is modestly derepressed when cells are grown at 37 degrees C relative to cells grown at 32 degrees C, suggesting that VraA is, in part, a temperature-inducible antigen. Homologues cross-reactive to B. burgdorferi B31 VraA, most with different molecular masses, were identified in several B. burgdorferi sensu lato isolates, including B. andersonii, suggesting that the immunogenic epitope(s) present in strain B31 VraA is conserved between Borrelia spp. In protection studies, only 8.3% of mice (1 of 12) immunized with full-length recombinant VraA fused to glutathione S-transferase (GST) were susceptible to infectious challenge with 10(2) B. burgdorferi strain B31, whereas naive mice or mice immunized with GST alone were infected 40% or 63 to 67% (depending on tissues assayed) of the time, respectively. As such, the partial protection elicited by VraA immunization provides an additional testable vaccine candidate to help protect against Lyme borreliosis.

FIG. 1

Sequence analysis of vraA repetitive domain. (A) Pustell DNA matrix analysis of vraA aligned against itself. Lines framing the main diagonal show the location of the repetitive domain from nucleotides 157 through 723 of vraA encoding the 21 consecutive 9-amino-acid repeat EEELKKKQQ. The number of lines above or below the diagonal indicates the number of repeat units in vraA. The absence of any breaks within the upper and lower diagonals demonstrates that the repeats are consecutive and invariant. The short perpendicular line in the lower right corner indicates a short imperfect inverted repeat sequence located at nucleotides 1222 to 1240 of vraA. (B) Comparison between the single region of homology of BBI28 and the vraA repeat unit. Underlined nucleotides and amino acid indicate differences in the vraA and BBI28 sequences, respectively.

FIG. 2

Overproduction and purification of full-length VraA fused to GST. (A) Coomassie blue stain of an SDS–10% polyacrylamide gel showing the overproduction of GST alone (lane 1) and full-length VraA fused to GST [GST-VraA(FL); lane 2, indicated by an arrow] following IPTG (isopropyl-β-d-thiogalactopyranoside) induction as outlined in Materials and Methods. (B) Solubility of recombinant GST-VraA(FL). After induction, E. coli cells synthesizing GST-VraA(FL) were subjected to breakage with a French pressure cell and the insoluble component cleared by centrifugation as indicated in the Methods section. The resulting membrane material (M) and soluble component (S) were resolved by SDS-PAGE, and the gel was stained with Coomassie blue. Note the appearance of an approximately 100-kDa species in the lane containing the soluble fraction (lane S, indicated by an arrow). (C) Purified GST-VraA(FL). GST-VraA(FL) was affinity purified with glutathione-Sepharose beads, electroeluted from an unfixed SDS-polyacrylamide gel to eliminate lower-molecular-mass contaminants, reseparated by SDS-PAGE, and stained with Coomassie blue. Purified GST-VraA(FL) was then used to immunize both rabbits and mice to generate anti-VraA serum and in protection studies, respectively. Numbers on the left refer to the molecular mass of protein markers (in kilodaltons).

FIG. 3

Specificity of antiserum directed against full-length VraA. Whole-cell lysates of protein derived from B. burgdorferi B31 MSK5 (wild type) and MSK7 (lp28-4⁻) were separated by SDS-PAGE and stained with Coomassie blue (A) or immunoblotted and probed with rabbit serum directed against full-length VraA fused to GST (B). Lane 1, MSK5; lane 2, MSK7. The numbers on the left refer to the molecular masses of the protein markers (in kilodaltons).

FIG. 4

Surface Localization of VraA. Intact B. burgdorferi B31 MSK5 either left untreated (lane 1) or treated with proteinase K (lane 2), resolved by SDS-PAGE, and immunoblotted. Identical blots were then probed with either anti-VraA (full-length VraA fused to GST) (A), anti-p66 (B), or anti-endoflagellum (C) serum. The numbers on the left refer to the molecular mass of protein markers (in kilodaltons).

FIG. 5

vraA induction at 37°C relative to 32°C. B. burgdorferi B31 MSK5 (passage 2) was inoculated at an initial cell density of 5 × 10⁵ per ml in BSK II medium and cultivated as outlined in Materials and Methods at both 32 and 37°C. At various phases of growth, samples were removed and protein from whole-cell lysates was subjected to SDS-PAGE and immunoblotting with anti-VraA serum. Lane 1, 1.25 × 10⁷ B. burgdorferi grown at 37°C; lane 2, 1.9 × 10⁷ B. burgdorferi grown at 32°C; lane 3, 1.22 × 10⁸ B. burgdorferi grown at 37°C; lane 4, 1.37 × 10⁸ B. burgdorferi grown at 32°C; lane 5, 3.14 × 10⁸ B. burgdorferi grown at 37°C; lane 6, 3.1 × 10⁸ B. burgdorferi grown at 32°C. The numbers on the left refer to the molecular mass of protein markers (in kilodaltons).

FIG. 6

Presence of VraA or VraA homologues in various B. burgdorferi sensu lato isolates. Whole-cell lysates from B. burgdorferi sensu stricto strain B31 passage 4 (lane 1), strain B31 passage 47 (lane 2), strain 297 (lane 3), strain CA-2-87 (lane 4), strain ECM-NY-86 (lane 5), strain JD-1 (lane 6), strain NT-1 (lane 7), and strain 2872-2 (lane 12) were resolved by SDS-PAGE along with B. andersonii strains MOD3, MOD5, and MOD6 (lanes 8 to 10) and B. garinii strain IP-90 (lane 11). All isolates with the exception of the strain B31 isolates were passaged two times in vitro. The resulting gels were either stained with Coomassie blue (A) or immunoblotted and probed with anti-VraA serum (B). The numbers on the left refer to the molecular mass of protein markers (in kilodaltons).