Article Figures & Data
Figures
- Fig. 1.
Purification of Oms66 from B. burgdorferiB313. (A) Coomassie blue-stained SDS-PAGE gel showing the purification of Oms66. Lane 1 shows whole organisms solubilized in SDS-PAGE sample buffer. Lanes 2, 3, and 4 show the soluble material after the sequential extraction of whole organisms with 0.5% sodium lauryl sarcosinate, 0.5% octyl polyoxyethylene, and 1.0% hydrogenated Triton X-100, respectively. Lane 5 is the insoluble pellet, and lane 6 is FPLC-purified Oms66. (B) Western blot of the samples described for panel A, with an additional lane (lane 7) containing FPLC-purified Oms66. Lanes 1 to 6 were probed with anti-Oms66 antisera, and lane 7 was probed with anti-B. burgdorferi immune serum from infection-immune rabbits.
- Fig. 2.
Western blot reactivities of serum from immunized animals. Each lane contains 5 × 106B. burgdorferi B31. Lanes 1 to 6 were probed with antisera from mice (mouse 1 to mouse 6, respectively) immunized with nOms66. Lane 7 was probed with anti-nOms66 generated in a rabbit, and lane 8 was probed with anti-hOms66 generated in a rabbit.
- Fig. 3.
Western blot of adsorbed serum. A total of 1 μg of recombinant Oms66 was electrophoresed in each lane. Lane 1 was probed with anti-nOms66, lane 2 was probed with anti-nOms66 adsorbed with Sepharose 4B beads, and lane 3 was probed with anti-nOms66 adsorbed with Sepharose 4B beads conjugated to recombinant Oms66. Sera were used at a 1:1,000 dilution.
- Fig. 4.
Dot blot of Oms66 probed with anti-Oms66 sera. (A) Dot blot probed with unadsorbed anti-nOms66 serum. (B) Dot blot probed with anti-nOms66 serum that had been adsorbed with recombinant Oms66 protein. N, native Oms66 protein; H, native Oms66 that had been heated to 95°C for 5 min; U, Oms66 denatured by the addition of 8 M urea; R, recombinant Oms66. Each spot contains 0.5 μg of the corresponding Oms66 protein.
- Fig. 5.
Immunoelectron microscopy. B. burgdorferi B31 and B313 were incubated with various sera. Sera used included NRS; hOms66, which is antiserum to denatured Oms66; IRS; Ads nOms66, which is antiserum to native Oms66 that had been adsorbed with recombinant Oms66; and nOms66, which is antiserum to native Oms66. After incubation, the cells were fixed to grids and were incubated with anti-rabbit immunoglobulin G conjugated to 10-nm colloidal gold. Each micrograph was taken at the same magnification. The bar represents 0.25 μm.
Tables
- Table 1.
Borreliacidal assay results: killing titers of immune sera against B. burgdorferi B31 and B313
Immune serum 100% killing titer (reciprocal)a B313 (basal) B313 (4th boost) B31 (4th boost) Mouseb Oms66-1 <20 320 <20 Oms66-2 <20 320 <20 Oms66-3 <20 320 <20 Oms66-4 <20 320 <20 Oms66-5 <20 320 <20 Oms66-6 <20 160 <20 Rabbit nOms66 <20 320 <20 Adsorbed nOms66 <20 160c <20 hOms66 <20 <20 <20 Infection immune <20 40 160 ↵a 100% killing indicates that after incubating organisms with immune serum and complement for 6 h, no viable organisms remained.
↵b Oms66-1 to -6 represent results for the six different mice that were immunized with nOms66 protein.
↵c The titer for the adsorbed serum was calculated with the assumption that the starting serum was diluted 1:4 compared to the unadsorbed serum.
- Table 2.
Enumeration of antibody binding to the surface of B. burgdorferi
Organism Antiserum No. of gold particles bound/μm B313 nOms66 17.1 B31 nOms66 5.1 B313 Adsorbed nOms66 9.8 B31 Adsorbed nOms66 3.4 B313 Infection immune 7.5 B31 Infection immune 13.7 B313 hOms66 1.0 B31 hOms66 0.5 B313 Basal 0.4 B31 Basal 0.7 - Table 3.
Mouse protection data: results from culture of extracted tissues at 3 weeks after challenge
Mouse Presence (+) or absence (−)a of B. burgdorferiin: Skin Ear Joint Spinal cord Bladder Oms66-1 − − − − − Oms66-2 − − − − − Oms66-3 + + + − + Oms66-4 − − − − − Oms66-5 + + + + − Oms66-6 − − − − − Naive-1 + + + + + Naive-2 + + + + + Naive-3 + + + − + Naive-4 + + + + + Naive-5 + + + + + Naive-6 + + + − − ↵a +, Tissue was culture positive forB. burgdorferi; −, tissue remained culture negative after 5 weeks of incubation.
