Investigating BB0405 as a novel Borrelia afzelii vaccination candidate in Lyme borreliosis

Abstract

BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

Figure 1

Alignment between the BB0405 protein sequence between Borrelia burgdorferi B31 and Borrelia afzelii PKO. Borrelia burgdorferi B31 corresponds to NCBI Reference Sequence: NP_212539. Borrelia afzelii PKO is identical to CB43 (GenBank: CP002933.1 translated in ExPASy translate tool). Identity between sequences is 88.2%. Additionally there are 17 similar positions bringing the similarity to 96.5%. Alignment was performed with Clustal Omega. * = identical, : = similar.

Figure 2

Enzyme-linked immunosorbent assay (ELISA) showing high-titer antibodies induced in recombinant BB0405 immunized mice. BB0405 specific total IgG responses were measured in mice sera of three vaccination groups with 8 mice each. IgG responses are presented as optical density (OD) 450–655 nm for multiple sera dilutions. The first group was immunized at three time points (0, 14 and 28 days) with recBB0405. The second groups received DNA vaccinations at the same time points with DNA BB0405 and the third group with DNA Empty (negative control). Plates were coated with recombinant BB0405 and incubated with mouse sera, which was collected from mice at time point 42 days, just before tick challenge and after three vaccinations. Sera was diluted in steps of 300 until 1: 218700.

Figure 3

BB0405 is expressed on the surface of B. burgdorferi sensu stricto and B. afzelii. Viable spirochetes were incubated with (+) or without (−) proteinase K for 30 min and 60 min and processed for immunoblot analyses with antibodies against BB0405. Flagellin B antibodies served as a subsurface control.

Figure 4

(A) Western blot showing expression of BB0405 in different B. burgdorferi sl strains (Borrelia burgdorferi strain B31 and Borrelia afzelii strain CB43) grown at different temperatures. A temperature of 33 °C, resembling (feeding) tick conditions, and 37 °C, resembling conditions in the mammalian host, was used to culture the spirochetes. Lysates of the in vitro cultured spirochetes were obtained, as described in the materials and method section, and subjected to Western blot. Three independent experiments were performed. The first experiment is displayed on the left, the second experiment in the middle and the third experiment is displayed on the right. The far left lane is the protein weight marker. Blots were loaded with 2.5 ug/well whole lysates of spirochetes and were cut in half just between the bands of Flagellin B (41 kDa) and BB0405 (22 kDa) to enable separate incubations. The blots displayed at the top are incubated with anti-flagellin rabbit IgG 1:1000 (as a loading control) and subsequently with secondary antibody anti-rabbit IgG-HRP 1:1000. Blots displayed at the bottom are incubated with pooled serum from mice vaccinated with recombinant BB0405 1:500 and subsequently with secondary antibody anti-mouse IgG-HRP 1:2000, respectively. Blot images were cropped (Image acquisition tools Microsoft Powerpoint). Imaging was performed using ImageQuant LAS 4000 and quantification using Image J (Wayne Rasband, National Institutes of Health, USA, Java 1.8.0_77(32-bit), http://imagej.nih.gov/ij). Full length blots are presented in Supplementary Fig. S1–S6. (B) Protein expression of BB0405 as determined by Western blot in panel A was quantified and normalized against the relative density of loading control Flagellin B. Relative density was determined using ImageJ software (National Institute of Health). A quantitative comparison between samples on the same blot and within the same experiment were made. Statistical differences between groups were calculated using an unpaired parametric t-test. Error bars represent mean ± s.e.m. n.s.: P > 0.05.

Figure 5

Schematic design of the vaccination study. The study consisted of 3 experimental groups of 8 mice each. The first group was vaccinated with recombinant BB0405, the second group was received a tattooed DNA vaccination with BB0405 and the third group received an empty vector DNA vaccination as a control. Mice were vaccinated at t = 0, t = 14 and t = 28 days. They were challenged with infected ticks 2 weeks after the last booster vaccination and sacrificed at day 63.