Michelle Klouwens

A novel strategy for an anti-Borrelia vaccine is described in chapter 4, making use of Outer Membrane Vesicles (OMVs), nanostructures that bud naturally from the outer membrane of gram-negative bacteria as a response to stress and to adapt in order to survive. OMVs have great immunogenic properties and can function both as adjuvant and as delivery vehicle, as antigens from other pathogens can be expressed on their surface. OMV-based vaccines could also be brought to the market relatively easily, as an OMV-based vaccine against meningococcal disease is already available for human use. As part of chapter 4, we have generated OMV constructs expressing OspA and immunized mice with meningococcal OMVs carrying OspA from B. burgdorferi ss strain BPN. We subsequently challenged the immunized mice with a subcutaneous injection of B. burgdorferi ss and assessed both the immunogenicity of the OMVs compared to aluminum (AlOH) and the protection against B. burgdorferi ss transmission. We showed reasonable antibody titers against the OspA-OMV construct, although lower compared to the antibody titers against recombinant OspA. More importantly, with regard to protection against B. burgdorferi ss transmission, we demonstrated that both the OspA-OMV construct as well as recombinant OspA with separate OMV as adjuvant, provided partial, yet significant protection. Our findings reveal that OMV-based vaccines expressing B. burgdorferi sl (lipo)proteins are a promising vaccination method protecting against B. burgdorferi sl infection and could be considered as a potential vaccination strategy in humans.

In chapter 5 we focus on a recently described B. burgdorferi sl protein, BB0405, which is a surface exposed protein that is highly conserved across different B. burgdorferi sl species. Therefore, this encompassed an interesting vaccine candidate potentially inducing cross-species protection. To investigate this further, we determined whether vaccination of mice with recombinant BB0405 or with bb0405 DNA tattoo vaccination from B. burgdorferi ss BPN protected against a challenge with B. afzelii CB4P infected Ixodes ricinus nymphs. We observed a high IgG antibody response when the recombinant BB0405 vaccine was applied, but not when the bb0405 DNA tattoo vaccine was used. Surprisingly, neither of the vaccines provided cross-species protection in the mice upon challenge of 8 B. afzelii strain CB4P-infected ticks. Next, we investigated the expression of BB0405 in the different B. burgdorferi sl species and we demonstrated a significant downregulation of BB0405 in the B. afzelii CB4P spirochetes at 37°C compared to 33°C. This was not observed for B. burgdorferi ss BPN. These observations could potentially explain the lack of protection in the mice vaccinated with the recombinant BB0405 vaccine, despite the high specific IgG antibody titers.

In Chapter 6 we used label-free quantitative proteomics to identify and select tick salivary gland proteins from I. ricinus that are differentially expressed during tick feeding and in response to B. afzelii CB4P infection. We have been able to identify 870 I. ricinus proteins from which 68 were upregulated upon 24 hour feeding in B. afzelii CB4P infected I. ricinus ticks. Next, we selected 7 tick salivary gland proteins, which we were able to technically and biologically validate, and we recombinantly expressed these in E. coli. We vaccinated both guinea pigs and mice with a cocktail of the 7 recombinant proteins and subsequently challenged them with I. ricinus ticks. We were able to show significant reduction in post-engorgement weights of I. ricinus ticks in both tick challenge models. Future research should investigate which of the tick proteins is responsible for the observed findings and focus on vaccine optimization. It would also be of interest to determine whether combining a selection of our newly identified antigens with other antigens from either tick or other tick-borne pathogens could protect against transmission of B. burgdorferi sl and other tick-borne diseases.

The last chapter of this thesis, the general discussion, reflects on the different vaccination strategies and vaccination candidates described in the previous chapters and puts our findings in broader perspective. We have shown that it remains a challenge to identify that one candidate, or a combination of candidates, that would prevent Lyme borreliosis and ideally also impairs tick feeding and transmission of other tick-borne diseases. Newer sophisticated techniques such as transcriptomics and proteomics proved to be instrumental to help identify potential vaccine candidates. However, more research is needed delineating the mechanisms by which these tick or B. burgdorferi sl proteins affect the intricate tick-host-pathogen interactions at the tick-bite site. Regardless, based on the findings described in this thesis, one could speculate that applying vaccination platforms that allow for combining antigens from both B. burgdorferi sl and the tick vector, constitute a promising approach in the quest for a broadly protective and efficacious vaccine to prevent Lyme borreliosis.