Final Report Summary - ANTIDOTE (Anti-tick Vaccines to Prevent Tick-borne Diseases in Europe)
Executive Summary:
The aim of the ANTIDotE project was to find tick salivary gland proteins (TSGPs) that block pathogen transmission and/or interfere with tick feeding. Two main antigen discovery strategies were employed. Firstly, a transcriptomic approach (MACE and RNAseq) was used to identify TSGPs upregulated upon infection with Borrelia, TBEV or Babesia, resulting in the identification of numerous TSGPs, of which a selection was technically and biologically validated. There are currently five manuscripts on the transcriptomes of I. ricinus upon feeding and infection in the pipeline. Secondly, an immunoscreening using a yeast surface display was used to identify and validate TSGPs that are recognized by antibodies of frequently tick-exposed humans, and/or tick immune animals. A selection of the thus identified TSGPs was investigated in downstream WPs. As part of WP2 we have established and improved tick-transmission models for Borrelia afzelii (submitted for publication), TBEV and Babesia microti (WP2). The infrastructure for RNAi in ticks was established and used to investigate the role of multiple TSGPs. Silencing of selected TSGPs did not affect Borrelia transmission, but silencing of one target significantly impaired both tick feeding and TBEV transmission. Our data show that one of the identified TSGPs promoted Borrelia survival in vivo by impairing phagocytosis. Finally, we have also generated fluorescent B. afzelii strain CB43 to study TSGP-Borrelia interactions. Production of TSGPs in E. coli and Drosophila was performed and a selection used for vaccination studies. Vaccination of mice with TSGPs did not result in a significant anti-tick effect and we shifted our focus to the tick-rabbit model to assess tick immunity (WP3). Also, six bovine vaccination experiments were performed, showing that (specific fractions of) tick tissue extracts resulted in a robust anti-tick immunity. The obtained cow sera were used to set-up an in vitro artificial tick feeding system, which demonstrated the need for combined cellular and humoral immune responses to induce tick immunity. As an additional effort, we identified and/or tested tick-borne pathogen antigens (from B. microti and B. afzelii) as candidates for transmission-blocking vaccines. In WP4 we identified one TSGP that, when used to actively immunize mice, protected against Lyme borreliosis and two TSGPs that, when tested as antigens for an anti-tick vaccine, partially protected mice from lethal TBEV-infection.
The ANTIDotE partners were actively engaged with stakeholders from medical and veterinary sciences, policy advisors, governmental institutions, patient interest groups and the pharmaceutical industry. We disseminated knowledge on (inter)national conferences, in peer-reviewed scientific journals and through the ANTIDotE website and (social) media. Indeed, 33 related ANTIDotE and ANTIDotE-related articles have currently been published in peer-reviewed scientific books or journals (of which the majority open access and including 2 PhD theses). These also included a technical report on preventive strategies to prevent TBDs, a review on anti-tick vaccines and a book chapter on the prevention of Lyme borreliosis and a publication on the cost-effectiveness analyses of an anti-tick vaccine in a Central Eastern European country where Lyme borreliosis and tick-borne encephalitis are endemic. Finally, the ANTIDotE project contributed to another four anticipated PhD theses and three manuscript have been submitted/accepted for publication. All in all, we significantly contributed to the understanding of, and steps towards the downscaling of, TBDs in Europe. All our initially planned discovery methods yielded vaccine candidates and we pursued multiple additional antigen discovery approaches and we have characterized and produced selected candidates, of which some indeed protected against LB or TBEV. We will continue our research and investigate whether combining multiple tick and pathogen antigens could lead to the development of a single vaccine to prevent bacterial, protozoal and viral tick-borne diseases. The antigens identified, described and characterized, as well as the assays, materials and methods developed as part of the ANTIDotE project are an excellent starting point.
Project Context and Objectives:
Ixodes ricinus transmit bacterial, protozoal and viral pathogens that cause Lyme borreliosis, babesiosis and tick-borne encephalitis, respectively and exceedingly affect Central and Eastern Europe. During feeding, ticks introduce salivary proteins into the skin that interfere with host defense mechanisms. However, in animals, repeated tick infestations as well as vaccination against selected tick proteins can lead to decreased pathogen transmission by inhibiting tick feeding - known as ‘tick immunity’ - or by neutralizing tick proteins that facilitate the transmission of tick-borne pathogens. Also humans with hypersensitivity to tick-bites have a lower risk of contracting tick-borne diseases. Therefore, anti-tick vaccines encompass an innovative strategy to prevent tick-borne diseases in humans, or animals and wildlife to indirectly reduce the risk of contracting tick-borne diseases for humans. Currently, although there is a veterinary vaccine available for another tick species, there is no vaccine available against I. ricinus, the main European vector for veterinary and human tick-borne diseases.
The ANTIDotE project aimed to identify and characterize tick proteins involved in ‘tick immunity’ and tick-borne pathogen transmission and use the acquired knowledge to investigate and develop an anti-tick vaccine to prevent multiple human tick-borne diseases.
To this end state of the art proteomic and transcriptomic approaches were used to identify and characterize novel tick salivary gland proteins, of which a selection was assessed as anti-tick vaccines to protect against LB, TBEV and babesiosis in animal models. In addition, through an integrated and multidisciplinary approach involving Central and Eastern European public health institutes, health organizations and industrial companies we examined how to develop anti-tick vaccines and implement these in public health systems. Thus, ANTIDotE delivered 1) essential knowledge on the biological mechanisms involved in the pathogenesis of tick-borne diseases, 2) experimental proof of concept of an anti-tick vaccine - based on single or multiple antigens - protecting against multiple human tick-borne pathogens and 3) a road map for the development exploitation and implementation of anti-tick vaccines. Collectively, the output of the ANTIDotE project could significantly contribute to downscale the severe medical and economic burden that tick-borne diseases have on societies in Europe and other parts of the world where Ixodes ticks are endemic.
As such, the ANTIDotE project had five key objectives, corresponding to five different work packages (WP). Since these are unaltered compared to the original proposal and all periodic reports thus far they are written in the present and simple future tense.
First key objective (WP1): To identify I. ricinus tick salivary gland proteins (TSGPs) that play a role in the transmission of tick-borne pathogens (TBPs) from the tick to the host, but also TSGPs that are recognized by anti-tick antibodies. Therefore, gene expression profiles will be analyzed of salivary glands (SGs) of uninfected and TBP-infected feeding I. ricinus ticks. We will also identify TSGPs that are recognized by mammalian anti-tick immune responses by an I. ricinus TSGP yeast surface display (YSD).
Objective 1.1 Identification of TSGPs involved in transmission of TBPs
Objective 1.2 Identification of TSGPs recognized by anti-tick immune responses
Second key objective (WP2): To functionally characterize the identified TSGPs. The TSGPs are characterized with the help of RNAi in ticks. Alongside, the functional activity of specific TSGPs (in relation to host defense mechanisms) will be investigated. As part of this objective we will provide in depth insights in the mechanisms involved in transmission of TBPs and will reveal candidates for anti-tick vaccine to prevent TBP transmission. For this objective in vivo animal models for the tick-borne diseases (TBDs) under study will be established or improved.
Objective 2.1 Describe the role of TSGPs in the transmission of TBPs in vivo using RNAi in ticks
Objective 2.2 Describe the function of TSGPs in in vitro and ex vivo functional assay
Third key objective (WP3): Aims to confirm which TSGPs induce ‘tick immunity’ in in vivo animal models and the artificial tick feeding system. Furthermore, the humoral and cellular immune response that these TSGPs elicit will be characterized. Thus, this objective will reveal candidates for anti-tick vaccine that are capable of interfering with tick feeding.
Objective 3.1 To confirm that TSGPs identified induce ‘tick immunity’ in mice
Objective 3.2 To characterize humoral and cellular anti-tick immune responses
Objective 3.3 To confirm that TSGPs identified induce ‘tick immunity’ in cows and to develop a robust in vitro artificial tick feeding system for research on anti-tick vaccines
Fourth key objective (WP4): As part of this objective we aim to deliver the proof of concept that a single anti-tick vaccine can prevent transmission of bacterial, protozoal as well as viral TBPs. Vaccine studies with promising candidates, or specific combinations of promising candidates, will be performed to identify an anti-tick vaccine that could interfere with all three TBPs under study. Both TSGPs that are crucial for the transmission of Borrelia, Babesia and tick-borne encephalitis virus (TBEV) from the tick to the mammalian host, and TSGPs that are of paramount importance for tick feeding, will be tested.
Objective 4.1 To identify an anti-tick vaccine that protects against multiple TBDs by specifically interfering with TSGPs crucial for transmission of TBDs
Objective 4.2 To identify an anti-tick vaccine that protects against multiple TBDs by interfering with tick feeding
Fifth key objective (WP5): The objective of this part of the project is to deliver plans for exploitation and implementation of our findings and concepts to contribute to downscaling of the burden of TBDs on Central and Eastern Europe and other endemic European societies, as well as to disseminate ANTIDotE results. We will also attend and arrange workshops and meetings.
Objective 5.1 To exchange knowledge on LB, TBE, human babesiosis and innovative strategies to prevent TBDs
Objective 5.2 To disseminate ANTIDotE results
Objective 5.3 To establish a road map for exploitation of novel anti-tick vaccines
Objective 5.4 To explore ways to implement anti-tick vaccines in health systems
Project Results:
WP1 Identification of tick salivary gland proteins (TSGPs)
In this work package we identified I. ricinus tick salivary gland proteins (TSGPs) that play a role in the transmission of tick-borne pathogens (TBPs) from the tick to the host. We also identified TSGPs that are recognized by anti-tick antibodies. These TSGPs comprise potential candidates for an anti-tick vaccine that could interfere with tick feeding and/or pathogen transmission. To this end we used different state of the art methods.
Objective/task 1.2: Identification of TSGPs recognized by anti-tick immune responses.
The main method used in task 1.1 was transcriptomics to identify TSGPs involved in transmission of tick-borne pathogens and/or tick feeding. The TBPs under study were Borrelia afzelii, the causative agent of Lyme borreliosis, TBEV, the virus that causes tick-borne encephalitis and Babesia microti, the protozoal causative agent of babesiosis. As part of subtask 1.1.1 we fed larval I. ricinus ticks on control animals or animals syringe-inoculated with B. afzelii or B. microti. Fed larval ticks were allowed to molt, giving rise to B. afzelii-infected, Babesia-infected or uninfected controls nymphal I. ricinus ticks. For TBEV we injected nymphal I. ricinus ticks with TBEV using a nano-injector. Ticks originating from at least three individual adult female I. ricinus ticks were pooled for the feeding experiments, in which ticks were allowed to feed for 0 hours, 24 hours or to repletion for B. afzelii or B. microti, and for 0, 6, 12, 24 or 48 hours for TBEV. After the indicated time salivary glands were dissected and RNA extracted for downstream sequencing and transcriptomic analysis. As part of subtask 1.1.2 and using a dual approach, combining RNA sequencing for reliable qualitative sequencing data and massive analysis of cDNA ends (MACE) for sensitive quantitative data, we obtained important insights into the transcriptome of I. ricinus in response to infection with the pathogens under study. This approach did not only yield multiple potential candidates for an anti-tick vaccine, but also unprecedented insights into the tick salivary gland genes differentially expressed during feeding and infection with B. afzelii, TBEV or B. microti. Indeed, two advanced manuscript drafts and three manuscript outlines have been drafted to describe these findings, which have been presented at multiple international conferences and are anticipated to be submitted in the near future. Importantly, TSGPs abundantly and significantly upregulated upon infection with B. afzelii, TBEV, B. microti or upon tick feeding were identified for further study as part of the ANTIDotE project. In total, a subset of approx. 20 TSGPs - both technically and biologically validated by quantitative RT-PCR - was subsequently selected for further study in downstream work packages (described elsewhere in this report). Due to technical reasons we were unable to biologically validate tick salivary gland gene expression induced by B. microti. However, the mRNA of B. microti, being a eukaryote, also has a poly-A tail, which allowed capturing by our methods for RNAseq and MACE. We were thus able to identify to B. microti sporozoites-specific transmembrane proteins that could serve as antigens for a vaccine blocking Babesia transmission from the tick to the host.
Objective/task 1.2: Identification of TSGPs recognized by anti-tick immune responses.
The main method used in task 1.2 was the Yeast Surface Display (YSD) to identify TSGPs involved in tick feeding and tick immunity. This specific library was made as part of subtask 1.2.1. Briefly, an I. ricinus salivary gland cDNA expression library was generated using the input of isolated RNA from 1350 nymphal I. ricinus ticks fed for 24, 48 and 72 hours, cloned into PYD1 expression vectors, which were transformed in EBY100 yeast cells. Induced yeast cells – expressing tick salivary proteins - were subsequently selected using sera from humans frequently exposed to tick bites (forestry workers) or controls or tick-exposed or tick-immune rabbit sera (or controls). Single yeast cells expressing a TSGP recognized by human or animal tick-exposed or tick-immune sera were selected, the recombinant PYD1 plasmids were isolated and the inserts (TSGPs) were sequenced. Differential expression between the animal tick-exposed or tick-immune sera on one hand and the control sera on the other hand was confirmed using flow cytometry on single yeast cells. Thus, we were able to identify 24 unique TSGPs that might be involved in tick-immunity, of which 13 were selected for further study (as described elsewhere in this report).
Overall, in WP1 we successfully identified TSGPs that are involved in tick feeding, transmission of different TBPs from the tick salivary gland to the host or in tick-immunity. A total of approx. 30 TSGPs, not including TSGPs identified or selected through different and additional approaches (as will be described later), were selected for further study in WP2, WP3 and WP4 as part of our quest for an anti-tick vaccine to prevent multiple human tick-borne diseases.
WP2 Functional characterization of tick salivary gland proteins (TSGPs)
Objective/task 2.1: The role of TSGPs in the transmission of TBPs in vivo using RNAi in ticks.
As part of this objective, transmission models for Borrelia afzelii, B. microti and TBEV were established and improved (subtask 2.1.1). In addition, the in vitro artificial tick feeding system was further modified to feed all life stages of I. ricinus ticks in vitro. Tick feeding units were made of borosilicate glass. Silicone membranes were prepared and attached to the tick feeding units and reinforced with mosquito netting for feeding I. ricinus adults, but not for I. ricinus larvae or nymphs. Additional attachment stimuli included finely clipped bovine hair and bovine hair extracts. Furthermore, we studied a selection of the vaccine candidates identified in WP1. In task 2.1.2 we have tested the role of 13 of these tick genes on B. afzelii transmission through knock down by RNAi (excluding two known targets from I. scapularis that were unsuccesfully tested as positive controls). Conformation of knockdown of these genes by q-RT-PCR showed that most of these genes were successfully knocked down, but unfortunately no evident phenotype could be observed for Borrelia transmission or tick feeding parameters. In addition, for TBEV, RNAi experiments have been performed for a total of 10 tick salivary gland genes that were feeding-induced and/or upregulated upon both Borrelia and TBEV infection, to test their importance in tick feeding and TBEV transmission. Again knockdown could be confirmed for most targets. Interestingly, knockdown of two tick salivary gland genes (gene 12 and gene 42) significantly impaired tick feeding and knockdown of gene 12 also protected mice from lethal TBEV infection (described in more detail in deliverable report DL2.2).
Objective/task 2.2: The function of TSGPs in in vitro and ex vivo functional assays
A part of subtask 2.2.1 as many as 28 tick salivary gland genes have been produced recombinantly in E. coli and/or Drosophila expression systems. A large subset of these proteins has been used in WP2, WP3 and WP4 for functional or vaccination studies. The selection of the proteins to be expressed was based on the different technologies used to identify tick proteins as part of mainly WP1, but also based on additional efforts in WP2 and to lesser extent WP3. These proteins were either upregulated during tick feeding, or were selected due to their recognition by immune sera. The identified tick proteins can largely be classified as follows:
A. Proteins corresponding to genes upregulated during tick feeding identified by MACE analysis. Two different analyses were performed, from which a set of genes was selected based on expression levels, upregulation upon feeding (particularly at the 24 h feeding time) and their upregulation in 4 independent experiments performed at two independent and geographically distinct locations. The selected genes were named Gene 1, Gene 2, Gene 3, Gene 4, Gene 8, Gene 11, Gene 12, Gene 17, Gene 42, Gene 44 and Gene 49.
B. Proteins corresponding to genes upregulated by the presence of Borrelia (GeneBb and Rdk) or TBEV (TVC), identified by MACE analysis. The selected genes were named GeneBb 1, GeneBb 2, GeneBb 6, GeneBb19, Rdk 1, Rdk 2, TVC2 and TVC4.
C. Several proteins recognized by human ‘tick exposed’ sera (Forestry workers, FW) or tick immune rabbits (MS, COMB, NS), identified by Yeast Surface Display (YSD). The selected proteins were named FW1, FW2, FW3, MS1, MS2, MS3, COMB1, COMB2, COMB3, NS1 and NS2.
D. Proteins identified by an additional approach consisting of immunoprecipitation and mass spectrometric analysis of cow sera immunized with salivary gland or midgut extracts. The selected proteins were named SG1, SG4, MG2, MG6 and MG9.
For the MACE analyses, the genes selected for expression were technically and biologically validated in independent groups of ticks and for the tick proteins identified by YSD and immunoprecipitation by different means, including FACS analysis and Western blots. A large selection of the genes encoding these proteins has also been tested in RNAi experiments, as described above, or in WP3, as described later. Further in silico analysis revealed that some identified proteins were highly identical. Highly overlapping proteins were omitted resulting in a final list of 28 uniquely identified tick proteins. The eight proteins identified by the comparative proteomic approach (described below) are not depicted in the above-described overview
As part of subtask 2.2.2 we developed a functional assay pipeline and developed and refined existing methods (the ANTIDotE pipeline). For example, we have successfully generated a fluorescently labeled B. afzelii strain CB43. We showed this isolate is both infectious to ticks and mice and can be used for microscopy work to assess interactions of Borrelia with tick salivary gland proteins. In addition, we characterized the function of one of the identified TSGPs, a putative secreted saliva protein of unknown function, referred to as COMB 2, which was highly conserved between different Ixodes tick species. Expression of the gene encoding this protein was upregulated upon Borrelia infection and co-incubation of Borrelia spirochetes with the Drosophila-expressed recombinant protein augmented infectivity in vivo in the mouse model. The recombinant protein could not prevent killing of Borrelia by the complement system, but it reduced phagocytosis of Borrelia by bone marrow derived mouse macrophages, whereas TNF-alpha production was unaffected. Another protein, for which the encoding gene is referred to as Gene 12 (see above), has been expressed in Drosophila for functional studies, because of its role in transmission of TBEV from the ticks to the host.
We also engaged in additional experiments as part of this work package. For instance, we investigated adaptation of the artificial tick feeding system to assess B. microti transmission (FUB). With the idea that this would greatly facilitate research on tick-host-pathogen interactions we tried to set this up in vitro, but were unsuccessful and decided not to pursue this any further. The artificial tick feeding system was however successfully used as part of WP3 (see elsewhere in this report). Notably, we successfully identified eight additional TSGPs by comparative proteomics that could serve as candidates for a B. afzelii-blocking anti-tick vaccine. Comparative proteomics was used in ticks under different feeding and B. afzelii-infection conditions. In line with the transcriptomic approach and experimental design, three different feeding times namely unfed, 24 hour fed and fully fed, as well as B. afzelii infected and uninfected nymphal ticks, were used. Tick protein expression levels between the different conditions were compared, enabling us to identify TSGPs abundantly expressed during the tick feeding process, as well as tick proteins that could be central for transmission of B. afzelii from the tick salivary glands to the host. From the approx. 1000 identified tick salivary gland proteins we have selected 8 TSGPs that were abundantly expressed and upregulated upon 24h feeding and upon B. afzelii infection. These 8 proteins have been generated in E. coli and two of them in Drosophila cells. For a subset of these proteins, we have performed a Western Blot on salivary gland extracts with specific antibodies that we had raised by immunization of mice to validate our data. We considered these proteins validated and they have been further studied in WP4. Finally, we have investigated the role of specific murine host factors in the course of experimental Lyme borreliosis.
In summary, we have generated multiple recombinant tick antigens, established and refined functional assays and methods - designated the ANTIDotE pipeline - that have been, and will be, used to investigate the functionality of tick proteins identified in the context of the ANTIDotE project. In addition, we identified a range of interesting tick proteins and unraveled the function and/or importance of a subset of these proteins in light of tick feeding or pathogen transmission from the tick to the host. Collectively, this knowledge will be used to rationally design an anti-tick vaccine to prevent multiple tick-borne diseases, as also discussed in deliverable report DL5.5 in more detail.
WP3 Characterization of immune response against TSGP’s
Objective/task 3.1: Induction of ‘tick immunity’ in mice by TSGPs
The generation of all recombinant tick proteins is reported as part of WP2 (and to avoid overlap and repetition not described in subtask 3.1.1). As part of subtask 3.1.2 we initially investigated whether we could induce tick immunity in mice. We proved to be unsuccessful, even with established strong candidate antigens. Alternative vaccine delivery strategies, such as DNA tattoo vaccination, were also not successful. We therefore switched to vaccination experiments in rabbits. Using antigens identified by the YSD, we vaccinated rabbits with 3 different cocktails of 2-3 tick salivary gland proteins, and 2 single antigens. Vaccination resulted in high antibody titers against all antigens. However, no significant reduction on tick feeding parameters (feeding time, engorgement weights and egg mass) for I. ricinus nymphs and adult females could be observed. In contrast, we showed that, when rabbits are exposed to adult I. ricinus for multiple times, this lead to drastically impaired tick feeding in subsequent challenges. Regardless, the sera from the cocktail vaccinations with the recombinant proteins were selected for passive transfer experiments to mice experimentally challenged with B. afzelii-infected ticks to assess their effect on Borrelia transmission in WP4. Besides the fact that mice represent an excellent model to study Borrelia transmission and infectivity, blocking this process with is different from trying to mimic tick immunity. Indeed, it is well known that cellular immune effectors play an important role in tick rejection and the subsequent tick immunity. However, from previous studies it appears that tick-directed immune responses preventing Borrelia transmission are largely antibody mediated. Indeed, antibodies against specific TSGPs could for instance neutralize the advantageous functions certain TSGPs have on Borrelia transmission and infectivity.
Objective/task 3.2 Cellular and humoral anti-tick immune responses against TSGPs
As part of subtask 3.2.1 and 3.2.2 we investigated antibody responses to tick salivary gland proteins.
Antibody subclasses are widely divergent among different animal species. Thus, while rabbits only contain one IgG type, humans contain four IgG subclasses (IgG1, IgG2, IgG3, IgG4) as do mice, albeit not coinciding in functionality. In contrast, bovines have only two subclasses of IgG (IgG1 and IgG2). Each antibody subclass is associated with differential functionalities, including the initiation of the complement cascade or the cellular activation through their interaction with Fc receptors. In the case of anti-tick vaccines, a predominant IgG1 response has been associated with better levels of protection using the commercial Bm86-based vaccines against Rhipicephalus (Boophilus) microplus. We performed ELISA using sera from cows vaccinated as part of this work package. In midgut extract vaccinated cows – which showed the strongest anti-tick responses (see below) – we showed a predominant IgG1, but also a robust IgG2 response. Interestingly, also in Fraction (F)1 vaccinated cows (see below), which showed protective effects against I. ricinus ticks too, we showed the same mixed response. Our results suggest that, in this model, although a predominant IgG1 response may be required, the presence of IgG2 antibodies specific for the protective antigens also appeared to be important for acquired resistance to ticks. Therefore, the generation of the proper balance between isotypes with differential functions may be required in order to generate a full, and protective response. This should be considered in future vaccination experiments.
Objective/task 3.3 To confirm that TSGPs identified induce ‘tick immunity’ in cows and to develop a robust ATFS for research on anti-tick vaccines
As part of subtask 3.3.1 we were able to show (and have published) that vaccination of cows with tick tissue homogenates strongly reduce tick feeding success. We thereafter compared the effects of salivary gland and midgut extract and showed that especially vaccination with midgut extract resulted in evident tick rejection. Elaborating on these findings we fractionated midgut extracts to identify the actual antigens involved in the observed tick immunity. Four different fractions (F) were created using size exclusion chromatography and used for vaccination-challenge experiments in cows. Although vaccination induced high antibody titers, no effect on adult female I. ricinus engorgement weights could be observed. However, F1 - containing proteins with the highest molecular weight - showed a reduction in egg mass also significantly reduced engorgement weights of nymphal ticks. The absence of the strong phenotype, as was observed following vaccination with total midgut extract, could be explained by a lower vaccination dose due to technical limitations while preparing the fractions. Indeed, a second experiment, in which we used a higher concentration of F1, did show an evident protective phenotype, suggesting that to be discovered proteins in F1 are responsible for the strong anti-tick immune responses in cows. In contrast, multiple cow vaccination trials with a total six of recombinant tick proteins identified in WP1 did not result in impaired tick feeding parameters.
Next to feeding ticks in vivo on cows, in subtask 3.3.2 we fed ticks in vitro using an optimized artificial tick feeding system. However, sera from three cow vaccination experiments did not show a marked reduction of tick feeding success in contrast to the in vivo results, suggesting that combined cellular and humoral responses are needed for proper tick immunity. Therefore, further in vitro feeding of sera from these experiments was considered irrelevant and was therefore not performed.
As a highly interesting additional effort in WP3, we have selected 15 tick salivary gland and tick midgut proteins from a list of proteins that we identified by an immunoprecipitation with tick-immune cow sera and salivary gland extracts or midgut extracts followed by identification of the precipitated antigens by LC-MS/MS. We subsequently, investigated the role of these proteins in the tick feeding process by RNAi in adult female I. ricinus ticks feeding on rabbits. Thus far, two of these proteins were made recombinantly in E. coli and investigated in an additional cow vaccination trial, but did not induce tick immunity.
WP4 Proof of concept
In WP4 we investigated whether we could prevent multiple tick-borne diseases by vaccination targeting the tick. We selected the candidates based on the identification and characterization described in WP1, WP2 and WP3. WP4 was the common intersection of all previous WPs. In WP4 we aimed to deliver proof of concept that anti-tick vaccines can prevent transmission of multiple tick-borne pathogens. We had planned to investigate tick antigens that are instrumental to transmission of tick-borne pathogens in WP4.1 and antigens that interfere with tick feeding and thereby indirectly interfere with transmission of tick-borne pathogens from the tick to the host. In reality, the difference was not as clear-cut and quite a few of the tick antigens identified through the different planned as well as additional approaches showed some degree of overlap in sequence, protein family, function or in their role in the tick feeding process or in transmission of TBPs from the tick salivary gland to the host. Therefore, we here report Objective/task 4.1 and Objective/task 4.2 together and for clarity we report our efforts, both planned and additional, per transmission model. In addition to the planned activities, we have performed additional experiments to identify potential tick antigens for an anti-tick vaccine targeting I. ricinus with the goal of preventing Lyme borreliosis, TBEV and human babesiosis. As a contingency plan we also investigated vaccine candidates targeting the pathogen itself. The transmission models for B. afzelii, B. microti and TBEV required for the WP4 experiments were established and refined as part of WP2.
Objective/task 4.1: Specifically interfering with transmission of tick-borne pathogens AND Objective/task 4.2: Interfering with tick feeding to prevent transmission of tick-borne pathogens
Borrelia afzelii transmission model
In the B. afzelii model we investigated 8 tick salivary gland antigens, either as single antigens or in combinations and identified by 3 different approaches, by active immunization. Furthermore, we passively transferred sera from rabbits that had been vaccinated with 3 different combination of tick antigens identified by the yeast surface display to mice, followed by B. afzelii-infected tick challenge. In addition, we also investigated whether a conserved Borrelia candidate could protect against Lyme borreliosis through conventional or an alternative vaccination vaccine delivery strategy (DNA tattoo). The main finding in the B. afzelii model was that we identified an anti-tick vaccine based on a tick salivary gland protein, COMB 2, that was able to significantly and robustly impair B. afzelii transmission from the tick to the host. This particular tick salivary gland protein was functionally described in WP2 and was shown to impair phagocytosis of Borrelia by murine macrophages in vitro and boost Borrelia infectivity in vivo. Interestingly, mice actively vaccinated with this tick protein were significantly less often Borrelia-culture positive and had significantly lower Borrelia loads, as determined by q-PCR in various tissues, as compared to controls. Although we will most definitely repeat this experiment, these data indicate that we have identified an antigen that can be used as an anti-tick vaccine to prevent Lyme borreliosis.
Babesia microti transmission model
As we have described in WP1 and WP2 we were unable to validate the tick salivary gland proteins that we identified as part of the Babesia MACE analyses due to the fact that we were unable to get highly B. microti-infected nymphal ticks. Indeed, preparation of cDNA for biological validations appeared to be complicated and for the last two years we did not get higher infection rates than 10-15%, which is too low for our validation experiments. At the time of reporting we had already infected larvae of 15 different tick populations, and we changed many conditions in an attempt to tweak the nymphal infection percentage. We for instance incubated larvae at different temperatures before and after feeding on B. microti-infected mice and tried to feed larvae on mice with different levels of parasitemia. Since we are of the opinion that, when biologically validated, the tick salivary genes identified in the Babesia MACE analysis could be very interesting, two ANTIDotE partners (BC ASCR and FUB) will keep trying to get B. microti-nymphal infection rates above 50%. As soon as we have these we can validate the already existing top-20 tick salivary gland genes that are induced upon B. microti infection at 24 hours of tick feeding. Interestingly, we did show that when we used 10 lowly B. microti-infected nymphs and fed them on BALB/c mice that the majority of mice became infected. This allows us to test vaccine candidates (either tick proteins or Babesia proteins) once they have been validated. Finally, as a contingency plan we identified two B. microti sporozoites-specific transmembrane proteins that were made in recombinant form (E. coli and/or Drosophila). Although the ANTIDotE project has come to an end, these vaccinations studies will be performed early 2019. As described in WP1, we were able to identify to B. microti sporozoites-specific transmembrane proteins. In addition, we have performed an additional analyses to identify more sporozoite-specific proteins.This additional approach could lead to the identification of additional promising Babesia proteins that could serve as candidates for a vaccine directly blocking B. microti early in the transmission from tick salivary gland to the host.
TBEV transmission model
The final vaccination experiment initially involved six immunisation groups (eight mice per group). However due to technical problems in two of the six groups, we were able to test two experimental groups (consisting of one tick salivary gland protein each) and two control groups, a negative control (PBS) and a positive control (TBEV-vaccine Pfizer). One tick protein was a tick salivary gland protein upregulated upon both B. afzelii and TBEV early during tick feeding by MACE analysis (and the corresponding gene was designated gene Bb2). The other was a feeding-induced tick salivary gland protein that was highly upregulated at 24 hours of tick feeding in uninfected nymphal I. ricinus ticks by MACE analysis (encoded by a tick salivary gene named gene 12). Interestingly, we showed in WP2 that when the encoding gene was silenced in TBEV-infected ticks that these were less able to feed and transmit TBEV to naive mice. For the vaccination experiments, mice were injected with 25 µg of recombinant protein in the final volume of 100 µl (50 µl of antigen with 50 µl of adjuvant and received two booster vaccinations. Blood samples were taken before the start of the experiment as well as about two weeks after every immunisation. The serum samples taken in the course of immunisation were analysed for the presence of specific antibodies by ELISA and showed that all vaccinated mice (expect for the control groups) developed high antibody titers against the tick protein they were vaccinated with. Two weeks after the third immunisation mice were challenged with TBEV-infected ticks. Three experimentally infected nymphal ticks were allowed to feed on the immunized mice for three days. To analyse the effect of the immunisation on the TBEV transmission during co-feeding, an additional 15 non-infected nymphs were allowed to feed together with the three infected nymphs. After three days, the nymphs were collected, weighed and stored for further analyses. The post-engorgement weight of ticks fed on the tick antigen vaccinated animals was not lower compared to the controls, although the feeding efficiency in one of the tick antigen vaccinated groups appeared to be lower. The mice were subsequently observed for the signs of tick-borne encephalitis or death for 21 days. Animals with severe symptoms were sacrificed to reduce animals’ suffering. Both control groups showed expected results: none of the PBS-immunized mice survived the challenge with three TBEV infected nymphs. The median survival was 10 days, which is compatible with previous experiments involving TBEV-infected nymphs in our hands. On the other hand, the commercially available human TBEV vaccine, FSME-IMMUN (Pfizer) protected all immunized mice against the challenge. In light of these highly significant results of the control, the data obtained for the two tick antigens were highly promising and encouraging: in both groups, the immunisation did not only lead to prolonged survival, but also to partial protection (approx. 40-50% protection) from lethal TBEV-infection. Serum and organs of surviving mice and TBEV titers and infection percentages of co-feeding ticks are currently being analysed. In summary, the immunization experiment with these two tick antigens followed by challenge with TBEV-infected nymphs showed very promising and encouraging results: both tick proteins were immunogenic and immunized animals were partially protected against TBEV when exposed to three TBEV-infected nymphs together with 15 additional uninfected ticks.
Overall conclusions WP4
In WP4 we have come a long way providing proof of principle that anti-tick vaccines can protect against multiple tick-borne pathogens. Indeed, we have obtained strong and highly encouraging experimental proof for B. afzelii and TBEV, the two most important tick-borne diseases in Europe. For Babesia we encountered some technical challenges, but we were able to establish the transmission model, identify other (Babesia) vaccine candidates and to generate a short-list with interesting TSGPs (that still requires biologically validation), which could serve as candidates for an anti-tick vaccine blocking B. microti transmission from the tick to the host. Although the ANTIDotE projected has ended, we will continue our research and investigate whether combining multiple tick and pathogen antigens could lead to the development of a single vaccine to prevent bacterial, protozoal and viral tick-borne diseases. The antigens identified, described and characterized, as well as the infrastructure, assays, materials and methods developed as part of the ANTIDotE project are an excellent starting point.
WP5 Valorization plans
In WP5 we delivered plans for exploitation and implementation of our findings and concepts to contribute to downscaling of the burden of TBDs on Central and Eastern European and other endemic European societies. Central to this approach has been the exchange of knowledge with our advisory board and relevant stakeholders as well as dissemination of our results and the rationale of anti-tick vaccines to our peers and the public.
Objective/task 5.1: Exchange of knowledge on Lyme borreliosis, tick-borne encephalitis, human babesiosis and innovative strategies to prevent tick-borne diseases.
All ANTIDotE partners - except for GenXPro - were highly actively involved in research into tick and tick-borne diseases. Meetings and conferences, that were both organized and attended with oral and poster presentations, as well as the number of pubmed-indexed publications on these topics by the directly involved researchers from the participating institutes in the ANTIDotE project are the main ways by which we exchanged our knowledge with the medical and scientific community and the public. The involved ANTIDotE researchers have published 170 unique pubmed-indexed publications on Lyme borreliosis, tick-borne encephalitis, human babesiosis and innovative strategies to prevent tick-borne diseases during the ANTIDotE project. In addition, we have organized three workshops on ticks and strategies to prevent ticks and tick-borne diseases, with a special emphasis on anti-tick vaccines. One workshop was in Potsdam, Germany (2016) and another in Cesky Krumlov, Czech Republic (2015), both in adjunction to our annual meetings, while the third one was in Wageningen, the Netherlands (2016). The workshops engaged all relevant stakeholders (patients, policy makers, junior as well as more senior scientists, public and one health experts and industry). In addition, ANTIDotE has come in contact with multiple pharmaceutical companies and discussed how to identify and develop an anti-tick vaccine or other ways to prevent ticks and tick-borne diseases for the human and veterinary market.
Objective/task 5.2: To disseminate ANTIDotE results
Dissemination tools included, but were not limited to I) a public ANTIDotE website (www.antidote-fp7.org) through which we informed the public by press releases on major breakthroughs or other newsworthy facts. The website contained relevant information on the consortium, the project and ANTIDotE publications; II) cross-linking with other related European initiatives. For example, Dr. Sprong WP5-leader (RIVM) has become a member of the WHO taskforce for the development of the WHO R&D Blueprint for a roadmap on Crimean-Congo Hemorrhagic Fever Virus, and has presented a One Health approach for the prevention of CCHF for example by anti-tick vaccines during a consultation meeting with experts; III) Data sharing. We disseminated our results in 33 peer-reviewed scientific publications (including two PhD theses), a multitude of national and international meetings and near publication of 4 additional academic PhD theses and three additional manuscripts. Part of the dissemination included important contributions to a free book on ticks and tick-borne diseases edited by one of the ANTIDotE partners (RIVM). In addition, GenXPro (SME) showcased the ANTIDotE project and results to clients. Furthermore, the ANTIDotE partners disseminated the ANTIDotE design, rational and data in various non-medical or non-scientific journals, as well as through (social) media, for example through interviews in newspapers, popular press, radio and television. One such dissemination highlight was our contribution to the news feature in Nature on the emergence of “Tick troubles” and the potential of anti-tick vaccines (Moyer et al., 2015); IV) open ANTIDotE end-conference. ANTIDotE organized an open-end conference incorporated in a plenary session of the 15th International Conference on Lyme borreliosis and other tick-borne diseases in Atlanta, GA, USA. Thus, our project and results were efficiently disseminated to our peers and the scientific community. At the meeting representatives from patient advocacy groups, industry and local and national governmental agencies were present. A meeting report has been submitted to the European Commission (deliverable report DL5.4). All of our dissemination efforts are listed in Table A1 of this report.
Objective/task 5.3: A road map for exploitation of anti-tick vaccines
An initial roadmap was made (deliverable report DL5.2) for the future exploitation of anti-tick vaccines identified within the ANTIDotE project. Furthermore, a technical report (deliverable report DL5.3) on the implementation of potential anti-tick vaccines in (inter)national strategies for the prevention and control of tick-borne diseases in Europe was published (Sprong et al., 2018). Our overall conclusions from these reports were that I. ricinus-borne diseases are diseases that emerge from interactions of humans and domestic animals with infected ticks in nature. Control strategies involving concerted actions from human and animal health sectors as well as from nature managers have not been formulated, let alone implemented. Control of TBDs asks for a “health in all policies” approach, both at the (inter)national level, but also at local levels. There, nature and health workers together can carry out tailor-made control options for the control of TBDs for humans and animals, with minimal effects on the environment. Anti-tick vaccines should most definitely be considered as one of the options in the future.
In addition, two novel milestones beyond the current ANTIDotE project have been set. Firstly, the ANTIDotE consortium has successfully developed and used innovative methodologies for the identification of novel targets for anti-tick vaccines. Due to the highly complex multi-angular interactions between microorganisms, tick vectors and their hosts, we realize that the search for the ‘magic bullet’ is not an easy task. Indeed, in the final roadmap (deliverable report DL5.5) we propose that the best way to bite back against tick-borne diseases is to obtain more knowledge on the many aspects of the interaction between ticks, pathogens and mammals, for example by making use of tools that we have developed and used within the ANTIDotE project. Therefore, the next step would be to gain more insights in the role of these targets in the interrelationship between ticks and their pathogens and hosts, including mechanisms of acquisition, persistence and transmission as this may lead to the identification of critical elements of the pathogens’ life cycle that could be targeted by vaccines. Indeed, we have performed mechanistic studies as part of ANTIDotE for some of the identified targets. However, considering the long list of novel vaccine targets we have identified as part of the ANTIDotE project, this requires further experimentation. To this end, members of the ANTIDotE consortium are jointly applying for novel grants, both at the national and international level.
Secondly, anti-tick vaccines can be powerful tools to safeguard human, animal and environmental health, and can be directed against other tick species of medical and veterinary relevance as well. In the final roadmap we put forward that sharing knowledge, tools and innovative screening methodologies among researchers working on anti-tick vaccines in different fields, on different tick species and on different aspects of anti-tick vaccines, is mandatory to come to new and successful preventive strategies. This can be facilitated by applying for a COST-action for networking activities, facilitating transfer of knowledge, data and skills and the formation of novel consortia that can successfully apply for future grants. This is exactly what members of the ANTIDotE consortium are currently doing. This will help to engage all relevant stakeholders including geneticists, epidemiologists, immunologists, vector biologists, bioinformaticians, physicians and veterinarians, public health specialists, and the pharmaceutical industry, amongst others. It will also help to engage the public, which is important in order to reach the populations at risk, especially with prevailing societal prejudices associated with vaccination. Increasing efforts to study tick-host-pathogen interactions will provide a higher chance of discovering new and more potent targets for anti-tick vaccines and might lead to the dawn of a new era where an anti-tick vaccine protecting against the most common tick-borne pathogens will come to fruition.
Objective/task 5.4: Ways to implement anti-tick vaccines in health systems
The workshops, the open-end conference (deliverable DL5.4) the technical report (deliverable DL5.3) and the roadmaps (deliverables DL5.2 and DL5.5) proved to be efficient means for us to discuss our idea’s and ANTIDotE data with policy makers and other relevant stakeholders. Another important outcome of this task was that we performed an early cost-effective analysis on a potential anti-tick vaccine. Besides presentations on Vaccine symposia, the manuscript describing the results of this study was published (Mihajlović et al., 2019). Our study assessed cost-effectiveness of a potential vaccine that would protect against both Lyme Borreliosis (LB) and tick-borne encephalitis (TBE) in the highly endemic setting of Slovenia. The analyses indicated that an anti-tick vaccine that would prevent both Lyme borreliosis and TBE could be a cost- effective option in Slovenia. Our analysis represented a rare - but increasingly important - example of cost-effectiveness assessment prior market authorization and might facilitate future support from governments and companies in the early stages of the development of vaccines. In addition, our model embodied a novel tool to analyze pharmacoeconomic criteria that can help development of a cost-effective health technology.
Ethics statement
ANTIDotE participants P1, P2, P3, P4 and P7 performed experiments with human biological samples or experiments in animal models (not including research on human embryo’s or fetuses, research on human being, processing of genetic information or personal data, generation of transgenic animal strains, or animal cloning). The involved personnel of each of the ANTIDotE participants had proven experience with working with animals (certificates) and all experiments complied with current regulations, legislation and codes of conduct in the countries where the experimentations have been carried out. All ANTIDotE partners obtained the written approval of the local human or animal ethics committees prior to start of the experimentation and had valid and approved protocols in place. As a result, research activities within this project complied with the international conventions and codes of conduct, in particular the Council Directive of 22 September 2010 on the protection of animals used for scientific purposes (2010/63/EU), which became law on January 1st 2013, and the Ethical rules of the Seventh Framework Program (Council Decision N° 1982/2006/EC, Article 6). The work also followed the Charter of Fundamental Rights of the EU (2000/C 364/01), the Council Decision regulating safety and ethics (99/167/EEC; Council Decision of 25/1/99), the Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of individuals with regard to the processing of personal data and on the free movement of such data, the 391L0628 Council Directive 91/628/EEC of 19 November 1991 on the protection of animals during transport, and the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (replacing former 386L069 Council Directive 86/609/EEC of 24 November 1986). The outcome, number of groups/animal and types of animals used are detailed within the different work packages elsewhere in this report.
Potential Impact:
The incidences of Lyme borreliosis (LB) and TBEV are on the rise in several European countries and diseases caused by other pathogens, such as Babesia spp. and Borrelia miyamotoi are emerging. Environmental, socio-economic and demographic factors synergistically increase the risk of acquiring tick-borne diseases (TBDs). Indeed, the European Center for Disease Prevention and Control (ECDC) predicted that the incidence of TBDs will rise in the future. Furthermore, the European Parliament has expressed its concerns about the spread of LB in the European population (November 2018). Therefore, and also because the societal fear for ticks and TBDs seems to be ever growing, the old adage ‘prevention is better than cure’ certainly holds true for tick-borne diseases.
Currently there are no human or animal vaccines against I. ricinus available. However, anti-tick vaccines are available against other tick species for the veterinary market and there is a body of experimental evidence that anti-tick vaccines could also work for Ixodes ticks. Our project aimed to show proof of concept that anti-I. ricinus vaccines can protect against multiple TBDs, which is innovative by itself. Moreover, we provided insights into the molecular mechanisms involved in transmission of tick-borne pathogens from the tick to the host, and in the process of tick feeding. Furthermore, we went one step further and also discussed with public health representatives and relevant industrial stakeholders how anti-I. ricinus vaccines could be exploited and implemented. This could lead to a paradigm shift in how public health institutes tackle TBDs in Europe and resulted in a road map for future use of anti-tick vaccines.
The ANTIDotE network (consortium and advisors) consisted of a large community of biomedical scientists, public health officials and private sector stakeholders. The ANTIDotE consortium put much effort in the dissemination of our results and we aimed to translate ANTIDotE’s innovative results as much as possible into tangible impact on health services. Moreover, ANTIDotE actively involved health organizations, health policy experts and end-users to further increase its impact. Importantly, ANTIDotE’s outcomes spanned from discovery science increasing fundamental biomedical knowledge, to translation of this new understandings into proof of concept studies, to valorization plans contributing to sustainable advances in the prevention of multiple tick-borne diseases.
ANTIDotE delivered new understandings on the biological mechanisms underlying tick feeding, anti-tick immune responses and transmission of the causative agents of TBE, LB and human babesiosis. These are all I. ricinus tick-transmitted infectious diseases with potential serious acute and chronic sequelae and disproportionately affect Europe. ANTIDotE embodied a multidisciplinary approach combining leading scientific and industrial experts on tick biology, genetics and bioinformatics, immunology, vaccinology, the three tick-borne pathogens under study and public health experts on tick-borne diseases, from Central and Eastern Europe as well as other European countries where tick-borne diseases are endemic. The ANTIDotE project was geared towards translating new knowledge into identification of novel anti-tick vaccines and providing proof of concept that such vaccines can cost-effectively prevent transmission of multiple human tick-borne pathogens. Furthermore, ANTIDotE also integrated basic science with industrial innovation and other (public) health disciplines to generate plans for product development and to explore how anti-tick vaccines could be implemented into health systems as part of novel preventive strategies to prevent tick-borne diseases in endemic European countries. Thereby ANTIDotE strongly adhered to the expected impacts as defined in the FP7 work program and the specific call text.
We maximized our impact by:
1. Establishment of new tools and technologies to study ticks and tick-borne diseases
2. Novel fundamental knowledge of ticks and tick-borne diseases and identification of candidates for anti-tick vaccines (WP1)
3. In depth insight in the function of tick salivary gland proteins and mammalian anti-tick immune responses (WP2 and WP3)
4. Proof of concept that anti-I. ricinus vaccines can prevent tick feeding/transmission of multiple tick-borne pathogens (WP4)
5. Exploitation and implementation plans for anti-I. ricinus vaccines in existing health services (WP5)
6. Dissemination of our results by a sustainable network, with currently 33 peer-reviewed manuscripts/publications published, including important contributions to a free book on ticks and tick-borne diseases, a published technical report on the implementation of potential anti-tick vaccines in (inter)national strategies for the prevention and control of tick-borne diseases in Europe and two PhD theses. In addition, we disseminated our results by the media (magazines, news papers, non-scientific journals, internet, radio and television), an open website, three workshops and an open end-conference. The ANTIDotE project also contributed to the anticipated PhD thesis of four additional PhD students that will defend their dissertations in the near future. Finally, at the time of writing of the final report 3 manuscripts have been submitted/accepted for publications. Of note, the vast majority of the ANTIDotE output was published open access.
Our concerted multidisciplinary integrated European approach lead to multiple breakthroughs in the field of ticks and tick-borne diseases and beyond in many ways and impacted a) the research community worldwide, b) health systems, societies, economies and individual patients in European countries where tick-borne diseases are endemic, and c) industry. The ANTIDotE outcomes contribute to a knowledge-based society and paved the way for the development of innovative ways to prevent tick-borne diseases by industry and health systems in Europe.
ANTIDotE’s impact on the research community
Firstly, ANTIDotE has established new in vitro and in vivo models to study tick-borne diseases. Secondly, ANTIDotE applied state of the art and innovative technologies and approaches to research on ticks and TBDs, including YSD technology, MACE and RNA sequencing and RNA interference. Not did this raise the standard of research on ticks and tick-borne diseases in Europe, it enabled us to provide the European scientific community with new and unique knowledge on the process of tick feeding, anti-I. ricinus immune responses and the transmission of B. burgdorferi sensu lato, B. microti and TBEV. Importantly, as we have put forward in the final road map, this new knowledge is essential for the identification and development of novel strategies, which include anti-I. ricinus vaccines, to prevent the fore-mentioned, but also other I. ricinus-transmitted, infectious diseases. Current preventive strategies for I. ricinus-transmitted infectious diseases fall short and clearly fail to stop the rise of TBDs in Europe, underscoring the need for action. The new ANTIDotE-derived knowledge on the processes involved in I. ricinus tick feeding and TBP transmission might also impact understanding of, and development of new preventive strategies against, other TBDs and other tick species in Europe, for example Crimean-Congo Hemorrhagic Fever transmitted by Hyalomma ticks. Indeed, one of the ANTIDotE partners became a member of the WHO taskforce for the development for the WHO R&D Blueprint for roadmap on Crimean-Congo Hemorrhagic Fever Virus, which is pursuing a One Health approach for the prevention of CCHF, including investigating the possibility of anti-tick vaccines. In addition, our findings will help the scientific community researching tick-borne diseases transmitted by other tick species around the world. For instance, the impact of ticks and tick-borne diseases on live stock in the (sub)tropics is considerable and anti-tick vaccines comprise a eco-friendly alternative to acaricides, which are becoming more and more problematic in light of acaricide resistance, environmental pollution and food security. Finally, ANTIDotE also revealed new insights into specific mammalian host defense mechanisms by investigating the function of the identified tick salivary gland proteins.
ANTIDotE’s impact on health systems and societies, individual patients and economies
By the international workshops, the open-end conference, a technical report, a cost-effective analyses on a potential anti-tick vaccine and two reviews on the prevention of tick-borne diseases we were able to influence policy makers and health systems. Through these we voiced our ideas, our findings, the importance of the prevention of tick-borne diseases and the potential of anti-tick vaccines. Although we provided unprecedented new knowledge and proof of concept that anti-tick vaccines could work against Lyme borreliosis and TBEV, there is no commercial available vaccine yet. This will require more research. This is badly needed, as the incidence of ticks and tick-borne diseases is on the rise with considerable economical and societal impact and sometimes devastating consequences for affected individuals. Therefore, protecting individuals at risk from contracting tick-borne diseases with a safe and effective anti-tick vaccine could save lives and improve quality of life by reducing the number of patients with TBDs. Hikers, campers, farmers, forestry workers, soldiers, gamekeepers, immunocompromised individuals and actually any person regularly entering I. ricinus tick habitats (forested areas) for work or recreational purposes in endemic European countries are at risk. Taken together, with the improved knowledge on tick feeding and the transmission of tick-borne pathogens, the identification of novel anti-tick vaccine candidates that could prevent multiple human tick-borne diseases, as well as plans on how to implement these in health systems and societies, ANTIDotE has had an important impact on health systems and societies, individual patients and economies and could contribute to diminishing the number of people at risk for, or suffering from, tick-borne diseases.
ANTIDotE’s impact on, and potential of, industrial innovation
We have involved industrial collaborators during the ANTIDotE project and there is clearly an interest in anti-tick vaccines, both from the human and veterinary pharmaceutical industry. The newly identified antigens and the knowledge obtained as part of the ANTIDotE project could directly lead to the development of novel anti-tick vaccines. Such a vaccine would be a paradigm-shifting medical and industrial innovation in itself. In this regard, ANTIDotE has not filed any patents at current (apart from an application for a patent on a biotechnical device by one of the ANTIDotE partners), but is investigating some interesting and very promising leads, which might to future patent applications.
List of Websites:
The ANTIDotE partners:
Partner # 1: Academisch Medisch Centrum, AMC, The Netherlands (coordinator)
Partner # 2: Biologicke centrum AV CR, v. v. i., BC ASCR , Czech Republic
Partner # 3: FREIE UNIVERSITAET BERLIN, FUB, Germany
Partner # 4: ASOCIACION CENTRO DE INVESTIGACION COOPERATIVA EN BIOCIENCIAS, CIC bioGUNE, Spain
Partner # 5: GENXPRO GMBH, GenXPro, Germany
Partner # 6 RIJKSINSTITUUT VOOR VOLKSGEZONDHEID EN MILIEU, RIVM, The Netherlands
Partner # 7: VIROLOGICKY USTAV SLOVENSKEJ AKADEMIE VIED (changed to BIOMEDICINSKE CENTRUM SLOVENSKEJ AKADEMIE VIED), IV SAS, Slovakia
Public website and contact details:
www.ANTIDotE-fp7.org
At the website our contact information can be found. Alternatively, contact info@antidote-fp7.org or lyme@amc.uva.nl
The aim of the ANTIDotE project was to find tick salivary gland proteins (TSGPs) that block pathogen transmission and/or interfere with tick feeding. Two main antigen discovery strategies were employed. Firstly, a transcriptomic approach (MACE and RNAseq) was used to identify TSGPs upregulated upon infection with Borrelia, TBEV or Babesia, resulting in the identification of numerous TSGPs, of which a selection was technically and biologically validated. There are currently five manuscripts on the transcriptomes of I. ricinus upon feeding and infection in the pipeline. Secondly, an immunoscreening using a yeast surface display was used to identify and validate TSGPs that are recognized by antibodies of frequently tick-exposed humans, and/or tick immune animals. A selection of the thus identified TSGPs was investigated in downstream WPs. As part of WP2 we have established and improved tick-transmission models for Borrelia afzelii (submitted for publication), TBEV and Babesia microti (WP2). The infrastructure for RNAi in ticks was established and used to investigate the role of multiple TSGPs. Silencing of selected TSGPs did not affect Borrelia transmission, but silencing of one target significantly impaired both tick feeding and TBEV transmission. Our data show that one of the identified TSGPs promoted Borrelia survival in vivo by impairing phagocytosis. Finally, we have also generated fluorescent B. afzelii strain CB43 to study TSGP-Borrelia interactions. Production of TSGPs in E. coli and Drosophila was performed and a selection used for vaccination studies. Vaccination of mice with TSGPs did not result in a significant anti-tick effect and we shifted our focus to the tick-rabbit model to assess tick immunity (WP3). Also, six bovine vaccination experiments were performed, showing that (specific fractions of) tick tissue extracts resulted in a robust anti-tick immunity. The obtained cow sera were used to set-up an in vitro artificial tick feeding system, which demonstrated the need for combined cellular and humoral immune responses to induce tick immunity. As an additional effort, we identified and/or tested tick-borne pathogen antigens (from B. microti and B. afzelii) as candidates for transmission-blocking vaccines. In WP4 we identified one TSGP that, when used to actively immunize mice, protected against Lyme borreliosis and two TSGPs that, when tested as antigens for an anti-tick vaccine, partially protected mice from lethal TBEV-infection.
The ANTIDotE partners were actively engaged with stakeholders from medical and veterinary sciences, policy advisors, governmental institutions, patient interest groups and the pharmaceutical industry. We disseminated knowledge on (inter)national conferences, in peer-reviewed scientific journals and through the ANTIDotE website and (social) media. Indeed, 33 related ANTIDotE and ANTIDotE-related articles have currently been published in peer-reviewed scientific books or journals (of which the majority open access and including 2 PhD theses). These also included a technical report on preventive strategies to prevent TBDs, a review on anti-tick vaccines and a book chapter on the prevention of Lyme borreliosis and a publication on the cost-effectiveness analyses of an anti-tick vaccine in a Central Eastern European country where Lyme borreliosis and tick-borne encephalitis are endemic. Finally, the ANTIDotE project contributed to another four anticipated PhD theses and three manuscript have been submitted/accepted for publication. All in all, we significantly contributed to the understanding of, and steps towards the downscaling of, TBDs in Europe. All our initially planned discovery methods yielded vaccine candidates and we pursued multiple additional antigen discovery approaches and we have characterized and produced selected candidates, of which some indeed protected against LB or TBEV. We will continue our research and investigate whether combining multiple tick and pathogen antigens could lead to the development of a single vaccine to prevent bacterial, protozoal and viral tick-borne diseases. The antigens identified, described and characterized, as well as the assays, materials and methods developed as part of the ANTIDotE project are an excellent starting point.
Project Context and Objectives:
Ixodes ricinus transmit bacterial, protozoal and viral pathogens that cause Lyme borreliosis, babesiosis and tick-borne encephalitis, respectively and exceedingly affect Central and Eastern Europe. During feeding, ticks introduce salivary proteins into the skin that interfere with host defense mechanisms. However, in animals, repeated tick infestations as well as vaccination against selected tick proteins can lead to decreased pathogen transmission by inhibiting tick feeding - known as ‘tick immunity’ - or by neutralizing tick proteins that facilitate the transmission of tick-borne pathogens. Also humans with hypersensitivity to tick-bites have a lower risk of contracting tick-borne diseases. Therefore, anti-tick vaccines encompass an innovative strategy to prevent tick-borne diseases in humans, or animals and wildlife to indirectly reduce the risk of contracting tick-borne diseases for humans. Currently, although there is a veterinary vaccine available for another tick species, there is no vaccine available against I. ricinus, the main European vector for veterinary and human tick-borne diseases.
The ANTIDotE project aimed to identify and characterize tick proteins involved in ‘tick immunity’ and tick-borne pathogen transmission and use the acquired knowledge to investigate and develop an anti-tick vaccine to prevent multiple human tick-borne diseases.
To this end state of the art proteomic and transcriptomic approaches were used to identify and characterize novel tick salivary gland proteins, of which a selection was assessed as anti-tick vaccines to protect against LB, TBEV and babesiosis in animal models. In addition, through an integrated and multidisciplinary approach involving Central and Eastern European public health institutes, health organizations and industrial companies we examined how to develop anti-tick vaccines and implement these in public health systems. Thus, ANTIDotE delivered 1) essential knowledge on the biological mechanisms involved in the pathogenesis of tick-borne diseases, 2) experimental proof of concept of an anti-tick vaccine - based on single or multiple antigens - protecting against multiple human tick-borne pathogens and 3) a road map for the development exploitation and implementation of anti-tick vaccines. Collectively, the output of the ANTIDotE project could significantly contribute to downscale the severe medical and economic burden that tick-borne diseases have on societies in Europe and other parts of the world where Ixodes ticks are endemic.
As such, the ANTIDotE project had five key objectives, corresponding to five different work packages (WP). Since these are unaltered compared to the original proposal and all periodic reports thus far they are written in the present and simple future tense.
First key objective (WP1): To identify I. ricinus tick salivary gland proteins (TSGPs) that play a role in the transmission of tick-borne pathogens (TBPs) from the tick to the host, but also TSGPs that are recognized by anti-tick antibodies. Therefore, gene expression profiles will be analyzed of salivary glands (SGs) of uninfected and TBP-infected feeding I. ricinus ticks. We will also identify TSGPs that are recognized by mammalian anti-tick immune responses by an I. ricinus TSGP yeast surface display (YSD).
Objective 1.1 Identification of TSGPs involved in transmission of TBPs
Objective 1.2 Identification of TSGPs recognized by anti-tick immune responses
Second key objective (WP2): To functionally characterize the identified TSGPs. The TSGPs are characterized with the help of RNAi in ticks. Alongside, the functional activity of specific TSGPs (in relation to host defense mechanisms) will be investigated. As part of this objective we will provide in depth insights in the mechanisms involved in transmission of TBPs and will reveal candidates for anti-tick vaccine to prevent TBP transmission. For this objective in vivo animal models for the tick-borne diseases (TBDs) under study will be established or improved.
Objective 2.1 Describe the role of TSGPs in the transmission of TBPs in vivo using RNAi in ticks
Objective 2.2 Describe the function of TSGPs in in vitro and ex vivo functional assay
Third key objective (WP3): Aims to confirm which TSGPs induce ‘tick immunity’ in in vivo animal models and the artificial tick feeding system. Furthermore, the humoral and cellular immune response that these TSGPs elicit will be characterized. Thus, this objective will reveal candidates for anti-tick vaccine that are capable of interfering with tick feeding.
Objective 3.1 To confirm that TSGPs identified induce ‘tick immunity’ in mice
Objective 3.2 To characterize humoral and cellular anti-tick immune responses
Objective 3.3 To confirm that TSGPs identified induce ‘tick immunity’ in cows and to develop a robust in vitro artificial tick feeding system for research on anti-tick vaccines
Fourth key objective (WP4): As part of this objective we aim to deliver the proof of concept that a single anti-tick vaccine can prevent transmission of bacterial, protozoal as well as viral TBPs. Vaccine studies with promising candidates, or specific combinations of promising candidates, will be performed to identify an anti-tick vaccine that could interfere with all three TBPs under study. Both TSGPs that are crucial for the transmission of Borrelia, Babesia and tick-borne encephalitis virus (TBEV) from the tick to the mammalian host, and TSGPs that are of paramount importance for tick feeding, will be tested.
Objective 4.1 To identify an anti-tick vaccine that protects against multiple TBDs by specifically interfering with TSGPs crucial for transmission of TBDs
Objective 4.2 To identify an anti-tick vaccine that protects against multiple TBDs by interfering with tick feeding
Fifth key objective (WP5): The objective of this part of the project is to deliver plans for exploitation and implementation of our findings and concepts to contribute to downscaling of the burden of TBDs on Central and Eastern Europe and other endemic European societies, as well as to disseminate ANTIDotE results. We will also attend and arrange workshops and meetings.
Objective 5.1 To exchange knowledge on LB, TBE, human babesiosis and innovative strategies to prevent TBDs
Objective 5.2 To disseminate ANTIDotE results
Objective 5.3 To establish a road map for exploitation of novel anti-tick vaccines
Objective 5.4 To explore ways to implement anti-tick vaccines in health systems
Project Results:
WP1 Identification of tick salivary gland proteins (TSGPs)
In this work package we identified I. ricinus tick salivary gland proteins (TSGPs) that play a role in the transmission of tick-borne pathogens (TBPs) from the tick to the host. We also identified TSGPs that are recognized by anti-tick antibodies. These TSGPs comprise potential candidates for an anti-tick vaccine that could interfere with tick feeding and/or pathogen transmission. To this end we used different state of the art methods.
Objective/task 1.2: Identification of TSGPs recognized by anti-tick immune responses.
The main method used in task 1.1 was transcriptomics to identify TSGPs involved in transmission of tick-borne pathogens and/or tick feeding. The TBPs under study were Borrelia afzelii, the causative agent of Lyme borreliosis, TBEV, the virus that causes tick-borne encephalitis and Babesia microti, the protozoal causative agent of babesiosis. As part of subtask 1.1.1 we fed larval I. ricinus ticks on control animals or animals syringe-inoculated with B. afzelii or B. microti. Fed larval ticks were allowed to molt, giving rise to B. afzelii-infected, Babesia-infected or uninfected controls nymphal I. ricinus ticks. For TBEV we injected nymphal I. ricinus ticks with TBEV using a nano-injector. Ticks originating from at least three individual adult female I. ricinus ticks were pooled for the feeding experiments, in which ticks were allowed to feed for 0 hours, 24 hours or to repletion for B. afzelii or B. microti, and for 0, 6, 12, 24 or 48 hours for TBEV. After the indicated time salivary glands were dissected and RNA extracted for downstream sequencing and transcriptomic analysis. As part of subtask 1.1.2 and using a dual approach, combining RNA sequencing for reliable qualitative sequencing data and massive analysis of cDNA ends (MACE) for sensitive quantitative data, we obtained important insights into the transcriptome of I. ricinus in response to infection with the pathogens under study. This approach did not only yield multiple potential candidates for an anti-tick vaccine, but also unprecedented insights into the tick salivary gland genes differentially expressed during feeding and infection with B. afzelii, TBEV or B. microti. Indeed, two advanced manuscript drafts and three manuscript outlines have been drafted to describe these findings, which have been presented at multiple international conferences and are anticipated to be submitted in the near future. Importantly, TSGPs abundantly and significantly upregulated upon infection with B. afzelii, TBEV, B. microti or upon tick feeding were identified for further study as part of the ANTIDotE project. In total, a subset of approx. 20 TSGPs - both technically and biologically validated by quantitative RT-PCR - was subsequently selected for further study in downstream work packages (described elsewhere in this report). Due to technical reasons we were unable to biologically validate tick salivary gland gene expression induced by B. microti. However, the mRNA of B. microti, being a eukaryote, also has a poly-A tail, which allowed capturing by our methods for RNAseq and MACE. We were thus able to identify to B. microti sporozoites-specific transmembrane proteins that could serve as antigens for a vaccine blocking Babesia transmission from the tick to the host.
Objective/task 1.2: Identification of TSGPs recognized by anti-tick immune responses.
The main method used in task 1.2 was the Yeast Surface Display (YSD) to identify TSGPs involved in tick feeding and tick immunity. This specific library was made as part of subtask 1.2.1. Briefly, an I. ricinus salivary gland cDNA expression library was generated using the input of isolated RNA from 1350 nymphal I. ricinus ticks fed for 24, 48 and 72 hours, cloned into PYD1 expression vectors, which were transformed in EBY100 yeast cells. Induced yeast cells – expressing tick salivary proteins - were subsequently selected using sera from humans frequently exposed to tick bites (forestry workers) or controls or tick-exposed or tick-immune rabbit sera (or controls). Single yeast cells expressing a TSGP recognized by human or animal tick-exposed or tick-immune sera were selected, the recombinant PYD1 plasmids were isolated and the inserts (TSGPs) were sequenced. Differential expression between the animal tick-exposed or tick-immune sera on one hand and the control sera on the other hand was confirmed using flow cytometry on single yeast cells. Thus, we were able to identify 24 unique TSGPs that might be involved in tick-immunity, of which 13 were selected for further study (as described elsewhere in this report).
Overall, in WP1 we successfully identified TSGPs that are involved in tick feeding, transmission of different TBPs from the tick salivary gland to the host or in tick-immunity. A total of approx. 30 TSGPs, not including TSGPs identified or selected through different and additional approaches (as will be described later), were selected for further study in WP2, WP3 and WP4 as part of our quest for an anti-tick vaccine to prevent multiple human tick-borne diseases.
WP2 Functional characterization of tick salivary gland proteins (TSGPs)
Objective/task 2.1: The role of TSGPs in the transmission of TBPs in vivo using RNAi in ticks.
As part of this objective, transmission models for Borrelia afzelii, B. microti and TBEV were established and improved (subtask 2.1.1). In addition, the in vitro artificial tick feeding system was further modified to feed all life stages of I. ricinus ticks in vitro. Tick feeding units were made of borosilicate glass. Silicone membranes were prepared and attached to the tick feeding units and reinforced with mosquito netting for feeding I. ricinus adults, but not for I. ricinus larvae or nymphs. Additional attachment stimuli included finely clipped bovine hair and bovine hair extracts. Furthermore, we studied a selection of the vaccine candidates identified in WP1. In task 2.1.2 we have tested the role of 13 of these tick genes on B. afzelii transmission through knock down by RNAi (excluding two known targets from I. scapularis that were unsuccesfully tested as positive controls). Conformation of knockdown of these genes by q-RT-PCR showed that most of these genes were successfully knocked down, but unfortunately no evident phenotype could be observed for Borrelia transmission or tick feeding parameters. In addition, for TBEV, RNAi experiments have been performed for a total of 10 tick salivary gland genes that were feeding-induced and/or upregulated upon both Borrelia and TBEV infection, to test their importance in tick feeding and TBEV transmission. Again knockdown could be confirmed for most targets. Interestingly, knockdown of two tick salivary gland genes (gene 12 and gene 42) significantly impaired tick feeding and knockdown of gene 12 also protected mice from lethal TBEV infection (described in more detail in deliverable report DL2.2).
Objective/task 2.2: The function of TSGPs in in vitro and ex vivo functional assays
A part of subtask 2.2.1 as many as 28 tick salivary gland genes have been produced recombinantly in E. coli and/or Drosophila expression systems. A large subset of these proteins has been used in WP2, WP3 and WP4 for functional or vaccination studies. The selection of the proteins to be expressed was based on the different technologies used to identify tick proteins as part of mainly WP1, but also based on additional efforts in WP2 and to lesser extent WP3. These proteins were either upregulated during tick feeding, or were selected due to their recognition by immune sera. The identified tick proteins can largely be classified as follows:
A. Proteins corresponding to genes upregulated during tick feeding identified by MACE analysis. Two different analyses were performed, from which a set of genes was selected based on expression levels, upregulation upon feeding (particularly at the 24 h feeding time) and their upregulation in 4 independent experiments performed at two independent and geographically distinct locations. The selected genes were named Gene 1, Gene 2, Gene 3, Gene 4, Gene 8, Gene 11, Gene 12, Gene 17, Gene 42, Gene 44 and Gene 49.
B. Proteins corresponding to genes upregulated by the presence of Borrelia (GeneBb and Rdk) or TBEV (TVC), identified by MACE analysis. The selected genes were named GeneBb 1, GeneBb 2, GeneBb 6, GeneBb19, Rdk 1, Rdk 2, TVC2 and TVC4.
C. Several proteins recognized by human ‘tick exposed’ sera (Forestry workers, FW) or tick immune rabbits (MS, COMB, NS), identified by Yeast Surface Display (YSD). The selected proteins were named FW1, FW2, FW3, MS1, MS2, MS3, COMB1, COMB2, COMB3, NS1 and NS2.
D. Proteins identified by an additional approach consisting of immunoprecipitation and mass spectrometric analysis of cow sera immunized with salivary gland or midgut extracts. The selected proteins were named SG1, SG4, MG2, MG6 and MG9.
For the MACE analyses, the genes selected for expression were technically and biologically validated in independent groups of ticks and for the tick proteins identified by YSD and immunoprecipitation by different means, including FACS analysis and Western blots. A large selection of the genes encoding these proteins has also been tested in RNAi experiments, as described above, or in WP3, as described later. Further in silico analysis revealed that some identified proteins were highly identical. Highly overlapping proteins were omitted resulting in a final list of 28 uniquely identified tick proteins. The eight proteins identified by the comparative proteomic approach (described below) are not depicted in the above-described overview
As part of subtask 2.2.2 we developed a functional assay pipeline and developed and refined existing methods (the ANTIDotE pipeline). For example, we have successfully generated a fluorescently labeled B. afzelii strain CB43. We showed this isolate is both infectious to ticks and mice and can be used for microscopy work to assess interactions of Borrelia with tick salivary gland proteins. In addition, we characterized the function of one of the identified TSGPs, a putative secreted saliva protein of unknown function, referred to as COMB 2, which was highly conserved between different Ixodes tick species. Expression of the gene encoding this protein was upregulated upon Borrelia infection and co-incubation of Borrelia spirochetes with the Drosophila-expressed recombinant protein augmented infectivity in vivo in the mouse model. The recombinant protein could not prevent killing of Borrelia by the complement system, but it reduced phagocytosis of Borrelia by bone marrow derived mouse macrophages, whereas TNF-alpha production was unaffected. Another protein, for which the encoding gene is referred to as Gene 12 (see above), has been expressed in Drosophila for functional studies, because of its role in transmission of TBEV from the ticks to the host.
We also engaged in additional experiments as part of this work package. For instance, we investigated adaptation of the artificial tick feeding system to assess B. microti transmission (FUB). With the idea that this would greatly facilitate research on tick-host-pathogen interactions we tried to set this up in vitro, but were unsuccessful and decided not to pursue this any further. The artificial tick feeding system was however successfully used as part of WP3 (see elsewhere in this report). Notably, we successfully identified eight additional TSGPs by comparative proteomics that could serve as candidates for a B. afzelii-blocking anti-tick vaccine. Comparative proteomics was used in ticks under different feeding and B. afzelii-infection conditions. In line with the transcriptomic approach and experimental design, three different feeding times namely unfed, 24 hour fed and fully fed, as well as B. afzelii infected and uninfected nymphal ticks, were used. Tick protein expression levels between the different conditions were compared, enabling us to identify TSGPs abundantly expressed during the tick feeding process, as well as tick proteins that could be central for transmission of B. afzelii from the tick salivary glands to the host. From the approx. 1000 identified tick salivary gland proteins we have selected 8 TSGPs that were abundantly expressed and upregulated upon 24h feeding and upon B. afzelii infection. These 8 proteins have been generated in E. coli and two of them in Drosophila cells. For a subset of these proteins, we have performed a Western Blot on salivary gland extracts with specific antibodies that we had raised by immunization of mice to validate our data. We considered these proteins validated and they have been further studied in WP4. Finally, we have investigated the role of specific murine host factors in the course of experimental Lyme borreliosis.
In summary, we have generated multiple recombinant tick antigens, established and refined functional assays and methods - designated the ANTIDotE pipeline - that have been, and will be, used to investigate the functionality of tick proteins identified in the context of the ANTIDotE project. In addition, we identified a range of interesting tick proteins and unraveled the function and/or importance of a subset of these proteins in light of tick feeding or pathogen transmission from the tick to the host. Collectively, this knowledge will be used to rationally design an anti-tick vaccine to prevent multiple tick-borne diseases, as also discussed in deliverable report DL5.5 in more detail.
WP3 Characterization of immune response against TSGP’s
Objective/task 3.1: Induction of ‘tick immunity’ in mice by TSGPs
The generation of all recombinant tick proteins is reported as part of WP2 (and to avoid overlap and repetition not described in subtask 3.1.1). As part of subtask 3.1.2 we initially investigated whether we could induce tick immunity in mice. We proved to be unsuccessful, even with established strong candidate antigens. Alternative vaccine delivery strategies, such as DNA tattoo vaccination, were also not successful. We therefore switched to vaccination experiments in rabbits. Using antigens identified by the YSD, we vaccinated rabbits with 3 different cocktails of 2-3 tick salivary gland proteins, and 2 single antigens. Vaccination resulted in high antibody titers against all antigens. However, no significant reduction on tick feeding parameters (feeding time, engorgement weights and egg mass) for I. ricinus nymphs and adult females could be observed. In contrast, we showed that, when rabbits are exposed to adult I. ricinus for multiple times, this lead to drastically impaired tick feeding in subsequent challenges. Regardless, the sera from the cocktail vaccinations with the recombinant proteins were selected for passive transfer experiments to mice experimentally challenged with B. afzelii-infected ticks to assess their effect on Borrelia transmission in WP4. Besides the fact that mice represent an excellent model to study Borrelia transmission and infectivity, blocking this process with is different from trying to mimic tick immunity. Indeed, it is well known that cellular immune effectors play an important role in tick rejection and the subsequent tick immunity. However, from previous studies it appears that tick-directed immune responses preventing Borrelia transmission are largely antibody mediated. Indeed, antibodies against specific TSGPs could for instance neutralize the advantageous functions certain TSGPs have on Borrelia transmission and infectivity.
Objective/task 3.2 Cellular and humoral anti-tick immune responses against TSGPs
As part of subtask 3.2.1 and 3.2.2 we investigated antibody responses to tick salivary gland proteins.
Antibody subclasses are widely divergent among different animal species. Thus, while rabbits only contain one IgG type, humans contain four IgG subclasses (IgG1, IgG2, IgG3, IgG4) as do mice, albeit not coinciding in functionality. In contrast, bovines have only two subclasses of IgG (IgG1 and IgG2). Each antibody subclass is associated with differential functionalities, including the initiation of the complement cascade or the cellular activation through their interaction with Fc receptors. In the case of anti-tick vaccines, a predominant IgG1 response has been associated with better levels of protection using the commercial Bm86-based vaccines against Rhipicephalus (Boophilus) microplus. We performed ELISA using sera from cows vaccinated as part of this work package. In midgut extract vaccinated cows – which showed the strongest anti-tick responses (see below) – we showed a predominant IgG1, but also a robust IgG2 response. Interestingly, also in Fraction (F)1 vaccinated cows (see below), which showed protective effects against I. ricinus ticks too, we showed the same mixed response. Our results suggest that, in this model, although a predominant IgG1 response may be required, the presence of IgG2 antibodies specific for the protective antigens also appeared to be important for acquired resistance to ticks. Therefore, the generation of the proper balance between isotypes with differential functions may be required in order to generate a full, and protective response. This should be considered in future vaccination experiments.
Objective/task 3.3 To confirm that TSGPs identified induce ‘tick immunity’ in cows and to develop a robust ATFS for research on anti-tick vaccines
As part of subtask 3.3.1 we were able to show (and have published) that vaccination of cows with tick tissue homogenates strongly reduce tick feeding success. We thereafter compared the effects of salivary gland and midgut extract and showed that especially vaccination with midgut extract resulted in evident tick rejection. Elaborating on these findings we fractionated midgut extracts to identify the actual antigens involved in the observed tick immunity. Four different fractions (F) were created using size exclusion chromatography and used for vaccination-challenge experiments in cows. Although vaccination induced high antibody titers, no effect on adult female I. ricinus engorgement weights could be observed. However, F1 - containing proteins with the highest molecular weight - showed a reduction in egg mass also significantly reduced engorgement weights of nymphal ticks. The absence of the strong phenotype, as was observed following vaccination with total midgut extract, could be explained by a lower vaccination dose due to technical limitations while preparing the fractions. Indeed, a second experiment, in which we used a higher concentration of F1, did show an evident protective phenotype, suggesting that to be discovered proteins in F1 are responsible for the strong anti-tick immune responses in cows. In contrast, multiple cow vaccination trials with a total six of recombinant tick proteins identified in WP1 did not result in impaired tick feeding parameters.
Next to feeding ticks in vivo on cows, in subtask 3.3.2 we fed ticks in vitro using an optimized artificial tick feeding system. However, sera from three cow vaccination experiments did not show a marked reduction of tick feeding success in contrast to the in vivo results, suggesting that combined cellular and humoral responses are needed for proper tick immunity. Therefore, further in vitro feeding of sera from these experiments was considered irrelevant and was therefore not performed.
As a highly interesting additional effort in WP3, we have selected 15 tick salivary gland and tick midgut proteins from a list of proteins that we identified by an immunoprecipitation with tick-immune cow sera and salivary gland extracts or midgut extracts followed by identification of the precipitated antigens by LC-MS/MS. We subsequently, investigated the role of these proteins in the tick feeding process by RNAi in adult female I. ricinus ticks feeding on rabbits. Thus far, two of these proteins were made recombinantly in E. coli and investigated in an additional cow vaccination trial, but did not induce tick immunity.
WP4 Proof of concept
In WP4 we investigated whether we could prevent multiple tick-borne diseases by vaccination targeting the tick. We selected the candidates based on the identification and characterization described in WP1, WP2 and WP3. WP4 was the common intersection of all previous WPs. In WP4 we aimed to deliver proof of concept that anti-tick vaccines can prevent transmission of multiple tick-borne pathogens. We had planned to investigate tick antigens that are instrumental to transmission of tick-borne pathogens in WP4.1 and antigens that interfere with tick feeding and thereby indirectly interfere with transmission of tick-borne pathogens from the tick to the host. In reality, the difference was not as clear-cut and quite a few of the tick antigens identified through the different planned as well as additional approaches showed some degree of overlap in sequence, protein family, function or in their role in the tick feeding process or in transmission of TBPs from the tick salivary gland to the host. Therefore, we here report Objective/task 4.1 and Objective/task 4.2 together and for clarity we report our efforts, both planned and additional, per transmission model. In addition to the planned activities, we have performed additional experiments to identify potential tick antigens for an anti-tick vaccine targeting I. ricinus with the goal of preventing Lyme borreliosis, TBEV and human babesiosis. As a contingency plan we also investigated vaccine candidates targeting the pathogen itself. The transmission models for B. afzelii, B. microti and TBEV required for the WP4 experiments were established and refined as part of WP2.
Objective/task 4.1: Specifically interfering with transmission of tick-borne pathogens AND Objective/task 4.2: Interfering with tick feeding to prevent transmission of tick-borne pathogens
Borrelia afzelii transmission model
In the B. afzelii model we investigated 8 tick salivary gland antigens, either as single antigens or in combinations and identified by 3 different approaches, by active immunization. Furthermore, we passively transferred sera from rabbits that had been vaccinated with 3 different combination of tick antigens identified by the yeast surface display to mice, followed by B. afzelii-infected tick challenge. In addition, we also investigated whether a conserved Borrelia candidate could protect against Lyme borreliosis through conventional or an alternative vaccination vaccine delivery strategy (DNA tattoo). The main finding in the B. afzelii model was that we identified an anti-tick vaccine based on a tick salivary gland protein, COMB 2, that was able to significantly and robustly impair B. afzelii transmission from the tick to the host. This particular tick salivary gland protein was functionally described in WP2 and was shown to impair phagocytosis of Borrelia by murine macrophages in vitro and boost Borrelia infectivity in vivo. Interestingly, mice actively vaccinated with this tick protein were significantly less often Borrelia-culture positive and had significantly lower Borrelia loads, as determined by q-PCR in various tissues, as compared to controls. Although we will most definitely repeat this experiment, these data indicate that we have identified an antigen that can be used as an anti-tick vaccine to prevent Lyme borreliosis.
Babesia microti transmission model
As we have described in WP1 and WP2 we were unable to validate the tick salivary gland proteins that we identified as part of the Babesia MACE analyses due to the fact that we were unable to get highly B. microti-infected nymphal ticks. Indeed, preparation of cDNA for biological validations appeared to be complicated and for the last two years we did not get higher infection rates than 10-15%, which is too low for our validation experiments. At the time of reporting we had already infected larvae of 15 different tick populations, and we changed many conditions in an attempt to tweak the nymphal infection percentage. We for instance incubated larvae at different temperatures before and after feeding on B. microti-infected mice and tried to feed larvae on mice with different levels of parasitemia. Since we are of the opinion that, when biologically validated, the tick salivary genes identified in the Babesia MACE analysis could be very interesting, two ANTIDotE partners (BC ASCR and FUB) will keep trying to get B. microti-nymphal infection rates above 50%. As soon as we have these we can validate the already existing top-20 tick salivary gland genes that are induced upon B. microti infection at 24 hours of tick feeding. Interestingly, we did show that when we used 10 lowly B. microti-infected nymphs and fed them on BALB/c mice that the majority of mice became infected. This allows us to test vaccine candidates (either tick proteins or Babesia proteins) once they have been validated. Finally, as a contingency plan we identified two B. microti sporozoites-specific transmembrane proteins that were made in recombinant form (E. coli and/or Drosophila). Although the ANTIDotE project has come to an end, these vaccinations studies will be performed early 2019. As described in WP1, we were able to identify to B. microti sporozoites-specific transmembrane proteins. In addition, we have performed an additional analyses to identify more sporozoite-specific proteins.This additional approach could lead to the identification of additional promising Babesia proteins that could serve as candidates for a vaccine directly blocking B. microti early in the transmission from tick salivary gland to the host.
TBEV transmission model
The final vaccination experiment initially involved six immunisation groups (eight mice per group). However due to technical problems in two of the six groups, we were able to test two experimental groups (consisting of one tick salivary gland protein each) and two control groups, a negative control (PBS) and a positive control (TBEV-vaccine Pfizer). One tick protein was a tick salivary gland protein upregulated upon both B. afzelii and TBEV early during tick feeding by MACE analysis (and the corresponding gene was designated gene Bb2). The other was a feeding-induced tick salivary gland protein that was highly upregulated at 24 hours of tick feeding in uninfected nymphal I. ricinus ticks by MACE analysis (encoded by a tick salivary gene named gene 12). Interestingly, we showed in WP2 that when the encoding gene was silenced in TBEV-infected ticks that these were less able to feed and transmit TBEV to naive mice. For the vaccination experiments, mice were injected with 25 µg of recombinant protein in the final volume of 100 µl (50 µl of antigen with 50 µl of adjuvant and received two booster vaccinations. Blood samples were taken before the start of the experiment as well as about two weeks after every immunisation. The serum samples taken in the course of immunisation were analysed for the presence of specific antibodies by ELISA and showed that all vaccinated mice (expect for the control groups) developed high antibody titers against the tick protein they were vaccinated with. Two weeks after the third immunisation mice were challenged with TBEV-infected ticks. Three experimentally infected nymphal ticks were allowed to feed on the immunized mice for three days. To analyse the effect of the immunisation on the TBEV transmission during co-feeding, an additional 15 non-infected nymphs were allowed to feed together with the three infected nymphs. After three days, the nymphs were collected, weighed and stored for further analyses. The post-engorgement weight of ticks fed on the tick antigen vaccinated animals was not lower compared to the controls, although the feeding efficiency in one of the tick antigen vaccinated groups appeared to be lower. The mice were subsequently observed for the signs of tick-borne encephalitis or death for 21 days. Animals with severe symptoms were sacrificed to reduce animals’ suffering. Both control groups showed expected results: none of the PBS-immunized mice survived the challenge with three TBEV infected nymphs. The median survival was 10 days, which is compatible with previous experiments involving TBEV-infected nymphs in our hands. On the other hand, the commercially available human TBEV vaccine, FSME-IMMUN (Pfizer) protected all immunized mice against the challenge. In light of these highly significant results of the control, the data obtained for the two tick antigens were highly promising and encouraging: in both groups, the immunisation did not only lead to prolonged survival, but also to partial protection (approx. 40-50% protection) from lethal TBEV-infection. Serum and organs of surviving mice and TBEV titers and infection percentages of co-feeding ticks are currently being analysed. In summary, the immunization experiment with these two tick antigens followed by challenge with TBEV-infected nymphs showed very promising and encouraging results: both tick proteins were immunogenic and immunized animals were partially protected against TBEV when exposed to three TBEV-infected nymphs together with 15 additional uninfected ticks.
Overall conclusions WP4
In WP4 we have come a long way providing proof of principle that anti-tick vaccines can protect against multiple tick-borne pathogens. Indeed, we have obtained strong and highly encouraging experimental proof for B. afzelii and TBEV, the two most important tick-borne diseases in Europe. For Babesia we encountered some technical challenges, but we were able to establish the transmission model, identify other (Babesia) vaccine candidates and to generate a short-list with interesting TSGPs (that still requires biologically validation), which could serve as candidates for an anti-tick vaccine blocking B. microti transmission from the tick to the host. Although the ANTIDotE projected has ended, we will continue our research and investigate whether combining multiple tick and pathogen antigens could lead to the development of a single vaccine to prevent bacterial, protozoal and viral tick-borne diseases. The antigens identified, described and characterized, as well as the infrastructure, assays, materials and methods developed as part of the ANTIDotE project are an excellent starting point.
WP5 Valorization plans
In WP5 we delivered plans for exploitation and implementation of our findings and concepts to contribute to downscaling of the burden of TBDs on Central and Eastern European and other endemic European societies. Central to this approach has been the exchange of knowledge with our advisory board and relevant stakeholders as well as dissemination of our results and the rationale of anti-tick vaccines to our peers and the public.
Objective/task 5.1: Exchange of knowledge on Lyme borreliosis, tick-borne encephalitis, human babesiosis and innovative strategies to prevent tick-borne diseases.
All ANTIDotE partners - except for GenXPro - were highly actively involved in research into tick and tick-borne diseases. Meetings and conferences, that were both organized and attended with oral and poster presentations, as well as the number of pubmed-indexed publications on these topics by the directly involved researchers from the participating institutes in the ANTIDotE project are the main ways by which we exchanged our knowledge with the medical and scientific community and the public. The involved ANTIDotE researchers have published 170 unique pubmed-indexed publications on Lyme borreliosis, tick-borne encephalitis, human babesiosis and innovative strategies to prevent tick-borne diseases during the ANTIDotE project. In addition, we have organized three workshops on ticks and strategies to prevent ticks and tick-borne diseases, with a special emphasis on anti-tick vaccines. One workshop was in Potsdam, Germany (2016) and another in Cesky Krumlov, Czech Republic (2015), both in adjunction to our annual meetings, while the third one was in Wageningen, the Netherlands (2016). The workshops engaged all relevant stakeholders (patients, policy makers, junior as well as more senior scientists, public and one health experts and industry). In addition, ANTIDotE has come in contact with multiple pharmaceutical companies and discussed how to identify and develop an anti-tick vaccine or other ways to prevent ticks and tick-borne diseases for the human and veterinary market.
Objective/task 5.2: To disseminate ANTIDotE results
Dissemination tools included, but were not limited to I) a public ANTIDotE website (www.antidote-fp7.org) through which we informed the public by press releases on major breakthroughs or other newsworthy facts. The website contained relevant information on the consortium, the project and ANTIDotE publications; II) cross-linking with other related European initiatives. For example, Dr. Sprong WP5-leader (RIVM) has become a member of the WHO taskforce for the development of the WHO R&D Blueprint for a roadmap on Crimean-Congo Hemorrhagic Fever Virus, and has presented a One Health approach for the prevention of CCHF for example by anti-tick vaccines during a consultation meeting with experts; III) Data sharing. We disseminated our results in 33 peer-reviewed scientific publications (including two PhD theses), a multitude of national and international meetings and near publication of 4 additional academic PhD theses and three additional manuscripts. Part of the dissemination included important contributions to a free book on ticks and tick-borne diseases edited by one of the ANTIDotE partners (RIVM). In addition, GenXPro (SME) showcased the ANTIDotE project and results to clients. Furthermore, the ANTIDotE partners disseminated the ANTIDotE design, rational and data in various non-medical or non-scientific journals, as well as through (social) media, for example through interviews in newspapers, popular press, radio and television. One such dissemination highlight was our contribution to the news feature in Nature on the emergence of “Tick troubles” and the potential of anti-tick vaccines (Moyer et al., 2015); IV) open ANTIDotE end-conference. ANTIDotE organized an open-end conference incorporated in a plenary session of the 15th International Conference on Lyme borreliosis and other tick-borne diseases in Atlanta, GA, USA. Thus, our project and results were efficiently disseminated to our peers and the scientific community. At the meeting representatives from patient advocacy groups, industry and local and national governmental agencies were present. A meeting report has been submitted to the European Commission (deliverable report DL5.4). All of our dissemination efforts are listed in Table A1 of this report.
Objective/task 5.3: A road map for exploitation of anti-tick vaccines
An initial roadmap was made (deliverable report DL5.2) for the future exploitation of anti-tick vaccines identified within the ANTIDotE project. Furthermore, a technical report (deliverable report DL5.3) on the implementation of potential anti-tick vaccines in (inter)national strategies for the prevention and control of tick-borne diseases in Europe was published (Sprong et al., 2018). Our overall conclusions from these reports were that I. ricinus-borne diseases are diseases that emerge from interactions of humans and domestic animals with infected ticks in nature. Control strategies involving concerted actions from human and animal health sectors as well as from nature managers have not been formulated, let alone implemented. Control of TBDs asks for a “health in all policies” approach, both at the (inter)national level, but also at local levels. There, nature and health workers together can carry out tailor-made control options for the control of TBDs for humans and animals, with minimal effects on the environment. Anti-tick vaccines should most definitely be considered as one of the options in the future.
In addition, two novel milestones beyond the current ANTIDotE project have been set. Firstly, the ANTIDotE consortium has successfully developed and used innovative methodologies for the identification of novel targets for anti-tick vaccines. Due to the highly complex multi-angular interactions between microorganisms, tick vectors and their hosts, we realize that the search for the ‘magic bullet’ is not an easy task. Indeed, in the final roadmap (deliverable report DL5.5) we propose that the best way to bite back against tick-borne diseases is to obtain more knowledge on the many aspects of the interaction between ticks, pathogens and mammals, for example by making use of tools that we have developed and used within the ANTIDotE project. Therefore, the next step would be to gain more insights in the role of these targets in the interrelationship between ticks and their pathogens and hosts, including mechanisms of acquisition, persistence and transmission as this may lead to the identification of critical elements of the pathogens’ life cycle that could be targeted by vaccines. Indeed, we have performed mechanistic studies as part of ANTIDotE for some of the identified targets. However, considering the long list of novel vaccine targets we have identified as part of the ANTIDotE project, this requires further experimentation. To this end, members of the ANTIDotE consortium are jointly applying for novel grants, both at the national and international level.
Secondly, anti-tick vaccines can be powerful tools to safeguard human, animal and environmental health, and can be directed against other tick species of medical and veterinary relevance as well. In the final roadmap we put forward that sharing knowledge, tools and innovative screening methodologies among researchers working on anti-tick vaccines in different fields, on different tick species and on different aspects of anti-tick vaccines, is mandatory to come to new and successful preventive strategies. This can be facilitated by applying for a COST-action for networking activities, facilitating transfer of knowledge, data and skills and the formation of novel consortia that can successfully apply for future grants. This is exactly what members of the ANTIDotE consortium are currently doing. This will help to engage all relevant stakeholders including geneticists, epidemiologists, immunologists, vector biologists, bioinformaticians, physicians and veterinarians, public health specialists, and the pharmaceutical industry, amongst others. It will also help to engage the public, which is important in order to reach the populations at risk, especially with prevailing societal prejudices associated with vaccination. Increasing efforts to study tick-host-pathogen interactions will provide a higher chance of discovering new and more potent targets for anti-tick vaccines and might lead to the dawn of a new era where an anti-tick vaccine protecting against the most common tick-borne pathogens will come to fruition.
Objective/task 5.4: Ways to implement anti-tick vaccines in health systems
The workshops, the open-end conference (deliverable DL5.4) the technical report (deliverable DL5.3) and the roadmaps (deliverables DL5.2 and DL5.5) proved to be efficient means for us to discuss our idea’s and ANTIDotE data with policy makers and other relevant stakeholders. Another important outcome of this task was that we performed an early cost-effective analysis on a potential anti-tick vaccine. Besides presentations on Vaccine symposia, the manuscript describing the results of this study was published (Mihajlović et al., 2019). Our study assessed cost-effectiveness of a potential vaccine that would protect against both Lyme Borreliosis (LB) and tick-borne encephalitis (TBE) in the highly endemic setting of Slovenia. The analyses indicated that an anti-tick vaccine that would prevent both Lyme borreliosis and TBE could be a cost- effective option in Slovenia. Our analysis represented a rare - but increasingly important - example of cost-effectiveness assessment prior market authorization and might facilitate future support from governments and companies in the early stages of the development of vaccines. In addition, our model embodied a novel tool to analyze pharmacoeconomic criteria that can help development of a cost-effective health technology.
Ethics statement
ANTIDotE participants P1, P2, P3, P4 and P7 performed experiments with human biological samples or experiments in animal models (not including research on human embryo’s or fetuses, research on human being, processing of genetic information or personal data, generation of transgenic animal strains, or animal cloning). The involved personnel of each of the ANTIDotE participants had proven experience with working with animals (certificates) and all experiments complied with current regulations, legislation and codes of conduct in the countries where the experimentations have been carried out. All ANTIDotE partners obtained the written approval of the local human or animal ethics committees prior to start of the experimentation and had valid and approved protocols in place. As a result, research activities within this project complied with the international conventions and codes of conduct, in particular the Council Directive of 22 September 2010 on the protection of animals used for scientific purposes (2010/63/EU), which became law on January 1st 2013, and the Ethical rules of the Seventh Framework Program (Council Decision N° 1982/2006/EC, Article 6). The work also followed the Charter of Fundamental Rights of the EU (2000/C 364/01), the Council Decision regulating safety and ethics (99/167/EEC; Council Decision of 25/1/99), the Directive 95/46/EC of the European Parliament and of the Council of 24 October 1995 on the protection of individuals with regard to the processing of personal data and on the free movement of such data, the 391L0628 Council Directive 91/628/EEC of 19 November 1991 on the protection of animals during transport, and the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (replacing former 386L069 Council Directive 86/609/EEC of 24 November 1986). The outcome, number of groups/animal and types of animals used are detailed within the different work packages elsewhere in this report.
Potential Impact:
The incidences of Lyme borreliosis (LB) and TBEV are on the rise in several European countries and diseases caused by other pathogens, such as Babesia spp. and Borrelia miyamotoi are emerging. Environmental, socio-economic and demographic factors synergistically increase the risk of acquiring tick-borne diseases (TBDs). Indeed, the European Center for Disease Prevention and Control (ECDC) predicted that the incidence of TBDs will rise in the future. Furthermore, the European Parliament has expressed its concerns about the spread of LB in the European population (November 2018). Therefore, and also because the societal fear for ticks and TBDs seems to be ever growing, the old adage ‘prevention is better than cure’ certainly holds true for tick-borne diseases.
Currently there are no human or animal vaccines against I. ricinus available. However, anti-tick vaccines are available against other tick species for the veterinary market and there is a body of experimental evidence that anti-tick vaccines could also work for Ixodes ticks. Our project aimed to show proof of concept that anti-I. ricinus vaccines can protect against multiple TBDs, which is innovative by itself. Moreover, we provided insights into the molecular mechanisms involved in transmission of tick-borne pathogens from the tick to the host, and in the process of tick feeding. Furthermore, we went one step further and also discussed with public health representatives and relevant industrial stakeholders how anti-I. ricinus vaccines could be exploited and implemented. This could lead to a paradigm shift in how public health institutes tackle TBDs in Europe and resulted in a road map for future use of anti-tick vaccines.
The ANTIDotE network (consortium and advisors) consisted of a large community of biomedical scientists, public health officials and private sector stakeholders. The ANTIDotE consortium put much effort in the dissemination of our results and we aimed to translate ANTIDotE’s innovative results as much as possible into tangible impact on health services. Moreover, ANTIDotE actively involved health organizations, health policy experts and end-users to further increase its impact. Importantly, ANTIDotE’s outcomes spanned from discovery science increasing fundamental biomedical knowledge, to translation of this new understandings into proof of concept studies, to valorization plans contributing to sustainable advances in the prevention of multiple tick-borne diseases.
ANTIDotE delivered new understandings on the biological mechanisms underlying tick feeding, anti-tick immune responses and transmission of the causative agents of TBE, LB and human babesiosis. These are all I. ricinus tick-transmitted infectious diseases with potential serious acute and chronic sequelae and disproportionately affect Europe. ANTIDotE embodied a multidisciplinary approach combining leading scientific and industrial experts on tick biology, genetics and bioinformatics, immunology, vaccinology, the three tick-borne pathogens under study and public health experts on tick-borne diseases, from Central and Eastern Europe as well as other European countries where tick-borne diseases are endemic. The ANTIDotE project was geared towards translating new knowledge into identification of novel anti-tick vaccines and providing proof of concept that such vaccines can cost-effectively prevent transmission of multiple human tick-borne pathogens. Furthermore, ANTIDotE also integrated basic science with industrial innovation and other (public) health disciplines to generate plans for product development and to explore how anti-tick vaccines could be implemented into health systems as part of novel preventive strategies to prevent tick-borne diseases in endemic European countries. Thereby ANTIDotE strongly adhered to the expected impacts as defined in the FP7 work program and the specific call text.
We maximized our impact by:
1. Establishment of new tools and technologies to study ticks and tick-borne diseases
2. Novel fundamental knowledge of ticks and tick-borne diseases and identification of candidates for anti-tick vaccines (WP1)
3. In depth insight in the function of tick salivary gland proteins and mammalian anti-tick immune responses (WP2 and WP3)
4. Proof of concept that anti-I. ricinus vaccines can prevent tick feeding/transmission of multiple tick-borne pathogens (WP4)
5. Exploitation and implementation plans for anti-I. ricinus vaccines in existing health services (WP5)
6. Dissemination of our results by a sustainable network, with currently 33 peer-reviewed manuscripts/publications published, including important contributions to a free book on ticks and tick-borne diseases, a published technical report on the implementation of potential anti-tick vaccines in (inter)national strategies for the prevention and control of tick-borne diseases in Europe and two PhD theses. In addition, we disseminated our results by the media (magazines, news papers, non-scientific journals, internet, radio and television), an open website, three workshops and an open end-conference. The ANTIDotE project also contributed to the anticipated PhD thesis of four additional PhD students that will defend their dissertations in the near future. Finally, at the time of writing of the final report 3 manuscripts have been submitted/accepted for publications. Of note, the vast majority of the ANTIDotE output was published open access.
Our concerted multidisciplinary integrated European approach lead to multiple breakthroughs in the field of ticks and tick-borne diseases and beyond in many ways and impacted a) the research community worldwide, b) health systems, societies, economies and individual patients in European countries where tick-borne diseases are endemic, and c) industry. The ANTIDotE outcomes contribute to a knowledge-based society and paved the way for the development of innovative ways to prevent tick-borne diseases by industry and health systems in Europe.
ANTIDotE’s impact on the research community
Firstly, ANTIDotE has established new in vitro and in vivo models to study tick-borne diseases. Secondly, ANTIDotE applied state of the art and innovative technologies and approaches to research on ticks and TBDs, including YSD technology, MACE and RNA sequencing and RNA interference. Not did this raise the standard of research on ticks and tick-borne diseases in Europe, it enabled us to provide the European scientific community with new and unique knowledge on the process of tick feeding, anti-I. ricinus immune responses and the transmission of B. burgdorferi sensu lato, B. microti and TBEV. Importantly, as we have put forward in the final road map, this new knowledge is essential for the identification and development of novel strategies, which include anti-I. ricinus vaccines, to prevent the fore-mentioned, but also other I. ricinus-transmitted, infectious diseases. Current preventive strategies for I. ricinus-transmitted infectious diseases fall short and clearly fail to stop the rise of TBDs in Europe, underscoring the need for action. The new ANTIDotE-derived knowledge on the processes involved in I. ricinus tick feeding and TBP transmission might also impact understanding of, and development of new preventive strategies against, other TBDs and other tick species in Europe, for example Crimean-Congo Hemorrhagic Fever transmitted by Hyalomma ticks. Indeed, one of the ANTIDotE partners became a member of the WHO taskforce for the development for the WHO R&D Blueprint for roadmap on Crimean-Congo Hemorrhagic Fever Virus, which is pursuing a One Health approach for the prevention of CCHF, including investigating the possibility of anti-tick vaccines. In addition, our findings will help the scientific community researching tick-borne diseases transmitted by other tick species around the world. For instance, the impact of ticks and tick-borne diseases on live stock in the (sub)tropics is considerable and anti-tick vaccines comprise a eco-friendly alternative to acaricides, which are becoming more and more problematic in light of acaricide resistance, environmental pollution and food security. Finally, ANTIDotE also revealed new insights into specific mammalian host defense mechanisms by investigating the function of the identified tick salivary gland proteins.
ANTIDotE’s impact on health systems and societies, individual patients and economies
By the international workshops, the open-end conference, a technical report, a cost-effective analyses on a potential anti-tick vaccine and two reviews on the prevention of tick-borne diseases we were able to influence policy makers and health systems. Through these we voiced our ideas, our findings, the importance of the prevention of tick-borne diseases and the potential of anti-tick vaccines. Although we provided unprecedented new knowledge and proof of concept that anti-tick vaccines could work against Lyme borreliosis and TBEV, there is no commercial available vaccine yet. This will require more research. This is badly needed, as the incidence of ticks and tick-borne diseases is on the rise with considerable economical and societal impact and sometimes devastating consequences for affected individuals. Therefore, protecting individuals at risk from contracting tick-borne diseases with a safe and effective anti-tick vaccine could save lives and improve quality of life by reducing the number of patients with TBDs. Hikers, campers, farmers, forestry workers, soldiers, gamekeepers, immunocompromised individuals and actually any person regularly entering I. ricinus tick habitats (forested areas) for work or recreational purposes in endemic European countries are at risk. Taken together, with the improved knowledge on tick feeding and the transmission of tick-borne pathogens, the identification of novel anti-tick vaccine candidates that could prevent multiple human tick-borne diseases, as well as plans on how to implement these in health systems and societies, ANTIDotE has had an important impact on health systems and societies, individual patients and economies and could contribute to diminishing the number of people at risk for, or suffering from, tick-borne diseases.
ANTIDotE’s impact on, and potential of, industrial innovation
We have involved industrial collaborators during the ANTIDotE project and there is clearly an interest in anti-tick vaccines, both from the human and veterinary pharmaceutical industry. The newly identified antigens and the knowledge obtained as part of the ANTIDotE project could directly lead to the development of novel anti-tick vaccines. Such a vaccine would be a paradigm-shifting medical and industrial innovation in itself. In this regard, ANTIDotE has not filed any patents at current (apart from an application for a patent on a biotechnical device by one of the ANTIDotE partners), but is investigating some interesting and very promising leads, which might to future patent applications.
List of Websites:
The ANTIDotE partners:
Partner # 1: Academisch Medisch Centrum, AMC, The Netherlands (coordinator)
Partner # 2: Biologicke centrum AV CR, v. v. i., BC ASCR , Czech Republic
Partner # 3: FREIE UNIVERSITAET BERLIN, FUB, Germany
Partner # 4: ASOCIACION CENTRO DE INVESTIGACION COOPERATIVA EN BIOCIENCIAS, CIC bioGUNE, Spain
Partner # 5: GENXPRO GMBH, GenXPro, Germany
Partner # 6 RIJKSINSTITUUT VOOR VOLKSGEZONDHEID EN MILIEU, RIVM, The Netherlands
Partner # 7: VIROLOGICKY USTAV SLOVENSKEJ AKADEMIE VIED (changed to BIOMEDICINSKE CENTRUM SLOVENSKEJ AKADEMIE VIED), IV SAS, Slovakia
Public website and contact details:
www.ANTIDotE-fp7.org
At the website our contact information can be found. Alternatively, contact info@antidote-fp7.org or lyme@amc.uva.nl
