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FP7

ANTIDotE Report Summary

Project ID: 602272
Funded under: FP7-HEALTH
Country: Netherlands

Periodic Report Summary 2 - ANTIDOTE (Anti-tick Vaccines to Prevent Tick-borne Diseases in Europe)

Project Context and Objectives:
The overall objective of ANTIDotE is to identify and characterize tick proteins involved in ‘tick immunity’ and transmission of tick-borne pathogens (TBPs) and to use this knowledge to develop anti-tick vaccines to prevent multiple human tick-borne diseases (TBDs) in the future. In addition, through an integrated and multidisciplinary approach involving public health institutes, health organizations and industrial companies we will examine how to develop anti-tick vaccines and how to implement these in public health systems. Because of its design, which combines state of the art basic science with translational research, ANTIDotE will also deliver essential knowledge on the biological mechanisms involved in the pathogenesis of TBDs. The key objectives are:

First Objective: To identify I.ricinus tick salivary gland proteins (TSGPs) that play a role in the transmission of 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).

Second Objective: Identified TSGPs (described above) will be 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 TBDs under study will be established and improved.

Third Objective: 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 responses that these TSGPs elicit will be characterized. Thus, third objective will reveal candidates for anti-tick vaccine that are capable of interfering with tick feeding.

Fourth Objective: 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 TBEV from the tick to the mammalian host, and TSGPs that are of paramount importance for tick feeding, will be tested.

Fifth Objective: 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 do so by attending and arranging workshops and meetings.

These key objective results in the following project objectives (OB):
OB 1.1 Identification of TSGPs involved in transmission of TBPs [M18]
OB 1.2 Identification of TSGPs recognized by anti-tick immune responses [M18]
OB 2.1 Describe the role of TSGPs in the transmission of TBPs in vivo using RNAi in ticks [M42]
OB 2.2 Describe the function of TSGPs in in vitro and ex vivo functional assay [M60]
OB 3.1 To confirm that TSGPs identified induce ‘tick immunity’ in mice [M42]
OB 3.2 To characterize humoral and cellular anti-tick immune responses [M48]
OB 3.3 To confirm that TSGPs identified induce ‘tick immunity’ in cows and to develop a robust ATFS for research on anti-tick vaccines [M48]
OB 4.1 To identify an anti-tick vaccine that protects against multiple TBDs by specifically interfering with TSGPs crucial for transmission of TBDs [M60]
OB 4.2 To identify an anti-tick vaccine that protects against multiple TBDs by interfering with tick feeding [M60]
OB 5.1 To exchange knowledge on LB, TBE, human babesiosis and innovative strategies to prevent TBDs
OB 5.2 To disseminate ANTIDotE results [M42, M60]
OB 5.3 To establish a road map for exploitation of novel anti-tick vaccines [M36]
OB 5.4 To explore ways to implement anti-tick vaccines in health systems [M60]

Project Results:
The aim of the ANTIDotE project is to find tick salivary gland proteins (TSGPs) that block pathogen transmission, either directly or by interfering with tick feeding. Two antigen discovery strategies were employed (WP1). Firstly, MACE and RNAseq to identify genes upregulated upon infection with Borrelia, TBEV or Babesia, resulting in the identification of 205 TSGPs, of which validation is ongoing. As an additional effort we identified and validated 20 TSGPs that are highly upregulated upon tick feeding. Secondly, the Yeast Surface Display (YSD) was used to identify and validate 24 TSGPs that are recognized by antibodies of frequently tick-exposed humans, 24 hr Ixodes ricinus infested rabbits, or Ixodes scapularis tick immune rabbits. The identified TSGPs are further characterized as part of WP2/3.

We have further refined the animal models for Borrelia and TBEV and are refining the Babesia transmission model (WP2). RNAi infrastructure was established and positive controls have been identified. For 6 feeding-induced genes RNAi experiments with TBEV and Borrelia infected ticks are ongoing; thus far no effect on feeding parameters was observed, but TBEV and Borrelia transmission has to be evaluated. Production of TSGPs in Drosophila has started; the first candidates are cloned and purification is underway. Preliminary data show that one of the identified TSGPs binds to Borrelia and promotes Borrelia survival in vivo. We also optimized cryo-scanning electron microscopy for studying TSGP-Borrelia interactions.

Vaccination experiments have started (WP3). Protein or DNA vaccination of mice with TSGPs did not result in a significant anti-tick effect. Passive immunization of mice with human and bovine sera did show a significant decrease in engorgement weights. Two bovine vaccination experiments were performed in search of a positive control, showing that using a prime-boost regime with tick protein extracts resulted in a robust anti-tick immunity. In addition, we used the obtained sera for additional immunoprecipitation experiments and with LC-MS label free analysis we identified 14 additional vaccine candidates. Also the first TSGPs from WP1 were expressed and used for vaccinations; C3, C4 and D4 (WP1.2) and TSGPs 1,2,3 and 4 (feeding-induced, WP1.1). Vaccination of cows with either C3, C4 and D4 or a combination of feeding induce TSGPs 1,2,3 and 4 did not show an effect on tick feeding, however we have used the obtained sera to set-up an artificial tick feeding system to feed nymphal and adult I. ricinus ticks in vitro. Cloning and expression of additional newly identified TSGPs is underway. The top candidates from WP2/3 will be further tested in WP4 starting in reporting period 3.

The ANTIDotE partners have been disseminating knowledge on (inter)national conferences, in peer-reviewed scientific journals, medical journals and through the ANTIDotE website. One example of our dissemination efforts was the news feature in Nature. An initial roadmap was created (DL5.2, M36) for the future exploitation of anti-tick vaccines. In this road map, the crucial steps in identification, characterization and assessment of anti-tick vaccines, as well as the manufacturing process, pre-clinical and clinical phases, and the demands of potential end-users were taken into account. Also, a Cost-Effectiveness Analyses of a hypothetical anti-tick vaccine was initiated in a Central Eastern European country where Lyme borreliosis and tick-borne encephalitis are endemic. Two workshops dedicated to innovative strategies to prevent tick-borne diseases were organized in Berlin (M30) and in Wageningen (M36), where stakeholders from medical and veterinary sciences, policy advisors, governmental institutions, patient interest groups and industry actively participated.

All in all we have made significant progress since our last periodic report: all discovery methods have yielded vaccine candidates and the vaccination and characterization studies have started.

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 Neoehrlichia mikurensis, Babesia spp. and Borrelia miyamotoi are emerging. Environmental, socio-economic and demographic factors synergistically increase the risk of acquiring tick-borne diseases. Indeed, the European Center for Disease Prevention and Control (ECDC) has predicted that the incidence of TBDs will rise in the near future. 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 aims to show proof of concept that anti-I. ricinus vaccines can protect against multiple TBDs, which is innovative by itself. Moreover, we will provide in depth insight 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 will go one step further and also discuss 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 result in a road map for future use of anti-tick vaccines.

Expected final results:
We expect to make major contributions in the prevention of TBDs in Europe by providing:
- unprecedented molecular insights into the process of tick feeding and pathogen transmission from the tick to the host.
- robust in vitro and in vivo animal models for research on transmission of Borrelia, Babesia and TBEV.
- identification of tick antigens that could serve as candidates for anti-tick vaccines preventing Lyme borreliosis, babesiosis and TBEV.
- proof of concept that such a vaccine works in animal models.
- plans for future product development and implementation of anti-tick vaccines in health systems in collaboration with industry, health institutes and other relevant stakeholders.

In summary, ANTIDotE aims to shed light on the mechanisms of I. ricinus tick feeding and TBP transmission and to identify, characterize and assess tick salivary gland proteins as candidates for anti-tick vaccines by developing and refining in vivo and in vitro models. Hereto, ANTIDotE applies state of the art and innovative technologies; these include the use of YSD technology, RNAseq, MACE and RNAi in ticks.
Potential impact/expected use:

ANTIDotE’s major contributions and concerted multidisciplinary integrated European approach will lead to a breakthrough in the field of ticks and TBDs in many ways, since these will impact a) the research community worldwide, b) health systems, societies, economies and individual patients in European countries where TBDs are endemic, and c) industrial innovation. The expected outcomes collectively contribute to a knowledge-based society and accelerate the establishment of innovative standards for prevention of TBDs by industry and health systems in Europe.
The models, technologies and approaches used within the project, enable us to achieve our ambitious objectives and will simultaneously raise the standard for research on ticks in Europe and throughout the world. Furthermore, ANTIDotE will provide the 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, Babesia and TBEV. This is of paramount importance for the understanding of the pathogenesis of these bacterial, protozoal and viral TBDs. The new ANTIDotE-derived knowledge on the processes involved in I. ricinus tick feeding and TBP transmission will impact the understanding and development of new preventive strategies against other TBDs and other tick species in Europe. It will also reveal novel insights into specific mammalian host defense mechanisms, such as innate and adaptive immune responses, fibrinolysis, inflammation and coagulation. Taken together, improved knowledge on tick feeding and TBP transmission and novel anti-tick vaccines that prevent multiple human TBDs as well as plans on how to implement anti-tick vaccines in health systems and societies will have a direct health and socioeconomic impact by diminishing the number of people at risk for TBDs.

List of Websites:
http://www.antidote-fp7.org/

Contact

Gülseren Yalvac, (Controller European Grants)
Tel.: +31 20 566 6265
E-mail

Subjects

Life Sciences
Record Number: 197041 / Last updated on: 2017-04-13
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