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Periodic Report Summary 2 - TRANSLINK (Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves)

Project Context and Objectives:
Cardiac valve disorders are widespread in the general population and represent the third most frequent cardiovascular illness after coronary disease and arterial hypertension. Accordingly, cardiac valve replacement represents the second most frequent cardiac operation after coronary artery bypass surgery and is increasingly performed worldwide, with approximately 430,000 such procedures being performed worldwide each year with a growing potential market, especially in developing countries.

Currently, two types of prosthetic valves are offered to patients needing heart valve replacement surgery. These are represented by mechanical valves and by their bioprosthetic counterparts, each presenting advantages and drawbacks. As a general approach, for their mechanical properties and biological compatibility, animal (bovine/porcine or, rarely, equine)–derived Bioprosthetic Heart Valves (BHV) are the preferred form of prosthesis. Indeed, BHV offer an optimal haemodynamic profile and, by contrast with mechanical valves, do not require life-long patient anticoagulation. Accordingly, nearly 300,000 patients worldwide undergoing cardiac valve replacement each year receive a BHV. Nonetheless, this number would greatly increase, especially in younger patients, if BHV did not suffer from premature structural valve deterioration (SVD). SVD is age-dependent, with <10 % occurring in patients >65 years of age, but almost uniform failure within 5 years in patients <35 years old. Indeed, BHV have a fairly limited life-span estimated at 12 to 15 years at most. As a consequence, despite providing a better mechanical performance and comfort, BHV cannot be offered to young individuals, who must thus receive a mechanical valve instead. Indeed, mechanical valves offer the long-term durability that is required for young individuals. On the other hand, in all cases, mechanical valves require lifelong anticoagulation, with risks of thrombosis, thromboembolism (e.g. stroke) or spontaneous bleeding. Therefore, in case of cardiac valve disorders, the biological characteristics of BHV would render them by far the preferable option over mechanical heart valves if durability were not a major drawback.

Based on the implementation of a clinical study that will entail the enrolment of a retrospective and prospective cohort of over 1,400 BHV recipients and 200 control patients from 4 large EU cardiac surgery groups, the TRANSLINK project aims primarily to establish the possible role of recipients' anti-BHV immune response as a major cause to mid- to-long term clinical dysfunction. Precise molecular analysis of preimplantation BVH sugar moieties will be performed. Possible indirect side-effects on BHV endocarditis and host vessels’ inflammation are secondary end points. Serial and trans-sectional blood samples will be despatched to a battery of highly specialised partner groups for testing anti-Gal, -Neu5Gc and -hyaluronic acid antibodies using both validated and newly-designed screening tools, glycan array patterns, and macrophages/NK responses. Data will be crossed with clinical outcome scores. The project design aims to deliver comprehensive recommendations in the timeframe of the grant. Fundamental basic science progress in the field of carbohydrate antigens is also expected. Furthermore, prevention (BHV from engineered animal source lacking major antigens) and treatment (bioabsorbants of deleterious Ig-oriented remedies) as well as prospective identification of biomarkers of long-term BHV deterioration will be set up by three first-class SMEs.

TRANSLINK is expected to strongly impact the treatment of heart valve diseases by improving morbidity/mortality in patients with heart valve diseases and increasing the applicability of BHV to younger patients.

Project Results:
Work package 1 Clinical data, biobanking and statistical analysis
TRANSLINK investigators began patient enrolment in the study in January 2014, which continued until June 2016. The reach of the project has been expanded with the recruitment of a North American centre, the University of Manitoba. A total of 1,615 patients have been recruited to the different cohorts, from whom 3,364 cardiac ultrasound scans have been recorded, 5,212 blood samples collected and 1,510 medical consultations performed. The target enrolment of the study has been exceeded. These patients will continue to be monitored throughout the remainder of the project.
Work package 2 Biochemical characterisation of engineered BHV and analysis of the anti-BHV humoral immune response
WP2 aimed to identify the various potential xeno-antigens in BHV-related complications. In parallel, the team developed new methods to investigate the immune response to BHV and initiated testing of sera. A method for investigating anti-Neu5Gc antibodies in serum has been developed and over 1,000 samples screened. Structural characterization of glycosphingolipid and glycoproteins from xeno-tissues has been performed. A new platform to express recombinant glycoproteins as potential anti-BHV targets has been refined and new targets identified. Data collection of humoral responses to BHV in patients and controls has begun.
Work package 3 Mechanisms of BHV damage and possible recipient disease mediated by the anti-BHV immune response
WP3 has continued its studies on the identification of the mechanisms leading to SVD of BHV and related pathological events. Activation of human complement triggered by three commercial BHV of different species (porcine, equine and bovine) has been quantified and evidence obtained for the participation of anti-αGal antibodies and Mannan-binding lectin (MBL) for specific BHV. Increases in NO and nitrotyrosine were also detected in the supernatants of monocytes co-cultured with BHV, in particular with Mitroflow valves without anti-calcification pretreatment. The role of anti-Neu5Gc antibodies in endothelial inflammation has been studied, comparing the effect of natural vs. elicited anti-Neu5Gc antibodies at the transcriptional level.
Work package 4 Potential infectious risk related to the anti-BHV immune response
Studies have been designed to describe the profile of anti-carbohydrate antibodies (ACA) associated with the haematological dissemination of the four microorganisms that most commonly cause heart bioprosthesis endocarditis. Natural ACA targeting substituted Galβ-structures seem to play an important role in the haematological spread of E. faecalis and S. aureus. Antibody binding assays have been developed for glycan antigens; work continues on assays to examine the effects of antibodies on virus infection.
Work package 5 Remedies: prevention and treatment
One Gal-KO and Neu5Gc-KO boars have been produced by cloning. Bovine gene editing of GGTA1 and CMAH genes was performed in fibroblasts and in IVF derived zygotes; but GGTA1 editing was challenging and only possible by the production of new double KO colonies for SCNT. Two pregnancies are still ongoing. Design, synthesis and biological evaluation of novel glycoconjugates with binding capacity for ACA is well underway. New linkers for the glycoconjugates are being explored in the lab and in silico to ensure patentability of the TRANSLINK results.
Work package 6 Dissemination and training
The website has been updated continuously ( with critical information on the project. Social media (e.g. Twitter and Facebook) have been used to provide “hot-off-the-press” updates to interested parties. The first TRANSLINK training event on Glycoimmunology was held at Tel Aviv University in October 2015; a second is planned for Cremona in spring 2017. The project is holding a dissemination event with the FP7 project XenoIslet in London in November 2016: plans for this are at an advanced stage.

Potential Impact:
The key advancements that the project will bring about are the following:

1. Identification of novel BHV carbohydrate determinants that may elicit or be the target of anti-BHV antibodies or activate human immune cells.
Carbohydrate determinants of BHV may be putative targets for the human immune system and, as such, contribute to the degenerative process that impairs BHV function over time. To investigate this further, TRANSLINK will rely on first-class scientists with extensive knowledge of the biochemical and analytical techniques required to reveal glycan structure, including technology allowing structural characterisation of both glycolipids and glycans carried by proteins. In addition, potential immunogens on clinically-used BHV will be characterised and screened for immune recognition by antibodies from BHV recipients. Therefore, the anticipated detailed characterisation of the targets for anti-BHV antibodies will allow the development of novel assays for detection and specificity-determination of such antibodies.

2. Characterisation of the natural and elicited anti-αGal but also anti non-αGal antibody repertoire in BHV recipients and demonstration of the detrimental correlation between appearance of such antibodies and Structural Valve Deterioration (SVD).

We believe that anti-BHV antibodies may contribute to premature BHV degeneration and inflammation in the recipients. Indeed, we speculate that pre-existing and/or de-novo anti-BHV antibodies may deposit in the BHV, activate complement and cause the inflammatory events eventually responsible for valve damage. In this light, TRANSLINK investigators are expected to unequivocally demonstrate a possible causative relationship between the appearance of such antibodies and SVD by characterising the immune response against xeno-antigens in a cohort of patients who have undergone valve replacement with a BHV. To this end, a two-phase TRANSLINK-dedicated clinical study will be conducted.

3. Demonstration of the potential detrimental role of anti-carbohydrate antibodies on the onset of endocarditis following BHV implantation

4. Demonstration of the possible detrimental role played in BHV recipients by the innate cellular immune response to BHV
TRANSLINK investigators will try to elucidate the mechanisms of the cellular immune response with an emphasis on innate immune cells and how they are activated in the presence of BHVs, antibody and complement.

5 Development of novel preventive and therapeutic strategies to combat BHV-related adverse immune reactions
Our preliminary results, efforts undertaken within TRANSLINK and the elucidation of the fine immunological mechanisms underlying premature BHV failure will enable our consortium to identify possible targets of intervention and the development of remedial strategies, as requested by the call. As a first step, a transgenic pig line lacking immunogenic epitopes possibly responsible for the production of anti-BHV antibodies will be produced. As a second approach, TRANSLINK scientists will be dedicated to the identification of novel glycoconjugates with binding capacity for several anti-carbohydrate antibodies.

In conclusion, at the end of this project TRANSLINK’s investigators will have demonstrated that the immune system leads to adverse immune reactions to BHVs and, possibly, to BHV recipients. Furthermore, newly engineered BHVs and compounds that mitigate these immune-mediated side-effects, ready to be tested in subsequent clinical trials will also be generated and patented. Indeed, TRANSLINK is expected to strongly impact the treatment of heart valve diseases by improving morbidity-mortality in patients with heart valves diseases and increasing the indication of BHV to younger patients.

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