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Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves

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

Executive Summary:
Much clinical evidence convincingly illustrates that, at least in theory, animal-derived bioprosthetic heart valves (BHV) represent the best treatment option for patients needing cardiac valve replacement. However, BHV are affected by premature failure due to structural valve deterioration (SVD), a condition that precludes their use in young patients.

Existing data have provided preliminary evidence that the anti-BHV immune response could represent a central event that determines the fate of the BHV. However, at the beginning of this project, solid data were still lacking to unequivocally demonstrate that such an immune response could indeed be responsible for this untoward event. In this light, the TRANSLINK project was designed to provide the necessary data to demonstrate beyond any reasonable doubt the central role of an immune response in the premature failure of BHV. Another TRANSLINK objective was to provide efficient strategies to enable safe implantation of BHV in currently unsuitable candidates.

To this end, TRANSLINK has conducted a prospective multi-centre clinical trial in five cardiothoracic units across Europe and Canada and has put together a multidisciplinary team of first-class investigators from academic and private institutions, whose complementary nature and synergy were indispensable to reach the ambitious goals of this project.
The results of this project are compelling and advance significantly the science in this area.

First, the TRANSLINK partners have successfully built the largest clinical and biological bank devoted to BHV recipients, with 1,697 patients recruited. At the end of the project, 4,381 blood samples have been collected and 388,118 clinical measurements have been included in the electronic case report forms and submitted for analysis. Second, the group has demonstrated that chemically-processed BHV currently used in the clinic still display large amounts of immunogenic xeno-carbohydrates. Third, the detailed characterisation of the elicited anti-αGal, anti-Neu5Gc and anti-hyaluronic acid antibodies in BHV recipients demonstrate the antigenicity of BHV and pave the way towards a hypothesis of a possible role of immunological events in the onset of SVD and of systemic vessel inflammation in BHV recipients. Fourth, TRANSLINK was able to demonstrate, both in vivo and in vitro, the role of an innate cellular immune response to BHV. Fifth, TRANSLINK provided a first exhaustive transcriptome analysis of the effect of anti-Neu5Gc antibodies on human endothelial cells (EC), showing a strong effect on EC, and raising safety concerns linked to the presence of Neu5Gc on clinically available BHV. Finally, the results generated have enabled the TRANSLINK SMEs to develop remedial strategies. In particular, engineered pig and cattle lines lacking Gal and Neu5Gc, have been produced. Furthermore, a TRANSLINK SME has identified glycoconjugates able to counteract potential effects of anti-BHV antibodies on BHV or on patients’ EC. In both circumstances, a patent application could be filed.

In addition, training young investigators and dissemination activities for lay people and stakeholders were two important parts of the project.

In conclusion, the TRANSLINK consortium has unquestionably demonstrated the immunogenicity of BHV and has set the scene for a consequent, deleterious role of such antibodies for the BHV itself and/or for the recipient vessels. Engineered BHV lacking xenoantigens and new compounds that mitigate these immune-mediated side effects could now be tested in the clinic. Indeed, TRANSLINK data have the potential to strongly impact the prevention and treatment of valve diseases by improving morbidity-mortality in patients with BHV, and extend BHV indication to younger patients.

Project Context and Objectives:
Much clinical evidence convincingly illustrates that animal-derived bioprosthetic heart valves (BHV) would represent the best treatment option for patients needing cardiac valve replacements if BHV were not affected by premature failure that precludes their use in young patients. Existing data provide preliminary evidence that the anti-BHV immune response could represent a central event that determines the fate of the BHV. However, solid data are still lacking to unequivocally demonstrate that this immune response may be responsible for this untoward event. Furthermore, the absence of solid data does not warrant the implementation of interventions to neutralize the suspected immune aggression and protect BHV or to undergo major engineering strategies to tailor the donor so that it lacks the major antigenic epitopes putatively involved in the premature structural valve deterioration (SVD) process.

In this light, the TRANSLINK project was essentially designed to provide the necessary data to demonstrate beyond any reasonable doubt the central role of the anti-BHV immune response in the premature failure of BHV; and to provide efficient strategies to enable safe implantation of BHV in currently unsuitable candidates.

To this end, the TRANSLINK Consortium was composed of a multidisciplinary team of first-class investigators working in six work packages (WP) whose complementary nature and synergy were felt indispensable to reach the ambitious goals of this project.

The main objectives of each WP and their relevance to reach the central goal of this innovative research effort are herewith briefly described.

WORK PACKAGE 1: Clinical data, biobanking and statistical analysis

The critical objective of WP1 was to enrol the crucial cohorts of patients indispensable to provide the clinical data and the biological specimens required to answer the pivotal questions to be addressed by the TRANSLINK Consortium.

To this end, the cohorts of patients expected to address the study goals were the following:

• Phase A (Cohort A): The objective of this cohort was to assess the immune response in patients with well-established structural valve deterioration (SVD). The number of SVD patients expected to be enrolled annually from each centre was 35, representing 140 patients over the 42-month inclusion time.

• Phase B (Cohorts B1 and B2): The aim of Phase B was to allow the identification of prognostic biomarkers related to echocardiographic parameters of SVD in a prospective cohort of patients operated with an aortic BHV. In order to cover at least 9 years of follow-up data, the prospective cohort was divided into two groups: B1 (de-novo BHV recipients) and B2 (patients operated on 5 years earlier). The entire Cohort B2 was designed to cover at least 9 years of echocardiographic and biological follow-up.

The B1 cohort was specifically designed to study the kinetics of the immune response against major potential animal-derived antigens before and after implantation of an aortic BHV. Porcine, bovine and equine valves were studied, to represent those most frequently used clinically. The total cohort size for B1 was originally estimated to be 500 patients (including 100 patients in the control group).
Due to the expected low incidence of SVD during the first four post-operative years, a second cohort, B2, constituted by patients who underwent biological aortic valve replacement at least 5 years before inclusion in the study, was evaluated in this project. The objective of this second cohort was to cover the period where the risk of SVD occurrence is potentially high. The total cohort size for B2 was originally estimated to be 820 patients (including 100 patients in the control group).

Partners in WP1 were expected to collect biological specimens at inclusion and at the time of follow-up visits at the various TransLink centres and transfer these, anonymised and frozen in dry ice, to the WP2 partner’s research laboratories for the immunological studies scheduled in WP2.

WORK PACKAGE 2: Biochemical characterization of engineered BHV and analysis of the anti-BHV humoral immune response

The overall objective of WP2 was to investigate the immune response against bioprosthetic heart valves (BHV) and to correlate the nature and magnitude of the response to the clinical outcome of the large and informative international cohort of BHV recipients and relevant controls of TransLink. State-of-the-art technologies were expected to be developed by WP2 partners to biochemically characterise the most commonly used BHVs and to identify the presence of various potential xeno-antigens in the BHV (i.e. antigens from animal derived tissues capable of producing an immune response in BHV recipients). Ultimately, WP2 was expected to characterise the xenoantigens of BHV and quantify the xeno-reactive antibodies in human blood samples that may be associated with BHV complications in humans.
To this end, diverse immunological high-throughput assays were expected to be established by WP2 partners. Furthermore, TAU was supposed to establish both an HPLC system to characterise sialic acids in BHV, and the mouse colony required for investigation of specific anti-carbohydrate antibodies in serum by ELISA. UCal was expected to synthesize glycans for an extensive glycan microarray. TAU and UGOT were to work closely to identify potential xeno-antigens in commercial BHVs and tissues that are used to produce them. Finally, the WP2 teams were expected to set up the groundwork for methods to investigate the immune response to BHV and undertake sera testing. In this context, ultimately WP2 partners were expected to undertake the measurements of the anti-alpha Gal, anti-Neu5GC and anti-hyaluronic acid (HA) antibodies in all the patients’ cohorts of the TRANSLINK project.

WORK PACKAGE 3: Mechanisms of BHV damage and possible recipient disease mediated by the anti-BHV immune response

The overall objective of WP3 was to elucidate the potential cellular mechanisms involved and the role of complement in triggering or contributing to SVD of bioprosthetic heart valves. It was also aimed to study other immune related pathologies possibly mediated by anti-Neu5Gc antibodies triggered by the implantation of a BHV and conceivably affecting the recipients’ own vessels, as normal individuals do express some amounts of diet-derived Neu5Gc on endothelial cells.

There is increasing evidence of a significant immune reaction to BHVs that may contribute to calcification, structural deterioration, and ultimately SVD. This is particularly relevant in young patients who have a more vigorous immune system. Thus, a better understanding of the molecular and cellular bases of rejection of BHVs was expected to help to identify relevant targets for intervention and develop new approaches to prevent the associated disease. In this regard, residual quantities of the carbohydrate antigens alpha Gal and Neu5Gc, the main xenoantigens recognised by human natural and elicited antibodies during immune responses against animal-derived antigens, have been found in commercial BHVs. These results are in accordance with early observations of deposition of complement activation products in commercial BHVs. Furthermore, the presence of an inflammatory infiltrate with a predominance of macrophages (CD68+ cells) but also with neutrophils polynucleated white blood cells has been described in BHVs explanted from patients after valve failure. Nevertheless, the mechanisms of recognition, activation and response to BHVs of the innate immune pathways remain unknown. With this in mind, IDIBELL was expected to work on elucidating these mechanisms against commercial BHVs with an emphasis on complement activation and macrophages stimulation by BHV tissues. Studies in α1,3-galactosyltransferase knock-out (alpha Gal KO) mice implanted with BHV pieces were included for further investigation of the relevance of these pathways in vivo.

Related to the potential deleterious effects of macrophages on BHVs, there is clinical and experimental evidence suggesting that nitrosative and oxidative stress can play an important role in cardiac valve deterioration. However, it remains unknown whether any of these factors induce or accelerate the degeneration of BHVs and this was the object of another line of research in TransLink.

Finally, the INSERM team studied the molecular basis of the eventual deleterious systemic effects (i.e. directed to the patient’s own tissues) of the immune response triggered by BHVs. Although there is yet no clear demonstration of clinical deleterious effects of diet-derived anti-Neu5Gc antibodies on autologous cells in human diseases, the use of BHV displaying Neu5Gc residues represents a theoretical risk of systemic inflammation for BHV recipients. The team at INSERM focused its work on studying: 1) the level of diet derived deposits of Neu5Gc on fresh aortic endothelial cells; 2) the changes of the anti-Neu5Gc antibody levels and repertoire in elicited responses after non-diet related immunisation (in collaboration with WP2); and 3) the potential effects of anti-Neu5Gc on endothelial cells. For this last task, the team was expected to analyse the exhaustive transcriptome profiling of a panel of 10 human primary endothelial cells, using deep RNA sequencing.

WORK PACKAGE 4: Potential infectious risk related to the anti-BHV immune response

It is well known that enveloped viruses, such as influenza, HIV and measles, incorporate host cell–dependent, surface antigens including glycoconjugates. HIV and the measles virus incorporate ABO blood group antigens that sensitise virions to complement-mediated inactivation in the presence of corresponding anti-ABO antibodies. The same is true for zoonotic viruses and, in this context, one of the investigators in WP4 has previously reported that humans’ natural anti-αGal antibodies, present in all patients, can inactivate several types of αGal containing enveloped viruses, but not αGal-free viruses, in a complement-dependent way. Alpha-galactosylation of virus particles or antigens had also been reported to enhance their immunogenicity, again signalling the protective effects of anti-carbohydrate antibodies (ACA).

It was a pivotal TRANSLINK issue to identify interactions between microbial pathogens and ACA induced by use of bioprosthetic devices and clarify their potential adverse effects and potential advantage in patients. An additional issue that experts in WP5 were keen to explore was the potential zoonoses from genetically engineered animals that may be utilised as BHV providers and lacking certain glycan antigens.

WORK PACKAGE 5: Remedies: prevention and treatment

The main purpose of this work package within the TRANSLINK project was the development of new technologies with Intellectual Property (IP) that could be used in the clinic for the prevention and treatment of SVD in patients undergoing heart valve replacement.

The working hypothesis of the project was to demonstrate that SVD was an immunologically-driven problem where immune events leads to calcification both of native valves and of the BHV that are implanted to patients and are manufactured with animal tissues, in particular pericardium. The major xenoantigens of potential interest are alpha Gal and Neu5Gc, which have previously been identified as highly immunogenic in xenotransplantation research and are antigens absent in human beings, who have lost the capacity to synthesise these sugars during evolution. Neu5Gc is also taken up by humans through the diet and accumulates in tissues, potentially triggering an autoimmune response. It is known that there could be other xenoantigens present in the animal tissues, yet to be identified, which are generically classified as non-alpha Gal antigens that could be the targets of the immune system.
To address this problem and find solutions that should significantly enhance the lifespan of BHV and decrease the risk of systemic side-effects in the recipients, two strategies were expected to be developed in TransLink: a) Reduction of xeno-antigens in the animal tissues used for valve manufacturing, by combining new genome editing technologies with somatic cell nuclear transfer (SCNT); b) synthesis of new molecules able to bind, thus depleting or inhibiting/neutralizing the patient antibodies directed against the xenoantigens accumulated either through the diet (Neu5Gc) or because still present on chemically processed BHV. To achieve these tasks different objectives were set and distributed between the WP5 partners, as follows:

• Generate, by somatic cell nuclear transfer and genetic engineering, a pig line KO for the CMAH enzyme on a currently available pig Gal-/- line
• Generate a bovine line by somatic cell nuclear transfer and engineering of a Gal / bovine line
• Generate, by somatic cell nuclear transfer and genetic engineering, a bovine line KO for the CMAH enzyme on the aforementioned bovine Gal-/- line.
• Generate, by somatic cell nuclear transfer and genetic engineering, a porcine or bovine line KO for a nhCA on the GGTA1/CMAH-KO genetic background.
• Design and synthesis of novel molecules (glycoconjugates) with binding capacity and neutralisation of several human anti-carbohydrate antibodies (hACA) in the sera of BHV patients.

WORK PACKAGE 6: Dissemination and training

WP6 was expected to build and develop the necessary tools for dissemination, which included brand and imaging, web site, social media and other dissemination tools, as needed. This WP was supposed to monitor and record the dissemination activities undertaken by all partners, making sure that the dissemination and publications of scientific articles were in accordance with the rules of the TRANSLINK Grant Agreement.

Finally, WP6 was expected to organise, with the aid of local partners, two training courses and two dissemination events for the scientific community and another dissemination activity targeting the broader public.

Project Results:
WORK PACKAGE 1: Clinical data, biobanking and statistical analysis

The work undertaken by clinicians in WP1 has been remarkable notwithstanding the difficulties related to the peculiarities of the patients enrolled. Indeed, WP1 investigators were expected to enrol in the various study cohorts 1460 patients whose clinical conditions were often precarious. In this context, it is worth reminding that the majority of the patients enrolled in TRANSLINK were elderly individuals, often physically debilitated and with financial constraints.

Nonetheless, as far as the enrolment, the WP1 results can be summarized as follows:

• Phase A: The enrolment of the patients started in January 2014. Patient enrolment was completed by December 31st, 2017 and exceeded the 140 patients originally scheduled. A total of 174 patients with SVD were enrolled in Cohort A. Cardiac echography and blood samples have been performed at the time of SVD diagnosis. Patients with different types of commercially available bioprosthetic heart valves (BHV) were enrolled (14 types).

• Phase B: At the end of the enrolment period (June 2016), 502 BHV recipients have been enrolled in the B1 cohort, which exceeded the 400 scheduled patients. Different types of BHV recipients have been enrolled in the B1 cohort (14 types). The most commonly implanted valves were bovine (86 %) followed by porcine (10%) and finally equine (4%), reflecting specific surgical practice followed at each centre. For the B1 control cohort, 120 patients were enrolled, which exceeded the target of 100. Of these, 48% had had a mechanical valve implanted and 52% had undergone coronary artery bypass graft (CABG) surgery. 71.4% of B1 control patients had at least 4 clinical visits with collection of blood samples.

As far as cohort B2, at the end of the enrolment period, 771 patients had been enrolled, exceeding the original target of 720 patients, with 130 controls. Each patient had at least two echocardiographic scans and clinical time-point evaluations (at the inclusion visit, at 12 months and 12 – 24 months). 72% of B2 patients underwent at least 2 clinical visits with collection of blood samples and echocardiogram.

Samples were delivered to TRANSLINK Partners in WP2 every 6 months by specialist courier companies authorised to transport human biological samples.

Therefore, enrolment of a large international and prospective cohort of 1,697 BHV patients has been achieved. The ambitious enrolment target of 1,460 patients was exceeded.

Clinical and biological data collected from the 1,697 patients enrolled were made available to all partners via an Excel file extracted from the study’s electronic database. In total, 4,381 blood samples were collected during the study and shipped to the TRANSLINK laboratories.

The TRANSLINK clinical trial was an exceptional opportunity to acquire a unique collection of samples that may form the basis for novel areas of future academic or private research related to the use of BHV implantations in humans. For this reason, a TRANSLINK bio-collection from each clinical partner (Nantes, Bellvitge, Vall d’Hebron, Padova and Manitoba) has been constituted and stored at each centre.

Overall, during the TRANSLINK project, 2,800 echocardiographic investigations have been carried out across the 3 different cohorts (A, B1 and B2). Data from all echocardiographic investigations for each centre (Padua, Barcelona, Nantes and Manitoba) were recorded in the eCRF and checked for consistency. Echo examinations were coded (anonymised) and transferred to Nantes’s CoreLab. All these echocardiographic investigations were analysed to determine the bioprosthesis score developed specifically within the TRANSLINK project.

Furthermore, the TRANSLINK clinically study has also resulted in the following unique output:
1. Enrolment of a large variety of surgical or TAVI implanted BHV worldwide (12 bovine valves, 8 porcine valves and 2 equine valves)
2. Constitution of a unique biological collection from 1,697 patients receiving an aortic BHV to identify immune biomarkers associated with SVD.
3. Implementation of a database (electronic Case Report Form, eCRF) which contains a large amount of clinical, biological and echocardiographic data. This database represents a potential opportunity for future BHV research. It is noteworthy that, in total and as of February 28th, 2018 (month 54), 388.118 measurements were present in the eCRF and have been monitored for all patients
4. Development of a new echocardiographic bioprosthesis score for early and standard SVD assessment.
5. Development of complementary imaging of SVD by using a CT-Scan, in order to better assess calcification rate of BHV (beyond the initial goals of the project).
6. First study suggesting an association between immunological response and SVD.

WORK PACKAGE 2: Biochemical characterization of engineered BHV and analysis of the anti-BHV humoral immune response

State-of-the-art technologies have been developed to biochemically characterise the most commonly used BHVs in order to identify the various potential xeno-antigens in BHV-related complications. This was achieved by the joint efforts of all WP2 participants.
Our initial goal was to analyse sialic acid targets in BHV and to determine the various sialic acid types and O-acetylation. The two common sialic acids on mammalian cell surfaces are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-glycolylneuraminic acid (Neu5Gc). Humans are unable to synthesise Neu5Gc, unlike other mammals, and therefore Neu5Gc is considered foreign in humans and recognised by circulating anti-Neu5Gc antibodies. In fact, humans have circulating antibodies against diverse glycans containing Neu5Gc due to function-loss mutation of the CMAH gene. Therefore, Neu5Gc may be a non-Gal carbohydrate xeno-antigen in BHV potentially recognised by circulating human anti-Neu5Gc antibodies, thereby likely contributing to BHV structural deterioration in time within human patients.
We investigated non-engineered animal-derived cardiac tissues and clinically used commercial BHV for expression of Neu5Gc by HPLC and by various immunostaining analysis. Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2-3>Siaα2-6 expression. To evaluate sialic acid content in tissues, sialic acid was released from samples with acid hydrolysis, then derivatised with 1,2-diamino-4,5-methylenedioxybenzene to generate a fluorescent derivative. Sialic acids were analysed by fluorescence detection using reverse-phase high-performance liquid chromatography (DMB-HPLC). Quantitative analysis of sialic acids by HPLC showed that porcine/bovine pericardium express 4-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6-fold over Neu5Gc. Likewise, we show that Neu5Gc was expressed on commercial BHV, with Neu5Ac at 8-fold over Neu5Gc.
Affinity-purified human anti-Neu5Gc IgG showing high specificity toward Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition of BHV. Overall, these data conclusively demonstrated Neu5Gc expression in native animal cardiac tissues, as well as in six commercial BHV. Altogether, these findings suggest that BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration, warranting further investigation.
We characterized the nature and magnitude of anti-Neu5Gc antibodies following non-diet immunization. Neu5Gc is abundant in red meat, xenografts and some biotherapeutics. Low levels of Neu5Gc accumulated on normal human endothelial cells from the diet, and together with circulating anti-Neu5Gc antibodies could potentially mediate “xenosialitis” chronic-inflammation. Rabbit anti-human thymocyte globulin (ATG) is a polyclonal IgG glycoprotein drug that is commonly used in human transplantation and autoimmune diseases as an immunosuppressant. It was previously shown that in type-1 diabetes patients, administration of Neu5Gc-glycosylated ATG resulted in an increased total anti-Neu5Gc response (collaboration with WP3). To elucidate eventual differences in ATG-elicited anti-Neu5Gc repertoire as a model of non-diet immunization shared with BHV implantation, we used a unique sialoglycan microarray with multiple glycans containing terminal Neu5Gc or matching pairs of control Neu5Ac-containing glycans. We examined samples before and after ATG administration and followed their kinetics with time (0, 1, 18 and 24 months). We found that the response of all basal-pre-existing Neu5Gc-specific antibodies had rapidly increased. This response peaked at one month post-ATG, with enhanced affinity, then resolved at 18 – 24 months. Furthermore, induced-antibodies revealed expanded diversity and de-novo recognition of different Neu5Gc-glycans, including those of endogenous glycolipids. These findings were further validated by affinity-purified anti-Neu5Gc antibodies from patients’ blood. Together, these data strongly suggest that ATG-induced anti-Neu5Gc IgGs, represent a secondary exposure to this dietary carbohydrate-antigen in humans, with immune memory. Given their modified recognition patterns, ATG-evoked anti-Neu5Gc antibodies could potentially mediate biological effects different from pre-existing antibodies (from work in WP3). It remains to be seen whether such responses also follow BHV administration and complications.
To expand our investigation of potential xenogeneic targets on BHVs, we aimed to characterise glycosphingolipid (GSL) targets on BHV-related tissues that are used as sources for BHV preparations. Glutaraldehyde-treated BHV that are made of porcine or bovine tissues are currently used for clinical replacement of diseased heart valves. However, the durability of these valve cusps is limited potentially due to the inception of immune responses against such grafts. The xenoantigen αGal and its corresponding circulating anti-Gal antibodies have been postulated to partially contribute to BHV complications. However, the presence of other non-Gal carbohydrate xeno-antigens, and the potential immune response against these non-Gal antigens had not been studied previously, as well as the inflammatory response that could potentially be generated by their interaction with the patients’ endothelial cells.
We have isolated and structurally defined both non-acid and acid glycosphingolipids from naïve porcine aortic and pulmonary valve cusps. Total non-acid and acid glycosphingolipids were isolated from porcine aortic and pulmonalis valve cusps of 20 animals. Glycosphingolipid components were structurally characterised by thin-layer chromatography, liquid chromatography-mass spectrometry and binding of monoclonal antibodies and lectins. The non-acid glycosphingolipids were characterised as globotetraosylceramide, H-type 2 pentaosylceramide, fucosyl-gangliotetraosylceramide, and Galα3neolactotetraosylceramide. The acid glycosphingolipid fractions had both sulphatide and gangliosides (GM3, GM2, GM1, fucosyl-GM1, GD3 and GD1a), and all gangliosides contained Neu5Ac. Interestingly, although Neu5Gc is a major component in many pig organs and humans have circulating antibodies against it, Neu5Gc was not detected within the purified gangliosides. In summary, pig valve cusps contain several complex lipid-bound carbohydrate structures that could potentially be targeted by the human immune system. Notably, the Neu5Gc determinant was absent in the cusp gangliosides. This work forms a platform for further characterizing the antibody reactivity of patients with porcine-derived BHV.
To investigate the complete humoural response in BHV recipients and controls of the TransLink cohorts, we have established diverse high-throughput assays to measure antibodies to diverse carbohydrate xeno-antigens. We used both ELISA as well as state-of the art glycan microarrays. Given the fact that antibody binding specificity could be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate, we investigated potential xeno-antigens as free carbohydrate chains (glycans), glycan polymers, glycoproteins and glycosphingolipids. Owing to the pathobiological significance of some glycans, we have also glyco-engineered cells to secrete mucin-type immunoglobulin-fused proteins carrying diverse glycans on defined core saccharide chains. Subsequent Western blot and liquid chromatography-mass spectrometry analysis allowed full characterisation of multiple recombinant mucins that carrying a highly diverse repertoire of glycosylated targets.

We investigated the immune response developed in BHV recipients and controls against the diverse xeno-antigens while blinded to the identities of samples. Once assays were completed, samples were de-identified to monitor and evaluate the developed immune responses in the different samples. All humoral response measurements of all WP2 groups on the samples received from WP1 have been completed by the end of the project, as planned. All humoral response measurements were conducted blind to case/control status and, once finalised, samples were de-identified for further statistical analysis. Initial statistical analysis provided a glimpse into those responses.

Initial statistical analysis revealed some potential xenogeneic targets, while leaving other targets uninvolved in BHV complications. We anticipate that the full investigation would reveal important insights into the factors that contribute to BHV deterioration in human patients with some practical implications such as using BHV from engineered donor lacking Gal or Neu5Gc antigens, and guidelines that could potentially be introduced to clinicians and patients.

The analyses conducted to date clearly show: 1) that the BHV are antigenic; 2) that not all carbohydrate antigens participate in the response to BHV; and 3) a hierarchy in the antibody response against carbohydrates. Together these data provide a molecular basis that gives credit to the hypothesis that the immune response against BHV may contribute to SVD. These findings will further be corroborated by an elaborated statistical analysis that is underway.

WORK PACKAGE 3: Mechanisms of BHV damage and possible recipient disease mediated by the anti-BHV immune response

As part of WP3, the team at IDIBELL focused on studying the innate immune responses against BHVs. To this end, in-vitro assays were set up to quantify and characterise complement activation triggered by BHVs when exposed to normal human serum. Three commercial BHVs were studied (one porcine, one bovine and one equine), which displayed different patterns of complement activation. The predominance of one complement pathway over another and the participation of anti-Gal antibodies were assessed in this process. The results from IDIBELL show that complement activation and the resulting anaphylatoxin generation may have an impact on the overall immune response to BHVs and may contribute to SVD in the long term.

Likewise, cell-based assays were developed at IDIBELL to study the immune response of human monocytes to commercial BHVs. Notably, incubation of human monocytes from healthy donors with pieces of five different BHVs in the absence of human serum or any other additional stimulus triggered a response that differed depending on the BHV and the donor. The study of the secretome showed that human monocytes release pro-inflammatory cytokines and chemokines in response to some BHVs. These studies also provide information on additional proteins that may play a role in the mechanisms of adaptation of the recipients to the BHV after implantation. This experimental setting was also used to assess the nitrosative and oxidative response of the monocytes/macrophages triggered by the various BHVs by measuring nitric oxide and nitrotyrosine in supernatants. The data generated provided very valuable information that could be related to clinical observations made in patients implanted with the assessed bioprostheses. In summary, the findings obtained through TRANSLINK showed that commercial BHVs trigger activation of human monocytes with various levels of intensity and complexity depending on the donor and the BHV. The induced inflammatory response may have an impact on the overall integrity of the BHV and may contribute to SVD.

A model of BHV degeneration was developed using Gal KO mice from the IDIBELL colony, which spontaneously generate natural anti-Gal antibodies. The model has been established using two age ranges (adult and old) and controlling within the same experimental cohort for gender and initial anti-Gal IgM and IgG antibody titres. The procedure involved the subcutaneous implantation of small pieces of BHV on the mouse’s back and follow up for 2 or 4 months. The histological analyses of the retrieved grafts provided a good tool for comparing the cellular immune response to different commercial BHVs, whereas the anti-carbohydrate antibody response was also measured for comparative studies. We studied four different commercial BHVs, one equine and three bovine. Our findings showed that commercial BHVs trigger a cellular immune response when implanted subcutaneously in Gal KO mice. This response varied depending on the recipient and the BHV, allowing identification of the BHV triggering the strongest cellular reactions. Notably, the data generated in this mouse model are consistent with the results obtained with in-vitro studies using human monocytes. Furthermore, a few BHVs have been retrieved from patients at IDIBELL due to SVD and the histological analysis demonstrated the presence of macrophages. In summary, these studies provided further evidence of the potential contribution of the immune response to SVD and provided a tool for comparative and mechanistic studies that set the bases for future improvement of BHVs.

Results also showed that patients with an implanted biological prosthesis develop progressive nitrosative stress. Changes were more significant when the prosthesis was made from bovine pericardium than when made from porcine aortic valves. However, oxidative stress, as indicated by the assessment of malondialdehyde (MDA) in plasma, was more prevalent in patients with porcine aortic valve prostheses than in those with bovine pericardium. Importantly, the antioxidant capacity, as assessed by the plasma uric acid, was not influenced by the type of prosthesis, as it was similar in all study groups, including controls. Taken together, the results from this study suggested that nitrosative and oxidative stress might play a role as effectors of SVD and that post-translational modification of bioprosthesis proteins might be an important mechanism of SVD. Additional laboratory and clinical studies will help to establish the relevance of this concept and set the bases for therapeutic intervention of these pathways.
Studies of the potential effects of anti-Neu5Gc antibodies also provided insight into possible side effects of the immune response induced by BHVs on recipients own endothelial cells that could generate systemic vascular inflammation. Using sialic acid arrays, in collaboration with TAU in WP2, INSERM documented for the first time that the antibody repertoire for the various Neu5Gc epitopes can be substantially altered in responses elicited by non-diet related immunisation, comprising also antibodies with high affinity against Neu5Gc.

INSERM investigated the effect of these elicited versus “natural” diet-derived anti-Neu5Gc antibodies on human endothelial cells at the whole transcriptome level by RNA deep sequencing using, for the first time, a source of elicited anti-Neu5Gc antibodies (immune-affinity purified by WP2). The study provided a first insight into whether elicited anti-Neu5Gc antibodies could trigger new signalling pathways on human Neu5Gc-positive endothelial cells. The current tentative conclusion suggested that, indeed, the non-dietary immunisation definitively mobilised a substantial number of genes in comparison with the response to “natural” diet-induced antibodies, but it did not produce a clear bias of the transcriptome pattern towards inflammation. In addition, this team provided the first analysis of the physiological loading of Neu5Gc onto the surface of 14 fresh human endothelial cell preparation and showed an unexpected heterogeneity in normal donor endothelial cells. In fact, one third of individuals analysed lacked detectable membrane Neu5Gc in aortic endothelial cells.

Altogether, the patterns produced by elicited anti-Neu5Gc antibodies on endothelial transcriptome did not suggest an activation profile classically associated with vascular diseases, although the data showed that these antibodies induced substantial changes in the endothelial transcriptome that might contribute to endothelial cells’ homeostasis. However, patterns of transcription do not equal in functional in-vivo effects. Nevertheless our in-vitro data were consistent with the lack of clear evidence of vascular inflammation induced in vivo by anti-Neu5Gc antibodies in a situation where a strong exposure to elicited anti-Neu5Gc antibodies is documented (at least for several months in the patients whose sera were used in our studies) and in whom no clinical symptoms of vascular toxicity were reported in these patients. Nevertheless the demonstration that purified anti-Neu5Gc antibodies mobilised a large set of transcripts, whatever the interpretation of their ultimate effect, supported the use of Neu5Gc-deficient BHVs to avoid any of these potential risk.

WORK PACKAGE 4: Potential infectious risk related to the anti-BHV immune response

• Determination of anti-carbohydrate antibodies involved in the haematological dissemination of bacteria.

The purpose of this part of study carried out at ICS was to determine the profile of anti-carbohydrate antibodies (ACA) associated with the haematological dissemination of the four microorganisms that most often cause biological heart valve endocarditis (BHVE) (Enterococcus faecalis, Staphylococcus aureus, Coagulase-negative staphylococci, and Streptococcus viridans). The methodology included in the case of E. faecalis was the intraperitoneal injection to BALB/c mice of different strains of the bacterium and the comparison of the ACA profile existing in animals before and after the infection using the printed glycan array (PGA) technology. For the other three pathogens, bacteria were incubated with a pooled human serum to promote antibody binding to the bacteria. Unbound fraction of antibodies was collected and analysed, taking baseline conditions as the reference of ACA levels.

Our study allowed the identification of ACA with particular affinities to antigens expressed on the bacterial surface of pathogens causing endocarditis. E. faecalis and S. aureus strains were poorly recognised by the repertoire of circulating anti-glycan antibodies of mouse and human, respectively. Interestingly, the Galβ residues identified in most E. faecalis and S. aureus samples were restricted and very similar, suggesting a potential role of the antibodies targeting these structures as facilitators of the infections caused by these two pathogens. By contrast, the recognition of coagulase-negative staphylococci and S. viridans by the human repertoire of ACA was more extended. The carbohydrate antigens bound by ACA in coagulase-negative staphylococci and S. viridans included different terminal glycan residues. There was not a restricted and conserved pattern as the one observed for E. faecalis and S. aureus, making the consideration of any of the described ACA as facilitators of infections caused by coagulase-negative staphylococci and S. viridans difficult.

• Removal of ACA involve in bacteraemia using WP5 compounds

In order to define the significance of these specific ACA on bacterial infections, we planned to develop a system for the removal of ACA involved in bacteraemia using WP5 compounds. As above, we identified antibodies targeting βGal antigens as potentially involved in the facilitation of infections caused by E. faecalis and S. aureus. Unfortunately, as result of technical difficulties in the production of polymeric glycoconjugates (see Deliverables 5.4 and 5.6) and delays in the identification of the participating antibodies, the production of specific compounds to remove anti-βGal has not been feasible within the project framework. However, we were able to test some intermediate molecules produced by IUCT in WP5.

In collaboration with WP5 partner IUCT we demonstrated in vitro the binding and “inhibition” of human anti-poly-N-acetylglucosamine (PNAG) and anti-hyaluronic acid antibodies by glycoconjugates containing a single NAG residue. The result supported, in the case of glycoconjugates bearing NAG, the TRANSLINK hypothesis that these glycoconjugates are useful to deplete antibodies against these moieties. More work should be done to improve the results with the available compounds, and to extend the concept to other anti-carbohydrate antibodies such as the anti-βGal identified to bind E. faecalis and S. aureus.

• Virus particle based ACA assays with an emphasis on SDa antigen

Assays to study biological properties of the anti-carbohydrate antibodies (ACA) responses using virus particles displaying relevant glycan antigens (αGal, Neu5Gc and SDa) were developed. First, a human cell line was genetically engineered to express these three glycan antigens followed by culture condition optimisation to ensure specific display of Neu5Gc on the surface of cells with the cmah gene. Retroviral particles that displayed corresponding glycan antigens were produced from the above cell lines, representing enveloped viruses. These virus particles encode GFP or luciferase marker genes, enabling simple quantification of virus titre and allow some biological function of corresponding antibodies to be determined. Three assays were developed, refined and established: (i) virus binding, (ii) complement-mediated virus killing and (iii) antibody mediated enhancement (ADE) of virus infection. However, the validation of these assays for anti-ACA antibodies has yet to be completed. Potential problems at this stage are (i) the sensitivity may be good enough for viruses bearing αGal, but not Neu5Gc and/or SDa-like glycans; (ii) these antibodies may not have ADE activity as, so far, ADE has not been detected.

As UCL focused on the SDa antigen and anti-SDa antibodies because the study of other two antigens, αGal and Neu5Gc, was covered by other consortium partners, the virus-based anti-ACA antibody binding assays turned out to be insufficiently sensitive for anti-SDa antibody. We hoped that our antibody binding assays could have identified anti-SDa antibody positive samples early on, as we expected we would need such positive samples to demonstrate usefulness of all developed assays for the TRANSLINK BHV patient studies and optimise these assays. We therefore put an effort to develop alternative ELISA assays for antibody binding in collaboration with UGOT. This is based on recombinant PSGL1/mIgG2b molecules and its development is still ongoing. The lack of positive serum samples has been problematic also for the other assays, complement killing and antibody dependent enhancement of infection, because these antibody actions would not be expected if there is no antibody binding to the antigen.

In summary, potentially useful reagents, e.g. genetically engineered, specific glycan-expressing cell line, virus particles and recombinant PSGL1/mIgG2b molecules produced from these cells, and assays were developed. Demonstration of their potential clinical usefulness and further optimisation will be made possible upon provision of positive control samples, such as serum samples from vets with frequent surgical exposure to animals.

WORK PACKAGE 5: Remedies: prevention and treatment

• Genome editing of the pig line to knock out (KO) Gal and Neu5Gc non-human carbohydrate antigens (nhCA). (AVT, collaboration with INSERM)

The generation of animals with less immunogenic tissues originates from a previous FP6 project (XENOME, LSHB-CT-2006-037377) in xenotransplantation where pigs KO for Gal were obtained and used in pig-to-primate xenotransplantation studies. Avantea in this project used the Gal KO line (obtained from a minipig line supplied by Dr. Sachs, Massachusetts General Hospital, USA) to add the KO of the Neu5Gc antigen by inactivating the enzyme CMAH encoding cytidine monophosphate-N-acetylneuraminic acid hydroxylase, an enzyme responsible for Neu5Gc biosynthesis. CMAH sequences were studied and validated in the GAL KO pig to be used. In this experiment, we elected to use the TALENs nucleases in combination with a targeting vector carrying puromycin as genome editing platform to facilitate the selection of edited cells. Male fibroblasts from Gal KO minipigs were nucleofected with TALENs and the targeting vector. After clonal selection cell clone were genotyped and sequenced and those carrying the biallelic mutation of CMAH were used in somatic cell nuclear transfer (SCNT) to generate live animals.

After birth, the piglets were confirmed again for the mutations expected (Gal and CMAH, defined as double KO, DKO), both by DNA sequencing and by immunophenotyping by FACS. These animals provided biological material (serum, red blood cells, valves, pancreatic islets, etc.) for the partners of TRANSLINK to complete their tasks. In particular, it was confirmed in a mouse model KO for Neu5Gc, that xenotransplantation of DKO pig islets did not elicit an immune response to Neu5Gc and that the islets were functional.

During the course of the project, thousands of sera from patients receiving BHV were screened to identify antibodies directed towards known antigens (Gal and Neu5Gc), but also to unknown potentially immunogenic nhCA and targets of the immune system. Because no novel nhCA were identified in TRANSLINK, we also decided to KO the recently discovered nhCA xenoantigen, B4GalNT2. For the KO of this nhCA, we used the CRISPR/Cas9 genome-editing platform. Because of the increased efficiency of genome editing using programmable nucleases, we decided to generate a completely new pig line. The pig line was selected for being PERV C negative and low in PERV A and B, as this is a safety requirement that will eventually be required for undertaking clinical trials with such tissues. To complete the targeting tools, we synthesised the TALENs for the GGTA1 (α-1,3-galactosyltransferase) responsible for the synthesis of Gal (galactose-α (1,3)-galactose). Pig fibroblasts were transfected with TALENs targeting the GGTA1 and CMAH as well as CRISPR targeting the B4GalNT2 without the use of a selectable marker as it was done in the first experiment. After transfection, cells were sorted using immunomagnetic beads carrying a lectin (IB4) that binds Gal. The cells that did not bind to the beads, presumably Gal KO, were plated for clonal selection. Growing colonies were split into two samples, one cryopreserved for SCNT and half used for genotyping. We first genotyped for CMAH KO and those that were KO for the CMAH were further genotyped for the KO of B4GalNT2. We also confirmed that the Gal was KO as expected. The colonies of cells that were triple KO (Gal, Neu5GC and B4GalNT2) were then used in SCNT to generate live pigs. After birth, the piglets were again genotyped but also phenotyped using FACS and confirmed to be triple KO for the three nhCA. The animals generated were male, did not show any abnormality, grew to adulthood and reached puberty within the normal range. Semen was collected from all of them and used for insemination and F1 generation obtained therefore confirming the absolute normality of these animals and the absence of any side effects attributable to the genome editing procedure using nucleases.

Breeding of these animals will continue to establish a nucleus herd to generate sufficient numbers of pigs from which to harvest the tissues required for the proof of principle and validation of the new property of triple KO tissues, i.e. which do not bind Gal, Neu5Gc and B4GalNT2 and lack of calcification after in-vivo transplantation into mouse models in collaboration with TRANSLINK partners (studies not included in the project).

Similar work has been undertaken in cattle as the majority of BHV available on the market are manufactured from bovine pericardium. AVANTEA has also filed a patent application to protect the intellectual property of these multiple KO animals for both the biomedical and food sectors, as Gal could be responsible for allergies through the consumption of red meat and Neu5Gc can be accumulated in human tissues through the diet.

• Design and synthesis of novel drug candidates with binding capacity and neutralisation of several human anti-carbohydrate antibodies (hACA) in the sera of patients receiving bioprosthetic hearth valves (BHV). (MtB, IUCT, ICS-HUB)

- Design of drug candidates

Computational models to design drug candidates aimed at depleting natural anticarbohydrate antibodies (NACA) were designed by MtB. These models have allowed the prediction of the “best” overall structure per epitope studied. These models have been developed following the main scheme introduced by IUCT, including the key elements in the structure. This in-silico design process affected all parts of the structure to keep the epitope as exposed as possible. An optimal design per epitope was achieved, taking into account necessary experimental conditions.

To ensure the validity of the results, all the simulations were performed three times with different simulation lengths (varying by a factor of 100, different simulation software and different simulation set-ups (force field and water models).

- Searches for biosimilar compounds

Although outside the scope of the project as it was initially conceived, MtB also sought to discover biosimilar compounds to the natural carbohydrates studied that might emulate their performance.

HA2: The first screening was based on the disaccharide of hyaluronic acid (HA2). A protein receptor, model was set up using different proteins that bind HA. This receptor model allowed us to predict the binding affinity of new molecules to a hypothetical HA antibody. Several compounds were found, but these were not progressed, as a biotechnological alternative to synthesising HA was found and they were no longer needed. MtB also supported IUCT on the biotechnological approach for the production of HA.

Neu5Gc: A second pharmacophore research was undertaken in order to find new compounds that emulate the Neu5Gc molecule. The process was done in two phases. First, a protein model was constructed based on a crystal structure of a sialic binding protein having Neu5Gc as a co-crystal. Using Chembl (a public library of chemical compounds) as a database, a virtual screening was performed. After discussions with IUCT, five compounds were selected as the most promising targets. The second phase was oriented to find commercial analogues of the selected compounds (as it would constitute a cheaper alternative, because commercial compounds are easy to buy and, theoretically, easier to synthesise) and to test the selectivity of these compounds, as they should block Neu5Gc antibodies but not Neu5Ac. To do that, we constructed a second model based on a crystal structure of sialic binding protein having Neu5Ac as a co-crystal. To ensure the results were robust, several models were also constructed, using different proteins having Neu5Gc/Ac as co-crystals. At the end a couple of compounds seemed to present certain selectivity; however further experiments would be needed to confirm this.

In deep synchronisation with compound design, the synthesis of the immunosorbent drug candidate compounds was defined, checked, implemented and optimised to fulfil some requested criteria such as being efficient; meeting industrial production criteria, economically viable and patentable, among others.

IUCT attempted to produce TRANSLINK drug candidates through a combination of organic synthesis and biotechnology, designing chemoenzymatic approaches to achieve the goals of constructing such high molecular weight compounds.

IUCT also developed analytical methods for in-process control during the reaction and post-processing, the development of purification methods and the verification of structural elucidation of these extremely challenging compounds. Last but not least, a synthetic method for certain non-available epitopes was also requested.

The anti-HA antibody drug candidates synthesised by IUCT based on MtB’s designs were sent to ICS-HUB for in-vitro screening. The main goal of this screening was to determine the binding activity against anti-HA antibodies for the available model glycoconjugates. According to what was expected, some model drug candidates showed relatively good inhibition against the anti-HA IgG that are present in the human serum.

Therefore, based on data achieved during TRANSLINK, even in the case of model drug candidates already obtained, we could consider that these compounds behave as immunosorbents useful to deplete anti-HA antibodies, although more work is needed to finalize these results.

• Work on drug candidates to deplete anti-Neu5Gc antibodies is ongoing and a patent is pending.

WORK PACKAGE 6: Dissemination and training

The main results of this WP can be synthesised as follows:
• A dedicated webpage, continuously updated throughout the project
• Dissemination materials for raising the project’s profile, including leaflets, presentation templates, stationary, etc.
• A specific intranet for sharing documentation related to the project
• Organisation of two conferences for the scientific community: one in London and other in Padua
• Organization of an event targeted at the broader public in Cremona
• Organisation of two specialist training courses in Tel Aviv and Cremona
• Publications of over 30 scientific articles
• Participation in more than 130 dissemination activities
• Establishment of metrics to quantify the effectiveness of the dissemination using Google Analytics.

Potential Impact:
WORK PACKAGE 1: Clinical data, biobanking and statistical analysis

With 1,697 patients enrolled from January 2014 to December 2017 at 5 different clinical centres, TRANSLINK has built a powerful clinical and scientific European network, with clinical data and biological specimens that will be invaluable for future cardiovascular research. In particular, TRANSLINK has enrolled the first prospective cohort of patients who underwent aortic valve replacement with an exhaustive clinical, echocardiographic and biological bank.

Furthermore, because pathophysiological mechanisms leading to SVD are probably multifactorial, the TRANSLINK biobank will have the potential to provide thousands of blood samples for other studies about oxidative stress, lipidic and/or calcic metabolism, coagulation disorders or other related investigations.

We also expect that the TRANSLINK cohort size will allow a new statistical predictive model of SVD to be established.

Although the study shows that BHV implantations result in a significant immunisation against Gal and Neu5Gc antigens in the B1 cohort, primary results from Cohort A showed that SVD, diagnosed at a mean follow up of eight years, does not express significant specific immune responses as anticipated. It confirms the design of the study, which allowed the project to assess immune response from the day before implantation to 24 months after implantation and making it possible to detect early specific immune profiles.

The impact of our results on routine patients care has been very effective. Our data clearly indicate that SVD has to be prospectively researched in patients who underwent aortic valve replacement before the 5th year post-surgery. SVD diagnosis can now be based on echocardiographic parameters, without the need to wait for clinical symptoms to develop, which can be too late to allow corrective measures to be taken. In TRANSLINK, 13 % of B2 patients were diagnosed with SVD during the study, which would probably not have happened without their enrolment in this trial. SVD is a tremendous risk factor for mortality after surgery, especially in some cases with “explosive” SVD and early diagnosis of SVD has a real impact on the patients’ prognosis. Indeed, in TRANSLINK such SVD patients were referred to their surgical or cardiology department for appropriate treatment (repeat surgery or TAVI) before life-threatening complications could occur. Therefore, TRANSLINK has potentially saved many patients whose lives would otherwise have been under threat; it has also developed an active and prospective echocardiographic detection of SVD

In this regard, it is noteworthy that, in order to better characterise SVD based on echocardiographic parameters, a new classification with echocardiographic items has been developed by TRANSLINK investigators, allowing better quantification of SVD disease. Efforts will be made after the project to have this method adopted as a standard approach by the clinical community.

Ultimately, TRANSLINK offers the potential to better evaluate SVD treatment and to define which approach (repeated surgery or TAVI) would be more suitable for older patients; in this light, Guidelines for the management and follow-up of bioprosthesis heart valve will have to be modified to allow for full uptake of the project’s results.

Research in “biological tissue valves” and especially pericardial tissue such as that undertaken within TRANSLINK is of utmost importance because a new generation of heart valve bioprotheses that will be implanted with no need for surgery (TAVI BHV) are made up with the same bovine or porcine tissue studied in TRANSLINK. On the other hand, new devices in mechanical cardiac support such total artificial hearts are also using biological tissue to improve biocompatibility of the artificial organ (Carmat TAH).

WORK PACKAGE 2: Biochemical characterization of engineered BHV and analysis of the anti-BHV humoral immune response

WP2 generated advanced technologies to fully characterise potential xeno-antigens in some of the most commonly used BHVs. Selected carbohydrate antigens were characterised in BHV and high-throughput assays have been developed to monitor the immune response in ~4500 samples from BHV recipients and controls. The TRANSLINK cohort was collected from multiple centres across Europe, covering both male and female patients. During the project, samples were collected by the WP1 team, which were then delivered to the WP2 research lab for monitoring of immune responses against diverse antigens. All samples were tested while blinded to case/controls status to avoid bias, then, once immunological analysis had been completed, samples were de-identified for initial statistical analysis. WP2’s immunoassays focused on several xenogeneic carbohydrate antigens, including free glycan chains, polysaccharides, glycoproteins and glycolipids.
Initial unblinded analysis reveals that BHV are antigenic and induce a significant increase in the level of anti-Gal and anti-Neu5Gc in implanted patients (B1 cohort), although their role in the onset of BHV complications is not yet demonstrated. In addition, we noticed that some dietary recommendations might be related to the developed immunological complications. Full statistical analysis is ongoing to monitor effects in individual patients and in concert with data generated in other WPs. These finding are expected to have immediate and important impact on both patients and clinicians, and are likely to generate important recommended guidelines to help alleviate BHV complications in patients.
In WP2, TRANSLINK has generated important outputs for both the scientific community as well as lay individuals that may be impacted by these studies. Our results are likely to find application in diverse areas such as food safety, general health guidelines, and most importantly, some will have a direct impact on public health.
WP2 also contributed to the education and training of scientists and the non-scientific community at various levels. During the project, WP2 members presented their research at important national and international conferences. WP2 organised an international training course for Ph.D. students and postdoctoral researchers from around the world, which included a full materials and methods kit, which received excellent feedback from participants. In addition to the impact on the scientific community, WP2 was also involved with scientific education and dissemination activities to lay audiences. Some WP2 members (e.g. TAU) routinely train undergraduate student in the lab; but, in addition, also train gifted high-school students on a yearly basis for a full year with hands-on research projects during the summer. Furthermore, we host school student classes (all levels) for a day visit in the lab.
The information generated by WP2 has been communicated to scientists as well as the general public in various ways. This included press releases, radio coverage, brochures, posters, flyers, coverage in specialist press, coverage in general non-specialist press, coverage in national press, the TRANSLINK web site for the general public, an event targeting the general public, school students’ open days etc.).
In summary, WP2 results have both scientific and societal impacts; these are likely to lead to further important implications on public health.

WORK PACKAGE 3: Mechanisms of BHV damage and possible recipient disease mediated by the anti-BHV immune response

Results from WP3 support the concept that the innate immune response plays a role in the pathogenesis of SVD and encourage a continuation of research in this field in order to develop new strategies to prevent SVD. Important modifications of transcriptome of aortic endothelial cells triggered by elicited anti-Neu5Gc suggest a possible risk of systemic activation in BHV recipients. We envisage that our investigations oriented towards the elucidation of the immune mechanisms that compromise the function and durability of commercial BHVs will be key for determining new targets for intervention. Importantly, the results from WP3, in collaboration with other work packages, will set the basis for the selection of the best BHVs. Considering that the quality of life of patients with BHVs is much higher than those with mechanical valves (allowing patients to undertake sporting activities, undergo pregnancies, etc.), the prolongation of the half-life of BHVs and the potential use of these prostheses in a wider population (younger and older patients through TAVI) will have a great impact at the clinical, public health and socio-economic levels.

The identification by the IDIBELL team of relevant pathways of the innate immune response contributing to the deterioration of commercial BHVs is of particular relevance for the advancement in the field. Furthermore, the identification of nitrosative and oxidative stress as potentially important players in SVD opens the door to the development of therapeutic approaches directed to reducing the levels of stress in patients with implanted bioprostheses and to effectively slow or abolish the occurrence of SVD. Indeed, the achievement of these goals will have an enormous clinical importance and offer clear economic advantages.

The preliminary conclusions of the RNA-sequencing study following incubation of human endothelial cells with immune affinity–purified anti-Neu5Gc antibodies shows that they induced significant differential expression on an important set of genes. These results thus suggest a safety advantage for using BHVs from genetically-engineered Neu5Gc-deficient animals, which would have a major potential impact by avoiding such potential deleterious immune responses.

Regarding dissemination activities, IDIBELL focused on laboratory training of research assistants, associates and students (including the supervision of a master’s thesis); and seminars for master’s students (including a highly successful exercise/activity related to TRANSLINK). The results from this research must be kept confidential at this stage, as consideration is currently being given to intellectual property protection. Some of the team’s work has been presented at two international congresses.

WORK PACKAGE 4: Potential infectious risk related to the anti-BHV immune response

The data obtained regarding the anti-carbohydrate antibodies reactivity to the different microorganisms involved in the development of endocarditis are already publishable, and a manuscript is currently in preparation. We expect to publish the results in one journal of the first quartile of Infectious Diseases/Microbiology. The particular role of anti-βGal antibodies as potential facilitators of infections caused by E. faecalis and S. aureus will require the production of polymeric glycoconjugates carrying these moieties. The production of these molecules has not been feasible during the project’s time frame; however, there is interest from ICS-HUB and IUCT to acquire additional funding to generate the polymeric glycoconjugates and to test their impact on infection models of E. faecalis and S. aureus, using opsonisation assays. The production of such new molecules would lead to the generation of potentially exploitable IP.

Reagents and assays developed at UCL may prove useful for ACA measurement and characterisation in the laboratory and clinical settings involving use of animal products beyond biological heart valves, e.g. cell, tissue and organ xenotransplantation. To this end further development and optimisation is required and a direction of interest is an ADE assay using flavivirus particles (Dengue, Zika, etc.), rather than HIV particles, because this particular assay has been optimised for Zika virus use. Efforts are underway to seek funding for resources elsewhere to continue this project with NIBSC.

UCL is finalizing data supporting publication in a scientific journal on the development of alternative ELISA assays for antibody binding based on recombinant PSGL1/mIgG2b molecules in collaboration with UGOT.

WORK PACKAGE 5: Remedies: prevention and treatment

Over 300,000 patients a year benefit from BHV replacement although animal-derived BHV can only be implanted in patients over 65 years of age.

As the project data suggest that antibody-mediated responses may contribute to BHV failure through SVD, there is likely to be greater consideration given to the source of the animals used for the manufacture of BHV. There was already evidence that the primary response to BHV implantation was to the Gal antigen and, as already suggested in xenotransplantation studies, more evidence has been provided that amongst the non-Gal antigens, there is a significant surge of antibodies against Neu5Gc.

At this stage, it is expected that future BHV will have a longer lifespan due to the reduced immunogenicity of these tissues, leading to a reduction of the valve leaflet calcifications that usually lead to SVD within 15 years of their transplant. The generation of low immunogenicity animals will potentially benefit not only the manufacturing of more innovative BHV but also xenotransplantation at large for cells, other tissues or solid organs.

Moreover, these animals could be used as a source of red meat for patients allergic to red meat, or, in general, to reduce the consumption of Neu5Gc that is accumulated in epithelia becoming the targets of the human immune system causing inflammation and degeneration.

The potential impact of new drug candidates on the prevention or treatment of SVD in BHV deterioration is noteworthy, since, to our knowledge, apart from the anti-HA antibodies and anti-Neu5Gc antibodies developed during TRANSLINK, the depletion of non-α-Gal deleterious antibodies using therapeutic drugs has never been attempted or reported. The effect of analogous drug candidates in the case of anti-α-Gal antibodies was successfully demonstrated by Novartis in primates, for which the compound GAS914 was deemed to be safe and efficacious. Therefore, taking GAS914 as a reference, we could expect that TRANSLINK’s immunosorbent drugs, once fully developed, could progress into the preclinical stage without major drawbacks.

During the course of the project, all partners have actively participated in dissemination activities and training courses for young investigators by participating at scientific conferences like the International Xenotrasplantation Association in Melbourne in 2015 and in Baltimore in 2017. One dissemination event was held in London in 2016, “Developing transgenic pigs for organ xenotransplantation into humans – Research on animal-derived products for use in the clinic”. Genome editing was also the subject of another dissemination event in Cremona, where a training course on this subject was organised and delivered. The basis of Drug Design, Discovery and Development in TRANSLINK was presented in two main dissemination activities performed during the project: “Drug Design, Discovery and Development” presentation in the “Le patologie cardiache: viaggio al cuore dell’innovazione terapeutica tra chirurgia, chimica e biotecnologie” (Cremona, May 17th 2017) and “New drug candidates to protect from SVD” in the “Animal-derived heart valves for the treatment of cardiac patients: state of the art. EU-funded dissemination conference” (Padova, January 23rd 2018).

The SME participants in TRANSLINK plan to exploit their results through the sales of the new goods, services and knowledge obtained during the project. Particularly preferred is licensing out patent exploitation. The KO animals generated in the TRANSLINK project have been patent protected. This will create the opportunity for industrial developments for the next generation of BHV and the creation of niche markets for safer and healthier red meat for some categories of citizens. Avantea plans to establish a breeding herd of these genome edited animals and making them available through partnerships or licensing to other companies.

Particularly, the removal of deleterious antibodies by immunoadsorption has never reached the market, nor been commercially exploited. Regarding the new drug candidates, anti-HA antibody glycoconjugates have been already protected by the patent application WO2014/02650 A1, through a research collaboration agreement among IUCT, MtB and ICS during TRANSLINK that allows its exploitation. The application entered the national phases in October 22th 2015, in Europe, USA, Canada and Japan (41 countries). In 2017, it has been granted in USA (US 9,719,987 B2). Currently, the applicants are waiting for European Patent Office communications.

Regarding the new drug candidates for anti-Neu5Gc antibody glycoconjugates, the products are still under development and therefore, their patent protection is pending.

WORK PACKAGE 6: Dissemination and training

Work Package 6 allowed TRANSLINK to disseminate its results to more 27,000 people on five continents. More than 7,000 people visited the web for news, updates and event information.
At dedicated events, 13 students were trained in glycoimmunology and genome editing; 118 scientists and students participated at TRANSLINK events in London and in Padova; and 250 secondary school students attended the dedicated TRANSLINK promotional event in Cremona.
Over 30 peer-reviewed publications were released during the lifetime of the project, directly referencing TRANSLINK. These have been cited 189 times at the time of writing this report.

List of Websites:
TransLink project website:

Attached documents:

-TransLink project logo
-TransLink project leaflet
- List of contributors and contact
- Leaflet of the Joint TRANSLINK-XENOISLET dissemination conference, London UK November 2016
- Leaflet of the Dissemination event, Cremona IT, May 2017
- Leaflet of the Final Dissemination event: Animal-derived heart valves for the treatment of cardiac patients: state of the art, Padova - IT January 2018
- Interview of Dr Cozzi during the final dissemination event in Padova - Italy