"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

The ability of human TIM1 to bind phosphatidylethanolamine enhances viral uptake and efferocytosis compared to rhesus and mouse orthologs.

T-cell immunoglobulin and mucin (TIM) family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals, such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and viral infection. Here, we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE, and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1.IMPORTANCEWe previously reported that human T-cell immunoglobulin and mucin protein 1 (TIM1) binds phosphatidylethanolamine (PE) as well as phosphatidylserine (PS), and that PE is exposed on the apoptotic cells and viral envelopes. Moreover, TIM1 recognition of PE contributes to phagocytic clearance of apoptotic cells and virus uptake. Here, we report that unlike human TIM1, murine and rhesus TIM1 orthologs bind only PS, and as a result, their ability to clear apoptotic cells or promote virus infection is less efficient. These findings are significant because they imply that the activity of TIM1 in humans is greater than what the studies conducted in common animal models would indicate.

Phosphatidylethanolamine and Phosphatidylserine Synergize To Enhance GAS6/AXL-Mediated Virus Infection and Efferocytosis.

Phosphatidylserine (PS) receptors mediate clearance of apoptotic cells-efferocytosis-by recognizing the PS exposed on those cells. They also mediate the entry of enveloped viruses by binding PS in the virion membrane. Here, we show that phosphatidylethanolamine (PE) synergizes with PS to enhance PS receptor-mediated efferocytosis and virus entry. The presence of PE on the same surface as PS dramatically enhances recognition of PS by PS-binding proteins such as GAS6, PROS, and TIM1. Liposomes containing both PE and PS bound to GAS6 and were engulfed by AXL-expressing cells much more efficiently than those containing PS alone. Further, infection of AXL-expressing cells by infectious Zika virus or Ebola, Chikungunya, or eastern equine encephalitis pseudoviruses was inhibited with greater efficiency by the liposomes containing both PS and PE compared to a mixture of liposomes separately composed of PS and PE. These data demonstrate that simultaneous recognition of PE and PS maximizes PS receptor-mediated virus entry and efferocytosis and underscore the important contribution of PE in these major biological processes.IMPORTANCE Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are usually sequestered to the inner leaflet of the plasma membrane of the healthy eukaryotic cells. During apoptosis, these phospholipids move to the cell's outer leaflet where they are recognized by so-called PS receptors on surveilling phagocytes. Several pathogenic families of enveloped viruses hijack these PS receptors to gain entry into their target cells. Here, we show that efficiency of these processes is enhanced, namely, PE synergizes with PS to promote PS receptor-mediated virus infection and clearance of apoptotic cells. These findings deepen our understanding of how these fundamental biological processes are executed.

The ability of human TIM1 to bind phosphatidylethanolamine enhances viral uptake and efferocytosis compared to rhesus and mouse orthologs.

T-cell Immunoglobulin and Mucin (TIM)-family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and virus infection. Here we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1.

An iterative and interdisciplinary categorisation process towards FAIRer digital resources for sensitive life-sciences data.

For life science infrastructures, sensitive data generate an additional layer of complexity. Cross-domain categorisation and discovery of digital resources related to sensitive data presents major interoperability challenges. To support this FAIRification process, a toolbox demonstrator aiming at support for discovery of digital objects related to sensitive data (e.g., regulations, guidelines, best practice, tools) has been developed. The toolbox is based upon a categorisation system developed and harmonised across a cluster of 6 life science research infrastructures. Three different versions were built, tested by subsequent pilot studies, finally leading to a system with 7 main categories (sensitive data type, resource type, research field, data type, stage in data sharing life cycle, geographical scope, specific topics). 109 resources attached with the tags in pilot study 3 were used as the initial content for the toolbox demonstrator, a software tool allowing searching of digital objects linked to sensitive data with filtering based upon the categorisation system. Important next steps are a broad evaluation of the usability and user-friendliness of the toolbox, extension to more resources, broader adoption by different life-science communities, and a long-term vision for maintenance and sustainability.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

AAV vectors engineered to target insulin receptor greatly enhance intramuscular gene delivery.

Adeno-associated virus (AAV) is one of the most commonly used vectors for gene therapy, and the applications for AAV-delivered therapies are numerous. However, the current state of technology is limited by the low efficiency with which most AAV vectors transduce skeletal muscle tissue. We demonstrate that vector efficiency can be enhanced by modifying the AAV capsid with a peptide that binds a receptor highly expressed in muscle tissue. When an insulin-mimetic peptide, S519, previously characterized for its high affinity to insulin receptor (IR), was inserted into the capsid, the AAV9 transduction efficiency of IR-expressing cell lines as well as differentiated primary human muscle cells was dramatically enhanced. This vector also exhibited efficient transduction of mouse muscle in vivo, resulting in up to 18-fold enhancement over AAV9. Owing to its superior transduction efficiency in skeletal muscle, we named this vector "enhanced AAV9" (eAAV9). We also found that the modification enhanced the transduction efficiency of several other AAV serotypes. Together, these data show that AAV transduction of skeletal muscle can be improved by targeting IR. They also show the broad utility of this modular strategy and suggest that it could also be applied to next-generation vectors that have yet to be engineered.

Zika Virus-Immune Plasmas from Symptomatic and Asymptomatic Individuals Enhance Zika Pathogenesis in Adult and Pregnant Mice.

Preexisting immunity against dengue virus or West Nile virus was previously reported to mediate antibody-dependent enhancement (ADE) of Zika virus (ZIKV) infection in a mouse model. We show here that ZIKV-immune plasma samples from both symptomatic and asymptomatic individuals mediated ZIKV ADE of infection in vitro and in mice. In a lethal infection model with a viral inoculum 10 times higher, both ADE and protection were observed, depending on the amount of infused immune plasma. In a vertical-transmission model, ZIKV-immune plasma infused to timed pregnant mice increased fetal demise and decreased the body weight of surviving fetuses. Depletion of IgG from an immune plasma abolished ADE of infection, and the presence of purified IgG alone mediated ADE of infection. Higher viral loads and proinflammatory cytokines were detected in mice treated with ZIKV-immune plasma samples compared to those receiving control plasma. Together, these data show that passive immunization with homotypic ZIKV antibodies, depending on the concentration, could either worsen or limit a subsequent ZIKV infection.IMPORTANCE Antibody-dependent enhancement (ADE) of virus infection is common to many viruses and is problematic when plasma antibody levels decline to subneutralizing concentrations. ADE of infection is especially important among flaviviruses, many of which are the cause of global health problems. Recently, human plasma samples immune to heterologous flaviviruses were shown to promote Zika virus (ZIKV) infection. Here we showed in immunocompromised mouse models that homologous immune plasma samples protect mice from subsequent infection at high antibody concentrations but that they mediate ADE of infection and increase ZIKV pathogenesis in adult mice and fetal demise during pregnancy at low concentrations.