Integrating artificial intelligence-based epitope prediction in a SARS-CoV-2 antibody discovery pipeline: caution is warranted.

BACKGROUND: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes. METHODS: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs. FINDINGS: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner. INTERPRETATION: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection. FUNDING: Full list of funders is provided at the end of the manuscript.

Radiolabeled gelatin type B analogues can be used for non-invasive visualisation and quantification of protein coatings on 3D porous implants.

This study covers the quantification of the covalent attachment of gelatin type B (GelB) and the subsequent adsorption of Fibronectin (Fn) on poly-ε-caprolactone (PCL) surfaces, functionalised with 2-aminoethyl methacrylate (AEMA) by means of post-plasma UV-irradiation grafting. As typical surface characterisation tools do not allow quantification of deposited amounts of GelB or Fn, radiolabeled analogues were used for direct measurement of the amount of immobilized material. Bolton-Hunter GelB (BHG) and Fn were radioiodinated with (131)I and (125)I respectively and S-Hynic GelB (SHG) was labeled with (99m)Tc. Immobilisation of (131)I-BHG or (99m)Tc-SHG on both PCL and PCL-AEMA scaffolds was performed in analogy with earlier work. SPECT images on scaffolds coated with (99m)Tc-SHG conjugates were acquired on a U-SPECT II camera. There was a clear difference in the amount of deposited (131)I-BHG between blanco and AEMA-grafted PCL on 2D samples. No significant differences in immobilization behaviour were observed between (99m)Tc-SHG and (131)I-BHG. Subsequent immobilisation of Fn was successful and depended on the amounts of deposited GelB. SPECT imaging on cylindrical 3D scaffolds confirmed these findings and showed that the amount of immobilized (99m)Tc-SHG was depth dependant. The architecture of the scaffolds strongly influences the distribution of GelB within these structures. Furthermore, there is a clear difference in the homogeneity of the protein coating when different GelB immobilization protocols were applied. This study shows that radiolabeled compounds are a rapid and accurate tool in the quantitative and qualitative evaluation of the biofunctionalisation of AEMA grafted PCL scaffolds.

Nonthermal plasma technology as a versatile strategy for polymeric biomaterials surface modification: a review.

In modern technology, there is a constant need to solve very complex problems and to fine-tune existing solutions. This is definitely the case in modern medicine with emerging fields such as regenerative medicine and tissue engineering. The problems, which are studied in these fields, set very high demands on the applied materials. In most cases, it is impossible to find a single material that meets all demands such as biocompatibility, mechanical strength, biodegradability (if required), and promotion of cell-adhesion, proliferation, and differentiation. A common strategy to circumvent this problem is the application of composite materials, which combine the properties of the different constituents. Another possible strategy is to selectively modify the surface of a material using different modification techniques. In the past decade, the use of nonthermal plasmas for selective surface modification has been a rapidly growing research field. This will be the highlight of this review. In a first part of this paper, a general introduction in the field of surface engineering will be given. Thereafter, we will focus on plasma-based strategies for surface modification. The purpose of the present review is twofold. First, we wish to provide a tutorial-type review that allows a fast introduction for researchers into the field. Second, we aim to give a comprehensive overview of recent work on surface modification of polymeric biomaterials, with a focus on plasma-based strategies. Some recent trends will be exemplified. On the basis of this literature study, we will conclude with some future trends for research.

The role of scaffold architecture and composition on the bone formation by adipose-derived stem cells.

Scaffold architecture and composition are crucial parameters determining the initial cell spatial distribution and consequently bone tissue formation. Three-dimensional poly-ε-caprolactone (PCL) scaffolds with a 0/90° lay-down pattern were plotted and subjected to (1) an oxygen plasma (PCL O) or (2) a postargon plasma modification with gelatin and fibronectin (PCL Fn). These scaffolds with an open pore structure were compared with more compact scaffolds fabricated by conventional processing techniques: oxidized polylactic acid (LA O) and collagen (COL) scaffolds. Human adipose tissue-derived stem cell/scaffold interactions were studied. The study revealed that the biomimetic surface modification of plotted scaffolds did not increase the seeding efficiency. The proliferation and colonization was superior for PCL Fn in comparison with PCL O. The plotted PCL Fn was completely colonized throughout the scaffold, whereas conventional scaffolds only at the edge. Protein-based scaffolds (PCL Fn and COL) enhanced the differentiation, although plotted scaffolds showed a delay in their differentiation compared with compact scaffolds. In conclusion, protein modification of plotted PCL scaffolds enhances uniform tissue formation, but shows a delayed differentiation in comparison with compact scaffolds. The present study demonstrates that biomimetic PCL scaffolds could serve as a guiding template to obtain a uniform bone tissue formation in vivo.

Synergistic effect of surface modification and scaffold design of bioplotted 3-D poly-ε-caprolactone scaffolds in osteogenic tissue engineering.

The hydrophobic nature and the regular scaffold architecture of bioplotted poly(ε-caprolactone) (PCL) scaffolds present some hurdles for homogeneous tissue formation and differentiation. The current hypothesis is that a synergistic effect of applied surface modification and scaffold design enhances colonization and osteogenic differentiation. First, PCL scaffolds with a 0/90° lay-down pattern (0/90) were plotted and subjected to an oxygen plasma (O2) or multistep surface modification, including post-argon 2-amino-ethylmethacrylate grafting (AEMA), followed by immobilization of gelatin type B (gelB) and physisorption of fibronectin (gelB Fn). Secondly, scaffolds of different designs were plotted (0/90° shift (0/90 S), 0/45° and 0/90° with narrow pores (0/90 NP)) and subjected to the double protein coating. Preosteoblasts were cultured on the scaffolds and the seeding efficiency, colonization and differentiation were studied. The data revealed that a biomimetic surface modification improved colonization (gelB Fn>gelB>AEMA>O2). Compact scaffold architectures (0/90 NP, 0/45, 0/90 S>0/90) positively influenced the seeding efficiency and differentiation. Interestingly, the applied surface modification had a greater impact on colonization than the scaffold design. In conclusion, the combination of a double protein coating with a compact design enhances tissue formation in the plotted PCL scaffolds.

In vitro cell-biological performance and structural characterization of selective laser sintered and plasma surface functionalized polycaprolactone scaffolds for bone regeneration.

In the present study a structural characterization and in vitro cell-biological evaluation was performed on polycaprolactone (PCL) scaffolds that were produced by the additive manufacturing technique selective laser sintering (SLS), followed by a plasma-based surface modification technique, either non-thermal oxygen plasma or double protein coating, to functionalize the PCL scaffold surfaces. In the first part of this study pore morphology by means of 2D optical microscopy, surface chemistry by means of hydrophilicity measurement and X-ray photoelectron spectroscopy, strut surface roughness by means of 3D micro-computed tomography (CT) imaging and scaffold mechanical properties by means of compression testing were evaluated before and after the surface modifications. The results showed that both surface modifications increased the PCL scaffold hydrophilicity without altering the morphological and mechanical properties. In the second part of this study the in vitro cell proliferation and differentiation of human osteoprogenitor cells, over 14 days of culture in osteogenic and growth medium were investigated. The O2 plasma modification gave rise to a significant lower in vitro cell proliferation compared to the untreated and double protein coated scaffolds. Furthermore the double protein coating increased in vitro cell metabolic activity and cell differentiation compared to the untreated and O2 plasma PCL scaffolds when OM was used.

Post-plasma grafting of AEMA as a versatile tool to biofunctionalise polyesters for tissue engineering.

In the last decade, substantial research in the field of post-plasma grafting surface modification has focussed on the introduction of carboxylic acids on surfaces by grafting acrylic acid (AAc). In the present work, we report on an alternative approach for biomaterial surface functionalisation. Thin poly-ε-caprolactone (PCL) films were subjected to a dielectric barrier discharge Ar-plasma followed by the grafting of 2-aminoethyl methacrylate (AEMA) under UV-irradiation. X-ray photoelectron spectroscopy (XPS) confirmed the presence of nitrogen. The ninhydrin assay demonstrated, both quantitatively and qualitatively, the presence of free amines on the surface. Confocal fluorescence microscopy (CFM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise the grafted surfaces, indicating the presence of pAEMA. Static contact angle (SCA) measurements indicated a permanent increase in hydrophilicity. Furthermore, the AEMA grafted surfaces were applied for comparing the physisorption and covalent immobilisation of gelatin. CFM demonstrated that only the covalent immobilisation lead to a complete coverage of the surface. Those gelatin-coated surfaces obtained were further coated using fibronectin. Osteosarcoma cells demonstrated better cell-adhesion and cell-viability on the modified surfaces, compared to the pure PCL films.