The Influence of Collaborative and Multi-Modal Mixed Reality: Cultural Learning in Virtual Heritage

Studies in the virtual heritage (VH) domain identify collaboration (social interaction), engagement, and a contextual relationship as key elements of interaction design that influence users’ experience and cultural learning in VH applications. The purpose of this study is to validate whether collaboration (social interaction), engaging experience, and a contextual relationship enhance cultural learning in a collaborative and multi-modal mixed reality (MR) heritage environment. To this end, we have designed and implemented a cloud-based collaborative and multi-modal MR application aiming at enhancing user experience and cultural learning in museums. A conceptual model was proposed based on collaboration, engagement, and relationship in the context of MR experience. The MR application was then evaluated at the Western Australian Shipwrecks Museum by experts, archaeologists, and curators from the gallery and the Western Australian Museum. Questionnaire, semi-structured interview, and observation were used to collect data. The results suggest that integrating collaborative and multi-modal interaction methods with MR technology facilitates enhanced cultural learning in VH.

NanoNet: Rapid end-to-end nanobody modeling by deep learning at sub angstrom resolution

Antibodies are a rapidly growing class of therapeutics. Recently, single domain camelid VHH antibodies, and their recognition nanobody domain (Nb) appeared as a cost-effective highly stable alternative to full-length antibodies. There is a growing need for high-throughput epitope mapping based on accurate structural modeling of the variable domains that share a common fold and differ in the Complementarity Determining Regions (CDRs). We develop a deep learning end-to-end model, NanoNet, that given a sequence directly produces the 3D coordinates of the Cα atoms of the entire VH domain. For the Nb test set, NanoNet achieves 1.7&Aring overall average RMSD and 3.0&Aring average RMSD for the most variable CDR3 loops. The accuracy for antibody VH domains is even higher: overall average RMSD < 1&Aring and 2.2&Aring RMSD for CDR3. NanoNet runtimes allow generation of ~1M nanobody structures in less than an hour on a standard CPU computer enabling high-throughput structure modeling.

Clouds-Based Collaborative and Multi-Modal Mixed Reality for Virtual Heritage

Recent technological advancements in immersive reality technologies have become a focus area in the virtual heritage (VH) domain. In this regard, this paper attempts to design and implement clouds-based collaborative and multi-modal MR application aiming at enhancing cultural learning in VH. The design and implementation can be adopted by the VH domain for various application themes. The application utilises cloud computing and immersive reality technologies. The use of cloud computing, collaborative, and multi-modal interaction methods is influenced by the following three issues. First, studies show that users’ interaction with immersive reality technologies and virtual environments determines their learning outcome and the overall experience. Second, studies also demonstrate that collaborative and multi-modal interaction methods enable engagement in immersive reality environments. Third, the integration of immersive reality technologies with traditional museums and cultural heritage sites is getting significant attention in the domain. However, a robust approach, development platforms (frameworks) and easily adopted design and implementation approaches, or guidelines are not commonly available to the VH community. This paper, therefore, will attempt to achieve two major goals. First, it attempts to design and implement a novel application that integrates cloud computing, immersive reality technology and VH. Second, it attempts to apply the proposed application to enhance cultural learning. From the perspective of cultural learning and users’ experience, the assumption is that the proposed approach (clouds-based collaborative and multi-modal MR) can enhance cultural learning by (1) establishing a contextual relationship and engagement between users, virtual environments and cultural context in museums and heritage sites, and (2) by enabling collaboration between users.

Engineering Disulphide-Free Autonomous Antibody VH Domains to modulate intracellular pathways

An attractive approach to target intracellular macromolecular interfaces is to design small high affinity proteins. In this manuscript a stable, autonomous, human derived non-immunogenic, disulphide-free VH domain, has been engineered for intracellular expression studies. VH domains can be designed to possess a large dynamic repertoire of binders, as opposed to other scaffolds types that are highly rigid and possess fewer sites of random variation. Picomolar inhibitors were identified using phage display against the eIF4F complex, which is commonly hyper-activated in many cancers. These molecules were also shown to impair cellular proliferation and to reduce the expression of malignancy related proteins. Structural characterization elucidated that these VH domains bound eIF4E at the eIF4G interaction interface via a novel binding pose. Molecules able to mimic this pose and interfere with the eIF4F complex are potentially important for wide-ranging tumour therapy applications.

Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains.

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

Fab Fragment of VHH-Based Antibody Netakimab: Crystal Structure and Modeling Interaction with Cytokine IL-17A

Interleukin 17A (IL-17A) is a proinflammatory cytokine produced by Th17 cells. Antibody BCD-085 (netakimab) against human IL-17A is one of the new inhibitors of this cytokine. In netakimab, the VH domain is replaced by the VHH domain of Lama glama possessing a long complementarity determining region (CDR-H3) in its heavy chain. Here we demonstrate the high affinity of IL-17A to the Fab fragment of netakimab and to its integral part, the VHH domain. We have determined the crystal structure of the Fab fragment of netakimab at 1.9 Å resolution. High variability in the orientation of light and heavy chains of the Fab fragment of netakimab was shown, which is determined by the peculiarity of the structural organization of the CDR-H3. As the high conformational plasticity of the molecule hampers modeling the Fab fragment of netakimab complexed to IL-17A, we have carried out modeling the complex between the antigen and the integral part of the Fab fragment, the VHH domain. We explain the high netakimab Fab fragment affinity for IL-17A by a large number of protein–protein contacts due to additional interactions between CDR-H3 and the cytokine dimer.