Design and Immunoinformatic Assessment of Candidate Multivariant mRNA Vaccine Construct against Immune Escape Variants of SARS-CoV-2.

To effectively counter the evolving threat of SARS-CoV-2 variants, modifications and/or redesigning of mRNA vaccine construct are essentially required. Herein, the design and immunoinformatic assessment of a candidate novel mRNA vaccine construct, DOW-21, are discussed. Briefly, immunologically important domains, N-terminal domain (NTD) and receptor binding domain (RBD), of the spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were assessed for sequence, structure, and epitope variations. Based on the assessment, a novel hypothetical NTD (h-NTD) and RBD (h-RBD) were designed to hold all overlapping immune escape variations. The construct sequence was then developed, where h-NTD and h-RBD were intervened by 10-mer gly-ala repeat and the terminals were flanked by regulatory sequences for better intracellular transportation and expression of the coding regions. The protein encoded by the construct holds structural attributes (RMSD NTD: 0.42 Å; RMSD RBD: 0.15 Å) found in the respective domains of SARS-CoV-2 immune escape variants. In addition, it provides coverage to the immunogenic sites of the respective domains found in SARS-CoV-2 variants. Later, the nucleotide sequence of the construct was optimized for GC ratio (56%) and microRNA binding sites to ensure smooth translation. Post-injection antibody titer was also predicted (~12000 AU) to be robust. In summary, the construct proposed in this study could potentially provide broad spectrum coverage in relation to SARS-CoV-2 immune escape variants.

Immunoinformatic analysis of structural and epitope variations in the spike and Orf8 proteins of SARS-CoV-2/B.1.1.7.

A newly emerged strain of SARS-CoV-2 of B.1.1.7 lineage has caused a significant surge in the SARS-CoV-2 infections in the UK. In this study, changes in the epitopes of spike and orf8 proteins in SARS-CoV-2 of B.1.1.7 lineage were investigated. Genomic alignment of the SARS-CoV-2/B.1.1.7 with SARS-CoV-2/Wuhan showed the presence of several mutations in orf1a/b, spike, orf8, and N proteins of SARS-CoV-2/B.1.1.7. Molecular models of spike and orf8 proteins were constructed by homology modeling. Superimposition between the spike proteins of SARS-CoV-2/Wuhan and SARS-CoV-2/B.1.1.7 showed noticeable variations in the spatial orientation in Val70-Asn74 and Thr250-Ser255 regions. This may have also resulted in the extension of the epitopic region at Ser244-Gly249 in the SARS-CoV-2/B.1.1.7 spike protein. Superimposition of the SARS-CoV-2/B.1.1.7 spike protein over Fab-spike protein complexes of SARS-CoV-2/Wuhan also showed subtle variations in the antibody binding affinity targeting the N-terminal domain of the spike protein. Epitopic variations were also observed between the corresponding orf8 regions of SARS-CoV-2/Wuhan and SARS-CoV-2/B.1.1.7. Moreover, the presence of a stop codon at position 27 in orf8 connotes the emergence of two frames (orf8a and orf8b) in SARS-CoV-2, which further hampers its extracellular secretion, and in turn, immunogenicity. The findings of the present study could further be used to develop targeted immunotherapeutics.

Molecular docking between human TMPRSS2 and SARS-CoV-2 spike protein: conformation and intermolecular interactions.

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational attributes of the molecular complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike protein. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provides structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

Dataset for homologous proteins in Drosophila melanogaster for SARS-CoV-2/human interactome.

Animal modelling for infectious diseases is critical to understand the biology of the pathogens including viruses and to develop therapeutic strategies against it. Herein, we present the sequence homology and expression data analysis of proteins found in Drosophila melanogaster that are orthologous to human proteins, reported as components of SARS-CoV-2/Human interactome. The dataset enlists sequence homology, query coverage, domain conservation, OrthoMCL and Ensembl Genome Browser support of 326 proteins in D.melanogaster that are potentially orthologous to 417 human proteins reported for their direct physical interactions with 28 proteins encoded by SARS-CoV-2 genome. Expression of these D.melanogaster orthologous genes in 26 anatomical positions are also plotted as heat maps in 27 sets, corresponding to the potential protein interactors for each viral protein. The data could be used to direct experiments and potentially predict their phenotypic and molecular outcome in order to dissect the biological roles and molecular functionality of SARS-CoV-2 proteins in a convenient animal model system like D.melanogaster.

Structural variations in human ACE2 may influence its binding with SARS-CoV-2 spike protein.

The recent pandemic of COVID-19, caused by SARS-CoV-2, is unarguably the most fearsome compared with the earlier outbreaks caused by other coronaviruses, SARS-CoV and MERS-CoV. Human ACE2 is now established as a receptor for the SARS-CoV-2 spike protein. Where variations in the viral spike protein, in turn, lead to the cross-species transmission of the virus, genetic variations in the host receptor ACE2 may also contribute to the susceptibility and/or resistance against the viral infection. This study aims to explore the binding of the proteins encoded by different human ACE2 allelic variants with SARS-CoV-2 spike protein. Briefly, coding variants of ACE2 corresponding to the reported binding sites for its attachment with coronavirus spike protein were selected and molecular models of these variants were constructed by homology modeling. The models were then superimposed over the native ACE2 and ACE2-spike protein complex, to observe structural changes in the ACE2 variants and their intermolecular interactions with SARS-CoV-2 spike protein, respectively. Despite strong overall structural similarities, the spatial orientation of the key interacting residues varies in the ACE2 variants compared with the wild-type molecule. Most ACE2 variants showed a similar binding affinity for SARS-CoV-2 spike protein as observed in the complex structure of wild-type ACE2 and SARS-CoV-2 spike protein. However, ACE2 alleles, rs73635825 (S19P) and rs143936283 (E329G) showed noticeable variations in their intermolecular interactions with the viral spike protein. In summary, our data provide a structural basis of potential resistance against SARS-CoV-2 infection driven by ACE2 allelic variants.