The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2.

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

Targeting the Conserved Sequence of the Substrate for the Proteinase of Severe-Acute-Respiratory-Syndrome-Coronavirus-2 (SARS-CoV-2) Using Nano-Networks: Efficacy, Stability, and No Cytotoxicity.

Herein, we designed a nano peptide that contains three important motifs for targeting the chemotrypsin-like cysteine protease (3CLpro) which is the enzyme responsible for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication. The novel nano peptide contains the Nap Phe-Phe motif that is responsible for peptide self-assembly, an octapeptide (Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe) motif where the enzyme recognizes the substrate and induces enzyme sensitivity, and a tetrapeptide motif which is positively charged containing the peptide (Lys)4 that facilitates penetration into a cell. The nano peptide was characterized using Proton Nuclear Magnetic Resonance (H-NMR) and Liquid Chromatography-Mass Spectrometry (LC-MS) to confirm its structure. In vitro results showed that the presently formulated nano peptide was not cytotoxic to fibroblasts for up to 72 hours, bound to 3CLpro, inhibited SARS-CoV-2 Omicron variant virus replication, and was stable for binding for up to one week in culture. In this manner, this timely study demonstrates that this novel nano peptide should be studied for a wide range of Coronavirus Disease (COVID-19) prophylactic or therapeutic applications.

Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus­host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2­3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

Limited Recognition of Highly Conserved Regions of SARS-CoV-2.

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.

Highly conserved s2m element of SARS-CoV-2 dimerizes via a kissing complex and interacts with host miRNA-1307-3p.

The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3' untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.

LinearTurboFold: Linear-time global prediction of conserved structures for RNA homologs with applications to SARS-CoV-2.

The constant emergence of COVID-19 variants reduces the effectiveness of existing vaccines and test kits. Therefore, it is critical to identify conserved structures in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes as potential targets for variant-proof diagnostics and therapeutics. However, the algorithms to predict these conserved structures, which simultaneously fold and align multiple RNA homologs, scale at best cubically with sequence length and are thus infeasible for coronaviruses, which possess the longest genomes (∼30,000 nt) among RNA viruses. As a result, existing efforts on modeling SARS-CoV-2 structures resort to single-sequence folding as well as local folding methods with short window sizes, which inevitably neglect long-range interactions that are crucial in RNA functions. Here we present LinearTurboFold, an efficient algorithm for folding RNA homologs that scales linearly with sequence length, enabling unprecedented global structural analysis on SARS-CoV-2. Surprisingly, on a group of SARS-CoV-2 and SARS-related genomes, LinearTurboFold's purely in silico prediction not only is close to experimentally guided models for local structures, but also goes far beyond them by capturing the end-to-end pairs between 5' and 3' untranslated regions (UTRs) (∼29,800 nt apart) that match perfectly with a purely experimental work. Furthermore, LinearTurboFold identifies undiscovered conserved structures and conserved accessible regions as potential targets for designing efficient and mutation-insensitive small-molecule drugs, antisense oligonucleotides, small interfering RNAs (siRNAs), CRISPR-Cas13 guide RNAs, and RT-PCR primers. LinearTurboFold is a general technique that can also be applied to other RNA viruses and full-length genome studies and will be a useful tool in fighting the current and future pandemics.

Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication.

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3'-to-5' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Independent infections of porcine deltacoronavirus among Haitian children.

Coronaviruses have caused three major epidemics since 2003, including the ongoing SARS-CoV-2 pandemic. In each case, the emergence of coronavirus in our species has been associated with zoonotic transmissions from animal reservoirs1,2, underscoring how prone such pathogens are to spill over and adapt to new species. Among the four recognized genera of the family Coronaviridae, human infections reported so far have been limited to alphacoronaviruses and betacoronaviruses3-5. Here we identify porcine deltacoronavirus strains in plasma samples of three Haitian children with acute undifferentiated febrile illness. Genomic and evolutionary analyses reveal that human infections were the result of at least two independent zoonoses of distinct viral lineages that acquired the same mutational signature in the genes encoding Nsp15 and the spike glycoprotein. In particular, structural analysis predicts that one of the changes in the spike S1 subunit, which contains the receptor-binding domain, may affect the flexibility of the protein and its binding to the host cell receptor. Our findings highlight the potential for evolutionary change and adaptation leading to human infections by coronaviruses outside of the previously recognized human-associated coronavirus groups, particularly in settings where there may be close human-animal contact.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Epitope Profiling Reveals the Critical Antigenic Determinants in SARS-CoV-2 RBD-Based Antigen.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 is a huge public health crisis for the globe. The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in viral infection and serves as a major target for developing neutralizing antibodies. In this study, the antibody response to the RBD of SARS-CoV-2 S protein was analyzed by a panel of sera from animals immunized with RBD-based antigens and four linear B-cell epitope peptides (R345, R405, R450 and R465) were revealed. The immunogenicity of three immunodominant peptides (R345, R405, R465) was further accessed by peptide immunization in mice, and all of them could induced potent antibody response to SARS-CoV-2 S protein, indicating that the three determinants in the RBD were immunogenic. We further generated and characterized monoclonal antibodies (15G9, 12C10 and 10D2) binding to these epitope peptides, and finely mapped the three immunodominant epitopes using the corresponding antibodies. Neutralization assays showed that all three monoclonal antibodies had neutralization activity. Results from IFA and western blotting showed that 12C10 was a cross-reactive antibody against both of SARS-CoV-2 and SARS-CoV. Results from conservative and structural analysis showed that 350VYAWN354 was a highly conserved epitope and exposed on the surface of SARS-CoV-2 S trimer, whereas 473YQAGSTP479 located in the receptor binding motif (RBM) was variable among different SARS-CoV-2 strains. 407VRQIAP412 was a highly conserved, but cryptic epitope shared between SARS-CoV-2 and SARS-CoV. These findings provide important information for understanding the humoral antibody response to the RBD of SARS-CoV-2 S protein and may facilitate further efforts to design SARS-CoV-2 vaccines and the target of COVID-19 diagnostic.

Potential Therapeutic Effect of Micrornas in Extracellular Vesicles from Mesenchymal Stem Cells against SARS-CoV-2.

Extracellular vesicles (EVs) are cell-released, nanometer-scaled, membrane-bound materials and contain diverse contents including proteins, small peptides, and nucleic acids. Once released, EVs can alter the microenvironment and regulate a myriad of cellular physiology components, including cell-cell communication, proliferation, differentiation, and immune responses against viral infection. Among the cargoes in the vesicles, small non-coding micro-RNAs (miRNAs) have received attention in that they can regulate the expression of a variety of human genes as well as external viral genes via binding to the complementary mRNAs. In this study, we tested the potential of EVs as therapeutic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. First, we found that the mesenchymal stem-cell-derived EVs (MSC-EVs) enabled the rescue of the cytopathic effect of SARS-CoV-2 virus and the suppression of proinflammatory responses in the infected cells by inhibiting the viral replication. We found that these anti-viral responses were mediated by 17 miRNAs matching the rarely mutated, conserved 3'-untranslated regions (UTR) of the viral genome. The top five miRNAs highly expressed in the MSC-EVs, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, were tested. They were bound to the complemented sequence which led to the recovery of the cytopathic effects. These findings suggest that the MSC-EVs are a potential candidate for multiple variants of anti-SARS-CoV-2.

Ferret Interferon (IFN)-Inducible Transmembrane Proteins Are Upregulated by both IFN-α and Influenza Virus Infection.

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.

Characterization of MW06, a human monoclonal antibody with cross-neutralization activity against both SARS-CoV-2 and SARS-CoV.

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.

CoVrimer: A tool for aligning SARS-CoV-2 primer sequences and selection of conserved/degenerate primers.

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.

Single-nucleotide conservation state annotation of the SARS-CoV-2 genome.

Given the global impact and severity of COVID-19, there is a pressing need for a better understanding of the SARS-CoV-2 genome and mutations. Multi-strain sequence alignments of coronaviruses (CoV) provide important information for interpreting the genome and its variation. We apply a comparative genomics method, ConsHMM, to the multi-strain alignments of CoV to annotate every base of the SARS-CoV-2 genome with conservation states based on sequence alignment patterns among CoV. The learned conservation states show distinct enrichment patterns for genes, protein domains, and other regions of interest. Certain states are strongly enriched or depleted of SARS-CoV-2 mutations, which can be used to predict potentially consequential mutations. We expect the conservation states to be a resource for interpreting the SARS-CoV-2 genome and mutations.

Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies.

BACKGROUND: The genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contact tracing. METHODS: Leveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies variants, and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints. RESULTS: We built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and a combination of mutations that appear in only a small number of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of 100 SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome. CONCLUSIONS: We conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients and determined their general prevalence when compared to over 70,000 other strains. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Immunogenicity and antigenicity based T-cell and B-cell epitopes identification from conserved regions of 10664 SARS-CoV-2 genomes.

The surge of SARS-CoV-2 has created a wave of pandemic around the globe due to its high transmission rate. To contain this virus, researchers are working around the clock for a solution in the form of vaccine. Due to the impact of this pandemic, the economy and healthcare have immensely suffered around the globe. Thus, an efficient vaccine design is the need of the hour. Moreover, to have a generalised vaccine for heterogeneous human population, the virus genomes from different countries should be considered. Thus, in this work, we have performed genome-wide analysis of 10,664 SARS-CoV-2 genomes of 73 countries around the globe in order to identify the potential conserved regions for the development of peptide based synthetic vaccine viz. epitopes with high immunogenic and antigenic scores. In this regard, multiple sequence alignment technique viz. Clustal Omega is used to align the 10,664 SARS-CoV-2 virus genomes. Thereafter, entropy is computed for each genomic coordinate of the aligned genomes. The entropy values are then used to find the conserved regions. These conserved regions are refined based on the criteria that their lengths should be greater than or equal to 60 nt and their corresponding protein sequences are without any stop codons. Furthermore, Nucleotide BLAST is used to verify the specificity of the conserved regions. As a result, we have obtained 17 conserved regions that belong to NSP3, NSP4, NSP6, NSP8, RdRp, Helicase, endoRNAse, 2'-O-RMT, Spike glycoprotein, ORF3a protein, Membrane glycoprotein and Nucleocapsid protein. Finally, these conserved regions are used to identify the T-cell and B-cell epitopes with their corresponding immunogenic and antigenic scores. Based on these scores, the most immunogenic and antigenic epitopes are then selected for each of these 17 conserved regions. Hence, we have obtained 30 MHC-I and 24 MHC-II restricted T-cell epitopes with 14 and 13 unique HLA alleles and 21 B-cell epitopes for the 17 conserved regions. Moreover, for validating the relevance of these epitopes, the binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles. Also, the physico-chemical properties of the epitopes are reported along with Ramchandran plots and Z-Scores and the population coverage is shown as well. Overall, the analysis shows that the identified epitopes can be considered as potential candidates for vaccine design.

Evaluation of molecular interaction, physicochemical parameters and conserved pattern of SARS-CoV-2 Spike RBD and hACE2: in silico and molecular dynamics approach.

OBJECTIVE: Recent pandemic virus SARS-CoV-2 is a global warning for the healthcare system. The spike protein of virus SARS-CoV-2 is significant because of two reasons. Firstly, the spike protein of this virus binds with the human ACE2 (hACE2) receptor. Secondly, it has several antigenic regions that might be targeted for vaccine development. However, the structural analytical data for the spike protein of this virus is not available. MATERIALS AND METHODS: Here, we performed an analysis to understand the structural two subunits of S glycoprotein (S gp) of SARS-CoV-2. Further, an analysis of secondary structure components and the tertiary structure analysis of RBD was carried out. We also performed molecular interaction analysis between S gp of this virus and hACE2 as well as between SARS-CoV S gp and hACE2 to compare the binding properties of these two viruses. RESULTS: We noted that the molecular interaction of SARS-CoV-2 S gp and hACE2 form eleven hydrogen bonds, while the molecular interaction of SARS-CoV S gp and hACE2 receptor form seven hydrogen bonds, indicating that the molecular interaction of SARS-CoV-2 S gp and hACE2 receptor is more stable than SARS-CoV S gp and hACE2 receptor. The pairwise sequence alignment of S gp SARS-CoV and SARS-CoV-2 shows several conserved residues of these two proteins. Besides, conserved pattern analysis of SARS-CoV-2 S gp and hACE2 revealed the presence of several highly conserved regions for these two proteins. The molecular dynamics simulation shows a stable interplay between SARS-CoV-2 S gp with the hACE2 receptor. CONCLUSIONS: The present study might help determine the SARS-CoV-2 virus entrance mechanism into the human cell. Moreover, the understanding of the conserved regions may help in the process of therapeutic development from the infection of the deadly virus.

Genetic variation analyses indicate conserved SARS-CoV-2-host interaction and varied genetic adaptation in immune response factors in modern human evolution.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a pandemic as of early 2020. Upon infection, SARS-CoV-2 attaches to its receptor, that is, angiotensin-converting enzyme 2 (ACE2), on the surface of host cells and is then internalized into host cells via enzymatic machineries. This subsequently stimulates immune response factors. Since the host immune response and severity of COVID-19 vary among individuals, genetic risk factors for severe COVID-19 cases have been investigated. Our research group recently conducted a survey of genetic variants among SARS-CoV-2-interacting molecules across populations, noting near absence of difference in allele frequency spectrum between populations in these genes. Recent genome-wide association studies have identified genetic risk factors for severe COVID-19 cases in a segment of chromosome 3 that involves six genes encoding three immune-regulatory chemokine receptors and another three molecules. The risk haplotype seemed to be inherited from Neanderthals, suggesting genetic adaptation against pathogens in modern human evolution. Therefore, SARS-CoV-2 uses highly conserved molecules as its virion interaction, whereas its immune response appears to be genetically biased in individuals to some extent. We herein review the molecular process of SARS-CoV-2 infection as well as our further survey of genetic variants of its related immune effectors. We also discuss aspects of modern human evolution.

Recognition of plausible therapeutic agents to combat COVID-19: An omics data based combined approach.

Coronavirus disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has become an immense threat to global public health. In this study, we performed complete genome sequencing of a SARS-CoV-2 isolate. More than 67,000 genome sequences were further inspected from Global Initiative on Sharing All Influenza Data (GISAID). Using several in silico techniques, we proposed prospective therapeutics against this virus. Through meticulous analysis, several conserved and therapeutically suitable regions of SARS-CoV-2 such as RNA-dependent RNA polymerase (RdRp), Spike (S) and Membrane glycoprotein (M) coding genes were selected. Both S and M were chosen for the development of a chimeric vaccine that can generate memory B and T cells. siRNAs were also designed for S and M gene silencing. Moreover, six new drug candidates were suggested that might inhibit the activity of RdRp. Since SARS-CoV-2 and SARS-CoV-1 have 82.30% sequence identity, a Gene Expression Omnibus (GEO) dataset of Severe Acute Respiratory Syndrome (SARS) patients were analyzed. In this analysis, 13 immunoregulatory genes were found that can be used to develop type 1 interferon (IFN) based therapy. The proposed vaccine, siRNAs, drugs and IFN based analysis of this study will accelerate the development of new treatments.