Multi-pathogen based chimeric vaccine to fight against COVID-19 and concomitant coinfections.

BACKGROUND: COVID-19 has proved to be a fatal disease of the year 2020, due to which thousands of people globally have lost their lives, and still, the infection cases are at a high rate. Experimental studies suggested that SARS-CoV-2 interacts with various microorganisms, and this coinfection is accountable for the augmentation of infection severity. METHODS AND RESULTS: In this study, we have designed a multi-pathogen vaccine by involving the immunogenic proteins from S. pneumonia, H. influenza, and M. tuberculosis, as they are dominantly associated with SARS-CoV-2. A total of 8 antigenic protein sequences were selected to predict B-cell, HTL, and CTL epitopes restricted to the most prevalent HLA alleles. The selected epitopes were antigenic, non-allergenic, and non-toxic and were linked with adjuvant and linkers to make the vaccine protein more immunogenic, stable, and flexible. The tertiary structure, Ramachandran plot, and discontinuous B-cell epitopes were predicted. Docking and MD simulation study has shown efficient binding of the chimeric vaccine with the TLR4 receptor. CONCLUSION: The in silico immune simulation analysis has shown a high level of cytokines and IgG after a three-dose injection. Hence, this strategy could be a better way to decrease the disease's severity and could be used as a weapon to prevent this pandemic.

Computational Protein Design for COVID-19 Research and Emerging Therapeutics.

As the world struggles with the ongoing COVID-19 pandemic, unprecedented obstacles have continuously been traversed as new SARS-CoV-2 variants continually emerge. Infectious disease outbreaks are unavoidable, but the knowledge gained from the successes and failures will help create a robust health management system to deal with such pandemics. Previously, scientists required years to develop diagnostics, therapeutics, or vaccines; however, we have seen that, with the rapid deployment of high-throughput technologies and unprecedented scientific collaboration worldwide, breakthrough discoveries can be accelerated and insights broadened. Computational protein design (CPD) is a game-changing new technology that has provided alternative therapeutic strategies for pandemic management. In addition to the development of peptide-based inhibitors, miniprotein binders, decoys, biosensors, nanobodies, and monoclonal antibodies, CPD has also been used to redesign native SARS-CoV-2 proteins and human ACE2 receptors. We discuss how novel CPD strategies have been exploited to develop rationally designed and robust COVID-19 treatment strategies.

A comprehensive protein design protocol to identify resistance mutations and signatures of adaptation in pathogens.

Most pathogens mutate and evolve over time to escape immune and drug pressure. To achieve this, they alter specific hotspot residues in their intracellular proteins to render the targeted drug(s) ineffective and develop resistance. Such hotspot residues may be located as a cluster or uniformly as a signature of adaptation in a protein. Identifying the hotspots and signatures is extremely important to comprehensively understand the disease pathogenesis and rapidly develop next-generation therapeutics. As experimental methods are time-consuming and often cumbersome, there is a need to develop efficient computational protocols and adequately utilize them. To address this issue, we present a unique computational protein design protocol that identifies hotspot residues, resistance mutations and signatures of adaptation in a pathogen's protein against a bound drug. Using the protocol, the binding affinity between the designed mutants and drug is computed quickly, which offers predictions for comparison with biophysical experiments. The applicability and accuracy of the protocol are shown using case studies of a few protein-drug complexes. As a validation, resistance mutations in severe acute respiratory syndrome coronavirus 2 main protease (Mpro) against narlaprevir (an inhibitor of hepatitis C NS3/4A serine protease) are identified. Notably, a detailed methodology and description of the working principles of the protocol are presented. In conclusion, our protocol will assist in providing a first-hand explanation of adaptation, hotspot-residue variations and surveillance of evolving resistance mutations in a pathogenic protein.

Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes.

Shortly after the onset of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired numerous variations in its intracellular proteins to adapt quickly, become more infectious, and ultimately develop drug resistance by mutating certain hotspot residues. To keep the emerging variants at bay, including Omicron and subvariants, FDA has approved the antiviral nirmatrelvir for mild-to-moderate and high-risk COVID-19 cases. Like other viruses, SARS-CoV-2 could acquire mutations in its main protease (Mpro) to adapt and develop resistance against nirmatrelvir. Employing a unique high-throughput protein design technique, the hotspot residues, and signatures of adaptation of Mpro having the highest probability of mutating and rendering nirmatrelvir ineffective were identified. Our results show that ∼40% of the designed mutations in Mpro already exist in the globally circulating SARS-CoV-2 lineages and several predicted mutations. Moreover, several high-frequency, designed mutations were found to be in corroboration with the experimentally reported nirmatrelvir-resistant mutants and are naturally occurring. Our work on the targeted design of the nirmatrelvir-binding site offers a comprehensive picture of potential hotspot sites and resistance mutations in Mpro and is thus crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving Mpro variations.

Molecular insights into the differential dynamics of SARS-CoV-2 variants of concern.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected the lives and livelihood of millions of individuals around the world. It has mutated several times after its first inception, with an estimated two mutations occurring every month. Although we have been successful in developing vaccines against the virus, the emergence of variants has enabled it to escape therapy. Few of the generated variants are also reported to be more infectious than the wild-type (WT). In this study, we analyze the attributes of all RBD/ACE2 complexes for the reported VOCs, namely, Alpha, Beta, Gamma, and Delta through computer simulations. Results indicate differences in orientation and binding energies of the VOCs from the WT. Overall, it was observed that electrostatic interactions play a major role in the binding of the complexes. Detailed residue level energetics revealed that the most prominent changes in interaction energies were seen particularly at the mutated residues which were present at RBD/ACE2 interface. We found that the Delta variant is one of the most tightly bound variants of SARS-CoV-2 with dynamics similar to WT. The high binding affinity of RBD towards ACE2 is indicative of an increase in viral transmission and infectivity. The details presented in our study provide additional information for the design and development of effective therapeutic strategies for the emerging variants of the virus in the future.

High-throughput design of symmetrical dimeric SARS-CoV-2 main protease: structural and physical insights into hotspots for adaptation and therapeutics.

Dimerization of SARS-CoV-2 main protease (Mpro) is a prerequisite for its processing activity. With >2000 mutations already reported in Mpro, SARS-CoV-2 may accumulate mutations in the Mpro dimeric interface to stabilize it further. We employed high-throughput protein design strategies to design the symmetrical dimeric interface of Mpro (300 000 designs) to identify mutational hotspots that render the Mpro more stable. We found that ∼22% of designed mutations that yield stable Mpro dimers already exist in SARS-CoV-2 genomes and are currently circulating. Our multi-parametric analyses highlight potential Mpro mutations that SARS-CoV-2 may develop, providing a foundation for assessing viral adaptation and mutational surveillance.

Scanning the RBD-ACE2 molecular interactions in Omicron variant.

The emergence of new SARS-CoV-2 variants poses a threat to the human population where it is difficult to assess the severity of a particular variant of the virus. Spike protein and specifically its receptor binding domain (RBD) which makes direct interaction with the ACE2 receptor of the human has shown prominent amino acid substitutions in most of the Variants of Concern. Here, by using all-atom molecular dynamics simulations we compare the interaction of Wild-type RBD/ACE2 receptor complex with that of the latest Omicron variant of the virus. We observed a very interesting diversification of the charge, dynamics and energetics of the protein complex formed upon mutations. These results would help us in understanding the molecular basis of binding of the Omicron variant with that of SARS-CoV-2 Wild-type.

Interface-based design of the favipiravir-binding site in SARS-CoV-2 RNA-dependent RNA polymerase reveals mutations conferring resistance to chain termination.

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19. Accumulation of mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp may facilitate antigenic drift, generating favipiravir resistance. Focussing on the chain-termination mechanism utilized by favipiravir, we used high-throughput interface-based protein design to generate > 100 000 designs of the favipiravir-binding site of RdRp and identify mutational hotspots. We identified several single-point mutants and designs having a sequence identity of 97%-98% with wild-type RdRp, suggesting that SARS-CoV-2 can develop favipiravir resistance with few mutations. Out of 134 mutations documented in the CoV-GLUE database, 63 specific mutations were already predicted as resistant in our calculations, thus attaining ˜ 47% correlation with the sequencing data. These findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir.

Accelerating COVID-19 Research Using Molecular Dynamics Simulation.

The COVID-19 pandemic has emerged as a global medico-socio-economic disaster. Given the lack of effective therapeutics against SARS-CoV-2, scientists are racing to disseminate suggestions for rapidly deployable therapeutic options, including drug repurposing and repositioning strategies. Molecular dynamics (MD) simulations have provided the opportunity to make rational scientific breakthroughs in a time of crisis. Advancements in these technologies in recent years have become an indispensable tool for scientists studying protein structure, function, dynamics, interactions, and drug discovery. Integrating the structural data obtained from high-resolution methods with MD simulations has helped in comprehending the process of infection and pathogenesis, as well as the SARS-CoV-2 maturation in host cells, in a short duration of time. It has also guided us to identify and prioritize drug targets and new chemical entities, and to repurpose drugs. Here, we discuss how MD simulation has been explored by the scientific community to accelerate and guide translational research on SARS-CoV-2 in the past year. We have also considered future research directions for researchers, where MD simulations can help fill the existing gaps in COVID-19 research.

Targeted design of drug binding sites in the main protease of SARS-CoV-2 reveals potential signatures of adaptation.

Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (Mpro), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro. Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future.

Unraveling the mechanism of arbidol binding and inhibition of SARS-CoV-2: Insights from atomistic simulations.

The COVID-19 pandemic has spread rapidly and poses an unprecedented threat to the global economy and human health. Broad-spectrum antivirals are currently being administered to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). China's prevention and treatment guidelines suggest the use of an anti-influenza drug, arbidol, for the clinical treatment of COVID-19. Reports indicate that arbidol could neutralize SARS-CoV-2. Monotherapy with arbidol is superior to lopinavir-ritonavir or favipiravir for treating COVID-19. In SARS-CoV-2 infection, arbidol acts by interfering with viral binding to host cells. However, the detailed mechanism by which arbidol induces the inhibition of SARS-CoV-2 is not known. Here, we present atomistic insights into the mechanism underlying membrane fusion inhibition of SARS-CoV-2 by arbidol. Molecular dynamics (MD) simulation-based analyses demonstrate that arbidol binds and stabilizes at the receptor-binding domain (RBD)/ACE2 interface with a high affinity. It forms stronger intermolecular interactions with the RBD than ACE2. Analyses of the detailed decomposition of energy components and binding affinities revealed a substantial increase in the affinity between the RBD and ACE2 in the arbidol-bound RBD/ACE2 complex, suggesting that arbidol generates favorable interactions between them. Based on our MD simulation results, we propose that the binding of arbidol induces structural rigidity in the viral glycoprotein, thus restricting the conformational rearrangements associated with membrane fusion and virus entry. Furthermore, key residues of the RBD and ACE2 that interact with arbidol were identified, opening the door for developing therapeutic strategies and higher-efficacy arbidol derivatives or lead drug candidates.

High-throughput rational design of the remdesivir binding site in the RdRp of SARS-CoV-2: implications for potential resistance.

The use of remdesivir to treat COVID-19 will likely continue before clinical trials are completed. Due to the lengthening pandemic and evolving nature of the virus, predicting potential residues prone to mutation is crucial for the management of remdesivir resistance. Using a rational ligand-based interface design complemented with mutational mapping, we generated a total of 100,000 mutations and provided insight into the functional outcomes of mutations in the remdesivir-binding site in nsp12 subunit of RdRp. After designing 46 residues in the remdesivir-binding site of nsp12, the designs retained 97%-98% sequence identity, suggesting that very few mutations in nsp12 are required for SARS-CoV-2 to attain remdesivir resistance. Several mutants displayed decreased binding affinity to remdesivir, suggesting drug resistance. These hotspot residues had a higher probability of undergoing selective mutation and thus conferring remdesivir resistance. Identifying the potential residues prone to mutation improves our understanding of SARS-CoV-2 drug resistance and COVID-19 pathogenesis.

Can SARS-CoV-2 Accumulate Mutations in the S-Protein to Increase Pathogenicity?

SARS-CoV-2 has developed a substantial number of mutations, especially in the S-protein. With the advancement of the pandemic, accumulations of further mutations at the S-protein receptor-binding domain could enhance the infectivity and pathogenicity of the virus. Prediction and evaluation of such mutations are essential for understanding the potential development of more pathogenic strains and for COVID-19 management.

Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease that was first reported in Wuhan, China, and has subsequently spread worldwide. In the absence of any antiviral or immunomodulatory therapies, the disease is spreading at an alarming rate. A possibility of a resurgence of COVID-19 in places where lockdowns have already worked is also developing. Thus, for controlling COVID-19, vaccines may be a better option than drugs. An mRNA-based anti-COVID-19 candidate vaccine has entered a phase 1 clinical trial. However, its efficacy and potency have to be evaluated and validated. Since vaccines have high failure rates, as an alternative, we are presenting a new, designed multi-peptide subunit-based epitope vaccine against COVID-19. The recombinant vaccine construct comprises an adjuvant, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes joined by linkers. The computational data suggest that the vaccine is non-toxic, non-allergenic, thermostable, with the capability to elicit a humoral and cell-mediated immune response. The stabilization of the vaccine construct is validated with molecular dynamics simulation studies. This unique vaccine is made up of 33 highly antigenic epitopes from three proteins that have a prominent role in host-receptor recognition, viral entry, and pathogenicity. We advocate this vaccine must be synthesized and tested urgently as a public health priority.