Spike Receptor-binding Domain Research Articles

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

BackgroundThe SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages.MethodsThe presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing.ResultsAll genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.ConclusionThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Kinetics of naturally induced binding and neutralising anti-SARS-CoV-2 antibody levels and potencies among SARS-CoV-2 infected Kenyans with diverse grades of COVID-19 severity: an observational study

Background: Given the low levels of coronavirus disease 2019 (COVID-19) vaccine coverage in sub-Saharan Africa (sSA), despite high levels of natural severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) exposures, strategies for extending the breadth and longevity of naturally acquired immunity are warranted. Designing such strategies will require a good understanding of natural immunity. Methods: We measured whole-spike immunoglobulin G (IgG) and spike-receptor binding domain (RBD) total immunoglobulins (Igs) on 585 plasma samples collected longitudinally over five successive time points within six months of COVID-19 diagnosis in 309 COVID-19 patients. We measured antibody-neutralising potency against the wild-type (Wuhan) SARS-CoV-2 pseudovirus in a subset of 51 patients over three successive time points. Binding and neutralising antibody levels and potencies were then tested for correlations with COVID-19 severities. Results: Rates of seroconversion increased from day 0 (day of PCR testing) to day 180 (six months) (63.6% to 100 %) and (69.3 % to 97%) for anti-spike-IgG and anti-spike-RBD binding Igs, respectively. Levels of these binding antibodies peaked at day 28 (p<0.0001) and were subsequently maintained for six months without significant decay (p>0.99). Similarly, antibody-neutralising potencies peaked at day 28 (p<0.0001) but declined by three-fold, six months after COVID-19 diagnosis (p<0.0001). Binding antibody levels were highly correlated with neutralising antibody potencies at all the time points analysed (r>0.6, p<0.0001). Levels and potencies of binding and neutralising antibodies increased with disease severity. Conclusions: Most COVID-19 patients generated SARS-CoV-2 specific binding antibodies that remained stable in the first six months of infection. However, the respective neutralising antibodies decayed three-fold by month-six of COVID-19 diagnosis suggesting that they are short-lived, consistent with what has been observed elsewhere in the world. Thus, regular vaccination boosters are required to sustain the high levels of anti-SARS-CoV-2 naturally acquired neutralising antibody potencies in our population.

Design of a bifunctional pan-sarbecovirus entry inhibitor targeting the cell receptor and viral fusion protein.

Development of highly effective antivirals that are robust to viral evolution is a practical strategy for combating the continuously evolved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inspired by viral multistep entry process, we here focus on developing a bispecific SARS-CoV-2 entry inhibitor, which acts on the cell receptor angiotensin converting enzyme 2 (ACE2) and viral S2 fusion protein. First, we identified a panel of diverse spike (S) receptor-binding domains (RBDs) and found that the RBD derived from Guangdong pangolin coronavirus (PCoV-GD) possessed the most potent antiviral potency. Next, we created a bispecific inhibitor termed RBD-IPB01 by genetically linking a peptide fusion inhibitor IPB01 to the C-terminal of PCoV-GD RBD, which exhibited greatly increased antiviral potency via cell membrane ACE2 anchoring. Promisingly, RBD-IPB01 had a uniformly bifunctional inhibition on divergent pseudo- and authentic SARS-CoV-2 variants, including multiple Omicron subvariants. RBD-IPB01 also showed consistently cross-inhibition of other sarbecoviruses, including SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus (PCoV-GX). RBD-IPB01 displayed low cytotoxicity, high trypsin resistance, and favorable metabolic stability. Combined, our studies have provided a tantalizing insight into the design of broad-spectrum and potent antiviral agent. IMPORTANCE Ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution and spillover potential of a wide variety of sarbecovirus lineages indicate the importance of developing highly effective antivirals with broad capability. By directing host angiotensin converting enzyme 2 receptor and viral S2 fusion protein, we have created a dual-targeted virus entry inhibitor with high antiviral potency and breadth. The inhibitor receptor-binding domain (RBD)-IPB01 with the Guangdong pangolin coronavirus (PCoV-GD) spike RBD and a fusion inhibitor IPB01 displays bifunctional cross-inhibitions on pseudo- and authentic SARS-CoV-2 variants including Omicron, as well as on the sarbecoviruses SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus. RBD-IPB01 also efficiently inhibits diverse SARS-CoV-2 infection of human Calu-3 cells and blocks viral S-mediated cell-cell fusion with a dual function. Thus, the creation of such a bifunctional inhibitor with pan-sarbecovirus neutralizing capability has not only provided a potential weapon to combat future SARS-CoV-2 variants or yet-to-emerge zoonotic sarbecovirus, but also verified a viable strategy for the designing of antivirals against infection of other enveloped viruses.

Adjuvanted-SARS-CoV-2 Spike Protein-Based Microparticulate Vaccine Delivered by Dissolving Microneedles Induces Humoral, Mucosal, and Cellular Immune Responses in Mice.

COVID-19 continues to cause an increase in the number of cases and deaths worldwide. Due to the ever-mutating nature of the virus, frequent vaccination against COVID-19 is anticipated. Most of the approved SARS-CoV-2 vaccines are administered using the conventional intramuscular route, causing vaccine hesitancy. Thus, there is a need for an effective, non-invasive vaccination strategy against COVID-19. This study evaluated the synergistic effects of a subunit microparticulate vaccine delivered using microneedles. The microparticles encapsulated a highly immunogenic subunit protein of the SARS-CoV-2 virus, such as the spike protein's receptor binding domain (RBD). Adjuvants were also incorporated to enhance the spike RBD-specific immune response. Our vaccination study reveals that a microneedle-based vaccine delivering these microparticles induced spike RBD-specific IgM, IgG, IgG1, IgG2a, and IgA antibodies. The vaccine also generated high levels of CD4+ and CD8a+ molecules in the secondary lymphoid organs. Overall, dissolving microneedles delivery spike RBD antigen in microparticulate form induced a robust immune response, paving the way for an alternative self-administrable, non-invasive vaccination strategy against COVID-19.

SARS-CoV-2 vaccination and infection in ozanimod-treated participants with relapsing multiple sclerosis.

To investigate the serologic response, predictors of response, and clinical outcomes associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and infection in ozanimod-treated participants with relapsing multiple sclerosis (RMS) from DAYBREAK. DAYBREAK (ClinicalTrials.gov-NCT02576717), an open-label extension study of oral ozanimod 0.92 mg, enrolled participants aged 18-55 years with RMS who completed phase 1-3 ozanimod trials. Participants who were fully vaccinated against SARS-CoV-2 with mRNA or non-mRNA vaccines, were unvaccinated, and/or had COVID-19-related adverse events (AEs, with or without vaccination) and postvaccination serum samples were included (n = 288). Spike receptor binding domain (RBD) antibody levels (seroconversion: ≥0.8 U/mL) and serologic evidence of SARS-CoV-2 infection (nucleocapsid IgG: ≥1 U/mL) were assessed (Roche Elecsys/Cobas e411 platform). In fully vaccinated participants (n = 148), spike RBD antibody seroconversion occurred in 90% (n = 98/109) of those without serologic evidence of prior SARS-CoV-2 exposure (100% [n = 80/80] seroconversion after mRNA vaccination) and in 100% (n = 39/39) of participants with serologic evidence of viral exposure. mRNA vaccination predicted higher spike RBD antibody levels, whereas absolute lymphocyte count (ALC), age, body mass index, and sex did not. COVID-19-related AEs were reported in 10% (n = 15/148) of fully vaccinated participants-all were nonserious and not severe; all participants recovered. Most ozanimod-treated participants with RMS mounted a serologic response to SARS-CoV-2 vaccination and infection, regardless of participant characteristics or ALC levels. In this analysis, all COVID-19-related AEs post-full vaccination in participants taking ozanimod were nonserious and not severe.

Investigating the role of glycans in Omicron sub-lineages XBB.1.5 and XBB.1.16 binding to host receptor using molecular dynamics and binding free energy calculations.

Omicron derived lineages viz. BA.2, BA.3, BA.4 BA.5, BF.7 and XBBs show prominence with improved immune escape, transmissibility, infectivity, and pathogenicity in general. Sub-variants, XBB.1.5 and XBB.1.16 have shown rapid spread, with mutations embedded throughout the viral genome, including the spike protein. Changing atomic landscapes in spike contributes significantly to modulate host pathogen interactions and infections thereof. In the present work, we computationally analyzed the binding affinities of spike receptor binding domains (RBDs) of XBB.1.5 and XBB.1.16 towards human angiotensin-converting enzyme 2 (hACE2) compared to Omicron. We have employed simulations and binding energy estimation of molecular complexes of spike-hACE2 to assess the interplay of interaction pattern and effect of mutations if any in the binding mode of the RBDs of these novel mutants. We calculated the binding free energy (BFE) of the RBD of the Omicron, XBB.1.5 and XBB.1.16 spike protein to hACE2. We showed that XBB.1.5 and XBB.1.16 can bind to human cells more strongly than Omicron due to the increased charge of the RBD, which enhances the electrostatic interactions with negatively charged hACE2. The per-residue decompositions further show that the Asp339His, Asp405Asn and Asn460Lys mutations in the XBBs RBD play a crucial role in enhancing the electrostatic interactions, by acquiring positively charged residues, thereby influencing the formation/loss of interfacial bonds and thus strongly affecting the spike RBD-hACE2 binding affinity. Simulation results also indicate less interference of heterogeneous glycans of XBB.1.5 spike RBD towards binding to hACE2. Moreover, despite having less interaction at the three interfacial contacts between XBB S RBD and hACE2 compared to Omicron, variants XBB.1.5 and XBB.1.16 had higher total binding free energies (ΔGbind) than Omicron due to the contribution of non-interfacial residues to the free energy, providing insight into the increased binding affinity of XBB1.5 and XBB.1.16. Furthermore, the presence of large positively charged surface patches in the XBBs act as drivers of electrostatic interactions, thus support the possibility of a higher binding affinity to hACE2.

Interchangeability of the Assays Used to Assess the Activity of Anti-SARS-CoV-2 Monoclonal Antibodies.

The recent global COVID-19 pandemic caused by SARS-CoV-2 lasted for over three years. A key measure in combatting this pandemic involved the measurement of the monoclonal antibody (mAb)-mediated inhibition of binding between the spike receptor-binding domain (RBD) and hACE2 receptor. Potency assessments of therapeutic anti-SARS-CoV-2 mAbs typically include binding or cell-based neutralization assays. We assessed the inhibitory activity of five anti-SARS-CoV-2 mAbs using ELISA, surface plasmon resonance (SPR), and four cell-based neutralization assays using different pseudovirus particles and 293T or A549 cells expressing hACE2 with or without TMPRSS2. We assessed the interchangeability between cell-based and binding assays by applying the Bland-Altman method under certain assumptions. Our data demonstrated that the IC50 [nM] values determined by eight neutralization assays are independent of the cell line, presence of TMPRSS2 enzyme on the cell surface, and pseudovirus backbone used. Moreover, the Bland-Altman analysis showed that the IC50 [nM] and KD [nM] values determined by neutralization/ELISA or by SPR are equivalent and that the anti-spike mAb activity can be attributed to one variable directly related to its tertiary conformational structure conformation, rate dissociation constant Koff. This parameter is independent from the concentrations of the components of the mAb:RBD:hACE2 complexes and can be used for a comparison between the activities of the different mAbs.

SARS-CoV-2 antibody response in SARS survivors with and without the COVID-19 vaccine

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. However, it remains unknown whether individuals with prior SARS-CoV-1 infection are protected from SARS-CoV-2 infection. This study assessed protective antibody levels in SARS survivors with and without the COVID-19 vaccine. We recruited 17 SARS survivors infected with SARS-CoV-1 in 2003, including 8 not vaccinated with the COVID-19 vaccine and 9 vaccinated with two doses of inactivated whole-virion COVID-19 vaccine (Sinopharm). In addition, 105 healthy adult volunteers without SARS-CoV-1 and SARS-CoV-2 infections were used as controls. The relative concentrations of three protective antibodies including anti-SARS-CoV-2 spike IgG (nCoV S-IgG), anti-SARS-CoV-2 spike receptor-binding domain IgG (nCoV RBD-IgG), and anti-SARS-CoV-2 neutralizing antibodies (nCoV NAbs) were measured to evaluate humoral immunity. We found that the positive rates of these antibodies in unvaccinated SARS survivors were 37.5%, 37.5%, and 62.5%, respectively. In contrast, the corresponding positive rates were all 0% in controls before vaccination. In controls, the levels of protective antibodies reached a peak ca. 28 days after the second dose of vaccine and then started to decline. Surprisingly, the levels of these antibodies were maintained at very high levels even 166 days after the second dose of vaccine in SARS survivors. Our study suggests that there are protective antibodies cross-reacting with SARS-CoV-2 in recovered SARS patients and that SARS survivors can generate a much stronger antibody response induced by the COVID-19 vaccine than can controls. These initial findings show the feasibility of developing novel pan-sarbecovirus vaccines.

Identification of terpenoids as potential inhibitors of SARS-CoV-2 (main protease) and spike (RBD) via computer-aided drug design

The scientific community has been faced with a major challenge in the fight against the SARS-CoV-2 virus responsible for the COVID-19 pandemic, due to the lack of targeted antiviral drugs. To address this issue, we used an in silico approach to screen 23 natural compounds from the terpenoid class for their ability to target key SARS-CoV-2 therapeutic proteins. The results revealed that several compounds showed promising interactions with SARS-CoV-2 proteins, specifically the main protease and the spike receptor binding domain. The molecular docking analysis revealed the importance of certain residues, such as GLY143, SER144, CYS145 and GLU166, in the main protease of the SARS-CoV-2 protein, which play a crucial role in interactions with the ligand. In addition, our study highlighted the importance of interactions with residues GLY496, ARG403, SER494 and ARG393 of the spike receptor-binding domain within the SARS-CoV-2 protein. ADMET and drug similarity analyses were also performed, followed by molecular dynamics and MM-GBSA calculations, to identify potential drugs could be repurposed to combat COVID-19. Indeed, the results suggest that certain terpenoid compounds of plant origin have promising potential as therapeutic targets for SARS-CoV-2. However, additional experimental studies are required to confirm their efficacy as drugs against COVID-19. Communicated by Ramaswamy H. Sarma

Running ahead of evolution—AI-based simulation for predicting future high-risk SARS-CoV-2 variants

The never-ending emergence of SARS-CoV-2 variations of concern (VOCs) has challenged the whole world for pandemic control. In order to develop effective drugs and vaccines, one needs to efficiently simulate SARS-CoV-2 spike receptor-binding domain (RBD) mutations and identify high-risk variants. We pretrain a large protein language model with approximately 408 million protein sequences and construct a high-throughput screening for the prediction of binding affinity and antibody escape. As the first work on SARS-CoV-2 RBD mutation simulation, we successfully identify mutations in the RBD regions of 5 VOCs and can screen millions of potential variants in seconds. Our workflow scales to 4096 NPUs with 96.5% scalability and 493.9× speedup in mixed-precision computing, while achieving a peak performance of 366.8 PFLOPS (reaching 34.9% theoretical peak) on Pengcheng Cloudbrain-II. Our method paves the way for simulating coronavirus evolution in order to prepare for a future pandemic that will inevitably take place. Our models are released at https://github.com/ZhiweiNiepku/SARS-CoV-2_mutation_simulation to facilitate future related work.

An intranasal vaccine comprising SARS-CoV-2 spike receptor-binding domain protein entrapped in mannose-conjugated chitosan nanoparticle provides protection in hamsters

We developed a novel intranasal SARS-CoV-2 subunit vaccine called NARUVAX-C19/Nano based on the spike protein receptor-binding domain (RBD) entrapped in mannose-conjugated chitosan nanoparticles (NP). A toll-like receptor 9 agonist, CpG55.2, was also added as an adjuvant to see if this would potentiate the cellular immune response to the NP vaccine. The NP vaccine was assessed for immunogenicity, protective efficacy, and ability to prevent virus transmission from vaccinated animals to naive cage-mates. The results were compared with a RBD protein vaccine mixed with alum adjuvant and administered intramuscularly. BALB/c mice vaccinated twice intranasally with the NP vaccines exhibited secretory IgA and a pronounced Th1-cell response, not seen with the intramuscular alum-adjuvanted RBD vaccine. NP vaccines protected Syrian hamsters against a wild-type SARS-CoV-2 infection challenge as indicated by significant reductions in weight loss, lung viral load and lung pathology. However, despite significantly reduced viral load in the nasal turbinates and oropharyngeal swabs from NP-vaccinated hamsters, virus transmission was not prevented to naïve cage-mates. In conclusion, intranasal RBD-based NP formulations induced mucosal and Th1-cell mediated immune responses in mice and protected Syrian hamsters against SARS-CoV-2 infection but not against viral transmission.

Label-free drug interaction screening via Raman microscopy

Development of a simple, label-free screening technique capable of precisely and directly sensing interaction-in-solution over a size range from small molecules to large proteins such as antibodies could offer an important tool for researchers and pharmaceutical companies in the field of drug development. In this work, we present a thermostable Raman interaction profiling (TRIP) technique that facilitates low-concentration and low-dose screening of binding between protein and ligand in physiologically relevant conditions. TRIP was applied to eight protein-ligand systems, and produced reproducible high-resolution Raman measurements, which were analyzed by principal component analysis. TRIP was able to resolve time-depending binding between 2,4-dinitrophenol and transthyretin, and analyze biologically relevant SARS-CoV-2 spike-antibody interactions. Mixtures of the spike receptor-binding domain with neutralizing, nonbinding, or binding but nonneutralizing antibodies revealed distinct and reproducible Raman signals. TRIP holds promise for the future developments of high-throughput drug screening and real-time binding measurements between protein and drug.

Repurposing the oncolytic virus VSV∆51M as a COVID-19 vaccine.

The coronavirus disease 2019 (COVID-19) pandemic imposes an urgent and continued need for the development of safe and cost-effective vaccines to induce preventive responses for limiting major outbreaks around the world. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we repurposed the VSV∆51M oncolytic virus platform to express the spike receptor-binding domain (RBD) antigen. In this study, we report the development and characterization of the VSV∆51M-RBD vaccine. Our findings demonstrate successful expression of the RBD gene by the VSV∆51M-RBD virus, inducing anti-RBD responses without attenuating the virus. Moreover, the VSV∆51M-RBD vaccine exhibited safety, immunogenicity, and the potential to serve as a safe and effective alternative or complementary platform to current COVID-19 vaccines.

ACE2 receptor polymorphism in humans and animals increases the risk of the emergence of SARS-CoV-2 variants during repeated intra- and inter-species host-switching of the virus.

Like other coronaviruses, SARS-CoV-2 has ability to spread through human-to-human transmission and to circulate from humans to animals and from animals to humans. A high frequency of SARS-CoV-2 mutations has been observed in the viruses isolated from both humans and animals, suggesting a genetic fitness under positive selection in both ecological niches. The most documented positive selection force driving SARS-CoV-2 mutations is the host-specific immune response. However, after electrostatic interactions with lipid rafts, the first contact between the virus and host proteins is the viral spike-cellular receptor binding. Therefore, it is likely that the first level of selection pressure impacting viral fitness relates to the virus's affinity for its receptor, the angiotensin I converting enzyme 2 (ACE2). Although sufficiently conserved in a huge number of species to support binding of the viral spike with enough affinity to initiate fusion, ACE2 is highly polymorphic both among species and within a species. Here, we provide evidence suggesting that when the viral spike-ACE2 receptor interaction is not optimal, due to host-switching, mutations can be selected to improve the affinity of the spike for the ACE2 expressed by the new host. Notably, SARS-CoV-2 is mutation-prone in the spike receptor binding domain (RBD), allowing a better fit for ACE2 orthologs in animals. It is possibly that this may also be true for rare human alleles of ACE2 when the virus is spreading to billions of people. In this study, we present evidence that human subjects expressing the rare E329G allele of ACE2 with higher allele frequencies in European populations exhibit a improved affinity for the SARS-CoV-2 spike N501Y variant of the virus. This may suggest that this viral N501Y variant emerged in the human population after SARS-CoV-2 had infected a human carrying the rare E329G allele of ACE2. In addition, this viral evolution could impact viral replication as well as the ability of the adaptive humoral response to control infection with RBD-specific neutralizing antibodies. In a shifting landscape, this ACE2-driven genetic drift of SARS-CoV-2 which we have named the 'boomerang effect', could complicate the challenge of preventing COVID with a SARS-CoV-2 spike-derived vaccine.

Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics.

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19-recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

Mouse model for pangolin-origin coronavirus GX/P2V/2017 infection and cross-protection from COVID-19 ZF2001 subunit vaccine

Many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–related coronaviruses have been discovered, constituting potential threats to human health. However, it remains unclear whether the currently available vaccines are effective against these coronaviruses. Here, we constructed a wild-type mouse model to evaluate pathogenicity of the SARS-CoV-2-related pangolin coronavirus GX/P2V/2017 and neutralization efficacy of the approved tandem-repeat SARS-CoV-2 spike receptor-binding domain (RBD) vaccine ZF2001. We found that ZF2001-induced cross-reactive and cross-neutralizing antibodies against GX/P2V/2017, and the vaccination alleviated the pathological lung damage caused by GX/P2V/2017 in mice. These results indicate that RBD may work as a promising candidate for pan-coronavirus vaccine development.

An Evidence of Drug Repurposing for COVID-19 Pandemic Based on <i>In silico</i> Investigation from Phenolic Derivatives of Silybum Marianum Against SARS-Cov-2 Proteins

The outbreak of coronavirus disease-2019 (COVID-19) had a striking impact on the worldwide healthcare system within a very short period. The availability of a large number of clinical data on SARS-CoV-2, conventional precautionary majors, and treatment strategies with the existing therapeutic antiviral drug molecules also fails to control progression and disease transmission among the population. Hence, we implemented pharmacoinformatics approaches to facilitate the drug discovery by repurposing naturally available therapeutic molecules as an effective intervention. The major phenolic derivatives of <em>Silybum marianum </em>(Milk thistle) have been identified and investigated for ADME (Absorption, Distribution, Metabolism and Excretion)/tox properties. Co-crystallized structure of three major proteins (i.e., main protease, RNA binding domain of nucleocapsid phosphoprotein and Spike receptor binding domain) from SARS-CoV-2 investigated with molecular docking (MD) interaction with the phenolic compounds from milk thistle. Furthermore, a 100 ns MD simulation was performed with silibinin molecule based on ADMET and MD interaction. Being less toxic in ADME, a good MD interaction and stability of silibinin molecule across the MD simulation trajectories with targeted proteins explicate that silibinin molecule can be a promising drug candidate against the main protease and will be helpful to cease the enzymatic activity in viral replication and transcription.

Targeting the Spike Receptor Binding Domain Class V Cryptic Epitope by an Antibody with Pan-Sarbecovirus Activity.

Novel therapeutic monoclonal antibodies (MAbs) must accommodate comprehensive breadth of activity against diverse sarbecoviruses and high neutralization potency to overcome emerging variants. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) in complex with MAb WRAIR-2063, a moderate-potency neutralizing antibody with exceptional sarbecovirus breadth, that targets the highly conserved cryptic class V epitope. This epitope overlaps substantially with the spike protein N-terminal domain (NTD) -interacting region and is exposed only when the spike is in the open conformation, with one or more RBDs accessible. WRAIR-2063 binds the RBD of SARS-CoV-2 WA-1, all variants of concern (VoCs), and clade 1 to 4 sarbecoviruses with high affinity, demonstrating the conservation of this epitope and potential resiliency against variation. We compare structural features of additional class V antibodies with their reported neutralization capacity to further explore the utility of the class V epitope as a pan-sarbecovirus vaccine and therapeutic target. IMPORTANCE Characterization of MAbs against SARS-CoV-2, elicited through vaccination or natural infection, has provided vital immunotherapeutic options for curbing the COVID-19 pandemic and has supplied critical insights into SARS-CoV-2 escape, transmissibility, and mechanisms of viral inactivation. Neutralizing MAbs that target the RBD but do not block ACE2 binding are of particular interest because the epitopes are well conserved within sarbecoviruses and MAbs targeting this area demonstrate cross-reactivity. The class V RBD-targeted MAbs localize to an invariant site of vulnerability, provide a range of neutralization potency, and exhibit considerable breadth against divergent sarbecoviruses, with implications for vaccine and therapeutic development.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein.

The COVID-19 pandemic has significantly impacted human health for three years. To mitigate the spread of SARS-CoV-2, the development of neutralizing antibodies has been accelerated, including the exploration of alternative antibody formats such as single-domain antibodies. In this study, we identified variable new antigen receptors (VNARs) specific for the receptor binding domain (RBD) of SARS-CoV-2 by immunizing a banded houndshark (Triakis scyllium) with recombinant wild-type RBD. Notably, the CoV2NAR-1 clone showed high binding affinities in the nanomolar range to various RBDs and demonstrated neutralizing activity against SARS-CoV-2 pseudoviruses. These results highlight the potential of the banded houndshark as an animal model for the development of VNAR-based therapeutics or diagnostics against future pandemics.

Differences in the organization of interface residues tunes the stability of the SARS-CoV-2 spike-ACE2 complex.

The continuous emergence of novel variants represents one of the major problems in dealing with the SARS-CoV-2 virus. Indeed, also due to its prolonged circulation, more than ten variants of concern emerged, each time rapidly overgrowing the current viral version due to improved spreading features. As, up to now, all variants carry at least one mutation on the spike Receptor Binding Domain, the stability of the binding between the SARS-CoV-2 spike protein and the human ACE2 receptor seems one of the molecular determinants behind the viral spreading potential. In this framework, a better understanding of the interplay between spike mutations and complex stability can help to assess the impact of novel variants. Here, we characterize the peculiarities of the most representative variants of concern in terms of the molecular interactions taking place between the residues of the spike RBD and those of the ACE2 receptor. To do so, we performed molecular dynamics simulations of the RBD-ACE2 complexes of the seven variants of concern in comparison with a large set of complexes with different single mutations taking place on the RBD solvent-exposed residues and for which the experimental binding affinity was available. Analyzing the strength and spatial organization of the intermolecular interactions of the binding region residues, we found that (i) mutations producing an increase of the complex stability mainly rely on instaurating more favorable van der Waals optimization at the cost of Coulombic ones. In particular, (ii) an anti-correlation is observed between the shape and electrostatic complementarities of the binding regions. Finally, (iii) we showed that combining a set of dynamical descriptors is possible to estimate the outcome of point mutations on the complex binding region with a performance of 0.7. Overall, our results introduce a set of dynamical observables that can be rapidly evaluated to probe the effects of novel isolated variants or different molecular systems.