Spike Receptor-binding Domain Research Articles

A statistically established reference value determined for the Vaxarray Coronavirus (CoV) seroassay to characterize vaccination and natural infection.

Serological diagnostic tests are available that measure antibody levels against SARS-CoV-2 antigens. We utilized the Vaxarray Coronavirus (CoV) seroassay, which measures SARS-CoV-2 IgG antibodies against the full-length spike protein (FLS), receptor binding domain (RBD), and S2 extracellular domain (ECD). Previous serological studies have used reference values that have not been validated and require many samples. Here, we show statistically established reference values determined using the upper tail of the Student t-distribution method. The target population was any personnel age 18years and older working on a U.S. Navy ship, and vaccinated with Wuhan variant. The relative fluorescence mean (RFM) reference values for the full-length spike protein, RBD, and S2 ECD were 17,731, 13,990 and 9096, respectively. By using generalized non-parametric regression and reference values for the RBD spike protein and S2 ECD of SARS-CoV-2, this study was able to distinguish vaccine-mediated immune responses from natural infections. We provide the method and statistical code as a resource to determine future reference values for other serological assays.

Distinct pathways for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses.

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014-CoV, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the S1 N-terminal domain, uncovered through the rescue and serial passage of a virus bearing the FPPR substitution, further enhanced spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles' heels that could be targeted with countermeasures.

Evidence of SARS-CoV-2 Spread in Rural Tanzania During the First 6 Months of the Global COVID-19 Pandemic.

In the first 6 months of the coronavirus disease 2019 pandemic, limited testing clouded understanding of the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in Africa. In particular, Tanzania halted all testing and reporting of SARS-CoV-2 cases after May 2020, not resuming until June 2021. In July-August 2020, we performed a seroprevalence survey in rural Bagamoyo district, 40 km outside Dar es Salaam. Among 347 asymptomatic children and adults, 64/347 (18.0%) demonstrated seroreactivity to SARS-CoV-2 spike receptor binding domain by ELISA. Given significant antibody cross-reactivity in malaria-endemic regions, seropositivity was additionally confirmed via a multitarget Luminex immunoassay. Thirty-seven, or 58% of initially seroreactive persons, were Luminex positive, leading to an estimated SARS-CoV-2 seroprevalence of 10.7% (37/347, 95% CI 7.6-14.4%). Working in health care appeared to be associated with seropositivity. Reporting of viral symptoms or health care-seeking behavior in the previous 3 months was not more frequent in seropositive individuals.

Super broad and protective nanobodies against Sarbecoviruses including SARS-CoV-1 and the divergent SARS-CoV-2 subvariant KP.3.1.1.

The ongoing evolution and immune escape of SARS-CoV-2, alongside the potential threat of SARS-CoV-1 and other sarbecoviruses, underscore the urgent need for effective strategies against their infection and transmission. This study highlights the discovery of nanobodies from immunized alpacas, which demonstrate exceptionally broad and potent neutralizing capabilities against the recently emerged and more divergent SARS-CoV-2 Omicron subvariants including JD.1.1, JN.1, KP.3, KP.3.1.1, as well as SARS-CoV-1 and coronaviruses from bats and pangolins utilizing receptor ACE2. Among these, Tnb04-1 emerges as the most broad and potent, binding to a conserved hydrophobic pocket in the spike's receptor-binding domain, distinct from the ACE2 binding site. This interaction disrupts the formation of a proteinase K-resistant core, crucial for viral-cell fusion. Notably, intranasal administration of Tnb04-1 in Syrian hamsters effectively prevented respiratory infection and transmission of the authentic Omicron XBB.1.5 subvariant. Thus, Thb04-1 holds promise in combating respiratory acquisition and transmission of diverse sarbecoviruses.

Quantitative SARS-CoV-2 Spike Receptor-Binding Domain and Neutralizing Antibody Titers in Previously Infected Persons, United States, January 2021-February 2022.

We studied SARS-CoV-2 binding and neutralizing antibody titers among previously infected persons in the United States over time. We assayed SARS-CoV-2 spike protein receptor-binding domain and neutralizing antibody titers for a convenience sample of residual clinical serum specimens that had evidence of prior SARS-CoV-2 infection gathered during January 2021-February 2022. We correlated titers and examined them by age group (<18, 18-49, 50-64, and >65 years) across 4 different SARS-CoV-2 variant epochs. Among selected specimens, 30,967 had binding antibody titers and 744 had neutralizing titers available. Titers in specimens from children and adults correlated. In addition, mean binding antibody titers increased over time for all age groups, and mean neutralization titers increased over time for persons 16-49 and >65 years of age. Incorporating binding and neutralization antibody titers into infectious disease surveillance could provide a clearer picture of overall immunity and help target vaccination campaigns.

Broad cross neutralizing antibodies against sarbecoviruses generated by SARS-CoV-2 infection and vaccination in humans

The outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 highlight the need for countermeasures to prevent future coronavirus pandemics. Given the unpredictable nature of spillover events, preparing antibodies with broad coronavirus-neutralizing activity is an ideal proactive strategy. Here, we investigated whether SARS-CoV-2 infection and vaccination could provide cross-neutralizing antibodies (nAbs) against zoonotic sarbecoviruses. We evaluated the cross-neutralizing profiles of plasma and monoclonal antibodies constructed from B cells from coronavirus disease 2019 (COVID-19) convalescents and vaccine recipients; against sarbecoviruses originating from bats, civets, and pangolins; and against SARS-CoV-1 and SARS-CoV-2. We found that the majority of individuals with natural infection and vaccination elicited broad nAb responses to most tested sarbecoviruses, particularly to clade 1b viruses, but exhibited very low cross-neutralization to SARS-CoV-1 in both natural infection and vaccination, and vaccination boosters significantly augmented the magnitude and breadth of nAbs to sarbecoviruses. Of the nAbs, several exhibited neutralization activity against multiple sarbecoviruses by targeting the spike receptor-binding domain (RBD) and competing with angiotensin-converting enzyme 2 (ACE2) binding. SCM12-61 demonstrated exceptional potency, with half-maximal inhibitory concentration (IC50) values of 0.001–0.091 μg/mL against tested sarbecoviruses; while VSM9-12 exhibited remarkable cross-neutralizing breadth against sarbecoviruses and SARS-CoV-2 Omicron subvariants, highlighting the potential of these two nAbs in combating sarbecoviruses and SARS-CoV-2 Omicron subvariants. Collectively, our findings suggest that vaccination with an ancestral SARS-CoV-2 vaccine, in combination with broad nAbs against sarbecoviruses, may provide a countermeasure for preventing further sarbecovirus outbreaks in humans.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Neutralisation resistance of SARS-CoV-2 spike-variants is primarily mediated by synergistic receptor binding domain substitutions

ABSTRACT The evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested that changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

A-235 Systemic Inflammation is associated with worse outcomes from SARS-CoV-2 infection but not neutralizing antibody

Abstract Background Numerous studies have demonstrated an association between systemic inflammation and mortality in patients with COVID-19. However, it is unclear how neutralizing antibody to severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) associates with outcomes. Further, how systemic inflammation impacts neutralizing antibody production is relatively poorly understood. The aim of this study was to determine the association between the neutralizing antibody response to SARS-CoV-2, inflammatory markers, and clinical outcomes in a cohort of hospitalized patients without prior exposure/vaccination. Methods This prospective observational study approved by the Washington University IRB included adults admitted through the ED at Barnes Jewish Hospital between 10/2020 and 10/2021 with symptomatic RT-PCR confirmed SARS-CoV-2 infection. A total of 395 serial samples were assessed from 208 patients without previous vaccination or documented exposure to SARS-CoV-2. Neutralization of SARS-CoV-2 was assessed using the Architect ACE2 binding inhibition assay (Abbott, Research Use only) which measures the % inhibition of interactions between antibodies and the SARS-COV-2 viral Spike Receptor Binding Domain. While 12.5% inhibition of ACE2 was considered positive &amp;gt;80% neutralization is the expected standard. CRP was assessed using the Architect Multigent CRP assay with 9.6mg/dL as the categorical cutoff for risk and interleukin 6 (IL-6) was assessed using the Alinity IL-6 assay (Abbott, Research Use Only) using 30 pg/mL as the threshold for risk in categorical analyses. The electronic medical record (EPIC) was interrogated for comorbidities, age, gender, 30-day mortality, 10-day intubation, and pharmacological interventions. Correlation between biomarkers was assessed using Spearman r. Kaplan-Meier curves and Cox proportional hazards models were constructed for 30-day mortality and 10-day intubation. Results 37 of 208 (18%) of patients died within thirty days of ED presentation and 59 (28%) required intubation within 10-days. There was a significant correlation between IL-6 and CRP (r=0.34) but not with ACE-2 binding (Spearman r &amp;lt;0.06 for both IL-6 and CRP). There was significantly higher CRP in those that died (median = 14 mg/dL, IQR = 8-21) than those that survived (5 mg/dL, 2-11). IL-6 was higher in patients that died (344 pg/mL, 0-870) than those that survived (65 pg/mL, 28-140). Median ACE-2 inhibition trended higher in those that survived (18%, 0-65%) than those that died (3%, 0-48%). Survival curves demonstrated that patients with IL-6 &amp;gt; 30 pg/mL, CRP &amp;gt; 9.6 mg/dL, and ACE2 binding &amp;lt;12.5% at baseline were more likely to die within 30-days. Multivariate modeling found that only IL-6 (Hazard Ratio, 1.28, 95%CI 1.08-1.52 for each doubling) and age (1.04, 1.01-1.08) were significantly associated with 30-day mortality. Conclusions Systemic inflammation measured by IL-6 and CRP upon admission is highly associated with 30-day mortality from SARS-CoV-2 infection. While ACE-2 inhibition was lower in those that died from COVID-19 at admission, it was not independently associated with mortality or correlated with inflammatory markers. This implies an importance of other aspects of the immune response for mediating inflammation and reducing risk of mortality from SARS-CoV-2.

Neonatal SARS-CoV-2 mRNA Vaccination Efficacy Is Influenced by Maternal Antibodies.

Vaccination in pregnancy guards against infection. Maternal antibodies, however, can inhibit antibody production in neonates. We sought to determine the effects of maternal vaccination on neonatal immune response to a SARS-CoV-2 mRNA vaccine. We hypothesized that mRNA-lipid nanoparticles (LNP) vaccination allows for a de novo neonatal antibody response even in the presence of vertically transmitted maternal antibodies. Female mice were vaccinated with SARS-CoV-2 spike receptor binding domain (RBD) mRNA-LNPs. Mice were then bred, and 21-day-old pups were inoculated with the same mRNA-LNPs. Spike-specific IgG ELISAs were performed using mouse serum. A SARS-CoV-2 spike protein peptide library to perform peptide ELISAs characterized high affinity binding domains within the spike protein. Results were analyzed with one-way ANOVAs with Tukey's multiple comparisons tests. Compared to pups of unvaccinated dams, there were high levels of spike-specific IgG detected in the pups of vaccinated dams at 3 weeks of life (p < 0.0001). After neonatal vaccination, pups of unvaccinated dams had higher spike-specific serum IgG than pups of vaccinated dams at 12 weeks of life (p < 0.001). Antibody specificity to peptide moieties within spike RBD were similar when comparing a vaccinated dam to her pup at Week 3 of life, with different binding affinities observed in the pups by Week 15 of life. Pre-existing maternal antibodies may partially blunt the initial neonatal antibody response to mRNA-LNPs vaccination. This vaccine strategy, however, does not prohibit the subsequent development of a broad range of RBD antibody specificities that may be protective.

Enhanced performance of impedimetric immunosensors to detect SARS-CoV-2 with bare gold nanoparticles and graphene acetic acid

Immunosensors based on electrical impedance spectroscopy allow for label-free, real-time detection of biologically relevant molecules and pathogens, without requiring electro-active materials. Here, we investigate the influence of bare gold nanoparticles (AuNPs), synthesized via laser ablation in solution, on the performance of an impedimetric immunosensor for detecting severe acute respiratory syndrome coronavirus (SARS-CoV-2). Graphene acetic acid (GAA) was used in the active layer for immobilizing anti-SARS-CoV-2 antibodies, owing to its high density of carboxylic groups. Immunosensors incorporating AuNPs exhibited superior performance compared to those relying solely on GAA, achieving a limit of detection (LoD) of 3 x 10−20 g/mL to detect the Spike Receptor Binding Domain (RBD) protein of SARS-CoV-2 and of 2 PFU/mL for inactivated virus. Moreover, these immunosensors presented high selectivity against the H1N1 influenza virus. We anticipate that this platform will be versatile and applicable in the early diagnosis of various diseases and viral infections, thereby facilitating Point-of-Care testing.

THE RECOMBINANT BACTERIA LACTOCOCCUS LACTIS EXPRESSING SARS-COV-2 SPIKE PROTEIN OR ITS RECEPTOR-BINDING DOMAIN ELICITS AN IMMUNE RESPONSE FOLLOWING ORAL IMMUNISATION OF RATS

Mucosal delivery of antigens represents an effective strategy for the development of vaccines to control diseases with the upper respiratory and/or gastrointestinal tracts as the main routes of transmission. Lactic acid bacteria (LAB) are food-grade bacteria used for the expression of target antigens in the development of mucosal vaccines. Over the last two decades, research on the use of LAB has focused on the construction of genetically modified strains. Current study aimed to develop and examine the recombinant Lactococcus lactis strains, expressing SARS-CoV-2 spike (S) protein or the receptor-binding domain (RBD) of S protein as immunizing antigens.The spike gene from the component II of Sputnik V vaccine and two synthesized coding sequences of the RBD region of SARS-CoV-2 spike protein (319–529 residues named HA-spike and 318–590 residues named mini-spike) were subcloned into expression vector pNZ8121. The resulting plasmids were verified by sequencing and subsequently transformed into competent L. lactis NZ3900 cells. The expression of recombinant proteins in obtained strains was analyzed by western blot using SARS-COV-2 Spike RBD antibodies. The results showed that the spike gene could be efficiently expressed in the cells of recombinant L. lactis. The major 150 kDa band detected in the cell lysate of L. lactis pNZ::spike represented the full-length S protein. Protein bands of 35 kDa and 23 kDa from the lysates of L. lactis pNZ::mini-spike and L. lactis pNZ::HA-spike respectively were detected indicating that the recombinant RBD protein was also successfully expressed. The mini-spike was expressed more abundantly than the HA-spike variant. Based on the growth kinetics and protein expression profiles of recombinant strains, the highest protein yield was obtained under the following conditions: overnight culture was inoculated (1/25 inoculum) and grown until reaching an optical density of 0,9 at 600 nm, then gene expression was induced by adding nisin (1 ng/ml), cells were harvested after 3 h incubation with the inducer. To examine the immunogenicity of recombinant strains, 1 ml of cell suspension (1,5×109 CFU) of L. lactis pNZ::spike, pNZ::mini-spike or L. lactis pNZ::HA-spike was orally administered to Wistar rats. The same volume of peptone water was administered to the control group. Serum was collected from the animals at 28 days and IgG level was determined by ELISA for the quantitative determination of IgG antibodies to SARS-CoV-2 (COVID-19) Spike RBD protein in rat serum. As expected, the serum from control animals lacked antibodies to the RBD antigen. The serum of rats immunised once with cell suspension of the recombinant strains showed the following levels of specific IgG: 124,24±12,56 ng/ml in group pNZ::mini-spike, 165,91±59,13 ng/ml in group pNZ::spike, and 213,60±54,08 ng/ml in group pNZ::HA-spike. The highest IgG level (545,56±230,24 ng/ml) was detected in the serum of rats immunized with ultrasound-treated cell suspension of L. lactis pNZ::spike. No adverse effects were observed in vaccinated animals at regular check-ups throughout the study period.The current results have demonstrated that the RBD and full spike protein were successfully expressed in L. lactis. Oral administration of the recombinant L. lactis expressing S protein or RBD to rats appeared to generate the anti-SARS-CoV-2 antibodies in serum, positioning this bacteria as promising basis for creating oral vaccine.

Synthesis, characterization, DFT and molecular docking study of an antiviral drug, the crystallized 6-methoxylated derivative of favipiravir, in the presence of an iron (III) Lewis acid.

Few years ago, the COVID-19 infection, caused by SARS-CoV-2 virus, presented a worldwide health pandemic that taken the life of millions of people. In this concern, Favipiravir (FAV) has attracted considerable attention as an effective antiviral drug against this disease. In this study, the favipiravir's derivative (3‑hydroxy-6-methoxypyrazine-2-carboxamide) was synthetized, in presence of iron (III), and fully characterized through various analytical techniques including single-XRay, IR, UV and 1H/13C NMR. The refinement show the exclusive existence of enol form in the solid state. In solution, UV and NMR data were used to highlight the tautomeric equilibrium of this molecule. Additionally, the density functional theory (DFT) calculations were achieved to estimate the chemical parameters, electronic affinity and molecular electrostatic potential of the compound. Mulliken atomic charges as well as the chemical reactivity descriptors were investigated. Finally, the two tautomer forms of synthetized derivative were docked on SARS-CoV-2 main protease (PDB: 6LU7) and spike receptor-binding domain (PDB: 7BZ5) to evaluate their binding affinity. The docking simulation studies was predicted that the two forms had high binding affinity the critical amino acids residues of the receptor. This affinity was attributed to the presence of several hydrogen bonds for both tautomer along with five different hydrophobic interactions with the enol form. These results conduct to conclude that the synthetized product could be an interest potential drug for that type of diseases.

Comparative Analysis of QCM and Electrochemical Aptasensors for SARS-CoV-2 Detection.

The rapid and accurate detection of SARS-CoV-2, particularly its spike receptor-binding domain (S-RBD), was crucial for managing the COVID-19 pandemic. This study presents the development and optimization of two types of aptasensors: quartz crystal microbalance (QCM) and electrochemical sensors, both employing thiol-modified DNA aptamers for S-RBD detection. The QCM aptasensor demonstrated exceptional sensitivity, achieved by optimizing aptamer concentration, buffer composition, and pre-treatment conditions, with a limit of detection (LOD) of 0.07 pg/mL and a linear range from 1 pg/mL to 0.1 µg/mL, and a significant frequency change was observed upon target binding. The electrochemical aptasensor, designed for rapid and efficient preparation, utilized a one-step modification process that reduced the preparation time to 2 h while maintaining high sensitivity and specificity. Electrochemical impedance spectroscopy (EIS) enabled the detection of S-RBD concentrations as low as 132 ng/mL. Both sensors exhibited high specificity, with negligible non-specific interactions observed in the presence of competing proteins. Additionally, the QCM aptasensor's functionality and stability were verified in biological fluids, indicating its potential for real-world applications. This study highlights the comparative advantages of QCM and electrochemical aptasensors in terms of preparation time, sensitivity, and specificity, offering valuable insights for the development of rapid, sensitive, and specific diagnostic tools for the detection of SARS-CoV-2 and other viruses.

Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant.

Deep mutational scanning experiments aid in the surveillance and forecasting of viral evolution by providing prospective measurements of mutational effects on viral traits, but epistatic shifts in the impacts of mutations can hinder viral forecasting when measurements were made in outdated strain backgrounds. Here, we report measurements of the impact of all single amino acid mutations on ACE2-binding affinity and protein folding and expression in the SARS-CoV-2 Omicron BA.2.86 spike receptor-binding domain. As with other SARS-CoV-2 variants, we find a plastic and evolvable basis for receptor binding, with many mutations at the ACE2 interface maintaining or even improving ACE2-binding affinity. Despite its large genetic divergence, mutational effects in BA.2.86 have not diverged greatly from those measured in its Omicron BA.2 ancestor. However, we do identify strong positive epistasis among subsequent mutations that have accrued in BA.2.86 descendants. Specifically, the Q493E mutation that decreased ACE2-binding affinity in all previous SARS-CoV-2 backgrounds is reversed in sign to enhance human ACE2-binding affinity when coupled with L455S and F456L in the currently emerging KP.3 variant. Our results point to a modest degree of epistatic drift in mutational effects during recent SARS-CoV-2 evolution but highlight how these small epistatic shifts can have important consequences for the emergence of new SARS-CoV-2 variants.

Novel antibody language model accelerates IgG screening and design for broad-spectrum antiviral therapy.

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline AbGen that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. Our AbGen centers around a novel antibody language model (AbLM) that is pretrained on 12 million generic protein domain sequences and fine-tuned on 4,000+ paired VH-VL sequences, with IgG-specific CDR-masking and VH-VL cross-attention. AbLM provides a latent space of IgG sequence embeddings for AbGen, including (a) landscapes of IgGs' activities in neutralizing the wild-type virus are analyzed through structure prediction for IgG and IgG-antigen (viral protein spike's receptor binding domain, RBD) interactions; and (b) landscapes of IgGs' susceptibility in neutralizing variant viruses are predicted through Gaussian process regression, despite that as few as 14 clinical antibodies' responses to variants of concern are available. The AbGen pipeline was applied to over 1300 IgG sequences we collected from RBD-binding B cells of convalescent patients. With experimental validations, AbGen efficiently prioritized IgG candidates against a broad spectrum of viral variants (wildtype, Delta, and Omicron), preventing the infection of host cells in vitro and hACE2 transgenic mice in vivo . Compared to other existing protein language models that require 10-100 times more model parameters, AbLM improved the precision from around 50% to 75% to predict IgGs with low variant susceptibility. Furthermore, AbGen enables structure-based computational protein redesign for selected IgG clones with single amino acid substitutions at the RBD-binding interface that doubled the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screen and re- design combining data-driven protein language models and Kriging for antibody sequence analysis and activity prediction, in synergy with physics-driven protein docking and design for antibody-antigen interface analyses and functional optimization.

Countercurrent chromatography isolation of green propolis biomarkers: Potential blockers of SARS-COV-2 RBD and ACE2 interaction

Propolis is a natural resinous mixture produced by honeybees with numerous biological activities. Considering the recently reported potential of propolis as an adjuvant in COVID-19 treatment, a methodology for the fractionation of the hexane extract of Brazilian green propolis (HEGP) was developed for the obtention of prenylated biomarkers by countercurrent chromatography. The inhibition of the interaction between the receptor binding domain (RBD) of spike and ACE2 receptor was evaluated by the Lumitᵀᴹ immunoassay. Fractionation of HEGP was performed by both normal (CCC1 and CCC2, with extended elution) and reversed (CCC3) phase elution-extrusion modes with the solvent system hexane–ethanol–water 4:3:1. The normal elution mode of CCC1 (471 mg HEGP in a 80 mL column volume, 1.6 mm id) was scaled-up (CCC5, 1211 mg HEGP in a 112 mL column volume, 2.1 mm id), leading to the isolation of 89.9 mg of artepillin C, 1; 52.7 mg of baccharin, 2; and 26.6 mg of chromene, with purities of 93 %, 83 % and 88 %, respectively, by HPLC-PDA. Among the isolated compounds, artepillin C, 1, and baccharin, 2, presented the best results in the Lumitᵀᴹ immunoassay, showing 67% and 51% inhibition, respectively, at the concentration of 10 μM. This technique proved to be of low operational cost and excellent reproducibility.

Biosensor-based active ingredient recognition system for screening potential small molecular Severe acute respiratory syndrome coronavirus 2 entry blockers targeting the spike protein from Rugosa rose.

The traditional formulation Hanchuan zupa granules (HCZPs) have been widely used for controlling coronavirus disease 2019 (COVID-19). However, its active components remain unknown. Here, HCZP components targeting the spike receptor-binding domain (S-RBD) of SARS-CoV-2 were investigated using a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). Recombinant S-RBD proteins were immobilized on the SPR chip by amine coupling for the prescreening of nine HCZP medicinal herbs. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) identified gallic acid (GA) and methyl gallate (MG) from Rosa rugosa as S-RBD ligands, with KD values of 2.69 and 0.95 μM, respectively, as shown by SPR. Molecular dynamics indicated that GA formed hydrogen bonds with G496, N501, and Y505 of S-RBD, and MG with G496 and Y505, inhibiting S-RBD binding to angiotensin-converting enzyme 2 (ACE2). SPR-based competition analysis verified that both compounds blocked S-RBD and ACE2 binding, and SPR demonstrated that GA and MG bound to ACE2 (KD = 5.10 and 4.05 μM, respectively), suggesting that they blocked the receptor and neutralized SARS-CoV-2. Infection with SARS-CoV-2 pseudovirus showed that GA and MG suppressed viral entry into 293T-ACE2 cells. These S-RBD inhibitors have potential for drug design, while the findings provide a reference on HCZP composition and its use for treating COVID-19.

Flexible Graphene-Based Sensors for Electrochemical Analysis of Human Saliva and Sweat

Flexible carbon nanomaterial sensors offer a low-cost, facile fabrication approach toward producing sensors at an industrial scale for electrochemical sensing. Graphene and carbon nanotube (CNT) based sensors have been studied, with various fabrication methods available, including producing high-quality thin film graphene and CNTs via chemical vapor deposition (CVD). However, CVD is a high-temperature vacuum process that is low-yielding and consequently costly, hindering its implementation into single-use test strip sensors. Other methods, such as inkjet or aerosol printing, are more cost-effective as they print graphene inks obtained from liquid-phase exfoliation of graphite, which can be performed using large batch processing techniques. Additionally, the laser-induced graphene (LIG) technique directly converts sp3 carbon found in carbon-rich substrates, such as polyimide, into conductive sp2-hybridized carbon found in graphene through a laser scribing technique. Saliva and sweat offer promising biological fluids for precision health monitoring and diagnostics due to the non-invasive nature of their sampling (no need for a blood draw at a laboratory or a fingerstick at home) and due to the abundance of medically relevant analytes. Saliva can also be used for monitoring infections such as COVID-19 infection. Herein, we introduce graphene-based flexible sensors fabricated using scalable manufacturing based on aerosol jet printing (AJP) using custom-formulated graphene inks and laser-induced graphene via direct writing. We report a label-free, high-sensitivity, and rapid-response-time graphene-based electrochemical immunosensor for quantitatively detecting the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) as well as SARS-CoV-2 Spike S1 protein. We demonstrate LIG-based electrochemical sensors for real-time monitoring of glucose, lactate, and sodium in sweat, and potassium and lactate in saliva. Different electrochemical methods are used, including amperometric, potentiometric, coulometric, and impedimetric, to quantify the analytes. All these electrochemical sensors were validated in human saliva and sweat and demonstrated to have limits of detection and linear sensing ranges that are relevant to their sensing applications. Flexibility studies were also performed to evaluate the ability of the sensors to operate under stresses caused by bending, such as those that would be encountered by skin and implanted oral sensors. Results demonstrate the utility of AJP-graphene and LIG for rapid and sensitive measurement of biological analytes and pathogen infection as simple methods for scalable production of non-invasive salivary and perspiration (sweat) health sensors.

Structural basis for mouse receptor recognition by bat SARS2-like coronaviruses

The animal origin of SARS-CoV-2 remains elusive, lacking a plausible evolutionary narrative that may account for its emergence. Its spike protein resembles certain segments of BANAL-236 and RaTG13, two bat coronaviruses considered possible progenitors of SARS-CoV-2. Additionally, its spike contains a furin motif, a common feature of rodent coronaviruses. To explore the possible involvement of rodents in the emergence of SARS-CoV-2 spike, we examined the crystal structures of the spike receptor-binding domains (RBDs) of BANAL-236 and RaTG13 each complexed with mouse receptor ACE2. Both RBDs have residues at positions 493 and 498 that align well with two virus-binding hotspots on mouse ACE2. Our biochemical evidence supports that both BANAL-236 and RaTG13 spikes can use mouse ACE2 as their entry receptor. These findings point to a scenario in which these bat coronaviruses may have coinfected rodents, leading to a recombination of their spike genes and a subsequent acquisition of a furin motif in rodents, culminating in the emergence of SARS-CoV-2.