Spike Receptor-binding Domain Research Articles

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

Many anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) neutralizing antibodies target the angiotensin-converting enzyme 2 (ACE2) binding site on viral spike receptor-binding domains (RBDs). Potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly emergent zoonotic sarbecoviruses and variants, but they usually show only weak neutralization potencies. Here, we characterize two class 4 anti-RBD antibodies derived from coronavirus disease 2019 (COVID-19) donors that exhibit breadth and potent neutralization of zoonotic coronaviruses and SARS-CoV-2 variants. C118-RBD and C022-RBD structures reveal orientations that extend from the cryptic epitope to occlude ACE2 binding and CDRH3-RBD main-chain H-bond interactions that extend an RBD β sheet, thus reducing sensitivity to RBD side-chain changes. A C118-spike trimer structure reveals rotated RBDs that allow access to the cryptic epitope and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

Convalescent plasma for COVID-19: a meta-analysis, trial sequential analysis, and meta-regression

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, particularly those preventing interaction between the viral spike receptor-binding domain and the host angiotensin-converting enzyme 2 receptor, may prevent viral entry into host cells and disease progression. MethodsWe performed a systematic review, meta-analysis, trial sequential analysis (TSA), and meta-regression of RCTs to evaluate the benefit of convalescent plasma for COVID-19. The primary outcome was 28–30 day mortality. Secondary outcomes included need for mechanical ventilation and ICU admission. Data sources were PubMed, Embase, MedRxiv, and the Cochrane library on July 2, 2021. ResultsWe identified 17 RCTs that recruited 15 587 patients with 8027 (51.5%) allocated to receive convalescent plasma. Convalescent plasma use was not associated with a mortality benefit (24.7% vs 25.5%; odds ratio [OR]=0.94 [0.85–1.04]; P=0.23; I2=4%; TSA adjusted confidence interval [CI], 0.84–1.05), or reduction in need for mechanical ventilation (15.7% vs 15.4%; OR=1.01 [0.92–1.11]; P=0.82; I2=0%; TSA adjusted CI, 0.91–1.13), or ICU admission (22.4% vs 16.7%; OR=0.80 [0.21–3.09]; P=0.75; I2=63%; TSA adjusted CI, 0.0–196.05). Meta-regression did not reveal association with titre of convalescent plasma, timing of administration, or risk of death and treatment effect (P>0.05). Risk of bias was high in most studies. ConclusionsIn patients with COVID-19, there was no clear mortality benefit associated with convalescent plasma treatment. In patients with mild disease, convalescent plasma did not prevent either the need for mechanical ventilation or ICU admission. Clinical trial registrationCRD42021234201 (PROSPERO).

In silico study of some selective phytochemicals against a hypothetical SARS-CoV-2 spike RBD using molecular docking tools

BackgroundThis world is currently witnessing a pandemic outbreak of ‘COVID-19′ caused by a positive-strand RNA virus ‘SARS-CoV-2’. Millions have succumbed globally to the disease, and the numbers are increasing day by day. The viral genome enters into the human host through interaction between the spike protein (S) and host angiotensin-converting enzyme-2 (ACE2) proteins. S is the common target for most recently rolled-out vaccines across regions. A recent surge in single/multiple mutations in S region is of great concern as it may escape vaccine induced immunity. So far, the treatment regime with repurposed drugs has not been too successful. HypothesisNatural compounds are capable of targeting mutated spike protein by binding to its active site and destabilizing the spike-host ACE2 interaction. Materials and methodsA hypothetical mutated spike protein was constructed by incorporating twelve different mutations from twelve geographical locations simultaneously into the receptor-binding domain (RBD) and docked with ACE2 and seven phytochemicals namely allicin, capsaicin, cinnamaldehyde, curcumin, gingerol, piperine and zingeberene. Molecular Dynamic (MD) simulation and Principal Component Analysis (PCA) were finally used for validation of the docking results. ResultThe docking results showed that curcumin and piperine were most potent to bind ACE2, mutated spike, and mutated spike-ACE2 complex, thereby restricting viral entry. ADME analysis also proved their drug candidature. The docking complexes were found to be stable by MD simulation. ConclusionThis result provides a significant insight about the phytochemicals' role, namely curcumin and piperine, as the potential therapeutic entities against mutated spike protein of SARS-CoV-2.

Molecular Docking Analysis of the Phytochemicals from Tinospora Cordifolia as Potential Inhibitor Against Multi Targeted SARS-CoV-2 & Cytokine Storm

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2, a novel coronavirus, is a member of the Coronoviridae family that has spread worldwide. Developing efficacious therapeutics for the treatment of SARS-CoV-2 is of high priority. Therefore, in this study, the chemical constituents obtained from Tinospora cordifolia are investigated for their in-silico interaction with protein targets crucial for SARSCoV-2 infection and cytokine storm. The five important targets chosen for SARSCoV-2 were the main protease (Mpro), Spike receptor binding domain (Spike-RBD), RNA-dependent RNA polymerase (RdRp or Nsp12), nonstructural protein 15 (Nsp15) of SARS-CoV-2 and the host angiotensin converting enzyme-2 (ACE-2) spike-RBD binding domain and cytokine receptors TNF-[Formula: see text] (Tumor Necrosis Factor-[Formula: see text]) and IL-6 (Interleukine-6). This was accomplished using Maestro 12.4 (Schrodinger Suite) to obtain docking scores. Also, the absorption, distribution, metabolism, elimination, and toxicity parameters (ADMET) were determined using Maestro QikProp modules. The results of computational study revealed that four constituents Cordifolioside-A, Palmatoside-E, Tinocordioside and Tinosporaside significantly antagonize the five targets of SARS-CoV-2 by binding in the binding pocket with docking score ranging from −9.664 to −6.488 kcal/mol and shows drug-like property and also effectively inhibit cytokine storm by antagonizing the TNF-[Formula: see text] and IL-6 receptors. Promising drug-like properties, excellent docking scores, and binding pose against each target makes the screened compounds as possible lead candidate which can be further evaluated in future studies to assess their in vitro and in vivo efficacy against SARS-CoV-2. The structure of these compounds can be used further for optimization and design of drugs against COVID-19.

Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics.

The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape.

The impact of spike N501Y mutation on neutralizing activity and RBD binding of SARS-CoV-2 convalescent serum.

BackgroundSeveral SARS-CoV-2 lineages with spike receptor binding domain (RBD) N501Y mutation have spread globally. We evaluated the impact of N501Y on neutralizing activity of COVID-19 convalescent sera and on anti-RBD IgG assays.MethodsThe susceptibility to neutralization by COVID-19 patients’ convalescent sera from Hong Kong were compared between two SARS-CoV-2 isolates (B117-1/B117-2) from the α variant with N501Y and 4 non-N501Y isolates. The effect of N501Y on antibody binding was assessed. The performance of commercially-available IgG assays was determined for patients infected with N501Y variants.FindingsThe microneutralization antibody (MN) titers of convalescent sera from 9 recovered COVID-19 patients against B117-1 (geometric mean titer[GMT],80; 95% CI, 47–136) were similar to those against the non-N501Y viruses. However, MN titer of these serum against B117-2 (GMT, 20; 95% CI, 11–36) was statistically significantly reduced when compared with non-N501Y viruses (P < 0.01; one-way ANOVA). The difference between B117-1 and B117-2 was confirmed by testing 60 additional convalescent sera. B117-1 and B117-2 differ by only 3 amino acids (nsp2-S512Y, nsp13-K460R, spike-A1056V). Enzyme immunoassay using 272 convalescent sera showed reduced binding of anti-RBD IgG to N501Y or N501Y-E484K-K417N when compared with that of wild-type RBD (mean difference: 0.1116 and 0.5613, respectively; one-way ANOVA). Of 7 anti-N-IgG positive sera from patients infected with N501Y variants (collected 9-14 days post symptom onset), 6 (85.7%) tested negative for a commercially-available anti-S1-IgG assay.InterpretationWe highlighted the importance of using a panel of viruses within the same lineage to determine the impact of virus variants on neutralization. Furthermore, clinicians should be aware of the potential reduced sensitivity of anti-RBD IgG assays.

An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global concern and necessitates efficient drug antagonists. Angiotensin-converting enzyme-2 (ACE2) is the main receptor of SARS-CoV-2 spike 1 (S1), which mediates viral invasion into host cells. Herein, we designed and prepared short peptide inhibitors containing 4–6 critical residues of ACE2 that contribute to the interaction with SARS-CoV-2 S1. Among the candidates, a peptide termed GK-7 (GKGDFRI), which was designed by extracting residues ranging from Gly353 to Ile359 in the ligand-binding domain of ACE2, exhibited the highest binding affinity (25.1 nM) with the SARS-CoV-2 spike receptor-binding domain (RBD). GK-7 bound to the RBD and decreased SARS-CoV-2 S1 attachment to A549 human alveolar epithelial cells. Owing to spike blockade, GK-7 inhibited SARS-CoV-2 spike pseudovirion infection in a dose-dependent manner, with a half-maximal inhibitory concentration of 2.96 μg/mL. Inspiringly, pulmonary delivery of GK-7 by intranasal administration did not result in toxicity in mice. This study revealed an easy-to-produce peptide inhibitor for SARS-CoV-2 spike blockade, thus providing a promising candidate for COVID-19 treatment.

Sequential Profiling of Anti-SARS CoV-2 IgG Antibody in Post COVID-19 Patients.

Upon SARS CoV-2 infection, humoral immune system triggers production of anti-SARS CoV-2 IgM and IgG antibodies. Currently, antibodies against SARS CoV-2 spike protein receptor binding domain play a central role in disease protection, making them potential target for in vitro diagnostics applications. This study determines the expression level and sustainability of anti-receptor binding domain (RBD) SARS CoV-2 IgG in post COVID-19 patients. Anti-RBD SARS CoV-2 IgG antibodies in patient serum were analysed by standardised indirect ELISA using SARS CoV-2 spike receptor binding domain protein and HRP conjugated anti-human IgG antibody (anti-h IgG). The study was conducted using 35 adult patient samples with confirmed SARS CoV-2 infection. Additionally, correlation between antibody response after each stage and disease symptoms in post COVID-19 patients were studied. Maximum antibody titre was seen at Day 40 and decreased relatively to Day 180 in antibody positive samples when compared with controls. Overall, more IgG antibody expression is observed in patients who suffered from loss of smell and taste at Day 40. 71% of the positive subjects in this study showed high SARS CoV-2 IgG antibody concentration of above 10 ng/mL and 37% showed strong antibody concentration above 20 ng/mL at the peak of seroconversion.

Impact of Age and Sex on Antibody Response Following the Second Dose of COVID-19 BNT162b2 mRNA Vaccine in Greek Healthcare Workers.

Anti-SARS-CoV-2 spike RBD (receptor-binding domain) IgG antibody levels were monitored in 1643 volunteer healthcare workers of Eginition, Evangelismos, and Konstantopoulio General Hospitals (Athens, Greece), who underwent vaccination with two doses of COVID-19 BNT162b2 mRNA vaccine (Pfizer) and had no history of SARS-CoV-2 infection. Venous blood was collected 20–30 days after the second vaccine dose and anti-RBD IgG levels were determined using CMIA SARS-CoV-2 IgG II Quant (Abbott) on ARCHITECT i System or ADVIA Centaur SARS-CoV-2 IgG (Siemens) on Centaur XP platform. From the total population of 1643 vaccinees (533 M/1110 F; median age = 49; interquartile range-IQR = 40–56), 1636 (99.6%) had anti-SARS-CoV-2 IgG titers above the positivity threshold of the assay used. One-Way ANOVA Kruskal-Wallis H test showed a statistically significant difference in the median of antibody titers between the different age groups (p < 0.0001). Consistently, Spearman’s correlation coefficient (r) for IgGs and age as continuous variables was −0.2380 (p = 1.98 × 10−17). Moreover, antibody titers were slightly higher by 1.2-mean fold (p = 3 × 10−6) in the total female population of the three hospitals (median = 1594; IQR = 875–2584) as compared to males (median = 1292; IQR = 671.9–2188). The present study supports that BNT162b2 vaccine is particularly effective in producing high anti-SARS-CoV-2 IgG levels in healthy individuals, and this humoral response is age- and gender-dependent.

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?

2020 Hindsight: Should evolutionary virologists have expected the unexpected during a pandemic?

Development of an in-house COVID-19 serology ELISA Test

Background: COVID-19 pandemic created an unprecedented demand for reagents and diagnostic tools to confirm COVID-19 cases. Thus, the development of a robust in-house diagnostic test is considered of high importance. Within a few days after exposure, the human body produces specific antibodies that recognize the surface proteins of the invading SARS-CoV-2 virus. Therefore, virus specific immunoglobulins are neutralizing antibodies and their appearance in the blood is a good sign of immunity. The aim of this study was to develop an in-house COVID-19 serology ELISA test to quantify induced antibody responses. This test can help identify convalescent plasma donors with high antibody titers that can be used to treat other patients. Methods: Spike protein antigen is highly expressed in SARS-CoV-2. Recombinant protein corresponding to the spike receptor-binding domain (RBD), which binds to specific antibodies circulating in COVID-19 patients’ blood was used as the antigen in this colorimetric ELISA test. Briefly, a 96-microtiter well plate was coated with RBD protein, where serum dilutions were added. Antibody titers were detected using an anti-human IgG- peroxidase labelled antibody and the substrate o-phenylenediamine dihydrochloride; measured at optical density (OD) of 450 nm. Results: The in-house quantitative serology test was validated using serum samples collected from severe COVID-19 patients (n = 282) admitted to the intensive care unit at Hamad General Hospital. Serum samples from non-COVID-19 (n = 10) were used as a negative control. We detected high antibody titers in ~90% of COVID-19 sera. In contrast, no SARS-CoV-2 specific antibodies were detected in the serum of non-infected subjects (n = 6), pooled human serum collected before 2019, or Middle East Respiratory Syndrome (MERS) infected subjects (n = 3) confirming the specificity and the sensitivity of this in-house serology test. Conclusion: This in-house quantitative serology test is sensitive, specific, and inexpensive. The test can address the rising issue of COVID-19 supply chain globally and foster the capacity-building efforts envisioned by Qatar University.

Walking down the memory lane with SARS-CoV-2 B cells.

The B-cell response to COVID-19 vaccines in convalescent individuals.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

The interaction of the severe acute respiratory syndrome coronavirus 2 spike protein with drug-inhibited angiotensin converting enzyme 2 studied by molecular dynamics simulation.

Hypertension has been identified as the most common comorbidity in coronavirus disease 2019 (COVID-19) patients, and has been suggested as a risk factor for COVID-19 disease outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host human cells via binding to host cell angiotensin-converting enzyme 2 (ACE2) receptors. Inhibition of ACE2 has been proposed as a potential therapeutic approach to block SARS-CoV-2 contagion. However, some experts suggest that ACE2 inhibition could worsen the infection. Here, we aimed to study the effect of ACE2 inhibition on the SARS-CoV-2 spike protein binding to ACE2. Crystallographic structures of the SARS-CoV-2 spike protein, the spike receptor-binding domain, native ACE2, and the ACE2 complexed with MLN-4760 were used as the study model structures. The spike proteins were docked to the ACE2 structures and the dynamics of the complexes, ligand-protein, and protein-protein interactions were studied by molecular dynamics simulation for 100 ns. Our result showed that inhibition of ACE2 by MLN-4760 increased the affinity of the SARS-CoV-2 spike protein binding to ACE2. Results also revealed that spike protein binding to the ACE2 inhibited by MLN-4760 restored the enzymatic active conformation of the ACE2 from closed/inactive to open/active conformation by removing MLN-4760 binding from the ligand-binding pocket of ACE2. We conclude that using ACE2 inhibitors can increase the risk of SARS-CoV-2 infection and worsen COVID-19 disease outcome. We also found that the SARS-CoV-2 can abrogate the function of ACE2 inhibitors and rescue the enzymatic activity of ACE2. Therefore, ACE2 inhibition is not a useful treatment against COVID-19 infection.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Cold atmospheric plasma: A non-negligible strategy for viral RNA inactivation to prevent SARS-CoV-2 environmental transmission.

Cold atmospheric plasma (CAP), regarded as a powerful physics technology, displays antimicrobial, antitumor, and even antiviral properties, but the underlying mechanism is rarely studied. In this study, four CAP exposure doses (30, 60, 120, and 240 s) were applied to inactivate a severe acute respiratory syndrome coronavirus 2 like pseudovirus on a stainless steel disk, which comprised spike protein on its membrane and can express a green fluorescent protein. In order to unravel the potential effects of CAP irradiation on pseudovirus, infection assay, optical emission spectra analysis, transmission electron microscopy (TEM), sodium dodecyl sulfate polyacrylamide gel electrophoresis, ELISA, and qPCR experiments were carried out. As a result, our study indicated that CAP irradiation can significantly decrease the infectivity of pseudovirus in a dose dependent manner through destroying the cell membrane and further damaging viral RNA, with the molecular weight and conformation of spike receptor binding domain protein unchanged.

Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Structure-based study of immune receptors as eligible binding targets of coronavirus SARS-CoV-2 spike protein

One of the most important challenges in the battle against contagious SARS-CoV-2 is subtle identification of the virus pathogenesis. The broad range of COVID-19 clinical manifestations may indicate diversity of virus-host cells. Amongst key manifestations, especially in severe COVID-19 patients, reduction and/or exhaustion of lymphocytes, monocytes, basophils, and dendritic cells are seen.; therefore, it is required to recognize that how the virus infects the cells. Interestingly, angiotensin-converting enzyme 2 (ACE2) as the well-known receptor of SARS-CoV-2 is low or non-expressed in these cells. Using computational approach, several receptor candidates including leukocyte surface molecules and chemokine receptors that expressed in most lineages of immune cells were evaluated as the feasible receptor of spike receptor-binding domain (RBD) of SARS-CoV-2. The results revealed the higher binding affinity of CD26, CD2, CD56, CD7, CCR9, CD150, CD4, CD50, XCR1 and CD106 compared to ACE2. However, the modes of binding and amino acids involved in the interactions with the RBD domain of spike were various. Overall, the affinity of immune receptor candidates in binding to SARS-CoV-2 RBD may offer insight into the recognition of novel therapeutic targets in association with COVID-19.

New Impedimetric Sandwich Immunosensor for Ultrasensitive and Highly Specific Detection of Spike Receptor Binding Domain Protein of SARS-CoV-2.

An impedance sensing platform-combined conducting nanocomposite layer was fabricated to develop an effective and rapid method for detection of coronavirus infection (COVID-19) specific spike receptor binding domain (RBD) protein, a precious antigen marker of COVID-19 disease. Coronavirus infection has spread globally and swiftly with major impacts on health, economy, and quality of life of communities. Fast and reliable detection of COVID-19 is a very significant issue for the effective treatment of this bad illness. For this aim, first, an Epoxy functional group-substituted thiophene monomer was synthesized and electrodeposited on a single-use indium tin oxide (ITO) platform in the presence of acetylene black by employing a cyclic voltammetry technique; thus, a conducting nanocomposite (C-NC) layer with high conductivity was obtained. This composite was electrodeposited for the first time on the ITO surface to generate a facile and cost-effective impedimetric biosensor. In addition, this composite provided proper attachment points for antibody binding and also supported the biosensor construction. The immuno-specific biointeractions between anti-RBD and RBD proteins hampered the electron transfer between the ITO substrate surface and electrolyte, and this reaction caused variations in impedance signals, and these signals were proportional to the immobilized RBD antigen amounts. The as-prepared immunosensor showed a wide linear dynamic range (0.0012-120 pg/mL), an ultra-low detection limit of 0.58 fg/mL with added superiorities of great selectivity, suitable repeatability, multiple reusability, and excellent reproducibility.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.