RBD Protein Research Articles

B-043 Q-nab™ Igg kit: verification and validation of a novel cell-free quantitative neutralizing antibody assay

Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.

Differential detection of SARS-CoV-2 variants and influenza A viruses utilizing a dual lateral flow strip based on colloidal gold-labeled monoclonal antibodies

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the seasonal influenza virus are spreading among humans concurrently, especially with the ongoing replacement of mutant strains. It is challenging to differentiate between symptoms for therapy due to the comparable symptoms following infection with the SARS-CoV-2 variants and influenza viruses. Meanwhile, in order to achieve rapid point-of-care testing (POCT) to manage the spread of the disease, we developed a dual lateral flow strip based on colloidal gold-labeled monoclonal antibodies that can perform differential detection of SARS-CoV-2 variants and influenza A viruses (IAV) in this study. High-affinity monoclonal antibodies (mAbs) targeting SARS-CoV-2 and IAV were prepared to capture antigens and labeled with colloidal gold nanoparticles (AuNPs). Based on high-affinity mAbs, two targets were immobilized on one nitrocellulose (NC) membrane to establish the lateral flow strip (LFS) for differential diagnosis of SARS-CoV-2 and IAV. With no reactivity to other viruses, this LFS is extremely specific and can only identify SARS-CoV-2 and IAV. The LFS showed a limit of detection (LOD) of 4.88 ng/mL for the Omicron BA.2 RBD protein and 2.44 ng/mL for the nucleoprotein (NP) protein of H1N1. When analyzing 16 SARS-CoV-2 positive clinical samples, eight IAV positive clinical samples, and six negative samples that had already been pre-confirmed by commercial kits, its clinical application is effectively and accurately proven. These results demonstrated that the LFS integrated with AuNPs has great potential to facilitate quick, easy, and reliable POCT diagnosis for promoting the control of infectious diseases.

Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19–29aa, RMSD 1–1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Leveraging the potential of bacterial lateral gene transfer in boosting the efficacy of an edible probiotic prototype yogurt vaccine for COVID-19

Administration of coronavirus disease-2019 (COVID-19) vaccines with appropriate booster doses through painful injections under clinical supervision was challenging during the recent COVID-19 pandemic. As an alternative solution, we designed a safer, edible probiotic yogurt vaccine prototype (YoVac) that can be orally consumed by circumventing painful injections and clinical supervision. We hypothesized that YoVac prepared using Lactobacillus carrying an antigen coding gene (donor) can transfer the same to other bacteria (recipients) in the human gut microbiome (hgMb) through lateral gene transfer (LGT) for boosted antigen levels potentially triggering a robust immune response. In this study we confirmed the in vitro LGT efficiency of a plasmid (pRBD-Ampr) containing severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) spike protein-receptor binding domain (RBD) coding gene along with an ampicillin-resistance gene (selection marker) from the probiotic Lactobacillus (donor) cultured from homemade yogurt to E. coli and Helicobacter pylori (recipients). Both the donor and recipient bacteria not only exhibited ampicillin-resistance from pRBD-Ampr but also expressed RBD protein. Furthermore, Lactobacillus isolated from YoVac consistently showed the expression of RBD protein over a period of one month confirming the shelf life of our prototype stored at 4 °C. Taken together, our in vitro results provide a preliminary basis for the potential in vivo transfer of RBD coding gene from YoVac to other bacterial species in the hgMb through LGT and may potentially boost the vaccine dosage by delegating them.

Fourth dose bivalent COVID-19 vaccines outperform monovalent boosters in eliciting cross-reactive memory B cells to Omicron subvariants

Bivalent COVID-19 vaccines comprising ancestral Wuhan-Hu-1 (WH1) and the Omicron BA.1 or BA.5 subvariant elicit enhanced serum antibody responses to emerging Omicron subvariants. Here, we characterized the RBD-specific memory B cell (Bmem) response following a fourth dose with a BA.1 or BA.5 bivalent vaccine, in direct comparison with a WH1 monovalent fourth dose. Healthcare workers previously immunized with mRNA or adenoviral vector monovalent vaccines were sampled before and one-month after a fourth dose with a monovalent or a BA.1 or BA.5 bivalent vaccine. Serum neutralizing antibodies (NAb) were quantified, as well as RBD-specific Bmem with an in-depth spectral flow cytometry panel including recombinant RBD proteins of the WH1, BA.1, BA.5, BQ.1.1, and XBB.1.5 variants. Both bivalent vaccines elicited higher NAb titers against Omicron subvariants compared to the monovalent vaccine. Following either vaccine type, recipients had slightly increased WH1 RBD-specific Bmem numbers. Both bivalent vaccines significantly increased WH1 RBD-specific Bmem binding of all Omicron subvariants tested by flow cytometry, while recognition of Omicron subvariants was not enhanced following monovalent vaccination. IgG1+ Bmem dominated the response, with substantial IgG4+ Bmem only detected in recipients of an mRNA vaccine for their primary dose. Thus, Omicron-based bivalent vaccines can significantly boost NAb and Bmem specific for ancestral WH1 and Omicron variants, and improve recognition of descendent subvariants by pre-existing, WH1-specific Bmem, beyond that of a conventional, monovalent vaccine. This provides new insights into the capacity of variant-based mRNA booster vaccines to improve immune memory against emerging SARS-CoV-2 variants and potentially protect against severe disease. One-sentence summaryOmicron BA.1 and BA.5 bivalent COVID-19 boosters, used as a fourth dose, increase RBD-specific Bmem cross-recognition of Omicron subvariants, both those encoded by the vaccines and antigenically distinct subvariants, further than a monovalent booster.

Development of Novel Peptide Inhibitors Adapted to the Surface Property and Morphology of S Protein RBD

Development of Novel Peptide Inhibitors Adapted to the Surface Property and Morphology of S Protein RBD

Trichinella spiralis Infection Inhibits the Efficacy of RBD Protein of SARS-CoV-2 Vaccination via Regulating Humoral and Cellular Immunity.

Vaccines are the most effective and feasible way to control pathogen infection. Helminths have been reported to jeopardize the protective immunity mounted by several vaccines. However, there are no experimental data about the effect of helminth infection on the effectiveness of COVID-19 vaccines. Here, a mouse model of trichinosis, a common zoonotic disease worldwide, was used to investigate effects of Trichinella spiralis infection on the RBD protein vaccine of SARS-CoV-2 and the related immunological mechanism, as well as the impact of albendazole (ALB) deworming on the inhibitory effect of the parasite on the vaccination. The results indicated that both the enteric and muscular stages of T. spiralis infection inhibited the vaccine efficacy, evidenced by decreased levels of IgG, IgM, sIgA, and reduced serum neutralizing antibodies, along with suppressed splenic germinal center (GC) B cells in the vaccinated mice. Pre-exposure to trichinosis promoted Th2 and/or Treg immune responses in the immunized mice. Furthermore, ALB treatment could partially reverse the inhibitory effect of T. spiralis infection on the efficiency of the vaccination, accompanied by a restored proportion of splenic GC B cells. Therefore, given the widespread prevalence of helminth infections worldwide, deworming therapy needs to be considered when implementing COVID-19 vaccination strategies.

Effect of apigetrin in pseudo-SARS-CoV-2-induced inflammatory and pulmonary fibrosis in vitro model

SARS-CoV-2 has become a global public health problem. Acute respiratory distress syndrome (ARDS) is the leading cause of death due to the SARS-CoV-2 infection. Pulmonary fibrosis (PF) is a severe and frequently reported COVID-19 sequela. In this study, an in vitro model of ARDS and PF caused by SARS-CoV-2 was established in MH-S, THP-1, and MRC-5 cells using pseudo-SARS-CoV-2 (PSCV). Expression of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and HIF-1α was increased in PSCV-infected MH-S and THP-1 cells, ARDS model, consistent with other profiling data in SARS-CoV-2-infected patients have been reported. Hypoxia-inducible factor-1 alpha (HIF-1α) siRNA and cobalt chloride were tested using this in vitro model. HIF-1α knockdown reduces inflammation caused by PSCV infection in MH-S and THP-1 cells and lowers elevated levels of CTGF, COLA1, and α-SMA in MRC-5 cells exposed to CPMSCV. Furthermore, apigetrin, a glycoside bioactive dietary flavonoid derived from several plants, including Crataegus pinnatifida, which is reported to be a HIF-1α inhibitor, was tested in this in vitro model. Apigetrin significantly reduced the increased inflammatory cytokine (IL-6, IL-1β, and TNF-α) expression and secretion by PSCV in MH-S and THP-1 cells. Apigetrin inhibited the binding of the SARS-CoV-2 spike protein RBD to the ACE2 protein. An in vitro model of PF induced by SARS-CoV-2 was produced using a conditioned medium of THP-1 and MH-S cells that were PSCV-infected (CMPSCV) into MRC-5 cells. In a PF model, CMPSCV treatment of THP-1 and MH-S cells increased cell growth, migration, and collagen synthesis in MRC-5 cells. In contrast, apigetrin suppressed the increase in cell growth, migration, and collagen synthesis induced by CMPSCV in THP-1 and MH-S MRC-5 cells. Also, compared to control, fibrosis-related proteins (CTGF, COLA1, α-SMA, and HIF-1α) levels were over two-fold higher in CMPSV-treated MRC-5 cells. Apigetrin decreased protein levels in CMPSCV-treated MRC-5 cells. Thus, our data suggest that hypoxia-inducible factor-1 alpha (HIF-1α) might be a novel target for SARS-CoV-2 sequela therapies and apigetrin, representative of HIF-1alpha inhibitor, exerts anti-inflammatory and PF effects in PSCV-treated MH-S, THP-1, and CMPVSC-treated MRC-5 cells. These findings indicate that HIF-1α inhibition and apigetrin would have a potential value in controlling SARS-CoV-2-related diseases.

The involvement of circulating miR-146a and miR-27a in patients with atherosclerotic cardiovascular disease after SARS-CoV-2 infection.

Atherosclerotic cardiovascular disease (ASCVD) is a group of clinical diseases based on pathology of atherosclerosis that is the leading cause of mortality worldwide. There is a bidirectional interaction between ASCVD and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alterations in circulating miRNAs levels are involved in the development of ASCVD in patients infected with SARS-CoV-2, however, the correlation between ASCVD co-infection with SARS-CoV-2 and alterations of cardiac-specific miRNAs is not well understood. The circulating miR-146a and miR-27a are involved in bidirectional interactions between ASCVD and SARS-CoV-2 infections. Circulating miR-146a and miR-27a levels were measured in serum and PBMCs deriving from ASCVD patients and controls after SARS-CoV-2 infection by qRT-PCR analysis. The levels of neutralizing antibodies-resistant SARS-CoV-2 in human serum was determined by competitive magnetic particle chemiluminescence method. Interleukin (IL)-6 levels were detected by automatic biochemical analyzer using electrochemiluminescence. Significant downregulation of circulating miR-146a and upregulation of miR-27a in ASCVD patients after infection with SARS-CoV-2 compared with controls were observed, among which the alterations were more evident in ASCVD patients comorbid with hyperlipidemia and diabetes mellitus. Consistently, correlation analysis revealed that serum miR-146a and miR-27a levels were associated with the levels of lipids and glucose, inflammatory response, and immune function in ASCVD patients. Remarkably, SARS-CoV-2 S protein RBD stimulation of PBMCs derived from both ASCVD and controls significantly downregulated miR-146a, upregulated miR-27a expression levels, and promoted IL-6 release in vitro. The circulating miR-146a and miR-27a are involved in metabolism, inflammation, and immune levels in patients with ASCVD after SARS-CoV-2 infection, laying the foundation for the development of strategies to prevent the risk of SARS-CoV-2 infection in ASCVD patients.

A Nasal Vaccine Candidate, Containing Three Antigenic Regions from SARS-CoV-2, to Induce a Broader Response.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

In vitro and in silico study of biosynthesized silver nanoparticles using Nigella sativa extract against SARS-CoV-2 and Candida albicans

Pathogens that are resistant to multiple drugs, commonly known as superbugs, including viruses and fungi, are posing a significant danger to human health. These superbugs are responsible for causing high rates of morbidity and mortality each year, and the numbers are continuing to rise. Therefore, the search for potent, safe, and viable alternative antimicrobial agents is imperative. In the current study, a rapid and eco-friendly method for synthesizing silver nanoparticles (AgNPs) using Nigella sativa seed extract was developed. Various characterization techniques, including ultraviolet–visible (UV–vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy associated with microanalysis by dispersive energy of X-rays (SEM-EDX), and Zeta-potential, were employed to confirm the formation of AgNPs. Agglomerated particles with a spherical shape and a size of approximately 50 nm were identified in the SEM images of AgNPs. The optical bandgap energy of the AgNPs was determined to be 2.43 eV for direct bandgap and 1.87 eV for allowable indirect band gap. The study also investigated the antiviral activity of AgNPs against the XBB.1.5 SARS-CoV-2 variant and assessed their Anti-Candida Assay against Candida albicans. Furthermore, we conducted molecular docking studies to explore the interactions between AgNPs and the Omicron receptor-binding motif (RBM). The spike protein RBD binds to the receptor angiotensin-converting enzyme 2 (ACE2) human cellular receptor (hACE2) that helps the virus enter the host cell. The results indicated that AgNPs can bind to the spike protein RBD, potentially elucidating their antiviral effects against SARS-CoV-2. The findings of this study suggest that silver nanoparticles (AgNPs) hold promise as potent agents for therapeutic applications, particularly in addressing multidrug-resistant strains of viruses and Candida. However, further research is essential to thoroughly elucidate the mode of action and assess the biosafety of AgNPs.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

In Silico Screening of Some Active Phytochemicals to Identify Promising Inhibitors Against SARS-CoV-2 Targets.

There are very few small-molecule drug candidates developed against SARS-CoV-2 that have been revealed since the epidemic began in November 2019. The typical medicinal chemistry discovery approach requires more than a decade of the year of painstaking research and development and a significant financial guarantee, which is not feasible in the challenge of the current epidemic. This current study proposes to find and identify the most effective and promising phytomolecules against SARS-CoV-2 in six essential proteins (3CL protease, Main protease, Papain- Like protease, N-protein RNA binding domain, RNA-dependent RNA polymerase, and Spike receptor binding domain target through in silico screening of 63 phytomolecules from six different Ayurveda medicinal plants. The phytomolecules and SARS-CoV-2 proteins were taken from public domain databases such as PubChem and RCSB Protein Data Bank. For in silico screening, the molecular interactions, binding energy, and ADMET properties were investigated. The structure-based molecular docking reveals some molecules' greater affinity towards the target than the co-crystal ligand. Our results show that tannic acid, cyanidin-3-rutinoside, zeaxanthin, and carbolactone are phytomolecules capable of inhibiting SARS-CoV-2 target proteins in the least energy conformations. Tannic acid had the least binding energy of -8.8 kcal/mol, which is better than the binding energy of its corresponding co-crystal ligand (-7.5 kcal/mol) against 3 CL protease. Also, it has shown the least binding energy of -9.9 kcal/mol with a more significant number of conventional hydrogen bond interactions against the RdRp target. Cyanidin-3-rutinoside showed binding energy values of -8.8 and -7.6 kcal/mol against Main protease and Papain-like protease, respectively. Zeaxanthin was the top candidate in the N protein RBD with a binding score of - 8.4 kcal/mol, which is slightly better when compared to a co-crystal ligand (-8.2 kcal/mol). In the spike, carbolactone was the suitable candidate with the binding energy of -7.2 kcal/mol and formed a conventional hydrogen bond and two hydrophobic interactions. The best binding affinity-scored phytomolecules were selected for the MD simulations studies. The present in silico screening study suggested that active phytomolecules from medicinal plants could inhibit SARS-CoV-2 targets. The elite docked compounds with drug-like properties have a harmless ADMET profile, which may help to develop promising COVID-19 inhibitors.

Effect of ultra-diluted ethanol extract of Rhus toxicodendron SARS-CoV-2 Spike protein RBD induced inflammation in chick embryo

Effect of ultra-diluted ethanol extract of Rhus toxicodendron SARS-CoV-2 Spike protein RBD induced inflammation in chick embryo

Synthesis of Zn(ii) coordination complexes, their molecular design and docking with SARS-CoV-2 RBD protein and Omicron spike protein

Two Zn(ii) complexes [Zn(L)(en)]ClO4 (1) and [Zn(L)2] (2) were synthesized and designed and their molecular docking with the SARS-CoV-2 RBD protein and Omicron spike protein were studied for assessing them as possible therapeutic drug candidates against SARS-CoV-2 and its Omicron variant.

A highly specific aptamer for the SARS-CoV-2 spike protein from the authentic strain.

DNA aptamers are oligonucleotides that specifically bind to target molecules, similar to how antibodies bind to antigens. We identified an aptamer named MEZ that is highly specific to the receptor-binding domain, RBD, of the SARS-CoV-2 spike protein from the Wuhan-Hu-1 strain. The SELEX procedure was utilized to enrich the initial 31-mer oligonucleotide library with the target aptamer. The aptamer identification was performed using the novel protocol based on nanopore sequencing developed in this study. The MEZ aptamer was chemically synthesized and tested for binding with the SARS-CoV-2 RBD of the spike protein from different strains. The Kd is 6.5 nM for the complex with the RBD from the Wuhan-Hu-1 strain, which is comparable with known aptamers; the advantage is that the MEZ aptamer is smaller than known analogs. The proposed aptamer is highly selective for the RBD protein from the Wuhan-Hu-1 strain and does not form complexes with the RBD from Beta, Delta and Omicron strains. Experimental and theoretical studies together revealed the molecular mechanism of aptamer binding. The aptamer occupies the same binding site as ACE2 when bound to the RBD. The 3'-end of the MEZ aptamer is important for complex formation and is responsible for the discrimination of the RBD protein from a specific strain. The 5'-end is responsible for the formation of a loop in the 3D structure of the aptamer, which is important for proper binding.

Design and synthesis of copper(ii) and nickel(ii) complexes as potential inhibitors against SARS-CoV-2 viral RNA binding protein and Omicron RBD protein

Two bis-Schiff base metal(ii) complexes [Cu(L)2](1) and [Ni(L)2](2) were designed and synthesized as potential inhibitors for the development of new anti-SARS-CoV-2 Omicron and anti-HIV drugs.

Structurally defined heparin octasaccharide domain for binding to SARS-CoV-2 Omicron BA.4/BA.5/BA.5.2 spike protein RBD

Heparin, a bio-molecule with the highest negative charge density, is pharmaceutically important to prevent SARS-CoV-2 infection due to its strong competitive binding to spike protein compared with cellular heparan sulfate, which was confirmed as a co-receptor for virus-host cell interaction. Hence, the refined structural characterization of heparin targeting viral protein-HS interaction was significant for developing antiviral pharmaceuticals. In our study, heparin oligomers (dp ≥ 4) were prepared using heparinase I. The affinity oligosaccharides binding to Omicron spike protein RBD were separated by affinity chromatography and size exclusion chromatography. HILIC-ESI-FTMS was used for chain mapping analysis. The basic building blocks were analyzed and the binding domain sequence was produced by Seq-GAG software and further measured by SAX chromatography. As results, heparin octasaccharide was found with significantly higher binding ability than hexasaccharide and tetrasaccharide, and the octasaccharide [ΔUA-GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S] with 12 sulfate groups showed high binding to RBD. The mechanism of this structurally well-defined octasaccharide binding to RBD was further investigated by molecular docking. The affinity energy of optimal pose was −6.8 kcal/mol and the basic amino acid residues in RBD sequence (Arg403, Arg452, Arg493 and His505) were identified as the major contribution factor to interacting with sulfate/carboxyl groups on saccharide chain. Our study demonstrated that heparin oligosaccharide with well-defined structure could be potentially developed as anti-SARS-CoV-2 drugs.

Microfluidic Diffusion Sizing Applied to the Study of Natural Products and Extracts That Modulate the SARS-CoV-2 Spike RBD/ACE2 Interaction.

The interaction between SARS-CoV-2 spike RBD and ACE2 proteins is a crucial step for host cell infection by the virus. Without it, the entire virion entrance mechanism is compromised. The aim of this study was to evaluate the capacity of various natural product classes, including flavonoids, anthraquinones, saponins, ivermectin, chloroquine, and erythromycin, to modulate this interaction. To accomplish this, we applied a recently developed a microfluidic diffusional sizing (MDS) technique that allows us to probe protein-protein interactions via measurements of the hydrodynamic radius (Rh) and dissociation constant (KD); the evolution of Rh is monitored in the presence of increasing concentrations of the partner protein (ACE2); and the KD is determined through a binding curve experimental design. In a second time, with the protein partners present in equimolar amounts, the Rh of the protein complex was measured in the presence of different natural products. Five of the nine natural products/extracts tested were found to modulate the formation of the protein complex. A methanol extract of Chenopodium quinoa Willd bitter seed husks (50 µg/mL; bisdesmoside saponins) and the flavonoid naringenin (1 µM) were particularly effective. This rapid selection of effective modulators will allow us to better understand agents that may prevent SARS-CoV-2 infection.

Application of a Receptor-Binding-Domain-Based Simple Immunoassay for Assessing Humoral Immunity against Emerging SARS-CoV-2 Virus Variants.

We have developed a simple, rapid, high-throughput RBD-based ELISA to assess the humoral immunity against emerging SARS-CoV-2 virus variants. The cDNAs of the His-tagged RBD proteins of the virus variants were stably engineered into HEK cells secreting the protein into the supernatant, and RBD purification was performed by Ni-chromatography and buffer exchange by membrane filtration. The simplified assay uses single dilutions of sera from finger-pricked native blood samples, purified RBD in 96-well plates, and a chromogenic dye for development. The results of this RBD-ELISA were confirmed to correlate with those of a commercial immunoassay measuring antibodies against the Wuhan strain, as well as direct virus neutralization assays assessing the cellular effects of the Wuhan and the Omicron (BA.5) variants. Here, we document the applicability of this ELISA to assess the variant-specific humoral immunity in vaccinated and convalescent patients, as well as to follow the time course of selective vaccination response. This simple and rapid assay, easily modified to detect humoral immunity against emerging SARS-CoV-2 virus variants, may help to assess the level of antiviral protection after vaccination or infection.