A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System.

Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an Escherichia coli protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a Kd of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.

Chromatographic characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc

At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices. © 2022 Walter de Gruyter GmbH, Berlin/Boston.

SARS-CoV-2 variants infectivity prediction and therapeutic peptide design using computational approaches.

The outbreak of severe acute respiratory coronavirus 2 (SARS-CoV-2) has created a public health emergency globally. SARS-CoV-2 enters the human cell through the binding of the spike protein to human angiotensin converting enzyme 2 (ACE2) receptor. Significant changes have been reported in the mutational landscape of SARS-CoV-2 in the receptor binding domain (RBD) of S protein, subsequent to evolution of the pandemic. The present study examines the correlation between the binding affinity of mutated S-proteins and the rate of viral infectivity. For this, the binding affinity of SARS-CoV and variants of SARS-CoV-2 towards ACE2 was computationally determined. Subsequently, the RBD mutations were classified on the basis of the number of strains identified with respect to each mutation and the resulting variation in the binding affinity was computationally examined. The molecular docking studies indicated a significant correlation between the Z-Rank score of mutated S proteins and the rate of infectivity, suitable for predicting SARS-CoV-2 infectivity. Accordingly, a 30-mer peptide was designed and the inhibitory properties were computationally analyzed. Single amino acid-wise mutation was performed subsequently to identify the peptide with the highest binding affinity. Molecular dynamics and free energy calculations were then performed to examine the stability of the peptide-protein complexes. Additionally, selected peptides were synthesized and screened using a colorimetric assay. Together, this study developed a model to predict the rate of infectivity of SARS-CoV-2 variants and propose a potential peptide that can be used as an inhibitor for the viral entry to human.Communicated by Ramaswamy H. Sarma.

Spike Protein Potential Receptors Study: Comparative Computational Analysis Approach on SARS-CoV-2-AC2/CD147 Complexes

SARS-CoV-2 invades host cells via interaction of its spike protein with the human angiotensin-converting enzyme 2 as the receptor. CD147, as a biomarker for hyperinflammation, was found to be the functional receptor for SARS-CoV-2 and an additional cell entry route. In this paper, we focused our analysis on the initial step of virus infection by comparing the affinity, stability, and specificity of the SARS-CoV-2 spike 1-AC2 and SARS-CoV-2 spike 1-CD147 complexes. Protein-protein docking was utilized for identifying the hotspot residues in the interface of spike protein with AC2 and CD147. The results of binding free energies showed a high affinity of SP1-AC2 complex (-52.97 kcal/mol) compared with SP1-CoV2/CD147 (-35.75 kcal/mol). RMSF values indicate that the spike protein of SARS-CoV-2 RBD is more compatible with binding to the human ACE2 with high flexibility. Computational analysis of binding modes and protein contacts reported that CD147 and ACE2 might be two complementary receptors mediating virus infection and confirmed the experimental results previously. © 2022 by the authors.

Simulation of Microfluidic Biosensor for Binding Kinetics of SARS-CoV-2 S Protein Using the Electrothermal Effect

To contribute to the fight versus the coronavirus disease 2019, great efforts have been made by scientists around the world to improve the performance of detection devices so that they can efficiently and quickly detect the virus responsible for this disease. In this context we performed a two-dimensional finite element simulation on the binding kinetics of SARS-CoV-2 S protein of a biosensor using the alternating current electrothermal (ACET) effect. The ACET flow can produce vortex patterns, thereby improving the transportation of the target analyte to the binding surface and thus enhancing the performance of the biosensor. The results showed that the detection time can be improved under the electrothermal effect. The effect of certain design parameters concerning the reaction surface, such as its length as well as its position on the top wall of the microchannel, on the biosensor efficiency were also presented. Results showed that the decrease in the length of the binding surface can lead to an increase in the rate of the binding reaction and therefore decrease the biosensor response time. Also, moving the sensitive surface from an optimal position, which is opposite the electrodes, decreases the performance of the biosensor. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Treatment of persistent COVID-19 in two B-cell-depleted patients with the monoclonal antibody Sotrovimab.

Background: Patients having undergone B-cell-depletion with anti-CD20-antibodies have a higher risk of mortality, delayed viral clearance and prolonged infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report two cases of patients with persistent coronavirus disease 2019 (COVID-19) in association with B-cell-depletion that were treated with the monoclonal antibody Sotrovimab. Case presentation: Both patients presented with chronic symptoms of COVID-19 such as dyspnea, fatigue, and chest pain. Nasopharyngeal swabs remained positive months after the initial infection with fluctuating cycle threshold (Ct) values around 30. Both patients received a single infusion with the monoclonal SARS-CoV-2 antibody Sotrovimab, which resulted in a rapid improvement of symptoms and inflammation markers as well as negative SARS-CoV-2 swabs. A follow-up after a month showed ongoing improvement of symptoms, persistent negative SARS-CoV-2 swabs, and positive serum antibodies. Conclusion: Infusion with the monoclonal SARS-CoV-2 antibody led to rapid improvement in two patients with persistent COVID-19 after B-cell depletion.

Design parameters optimization of an electrothermal flow biosensor for the SARS-CoV-2 S protein immunoassay.

To combat the coronavirus disease 2019 (COVID-19), great efforts have been made by scientists around the world to improve the performance of detection devices so that they can efficiently and quickly detect the virus responsible for this disease. In this context we performed 2D finite element simulation on the kinetics of SARS-CoV-2 S protein binding reaction of a biosensor using the alternating current electrothermal (ACET) effect. The ACET flow can produce vortex patterns, thereby improving the transportation of the target analyte to the binding surface and thus enhancing the performance of the biosensor. Optimization of some design parameters concerning the microchannel height and the reaction surface, such as its length as well as its position on the top wall of the microchannel, in order to improve the biosensor efficiency, was studied. The results revealed that the detection time can be improved by 55% with an applied voltage of 10 V rms and an operating frequency of 150 kHz and that the decrease in the height of the microchannel and in the length of the binding surface can lead to an increase in the rate of the binding reaction and therefore decrease the biosensor response time. Also, moving the sensitive surface from an optimal position, located in front of the electrodes, decreases the performance of the device.

Seroreactivity of the Severe Acute Respiratory Syndrome Coronavirus 2 Recombinant S Protein, Receptor-Binding Domain, and Its Receptor-Binding Motif in COVID-19 Patients and Their Cross-Reactivity With Pre-COVID-19 Samples From Malaria-Endemic Areas.

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM436-507). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM (p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD (p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated (p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study.

Chromatographic characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc

At the Center of Molecular Immunology (Havana, Cuba), the fusion protein SARS-CoV-2 S protein (RBD)-hFc was synthesized linking the receptor-binding domain (RBD) of the SARS-CoV-2 virus and the crystallizable fragment of a human immunoglobulin. This fusion protein was used in the construction of a diagnostic device for COVID-19 called UMELISA SARS-CoV-2-IgG. Given the relevance of this protein, the characterization of three batches (A1, A2 and A3) was carried out. The molecular weight of the protein was determined to be 120 kDa, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its isoelectric point was estimated between 8.3 and 9 by isoelectric focusing. The molecular integrity was evaluated by size exclusion liquid chromatography and SDS-PAGE after one year of the production of the protein;the presence of aggregates and fragments was detected. Batches A1 and A2 have a purity percentage higher than 95% and they can be used for the construction of new diagnostic devices.

Use of Circular Dichroism in the characterization of the fusion protein SARS-CoV-2 S protein (RBD)-hFc

From the receptor-binding domain (RBD) of the SARS-CoV-2 virus, which causes coronavirus disease 2019 (COVID-19), a RBD-hFc fusion protein was obtained at the Center of Molecular Immunology (Havana, Cuba). This fusion protein was used in the construction of a diagnostic device for COVID-19 called Ultramicroenzyme-Linked Immunosorbent Assay (UMELISA)-SARS-CoV-2-IgG and it is currently been used in the studies of biological activity of the Cuban vaccine Abdala (CIGB-66). In this work, Circular Dichroism (CD) is used to characterize this protein. Using Far Ultraviolet Circular Dichroism (FAR-UV CD), it was determined that the protein has a secondary structure in the form of a sheet-beta fundamentally. Using this technique, a thermodynamic study was carried out and it was determined that the melting temperature (Tm) of the protein is 71.5 degrees C. Information about the tertiary structure of the protein was obtained using Near Ultraviolet Circular Dichroism (NEAR-UV CD) and Molecular Fluorescence;they indicates that the protein has a three-dimensional folding associated with the aromatic amino acids in its structure, where tryptophan (Trp) is located inside the folded structure of the protein while tyrosine (Tyr) is exposed to the solvent.

Performance comparison of a flow cytometry immunoassay for intracellular cytokine staining and the QuantiFERON® SARS-CoV-2 test for detection and quantification of SARS-CoV-2-Spike-reactive-IFN-γ-producing T cells after COVID-19 vaccination.

PURPOSE: We compared the performance of an in-house-developed flow cytometry assay for intracellular cytokine staining (FC-ICS) and a commercially-available cytokine release assay (the QuantiFERON® SARS-CoV-2 Test [QF]) for detection and quantification of SARS-CoV-2-Spike (S)-reactive-IFN-γ-producing T cells after COVID-19 vaccination. PATIENTS AND METHODS: The sample included 141 individuals (all male; median age, 42 years; 20-72) who had been fully vaccinated with the Comirnaty® COVID-19 vaccine (at a median of 114 days; 34-145). Prior to vaccination, 91 were categorized as being SARS-CoV-2-naïve and 50 as SARS-CoV-2-experienced. A whole blood-based FC-ICS using 15-mer overlapping peptides encompassing the entire SARS-CoV-2 S protein was used for enumeration of virus-specific IFN-γ-producing CD4+ and CD8+ T cells. The QF test (Ag1 for CD4+ T cells and Ag2 for CD4+ and CD8+ T cells in combination) was carried out following the manufacturer's instructions. RESULTS: The FC-ICS and the QF assays returned significantly discordant qualitative results in both the entire cohort (P<0.001 with QF Ag1 and QF Ag2) and in SARS-CoV-2-naïve participants alone (P=0.005 and P=0.01, respectively). Discrepant results mostly involved FC-ICS positive/QF negative specimens. Overall, no correlation was found either between SARS-CoV-2 IFN-γ- CD4+ T-cell frequencies and IFN-γ levels measured in the QF Ag1 tube (P=0.78) or between the sum of SARS-CoV-2 IFN-γ CD4+ and CD8+ T-cell frequencies and IFN-γ levels quantified in the QF Ag2 tube. CONCLUSION: The data suggest a greater sensitivity for the FC-ICS assay than the QF test, and urge caution when comparing SARS-CoV-2 T-cell immune responses assessed using different analytical platforms.

SARS-CoV-2 deregulates the vascular and immune functions of brain pericytes via Spike protein.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Development of flexible electrochemical impedance spectroscopy-based biosensing platform for rapid screening of SARS-CoV-2 inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the cells through the binding of its spike protein (S-protein) to the cell surface-expressing angiotensin-converting enzyme 2 (ACE2). Thus, inhibition of S-protein-ACE2 binding may impede SARS-CoV-2 cell entry and attenuate the progression of Coronavirus disease 2019 (COVID-19). In this study, an electrochemical impedance spectroscopy-based biosensing platform consisting of a recombinant ACE2-coated palladium nano-thin-film electrode as the core sensing element was fabricated for the screening of potential inhibitors against S-protein-ACE2 binding. The platform could detect interference of small analytes against S-protein-ACE2 binding at low analyte concentration and small volume (0.1 µg/mL and ~1 µL, estimated total analyte consumption < 4 pg) within 21 min. Thus, a few potential inhibitors of S-protein-ACE2 binding were identified. This includes (2S,3aS,6aS)-1-((S)-N-((S)-1-Carboxy-3-phenylpropyl)alanyl)tetrahydrocyclopenta[b] pyrrole-2-carboxylic acid (ramiprilat) and (2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-Carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid (perindoprilat) that reduced the binding affinity of S-protein to ACE2 by 72% and 67%; and SARS-CoV-2 in vitro infectivity to the ACE2-expressing human oral cavity squamous carcinoma cells (OEC-M1) by 36.4 and 20.1%, respectively, compared to the PBS control. These findings demonstrated the usefulness of the developed biosensing platform for the rapid screening of modulators for S-protein-ACE2 binding.

Withanone from Withania somnifera Attenuates SARS-CoV-2 RBD and Host ACE2 Interactions to Rescue Spike Protein Induced Pathologies in Humanized Zebrafish Model.

PURPOSE: SARS-CoV-2 engages human ACE2 through its spike (S) protein receptor binding domain (RBD) to enter the host cell. Recent computational studies have reported that withanone and withaferin A, phytochemicals found in Withania somnifera, target viral main protease (MPro) and host transmembrane TMPRSS2, and glucose related protein 78 (GRP78), respectively, implicating their potential as viral entry inhibitors. Absence of specific treatment against SARS-CoV-2 infection has encouraged exploration of phytochemicals as potential antivirals. AIM: This study aimed at in silico exploration, along with in vitro and in vivo validation of antiviral efficacy of the phytochemical withanone. METHODS: Through molecular docking, molecular dynamic (MD) simulation and electrostatic energy calculation the plausible biochemical interactions between withanone and the ACE2-RBD complex were investigated. These in silico observations were biochemically validated by ELISA-based assays. Withanone-enriched extract from W. somnifera was tested for its ability to ameliorate clinically relevant pathological features, modelled in humanized zebrafish through SARS-CoV-2 recombinant spike (S) protein induction. RESULTS: Withanone bound efficiently at the interacting interface of the ACE2-RBD complex and destabilized it energetically. The electrostatic component of binding free energies of the complex was significantly decreased. The two intrachain salt bridge interactions (K31-E35) and the interchain long-range ion-pair (K31-E484), at the ACE2-RBD interface were completely abolished by withanone, in the 50 ns simulation. In vitro binding assay experimentally validated that withanone efficiently inhibited (IC50=0.33 ng/mL) the interaction between ACE2 and RBD, in a dose-dependent manner. A withanone-enriched extract, without any co-extracted withaferin A, was prepared from W. somnifera leaves. This enriched extract was found to be efficient in ameliorating human-like pathological responses induced in humanized zebrafish by SARS-CoV-2 recombinant spike (S) protein. CONCLUSION: In conclusion, this study provided experimental validation for computational insight into the potential of withanone as a potent inhibitor of SARS-CoV-2 coronavirus entry into the host cells.

Charge-Transfer Resonance and Electromagnetic Enhancement Synergistically Enabling MXenes with Excellent SERS Sensitivity for SARS-CoV-2 S Protein Detection.

The outbreak of coronavirus disease 2019 has seriously threatened human health. Rapidly and sensitively detecting SARS-CoV-2 viruses can help control the spread of viruses. However, it is an arduous challenge to apply semiconductor-based substrates for virus SERS detection due to their poor sensitivity. Therefore, it is worthwhile to search novel semiconductor-based substrates with excellent SERS sensitivity. Herein we report, for the first time, Nb2C and Ta2C MXenes exhibit a remarkable SERS enhancement, which is synergistically enabled by the charge transfer resonance enhancement and electromagnetic enhancement. Their SERS sensitivity is optimized to 3.0 × 106 and 1.4 × 106 under the optimal resonance excitation wavelength of 532 nm. Additionally, remarkable SERS sensitivity endows Ta2C MXenes with capability to sensitively detect and accurately identify the SARS-CoV-2 spike protein. Moreover, its detection limit is as low as 5 × 10-9 M, which is beneficial to achieve real-time monitoring and early warning of novel coronavirus. This research not only provides helpful theoretical guidance for exploring other novel SERS-active semiconductor-based materials but also provides a potential candidate for the practical applications of SERS technology.

Binding of the SARS-CoV-2 spike protein to glycans.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a high number of deaths in the world. To combat it, it is necessary to develop a better understanding of how the virus infects host cells. Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (HS) and sialic acid-containing glycolipids/glycoproteins. In this study, we examined and compared the binding of the subunits and spike (S) proteins of SARS-CoV-2, SARS-CoV, and Middle East respiratory disease (MERS)-CoV to these glycans. Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner and no binding with sialic acid residues was detected. Overall, this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.