Indonesian vaccine producer (PT BIOFARMA), conducted a study to assess the safety and immunogenicity of a new COVID-19 vaccine candidate. This vaccine is based on a recombinant subunit protein platform, with the SARS-CoV-2 receptor-binding domain (RBD) as its target antigen. The study compared the candidate's safety and immunogenicity to the control group vaccine, the Sinovac vaccine, 28 days after administration. This was an observer-blinded and randomized Phase 1 trial which recruited 175 subjects. The subject received 0.5 ml of vaccine in two doses. The subjects were split into five treatment groups, consisting of different combinations of doses between RBD and CpG. The safety of this vaccine was evaluated within 7 days after the first dose and for 6 months after the second dose, while the immunogenicity was evaluated on days 14 and 28 after the second dose. The overall incidence of AEs was 54.86% from the beginning of the vaccination to 28 days after each injection. Most AEs were local pain and had no serious AEs. The study revealed a significant rise in the Geometric Mean Titer (GMT) of IgG antibodies in every group, indicating a strong immune response. The phase I data demonstrated that the new vaccine candidate showed promising results in safety and immunogenicity.

The aerial parts of Nuxia congesta R.Br. ex Fresen were chromatographically purified, yielding five new compounds: two iridoids (1,2), one phenylpropanoid glycoside (3), and two rhamnosyl esters (4,5). The compounds were characterised via NMR and HRESIMS. The isolated compounds inhibitory action towards the COVID-19 major protease (Mpro) and receptor-binding domain (RBD) enzymes was evaluated. Comparative molecular docking and binding free energy analyses were conducted based on the binding affinities of the ligand binding domains of the therapeutic targets Mpro and RBD. The results indicated that all compounds effectively interacted with the binding sites of Mpro and RBD receptors, exhibiting a greater affinity for the Mpro receptor. The binding energy to Mpro ranged between -7.8 and -8.1 kcal/mol, however for RBD receptors it ranged from -7.0 to -7.3 kcal/mol. This research supports the efficacy of isolated compounds as antiviral medications, especially in the context of COVID-19.

Phycocyanobilin (PCB), tetrapyrrole chromophore of Spirulina phycocyanin, is bilirubin analog and weak thiol-modifying agent. SARS-CoV-2 spike protein (SP) has bilirubin binding pocket, lacks free sulfhydryl, but it has two pairs of functionally important semi-stable disulfides reactive towards thiol-modifying agents. We investigated covalent and noncovalent binding of PCB to SP and its receptor-binding domain (RBD) and impact of covalent PCB conjugation to RBD on structure and binding to human angiotensin-converting enzyme 2 (ACE-2). PCB shows high-affinity for SP (Ka = 2.1 × 107 M-1), moderate-affinity for RBD (Ka = 8.4 × 104 M-1) and binds covalently to SP and RBD in reaction involving thiols. PCB binding alters RBD conformation. Molecular docking identified two binding sites of PCB to SP, bilirubin/biliverdin binding site and hydrophobic pocket of RBD in vicinity of Cys432, preferential target for covalent binding in in silico covalent docking of PCB to RBD. Redox proteomics mapped reactive Cys432, Cys391 and Cys525 in RBD. PCB-modified RBD exhibited reduced ability to bind to ACE-2. This is the first study demonstrating PCB reactivity towards semi-stable disulfides of proteins lacking free sulfhydryl groups. PCB may affect functionality and structure of SP and its RBD by noncovalent and covalent binding.

Introduction: Coronavirus (CoV) is an extremely contagious, enveloped positive-single-stranded RNA virus, which has become a global pandemic that causes several illnesses in humans and animals. Hence, it is necessary to investigate viral-induced reactions across diverse hosts. Herein, we propose utilizing naturally secreted extracellular vesicles (EVs), mainly focusing on exosomes to examine virus-host responses following CoV infection. Exosomes are small membrane-bound vesicles originating from the endosomal pathway, which play a pivotal role in intracellular communication and physiological and pathological processes. We suggested that CoV could impact EV formation, content, and diverse immune responses in vitro. Methods: In this study, we infected A-72, which is a canine fibroblast cell line, with a feline coronavirus (FCoV) and canine coronavirus (CCoV) independently in an exosome-free media at 0.001 multiplicity of infection (MOI), with incubation periods of 48 and 72 h. The cell viability was significantly downregulated with increased incubation time following FCoV and CCoV infection, which was identified by performing the 3-(4,5-dimethylthiazo-1-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the infection, EVs were isolated through ultracentrifugation, and the subsequent analysis involved quantifying and characterizing the purified EVs using various techniques. Results: NanoSight particle tracking analysis (NTA) verified that EV dimensions fell between 100 and 200 nm at both incubation periods. At both periods, total protein and RNA levels were significantly upregulated in A-72-derived EVs following FCoV and CCoV infections. However, total DNA levels were gradually upregulated with increased incubation time. Dot blot analysis indicated that the expression levels of ACE2, IL-1β, Flotillin-1, CD63, caspase-8, and Hsp90 were modified in A-72-derived EVs following both CoV infections. Conclusions: Our results indicated that FCoV and CCoV infections could modulate the EV production and content, which could play a role in the development of viral diseases. Investigating diverse animal CoV will provide in-depth insight into host exosome biology during CoV infection. Hence, our findings contribute to the comprehension and characterization of EVs in virus-host interactions during CoV infection.

Accurate modeling of protein-protein complex structures is essential for understanding biological mechanisms. Hydrogen-deuterium exchange (HDX) experiments provide valuable insights into binding interfaces. Incorporating HDX data into protein complex modeling workflows offers a promising approach to improve prediction accuracy. Here, we developed HDXRank, a graph neural network (GNN)-based framework for candidate structure ranking utilizing alignment with HDX experimental data. Trained on a newly curated HDX data set, HDXRank captures nuanced local structural features critical for accurate HDX profile prediction. This versatile framework can be integrated with a variety of protein complex modeling tools, transforming the HDX profile alignment into a model quality metric. HDXRank demonstrates effectiveness at ranking models generated by rigid docking or AlphaFold, successfully prioritizing functionally relevant models and improving prediction quality across all tested protein targets. These findings underscore HDXRank's potential to become a pivotal tool for understanding molecular recognition in complex biological systems.

Antibiotics are essential chemicals for medicine and agritech. However, all antibiotics are small molecules that pathogens evolve antimicrobial resistance (AMR). Alternatively, antimicrobial peptides (AMPs) offer potential to overcome or evade AMR. AMPs provide broad-spectrum activity, favourable biosafety profiles, and a rapid and efficient mechanism of action with low resistance incidence. These properties have driven innovative applications, positioning AMPs as promising contributors to advancements in various industrial sectors. This review evaluates the multifaceted nature of AMPs and their biotechnological applications in underexplored sectors. In the food industry, the application of AMPs helps to suppress the growth of microorganisms, thereby decreasing foodborne illnesses, minimizing food waste, and prolonging the shelf life of products. In animal husbandry and aquaculture, incorporating AMPs into the diet reduces the load of pathogenic microorganisms and enhances growth performance and survival rates. In agriculture, AMPs provide an alternative to decrease the use of chemical pesticides and antibiotics. We also review current methods for obtaining AMPs, including chemical synthesis, recombinant DNA technology, cell-free protein synthesis, and molecular farming, are also reviewed. Finally, we look to the peptide market to assess its status, progress, and transition from the discovery stage to benefits for society and high-quality products. Overall, our review exemplifies the other side of the coin of AMPs and how these molecules provide similar benefits to conventional antibiotics and pesticides in the agritech sector.

SARS-CoV-2 high transmission and genomic mutations result in the emergence of new variants that impact COVID-19 vaccine efficacy and virus transmission by evading the host immune system. Wastewater-based epidemiology is an effective approach to monitor SARS-CoV-2 variants circulation in the population but is a challenge due to the presence of reaction inhibitors and the low concentrations of SARS-CoV-2 in this environment. Here, we aim to improve SARS-CoV-2 variant detection in wastewater by employing nested PCR followed by next-generation sequencing (NGS) of small amplicons of the S gene. Eight SARS-CoV-2 wastewater samples from Alegria Wastewater Treatment Plant, in Rio de Janeiro, Brazil, were collected monthly from February to September 2021. Samples were submitted to virus concentration, RNA extraction and nested PCR followed by NGS. The small amplicons were used to prepare libraries for sequencing without the need to perform any fragmentation step. We identified and calculated the frequencies of 29 mutations matching the Alpha, Beta, Gamma, Delta, Omicron, and P.2 variants. Omicron matching-mutations were detected before the lineage was classified as a variant of concern. SARS-CoV-2 wastewater sequences clustered with SARS-CoV-2 variants detected in clinical samples that circulated in 2021 in Rio de Janeiro. We show that sequencing of selected small amplicons of SARS-CoV-2 S gene allows the identification of SARS-CoV-2 variants matching mutations and their frequencies' calculation. This approach may be expanded using customizing primers for additional genomic regions, in order to differentiate current variants. Approaches that allow us to learn how variants emerge and how they relate to clinical outcomes are crucial for our understanding of the dynamics of virus variants circulation, providing valuable data for public health management.

Introduction. Pre-existing immunity to human coronaviruses (HCoVs) may shape the immune response in COVID-19 patients. Increasing evidence suggests that immune cross-reactivity between SARS-CoV-2 and other coronaviruses may determine clinical prognosis.Hypothesis. SARS-CoV-2 disease severity is influenced by pre-existing immunity to HCoVs, with distinct antibody profiles and cross-reactivity patterns.Aim. To investigate the antibody response of ICU and non-ICU SARS-CoV-2 patients against different HCoV proteins and assess the potential impact of pre-existing immunity on SARS-CoV-2 disease outcomes.Methodology. This study used a comprehensive HCoVs antigen bead array to measure antibody response to pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, SARS-CoV-2 and the four seasonal HCoVs in 70 ICU and 63 non-ICU COVID-19 patients.Results. Our analysis demonstrates an overall higher antibody response in ICU than in non-ICU COVID-19 patients. Interestingly, the anti-S1 IgG and IgA were significantly higher among ICU than in non-ICU patients. Similarly, the anti-S1 IgG against NL63 showed a lower response among ICU compared to non-ICU. Cross-reactivity was evident between SARS-CoV-2 and SARS-CoV antibodies but not with MERS-CoV and seasonal HCoVs. The subclass analysis of antibodies recognizing SARS-CoV-2 revealed that anti-S1 IgG1, IgG3, IgA1 and IgA2 were significantly higher in ICU compared to non-ICU. The predominant IgA subtype among SARS-CoV-2 patients was IgA1. We applied machine learning algorithms to subclass serological responses to build classifiers that could distinguish between ICU patients and patients with milder COVID-19. Out of 90 variables used in two different types of models, the variable of highest influence in determining the ICU status was IgG3 against SARS-CoV-2 S, and the top 8 variables of influence included the presence of IgG3 against S-trimer as well as IgA against SARS-CoV-2 S.Conclusion. Understanding the complexities of humoral immunity in various patients is critical for early medical intervention, disease management, selective vaccination and passive immunotherapy.

The SARS-CoV-2 virus mutates rapidly, reducing the effectiveness of antibodies. The novel Class IV antibody IY2A partially unfolds the receptor-binding domain (RBD), allowing tolerance of antigenic variations and effectively neutralizing Omicron variants. In this study, we used molecular dynamics simulations and alanine scanning combined with interaction entropy method to elucidate how IY2A maintains its binding affinity across Omicron variants. We compared IY2A with EY6A and evaluated how mutations affect IY2A inhibition. The findings revealed that the IY2A adopted a closer conformation when binding to Omicron variants than to the WT. Energy calculations indicate that van der Waals interactions primarily drive IY2A binding to the RBD. Following unfolding, IY2A interacts with the RBD via interatomic hydrogen bonds and hydrophobic contacts involving LEU368, PHE377, LYS378, and SER383. This study provides theoretical insights to guide the development of Class IV antibodies against emerging and future Omicron variants.

The emergence of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global issue since 2019. The prominent characteristic of SARS-CoV-2 is the presence of the spike (S) protein protruding from the virus particle envelope. The S protein is a major drug and vaccine target because it initiates the key step in infection. Medicinal herbs are a potential treatment option to enhance immunity to fight viral infections. Caesalpinia sappan L. has been reported to display promising anti-viral activities. Specifically, brazilin (BRA), a major bioactive compound in C. sappan, was reported to play a role in inhibiting viral infection. Thus, the ability of BRA as a COVID-19 treatment was tested. The S protein was used as the BRA target of this work. Understanding the binding mechanism of BRA to the S protein is crucial for future utilisation of C. sappan as a COVID-19 treatment or other coronavirus-caused pandemics. Here, we performed molecular docking of BRA onto the S protein receptor binding domain (RBD) and multimerisation (MM) pockets. Molecular dynamics (MD) simulations were conducted to study the stability of binding to glycosylated and non-glycosylated S protein constructs. BRA can bind to the Receptor-binding motif (RBM) on an RBD surface stably; however, it is too large to fit into the MM pocket, resulting in dissociation. Nonetheless, BRA is bound by residues near the S1/S2 interface. We found that glycosylation has no effect on BRA binding, as the proposed binding site is far from any glycans. Our results thus indicate that C. sappan may act as a promising preventive and therapeutic alternative for COVID-19 treatment.

We have developed a robust method for efficiently tracking multiple co-occurring mutations in a sequence database. Evolution often hinges on the interaction of several mutations to produce significant phenotypic changes that lead to the proliferation of a variant. However, identifying numerous simultaneous mutations across a vast database of sequences poses a significant computational challenge. Our approach leverages a matrix factorization technique to automatically and efficiently pinpoint subsets of positions where co-mutations occur, appearing in a substantial number of sequences within the database. We validated our method using SARS-CoV-2 receptor-binding domains, comprising approximately seven hundred thousand sequences of the Spike protein, demonstrating superior performance compared to a reasonably exhaustive brute-force method. Furthermore, we explore the biological significance of the identified co-mutational positions (CMPs) and their potential impact on the virus's evolution and functionality, identifying key mutations in Delta and Omicron variants. This analysis underscores the significant role of identified CMPs in understanding the evolutionary trajectory. By tracking the "birth" and "death" of CMPs, we can elucidate the persistence and impact of specific groups of mutations across different viral strains, providing valuable insights into the virus' adaptability and thus, possibly aiding vaccine design strategies.

The structural integrity of viral envelopes is a critical determinant of infectivity for enveloped viruses, directly influencing vector stability, functional accuracy of surface-displayed epitopes, and preservation of native conformational states required for membrane protein studies. However, conventional purification methods often disrupt envelope integrity and cause envelope proteins to lose their activity. Here, we systematically compared discontinuous, continuous, and optimized continuous sucrose density gradient centrifugation protocols for purifying Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Through cryo-EM, we demonstrated that our optimized continuous sucrose gradient protocol significantly increased the proportion of AcMNPV budded virions with intact envelopes from 36% to 81%, while preserving the metastable prefusion conformation of the fusion protein GP64. This advancement should prove useful for structural studies of viral envelope proteins and may enhance applications in gene therapy and vaccine development utilizing enveloped viruses.

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic, which caused over 6.7 million deaths worldwide. The Spike protein plays a crucial role in the infection process, mediating the binding of the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2), and its subsequent entry into target cells. Previous studies identified, through virtual screening, several natural products capable of binding to two distinct pockets of the Spike protein: triterpenoids binding to pocket 1 and bile acid derivatives binding to pocket 5. Building on these findings, our study advances the field by developing bivalent compounds 1-4 that through a spacer combine a triterpenoid (betulinic acid or glycyrrhetinic acid) with a semisynthetic bile acid derivative (obeticholic acid). These bivalent compounds are designed to simultaneously bind both pockets of the Spike protein, offering significant advantages over single molecules or the combination of the two natural products. In vitro cell assays using pseudotyped recombinant lentiviral particles with selected SARS-CoV-2 Spike proteins demonstrated that 1 and 2 exhibit enhanced activity in reducing viral entry into target cells compared to individual natural products, thus highlighting their potential as superior antiviral agents with reduced side effects.

During the coronavirus disease 2019 (COVID-19) pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here, we report an analysis of 164 serum samples isolated from patients with breakthrough infection in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wild-type (WT), Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping, using charged scanning mutagenesis coupled with yeast surface display and next-generation sequencing. The mapped sera primarily targeted the recently identified class 5 cryptic epitope and, to a lesser extent, the class 1 and class 4 epitopes. The class 5 epitope is completely conserved across all severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and for most sarbecoviruses. Based on these observations, an additional 26 sera were characterized, and all showed a broad neutralizing activity, including against XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variants of concern (VOCs) were observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population were sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern. These data highlight the important role of hybrid immunity in conferring broad protection and inform future vaccine strategies to protect against rapidly mutating viruses.

Worldwide implementation of coronavirus disease 2019 (COVID-19) vaccines and the parallel emergence of newer severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have shaped the humoral immune response in a population-specific manner. While characterizing this immune response is important for monitoring disease progression at the population level, it is also imperative for developing effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the world's second largest COVID-19 vaccination drive and encountered a large number of post-vaccination "breakthrough" infections. From a cohort of patients with breakthrough infection, we identified individuals whose sera showed broadly neutralizing immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope, which was not identified in previous population-level studies conducted in Western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants, including recently emerged ones and for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

This study presents an optimised cultured ELISpot protocol for detecting central memory T-cell interferon gamma (IFNγ) responses against SARS-CoV-2 peptides following an initial priming with either peptides, or whole spike protein.

Key variations optimised include the culture length, timing of exogenous survival signals (IL-2), and endpoint analysis modality and cell density to enhance assay sensitivity without compromising specificity for central memory T-cell IFNγ recall responses to cognate antigen.

We noted a culture duration of 10 days, combined with a delayed IL-2 administration on day 5 to enhance assay sensitivity while maintaining response specificity towards cognate antigen when compared with shorter culture periods or earlier exogenous survival signal provision. With regards to lower-frequency T-cell interactions, as we observed with our donor SARS-CoV-2 epitope responses, our findings suggest Fluorospot to be preferable to the chromogenic ELISpot modality, and an immediate cell washing after culture collection to better facilitate cognate antigen responses. Fluorospot enabled a higher cell density while minimising the generation of visual artefacts, meanwhile immediate cell washing was critical for improving endpoint assay sensitivity. CCR7+ cell depletion was used to demonstrate our optimised protocol to selectively demonstrate central memory T-cell responses. Lastly, we provide evidence for the capacity of our assay to delineate individual responding peptides following peptide pool priming, and to explore cross-reactivity between viral variant peptides.

This work advances the methodology for investigating T-cell immunity, particularly in the context of SARS-CoV-2, and emphasises the balance between enhancing specific cognate central memory responses while limiting non-specific activation.

The ongoing COVID-19 has caused a global pandemic, resulting in millions of infections and deaths. While current vaccines target the SARS-CoV-2 spike (S) protein, its high mutation rate significantly compromises vaccine efficacy. We aimed to evaluate the potential of epitope-based nanoparticles (NPs) to induce broad cross-protection and durable immune responses against SARS-CoV-2.

Four conserved epitopes derived from the receptor-binding domain (RBD) and S2 subunit of the spike protein were integrated into Helicobacter pylori ferritin to create epitope-based NPs named S18-F, RBM-F, UH-F, and HR2-F. The immunogenicity of the epitope-based NPs was evaluated through animal experiments to measure epitope-specific antibody titers and assess neutralizing activity against SARS-CoV-2 pseudovirus. To characterize cellular immune responses, splenic lymphocyte proliferation following epitope stimulation was measured, and cytokine secretion profiles including IFN-γ, IL-2, IL-4, and IL-10 were analyzed to determine Th1/Th2 immune polarization. Antibody-dependent cellular cytotoxicity (ADCC) assays were performed to evaluate NP-enhanced recognition and elimination of infected target cells.

These NPs induced high titers of epitope-specific antibodies lasting three months post-immunization. Sera from the RBM-F, UH-F, and HR2-F groups exhibited neutralizing activity against the SARS-CoV-2 pseudovirus WH-1 in vitro. Splenic lymphocytes from the S18-F, RBM-F, and UH-F groups showed significantly increased proliferation. Lymphocytes from the RBM-F group demonstrated increased secretion of IFN-γ, IL-2, IL-4, and IL-10 cytokines, indicating a balanced Th1 and Th2 immune response. Immune sera from the S18-F and mixed-immunized groups exhibited antibody-dependent cellular cytotoxicity.

The results indicate that these NPs induce robust humoral and cellular immune responses, potentially offering a promising strategy for effective vaccine development against SARS-CoV-2.

This study explores the role of intrinsically disordered regions (IDRs) in the SARS-CoV-2 proteome and their potential as targets for small-molecule drug discovery. Experimentally validated intrinsic disordered regions from the literature were utilized to assess the prediction of intrinsic disorder across a selection of SARS-CoV-2 proteins. The disorder propensities of proteins using four deep learning-based disorder prediction models: ADOPT, PONDR®VLXT, PONDR®VSL2, and flDPnn, were analyzed. ADOPT, VSL2, and VLXT identified a flexible linker (129-147), while VSL2 and VLXT predicted disorder in the Cu(II) binding region (163-167) of NSP1. ADOPT did not predict disordered regions in NSP11; however, VSL2 and VLXT identified disorder in the experimentally validated regions. The IDR in ORF3a is crucial for protein localization and immune modulation, affecting inflammatory pathways. VSL2 predicted significant disorder in the N-terminal domain (18-23), which aligns with experimental data (1-41), overlapping with the TRAF-binding motif, while ADOPT indicated high disorder in the C-terminal domain (255-275), consistent with VSL2 and flDPnn. All tools identified disorder in the N-terminal (1-68), central linker (181-248), and C-terminal (370-419) regions of the nucleocapsid (N) protein, suggesting flexibility and accuracy. The S2 subunit of the spike protein displayed more predicted disorder than the S1 subunit across ADOPT, VSL2, and flDPnn. These IDRs are essential for viral functions, like protein localization, immune modulation, receptor binding, and membrane fusion. This study highlights the importance of IDR in modulating key inflammatory pathways, suggesting that they could serve as promising targets for small-molecule drug development to combat COVID-19.

Bacteria-based vaccines have received increasing attention given the ability to induce strong systemic immune responses. However, the application of bacteria as therapeutic agents inevitably suffers from infection-associated side effects due to the living characteristics. Here, the use of bacteria-derived flagella is described to construct self-adjuvated nanofiber vaccines. With the help of charge-reversal mediated by decoration with cationic polymers, the flagella can be coated with negatively charged antigens through electrostatic interaction. By virtue of the large aspect ratio, the resulting nanofiber vaccines show prolonged retention at the injection site and increased uptake by dendritic cells and macrophages. Thanks to the innate immunogenicity, self-adjuvated flagella robustly promote dendritic cell maturation and macrophage polarization, resulting in the elicitation of antigen-specific T-cell and B-cell immune responses. In ovalbumin-overexpressing melanoma-bearing mice, immunization with ovalbumin-carried vaccines not only exhibits a favorable tolerance, but also displays superior inhibition efficacies on tumor growth and metastasis separately under the therapeutic and prophylactic settings. The flexibility of this approach is further demonstrated for vaccine fabrication by coating with the SARS-CoV-2 Spike protein S1 subunit. Bacterial flagella-based self-adjuvated nanofiber platform proposes a versatile strategy to develop various vaccines for disease prevention and treatment.

The COVID-19 pandemic began in March 2020 and has affected many countries and infected over a million people. It has had a serious impact on people's physical and mental health, daily life and the global economy. Today, many drugs show limited efficacy in the treatment of COVID-19 and studies to develop effective drugs continue. Here, we aim to the synthesise and characterise of the flavonol-3-O-glycoside derivatives, the following and evaluated molecular docking studies with antimicrobial activity, inhibition of SARS-CoV-2 main protease enzyme (3CLpro) and nuclease activity. Molecular docking simulations of the synthesized flavonol-3-O-glycoside derivatives, especially compounds 5a, 5d, 5h, 5i and 5m, showed a stronger interaction with SARS-CoV-2 3CLpro in the active site. Two compounds from the target compounds, 5h and 5m, were found to be specifically effective against M. smegmatis and yeasts. In particular, compounds 5a, 5d, 5h, 5i and 5m, which exhibited high activity against the SARS-CoV-2 main protease enzyme, were found to be effective at low concentrations. We determined the IC50 values for the compounds that showed an inhibitory effect as well as their nuclease activities, which further emphasising the potential of our results. Among these, compound 5d showed a significant competitive inhibitor of 3CLpro. Furthermore, nuclease activity studies identified compound 5d as the most potent. The above results suggest that the flavonol-3-O-glycoside derivatives could be promising new antiviral agents for the development of 3CLpro inhibitors to combat COVID-19.

The COVID-19 pandemic has underscored the urgent need for a vaccine strategy that is safe, effective, rapid, cost-efficient, and scalable for large-scale deployment during widespread infectious outbreaks. Here, we present a new vaccination strategy that meets these critical requirements. The SARS-CoV-2 S protein consists of the S1 and S2 subunits. The S2 subunit acts as the viral cell membrane fusion protein, and mutations in its C-terminal region facilitate the transport of the entire S protein to the cell membrane. When we expressed the SARS-CoV-2 S protein with a deletion of 21 amino acids from its C-terminal region in various cell types, we observed a dense presence of the protein in the cell membrane, as determined by IHC, dot blot, and ELISA. In the cell membrane-SARS-CoV-2 S protein complex, the cell membrane functions as an exosome mimic, carrying protein antigens (S protein) in their most natural form, as no further protocols are used to attach antigens to the membrane. We demonstrate that using the membrane-S protein component as a vaccine yields a more robust and protective immune response, with enhanced safety compared to mRNA-based or inactivated virus-based vaccines against SARS-CoV-2. Additionally, we show that fusing the transmembrane domain of the Vesicular Stomatitis Virus (VSV) G protein with the SARS-CoV-2 S1 protein effectively transports the S1 protein to the cell membrane, similar to SARS-CoV-2 S Δ21. We propose that designing the S2 subunit of the SARS-CoV-2 S protein, or its analogues such as the VSV-G protein, as carriers for fusing bacterial, viral, or tumor proteins with antigenic properties-and transporting them to the cell membrane-could result in a comprehensive vaccination protocol applicable to all bacteria, viruses, and even tumors.

Hypertension is one of the most common risk factors for COVID-19 clinical progression. The identification of plasma biomarkers for anticipating worse clinical outcomes and to better understand the shared mechanisms between hypertension and COVID-19 are needed. A hypothesis-generating study was designed to compare plasma proteomics and metabolomics between 22 hypertensives (HT) and 41 non-hypertensives (nHT) patients with the most unfavorable COVID-19 progression. A total of 43 molecules were significantly differed between HT (n = 22) and nHT (n = 41). Random Forest (RF) analysis identified myo-inositol, gelsolin and phosphatidylcholine (PC) 32:1 as the top molecules for distinguishing between HT and nHT. Plasma myo-inositol and gelsolin were higher (P = 0.03 and P = 0.02, respectively) and plasma PC 32:1 was lower (P = 0.03) in HT compared to nHT. Biological processes like stress response and blood coagulation, along with KEGG pathways including ascorbate and aldarate metabolism (P = 0.021) and linoleic acid metabolism (P = 0.028), were altered in hypertensive patients with the most unfavorable COVID-19 progression. There is a clear link between hypertension and severe COVID-19. Key biological pathways to consider for improving the prognosis and quality of life of hypertensive patients who become infected with SARS-CoV-2 include oxidative stress, ascorbate and aldarate metabolism, lipid metabolism, immune system and inflammation.

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Anti-SARS-CoV-2 antibodies can provide information on patient immunity, identify asymptomatic patients, and track the spread of COVID-19. Efforts have been made to develop methods to detect anti-SARS-CoV-2 antibodies in humans. Here, we describe a flow cytometric assay for the simultaneous detection of anti-SARS-CoV-2 IgG and IgM in human plasma. To assess the antibody response against the different SARS-CoV-2 structural proteins, five viral recombinant proteins, including spike protein subunit 1 (S1), N-terminal domain of S1 (S1A), spike receptor-binding domain (RBD), spike protein subunit 2 (S2), and nucleocapsid protein (N), were generated. A comparison of the antibody profiles detected by the assay with plasma from 100 healthy blood donors collected prior to the COVID-19 pandemic and plasma from 100 virologically confirmed COVID-19 patients demonstrated a clear discrimination between the two groups. Among the COVID-19 patients, the antibody responses for the viral proteins, as determined by their prevalence, were anti-RBD IgG = anti-N IgG > anti-S1 IgG > anti-S1A IgG > anti-S2 IgG, and anti-RBD IgM > anti-S1 IgM > anti-N IgM > anti-S2 IgM. The prevalence of anti-SARS-CoV-2 IgG and IgM was not associated with sex, age, race, days after the onset of symptoms, or severity of illness, except for a higher prevalence of anti-S2 IgG being observed in men than in women. The levels of anti-RBD IgG were higher in patients 65 years and older and in patients who had severe symptoms. Similarly, patients who had severe symptoms exhibited higher levels of anti-S1 and anti-S1A IgG than patients who had mild or moderate symptoms. The levels of anti-RBD IgM tended to be higher in men but did not differ among age, race, days after the onset of symptoms, or severity of illness. Our study indicates that the flow cytometric assay, especially using RBD as target antigen, can be used to detect simultaneously anti-SARS-CoV-2 IgG and IgM antibodies in human plasma.

The coronavirus disease 2019 (COVID-19) global pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, our understanding of SARS-CoV-2-induced inflammation in alveolar epithelial cells remains very limited. The contributions of intracellular insulin-like growth factor binding protein-2 (IGFBP2) to SARS-CoV-2 pathogenesis are also unclear. In this study, we have uncovered a critical role for IGFBP2, specifically in alveolar epithelial type 2 cells (AEC2), in the immunopathogenesis of COVID-19. Using bulk RNA sequencing, we show that IGFBP2 mRNA expression is significantly downregulated in primary AEC2 cells isolated from fibrotic lung regions from patients with COVID-19-acute respiratory distress syndrome (ARDS) compared to those with idiopathic pulmonary fibrosis (IPF) alone or IPF with a history of COVID-19. Using multicolor immunohistochemistry, we demonstrated that IGFBP2 and its selective ligands IGF1 and IGF2 were significantly reduced in AEC2 cells from patients with COVID-ARDS, IPF alone, or IPF with COVID history than in those from age-matched donor controls. Further, we demonstrated that lentiviral expression of Igfbp2 significantly reduced mRNA expression of proinflammatory cytokines-Tnf-α, Il1β, Il6, Stat3, Stat6 and chemokine receptors-Ccr2 and Ccr5-in mouse lung epithelial cells challenged with SARS-CoV-2 spike protein injury (S2; 500 ng/mL). Finally, we demonstrated higher levels of cytokines-TNF-α; IL-6 and chemokine receptor-CCR5 in AEC2 cells from COVID-ARDS patients compared to the IPF alone and the IPF with COVID history patients. Altogether, these data suggest that anti-inflammatory properties of IGFBP2 in AEC2 cells and its localized delivery may serve as potential therapeutic strategy for patients with COVID-19.

Viral infectious diseases pose a serious threat to global public health due to their high transmissibility, rapid mutation rates, and limited treatment options. Recent outbreaks of diseases such as plague, monkeypox, avian influenza, and coronavirus disease 2019 (COVID-19) have underscored the urgent need for efficient diagnostic and surveillance technologies. Focusing on viral infectious diseases that seriously threaten human health, this review summarizes and analyzes detection techniques from the perspective of combining viral surveillance and prevention advice, and discusses applications in improving diagnostic sensitivity and specificity. One of the major innovations of this review is the systematic integration of advanced biorecognition and detection technologies, such as bionanosensors, rapid detection test strips, and microfluidic platforms, along with the exploration of artificial intelligence in virus detection. These technologies address the limitations of traditional methods and enable the real-time monitoring and early warning of viral outbreaks. By analyzing the application of these technologies in the detection of pathogens, new insights are provided for the development of next-generation diagnostic tools to address emerging and re-emerging viral threats. In addition, we analyze the current progress of developed vaccines, combining virus surveillance with vaccine research to provide new ideas for future viral disease prevention and control and vaccine development, and call for global attention and the development of new disease prevention and detection technologies.

The COVID-19 pandemic highlighted SARS-CoV-2 variants with increased transmissibility and immune evasion. In Ethiopia, where cases surged, the understanding of the virus's dynamics was limited. This study analyzed SARS-CoV-2 variants during the fifth wave, crucial for guiding vaccines, therapeutics, diagnostics, and understanding disease severity.

From June to August 2022, 150 SARS-CoV-2-positive samples were randomly selected from the Ethiopian Public Health Institute repository. Sixty-three high-quality genome sequences were analyzed.

Of the 63 sequences, 70% were from males and 30% from females, with a median age of 34. Omicron dominated (97%, 61/63), primarily clade 22A (64%, 40/63), followed by 22B (18%, 11/63) and 21K (14%, 9/63). Delta accounted for 3.2% (2/63). Omicron was identified in all (25) vaccinated study participants. Ethiopian sequences showed limited evolutionary divergence and lower genetic diversity compared to global sequences.

Omicron was the predominant variant during Ethiopia's fifth wave, indicating recent community transmission. Despite minor genetic diversity differences, ongoing surveillance remains critical for tracking variants and informing public health interventions.

Background/Objectives: As with many viral fusion proteins, the native conformation of SARS-CoV-2 Spike is metastable. Most COVID-19 vaccines utilize a stabilized Spike (Spike-2P) containing two proline substitutions, and subsequently, a further stabilized variant with four additional proline substitutions, Spike-6P, has been developed. In an alternative approach, we introduced two aspartic acid residues (2D) in the HR1 region of Spike at positions that are exposed and buried in the pre- and postfusion states, respectively, to destabilize the postfusion conformation. Methods: The recombinant protein constructs were expressed in a mammalian cell culture and characterized for their yield and antigenicity, and the formulations were then used to immunize hamsters. After two immunizations, the hamsters were challenged with live B.1.351 SARS-CoV-2 virus for an evaluation of the protective efficacy. Results: The introduction of the two aspartic acid mutations resulted in an approximately six-fold increase in expression, comparable to that in Spike-2P. When the 2D mutations were combined with the above four proline mutations (Spike-4P-2D), this led to a further three- to four-fold enhancement of protein expression, similar to that seen in Spike-6P. When formulated with the oil-in-water emulsion adjuvant Sepivac SWE, the 2P, 2D, 6P, and 4P-2D Spike variants all protected female hamsters against heterologous challenge with the B.1.351 SARS-CoV-2 virus and elicited high titers of neutralizing antibodies. Conclusions: We suggest that destabilization of the postfusion conformation through the introduction of charged amino acids at sites that are exposed in the pre- and buried in the postfusion conformation offers a general strategy to enhance the yield and stability of the native, prefusion conformation of viral surface proteins.

The SARS-CoV-2 Spike protein is the primary target for vaccine design, with immunogens typically engineered to enhance stability by introducing proline mutations (2P) and mutating or deleting the Furin Cleavage Site (FCS). While these modifications improve structural integrity, studies suggest that furin cleavage can play a functional role in Spike protein dynamics, potentially enhancing ACE2 receptor binding. However, the impact of this cleavage on the unbound form of the Spike protein remains unclear. In this study, we use extensive all-atom molecular dynamics (MD) simulations to compare the structural and dynamic properties of cleaved and uncleaved Spike proteins in their pre-fusion, unbound state. Our results show that Furin cleavage significantly alters allosteric communication within the protein, increasing correlated motions between the Receptor Binding Domain (RBD) and N-terminal Domain (NTD), which may facilitate receptor engagement. Principal Component Analysis (PCA) reveals that the cleaved and uncleaved Spike proteins sample distinct conformational landscapes, with the cleaved form displaying enhanced flexibility and a broader range of RBD tilt angles. Additionally, Furin cleavage primes the S2 subunit by expanding the central helix, potentially influencing the transition to the post-fusion state. Glycan clustering patterns further suggest an adaptive structural response to cleavage, particularly in the NTD and RBD regions. These findings highlight the potential functional consequences of FCS deletion in immunogen design and underscore the importance of considering the native cleavage state in vaccine and therapeutic development.

SARS-CoV-2 binds to its obligatory receptor, angiotensin-converting enzyme 2 (ACE2) and capitalizes on decreasing endosomal acidity and cathepsin-mediated spike protein cleavage to enter cells. Endosomal acidification is driven by V-ATPase which pumps protons (H + ) into the lumen. The driving force for H + is maintained by the import of chloride (Cl - ) which is mediated by intracellular CLC transporters. We have recently identified the Proton-Activated Chloride (PAC) channel as a negative regulator of endosomal acidification. PAC responds to low pH and releases Cl - from the lumen to prevent endosomal hyperacidification. However, its role in SARS-CoV-2 viral entry remains unexplored. Here, we show that overexpressing the PAC channel in ACE2 expressing HEK 293T cells markedly inhibited the SARS-CoV-2 spike-mediated viral entry. Several lines of evidence suggest that this effect was due to the suppression of the endosomal entry pathway. First, the abilities of PAC to regulate endosomal acidification and inhibit pseudoviral entry were both dependent on its endosomal localization and channel activity. Second, the inhibitory effect on viral entry was similar to the suppression mediated by E64-d, a cathepsin inhibitor, while no major additive effect for both treatments was observed. Third, this inhibition was also attenuated in cells expressing TMPRSS2, which provides the alternative entry pathway through cell surface. Importantly, PAC overexpression also inhibited the number and size of plaques formed by two live SARS-CoV-2 isolates (B.1 and Omicron XBB.1.16) in Vero E6 cells. Altogether, our data indicates that PAC plays a vital role in inhibiting SARS-CoV-2 viral entry and identifies this endosomal channel as a potential novel target against the infection of SARS-CoV-2 and other viruses, which rely on the endosomal pathway.

Effective management of COVID-19 requires clinical tools to treat the disease in addition to preventive vaccines. Several recombinant mAbs and their cocktails have been developed to treat COVID-19 but these have limitations. Here, we evaluate small ankyrin repeat proteins called Ankyrons that were generated to bind with high affinity to the SARS-CoV-2 virus. Ankyrons are ankyrin repeat proteins comprised of repetitions a structural module. Each module consists of a β-turn followed by two antiparallel α-helices. The Ankyrons™ are directly selected in vitro from a highly diverse library of around a trillion clones in ribosome display and like antibodies can bind with high affinity to almost any target. We assessed Ankyrons that were generated against the wild-type SARS-CoV-2 and the Delta (B.1.617.2) and Omicron (BA.1) variants in a binding assay. We determined that all Ankyrons were specific in that they did not bind to MERS. While all Ankyrons bound with high affinity to the variant they were generated against, some also showed cross-reactivity to all three SARS-CoV-2 variants. Binding assays are useful for screening analytes but do not provide information about clinical effectiveness. Therefore, we used a pseudovirus-based neutralization assay to show that five of the Ankyrons evaluated neutralized all three strains of SARS-CoV-2. We have provided a workflow for the evaluation of novel Ankyrons against a viral target. This suggests that Ankyrons could be useful for rapidly developing new research tools for studying other emerging infectious diseases rapidly with the optional further potential for developing Ankyrons into diagnostic and even therapeutic applications.

Noninvasive visualization of the distribution and persistence of mRNA vaccine antigen expression in mammalian systems has implications for the development and evaluation of future mRNA vaccines. Here, we genetically fuse E. coli dihydrofolate reductase (eDHFR) to the delta furin diproline modified SARS-CoV-2 spike glycoprotein (S2P∆f) mRNA vaccine and image its expression in female mice and male non-human primates using [18F]fluoropropyl-trimethoprim ([18F]FP-TMP). Whole body positron emission tomography (PET) imaging revealed transient expression of the vaccine antigen in the injection site and draining lymph nodes (dLNs). Fusion of eDHFR did not impact S2P immunogenicity and no humoral or cellular immune response was detected against eDHFR in either species. In this work, we show that eDHFR can be used as an mRNA-encoded PET reporter gene to monitor the spatiotemporal dynamics of mRNA vaccine antigen expression in vivo. This technique could be applied in clinical translation of future mRNA vaccines or therapeutics.

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

The emergence of the COVID-19 pandemic has pointed out the urgent need for rapid and accurate diagnostic tools to detect the SARS-CoV-2 virus. Nanotechnology-based biosensors have emerged as a promising solution due to their high sensitivity, specificity, and speed in detecting biological molecules. This article focuses on the advancements in using nanotechnology for the development of modern biosensors tailored for the detection of the SARS-CoV-2 virus. Various nanomaterials, such as quantum dots, metallic nanoparticles, and nanowires, have been harnessed to enhance the performance of biosensors, offering improved detection limits and specificity. Besides this, innovative detection platforms, such as field-effect transistors, surface plasmon resonance, and electrochemical sensors, have revolutionized the landscape of SARS-CoV-2 diagnostics. These nanotechnology-based biosensors demonstrate the potential for point-of-care testing, enabling rapid and on-site detection with minimal sample preparation. The scalability, cost-effectiveness, and portability of these biosensors make them suitable for mass screening efforts in various healthcare settings, including hospitals, clinics, and community centers. The development of reliable biosensors for SARS-CoV-2 detection aligns with global efforts to curb the spread of the virus through early identification and containment strategies.

The development of vaccines against infectious diseases is of the utmost importance to prevent global pandemics such as COVID-19. The application of antigens and adjuvants to efficiently target antigen presenting cells (APCs) is paramount for the development of efficient vaccines. In our previous study, we showed that splenic marginal zone-B (MZ-B) cells are promising APCs in addition to dendritic cells (DCs). In this study we achieved the targeted delivery of sufficient antigen to MZ-B cells by utilizing an intravenous (IV) immunization system we originally developed. This system involves the sequential injection of empty PEGylated liposomes (PEG-Lip) and antigen-containing PEG-Lip within a prescribed interval. Herein, we describe the application of this IV immunization system as a COVID-19 vaccine to induce specific antibodies against SARS-CoV-2. To establish efficacy, SARS-CoV-2 spike proteins were used as an antigen, and α-galactosylceramide (GC) was used as an adjuvant in this study. Three days after priming with empty PEG-Lip, we injected PEG-Lip containing spike protein and α-GC. Our IV immunization system successfully induced higher levels of anti-spike antibodies when spike protein derived from HEK-293, but not E. coli., was injected into mice. The levels were less produced using conventional immunization via subcutaneous (s.c.) injections of complete Freund's adjuvant without priming. Interestingly, a lower dose (0.2 µg) of spike protein antigen encapsulated into PEG-Lip induced a higher level of anti-spike antibodies than that produced using a significantly higher dose (5 µg). The induced anti-spike antibodies inhibited the interaction between the receptor binding domain of the spike protein and the angiotensin-converting enzyme 2. This indicates that the induced antibodies tend to neutralize SARS-CoV-2. Collectively, the specific delivery of spike proteins to spleen, probably MZ-B cells, via nano-carriers could be a promising approach for the development of global pandemic vaccines that require only minimum dosages of antigen.

BackgroundEvolution of SARS-CoV-2 is continuous.AimBetween 01/2020 and 02/2022, we studied SARS-CoV-2 variant epidemiology, evolution and association with COVID-19 severity.MethodsIn nasopharyngeal swabs of COVID-19 patients (n = 1,762) from France, Italy, Spain, and the Netherlands, SARS-CoV-2 was investigated by reverse transcription-quantitative PCR and whole-genome sequencing, and the virus variant/lineage (NextStrain/Pangolin) was determined. Patients' demographic and clinical details were recorded. Associations between mild/moderate or severe COVID-19 and SARS-CoV-2 variants and patient characteristics were assessed by logistic regression. Rates and genomic locations of mutations, as well as quasi-species distribution (≥ 2 heterogeneous positions, ≥ 50× coverage) were estimated based on 1,332 high-quality sequences.ResultsOverall, 11 SARS-CoV-2 clades infected 1,762 study patients of median age 59 years (interquartile range (IQR): 45-73), with 52.5% (n = 925) being male. In total, 101 non-synonymous substitutions/insertions correlated with disease prognosis (severe, n = 27; mild-to-moderate, n = 74). Several hotspots (mutation rates ≥ 85%) occurred in Alpha, Delta, and Omicron variants of concern (VOCs) but none in pre-Alpha strains. Four hotspots were retained across all study variants, including spike:D614G. Average number of mutations per open-reading-frame (ORF) increased in the spike gene (average < 5 per genome in January 2020 to > 15 in 2022), but remained stable in ORF1ab, membrane, and nucleocapsid genes. Quasi-species were most prevalent in 20A/EU2 (48.9%), 20E/EU1 (48.6%), 20A (38.8%), and 21K/Omicron (36.1%) infections. Immunocompromised status and age (≥ 60 years), while associated with severe COVID-19 or death irrespective of variant (odds ratio (OR): 1.60-2.25; p ≤ 0.014), did not affect quasi-species' prevalence (p > 0.05).ConclusionSpecific mutations correlate with COVID-19 severity. Quasi-species potentially shaping VOCs' emergence are relevant to consider.

The novel coronavirus SARS-CoV-2, which wrecked havoc around the world in the recent years through COVID-19, gains entry into the host cell through various receptors. Development of therapies targeting host-pathogen interaction will be a key to curb the infection as it potentially suppresses viral attachment and entry into the host. Boundless bioactives abundant in natural resources are the important source of new as well as safer alternatives. Tannic acid, a polyphenolic compound found abundantly in various plant sources, has gained much attention owing to its multifaceted pharmacological properties. This research paper presents a comprehensive investigation on antioxidant, anti-inflammatory and anti-viral abilities of tannic acid, substantiated through a triad of methodologies: in silico, in vitro and in vivo approaches. In vitro experiments, confirmed the antioxidant and anti-inflammatory efficacy as well as the host receptor modulating potential of tannic acid. In silico docking analyses elucidated the molecular interactions between tannic acid and key host receptors involved in inflammation and viral pathogenesis. Furthermore, the in vivo studies involving Danio rerio provided a holistic understanding of the systemic impact of tannic acid, including its antioxidant effects by mitigating the oxidative stress.

The N234 and N343-linked glycans of the SARS-CoV 2 spike protein are known to stabilize the up-conformation of its receptor-binding domains (RBDs), enabling human angiotensin enzyme 2 (hACE2) receptor binding. However, the effect of spike-hACE2 binding on these important glycans remains poorly understood, and these changes could have implications in the development of drugs that inhibit viral entry. In this study, Gaussian accelerated molecular dynamics (GaMD) simulations of the hACE2-free and hACE2-bound spike protein are performed. Biophysical analyses were focused on the accessibility of three previously suggested druggable pockets underneath the three RBD subunits. A shielding effect by N234-linked glycans on the components of their adjacent pockets was observed. Although deshielding of central scaffold residues was observed in the hACE2-bound state, pocket A's accessibility was reduced due to an increase in NTDB-RBDB contacts, restricting entry into the pocket. For pocket B, changes in N234C and N343C expose the central scaffold residues in the bound state, increasing accessibility. In Pocket C, increased shielding due to N234A was found in the bound state, reducing accessibility. Despite these changes, the pockets remain accessible to ligands in both states and are still valid targets for drug development studies.