The sudden global crisis of COVID-19, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demands swift containment measures due to its rapid spread and numerous problematic mutations, which complicate the establishment of herd immunity. With escalating fatalities across various nations no foreseeable end in sight, there is a pressing need to create swiftly deployable, rapid, cost-effective detection, and treatment methods. While various steps are taken to mitigate the transmission and severity of the disease, vaccination is proven throughout mankind history as the best method to acquire immunity and circumvent the spread of infectious diseases. Nonetheless, relying solely on vaccination might not be adequate to match the relentless viral mutations observed in emerging variants of SARS-CoV-2, including alterations to their RBD domain, acquisition of escape mutations, and potential resistance to antibody binding. Beyond the immune system activation achieved through vaccination, it is crucial to develop new medications or treatment methods to either impede the infection or enhance existing treatment modalities. This review emphasizes innovative treatment strategies that aim to directly disrupt the virus's ability to replicate and spread, which could play a role in ending the SARS-CoV-2 pandemic.

The persistent emergence of new SARS-CoV-2 variants has presented significant challenges to vaccines and antiviral therapeutics, highlighting the need for the development of methods that ensure variant-independent responses. This study introduces a unique sensor capable of electrochemically detecting SARS-CoV-2 across a wide range of variants. The comprehensive detection is achieved by using a peptide-DNA hybrid, R7-02, as the capture probe, mimicking the binding interface between a SARS-CoV-2 spike protein and a host cell receptor, hACE2. Since the first step of viral infection is the binding of the spike protein to hACE2 regardless of variant type, the hACE2-mimicking probe can naturally acquire the pan-variant recognition capability. In constructing the sensor, the R7-02 probes are positioned on electrodes via a tetrahedral DNA nanostructure for enhanced detection efficiency. Since R7-02 directly captures the externally-exposed spike protein, our approach does not require sample pretreatments, such as virus particle lysis, unlike conventional diagnostic methods. The R7-02-embedded sensor demonstrated high sensitivity towards Omicron and its major subvariants-commonly known as 'stealth Omicron' (BA.5, BA.2.75, BQ.1.1, and XBB.1.5)-with a detection limit as low as 811.9 pM, along with robust specificity for SARS-CoV-2 against influenza and other human coronaviruses. The sensor also successfully detected SARS-CoV-2 directly from non-treated saliva samples of COVID-19-positive patients. Given the comprehensive and sensitive detection capability, combined with its simple operation, our receptor-mimicking probe-based electrochemical sensor holds the potential to be a sustainable and effective point-of-care diagnostic tool, offering a promising solution to the constant challenges posed by the endemic presence of SARS-CoV-2.

Machine learning (ML) techniques have become powerful tools in both industrial and academic settings. Their ability to facilitate analysis of complex data and generation of predictive insights is transforming how scientific problems are approached across a wide range of disciplines. In this tutorial, we present a cursory introduction to three widely used ML techniques─logistic regression, random forest, and multilayer perceptron─applied toward analyzing molecular dynamics (MD) trajectory data. We employ our chosen ML models to the study of the SARS-CoV-2 spike protein receptor binding domain interacting with the receptor ACE2. We develop a pipeline for processing MD simulation trajectory data and identifying residues that significantly impact the stability of the complex.

Neurological symptoms involving the central nervous system (CNS) and peripheral nervous system (PNS) are common complications of acute COVID-19 as well as post-COVID conditions. Most research into these neurological sequalae focuses on the CNS, disregarding the PNS. Guinea pigs were previously shown to be useful models of disease during the SARS-CoV-1 epidemic. However, their suitability for studying SARS-CoV-2 has not been experimentally demonstrated. To assess the suitability of guinea pigs as models for SARS-CoV-2 infection and the impact of SARS-CoV-2 infection on the PNS, and to determine routes of CNS invasion through the PNS, we intranasally infected wild-type Dunkin-Hartley guinea pigs with ancestral SARS-CoV-2 USA-WA1/2020. We assessed PNS sensory neurons (trigeminal ganglia, dorsal root ganglia), autonomic neurons (superior cervical ganglia), brain regions (olfactory bulb, brainstem, cerebellum, cortex, hippocampus), lungs, and blood for viral RNA (RT-qPCR), protein (immunostaining), and infectious virus (plaque assay) at three- and six-days post infection. We show that guinea pigs, which have previously been used as a model of SARS-CoV-1 pulmonary disease, are not susceptible to intranasal infection with ancestral SARS-CoV-2, and are not useful models in assessing neurological impacts of infection with SARS-CoV-2 isolates from the early pandemic.

Glycoproteins, which are conjugates of glycans and proteins, are crucial in a wide variety of physiological and disease processes. Understanding the structures and functions of glycoproteins, as well as the regulation of glycosylation, is essential for studying the causes of diseases for intervention therapy. However, the detailed structure-function relationships and therapeutic applications of glycoproteins are hindered by their structural complexity and heterogeneity. Chemical protein synthesis is a powerful and effective strategy for producing homogeneous glycoforms of glycoproteins. The chemical synthesis of glycoproteins involves ligating different peptide and/or glycopeptide fragments, and the preparation of glycopeptide fragments requires the assembly of amino acid and glyco-based building blocks. This review provides a comprehensive and systematic survey of glyco-based building blocks for synthesizing homogeneous glycopeptides and glycoproteins, encompassing glyco-amino acids for direct SPPS and glyco-based donors for convergent sugar assembly. Additionally, an analysis of the applications of these building blocks in the chemical synthesis of representative glycoproteins with therapeutic potential is presented.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor to enter host cells, and primary receptor recognition of the spike protein is a major determinant of the host range of SARS-CoV-2. Since the emergence of SARS-CoV-2, a considerable number of variants have emerged. However, the determinants of host tropism of SARS-CoV-2 remain elusive. We conducted infection assays with chimeric recombinant SARS-CoV-2 carrying the spike protein from 10 viral variants, assessing their entry efficiency using mammalian ACE2 orthologs from species that have close contact with humans. We found that only murine ACE2 exhibited different susceptibilities to infection with the SARS-CoV-2 variants. Moreover, we revealed that the mutation N501Y in the viral spike protein has a crucial role in determining the infectivity of cells expressing murine ACE2 and of mice in vivo. Next, we identified six amino acid substitutions at 24, 30, 31, 82, 83, and 353 in murine ACE2 that allowed for viral entry of the variants to which murine ACE2 was previously resistant. Furthermore, we showed that ACE2 from a species closely related to mice, Mus caroli, is capable of supporting entry of the viral variants that could not use murine ACE2. These results suggest that few ACE2 orthologs have different susceptibility to infection with SARS-CoV-2 variants as observed for murine ACE2. Collectively, our study reveals critical amino acids in ACE2 and the SARS-CoV-2 spike protein that are involved in the host tropism of SARS-CoV-2, shedding light on interspecies susceptibility to infection.IMPORTANCESARS-CoV-2 can infect many species besides humans, leading to the evolution of the virus and adaptation to other animal hosts, which could trigger a new COVID-19 wave. The SARS-CoV-2 spike protein utilizes ACE2 as a receptor for entry into host cells. The interaction of ACE2 with the spike protein determines the host range of SARS-CoV-2. In this study, using chimeric viruses carrying the spike protein of SARS-CoV-2 variants to infect cells expressing different ACE2 orthologs from species humans come in close contact with, we confirmed murine ACE2 alone showed different susceptibility to the variants. We identified residues in murine ACE2 and the viral spike that restrict viral entry. Furthermore, an ACE2 ortholog from a species genetically close to mice mediated entry of SARS-CoV-2 variants incapable of infecting mice. This research highlights the uniquely limited susceptibility of mice to different SARS-CoV-2 variants and provides invaluable insights into the host tropism of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, employs the viral spike (S) protein to associate with host cells. While angiotensin-converting enzyme 2 (ACE2) is a major receptor for the SARS-CoV-2 spike protein, evidence reveals that other cellular receptors may also contribute to viral entry. We interrogated the role of the low-density lipoprotein receptor-related protein 1 (LRP1) in the involvement of SARS-CoV-2 viral entry. Employing surface plasmon resonance studies, we demonstrated high affinity binding of the trimeric SARS-CoV-2 spike protein to purified LRP1. Further, we observed high affinity interaction of the SARS-CoV-2 spike protein with other low-density lipoprotein receptor (LDLR) family members as well, including LRP2 and the very low-density lipoprotein receptor (VLDLR). Binding of the SARS-CoV-2 spike protein to LRP1 was mediated by its receptor binding domain (RBD). Several LRP1 ligands require surface exposed lysine residues for their interaction with LRP1, and chemical modification of lysine residues on the RBD with sulfo-NHS-acetate ablated binding to LRP1. Using cellular model systems, we demonstrated that cells expressing LRP1, but not those lacking LRP1, rapidly internalized purified 125I-labeled S1 subunit of the SARS-CoV-2 spike protein. LRP1-mediated internalization of the 125I-labeled S1 subunit was enhanced in cells expressing ACE2. By employing pseudovirion particles containing a murine leukemia virus core and luciferase reporter that express the SARS-CoV-2 spike protein on their surface, we confirmed that LRP1 facilitates ACE2-mediated psuedovirion endocytosis. Together, these data implicate LRP1, and perhaps other LDLR family members as host factors for SARS-CoV-2 infection.

The blood-brain barrier (BBB) presents a major challenge in the theranostics of brain diseases by impeding the delivery of drugs to the brain. Currently the most common strategy for transferring substances across the BBB is receptor-mediated transcytosis, which is restricted by several key factors, including insufficient endocytosis by brain microvessel endothelial cells (BMECs) due to underexpressed pinocytotic vesicles, lysosomal retention, and limited exocytosis to the brain parenchyma. We report a hybrid cell membrane (HCM)-coated and 2-methacryloyloxyethyl phosphorylcholine (MPC)-modified nanocarrier to promote drug delivery across the BBB by modulating the transcytosis process. The HCM incorporates a brain metastatic tumor cell membrane for recognition of BMECs and a GFP-293-S cell membrane expressing Spike protein to facilitate membrane fusion between the nanocarrier and BMECs, thereby bypassing vesicle-dependent endocytosis and enhancing cellular uptake. Membrane fusion reduces the chance of lysosomal retention, and MPC modification enhances exocytosis into the brain parenchyma via the interaction of MPC with transporters expressed on the abluminal endothelial membrane. The nanocarrier achieves significantly improved delivery of CuS, a photothermal agent, to the brain and thus enables highly efficient therapy of brain glioma.

Peptides have become a powerful frontier in modern medicine, offering a promising therapeutic solution for various diseases and advancing rapidly in pharmaceutical development. These small amino acid chains, with their innovative design, have attracted significant attention due to their versatility and high receptor specificity, which minimizes off-target effects, along with enhanced therapeutic efficacy, biodegradability, low toxicity, and minimal immunogenicity. They are being explored for use in several clinical domains, like metabolic diseases, immunomodulation, and cancer. Furthermore, antimicrobial peptides (AMPs) have grown to be a promising strategy to combat the worldwide challenge of antibiotic resistance, demonstrating promising results against multidrug-resistant organisms. Both natural and engineered peptides have been discovered and investigated, whereas numerous others are progressing toward clinical trials in a number of therapeutic domains. Recent improvements with surface modification, such as peptide engineering, peptide cyclization, PEGylation, and the utilization of synthetic amino acids to enhance their pharmacokinetic profiles and overcome the inherent disadvantages of these peptides have made it possible for the area to continue to advance. Moreover, their therapeutic potential has been further enhanced by innovative delivery methods, such as self-assembling peptides, nanocarriers, and alternate routes of administration. This Review critically states the potential of peptides as versatile therapeutics along with their modifications and advancements to drive the significant progress to treat infections and chronic diseases, along with their potential benefits and challenges.

This work aimed to evaluate the antiviral potential of compounds from Asian medicinal plants against SARS-CoV-2's main protease and spike glycoprotein, identifying dual inhibitors from these plants that target both proteins through advanced virtual screening, molecular dynamics simulations, and pharmacophore analysis. An in-house library of 335 antiviral natural products was prepared from the selected medicinal plants. Following the virtual screening of this library against the main protease and spike glycoprotein, top compounds were subjected to downstream analysis for evaluating druggability potential and toxicity analysis. Molecular dynamic simulations were performed to confirm the stability of interactions between the ligands and target proteins. Our analysis demonstrated 67 compounds as dual inhibitors. The six top dual inhibitors, namely trans-delta-viniferin, trans-E-viniferin, 3,4-DHPEA-EDA, oleuropein aglycone, lactucopicrin, and 11β,13-dihydrolactucopicrin, exhibited superior docking scores and met drug-likeness criteria, including Lipinski's rule, bioavailability, and favorable ADME and toxicity profiles. Trans-delta-viniferin and trans-E-viniferin, featuring a stilbene scaffold, emerged as the most promising candidates due to their stable interactions, minimal fluctuations, and consistent hydrogen bonding across SARS-CoV-2's Mpro and S-protein in MD simulations, while 3,4-DHPEA-EDA displayed comparatively less stability. All compounds demonstrated key pharmacophoric features and lacked mutagenicity or PAINS alerts, although lactucopicrin and 11β,13-dihydrolactucopicrin showed risks for hepatotoxicity. Overall, the critical bonding and drug-like features, biological activity spectra, and favorable medicinal characteristics predict their biological behavior in laboratory testing. Although additional experimental validations are necessary, our findings indicate that the three lead compounds-namely, trans-delta-viniferin, trans-E-viniferin, and 3,4-DHPEA-EDA, isolated from traditional medicinal plants-are promising novel dual inhibitors of two critical SARS-CoV-2 proteins.

Despite the global impact caused by the most recent SARS-CoV-2 pandemic, our knowledge of the molecular underpinnings of its highly infectious nature remains incomplete. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. In addition to catalyzing CTP synthesis, CTPS1 also deamidates interferon regulatory factor 3 (IRF3) to dampen interferon induction. Screening a SARS-CoV-2 expression library, we identified several viral proteins that interact with CTPS1. Functional analyses demonstrate that ORF8 and Nsp8 activate CTPS1 to deamidate IRF3 and negate IFN induction, whereas ORF7b and ORF8 activate CTPS1 to promote CTP synthesis. These results highlight CTPS1 as a signaling node that integrates cellular metabolism and innate immune response. Indeed, small-molecule inhibitors of CTPS1 deplete CTP and boost IFN induction in SARS-CoV-2-infected cells, thus effectively impeding SARS-CoV-2 replication and pathogenesis in mouse models. Our work uncovers an intricate mechanism by which a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means to develop antiviral therapy against highly mutagenic viruses.IMPORTANCEOur understanding of the underpinnings of highly infectious SARS-CoV-2 is rudimentary at best. We report here that SARS-CoV-2 activates CTPS1 to promote CTP synthesis and suppress IFN induction, thus coupling immune evasion to activated nucleotide synthesis. Inhibition of the key metabolic enzyme not only depletes the nucleotide pool but also boosts host antiviral defense, thereby impeding SARS-CoV-2 replication. Targeting cellular enzymes presents a strategy to counter the rapidly evolving SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus of the coronaviridae family. The virus enters the cell through binding to the corresponding receptor angiotensin-converting enzyme 2 (ACE2) on host cell membrane with the spike protein (S protein) on its envelope; thus, we can design inhibitors that bind the S protein to block the entry of the virus into cells. Aptamers are single stranded DNA or RNA molecules that can form specific three-dimensional structures and bind their target molecules with high affinity and specificity and thus are promising candidates for S protein inhibitors. This paper reviews the replication cycle and cell entry mechanisms of SARS-CoV-2 as well as the preparation principle and characteristics of aptamers, features a discussion of the advantages of using aptamers to target the S protein to prevent SARS-CoV-2 from infecting cells, and finally summarizes the research progress in S protein-blocking aptamers.

SARS-CoV-2 Omicron sub-variants continuously evolve under the pressure of neutralizing antibodies (nAbs), eliminating numerous potential elite monoclonal nAbs. The IGHV3-53/3-66 public nAbs have great potential for neutralizing SARS-CoV-2. However, it has been unclear whether a primary Omicron infection could also induce IGHV3-53/3-66 nAbs. In this study, we report an IGHV3-66-encoding monoclonal nAb, ConBA-998, that was elicited by primary infection with BA.1. ConBA-998 is an Omicron-dependent nAb with high binding affinity that triggers the shedding of the S1 subunit from the spike protein. The cryo-electron microscopy (cryo-EM) structure revealed the interactions between ConBA-998 and the Omicron BA.1 spike protein. ConBA-998 has a distinctive binding mode to receptor-binding domain (RBD) that differs from canonical IGHV3-53/3-66 nAbs. Overall, our findings indicate that Omicron may elicit unique specific nAbs distinct from those induced by pre-Omicron variants, providing further insights into SARS-CoV-2 variant-specific antibody responses.

The 3C-like protease (3CLpro) is essential in the SARS-CoV-2 life cycle and a promising target for antiviral drug discovery, as no similar proteases exist in humans. This study aimed to identify effective SARS-CoV-2 inhibitors among FDA-approved drugs. Previous computational analysis revealed several drugs with high binding affinity to the 3CLpro active site. In vitro enzymatic assays confirmed that ten of these drugs effectively inhibited the enzyme. To evaluate their impact on viral replication, we used non-infectious SARS-CoV-2 sub-genomic replicons in lung and intestinal cells. Amcinonide, eltrombopag, lumacaftor, candesartan, and nelfinavir inhibited replication at low micromolar concentrations. Lumacaftor showed IC50 values of 964 nM in Caco-2 cells and 458 nM in Calu-3 cells, while candesartan had IC50 values of 714 nM and 1.05 µM, respectively. Furthermore, dual combination experiments revealed that amcinonide, pimozide, lumacaftor, and eltrombopag acted as potent inhibitors at nanomolar concentrations when combined with candesartan. This study highlights lumacaftor, candesartan, and nelfinavir as effective inhibitors of SARS-CoV-2 replication in vitro and emphasizes their potential for repurposing as antiviral treatments. These findings support future clinical trials and may lead to breakthroughs in COVID-19 treatment strategies.

Congregate animal settings can serve as foci for the increased transmission of pathogens, including zoonoses. Domestic cats have been shown to be reservoirs for SARS-CoV-2 but the public health importance of infected cats has not yet been determined. A population of indoor-only residential cats at a cat café in central Texas with a high level of human interaction was evaluated for infection with SARS-CoV-2 in a longitudinal study in 2021-2022. Among 25 cats, none were qRT-PCR-positive, while 50% harbored SARS-CoV-2-neutralizing antibodies, including 1 that remained seropositive for >8 months. The high level of human exposure in this unique congregate cat setting-in which dozens of new visitors interact with the cats every day-likely facilitated the human-to-cat transmission of SARS-CoV-2 that led to a 50% infection prevalence in cats. This work was conducted when the Delta and Omicron variants predominated. Given that feline susceptibility to infection and shedding of a virus may vary across different viral variants, veterinary surveillance may be an important component of veterinary and human health risk assessments.

Evolution of SARS-CoV-2 has led to the emergence of variants with increased immune evasion capabilities, posing significant challenges to antibody-based therapeutics and vaccines. The cross-neutralization activity of antibodies against Omicron variants is governed by a complex and delicate interplay of multiple energetic factors and interaction contributions. In this study, we conducted a comprehensive analysis of the interactions between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and four neutralizing antibodies S309, S304, CYFN1006, and VIR-7229. Using integrative computational modeling that combined all-atom molecular dynamics (MD) simulations, mutational scanning, and MM-GBSA binding free energy calculations, we elucidated the structural, energetic, and dynamic determinants of antibody binding. Our findings reveal distinct dynamic binding mechanisms and evolutionary adaptation driving broad neutralization effect of these antibodies. We show that S309 targets conserved residues near the ACE2 interface, leveraging synergistic van der Waals and electrostatic interactions, while S304 focuses on fewer but sensitive residues, making it more susceptible to escape mutations. The analysis of CYFN-1006.1 and CYFN-1006.2 antibody binding highlights broad epitope coverage with critical anchors at T345, K440, and T346, enhancing its efficacy against variants carrying the K356T mutation which caused escape from S309 binding. Our analysis of broadly potent VIR-7229 antibody binding to XBB.1.5 and EG.5 Omicron variants emphasized a large and structurally complex epitope, demonstrating certain adaptability and compensatory effects to F456L and L455S mutations. Mutational profiling identified key residues crucial for antibody binding, including T345, P337, and R346 for S309, and T385 and K386 for S304, underscoring their roles as evolutionary "weak spots" that balance viral fitness and immune evasion. The results of this energetic analysis demonstrate a good agreement between the predicted binding hotspots and critical mutations with respect to the latest experiments on average antibody escape scores. The results of this study dissect distinct energetic mechanisms of binding and importance of targeting conserved residues and diverse epitopes to counteract viral resistance. Broad-spectrum antibodies CYFN1006 and VIR-7229 maintain efficacy across multiple variants and achieve neutralization by targeting convergent evolution hotspots while enabling tolerance to mutations in these positions through structural adaptability and compensatory interactions at the binding interface. The results of this study underscore the diversity of binding mechanisms employed by different antibodies and molecular basis for high affinity and excellent neutralization activity of the latest generation of antibodies.

Pregnant women are considered a high-risk population for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as the virus can infect the placenta and embryos. Recently, SARS-CoV-2 has been widely reported to cause retinal pathological changes and to infect the embryonic retina. The infection of host cells by SARS-CoV-2 is primarily mediated through spike (S) protein, which also plays a crucial role in the pathogenesis of SARS-CoV-2. However, it remains poorly understood how the S protein of SARS-CoV-2 affects retinal development, and the underlying mechanism has not yet been clarified.

We used human embryonic stem cell-derived retinal organoids (hEROs) as a model to study the effect of S protein exposure at different stages of retinal development. hEROs were treated with 2 μg/mL of S protein on days 90 and 280. Immunofluorescence staining, RNA sequencing, and RT-PCR were performed to assess the influence of S protein exposure on retinal development at both early and late stages.

The results showed that ACE2 and TMPRSS2, the receptors facilitating SARS-CoV-2 entry into host cells, were expressed in hEROs. Exposure to the S protein induced an inflammatory response in both the early and late stages of retinal development in the hEROs. Additionally, RNA sequencing indicated that early exposure of the S protein to hEROs affected nuclear components and lipid metabolism, while late-stages exposure resulted in changes to cell membrane components and the extracellular matrix.

This work highlights the differential effects of SARS-CoV-2 S protein exposure on retinal development at both early and late stages, providing insights into the cellular and molecular mechanisms underlying SARS-CoV-2-induced developmental impairments in the human retina.

The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.

Melissa officinalis is a perennial medicinal plant traditionally used for its diverse biological activities, including antiviral properties. This study investigates the antiviral efficacy of various extracts, including water, acetone, alkaloid, non-alkaloid, ethanol, and methanol extracts, against influenza A (H1N1), SARS-CoV-2, and MERS-CoV. The water extract demonstrated significant inhibitory effects on SARS-CoV-2 (IC50 = 421.9 µg/mL) and MERS-CoV (IC50 = 222.1 µg/mL) in Vero E6 cells (an African green monkey kidney cell line), with a CC50 of 4221 µg/mL, indicating a favorable selectivity index. Additionally, it exhibited strong activity against H1N1 in Madin-Darby canine kidney cell line (MDCK cells) (IC50 = 57.30 µg/mL, CC50 = 3073 µg/mL). Among all the extracts, the methanol extract showed the highest antiviral activity. It has IC50 = 2.549 µg/ml and selectivity index (SI) = 230 against H1N1.While it showed IC50 = 10.83 µg/ml against SARS-CoV-2 and 9.82 µg/ml against MERS-CoV with SI values of 54.2 and 59.77, respectively. Molecular docking studies revealed that 5-Methyl-5 H-naphtho[2,3-c]carbazole,7 H-Dibenzo[b, g]carbazole, 7-methyl, hesperidin, luteolin-7-glucoside-3'-glucuronide, Melitric acid A, and other compounds exhibited high binding affinities to the receptor-binding domains (RBDs) of SARS-CoV-2 and MERS-CoV spike glycoproteins, suggesting their potential to interfere with viral entry. Furthermore, GC-MS-identified bioactive compounds, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), paromomycin, and phenolic acids, demonstrated additional antiviral potential. These results underscore the potential of M. officinalis extracts as natural antiviral agents, offering a foundation for further in vitro and in vivo validation and potential therapeutic applications against respiratory viral infections, including coronaviruses and influenza viruses.

The SARS-CoV-2 Omicron variants show different behavior compared to the previous variants, especially with respect to the Delta variant, which promotes a lower morbidity despite being much more contagious. In this perspective, we performed molecular dynamics (MD) simulations of the different spike RBD/hACE2 complexes corresponding to the WT, Delta and four Omicron variants. Carrying out a comprehensive analysis of residue interactions within and between the two partners allowed us to draw the profile of each variant by using complementary methods (PairInt, hydrophobic potential, contact PCA). PairInt calculations highlighted the residues most involved in electrostatic interactions, which make a strong contribution to the binding with highly stable interactions between spike RBD and hACE2. Apolar contacts made a substantial and complementary contribution in Omicron with the detection of two hydrophobic patches. Contact networks and cross-correlation matrices were able to detect subtle changes at point mutations as the S375F mutation occurring in all Omicron variants, which is likely to confer an advantage in binding stability. This study brings new highlights on the dynamic binding of spike RBD to hACE2, which may explain the final persistence of Omicron over Delta.

The spike protein encoded by the SARS-CoV-2 has become one of the most studied macromolecules in recent years due to its central role in COVID-19 pathogenesis. The spike protein's receptor-binding domain (RBD) directly interacts with the host-encoded receptor protein, ACE2. This review critically examines computational insights into RBD's interaction with ACE2 and with therapeutic antibodies designed to interfere with this interaction. We begin by summarizing insights from early computational studies on pre-pandemic SARS-CoV-1 RBD interactions and how these early studies shaped the understanding of SARS-CoV-2. Next, we highlight key theoretical contributions that revealed the molecular mechanisms behind the binding affinity of SARS-CoV-2 RBD against ACE2, and the structural changes that have enhanced the infectivity of emerging variants. Special attention is given to the "RBD charge rule", a predictive framework for determining variant infectivity based on the electrostatic properties of the RBD. Towards applying the computational insights to therapy, we discuss a multiscale computational protocol for optimizing monoclonal antibodies to improve binding affinity across multiple spike protein variants, including representatives from the Omicron family. Finally, we explore how these insights can inform the development of future vaccines and therapeutic interventions for combating future coronavirus diseases.

The persistent emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants highlights the need for developing broad-spectrum antiviral agents. Here, we report the identification of two sarbecovirus S2-specific alpaca nanobodies, namely H17 and H145, that effectively neutralize known SARS-CoV-2 variants (including the Omicron subvariants) and other sarbecoviruses (such as SARS-CoV, PANG/GD, WIV1, and HKU3). The two nanobodies recognize a linear epitope (D1139PLQPELDSFKEEL1152) in the upper region of the S2 stem-helix (SH), which is highly conserved among SARS-CoV-2 variants and other sarbecoviruses. The complex structure of the nanobody bound to the epitope SH-peptide reveal that nanobody binding will impede the refolding of S2, effectively neutralizing the virus. Moreover, the nanobodies bind viral S2 in an acidification-insensitive manner, demonstrating their capacity for entry inhibition especially when viruses enter via the endosomal route. Finally, H17 and H145 possess a better taking-action window for virus neutralization, superior to the RBD-targeting nanobodies that exert neutralization by competing against ACE2 binding. Taken together, the results suggest that anti-SH nanobodies H17 and H145 are promising broad-spectrum drug candidates for preventing and treating the pandemic infections by SARS-CoV-2 variants and other sarbecoviruses.

Accurate diagnosis and effective antiviral strategies are critical to combat acute infection and to avoid damage to the host. Due to their restricted radiation range and energy, Auger electron emitters have shown potential as a RNA-destructing radionuclide therapy in oncology and infection. Focusing on the process of angiotensin-converting enzyme 2 (ACE2)-mediated endocytosis, Technetium-99m-labeled DX600 (99mTc-DX600) was synthesized as an Auger electron vector to specifically bind to surface-expressed ACE2 proteins on 293T-hACE2 cells (293T cells stably expressing human ACE2), and Technetium-99m-loaded microvesicles (99mTc-MVs) served as an antiviral tracer and effector in pseudovirus infection. The whole-body ACE2 expression evaluation was non-invasive, meanwhile, the enhanced green fluorescent protein expression of pseudoviruses was substantially inhibited as a result of the 99mTc-DX600 loading of microvesicles, though the mitochondrial and DNA stabilities of the host cells were not affected. Furthermore, the in vivo distribution of 99mTc-DX600 in humanized ACE2 mice was demonstrated to be both ACE2-specific and long-lasting, and an antiviral effect was fully exhibited with two cycles of intravenous injection at a dosage of 37 MBq. Taking advantage of the ACE2-mediated interaction and natural trigger mechanism of virus-induced endocytosis, 99mTc-MV represents a theranostic biosensor of Auger electrons that can expose viral RNA to lethal amounts of radiation, with the host cells receiving no detrimental radiation.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone significant genomic mutations, contributing to resistance against existing 2019 coronavirus disease (COVID-19) treatments. In a previous study, we identified N-0385, a potent host-directed inhibitor of transmembrane serine protease 2 (TMPRSS2), which has therapeutic efficacy towards SARS-CoV-2 infection. However, in further evaluation of its preclinical druggability, N-0385 displayed unfavorable pharmacokinetic properties, including high bioavailability (99 %) following intranasal (IN) administration. This can lead to substantial systemic exposure and potential adverse effects due to off-target interactions. Here, we designed a library of peptidomimetic compounds with P3 site modifications on an optimized scaffold. We sought to maintain sub-nanomolar potency against TMPRSS2 (Kis < 2 nM), reduce pseudovirus infection, while addressing the lack of selectivity and excessive lung uptake. Notably, inhibitor 9, which contains Asp at the P3 position, achieved a two-fold increase in TMPRSS2 inhibitory potency (Ki = 0.13 ± 0.03 nM), a >700-fold selectivity over Factor Xa (FXa), and showed superior selectivity against other proteases (matriptase, transmembrane serine protease 6 (TMPRSS6), thrombin, furin, and tPA). Despite concerns about the role of FXa in the coagulation cascade, compound 9 had no impact on coagulation or thrombolysis 2 h after in vitro treatment. In the air-liquid interface (ALI) model of the lung epithelium, compound 9 displayed a 1.5-fold decrease in permeability compared to N-0385 and demonstrated sustained stability in lungs (11 h) and plasma (13 h). Taken together, our data demonstrate that continued optimization of this type of inhibitors will lead to improved therapeutics for the treatment of SARS-CoV-2 infection by IN administration.

Early antibody therapy can prevent severe SARS-CoV-2 infection (COVID-19). However, the effectiveness of COVID-19 convalescent plasma (CCP) therapy in treating severe COVID-19 remains inconclusive. To test a hypothesis that some CCP units are associated with a coagulopathy hazard in severe disease that offsets its benefits, we tracked 304 CCP units administered to 414 hospitalized COVID-19 patients to assess their association with the onset of unfavorable post-transfusion D-dimer trends. CCP recipients with increasing or persistently elevated D-dimer trajectories after transfusion experienced higher mortality than those whose D-dimer levels were persistently low or decreasing after transfusion. Within the CCP donor-recipient network, recipients with increasing or persistently high D-dimer trajectories were skewed toward association with a minority of CCP units. In in vitro assays, CCP from "higher-risk" units had higher cross-reactivity with the spike protein of human seasonal betacoronavirus OC43. "Higher-risk" CCP units also mediated greater Fcγ receptor IIa signaling against cells expressing SARS-CoV-2 spike compared with "lower-risk" units. This study finds that post-transfusion activation of coagulation pathways during severe COVID-19 is associated with specific CCP antibody profiles and supports a potential mechanism of immune complex-activated coagulopathy.

A multiplexed microarray chip (Immuno-μSARS2) aiming at providing information on the prognosis of the COVID-19 has been developed. The diagnostic technology records information related to the profile of the immunological response of patients infected by the SARS-CoV-2 virus. The diagnostic technology delivers information on the avidity of the sera against 28 different peptide epitopes and 7 proteins printed on a 25 mm2 area of a glass slide. The peptide epitopes (12-15 mer) derived from structural proteins (Spike and Nucleocapsid) have been rationally designed, synthesized, and used to develop Immuno-μSARS2 as a multiplexed and high-throughput fluorescent microarray platform. The analysis of 755 human serum samples (321 from PCR+ patients; 288 from PCR- patients; 115 from prepandemic individuals and classified as hospitalized, admitted to intensive-care unit (ICU), and exitus) from three independent cohorts has shown that the chips perform with a 98% specificity and 91% sensitivity identifying RT-PCR+ patients. Computational analysis utilized to correlate the immunological signatures of the samples analyzed indicate significant prediction rates against exitus conditions with 82% accuracy, ICU admissions with 80% accuracy, and 73% accuracy over hospitalization requirement compared to asymptomatic patients' fingerprints. The miniaturized microarray chip allows simultaneous determination of 96 samples (24 samples/slide) in 90 min and requires only 10 μL of sera. The diagnostic approach presented for the first time here could have a great value in assisting clinicians in decision-making based on the information provided by the Immuno-μSARS2 regarding progression of the disease and could be easily implemented in diagnostics of other infectious diseases.

Engineering of adeno-associated virus (AAV) capsids allowed for the development of gene therapy vectors with improved tropism and enhanced transduction efficiency. Capsid engineering can also be used to adapt the AAV technology for applications outside gene therapy. Here, we investigated modified AAV capsids as scaffolds for the presentation of large immunogenic antigens to elicit a strong and specific immune response against pathogens. Using SARS-CoV-2 as a model pathogen, we introduced ∼200 amino acids of the SARS-CoV-2 receptor-binding domain (RBD) into a surface-exposed variable loop region of AAV2 and AAV9, resulting in AAV2.RBD and AAV9.RBD capsids (AAV.RBDs). This engineering endowed AAV.RBDs with SARS-CoV-2-like properties, such as angiotensin-converting enzyme 2 receptor affinity. In line with this, AAV.RBDs were neutralized by sera from human donors vaccinated against SARS-CoV-2. When administered subcutaneously to rabbits, AAV.RBDs elicited a strong humoral response against SARS-CoV-2 RBD. Moreover, the AAV.RBDs were able to trigger RBD-specific cellular immune responses in peripheral human lymphocytes. In conclusion, this novel AAV-based next-generation vaccine platform allows for the presentation of large antigenic sequences to elicit strong and specific immune responses. This versatile vaccine technology could be explored in the context of diseases where conventional immunization approaches have been unsuccessful.

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7F's ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region, though concentration dependent. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of bivalent 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of bivalent 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses.

This study presents a novel computational approach for engineering nanobodies (Nbs) for improved interaction with receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Using Protein Structure Reliability reports, RBD (7VYR_R) was selected and refined for subsequent Nb-RBD interactions. By leveraging electrostatic complementarity (EC) analysis, we engineered and characterized five Electrostatically Complementary Nbs (ECSb1-ECSb5) based on the CeVICA library's SR6c3 Nb. Through targeted modifications in the complementarity-determining regions (CDR) and framework regions (FR), we optimized electrostatic interactions to improve binding affinity and specificity. The engineered Nbs (ECSb3, ECSb4, and ECSb5) demonstrated high binding specificity for AS3, CA1, and CA2 epitopes. Interestingly, ECSb1 and ECSb2 selectively engaged with AS3 and CA1 instead of AS1 and AS2, respectively, due to a preference for residues that conferred superior binding complementarities. Furthermore, ECSbs significantly outperformed SR6c3 Nb in MM/GBSA results, notably, ECSb4 and ECSb3 exhibited superior binding free energies of -182.58 kcal.mol-1 and -119.07 kcal.mol-1, respectively, compared to SR6c3 (-105.50 kcal.mol-1). ECSbs exhibited significantly higher thermostability (100.4-148.3 kcal·mol⁻1) compared to SR6c3 (62.6 kcal·mol⁻1). Similarly, enhanced electrostatic complementarity was also observed for ECSb4-RBD and ECSb3-RBD (0.305 and 0.390, respectively) relative to SR6c3-RBD (0.233). Surface analyses confirmed optimized electrostatic patches and reduced aggregation propensity in the engineered Nb. This integrated EC and structural engineering approach successfully developed engineered Nbs with enhanced binding specificity, increased thermostability, and reduced aggregation, laying the groundwork for novel therapeutic applications targeting the SARS-CoV-2 spike protein.

Pulmonary fibrosis due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the leading cause of death in patients with COVID-19. β-catenin, a key molecule in the Wnt/β-catenin signaling pathway, has been shown to be involved in the development of pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, silicosis). In this study, we developed a SARS-CoV-2-infected A549-hACE2 cell model to evaluate the efficacy of the A549-hACE2 monoclonal cell line against SARS-CoV-2 infection. The A549-hACE2 cells were then subjected to either knockdown or overexpression of the effector β-catenin, and the modified cells were subsequently infected with SARS-CoV-2. Additionally, we employed transcriptomics and raw letter analysis approaches to investigate other potential effects of β-catenin on SARS-CoV-2 infection. We successfully established a model of cellular fibrosis induced by SARS-CoV-2 infection in lung-derived cells. This model can be utilized to investigate the molecular biological mechanisms and cellular signaling pathways associated with virus-induced lung fibrosis. The results of our mechanistic studies indicate that β-catenin plays a significant role in lung fibrosis resulting from SARS-CoV-2 infection. Furthermore, the inhibition of β-catenin mitigated the accumulation of mesenchymal stroma in A549-hACE2 cells. Additionally, β-catenin knockdown was found to facilitate multi-pathway crosstalk following SARS-CoV-2 infection. The fact that β-catenin overexpression did not exacerbate cellular fibrosis may be attributed to the activation of PPP2R2B.

The COVID-19 pandemic has significantly impacted global healthcare systems, revealed vulnerabilities and prompted a re-evaluation of medical practices. Acute complications from the virus, including cardiovascular and neurological issues, have underscored the necessity for timely medical interventions. Advances in diagnostic methods and personalized therapies have been pivotal in mitigating severe outcomes. Additionally, Long COVID has emerged as a complex challenge, affecting various body systems and leading to respiratory, cardiovascular, neurological, psychological, and musculoskeletal problems. This broad spectrum of complications highlights the importance of multidisciplinary management approaches that prioritize therapy, rehabilitation, and patient-centered care. Vulnerable populations such as paediatric patients, pregnant women, and immunocompromised individuals face unique risks and complications, necessitating continuous monitoring and tailored management strategies to reduce morbidity and mortality associated with COVID-19.

Severe acute respiratory syndrome coronavirus (SARS-CoV), the virus responsible for COVID-19, interacts with the host immune system through complex mechanisms that significantly influence disease outcomes, affecting both innate and adaptive immunity. These interactions are crucial in determining the disease's severity and the host's ability to clear the virus. Given the virus's substantial socioeconomic impact, high morbidity and mortality rates, and public health importance, understanding these mechanisms is essential. This article examines the diverse innate immune responses triggered by SARS-CoV-2's structural proteins, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, along with nonstructural proteins (NSPs) and open reading frames. These proteins play pivotal roles in immune modulation, facilitating viral replication, evading immune detection, and contributing to severe inflammatory responses such as cytokine storms and acute respiratory distress syndrome (ARDS). The virus employs strategies like suppressing type I interferon production and disrupting key antiviral pathways, including MAVS, OAS-RNase-L, and PKR. This study also explores the immune pathways that govern the activation and suppression of immune responses throughout COVID-19. By analyzing immune sensing receptors and the responses initiated upon recognizing SARS-CoV-2 structural proteins, this review elucidates the complex pathways associated with the innate immune response in COVID-19. Understanding these mechanisms offers valuable insights for therapeutic interventions and informs public health strategies, contributing to a deeper understanding of COVID-19 immunopathogenesis.

The genome-based surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the past nearly 5 years since its emergence has refreshed our understanding of virus evolution, especially on convergent co-evolution with the host. SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations that affect the functional properties of the virus by altering its infectivity, virulence, transmissibility, and interactions with host immunity. This poses a huge challenge to global prevention and control measures based on drug treatment and vaccine application. As one of the key evasion strategies in response to the immune profile of the human population, there are overwhelming amounts of evidence for the reduced antibody neutralization of SARS-CoV-2 variants. Additionally, data also suggest that the levels of CD4+ and CD8+ T-cell responses against variants or sub-variants decrease in the populations, although non-negligible cross-T-cell responses are maintained. Herein, from the perspectives of structural immunology, we outline the characteristics and mechanisms of the T cell and antibody responses to SARS-CoV and its variants/sub-variants. The molecular bases for the impact of the immune escaping variants on the interaction of the epitopes with the key receptors in adaptive immunity, that is, major histocompatibility complex (MHC), T-cell receptor (TCR), and antibody are summarized and discussed, the knowledge of which will widen our understanding of this pandemic-threatening virus and assist the preparedness for Pathogen X in the future.

SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM has revealed stable prefusion and postfusion conformations of the spike, the transient intermediate states during the fusion process have remained poorly understood. Here, we designed a near-native viral fusion system that recapitulates SARS-CoV-2 entry and used cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that is dependent on protease cleavage and inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2' cleavage concurrently transition to postfusion conformations encircling the hemifusion and pre-fusion pores in a distinct conical arrangement. Subtomogram averaging revealed that the WS6 S2 antibody binds to the spike's stem-helix, crosslinks and clusters prefusion spikes and inhibits refolding of fusion intermediates. These findings elucidate the complete process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.

The SARS-CoV-2 Spike (S) protein plays a central role in viral entry into host cells, making it a key target for therapeutic interventions. Oxidative stress, often triggered during viral infections, can cause oxidation of cysteine in this protein. Here we investigate the impact of cysteine oxidation, specifically the formation of cysteic acid, on the conformational dynamics of the SARS-CoV-2 S protein using atomistic simulations. In particular, we examine how cysteine oxidation influences the transitions of the S protein's receptor-binding domain (RBD) between "down" (inaccessible) and "up" (accessible) states, which are critical for host cell receptor engagement. Using solvent-accessible surface area (SASA) analysis, we identify key cysteine residues susceptible to oxidation. The results of targeted molecular dynamics (TMD) and umbrella sampling (US) simulations reveal that oxidation reduces the energy barrier for RBD transitions by approximately 30 kJ mol-1, facilitating conformational changes and potentially enhancing viral infectivity. Furthermore, we analyze the interactions between oxidized cysteine residues and glycans, as well as alterations in hydrogen bonds and salt bridges. Our results show that oxidation disrupts normal RBD dynamics, influencing the energy landscape of conformational transitions. Our work provides novel insights into the role of cysteine oxidation in modulating the structural dynamics of the SARS-CoV-2 S protein, highlighting potential targets for antiviral strategies aimed at reducing oxidative stress or modifying post-translational changes. These findings contribute to a deeper understanding of viral infectivity and pathogenesis under oxidative conditions.

Affinity proteins based on a three-helix bundle (affibodies, alphabodies, and computationally de novo designed ones) have been shown to be a general platform to discover binders with properties reminiscent of antibodies, combining high target specificity with affinities reaching well below the nanomolar. Herein, we report a strategy, coined self-assembled proteomimetic (SAP), to mimic such three-helix bundle architecture with a hybridization-enforced two-helix coiled coil that is obtained by templated native chemical ligation (T-NCL) of PNA-peptide conjugates. This SAP strategy stands out by its synthetic accessibility, reducing the length on the longest synthetic peptide to less than 30 amino acids which is readily attainable by standard SPPS methodologies. We show that the T-NCL dramatically accelerates the ligation, enabling this chemistry to proceed in a combinatorial fashion at low micromolar concentrations. We demonstrate that small combinatorial libraries of SAPs can be prepared in one operation and used directly in affinity selections against a target of interest with an LC-MS analysis of the fittest binders. Moreover, we show that the underlying design paradigm is functional for SAPs based on structurally distinct three-helix peptides aimed at different therapeutic targets, namely HER2 and spike's RBD, reaching picomolar affinities. We further illustrate that the affinity of the SAP can be allosterically regulated using a toehold displacement of the hybridizing PNAs to disrupt the coiled coil stabilization. Finally, we show that an RBD-targeting SAP effectively inhibits viral entry of SARS-CoV-2 with an IC50 of 2.8 nM.

Needle-free injection system (NFIS) is easy to operate and can decrease needle phobia. Besides, NFIS can increase the interaction of antigens in a more dispersed manner with immune cell at local injection site, which may improve the immune responses of mRNA vaccines. Although SARS-CoV-2 mRNA vaccines have great success, universal vaccines are urgently needed. Delivering universal mRNA vaccines by NFIS is preferred to combat COVID-19.

RBD3-Fc mRNA expressing BA.4, Delta, and prototype RBD, and human IgG Fc with YTE mutation was designed and synthesized. The safety and immune responses of universal RBD3-Fc naked mRNA and mRNA-LNP vaccines delivered intradermally using NFIS (named GV-01) and intramuscularly via needles were evaluated and compared in rats.

The prime-boost regimen administered by two routes resulted in potent immune responses and intradermal delivery displays comparable or better performance in terms of binding antibodies, neutralizing antibodies and T cell responses. Naked mRNA vaccines were functional, but less effective than mRNA-LNP vaccines.

The above results suggest that RBD3-Fc vaccines are safe and immunogenic and NFIS can be used as an alternative to needles/syringes for the inoculation of mRNA-LNP vaccines to elicit robust systematic immune responses.

The rapid evolution of SARS-CoV-2 has led to the emergence of variants with increased immune evasion capabilities, posing significant challenges to antibody-based therapeutics and vaccines. In this study, we conducted a comprehensive structural and energetic analysis of SARS-CoV-2 spike receptor-binding domain (RBD) complexes with neutralizing antibodies from four distinct groups (A-D), including group A LY-CoV016, group B AZD8895 and REGN10933, group C LY-CoV555, and group D antibodies AZD1061, REGN10987, and LY-CoV1404. Using coarse-grained simplified simulation models, rapid energy-based mutational scanning, and rigorous MM-GBSA binding free energy calculations, we elucidated the molecular mechanisms of antibody binding and escape mechanisms, identified key binding hotspots, and explored the evolutionary strategies employed by the virus to evade neutralization. The residue-based decomposition analysis revealed energetic mechanisms and thermodynamic factors underlying the effect of mutations on antibody binding. The results demonstrate excellent qualitative agreement between the predicted binding hotspots and the latest experiments on antibody escape. These findings provide valuable insights into the molecular determinants of antibody binding and viral escape, highlighting the importance of targeting conserved epitopes and leveraging combination therapies to mitigate the risk of immune evasion.