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The SARS-CoV-2, responsible for the COVID-19 pandemic has wrecked devastation throughout the globe. The SARS-CoV-2 spike (S) glycoprotein plays crucial role in virus attachment, fusion, and entry. This study aims to identify inhibitors targeting the receptor binding domain (RBD) of the S protein using computational and experimental techniques. We carried out virtual screening of four datasets against the S-RBD. Six potential candidate inhibitors were selected for experimental evaluation. Here, we provide experimental evidence that the molecules 9‴-MethyllithosperMate, Epimedin A, Pentagalloylglucose, and Theaflavin-3-gallate have a high binding affinity towards SARS-CoV-2 S-RBD. 9‴-MethyllithosperMate with a K D value of 1.3 nM serves as the best inhibitor, followed by others with K D values in micromolar range. We performed molecular dynamics simulation to assess the binding stability of these inhibitors. Hence, our study reports novel inhibitors against the SARS-CoV-2 S-RBD and their predicted binding mode also suggest the possibility to interfere with the ACE2 binding.Communicated by Ramaswamy H. Sarma.

A fundamental problem in the field of protein evolutionary biology is determining the degree and nature of evolutionary relatedness among homologous proteins that have diverged to a point where they share less than 30% amino acid identity yet retain similar structures and/or functions. Such proteins are said to lie within the "Twilight Zone" of amino acid identity. Many researchers have leveraged experimentally determined structures in the quest to classify proteins in the Twilight Zone. Such endeavors can be highly time consuming and prohibitively expensive for large-scale analyses. Motivated by this problem, here we use molecular weight-hydrophobicity physicochemical dynamic time warping (MWHP DTW) to quantify similarity of simulated and real-world homologous protein domains. MWHP DTW is a physicochemical method requiring only the amino acid sequence to quantify similarity of related proteins and is particularly useful in determining similarity within the Twilight Zone due to its resilience to primary sequence substitution saturation. This is a step forward in determination of the relatedness among Twilight Zone proteins and most notably allows for the discrimination of random similarity and true homology in the 0%-20% identity range. This method was previously presented expeditiously just after the outbreak of COVID-19 because it was able to functionally cluster ACE2-binding betacoronavirus receptor binding domains (RBDs), a task that has been elusive using standard techniques. Here we show that one reason that MWHP DTW is an effective technique for comparisons within the Twilight Zone is because it can uncover hidden homology by exploiting physicochemical conservation, a problem that protein sequence alignment algorithms are inherently incapable of addressing within the Twilight Zone. Further, we present an extended definition of the Twilight Zone that incorporates the dynamic relationship between structural, physicochemical, and sequence-based metrics., (© 2024 Wiley Periodicals LLC.)

The omicron (B.1.19) variant of contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered a variant of concern (VOC) due to its increased transmissibility and highly infectious nature. The spike receptor-binding domain (RBD) is a hotspot of mutations and is regarded as a prominent target for screening drug candidates owing to its crucial role in viral entry and immune evasion. To date, no effective therapy or antivirals have been reported; therefore, there is an urgent need for rapid screening of antivirals. An extensive molecular modelling study has been performed with the primary goal to assess the inhibition potential of natural flavonoids as inhibitors against RBD from a manually curated library. Out of 40 natural flavonoids, five natural flavonoids, namely tomentin A (-8.7 kcal/mol), tomentin C (-8.6 kcal/mol), hyperoside (-8.4 kcal/mol), catechin gallate (-8.3 kcal/mol), and corylifol A (-8.2 kcal/mol), have been considered as the top-ranked compounds based on their binding affinity and molecular interaction profiling. The state-of-the-art molecular dynamics (MD) simulations of these top-ranked compounds in complex with RBD exhibited stable dynamics and structural compactness patterns on 200 nanoseconds. Additionally, complexes of these molecules demonstrated favorable free binding energies and affirmed the docking and simulation results. Moreover, the post-simulation validation of these interacted flavonoids using principal component analysis (PCA) revealed stable interaction patterns with RBD. The integrated results suggest that tomentin A, tomentin C, hyperoside, catechin gallate, and corylifol A might be effective against the emerging variants of SARS-CoV-2 and should be further evaluated using in-vitro and in-vivo experiments.Communicated by Ramaswamy H. Sarma.

The initial interaction between COVID-19 and the human body involves the receptor-binding domain (RBD) of the viral spike protein with the angiotensin-converting enzyme 2 (ACE2) receptor. Likewise, the spike protein can engage with immune-related proteins, such as toll-like receptors (TLRs) and pulmonary surfactant proteins A (SP-A) and D (SP-D), thereby triggering immune responses. In this study, we utilize computational methods to investigate the interactions between the spike protein and TLRs (specifically TLR2 and TLR4), as well as (SP-A) and (SP-D). The study is conducted on four variants of concern (VOC) to differentiate and identify common virus behaviours. An assessment of the structural stability of various variants indicates slight changes attributed to mutations, yet overall structural integrity remains preserved. Our findings reveal the spike protein's ability to bind with TLR4 and TLR2, prompting immune activation. In addition, our in-silico results reveal almost similar docking scores and therefore affinity for both ACE2-spike and TLR4-spike complexes. We demonstrate that even minor changes due to mutations in all variants, surfactant A and D proteins can function as inhibitors against the spike in all variants, hindering the ACE2-RBD interaction.Communicated by Ramaswamy H. Sarma.

The outbreak of COVID-19 coronavirus disease around the end of 2019 was a pandemic. The virus has been mutated and so many strains like Alpha, Beta, and Omicron are present in different parts of the world. Hence, timely detection technique is important to overcome the diagnostic challenges. Considering the need for this pandemic situation, we used a spectroscopy test methodology to distinguish different strains of Covid-19 by computing the infrared & Raman frequencies and the optical absorption data. Optimization of spike protein of Alpha, and Omicron mutations showed the high value of dipole moment (4.44, and 4.36) Debye, and polarizability [Alpha (233.62), Omicron (228.65)] indicating more bioactivity of Alpha and Omicron instead of Beta. Molecular electrostatic potential map exhibits the presence of electrophilic and nucleophilic region suggesting charge transfer of spikes to accept/donate electrons and hence the system increased reactiveness. UV-Vis absorption analysis also shows electronic transitions (σ to π∗ and π to π∗) due to that protein probe mechanism of Alpha and Omicron becomes increasingly become unsaturated thus confirming its easy binding ability to the target human protein as compared to binding affinity of Beta spike protein., Competing Interests: Declaration of competing interest ƴ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pathogen mutations present an inevitable and challenging problem for therapeutics and the development of mutation-tolerant anti-infective drugs to strengthen global health and combat evolving pathogens is urgently needed. While spike proteins on viral surfaces are attractive targets for preventing viral entry, they mutate frequently, making it difficult to develop effective therapeutics. Here, we used a structure-guided strategy to engineer an inhibitor peptide against the SARS-CoV-2 spike, called CeSPIACE, with mutation-tolerant and potent binding ability against all variants to enhance affinity for the invariant architecture of the receptor-binding domain (RBD). High-resolution structures of the peptide complexed with mutant RBDs revealed a mechanism of mutation-tolerant inhibition. CeSPIACE bound major mutant RBDs with picomolar affinity and inhibited infection by SARS-CoV-2 variants in VeroE6/TMPRSS2 cells (IC 50 4 pM to 13 nM) and demonstrated potent in vivo efficacy by inhalation administration in hamsters. Mutagenesis analyses to address mutation risks confirmed tolerance against existing and/or potential future mutations of the RBD. Our strategy of engineering mutation-tolerant inhibitors may be applicable to other infectious diseases., Competing Interests: Competing interests statement:Y.F. is the Director of CeSPIA Inc. S.N., A.K., and K.N. are members of CeSPIA Inc., Yes, the authors have stock ownership to disclose, Y.F. owns 73% of the $300,000 stock in CeSPIA Inc., Yes, the authors have patent filings to disclose, Y.F., S.N., Y.T., R.N., A.K., N.N., and Y.S. are co-inventors on provisional patents covering the anti-SARS-CoV-2 peptides described in this article.

The recent outbreak of the coronavirus (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has posed serious threats to global health systems. Although several directions have been put by the WHO for effective treatment, use of antibiotics, particularly ciprofloxacin, in suspected and acquired Covid-19 patients has raised an even more serious concern of antibiotic resistance. Ciprofloxacin has been reported to inhibit entry of SARS-CoV-2 into the host cells via interacting with the spike (S) protein. However, a proper structure-activity relationship study of ciprofloxacin with the S-protein is lacking, which inhibits researchers from developing a more potent fluoroquinolone analogue, specific for inhibition of SARS-CoV-2 viral entry. Herein, in order to have a structure-activity relationship study, we have accomplished a short and convergent synthesis of different derivatives of ciprofloxacin and a detailed in-silico study using molecular docking to explore the interactions of the derivatives with S-protein. The ADMET studies also indicated the drug likeliness and nontoxicity of the derivatives. Furthermore, the molecular dynamics simulation approach was used to study the dynamical behavior after the best docked derivative binds to the protein, and the MM-PBSA approach was adopted to calculate the binding energies. This has led to a derivative that has higher interactions with the S-protein compared to ciprofloxacin, without hampering the dynamics of the interactions. The strong affinity of compound 5 with the SARS-CoV-2 spike RBD protein was further evaluated experimentally using biolayer interferometry (BLI). Furthermore, molecular docking and molecular dynamics simulation were extended to evaluate its binding with the mutated variants Delta and Omicron. We anticipate that the current study could lead to an alternative therapeutic viral inhibitor with a better efficacy than ciprofloxacin.

Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics., (© The Author(s) 2025. Published by Oxford University Press.)

Passive antibody therapies, typically administered via parenteral routes, have played a crucial role in the initial response to the COVID-19 pandemic. However, the ongoing evolution of SARS-CoV-2 has revealed significant limitations of this approach, primarily due to mutational escape and the inadequate delivery of antibodies to the upper respiratory tract. To overcome these challenges, we propose a novel prophylactic strategy involving the intranasal delivery of an antibody in combination with an Fc-binding nanodisc. This nanodisc, engineered to specifically bind to the Fc regions of IgG antibodies, served two key functions: extending the antibody's half-life in the larynx and trachea, and enhancing its neutralization efficacy. Notably, Sotrovimab, an FDA-approved monoclonal antibody that has experienced a significant decline in neutralizing potency due to viral evolution, exhibited robust antiviral activity when complexed with the nanodisc against all tested Omicron variants. Furthermore, the Fc-binding nanodisc significantly boosted the antiviral efficacy of the soluble angiotensin-converting enzyme 2 (sACE2) Fc fusion protein, which possesses broad but modest antiviral activity. In ACE2 transgenic mice, the Fc-binding nanodisc protected better than sACE2-Fc alone with two more log reduction in lung viral titer. Therefore, the intranasal Fc-binding nanodisc offers a promising and powerful approach to counteract the diminished antiviral activity of neutralizing antibodies caused by mutational escape, effectively restoring antiviral efficacy against various evolving SARS-CoV-2 variants., Competing Interests: Declarations. Ethics approval and consent to participate: Ethics and Consent to Participate declarations: not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The emergence of mRNA vaccines offers great promise and a potent platform in combating various diseases, notably COVID-19. Nevertheless, challenges such as inherent instability and potential side effects of current delivery systems underscore the critical need for the advancement of stable, safe, and efficacious mRNA vaccines. In this study, a robust mRNA vaccine (cmRNA-1130) eliciting potent immune activation has been developed from a biodegradable lipid with eight ester bonds in the branched tail (AX4) and synthetic circular mRNA (cmRNA) encoding the trimeric Delta receptor binding domain of the SARS-CoV-2 spike protein. Notably, the cmRNA-1130 vaccine exhibits outstanding stability, remaining effective after six months of storage at 4 °C and multiple freeze-thaw cycles. In comparison with the commercial MC3 lipid, the nanoparticles formed from the degradable AX4 lipid revealed a much faster metabolic rate from the liver and spleen, affording negligible impairment to the hepatorenal function. Following intramuscular administration, cmRNA-1130 generates robust and sustained neutralizing antibodies and induces the activation of Delta RBD-specific CD4 + and CD8 + T effector memory cells (TEM) and Th1-biased T cells in mice. Featured with potent immune activation, high stability, and decent safety, vaccines formed from cmRNA and AX4 hold a huge clinical potential for the prophylaxis and treatment of different diseases.

In this study, the vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed using ferritin complexes with the immunogenic sequences screened against the SARS-CoV-2 spike protein (SP) from the Fv-antibody library. The Fv-antibody library was prepared on the outer membrane of E. coli by the expression of the V H region of immunoglobulin G (IgG) with a randomized complementarity-determining region 3 (CDR3). Four Fv-antibodies to the receptor-binding domain (RBD) were screened from the Fv-antibody library, which had a comparable binding constant ( K D ) between SARS-CoV-2 SP and the angiotensin-converting enzyme 2 (ACE2) receptor. The binding sites of screened Fv-antibodies on the RBD were analyzed using a docking analysis, and these binding sites were used as immunogenic sequences for the vaccine. The four immunogenic sequences were modified and co-expressed as a part of ferritin which was assembled into a ferritin complex. After the vaccination of ferritin complexes to mice, the anti-sera were analyzed to have a high enough titer. Additionally, the immune responses were found to be activated by vaccination, such as the expression of IgG subclasses and the increased level of cytokines. The neutralizing activity of the anti-sera was estimated using a cell-based infection assay based on pseudo-virus expressing the SP of SARS-CoV-2 variants.

Predicting novel mutations has long-lasting impacts on life science research. Traditionally, this problem is addressed through wet-lab experiments, which are often expensive and time consuming. The recent advancement in neural language models has provided stunning results in modeling and deciphering sequences. In this paper, we propose a Deep Novel Mutation Search (DNMS) method, using deep neural networks, to model protein sequence for mutation prediction. We use SARS-CoV-2 spike protein as the target and use a protein language model to predict novel mutations. Different from existing research which is often limited to mutating the reference sequence for prediction, we propose a parent-child mutation prediction paradigm where a parent sequence is modeled for mutation prediction. Because mutations introduce changing context to the underlying sequence, DNMS models three aspects of the protein sequences: semantic changes, grammatical changes, and attention changes, each modeling protein sequence aspects from shifting of semantics, grammar coherence, and amino-acid interactions in latent space. A ranking approach is proposed to combine all three aspects to capture mutations demonstrating evolving traits, in accordance with real-world SARS-CoV-2 spike protein sequence evolution. DNMS can be adopted for an early warning variant detection system, creating public health awareness of future SARS-CoV-2 mutations., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has imposed substantial challenges on our society due to the COVID-19 pandemic. This virus relies heavily on its surface glycoprotein (S-glycoprotein) to facilitate attachment, fusion, and entry into host cells. While the nucleoprotein (N) in the ribonucleoprotein core binds to the viral RNA genome. Therefore, our objective is to develop a novel vaccine candidate targeting the dominant T-cell and B-cell epitopes of the immune system. On the S-glycoprotein and nucleoprotein. Employing an immunoinformatic approach, we constructed a vaccine candidate with 13 highly antigenic B-cell epitopes, 19 HTL antigenic epitopes, and 18 CTL epitopes following a rigorous assessment. The multi-epitope construct successfully passed three-fold toxicity, allergenicity, and antigenicity tests, affirming its non-toxic, non-allergenic, and antigenic nature. This demonstrates the potentiality of the vaccine design to trigger an immunological response. Furthermore, the vaccine-ACE-2 receptor complex was tested, confirming its ability to interact with ACE-2's core pocket and induce an immunological response. Additionally, the vaccine's binding prowess for human toll-like receptors (TLR) (1, 3, 4, and 8) was investigated. According to the Ramachandran plot, 77.46% of the construct's amino acid residues fall within a favorable zone, establishing it as a viable vaccine candidate., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: This is an observable study; the laboratory research ethics committee has confirmed that no ethical approval is required., (© 2024. The Author(s).)

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors.

SARS-CoV-2 viral entry requires membrane fusion, which is facilitated by the fusion peptides within its spike protein. These predominantly hydrophobic peptides insert into target membranes; however, their precise mechanistic role in membrane fusion remains incompletely understood. Here, we investigate how FP1 (SFIEDLLFNKVTLADAGFIK), the N-terminal fusion peptide, modulates membrane stability and barrier function across various model membrane systems. Through a complementary suite of biophysical techniques-including electrophysiology, fluorescence spectroscopy, and atomic force microscopy-we demonstrate that FP1 significantly promotes pore formation and alters the membrane's mechanical properties. Our findings reveal that FP1 reduces the energy barrier for membrane defect formation and stimulates the appearance of stable conducting pores, with effects modulated by membrane composition and mechanical stress. The observed membrane-destabilizing activity suggests that, beyond its anchoring function, FP1 may facilitate viral fusion by locally disrupting membrane integrity. These results provide mechanistic insights into SARS-CoV-2 membrane fusion mechanisms and highlight the complex interplay between fusion peptides and target membranes during viral entry.

Electrochemical biosensors hold great promise for the rapid screening of viral infectious diseases. However, the recognition elements of these biosensors are typically limited to antibodies, aptamers, and molecularly imprinted polymers. In this study, acetylated sialic acids were explored as recognition elements because they serve as natural viral receptors expressed on host cells. Specifically, 4-O-acetylated-SA (4-O-Ac-SA) and 9-O-Ac-SA, were synthesized selectively, and their binding affinity with the SARS-CoV-2 S antigen was examined. The S antigen tended to bind to 9-O-Ac-SA. Additionally, the biocompatibility and neutralizing effects of 4/9-O-Ac-SA on the S antigen were validated. The validation demonstrated that 9-O-Ac-SA could efficiently inhibit S antigen binding to host cells. The cluster glycoside effect of the recognition between the S antigen and 9-O-Ac-SA was validated. Subsequently, an electrochemical biosensor for the rapid screening of viral antigens was developed using 9-O-Ac-SA as the recognition element. The application of electrochemical impedance spectroscopy as a readout method allowed for the identification of the S antigen at concentrations of 10 ng/mL with acceptable stability and repeatability. The biosensor demonstrated a strong linear response over the range of 10∼1 × 10 4  ng/mL. In summary, the study presented a promising recognition element for the development of electrochemical biosensors for rapid viral infection screening. The utilization of glycans for viral antigen detection could pave the way for innovative advances in electrochemical biosensor technology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An on-going challenge with COVID-19, which has huge implications for future pandemics, is the rapid emergence of viral variants that makes diagnostic tools less accurate, calling for rapid identification of recognition elements for detecting new variants caused by mutations. We hypothesize that we can fight mutations of the viruses with mutations of existing recognition elements. We demonstrate this concept via rapidly evolving an existing DNA aptamer originally selected for the spike protein (S-protein) of wildtype SARS-CoV-2 to enhance the interaction with the same protein of the Omicron variants. The new aptamer, MBA5SA1, has acquired 22 mutations within its 40-nucleotide core sequence and improved its binding affinity for the S-proteins of diverse Omicron subvariants by >100-fold compared to its parental aptamer (improved from nanomolar to picomolar affinity). Deep sequencing analysis reveals dynamic competitions among several MBA5SA1 variants in response to increasing selection pressure imposed during in vitro selection, with MBA5SA1 being the final winner of the competition. Additionally, MBA5SA1 was implemented into an enzyme-linked aptamer binding assay (ELABA), which was applied for detecting Omicron variants in the saliva of infected patients. The assay produced a sensitivity of 86.5 % and a specificity of 100 %, which were established with 83 clinical samples., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The COVID-19 pandemic has caused over 7 million deaths globally in the past four years. Siparuna spp. (Siparunaceae), which is used in Brazilian folk medicine, is considered a genus with potential antiviral alternatives. This study explored the correlation between phytochemicals in Siparuna leaf extracts ( S. ficoides , S. decipiens , S. glycycarpa , S. reginae , and S. cymosa ) and their potential against various SARS-CoV-2 targets. In vitro assays examined interactions between the spike protein and the ACE2 receptor, protease activity, and viral replication inhibition in Calu-3 cell models. UHPLC-MS/MS analysis, processed with MZmine and evaluated chemometrically, revealed isoquinoline alkaloids with bulbocapnine, showing promising therapeutic potential. Predictions regarding absorption, distribution, metabolism, excretion, and toxicity were conducted, along with molecular docking and dynamics simulations, to evaluate protein-ligand interaction stability. The results confirmed the antiviral activity of the Siparuna genus against SARS-CoV-2 targets, with 92% of the extracts maintaining over 70% cellular viability at 200 μg·mL -1 and 80% achieving more than 50% viral activity suppression at 50 μg·mL -1 . These findings highlight the potential of isoquinoline alkaloids as novel anti-coronavirus agents and support the need for further exploration, isolation, and testing of Siparuna compounds in the fight against COVID-19.

The global spread of Severe Acute Respiratory Syndrome Coronavirus 2. (SARS-CoV-2) and its variant strains, including Alpha, Beta, Gamma, Delta, and now Omicron, pose a significant challenge. With the constant evolution of the virus, Omicron and its subtypes BA.1, BA.2, BA.3, BA.4, and BA.5 have developed the capacity to evade neutralization induced by previous vaccination or infection. This evasion highlights the urgency in discovering new monoclonal antibodies (mAbs) with neutralizing activity, especially broadly neutralizing antibodies (bnAbs), to combat the virus.In the current study, we introduced a fully human neutralizing mAb, CR9, that targets Omicron variants. We demonstrated the mAb's effectiveness in inhibiting Omicron replication both in vitro and in vivo. Structural analysis using cryo-electron microscopy (cryo-EM) revealed that CR9 binds to an epitope formed by RBD residues, providing a molecular understanding of its neutralization mechanism. Given its potency and specificity, CR9 holds promise as a potential adjunct therapy for treating Omicron infections. Our findings highlight the importance of continuous mAb discovery and characterization in addressing the evolving threat of COVID-19., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Previous works show the key role of electrostatics in the SARS-CoV-2 virus in aspects such as virus-cell interactions or virus inactivation by ionic surfactants. Electrostatic interactions depend strongly on the variant since the charge of the Spike protein (responsible for virus-environment interactions) evolved across the variants from the highly negative Wild Type (WT) to the highly positive Omicron variant. The distribution of the charge also evolved from diffuse to highly localized. These facts suggest that SARS-CoV-2 should interact strongly with charged surfaces in a way that changed during the virus evolution. This question is studied here by computing the electrostatic interaction between WT, Delta and Omicron Spike proteins with charged surfaces using a new method (based on Debye-Hückel theory) that provides efficiently general results as a function of the surface charge density σ. We found that the interaction of the WT and Delta variant spikes with charged surfaces is dominated by repulsive image forces proportional to σ 2 originating at the protein/water interface. On the contrary, the Omicron variant shows a distinct behavior, being strongly attracted to negatively charged surfaces and repelled from positively charged ones. Therefore, the SARS-CoV-2 virus has evolved from being repelled by charged surfaces to being efficiently adsorbing to negatively charged ones.

Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with Escherichia coli by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of E. coli cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by E. coli retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, E. coli cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones.

The purification of large biotherapeutic modalities such as viral coat proteins, plasmid DNA, mRNA, therapeutic viruses and vesicles is more challenging than the purification of smaller and more established products such as monoclonal antibodies. This is because these entities, due to their large size, have limited access to binding sites present in the pores of conventional resin-based chromatographic media. However, this transport limitation could potentially be exploited for their purification using size exclusion chromatography (SEC). Here, the strategy is to isolate these in the void fraction of an appropriate SEC column. However, challenges such as low capacity, low productivity and poor scalability typically associated with SEC would first need to be addressed. In this study, we propose an alternating flow-direction-based SEC technique as an approach for increasing the productivity of preparative SEC. The feed is introduced into the SEC device from opposite directions in an alternating manner. By doing so, the separation time could be significantly reduced. Proof of concept for this technique was obtained using a z 2 cuboid SEC device, having a volume of 24 mL, and packed with Sephacryl S 200 resin. The effect of alternating flow direction on the separation time was examined based on a case study for the purification SARS-CoV-2 delta spike protein from small molecular weight impurities present in cell-free supernatant. Compared to conventional unidirectional SEC, the time (or volume of mobile phase) required for purifying the spike protein could be reduced by about 42 %., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Messenger RNA (mRNA) vaccines against COVID-19 have demonstrated high efficacy and rapid deployment capability to target emerging infectious diseases. However, the need for ultra-low temperature storage made the distribution of LNP/mRNA vaccines to regions with limited resources impractical. This study explores the use of lyophilization to enhance the stability of self-replicating mRNA (repRNA) vaccines, allowing for their storage at non-freezing temperatures such as 2-8 °C or room temperature (25 °C). We lyophilized repRNA molecules complexed to a novel cationic emulsion delivery system, LION™, with different sugar-based lyoprotectants to identify candidates that provided the best vaccine integrity and effectiveness after being thermally stressed. For screening, we used repRNA encoding the reporter protein secreted embryonic alkaline phosphatase (SEAP) and for proof-of-concept, we used repRNA vaccines encoding SARS-CoV-2 full-length spike (WA-1 isolate) or full-length surface protein circumsporozoite (CS) of Plasmodium yoelii (Py). We found that lyophilization of LION/repRNA with sucrose provided the best colloidal stability, preserved in vitro expression, and induced equivalent antigen-specific antibody responses in mice compared to freshly prepared liquid LION/repRNA. Furthermore, lyophilized vaccines were stable for at least one week at 25 °C and at least one year at 2-8 °C. The cumulative analysis of stability-determining physicochemical data, in vitro potency, and in vivo immunogenicity in mice enabled the selection of a lead lyophilized composition containing 10 % w/v sucrose as the lyoprotectant. The data presented here provide a foundation for the clinical evaluation of next-generation thermostable repRNA vaccines that will enable more equitable vaccine access globally., Competing Interests: Declaration of competing interest AK, AS, JE, SR are co-inventors on patent applications related to the LION materials described in this work. GG, AS, KK, SS, SR, and AK have equity interests in HDT Bio. MA, ZM and JD have equity interests in MalarVx, Inc., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Addressing the frequent emergence of SARS-CoV-2 mutant strains requires therapeutic approaches with innovative neutralization mechanisms. The targeting of multivalent nanobodies can enhance potency and reduce the risk of viral escape, positioning them as promising drug candidates. Here, the synergistic mechanisms of the two types of nanobodies are investigated deeply. Our research revealed that the Fu2-1-Fu2-2 system exhibited significant synergy, whereas the Sb#15-Sb#68 system demonstrated antagonism, in which entropy was the dominant contributor to antagonism. Conformational analysis further demonstrated that the presence of a monomeric nanobody influenced the flexibility of residues near other epitopes, thereby affecting the overall synergy of the systems. Moreover, we identified that changes in the hydrogen bond network and the charge of residues played a critical role in the binding between nanobodies and spike. We hope this study will provide novel insights into the development of multivalent nanobody combinations.

The homotrimeric SARS-CoV-2 spike protein enables viral infection by undergoing a large conformational transition, which facilitates the fusion of the viral envelope with the host cell membrane. The spike protein is anchored to the SARS-CoV-2 envelope by its transmembrane domain (TMD), composed of three TM helices, each contributed by one of the protomers of spike. Although the TMD is known to be important for viral fusion, whether it is a passive anchor of the spike or actively promotes fusion remains unknown. Specifically, it is unclear if the TMD and its dynamics facilitate the prefusion to postfusion conformational transition of the spike. Here, we computationally study the dynamics and self-assembly of the SARS-CoV-2 spike TMD in homogeneous POPC and cholesterol containing membranes. Atomistic simulations of a long TM helix-containing protomer segment show that the membrane-embedded segment bobs, tilts and gains and loses helicity, locally thinning the membrane. Coarse-grained multimerization simulations using representative TM helix structures from the atomistic simulations exhibit diverse trimer populations whose architecture depends on the structure of the TM helix protomer. While a symmetric conformation reflects the symmetry of the resting spike, an asymmetric TMD conformation could promote membrane fusion through the stabilization of a fusion intermediate. Together, our simulations demonstrate that the sequence and length of the SARS-CoV-2 spike TM segment make it inherently dynamic, that trimerization does not abrogate these dynamics and that the various observed TMD conformations may enable viral fusion.

Background: Although immunoglobulin (Ig) alleles play a pivotal role in the antibody response to pathogens, research to understand their role in the humoral immune response is still limited., Methods: We retrieved the germline sequences for the IGHV from the IMGT database to illustrate the amino acid polymorphism present within germline sequences of IGHV genes. We aassembled the sequences of IgM and IgD repertoire from 130 people to investigate the genetic variations in the population. A dataset comprising 10,643 SARS-CoV-2 spike-specific antibodies, obtained from COV-AbDab, was compiled to assess the impact of SARS-CoV-2 infection on allelic gene utilization. Binding affinity and neutralizing activity were determined using bio-layer interferometry and pseudovirus neutralization assays. Primary docking was performed using ZDOCK (3.0.2) to generate the initial conformation of the antigen-antibody complex, followed by simulations of the complete conformations using Rosetta SnugDock software. The original and simulated structural conformations were visualized and presented using ChimeraX (v1.5)., Results: We present an allelic atlas of immunoglobulin heavy chain (IgH) variable regions, illustrating the diversity of allelic variants across 33 IGHV family germline sequences by sequencing the IgH repertoire of in the population. Our comprehensive analysis of SARS-CoV-2 spike-specific antibodies revealed the preferential use of specific Ig alleles among these antibodies. We observed an association between Ig alleles and antibody binding epitopes. Different allelic genotypes binding to the same RBD epitope on the spike show different neutralizing potency and breadth. We found that antibodies carrying the IGHV1-69*02 allele tended to bind to the RBD E2.2 epitope. The antibodies carrying G50 and L55 amino acid residues exhibit potential enhancements in binding affinity and neutralizing potency to SARS-CoV-2 variants containing the L452R mutation on RBD, whereas R50 and F55 amino acid residues tend to have reduced binding affinity and neutralizing potency. IGHV2-5*02 antibodies using the D56 allele bind to the RBD D2 epitope with greater binding and neutralizing potency due to the interaction between D56 on HCDR2 and K444 on RBD of most Omicron subvariants. In contrast, IGHV2-5*01 antibodies using the N56 allele show increased binding resistance to the K444T mutation on RBD., Discussion: This study provides valuable insights into humoral immune responses from the perspective of Ig alleles and population genetics. These findings underscore the importance of Ig alleles in vaccine design and therapeutic antibody development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Deng, Niu, He, Yan, Liang, Wang, Ning, Lin, Zhang, Zhao, Feng, Qu and Chen.)

The emergence of SARS-CoV-2 variants of concern (VOCs) has greatly diminished the neutralizing activity of previously FDA-approved monoclonal antibodies (mAbs), including that of antibody cocktails and of first-generation broadly neutralizing antibodies such as S309 (Sotrovimab). In contrast, antibodies targeting cryptic conformational epitopes of the receptor binding domain (RBD) have demonstrated broad activity against emerging variants, but exert only moderate neutralizing activity, which has so far hindered clinical development. Here, we utilize in vitro display technology to identify and affinity-mature antibodies targeting the cryptic class 6 epitope, accessible only in the "up" conformation of the SARS-CoV-2 spike trimer. Increasing antibody affinity into the low picomolar range endowed potent neutralization of VOCs and protection of hACE2 mice from viral challenge. Cryoelectron microscopy and crystal structures of two affinity-matured antibodies (4C12-B12 and 4G1-C2) in complex with RBD highlighted binding modes and epitopes distal from mutational hotspots commonly overserved in VOCs, providing direct structural insights into the observed mutational resistance. Moreover, we further demonstrate that antibodies targeting the class 6 epitope, rather than being an artifact of in vitro selection, are common in the IgG1 + memory B cell repertoire of convalescent patients and can be induced in human antibody V-gene transgenic mice through immunization. Our results highlight the importance of very high (picomolar) affinity in the development of neutralizing antibodies and vaccines and suggest an affinity threshold in the provision of broad and long-lasting immunity against SARS-CoV-2., Competing Interests: Competing interests statement:O.M., D.B.L., P.S., and D.C. are named inventors on patent application WO2022133545A1 and declare competing interests.

Severe COVID-19 can trigger a cytokine storm, leading to acute respiratory distress syndrome (ARDS) with similarities to superantigen-induced toxic shock syndrome. An outstanding question is whether SARS-CoV-2 protein sequences can directly induce inflammatory responses. In this study, we identify a region in the SARS-CoV-2 S2 spike protein with sequence homology to bacterial super-antigens (termed P3). Computational modeling predicts P3 binding to sites on MHC class I/II and the TCR that partially overlap with sites for the binding of staphylococcal enterotoxins B and H. Like SEB and SEH derived peptides, P3 stimulated 25-40% of human CD4+ and CD8 + T-cells, increasing IFN-γ and granzyme B production. viSNE and SPADE profiling identified overlapping and distinct IFN-γ+ and GZMB+ subsets. The super-antigenic properties of P3 were further evident by its selective expansion of T-cells expressing specific TCR Vα and Vβ chain repertoires. In vivo experiments in mice revealed that the administration of P3 led to a significant upregulation of proinflammatory cytokines IL-1β, IL-6, and TNF-α. While the clinical significance of P3 in COVID-19 remains unclear, its homology to other mammalian proteins suggests a potential role for this peptide family in human inflammation and autoimmunity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Prediction of protein fitness from computational modeling is an area of active research in rational protein design. Here, we investigated whether protein fluctuations computed from molecular dynamics simulations can be used to predict the expression levels of SARS-CoV-2 receptor binding domain (RBD) mutants determined in the deep mutational scanning experiment of Starr et al. [Science (New York, N.Y.) 2022, 377, 420] Specifically, we performed more than 0.7 milliseconds of molecular dynamics (MD) simulations of 557 mutant RBDs in triplicate to achieve statistical significance under various simulation conditions. Our results show modest but significant anticorrelation in the range [-0.4, -0.3] between expression and RBD protein flexibility. A simple linear regression machine learning model achieved correlation coefficients in the range [0.7, 0.8], thus outperforming MD-based models, but required about 25 mutations at each residue position for training., (© 2024 Wiley Periodicals LLC.)

SARS-CoV-2 has continued spreading around the world in recent years since the initial outbreak in 2019, frequently developing into new variants with greater human infectious capacity. SARS-CoV-2 and its mutants use the angiotensin-converting enzyme 2 (ACE2) as a cellular entry receptor, which has triggered several therapeutic strategies against COVID-19 relying on the use of ACE2 recombinant proteins as decoy receptors. In this work, we propose an ACE2 silent Fc fusion protein (ACE2-hFcLALA) as a candidate therapy against COVID-19. This fusion protein was able to block the binding of SARS-CoV-2 RBD to ACE2 receptor as measured by ELISA and flow cytometry inhibition assays. Moreover, we used classical neutralization assays and a progeny neutralization assay to show that the ACE2-hFcLALA fusion protein is capable of neutralizing the authentic virus. Additionally, we found that this fusion protein was more effective in preventing in vitro infection with different variants of interest (alpha, beta, delta, and omicron) compared to the D614G strain. Our results suggest the potential of this molecule to be used in both therapeutic and preventive settings against current and emerging mutants that use ACE2 as a gateway to human cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Bermúdez-Abreut et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The SARS-CoV-2 spike protein's membrane-binding domain bridges the viral and host cell membrane, a critical step in triggering membrane fusion. Here, we investigate how the SARS-CoV-2 spike protein interacts with host cell membranes, focusing on a membrane-binding peptide (MBP) located near the TMPRSS2 cleavage site. Through in vitro and computational studies, we examine both primed (TMPRSS2-cleaved) and unprimed versions of the MBP, as well as the influence of its conserved disulfide bridge on membrane binding. Our results show that the MBP preferentially associates with cholesterol-rich membranes, and we find that cholesterol depletion significantly reduces viral infectivity. Furthermore, we observe that the disulfide bridge stabilizes the MBP's interaction with the membrane, suggesting a structural role in viral entry. Together, these findings highlight the importance of membrane composition and peptide structure in SARS-CoV-2 infectivity and suggest that targeting the disulfide bridge could provide a therapeutic strategy against infection., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Key functions of antibodies, such as viral neutralisation, depend on high-affinity binding. However, viral neutralisation poorly correlates with antigen affinity for reasons that have been unclear. Here, we use a new mechanistic model of bivalent binding to study  >45 patient-isolated IgG1 antibodies interacting with SARS-CoV-2 RBD surfaces. The model provides the standard monovalent affinity/kinetics and new bivalent parameters, including the molecular reach: the maximum antigen separation enabling bivalent binding. We find large variations in these parameters across antibodies, including reach variations (22-46 nm) that exceed the physical antibody size (~15 nm). By using antigens of different physical sizes, we show that these large molecular reaches are the result of both the antibody and antigen sizes. Although viral neutralisation correlates poorly with affinity, a striking correlation is observed with molecular reach. Indeed, the molecular reach explains differences in neutralisation for antibodies binding with the same affinity to the same RBD-epitope. Thus, antibodies within an isotype class binding the same antigen can display differences in molecular reach, substantially modulating their binding and functional properties., Competing Interests: Competing interests: G.R.S. is on the GSK Vaccines Scientific Advisory Board, a founder shareholder of RQ Biotechnology, and a Jenner investigator. Oxford University holds intellectual property related to the Oxford-AstraZeneca vaccine. The remaining authors declare no competing interests., (© 2025. The Author(s).)

The ongoing global pandemic caused by the SARS-CoV-2 virus has demanded the urgent search for effective therapeutic interventions. In response, our research aimed at identifying natural products (NPs) with potential inhibitory effects on the entry of the SARS-CoV-2 spike (S) protein into host cells. Utilizing the Protein Data Bank Japan (PDBJ) and BindingDB databases, we isolated 204 S-glycoprotein sequences and conducted a clustering analysis to identify similarities and differences among them. We subsequently identified 33,722 binding molecules (BMs) by matching them with the sequences of 204 S-glycoproteins and compared them with 52,107 secondary metabolites (SMs) from the KNApSAcK database to identify potential inhibitors. We conducted docking and drug-likeness property analyses to identify several SMs with potential as drug candidates based on binding energy (BE), no Lipinski's rule violation (LV), psychochemical properties within the pink area of the bioavailability radar, and a bioavailability score (BAS) not less than 0.55. Fourteen SMs were predicted through computational analysis as potential candidates for inhibiting the three major types of S proteins. Our study provides a foundation for further experimental validation of these compounds as potential therapeutic agents against SARS-CoV-2., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

One of the most important and challenging biological events of recent times has been the pandemic caused by SARS-CoV-2. Since the underpinning argument behind this book is the ubiquity of electrical forces driving multiple disparate biological events, consideration of key aspects of the SARS-CoV-2 structural proteins is included. Electrical regulation of spike protein, nucleocapsid protein, membrane protein, and envelope protein is included, with several of their activities regulated by LLPS and the multivalent and π-cation and π-π electrical forces that drive phase separation., (© 2025. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Low-cost and safe vaccines are needed to fill the vaccine inequity gap for future pandemics. Pichia pastoris is an ideal expression system for recombinant protein production due to its cost-effective and easy-to-scale-up process. Here, we developed a next-generation SARS-CoV2 Omicron BA.1-based recombinant vaccine candidate expressed in P. pastoris. The receptor binding domain of Omicron BA.1 spike protein (RBD-Omicron) was produced at 0.35 g/L in supernatant. With a 60% recovery after two-step purification, RBD-Omicron showed 99% purity. After in vitro characterisation of purified RBD-Omicron via chromatography, mass spectrometry, calorimetry and surface plasmon resonance-based methods, it was injected into mice for immunization studies. Three different doses of Alum and CpG adjuvanted RBD-Omicron were investigated and 10 μg RBD-Omicron gave the highest antigenicity. After two doses of vaccination, IgG titers in mice serum reached to more than 10 6 . These serum antibodies also recognized earlier (Delta Plus: B.1.617.2) and later (Eris: EG.5, Pirola: BA.2.86) SARS-CoV2 variants. The long-term immunological response in mice was measured by analyzing serum antibody titers and T-cell response of splenocytes after 60 weeks. Interestingly, IgG titers and Th1 response were significantly high even after a year. Omicron subvariants are dominantly circulating in the world, so Omicron sub-lineage-based vaccines can be used for future pandemics. The RBD-Omicron-based vaccine candidate developed in this study is suitable for technology transfer and transition into the clinic., (© 2025 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The COVID-19 epidemic and its continuous spread pose a serious threat to public health. Coronavirus strains known as SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) variants have undergone genomic changes. The severity of the symptoms, the efficiency of vaccinations, and the transmission capacity of the virus can be impacted by these alterations. Point-of-care diagnostic assays can identify particular genetic or protein sequences that are exclusive to each variety. Currently, ultrafast, responsive, and accurate antibody detection faces several challenges. Here, we outline the fabrication, implementation, and sensing performance benchmarking of an ultrafast (5 s) and inexpensive (0.15 USD) assay with label-free sensing of SARS-CoV-2 S (Spike)/N (Nucleocapsid) protein and other variants in real patient samples. A label-free DNA aptameric capacitive bio-sensing device was used to detect SARS-CoV-2 variants. Our novel, cutting-edge bio-sensing device contains a Wooden quoits conformation structural aptamer (WQCSA)-based inter-digitated capacitor electronic (WQCSA-IDCE) system. WQCSA-aptamer was used as a switch-turn on response to achieve ultrasensitivity in the variable area of the SARS-CoV-2. The molecular beacon (MB) method was also used to measure the fluorescently colored SARS-CoV-2 S/N protein. These sensors can be used with several types of label-free DNA aptamers to act as rapid, affordable, and label-free biosensors for a variety of critical acute respiratory virus syndrome disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) transduction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer-associated fluorophore methylene blue (MB). We observed a five and seven-times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon-enhanced fluorescence, and the development of an advanced but universal aptasensor platform., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Herein, we present the first experimental study of individual water-soluble fullerene derivatives proving their ability to inhibit SARS-CoV-2 in vitro. The initial screening allowed us to identify a few new compounds that have demonstrated pronounced antiviral activity with IC 50 values as low as 390 nM and selectivity indexes reaching 214. Time-of-addition analysis and molecular docking results suggested that the viral protease and/or the spike protein are the most probable targets inhibited by the fullerene derivatives. Further rational design of fullerene derivatives might lead to the development of compounds with further enhanced antiviral activity and decreased toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic is casting a long shadow, and the appearance of the JN.1 variety calls attention to the necessity of maintaining heightened awareness. It considers the strength that has been developed via immunization programs and the necessity of global collaboration to find a solution in light of the emergence of new strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Phylogenetically, the SARS-CoV-2 Omicron XBB lineages, which include EG.5.1 and HK.3, are different from the SARS-CoV-2 BA.2.86 lineage, which was initially discovered in August 2023. More than 30 mutations in the spike (S) protein are carried by BA.2.86 compared to XBB and BA.2, suggesting a high potential for immune evasion. JN.1 (BA.2.86.1.1), appeared in late 2023 after the format had undergone evolution. JN.1 carries three mutations in proteins that do not include S, as well as S: L455S. As previously demonstrated, the HK.3 and other "FLip" variations possess the S: L455F mutation, which enhances transmissibility and immune escape capacity in comparison to the parental EG.5.1 variety. This mutation is a characteristic of JN.1. The COVID-19 virus is dynamic and evolves over time. New varieties can sometimes spread more quickly or effectively after these alterations. If that happens, the new variant has a chance to outpace the current varieties in terms of frequency., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The continuous evolution of SARS-CoV-2, particularly the emergence of the BA.2.86/JN.1 lineage replacing XBB, necessitates re-evaluation of vaccine compositions 1-3 . Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposure. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals and show that infection with JN.1 elicits superior plasma neutralization against its subvariants. We highlight the strong immune evasion and receptor-binding capability of KP.3, supporting its foreseeable prevalence. Extensive analysis of the B cell receptor repertoire, in which we isolate approximately 2,000 receptor-binding-domain-specific antibodies, with targeting epitopes characterized by deep mutational scanning, underscores the superiority of JN.1-elicited memory B cells 4,5 . Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) are important contributors to the wild-type reactivity of NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset of these antibodies, even those induced by JN.1, supporting a need for booster updates. JN.1-induced Omicron-specific antibodies also demonstrate high potency across Omicron. Escape hotspots for these NAbs have already been mutated, resulting in a higher immune barrier to escape and indicating probable recovery of escaped NAbs. In addition, the prevalence of IGHV3-53/3-66-derived antibodies and their ability to compete with all Omicron-specific NAbs suggests that they have an inhibitory effect on the activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals 6-8 . These findings delineate the evolving antibody response to the antigenic shift of Omicron from XBB to JN.1 and highlight the importance of developing the JN.1 lineage, especially KP.2- and KP.3-based vaccine boosters., Competing Interests: Competing interests: Y.C. is listed as an inventor of provisional patent applications of SARS-CoV-2 RBD-specific antibodies. Y.C. is a cofounder of Singlomics Biopharmaceuticals. The remaining authors declare no competing interests., (© 2024. The Author(s).)

The research aims to identify and characterize the antibody escape mutations of NTD and RBD regions of SARS-CoV-2 using prompt engineering-enabled combined LLMs (large language models) and instigative bioinformatics techniques. We used two LLMs (ChatGPT and Mistral 7B) and one MLLM (Gemini model) to retrieve the significant NTD and RBD mutations. The retrieved significant mutations were characterized through the in silico servers. The retrieved 15 NTD significant mutations (six deletions and nine-point mutations) and 17 RBD point mutations were noted. We further characterized them in terms of distribution, count, ΔΔG of mutation (ΔΔG stability mCSM, ΔΔG stability DUET, ΔΔG stability SDM) to understand the stabilized or destabilized mutation, interaction interface, distance to PPI interface, Δvibrational entropy energy (ΔΔSVib ENCoM), and change in the flexibility. Here, we analyzed every mutation's ΔΔG, interaction, and related parameters using the trimeric Spike protein complex. In NTD mutations, our five analyzed mutations show two destabilising (G142D, R190S) and three showing stabilising properties (D215G, A222V, and R246I). Some RBD mutations are noted as entirely destabilising (K417N, K417T, L452R, F490S). N440K, N460K, and Q493R show stabilising and neutral properties. Combined LLMs and instigative bioinformatics techniques were used to identify and characterize the antibody escape mutations. With our strategy, the LLM and MLLM can help to fight future pandemic viruses by quickly identifying mutations and their significance., Competing Interests: Declaration of competing interest All authors declared no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The SARS-CoV-2 spike (S) protein has undergone significant evolution, enhancing both receptor binding and immune evasion. In this review, we summarize ongoing efforts to develop antibodies targeting various epitopes of the S protein, focusing on their neutralization potency, breadth, and escape mechanisms. Antibodies targeting the receptor-binding site (RBS) typically exhibit high neutralizing potency but are frequently evaded by mutations in SARS-CoV-2 variants. In contrast, antibodies targeting conserved regions, such as the S2 stem helix and fusion peptide, exhibit broader reactivity but generally lower neutralization potency. However, several broadly neutralizing antibodies have demonstrated exceptional efficacy against emerging variants, including the latest omicron subvariants, underscoring the potential of targeting vulnerable sites such as RBS-A and RBS-D/CR3022. We also highlight public classes of antibodies targeting different sites on the S protein. The vulnerable sites targeted by public antibodies present opportunities for germline-targeting vaccine strategies. Overall, developing escape-resistant, potent antibodies and broadly effective vaccines remains crucial for combating future variants. This review emphasizes the importance of identifying key epitopes and utilizing antibody affinity maturation to inform future therapeutic and vaccine design., (© 2024 The Author(s). Immunological Reviews published by John Wiley & Sons Ltd.)

The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S) protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. More recent variants, notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the interaction of the S-protein trimer with the negatively charged HS. However, these enhanced interactions may reduce Omicron's ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, raising the question of how to mechanistically explain HS-associated viral movement. In this work, we show that Omicron S proteins have evolved to balance HS interaction stability and dynamics, resulting in enhanced mobility on an HS-functionalized artificial matrix. This property is achieved by the ability of Omicron S-proteins to cross-link at least two HS chains, allowing direct S-protein switching between chains as a prerequisite for cell surface mobility. Optimized HS interactions can be targeted pharmaceutically, as an HS mimetic significantly suppressed surface binding and cellular infection specifically of the Omicron variant. These findings suggest a robust way to interfere with SARS-CoV-2 Omicron infection and potentially future variants., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

SARS-CoV-2, the cause of COVID-19, primarily targets lung tissue, leading to pneumonia and lung injury. The spike protein of this virus binds to the common receptor on susceptible tissues and cells called the angiotensin-converting enzyme-2 (ACE2) of the angiotensin (ANG) system. In this study, we produced chimeric Macrobrachium rosenbergii nodavirus virus-like particles, presenting a short peptide ligand (ACE2tp), based on angiotensin-II (ANG II), on their outer surfaces to allow them to specifically bind to ACE2-overexpressing cells called ACE2tp-MrNV-VLPs. Replacing the ACE2tp at the protruding domains (P-domain) of the MrNV capsid proteins did not affect their normal assembly into icosahedral VLPs. The presentation of the ACE2tp on the P-domains significantly improved the binding and internalization of ACE2tp-MrNV-VLPs to hACE2-overexpressing HEK293T cells in a concentration-dependent manner. Furthermore, ACE2tp-MrNV-VLPs exhibited the ability to block the binding and infection of SARS-CoV-2 pseudovirus variants, including Wuhan, BA.2 Omicron, and Delta subtypes. Our results suggest that chimeric ACE2tp-MrNV-VLPs can serve as a blocking agent against various SARS-CoV-2 mutated variants and could also potentially serve as target-specific nano-containers to carry therapeutic agents to combat SARS-CoV-2 infections in the future., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that emerged in late 2019 and rapidly spread worldwide, causing the COVID-19 pandemic. The spike glycoprotein (S protein) plays a crucial role in viral target recognition and entry by interacting with angiotensin, converting enzyme 2 (ACE2), the functional receptor for the virus, via its receptor binding domain (RBD). The RBD availability for this interaction can be influenced by external factors, such as fatty acids. Linoleic acid (LA), a free fatty acid, has been shown to bind the S protein, modulating the viral infection by reducing initial target recognition. LA interacts with the fatty acid binding pocket (FABP), a potential drug target against SARS-CoV-2. In this study, we aimed to exploit the FABP as a drug target by performing a docking-based virtual screening with a library of commercially available, drug-like compounds. The virtual hits identified were then assessed in in vitro assays for the inhibition of the virus-host interaction and cytotoxicity. Binding assays targeting the spike-ACE2 interaction identified multiple compounds with inhibitory activity and low cytotoxicity.

Developing intranasal vaccines against pandemics and devastating airborne infectious diseases is imperative. The superiority of intranasal vaccines over injectable systemic vaccines is evident, but developing effective intranasal vaccines presents significant challenges. Fusing a protein antigen with the catalytic domain of cholera toxin (CTA1) and the two-domain D of staphylococcal protein A (DD) has significant potential for intranasal vaccines. In this study, we constructed two fusion proteins containing CTA1, tandem repeat linear epitopes of the SARS-CoV-2 spike protein (S14P5 or S21P2), and DD. Structural predictions indicated that each component of the fusion proteins was compatible with its origin. In silico analyses predicted high solubility for both fusion proteins when overexpressed in Escherichia coli. However, contrary to these predictions, the constructs exhibited limited solubility. Lowering the cultivation temperature from 37°C to 18°C did not improve solubility. Inducing expression with IPTG at the early log phase significantly increased soluble CTA1-(S21P2)4-DD but not CTA1-(S14P5)4-DD. Adding non-denaturing detergents (Nonidet P40, Triton X100, or Tween 20) to the extraction buffer significantly enhanced solubility. Despite this, purification experiments yielded low amounts, only 1-2 mg/L of culture, due to substantial losses during the purification stages. These findings highlight the challenges and potential strategies for optimizing soluble expression of CTA1-DD fusion proteins for intranasal vaccines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tarigan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

SARS-CoV-2, a β-coronavirus responsible for the COVID-19 pandemic, has resulted in approximately 4.9 million fatalities worldwide. Despite the urgent need, there is currently no specific therapeutic developed for treating or preventing SARS-CoV-2 infections. The virus enters the host by engaging in a molecular interaction between the viral Spike glycoprotein (S protein) and the host ACE2 receptor, facilitating membrane fusion and initiating infection. Inhibiting this interaction could impede viral activity. Therefore, this study aimed to identify natural small molecules from perennial rue herb (Ruta graveolens) as potential inhibitors against the S protein, thus preventing virus infection. Initially, a screening process was conducted on 53 compounds identified from rue herbs, utilizing pharmacophore-based virtual screening approaches. This analysis resulted in the identification of 12 hit compounds. Four compounds, namely Amentoflavone (CID: 5281600), Agathisflavone (CID: 5281599), Vitamin P (CID: 24832108), and Daphnoretin (CID: 5281406), emerged as potential S protein inhibitors through molecular docking simulations, exhibiting binding energies in kcal/mol of -9.2, -8.8, -8.2, and -8.0, respectively. ADMET analysis revealed favorable pharmacokinetics and toxicity profiles for these compounds. The compounds' stability with respect to the target S protein was evaluated using MD simulation and MM-GBSA approaches. The analysis revealed the stability of the selected compounds with the target protein. Also, PCA revealed distinctive movement patterns in four selected compounds, offered valuable insights into their functional behaviors and potential interactions. In-vitro assays revealed that rue herb extracts containing these compounds displayed potential inhibitory properties against the virus, with an IC 50 value of 1.299 mg/mL and a cytotoxic concentration (CC 50 ) value of 11.991 mg/mL. The compounds derived from rue herb, specifically Amentoflavone, Agathisflavone, Vitamin P, and Daphnoretin, show promise as candidates for the therapeutic intervention of SARS-CoV-2-related complications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Study of mechanisms by which antibodies recognize different viral strains is necessary for the development of new drugs and vaccines to treat COVID-19 and other infections. Here, we report 2.5 Å cryo-EM structure of the SARS-CoV-2 Delta trimeric S-protein in complex with Fab of the recombinant analog of REGN10987 neutralizing antibody. S-protein adopts "two RBD-down and one RBD-up" conformation. Fab interacts with RBDs in both conformations, blocking the recognition of angiotensin converting enzyme-2. Three-dimensional variability analysis reveals high mobility of the RBD/Fab regions. Interaction of REGN10987 with Wuhan, Delta, Omicron BA.1, and mutated variants of RBDs is analyzed by microscale thermophoresis, molecular dynamics simulations, and ΔG calculations with umbrella sampling and one-dimensional potential of mean force. Variability in molecular dynamics trajectories results in a large scatter of calculated ΔG values, but Boltzmann weighting provides an acceptable correlation with experiment. REGN10987 evasion of the Omicron variant is found to be due to the additive effect of the N440K and G446S mutations located at the RBD/Fab binding interface with a small effect of Q498R mutation. Our study explains the influence of known-to-date SARS-CoV-2 RBD mutations on REGN10987 recognition and highlights the importance of dynamics data beyond the static structure of the RBD/Fab complex., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

An unprecedented amount of SARS-CoV-2 data has been accumulated compared with previous infectious diseases, enabling insights into its evolutionary process and more thorough analyses. This study investigates SARS-CoV-2 features as it evolved to evaluate its infectivity. We examined viral sequences and identified the polarity of amino acids in the receptor binding motif (RBM) region. We detected an increased frequency of amino acid substitutions to lysine (K) and arginine (R) in variants of concern (VOCs). As the virus evolved to Omicron, commonly occurring mutations became fixed components of the new viral sequence. Furthermore, at specific positions of VOCs, only one type of amino acid substitution and a notable absence of mutations at D467 were detected. We found that the binding affinity of SARS-CoV-2 lineages to the ACE2 receptor was impacted by amino acid substitutions. Based on our discoveries, we developed APESS, an evaluation model evaluating infectivity from biochemical and mutational properties. In silico evaluation using real-world sequences and in vitro viral entry assays validated the accuracy of APESS and our discoveries. Using Machine Learning, we predicted mutations that had the potential to become more prominent. We created AIVE, a web-based system, accessible at https://ai-ve.org to provide infectivity measurements of mutations entered by users. Ultimately, we established a clear link between specific viral properties and increased infectivity, enhancing our understanding of SARS-CoV-2 and enabling more accurate predictions of the virus., Competing Interests: JP, WC, DS, SJ, JL, HP, DC, KY, UK, GY, HK, TK, SK, EM, HC, NC, DH No competing interests declared, (© 2024, Park, Choi, Seong et al.)

Systems serology aims to broadly profile the antigen binding, Fc biophysical features, immune receptor engagement, and effector functions of antibodies. This experimental approach excels at identifying antibody functional features that are relevant to a particular disease. However, a crucial limitation of this approach is its incomplete description of what structural features of the antibodies are responsible for the observed immune receptor engagement and effector functions. Knowing these antibody features is important for both understanding how effector responses are naturally controlled through antibody Fc structure and designing antibody therapies with specific effector profiles. Here, we address this limitation by modeling the molecular interactions occurring in these assays and using this model to infer quantities of specific antibody Fc species among the antibodies being profiled. We used several validation strategies to show that the model accurately infers antibody properties and then applied the model to infer previously unavailable antibody fucosylation information from existing systems serology data. Using this capability, we find that COVID-19 vaccine efficacy is associated with the induction of afucosylated spike protein-targeting IgG. Our results also question an existing assumption that controllers of HIV exhibit gp120-targeting IgG that are less fucosylated than those of progressors. Additionally, we confirm that afucosylated IgG is associated with membrane-associated antigens for COVID-19 and HIV, and present new evidence indicating that this relationship is specific to the host cell membrane. Finally, we use the model to identify redundant assay measurements and subsets of information-rich measurements from which Fc properties can be inferred. In total, our modeling approach provides a quantitative framework for the reasoning typically applied in these studies, improving the ability to draw mechanistic conclusions from these data., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Abraham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)