Coronavirus Spike Protein Research Articles

A novel neutralizing monoclonal antibody recognizes a linear antigenic epitope of the spike protein of swine acute diarrhoea syndrome coronavirus

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) causes vomiting, severe diarrhoea and death in newborn piglets. The spike (S) protein plays a crucial role in promoting virus invasion and inducing neutralizing antibody production. In this study, the extracellular region of the S protein was used as an immunogen to immunize BALB/c mice. After immunization, B cells were collected, fused with SP2/0 myeloma cells, cultured and subcloned, and a cell line capable of secreting neutralizing antibodies was obtained and named as 5D6. Additionally, it was determined that the 5D6 mAb could be used as the primary antibody for western blotting and indirect immunofluorescence assay (IFA) to detect SADS-CoV. Further studies indicated that the 5D6 mAb binds to the 136STSHAAD142 motif, which located in the N-terminal domain (NTD) of the spike protein. This result suggested that the NTD of the S protein can induce the production of neutralizing antibodies. Amino acid sequence alignment revealed that the epitope of the 5D6 mAb was conserved among SADS-CoV strains. This study helps elucidate the S protein function of SADS-CoV, and the 5D6 mAb may be used to develop diagnostic and treatment tools for detecting SADS-CoV infection.

Cross-Reactive Immune Response of Bovine Coronavirus Spike Glycoprotein to SARS-CoV-2 Variants of Concern.

The high variability observed in the clinical symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has been attributed to the presence, in a proportion of infection-naive subjects, of pre-existing cross-reactive immune responses. Here, we demonstrate that the bovine coronavirus spike protein (BoS) may represent a source of protective immunity to SARS-CoV-2. Indeed, vaccination of BALB/c mice with a Bovine herpesvirus 4 (BoHV-4)-based vector expressing BoS induced both cell-mediated and humoral immune responses that cross-react with SARS-CoV-2 spike protein. Although the spike-specific antibodies induced by BoS did not neutralize SARS-CoV-2, the T lymphocytes activated by BoS were able to induce cytotoxicity of cells expressing spike proteins derived from several SARS-CoV-2 variants. These results demonstrate that immunization with BoS may represent a source of cross-reactive immunity to SARS-CoV-2, and that these cross-reactive immune responses may exert protective functions. These results contribute to deciphering the mechanisms responsible for lack or mildness of symptoms observed in many individuals upon SARS-CoV-2 infection and may open new ways for the development of new vaccines for coronaviruses.

Biochemical analysis of packing and assembling heptad repeat motifs in the coronavirus spike protein trimer.

During a coronavirus infection, the spike protein undergoes sequential structural transitions triggered by its receptor and the host protease at the interface between the virus and cell membranes, thereby mediating membrane fusion. After receptor binding, the heptad repeat motif (HR1/HR2) within the viral spike protein bridges the viral and cellular membranes; however, the intermediate conformation adopted by the spike protein when drawing the viral and cellular membranes into close proximity remains unclear due to its transient and unstable nature. Here, we experimentally induced conformational changes in the spike protein of a murine coronavirus by incubating the virus with its receptor, followed by exposure to trypsin. We then treated the virus/receptor complex with proteinase K to probe the tightly packed core structure of the spike protein. The conformations of the spike protein were predicted from the sizes of the protease digestion products detected by western blot analysis. Upon receptor binding, two bands (each showing different reactivity with a fusion-inhibiting HR2-peptide) were detected; we propose that these bands correspond to the packed and unpacked HR1/HR2 motifs. After trypsin-mediated triggering, measurement of temperature and time dependency revealed that packing of the remaining unpacked HR1/HR2 motifs and assembly of three HR1 motifs in a trimer occur almost simultaneously. Thus, the trimeric spike protein adopts an asymmetric-unassembled conformation after receptor binding, followed by direct assembly into the post-fusion form triggered by the host protease. This biochemical study provides mechanistic insight into the previously unknown intermediate structure of the viral fusion protein.IMPORTANCEDuring infection by an enveloped virus, receptor binding triggers fusion between the cellular membrane and the virus envelope, enabling delivery of the viral genome to the cytoplasm. The viral spike protein mediates membrane fusion; however the molecular mechanism underlying this process is unclear. This is because using structural biology methods to track the transient conformational changes induced in the unstable spike trimer is challenging. Here, we harnessed the ability of protease enzymes to recognize subtle differences on protein surfaces, allowing us to detect structural differences in the spike protein before and after conformational changes. Differences in the size of the degradation products were analyzed by western blot analysis. The proposed model explaining the conformational changes presented herein is a plausible candidate that provides valuable insight into unanswered questions in the field of virology.

Higher Affinity Enables More Accurate Detection of SARS-CoV-2 in Human Saliva Using Aptamer-Based Litmus Test.

Many aptamers have been generated by systematic evolution of ligands by exponential enrichment (SELEX) to recognize spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2&ek), some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000-fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd≈10 pM) can identify the SARS-CoV-2 infection much more accurately than the dimeric aptamer assay (Kd≈100 pM) and monomeric aptamer assay (Kd≈10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80-100 % sensitivity and 100 % specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.

Higher Affinity Enables More Accurate Detection of SARS‐CoV‐2 in Human Saliva Using Aptamer‐Based Litmus Test

AbstractMany aptamers have been generated by systematic evolution of ligands by exponential enrichment (SELEX) to recognize spike proteins of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2&ek), some of which have been engineered into dimeric and trimeric versions for enhanced affinity for diagnostic applications. However, no studies have been conducted to compare the utilities of monomeric, dimeric and trimeric aptamers in diagnostic assays with real clinical samples to answer the question of what levels of affinity an aptamer must have for accurate clinical diagnostics. Herein, we carried out a comparative study with two monomeric aptamers MSA1 and MSA5, one dimeric aptamer and two homotrimeric aptamers constructed with MSA1 and MSA5, with affinity varying by 1000‐fold. Using a colorimetric sandwich assay to analyze 48 human saliva samples, we found that the trimeric aptamer assay (Kd≈10 pM) can identify the SARS‐CoV‐2 infection much more accurately than the dimeric aptamer assay (Kd≈100 pM) and monomeric aptamer assay (Kd≈10,000 pM). Based on the experimental data, we theoretically predict the levels of affinity an aptamer needs to possess to achieve 80–100 % sensitivity and 100 % specificity. The findings from this study highlight the need for deriving very high affinity aptamers to enable highly accurate detection of viral infection for future pandemics.

Insilco vaccine design of spike and hemagglutinin esterase proteins of bovine coronavirus

BackgroundBovine coronavirus (BCoV) is a widespread, fatal illness in cattle that has a large economic impact in particularly disease-prone hosts. BCoV does not have an effective vaccine. As a result, the objective of this study was to use immunoinformatics and computational tools to design a multi-epitope vaccine against Spike and haemagglutinin proteins of Bovine coronavirus. BCoV proteins were also subjected to protein analysis.MethodsA variety of tools of bioinformatics were used for data analysis. Conserved B and T cell epitopes against BCoV target proteins were predicted using the Immune Epitope Database (IEDB). Proteins were characterized utilizing a variety of servers, including Protparam, PSIPRED and the GOR IV servers. The antigenicity, allergenicity, and toxicity of the anticipated epitopes were assessed as well.ResultsSeveral MHCI epitopes were predicted from S and HE proteins. As top epitopes, the peptides 77NMALKGTLL85 and 56SYMDLNPAL65 were proposed from Spike and hemagglutinin proteins, respectively. These epitopes exhibited high scores of antigenicity, no allergenicity, no toxicity, and a strong connection to Bola alleles. Moreover, three epitopes (1204YYYPE1208, 379TCQPQ384, and 720QLQPINY726) from Spike glycoprotein were selected as surface, linear, and antigenic epitopes using B cell scales. The methods dropped to anticipate effective and safe epitope(s) to cover all B cell scales from HE protein.ConclusionThree B cell epitopes (1204YYYPE1208, 379TCQPQ384, and 720QLQPINY726) were predicted from Spike protein (S) of BCoV only. MHC I epitopes of S and HE proteins of BCoV predicted two epitopes (77NMALKGTLL85 and 56SYMDLNPAL65 respectively) to have a strong link to Bola alleles, as well as high antigenicity and safety. The predicted epitopes’ activity should be tested experimentally as a multi-epitope vaccine against BCoV using in vitro and in vivo trials.

Comparison of the Neutralization Power of Sotrovimab Against SARS-CoV-2 Variants: Development of a Rapid Computational Method.

The rapid evolution of SARS-CoV-2 imposed a huge challenge on disease control. Immune evasion caused by genetic variations of the SARS-CoV-2 spike protein's immunogenic epitopes affects the efficiency of monoclonal antibody-based therapy of COVID-19. Therefore, a rapid method is needed to evaluate the efficacy of the available monoclonal antibodies against the new emerging variants or potential novel variants. The aim of this study is to develop a rapid computational method to evaluate the neutralization power of anti-SARS-CoV-2 monoclonal antibodies against new SARS-CoV-2 variants and other potential new mutations. The amino acid sequence of the extracellular domain of the spike proteins of the severe acute respiratory syndrome coronavirus (GenBank accession number YP_009825051.1) and SARS-CoV-2 (GenBank accession number YP_009724390.1) were used to create computational 3D models for the native spike proteins. Specific mutations were introduced to the curated sequence to generate the different variant spike models. The neutralization potential of sotrovimab (S309) against these variants was evaluated based on its molecular interactions and Gibbs free energy in comparison to a reference model after molecular replacement of the reference receptor-binding domain with the variant's receptor-binding domain. Our results show a loss in the binding affinity of the neutralizing antibody S309 with both SARS-CoV and SARS-CoV-2. The binding affinity of S309 was greater to the Alpha, Beta, Gamma, and Kappa variants than to the original Wuhan strain of SARS-CoV-2. However, S309 showed a substantially decreased binding affinity to the Delta and Omicron variants. Based on the mutational profile of Omicron subvariants, our data describe the effect of the G339H and G339D mutations and their role in escaping antibody neutralization, which is in line with published clinical reports. This method is rapid, applicable, and of interest to adapt the use of therapeutic antibodies to the treatment of emerging variants. It could be applied to antibody-based treatment of other viral infections.

An Analysis of Combined Molecular Weight and Hydrophobicity Similarity between the Amino Acid Sequences of Spike Protein Receptor Binding Domains of Betacoronaviruses and Functionally Similar Sequences from Other Virus Families

Recently, we proposed a new method, based on protein profiles derived from physicochemical dynamic time warping (PCDTW), to functionally/structurally classify coronavirus spike protein receptor binding domains (RBD). Our method, as used herein, uses waveforms derived from two physicochemical properties of amino acids (molecular weight and hydrophobicity (MWHP)) and is designed to reach into the twilight zone of homology, and therefore, has the potential to reveal structural/functional relationships and potentially homologous relationships over greater evolutionary time spans than standard primary sequence alignment-based techniques. One potential application of our method is inferring deep evolutionary relationships such as those between the RBD of the spike protein of betacoronaviruses and functionally similar proteins found in other families of viruses, a task that is extremely difficult, if not impossible, using standard multiple alignment-based techniques. Here, we applied PCDTW to compare members of four divergent families of viruses to betacoronaviruses in terms of MWHP physicochemical similarity of their RBDs. We hypothesized that some members of the families Arteriviridae, Astroviridae, Reoviridae (both from the genera rotavirus and orthoreovirus considered separately), and Toroviridae would show greater physicochemical similarity to betacoronaviruses in protein regions similar to the RBD of the betacoronavirus spike protein than they do to other members of their respective taxonomic groups. This was confirmed to varying degrees in each of our analyses. Three arteriviruses (the glycoprotein-2 sequences) clustered more closely with ACE2-binding betacoronaviruses than to other arteriviruses, and a clade of 33 toroviruses was found embedded within a clade of non-ACE2-binding betacoronaviruses, indicating potentially shared structure/function of RBDs between betacoronaviruses and members of other virus clades.

Optimization of Cellular Transduction by the HIV-Based Pseudovirus Platform with Pan-Coronavirus Spike Proteins.

Over the past three years, new SARS-CoV-2 variants have continuously emerged, evolving to a point where an immune response against the original vaccine no longer provided optimal protection against these new strains. During this time, high-throughput neutralization assays based on pseudoviruses have become a valuable tool for assessing the efficacy of new vaccines, screening updated vaccine candidates against emerging variants, and testing the efficacy of new therapeutics such as monoclonal antibodies. Lentiviral vectors derived from HIV-1 are popular for developing pseudo and chimeric viruses due to their ease of use, stability, and long-term transgene expression. However, the HIV-based platform has lower transduction rates for pseudotyping coronavirus spike proteins than other pseudovirus platforms, necessitating more optimized methods. As the SARS-CoV-2 virus evolved, we produced over 18 variants of the spike protein for pseudotyping with an HIV-based vector, optimizing experimental parameters for their production and transduction. In this article, we present key parameters that were assessed to improve such technology, including (a) the timing and method of collection of pseudovirus supernatant; (b) the timing of host cell transduction; (c) cell culture media replenishment after pseudovirus adsorption; and (d) the centrifugation (spinoculation) parameters of the host cell+ pseudovirus mix, towards improved transduction. Additionally, we found that, for some pseudoviruses, the addition of a cationic polymer (polybrene) to the culture medium improved the transduction process. These findings were applicable across variant spike pseudoviruses that include not only SARS-CoV-2 variants, but also SARS, MERS, Alpha Coronavirus (NL-63), and bat-like coronaviruses. In summary, we present improvements in transduction efficiency, which can broaden the dynamic range of the pseudovirus titration and neutralization assays.

In silico Investigation of the Interactions of Thymol and Carvacrol on the Spike Protein of Omicron Variant and MPro Enzyme of Coronavirus

Many drug studies have been conducted against the coronavirus disease, which has affected the whole world since December 2019, and some studies have been carried out on natural treatment methods. Many ideas for curing coronavirus disease of T. vulgaris known as thyme plant have been presented, although there are gaps in the literature on the subject. In this work, the anti-severe acute respiratory syndrome coronavirus 2 potential of the major compounds of the T. vulgaris plant’s essential oil was investigated in silico. The major components of the T. vulgaris plant's essential oil are thymol and carvacrol. Using molecular docking experiments, we evaluated the effects of thymol and carvacrol in thyme essential oil on Omicron variant spike protein and main protease enzyme (Mpro) of severe acute respiratory syndrome coronavirus 2. We also used online databases to investigate the adsorption, distribution, metabolism, absorption, and toxic (ADMET) aspects of these two compounds. It was determined that thymol and carvacrol have strong binding affinity to the spike protein of the Omicron variant and the main protease enzyme. The compounds interact with target proteins through electrostatic, hydrogen bonds, and hydrophobic interactions. More promising findings are obtained when the contacts of carvacrol with target proteins are assessed in terms of the structure-activity relationship.

Specific kinesin and dynein molecules participate in the unconventional protein secretion of transmembrane proteins.

Secretory proteins, including plasma membrane proteins, are generally known to be transported to the plasma membrane through the endoplasmic reticulum- to-Golgi pathway. However, recent studies have revealed that several plasma membrane proteins and cytosolic proteins lacking a signal peptide are released via an unconventional protein secretion (UcPS) route, bypassing the Golgi during their journey to the cell surface. For instance, transmembrane proteins such as the misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein and the Spike protein of coronaviruses have been observed to reach the cell surface through a UcPS pathway under cell stress conditions. Nevertheless, the precise mechanisms of the UcPS pathway, particularly the molecular machineries involving cytosolic motor proteins, remain largely unknown. In this study, we identified specific kinesins, namely KIF1A and KIF5A, along with cytoplasmic dynein, as critical players in the unconventional trafficking of CFTR and the SARS-CoV-2 Spike protein. Gene silencing results demonstrated that knockdown of KIF1A, KIF5A, and the KIF-associated adaptor protein SKIP, FYCO1 significantly reduced the UcPS of △F508-CFTR. Moreover, gene silencing of these motor proteins impeded the UcPS of the SARS-CoV-2 Spike protein. However, the same gene silencing did not affect the conventional Golgimediated cell surface trafficking of wild-type CFTR and Spike protein. These findings suggest that specific motor proteins, distinct from those involved in conventional trafficking, are implicated in the stress-induced UcPS of transmembrane proteins.

The Role of Coronavirus Spike Protein in Inducing Optic Neuritis in Mice: Parallels to the SARS-CoV-2 Virus.

Optic neuritis (ON), one of the clinical manifestations of the human neurological disease multiple sclerosis (MS), was also reported in patients with COVID-19 infection, highlighting one potential neurological manifestation of SARS-CoV-2. However, the mechanism of ON in these patients is poorly understood. Insight may be gained by studying the neurotropic mouse hepatitis virus (MHV-A59), a β-coronavirus that belongs to the same family as SARS-CoV-2. Mouse hepatitis virus-A59, or its isogenic spike protein recombinant strains, inoculation in mice provides an important experimental model to understand underpinning mechanisms of neuroinflammatory demyelination in association with acute stage optic nerve inflammation and chronic stage optic nerve demyelination concurrent with axonal loss. Spike is a surface protein that mediates viral binding and entry into host cells, as well as cell-cell fusion and viral spread. Studies have implicated spike-mediated mechanisms of virus-induced neuroinflammatory demyelination by comparing naturally occurring demyelinating (DM) and nondemyelinating (NDM) MHV strains. Here, we summarize findings in MHV-induced experimental ON and myelitis, using natural DM and NDM strains as well as engineered recombinant strains of MHV to understand the role of spike protein in inducing ON and demyelinating disease pathology. Potential parallels in human coronavirus-mediated ON and demyelination, and insight into potential therapeutic strategies, are discussed.

Identification of two novel B-cell epitopes located on the spike protein of swine acute diarrhea syndrome coronavirus

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging alpha-coronavirus that causes diarrhea in piglets and results in serious economic losses. During SADS-CoV infection, the spike protein (S) serves as a crucial structural component of the virion, interacting with receptors and eliciting the production of neutralizing antibodies. Due to the potential risk of zoonotic transmission of SADS-CoV, the identification and screening of epitopes on the S glycoproteins will be crucial for development of sensitive and specific diagnostic tools. In this study, we immunized BALB/c mice with recombinant SADS-CoV S trimer protein and generated two S1-specific monoclonal antibodies (mAbs): 8D6 and 6E9, which recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 8D6 was mapped to 311NPDQRD316, the minimal fragment recognized by mAb 6E9 was mapped to 492ARFVDRL498. Homology analysis of the regions corresponding to 13 typical strains of different SADS-CoV subtypes showed high conservation of these two epitopes. These findings contribute to a deeper understanding of the structure of the SADS-CoV S protein, which is valuable for vaccine design and holds potential for developing diagnostic methods to detect SADS-CoV.

Intestinal helminth infection impairs vaccine-induced T cell responses and protection against SARS-CoV-2 in mice.

Although vaccines have reduced the burden of COVID-19, their efficacy in helminth infection-endemic areas is not well characterized. We evaluated the impact of infection by Heligmosomoides polygyrus bakeri (Hpb), a murine intestinal roundworm, on the efficacy of an mRNA vaccine targeting the Wuhan-1 spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice. Although immunization generated similar B cell responses in Hpb-infected and uninfected mice, polyfunctional CD4+ and CD8+ T cell responses were markedly reduced in Hpb-infected mice. Hpb-infected and mRNA-vaccinated mice were protected against the ancestral SARS-CoV-2 strain WA1/2020, but control of lung infection was diminished against an Omicron variant compared with animals immunized without Hpb infection. Helminth-mediated suppression of spike protein-specific CD8+ T cell responses occurred independently of signal transducer and activator of transcription 6 (STAT6) signaling, whereas blockade of interleukin-10 (IL-10) rescued vaccine-induced CD8+ T cell responses. Together, these data show that, in mice, intestinal helminth infection impaired vaccine-induced T cell responses through an IL-10 pathway, which compromised protection against antigenically drifted SARS-CoV-2 variants.

Spike Protein of SARS-CoV-2 Activates Cardiac Fibrogenesis through NLRP3 Inflammasomes and NF-κB Signaling.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood. We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor. S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β. S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Coronavirus spike protein fragment-containing chimeric virus-like particles stimulate human dendritic cell maturation

Introduction. Viral capsid proteins can assemble into virus-like particles lacking infectivity and bearing parental virus antigens or artificially introduced antigens from other pathogens. At least some of such particles are highly immunogenic and could serve as a platform for promising vaccines. In this work, we assessed an effect of virus-like particles decorated with a SARS-CoV-2 spike protein fragment on human dendritic cell phenotype and functional properties. Materials and methods. The virus-like particles were assembled using chimeric molecules obtained by fusing genetic sequences encoding a norovirus major capsid protein VP1 fragment and a coronavirus spike protein fragment, including the receptor-binding domain. Dendritic cells were obtained from monocytes in vitro. Results. Incubation of immature dendritic cells with virus-like particles induced their phenotypic and functional maturation. The former was revealed by significantly increased expression of HLA-DR, CD80, CD86 and CD83. Dendritic cell phenotype after incubation with virus-like particles at the maximum concentration of 10 μg/ml did not differ significantly from that of mature dendritic cells in positive control. Along with phenotypic maturation, virus-like particles caused a manifold increase in the production of pro-inflammatory tumor necrosis factor-α, anti-inflammatory interleukin-10, as well as interleukin-6, which can stimulate both antibody synthesis and cellular pro-inflammatory reactions. The pronounced stimulation of dendritic cells by virus-like particles coated with coronavirus antigens evidence about successful particle recognition. Finally, we discuss plausible mechanisms for recognition of such virus-like particles by dendritic cell receptors. Conclusion. It has been shown that chimeric virus-like particles induced phenotypic and functional dendritic cell maturation, which is manifested by markedly elevated expression of functionally important membrane molecules, as well as a manifold rise in production of cytokines with a wide functional range. In our opinion, the data obtained indicate a promise of using virus-like particles based on norovirus proteins to display SARS-CoV-2 antigens.

A Machine Learning Approach to Identify Key Residues Involved in Protein-Protein Interactions Exemplified with SARS-CoV-2 Variants.

Human infection with the coronavirus disease 2019 (COVID-19) is mediated by the binding of the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the human angiotensin-converting enzyme 2 (ACE2). The frequent mutations in the receptor-binding domain (RBD) of the spike protein induced the emergence of variants with increased contagion and can hinder vaccine efficiency. Hence, it is crucial to better understand the binding mechanisms of variant RBDs to human ACE2 and develop efficient methods to characterize this interaction. In this work, we present an approach that uses machine learning to analyze the molecular dynamics simulations of RBD variant trajectories bound to ACE2. Along with the binding free energy calculation, this method was used to characterize the major differences in ACE2-binding capacity of three SARS-CoV-2 RBD variants-namely the original Wuhan strain, Omicron BA.1, and the more recent Omicron BA.5 sublineages. Our analyses assessed the differences in binding free energy and shed light on how it affects the infectious rates of different variants. Furthermore, this approach successfully characterized key binding interactions and could be deployed as an efficient tool to predict different binding inhibitors to pave the way for new preventive and therapeutic strategies.

Distribusi Mutasi Asp614Gly pada Spike Protein SARS-CoV-2 di Sumatera Barat

Objective: This study aims to map the distribution of the Aspartate 614 to Glycine (Asp614Gly) mutation in the spike protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) circulating in West Sumatra, to understand the extent of its spread within the local population. Methods: RNA samples were taken from 98 confirmed COVID-19 cases across various area in West Sumatra. Genetic amplification and next-generation sequencing (NGS) were performed to identify the presence of the Asp614Gly mutation. The frequency of mutations was analyzed using bioinformatics software. Results: Out of the 98 samples analyzed, approximately 97% exhibited the Asp614Gly mutation. The geographic distribution of this mutation showed significant variation among the districts of West Sumatra, with the highest prevalence in urban areas. Conclusion: These findings indicate that the Asp614Gly mutation is highly prevalent in West Sumatra. Associated with increased virus transmissibility, this mutation dominates the circulating virus strains in the region, highlighting the need for ongoing genetic surveillance to guide public health interventions and vaccination strategies.

Variation in structural motifs within SARS-related coronavirus spike proteins.

SARS-CoV-2 is the third known coronavirus (CoV) that has crossed the animal-human barrier in the last two decades. However, little structural information exists related to the close genetic species within the SARS-related coronaviruses. Here, we present three novel SARS-related CoV spike protein structures solved by single particle cryo-electron microscopy analysis derived from bat (bat SL-CoV WIV1) and civet (cCoV-SZ3, cCoV-007) hosts. We report complex glycan trees that decorate the glycoproteins and density for water molecules which facilitated modeling of the water molecule coordination networks within structurally important regions. We note structural conservation of the fatty acid binding pocket and presence of a linoleic acid molecule which are associated with stabilization of the receptor binding domains in the "down" conformation. Additionally, the N-terminal biliverdin binding pocket is occupied by a density in all the structures. Finally, we analyzed structural differences in a loop of the receptor binding motif between coronaviruses known to infect humans and the animal coronaviruses described in this study, which regulate binding to the human angiotensin converting enzyme 2 receptor. This study offers a structural framework to evaluate the close relatives of SARS-CoV-2, the ability to inform pandemic prevention, and aid in the development of pan-neutralizing treatments.