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Abstract The spread of SARS-CoV-2 to animals has the potential to evolve independently. In this study, we distinguished several sentinel animal species and genera for monitoring the re-emergence of COVID-19 or the new outbreak of COVID-19-like disease. We analyzed SARS-CoV-2 genomic data from human and nonhuman mammals in the taxonomic hierarchies of species, genus, family and order of their host. We find that SARS-CoV-2 carried by domestic dog (Canis lupus familiaris), domestic cat (Felis catus), mink (Neovison vison), and white-tailed deer (Odocoileus virginianus) cluster closely to human-origin viruses and show no differences in the majority of amino acids, but have the most positively selected sites and should be monitored to prevent the re-emergence of COVID-19 caused by novel variants of SARS-CoV-2. Viruses from the genera Panthera (especially lion (Panthera leo)), Manis and Rhinolophus differ significantly from human-origin viruses, and long-term surveillance should be undertaken to prevent the future COVID-19-like outbreaks. Investigation of the variation dynamics of sites 142, 501, 655, 681 and 950 within the S protein may be necessary to predict the novel animal SARS-CoV-2 variants.

Abstract In December 2023, we observed a notable shift in the COVID-19 landscape, when JN.1 omicron emerged as the predominant SARS-CoV-2 variant with a 95% incidence. We characterized the clinical profile, and genetic changes in JN.1, an emerging SARS-CoV-2 variant of interest. Whole genome sequencing was performed on SARS-CoV-2 positive clinical specimens, followed by sequence analysis. Mutations within the spike protein sequences were analysed and compared with the previously reported lineages and sub-lineages, to identify the potential impact of the unique mutations on protein structure and possible alterations in the functionality. Several unique and dynamic mutations were identified herein. Molecular docking analysis showed changes in the binding affinity, and key interacting residues of wild-type and mutated structures with key host cell receptors of SARS-CoV-2 entry viz., ACE2, CD147, CD209L and AXL. Our data provides key insights on the emergence of newer variants and highlights the necessity for robust and sustained global genomic surveillance of SARS-CoV-2.

Summary: Background: Coronavirus disease 2019 (COVID-19) is an immune-related disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The complete pathogenesis of the virus remains to be determined. Unraveling the molecular mechanisms governing SARS-CoV-2 interactions with host cells is crucial for the formulation of effective prophylactic measures and the advancement of COVID-19 therapeutics. Methods: We analyzed human lung single-cell RNA sequencing dataset to discern the association of butyrophilin subfamily 3 member A2 (BTN3A2) expression with COVID-19. The BTN3A2 gene edited cell lines and transgenic mice were infected by live SARS-CoV-2 in a biosafety level 3 (BSL-3) laboratory. Immunoprecipitation, flow cytometry, biolayer interferometry and competition ELISA assays were performed in BTN3A2 gene edited cells. We performed quantitative real-time PCR, histological and/or immunohistochemical analyses for tissue samples from mice with or without SARS-CoV-2 infection. Findings: The BTN3A2 mRNA level was correlated with COVID-19 severity. BTN3A2 expression was predominantly identified in epithelial cells, elevated in pathological epithelial cells from COVID-19 patients and co-occurred with ACE2 expression in the same lung cell subtypes. BTN3A2 targeted the early stage of the viral life cycle by inhibiting SARS-CoV-2 attachment through interactions with the receptor-binding domain (RBD) of the Spike protein and ACE2. BTN3A2 inhibited ACE2-mediated SARS-CoV-2 infection by reducing ACE2 in vitro and in vivo. Interpretation: These results reveal a key role of BTN3A2 in the fight against COVID-19. Identifying potential monoclonal antibodies which mimic BTN3A2 may facilitate disruption of SARS-CoV-2 infection, providing a therapeutic avenue for COVID-19. Funding: This study was supported by the National Natural Science Foundation of China (32070569, U1902215, and 32371017), the CAS “Light of West China” Program, and Yunnan Province (202305AH340006).

Background Post-acute COVID-19 syndrome (long COVID) refers to the persistence of COVID-19 symptoms or exceptional symptoms following recovery. Even without conferring fatality, it represents a significant global public health burden. Despite many reports on long COVID, the prevalence and data on associated biological factors remain unclear and limited. This research aimed to determine the prevalence of long COVID during the two distinct epidemic periods in Thailand, due to the Delta and Omicron variants of SARS-CoV-2, and to investigate the biological factors associated with long COVID. In addition, the spike protein amino acid sequences of the Delta and Omicron variants were compared to determine the frequency of mutations and their potential biological implications. Methods A retrospective cross-sectional study was established to recruit confirmed COVID-19 participants at Maharat Nakhon Ratchasima Hospital who had recovered for at least three months and were infected between June 2021 and August 2022. The demographic data and long COVID experience were collected via telephone interview. The biological factors were analyzed through binary logistic regression. The datasets of the SARS-CoV-2 spike protein amino acid sequence of the Delta and Omicron variants in Thailand were retrieved from GIDSAID to determine mutation frequencies and to identify possible roles of the mutations based on published data. Results Data was collected from a total of 247 participants comprising 106 and 141 participants of the Delta and Omicron epidemic periods, respectively. Apart from the COVID-19 severity and health status, the baseline participant data of the two time periods were remarkably similar. The prevalence of long COVID observed in the Omicron period was higher than in the Delta period (74.5% vs. 66.0%). The biological factors associated with long COVID were epidemic variant, age, treatment with symptomatic medicines, and vaccination status. When the spike protein sequence data of the two variants were compared, it was observed that the Omicron variant exhibited a greater quantity of amino acid changes in its receptor-binding domain (RBD) and receptor-binding motif (RBM). The critical changes of the Omicron variant within these regions had a significant function in enhancing virus transmissibility and host immune response resistance. Conclusion This study revealed informative data associated with long COVID in Thailand. More attention should be given to long COVID caused by unique virus variants and other biological factors to prepare a healthcare management strategy for COVID-19 patients after recovery.

Abstract The southernmost part of Thailand is a unique and culturally diverse region that has been greatly affected by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak during the coronavirus disease-2019 pandemic. To gain insights into this situation, we analyzed 1942 whole-genome sequences of SARS-CoV-2 obtained from the five southernmost provinces of Thailand between April 2021 and March 2022, together with those publicly available in the Global Initiative on Sharing All Influenza Data database. Our analysis revealed evidence for transboundary transmissions of the virus in and out of the five southernmost provinces during the study period, from both domestic and international sources. The most prevalent viral variant in our sequence dataset was the Delta B.1.617.2.85 variant, also known as the Delta AY.85 variant, with many samples carrying a non-synonymous mutation F306L in their spike protein. Protein–protein docking and binding interface analyses suggested that the mutation may enhance the binding between the spike protein and host cell receptor protein angiotensin-converting enzyme 2, and we found that the mutation was significantly associated with an increased fatality rate. This mutation has also been observed in other SARS-CoV-2 variants, suggesting that it is of particular interest and should be monitored.

Abstract Background SARS-CoV-2 belongs to the coronaviridae family and infects human cells by directly interacting with the angiotensin-converting enzyme-2 (ACE-2) through the viral Spike Protein (SP). While vaccines are crucial, much attention has been directed towards managing the symptoms of acute respiratory distress syndrome. Our present study highlights the potential in counteracting lung inflammation triggered by SARS-CoV-2 SP of the intranasal administration of the engineered probiotic Lactobacillus paracasei F19 expressing the enzyme NAPE-PLD (pNAPE-LP) able to in situ release palmitoylethanolamide (PEA) under a super-low boost of palmitate. Methods C57BL/6J mice undergo prophylactic treatment with intranasal pNAPE-LP/palmitate for 7 days before a 7 days challenge with intranasal SARS-CoV-2 SP. Then the capability of pNAPE-LP of colonizing the lungs and actively release PEA in situ have been determined by immunofluorescence, western blot and HPLC-MS. Moreover, the innate immune system downregulation and the histological damage rescue exerted by pNAPE-LP have been tested by immunofluorescence, hematoxylin and eosin staining, western blot analysis and ELISA test for the release of the pro-inflammatory mediators. Results pNAPE-LP effectively colonizes mice lungs and releases the anti-inflammatory compound PEA. Moreover, pNAPE-LP exhibits a protective effect on alveolar morphology, innate immune cells infiltration and in the reduction of neutrophil count, effectively reducing lung injury induced by SARS-CoV-2 SP. This is achieved by mitigating TLR4-mediated NLRP3 activation and the downstream pro-inflammatory products such as ILs, TNFα, C-reactive protein and the myeloperoxidase activity. Interestingly we observed a global reduction ACE2 expression in the lungs. Conclusion pNAPE-LP actively protect from severe inflammatory-related symptoms in SP-challenged mice. Also, it can downregulate the expression of ACE-2 receptors at the lung site potentially preventing the spreading of the infection.

Abstract COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2’s spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72–90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein’s conformational arrangements. The binding free energy ΔGTOTAL of C3 (−38.0 ± 0.08 kcal/mol) and C6E (−41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2.

Abstract The spike protein of SARS-CoV-2 is critical to viral infection of human host cells which ultimately results in COVID-19. In this study we analyzed the behavior of dihedral angles (phi and psi) of the wild-type spike protein over time from molecular dynamics and identified that their oscillations are dominated by a few discrete, relatively low frequencies in the 23–63 MHz range with 42.969 MHz being the most prevalent frequency sampled by the oscillations. We thus observed the spike protein to favor certain frequencies more than others. Gaps in the tally of all observed frequencies for low-abundance amino acids also suggests that the frequency components of dihedral angle oscillations may be a function of position in the primary structure since relatively more abundant amino acids lacked gaps. Lastly, certain residues identified in the literature as constituting the inside of a druggable pocket, as well as others identified as allosteric sites, are observed in our data to have distinctive time domain profiles. This motivated us to propose additional residues with similar time domain profiles, which may be of potential interest to the vaccine and drug design communities for further investigation. Thus these findings indicate that there is a particular frequency domain profile for the spike protein hidden within the time domain data and this information, perhaps with the suggested residues, might provide additional insight into therapeutic development strategies for COVID-19 and beyond.

We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.

Lung epithelial cells and fibroblasts poorly express angiotensin-converting enzyme 2, and the study aimed to investigate the role of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on inflammation and epithelial–mesenchymal transition (EMT) in two lung cell lines and to understand the potential mechanism. Lung epithelial cells (BEAS-2B) and fibroblasts (MRC-5) were treated with the spike protein, then inflammatory and EMT phenotypes were detected by enzyme-linked immunosorbent assay, Transwell, and western blot assays. RNA-sequence and bioinformatic analyses were performed to identify dysregulated genes. The roles of the candidate genes were further investigated. The results showed that treatment with 1,000 ng/mL of spike protein in two lung cell lines caused increased levels of IL-6, TNF-α, CXCL1, and CXCL3, and the occurrence of EMT. RNA-sequence identified 4,238 dysregulated genes in the spike group, and 18 candidate genes were involved in both inflammation- and EMT-related processes. GADD45A had the highest verified fold change (abs), and overexpression of GADD45A promoted the secretion of cytokines and EMT in the two lung cell lines. In conclusion, the spike protein induces inflammation and EMT in lung epithelial cells and fibroblasts by upregulating GADD45A, providing a new target to inhibit inflammation and EMT.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen–deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein.

Cancer patients, prone to severe COVID-19, face immune challenges due to their disease and treatments. Identifying biomarkers, particularly extracellular vesicle (EV)-derived microRNAs (miRNAs), is vital for comprehending their response to COVID-19 vaccination. Therefore, this study aimed to investigate specific EV-miRNAs in the plasma of cancer patients under active treatment who received the COVID-19 booster vaccine. The selected miRNAs (EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, EV-hsa-miR-145- 5p, and EV-hsa-miR-223-3p) are involved in regulating SARS-CoV-2 spike protein and cytokine release, making them potential biomarkers for vaccination response. The study involved 54 cancer patients. Plasma and serum samples were collected at pre-boost vaccination, and at 3 and 6 months post-boost vaccination. Anti-spike antibody levels were measured. Additionally, RNA was extracted from EVs isolated from plasma and the expression levels of miRNAs were assessed. The results showed a significantly positive antibody response after COVID-19 boost vaccination. The expression levels of EV-hsa-miR-7-5p, EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p increased significantly after 6 months of COVID-19 booster vaccination. Interestingly, an increased expression of certain EV-hsa-miRNAs was positively correlated. Bioinformatic analysis revealed that these correlated miRNAs play a critical role in regulating the targets present in antiviral responses and cytokine production. These findings suggest that EV-hsa-miR-15b-5p, EV-hsa-miR-24-3p, and EV-hsa-miR-223-3p may be crucial in immune response induced by mRNA vaccines.

Within the last two decades, SARS-CoV-2 was the third zoonotic severe acute respiratory betacoronavirus (sarbecovirus) to infect humans, following SARS and MERS. The disruptions caused by the pandemic underscore the need for a universal vaccine against respiratory betacoronaviruses. Our group previously developed the universal platform for vaccine development, MultiTEP, which has been utilized in this study to generate a range of SARS-CoV-2 epitope vaccine candidates. We prepared and characterized 18 vaccines incorporating small peptide fragments from SARS-CoV-2 Spike protein fused with the MultiTEP sequence using overlapping PCR. Wild-type mice were immunized intramuscularly with the immunogen formulated in AdvaxCpG adjuvant. Serum antibodies were detected by ELISA, surrogate neutralization, and pseudovirus neutralization assays. Finally, the most promising vaccine candidate was administered to three non-human primates. All vaccines generated high titers of spike-binding IgG antibodies. However, only three vaccines generated antibodies that blocked RBD binding to the ACE2 receptor in a surrogate virus neutralization assay. However, none of the vaccines induced antibodies able to neutralize pseudotype viruses, including after the administration of the lead vaccine to NHPs. MultiTEP-based COVID-19 vaccines elicited robust, IgG-binding responses against the Spike protein in mice and non-human primates, but these antibodies were not neutralizing, underscoring the need to refine this approach further.

The highly pathogenic coronaviruses SARS-CoV-2 and SARS-CoV have led to the COVID-19 pandemic and SARS outbreak, respectively. The receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2, particularly the Omicron variant, has frequent mutations, resulting in the reduced efficiency of current COVID-19 vaccines against new variants. Here, we designed two lipid nanoparticle-encapsulated mRNA vaccines by deleting the mutant RBD of the SARS-CoV-2 Omicron variant (SARS2-S (RBD-del)) or by replacing this mutant RBD with the conserved and potent RBD of SARS-CoV (SARS2-S (SARS-RBD)). Both mRNA vaccines were stable at various temperatures for different time periods. Unlike SARS2-S (RBD-del) mRNA, SARS2-S (SARS-RBD) mRNA elicited effective T-cell responses and potent antibodies specific to both SARS-CoV-2 S and SARS-CoV RBD proteins. It induced strong neutralizing antibodies against pseudotyped SARS-CoV-2 and SARS-CoV infections and protected immunized mice from the challenge of the SARS-CoV-2 Omicron variant and SARS-CoV by significantly reducing the viral titers in the lungs after Omicron challenge and by completely preventing SARS-CoV-induced weight loss and death. SARS2-S (SARS-RBD)-immunized serum antibodies protected naïve mice from the SARS-CoV challenge, with its protective efficacy positively correlating with the neutralizing antibody titers. These findings indicate that this mRNA vaccine has the potential for development as an effective vaccine against current and future SARS-CoV-2 variants and SARS-CoV.

Transmissibility and immune evasion of the recently emerged, highly mutated SARS-CoV-2 BA.2.87.1 are unknown. Here, we report that BA.2.87.1 efficiently enters human cells but is more sensitive to antibody-mediated neutralization than the currently dominating JN.1 variant. Acquisition of adaptive mutations might thus be needed for efficient spread in the population.

Aims: To investigate the role of phosphorylation in SARS-CoV-2 infection, potential therapeutic targets and its harmful genetic sequences. Materials & Methods: Data mining techniques were employed to identify upregulated kinases responsible for proteomic changes induced by SARS-CoV-2. Spike and nucleocapsid proteins' sequences were analyzed using predictive tools, including SNAP2, MutPred2, PhD-SNP, SNPs&Go, MetaSNP, Predict-SNP and PolyPhen-2. Missense variants were identified using ensemble-based algorithms and homology/structure-based models like SIFT, PROVEAN, Predict-SNP and MutPred-2. Results: Eight missense variants were identified in viral sequences. Four damaging variants were found, with SNPs&Go and PolyPhen-2. Promising therapeutic candidates, including gilteritinib, pictilisib, sorafenib, RO5126766 and omipalisib, were identified. Conclusion: This research offers insights into SARS-CoV-2 pathogenicity, highlighting potential treatments and harmful variants in viral proteins.

SARS-CoV-2 spike protein plays a key role in mediating viral entry and inducing host immune responses. It can adopt either an open or closed conformation based on the position of its receptor-binding domain (RBD). It is yet unclear what causes these conformational changes or how they influence the spike’s functions. Here, we show that Lys417 in the RBD plays dual roles in the spike’s structure: it stabilizes the closed conformation of the trimeric spike by mediating inter-spike–subunit interactions; it also directly interacts with ACE2 receptor. Hence, a K417V mutation has opposing effects on the spike’s function: it opens up the spike for better ACE2 binding while weakening the RBD’s direct binding to ACE2. The net outcomes of this mutation are to allow the spike to bind ACE2 with higher probability and mediate viral entry more efficiently, but become more exposed to neutralizing antibodies. Given that residue 417 has been a viral mutational hotspot, SARS-CoV-2 may have been evolving to strike a balance between infection potency and immune evasion, contributing to its pandemic spread.

Summary: Background: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18–55 years and ≥56 years. Methods: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020–December 2021; ClinicalTrials.gov NCT04495933; active, not recruiting). Healthy adults (Part 1: 18–55 years; Part 2: ≥56 years) received two doses of placebo, 5 μg, 15 μg, or 45 μg vaccine, or one 45 μg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. Findings: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. Interpretation: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. Funding: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

Essential in halting the COVID-19 pandemic caused by SARS-CoV-2, it is crucial to have stable, effective, and easy-to-manufacture vaccines. We developed a potential vaccine using a tobacco mosaic virus (TMV) epitope display model presenting peptides derived from the SARS-CoV-2 spike protein. The TMV-epitope fusions in laboratory tests demonstrated binding to the SARS-CoV-2 polyclonal antibodies. The fusion constructs maintained critical epitopes of the SARS-CoV-2 spike protein, and two in particular spanned regions of the receptor-binding domain that have mutated in the more recent SARS-CoV-2 variants. This would allow for the rapid modification of vaccines in response to changes in circulating variants. The TMV-peptide fusion constructs also remained stable for over 28 days when stored at temperatures between −20 and 37 °C, an ideal property when targeting developing countries. Immunogenicity studies conducted on BALB/c mice elicited robust antibody responses against SARS-CoV-2. A strong IFNγ response was also observed in immunized mice. Three of the six TMV-peptide fusion constructs produced virus-neutralizing titers, as measured with a pseudovirus neutralization assay. These TMV-peptide fusion constructs can be combined to make a multivalent vaccine that could be adapted to meet changing virus variants. These findings demonstrate the development of a stable COVID-19 vaccine candidate by combining SARS-CoV-2 spike protein-derived peptides presented on the surface of a TMV nanoparticle.

Chitosan, a natural polysaccharide derived from chitin, possesses biocompatibility, biodegradability, and mucoadhesive characteristics, making it an attractive material for the delivery of mRNA payloads to the nasal mucosa and promoting their uptake by target cells such as epithelial and immune cells (e.g., dendritic cells and macrophages). In this project, we aimed at developing novel lipid-based nanoformulations for mRNA delivery to counteract the pandemic caused by SARS-CoV-2 virus. The formulations achieved a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay. Furthermore, the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for our vaccine formulations showed transfection levels in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively. Our most promising vaccine formulation was also demonstrated to be amenable to lyophilization with minimal degradation of loaded mRNA, paving the way towards a more accessible and stable vaccine. Preliminary in vivo studies in mice were performed to assess the systemic and local immune responses. Nasal bronchoalveolar lavage fluid (BALF) wash showed that utilizing the optimized formulation resulted in local antibody concentrations and did not trigger any systemic antibody response. However, if further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.

The coronavirus 2019 disease (COVID-19) course and serological statuses of patients with relapsing–remitting multiple sclerosis (RRMS), treated with disease-modifying therapies (DMTs) are generally parallel that of the general population. Over the pandemic’s course, however, a notable increase in the number of RRMS patients who received vaccination against severe acute respiratory coronavirus 2 (SARS-CoV-2) and those who had COVID-19 (symptomatic and asymptomatic) was reported. This virus and/or vaccination likely influenced DMT-treated RRMS patients’ serological statuses regarding the presence of SARS-CoV-2 antibodies and their quantitative expression. This investigation assesses the presence and levels of the antibody directed against the S1 protein receptor binding domain (SRBD) and against the N protein of SARS-CoV-2 in 38 DMT-treated RRMS patients. The findings indicate that people vaccinated against SARS-CoV-2 exhibited significantly higher levels of IgG antibodies against S1-RBD at both assessment points. Patients with a prior history of COVID-19 demonstrated statistically significant increases in anti-N antibodies at visit 1, whereas such statistical significance was not observed at visit 2. DMT-treated RRMS patients generated neutralizing antibodies following vaccination and/or COVID-19 infection. Nevertheless, it is noteworthy that antibody levels more accurately reflect the serological status and exhibit a stronger correlation with vaccination than just the presence of antibodies.

The severe acute respiratory illness caused by the recent global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), a new strain, is a serious health crisis. Since new virus strains are continuously developing, the 2019 coronavirus disease pandemic, also known as COVID-19, is a major concern around the world. Human mortality and economic loss have been significantly impacted by the varying degrees of severity of COVID-19 symptoms, which can range from no symptoms at all to the lethal acute respiratory distress syndrome (ARDS). Scientists around the world have taken a ''war footing'' in their search for a method to detect, treat, and immunise against the extremely dangerous COVID-19 virus so that society can return to its pre-pandemic state. Since its first publication, the number of laboratory-based and point-of-care (POC) techniques for detecting and monitoring the outbreak status of COVID-19 viral infection has increased. A rapidly growing and increasingly used approach for detecting COVID-19 is the RT-PCR-based viral nucleic acid test (NAT). Due to the urgent situation caused by the spread of COVID-19, the rapid development of effective, safe immunisations is urgently required. Rapid implementation of mass immunisation measures is the greatest challenge against the spread of the COVID-19 pandemic. The extremely contagious COVID-19 virus originated during the SARS-CoV-2 virus pandemic, and the development of diagnostics and possible vaccine candidates has been greatly aided by new biotechnologies and the interdisciplinary approaches they permit. Phase III clinical trials have shown promise for several potential vaccinations against COVID-19, and these vaccines have been granted emergency use authorisation (EUA) for distribution. However, it is critical to find most promising vaccine candidates to protect against the harmful strains that are anticipated to arise in the future and the most effective ways to give them. This review article will aid in the development of diagnostic tests and immunisations for COVID-19, which may contribute to the global eradication of virus

Among the multiple SARS-CoV-2 variants recently reported, the Delta variant has generated the most perilous and widespread effects. Another variant, Omicron, has been identified specifically for its high transmissibility. Omicron contains numerous spike (S) protein mutations and numbers much larger than those of its predecessor variants. In this report, the author has discussed some essential structural aspects and time-based structure changes of a selected set of spike protein mutations within the Delta and Omicron variants. The expected impact of multiple point mutations within the spike protein’s receptor-binding domain (RBD) and S1 of these variants are examined. Additionally, the RBDs of the more recently emerged subvariants BA.4, BA.5, and BA.2.12.1 are discussed. Within the latter group, BA.5 represents the most prevalent form of SARS-CoV-2 globally until recently. This computational work also briefly explores the temporal mutation profile for the currently circulating variants of interest (VOIs), variants under monitoring (VUMs), and variants being monitored (VBMs) including XBB.1.5, BQ.1, BA.2.75, CH.1.1, XBB, XBF, EG.5 (or Eris), and BA.2.86 (or Pirola). It is expected that these structural data can facilitate the tasks of identifying drug targets and neutralizing antibodies for the evolving variants/subvariants of SARS-CoV-2.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.

Abstract Background The outbreak of SARS-CoV-2 continues to pose a serious threat to human health and social. The ongoing pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made a serious threat to public health and economic stability worldwide. Given the urgency of the situation, researchers are attempting to repurpose existing drugs for treating COVID-19. Methods We first established an anti-coronavirus drug screening platform based on the Homogeneous Time Resolved Fluorescence (HTRF) technology and the interaction between the coronavirus spike protein and its host receptor ACE2. Two compound libraries of 2,864 molecules were screened with this platform. Selected candidate compounds were validated by SARS-CoV-2_S pseudotyped lentivirus and ACE2-overexpressing cell system. Molecular docking was used to analyze the interaction between S protein and compounds. Results We identified three potential anti-coronavirus compounds: tannic acid (TA), TS-1276 (anthraquinone), and TS-984 (9-Methoxycanthin-6-one). Our in vitro validation experiments indicated that TS-984 strongly inhibits the interaction of the coronavirus S protein and the human cell ACE2 receptor. Additionally, tannic acid showed moderate inhibitory effect on the interaction of S protein and ACE2. Conclusion This platform is a rapid, sensitive, specific, and high throughput system, and available for screening large compound libraries. TS-984 is a potent blocker of the interaction between the S-protein and ACE2, which might have the potential to be developed into an effective anti-coronavirus drug.

Abstract The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading globally and continues to rage, posing a serious threat to human health and life quality. Antibody therapy and vaccines both have shown great efficacy in the prevention and treatment of COVID-19, whose development progress and adaptation range have attracted wide attention. However, with the emergence of variant strains of SARS-CoV-2, the neutralization activity of therapeutic or vaccine-induced antibodies may be reduced, requiring long-term virus monitoring and drug upgrade in response to its evolution. In this paper, conformational changes including continuous epitopes (CPs), discontinuous epitopes (DPs) and recognition interfaces of the three representative SARS-CoV-2 spike protein (SP) mutants (i.e., the Delta (B.1.617.2), Mu (B.1.621) and Omicron (B.1.1.529) strains), were analyzed to evaluate the effectiveness of current mainstream antibodies. The results showed that the conformation of SP wild type (WT) and mutants both remained stable, while the local antigenic epitopes underwent significant changes. Sufficient flexibility of SP CPs is critical for effective antibody recognition. The DPs of Delta, Mu and Omicron variants have showed stronger binding to human angiotensin converting enzyme-2 (hACE2) than WT; the possible drug resistance mechanisms of antibodies against three different epitopes (i.e., NTD_DP, RBD1_DP and RBD2_DP) were also proposed, respectively; the RBD2 of Delta, NTD of Mu, NTD and RBD2 of Omicron are deserve more attention in the subsequent design of next-generation vaccines. The simulation results not only revealed structural characteristics of SP antigenic epitopes, but also provided guidance for antibody modification, vaccine design and effectiveness evaluation.

COVID-19 continues to cause an increase in the number of cases and deaths worldwide. Due to the ever-mutating nature of the virus, frequent vaccination against COVID-19 is anticipated. Most of the approved SARS-CoV-2 vaccines are administered using the conventional intramuscular route, causing vaccine hesitancy. Thus, there is a need for an effective, non-invasive vaccination strategy against COVID-19. This study evaluated the synergistic effects of a subunit microparticulate vaccine delivered using microneedles. The microparticles encapsulated a highly immunogenic subunit protein of the SARS-CoV-2 virus, such as the spike protein’s receptor binding domain (RBD). Adjuvants were also incorporated to enhance the spike RBD-specific immune response. Our vaccination study reveals that a microneedle-based vaccine delivering these microparticles induced spike RBD-specific IgM, IgG, IgG1, IgG2a, and IgA antibodies. The vaccine also generated high levels of CD4+ and CD8a+ molecules in the secondary lymphoid organs. Overall, dissolving microneedles delivery spike RBD antigen in microparticulate form induced a robust immune response, paving the way for an alternative self-administrable, non-invasive vaccination strategy against COVID-19.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a pandemic and has caused damage to the lives of the people and economy of countries. However, the therapeutic reagents against SARS-CoV-2 remain unclear. The spike (S) protein of SARS-CoV-2 contains a cleavage motif at the S1/S2 boundary, known to be cleaved by furin. As cleavage is essential for S protein activation and viral entry, furin was selected as the target compound. In this study, we examined the inhibitory effects of two lignans (honokiol and magnolol) on furin-like enzymatic activity using a fluorogenic substrate with whole-cell lysates. Of two compounds tested, honokiol partially inhibited furin-like enzymatic activity. We further examined the anti-SARS-CoV-2 activity of honokiol using VeroE6 cell line, which is stably expressing a transmembrane protease serine 2 (TMPRSS2). It was shown that honokiol exhibited remarkable inhibition of SARS-CoV-2 infection. Therefore, honokiol and crude drugs which contain honokiol such as Magnolia species have a potential therapeutic reagents for SARS-CoV-2.

Background and aim: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells through the binding of the viral spike protein with human angiotensin-converting enzyme 2 (ACE2), resulting in the development of coronavirus disease 2019 (COVID-19). To date, few antiviral drugs are available that can effectively block viral infection. This study aimed to identify potential natural products from Taiwan Database of Extracts and Compounds (TDEC) that may prevent the binding of viral spike proteins with human ACE2 proteins. Methods: The structure-based virtual screening was performed using the AutoDock Vina program within PyRX software, the binding affinities of compounds were verified using isothermal titration calorimetry (ITC), the inhibitions of SARS-CoV-2 viral infection efficacy were examined by lentivirus particles pseudotyped (Vpp) infection assay, and the cell viability was tested by 293T cell in MTT assay. Results and conclusion: We identified 39 natural products targeting the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in silico. In ITC binding assay, dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone showed dissociation constant (Kd) = 0.468 μM, 1.712 μM, 6.650 μM, 2.86 μM, 3.761 μM and 4.27 μM, respectively. In Vpp infection assay, the compounds have significantly and consistently inhibition with the 50–90% inhibition of viral infection efficacy. In cell viability, torvoside K, epimedin, amentoflavone, and saikosaponin C showed IC50 > 100 μM; dioscin and celastrol showed IC50 = 1.5625 μM and 0.9866 μM, respectively. These natural products may bind to the viral spike protein, preventing SARS-CoV-2 from entering cells. Section: 1: Natural Products. Taxonomy (classification by evise): SARS-CoV-2, Structure-Based Virtual Screening, Isothermal Titration Calorimetry and Lentivirus Particles Pseudotyped (Vpp) Infection Assay, in silico and in vitro study.

OBJECTIVE: To analyze SARS-CoV-2 whole genome reported of Pakistan and compare them with other viral strains collected from other world region to better understand the origin and genetic characterization of the virus. METHODS: All the available genomic information of SARS-CoV-2 including Pakistani strains were collected from various online sources. Phylogenetic analysis of 131 sequences from 11 countries (Brazil, China, India, Italy, Nepal, Pakistan, Spain, Sweden, Taiwan, USA and Viet-Nam) were performed and compared with other related coronaviruses to find the evolution of virus and its origin. Individual SARS-CoV-2 gene, spike (S) glycoprotein and the receptor binding domain (RBD) with closely related coronaviruses were compared to further explore genetic variations and the likely RBD properties of the virus. RESULTS: The analysis shows that genome of all analyzed 131 SARS-CoV-2 strains collected from different geographical area were extremely similar, exhibiting >99% sequence identity. Notably, genome of the SARS-CoV-2 has high similarity (89.1% sequence identity) with the two bat-derived severe acute respiratory syndrome (SARS) like betacoronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21 but was sufficiently divergent from SARS-CoV (82.3% similarity) and MERS-CoV (50% similarity). Phylogenetic analysis shows that the SARS-CoV-2 has relatively similar spike glycoprotein with bat-SL-CoVZC45, however, the RBD was more like that of SARS-CoVGZ02. CONCLUSION: Using different bioinformatics tools, we determined that SARS-CoV-2 has high similarities to bat-derived SARS like betacoronaviruses than SARS-CoV at the whole genome level, however, the RBD was more like that of SARS-CoVGZ02, which shows that they use similar ACE2 as a cell receptor.

Background and Aim: The human coronavirus is a member of the Coronaviridae family and causes upper respiratory tract infections. Despite repeated severe epidemics and the lack of appropriate antiviral drugs, not much progress has been made on the epitope-based vaccine designed for HCoV. Methods & Materials: The method of this study was to select the spike corona virus protein sequence from NCBI, retrieve the protein sequence and determine the T, B epitopes required to produce the chimer vaccine, evaluate the antigenicity and allergenicity and toxicity of the selected epitopes, respectively. Different servers were designed to configure the primary chimer composition of the epitope vaccine. Then, the chimer vaccine was evaluated in terms of structure and connectivity to B cells and MHCI and II compounds, and the two-dimensional structure and position of amino acids and bonds in the immunogenic model were studied, as well as the physicochemical and stability of the model vaccine by some other servers. Finally, it was tested for binding against HLA molecules using silico docking techniques to investigate the interaction with the epitope. Ethical Considerations: All ethical principles are considered in this article. Participants were informed about the research objective and its implementation stages. They also made sure their information was confidential. The principles of the Helsinki Convention were also observed. Results: The results showed that the immunogenic construct created in terms of two-dimensional and three-dimensional structure and the position of amino acids and bonds in the model of immunogenic structure, toxicity and allergenicity and antigenicity were in good condition. And had stability (instability index 33.93) and favorable half-life and suitable physicochemical conditions. Conclusion: In general, the immunogenic structure that was prepared in this research process could have a favorable interaction with some components of the immune system (HLA) in the docking process, which indicates the optimal identification of this structure by the humoral and cellular immune system and stimulation in In order to produce immunity in the body of the host, of course, more reliable proof of it requires clinical phase processes.

Summary: Background: We assessed the efficacy of a receptor-binding domain (RBD)-based protein subunit COVID-19 vaccine. Methods: A randomised Phase-1/2 trial followed by a Phase-2 trial were conducted to assess the safety and immunogenicity of the COVID-19 vaccine Corbevax and select to an optimum formulation. Healthy adults (n=460) without COVID-19 vaccination or SARS-CoV-2 infection in the Phase-1/2 study were randomly divided into four vaccine formulation groups. Findings: A low incidence of adverse events was reported post-vaccination. All formulations showed similar profiles of humoral and cellular immune responses that were associated with the content of CpG1018 adjuvant in the vaccine. In the Phase-2 study, 750 µg of CpG1018 showed significant improvement (> 4-fold increase from baseline) in immune responses, including the titres of anti-RBD IgG and neutralising antibody (nAb), and cellular immune responses, while maintaining the safety profile. Antibodies persisted consistently for 12 months after the second dose of vaccine. Interpretations: Corbevax (two-dose schedule with 28 days of interval between doses) was well tolerated with no observed safety concerns. Previous observations from efficacy studies by Moderna and AstraZeneca and the correlation between nAb titres post-vaccination and a human convalescent serum panel showed that Corbevax induced significantly high nAb titres. These studies were prospectively registered with the Clinical Trial Registry of India (CTRI/2021/06/034014 and CTRI/2020/11/029032). Funding: Bill & Melinda Gates Foundation, BIRAC-Division of Department of Biotechnology, Govt of India, and the Coalition for Epidemic Preparedness Innovations funded this study.

Introduction: The hallmark of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is high infectivity, pre symptomatic transmission and asymptomatic prevalence which could result in high cumulative numbers of infections, hospitalisations, and deaths. Kerala was the first state to confirm community transmission in July 2020. Healthcare Workers (HCWs) being in the forefront in the war against Coronavirus Disease-2019 (COVID-19) are more prone to acquire the infection and could possibly be asymptomatic sources for cluster formation. Knowing the development of immunity as shown by the presence of anti COV-2 antibodies in the population contributes to the epidemiological understanding of the disease. Aim: To determine the pattern of seropositivity of SARS-CoV-2 among the HCWs at Jubilee Mission Medical College and Research Institute, Thrissur, Kerala, India, six months after revoking the lockdown. Materials and Methods: This cross-sectional study was carried out among 423 HCWs of the medical college from September 5th to December 15th, 2020. Multistage sampling was done with the hospital block as the first stage and departments as the second stage. Blood sample was collected and Anti SARS COV-2 IgG antibody testing which targets the Spike Protein 1 (SP1) was done using the vitros chemiluminescence platform (Orthoclinical diagnostics, USA). For the summary of demographic characteristics, continuous variables were summarised as mean values and Standard Deviation (SD) while categorical variables were summarised as proportions. The χ2 test was used for comparing the epidemiological features between positive and negative cases. Chi-square test for trend analysis was done for exploring the relationship of the degree of severity with test positivity. All analyses were conducted using Statistical Package for the Social Sciences (SPSS) version 25.0. Results: Jubilee Mission Medical College had 2785 working staff at the time of study. A total of 423 staff consented and their samples were tested. Thirty seven staff members tested positive for COVID-19 antibody, yielding an overall prevalence of 8.75% (95% CI, 6.23-11.86). A 86.5% (32/37) of them were having a history of COVID-19 Antigen/Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Positivity. A statistically significant linear trend (p-value=0.00001) was observed, between seropositivity and the degree of severity of COVID-19. Among the various factors which increase the risk of seroconversion, history of undergoing quarantine (p-value

Viruses require the host cellular machinery for protein translation and replication. Upon proliferation, virions damage cells and are released from the infected cells before infecting other cells. Acute inflammation occurs when host cells are damaged by infection. The cell receptors to which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds, are widely distributed compared to those for other viruses, thereby resulting in various symptoms such as rhinitis, pneumonia, and enteritis. In general, RNA viruses, including SARS-CoV-2, exhibit a high frequency of gene mutations. Antigenic modulation due to genetic mutations of the spike protein causes cytokine storms because of strong activation of the innate immune system, similar to the phenomenon previously observed in highly pathogenic avian influenza. The proportion of severely ill patients due to coronavirus disease 2019 (COVID-19) varies from country to country, and factors that are responsible for the severity of the disease include antibody-dependent enhancement (ADE), Bacillus Calmette-Guérin (BCG) vaccination, and human leukocyte antigen (HLA) type. ADE and HLA types may also influence the protective effect of immunity, including its vaccine response against SARS-CoV-2. This report is a secondary publication of our previous review report "J Tokyo Wom Med Univ 91: 2-10, 2021."

Background The COVID-19 pandemic is still a global public health issue. Omicron, a SARS-CoV-2 B.1.1.529 variant, has raised concerns about transmission and vaccine effectiveness. Omicron currently has the greatest number of variantions. Methods To gain a better understanding of the significance of these variations and the dynamics of the interaction between the Omicron spike (S) protein and its human host factor angiotensin-converting enzyme 2 (ACE2), triplicate 500 ns molecular dynamics simulations were run using the structure of the S protein’s receptor-binding domain (RBD) in complex with ACE2. The interaction and binding energy, determined using the molecular mechanics—generalized Born surface area approach, were compared to the original SARS-CoV-2 and the B.1.617 variant. Results Though mutations K417N and G496S in the S protein RBD disrupt interactions found in the original SARS-CoV-2 complex, mutations Q493R and N501Y introduce interactions not found in the original complex. Interaction at a key viral hotspot and hydrophobic contacts at ACE2’s N-terminus were preserved, but intermolecular hydrogen bonds and polar contacts in the S-ACE2 interface were lower than in the original SARS-CoV-2 interface.

Abstract In view of the rapid development of the COVID‐19 pandemic and SARS‐CoV‐2 mutation, we characterized the emerging SARS‐CoV‐2 variants of concern (VOCs) by both bioinformatics methods and experiments. The representative genomic sequences of SARS‐CoV‐2 VOCs were first downloaded from NCBI, including the prototypic strain, Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Delta (B.1.617.2), and Omicron (B1.1.529) strain. Bioinformatics analysis revealed that the D614G mutation led to formation of a protruding spike (S) in the tertiary structure of spike protein, which could be responsible for the enhanced binding to angiotensin‐converting enzyme 2 (ACE2) receptor. The epitope analysis further showed that the S protein antigenicity of the Omicron variant changed dramatically, which was possibly associated with its enhanced ability of immune escape. To verify the bioinformatics results, we performed experiments of pseudovirus infection and protein affinity assay. Notably, we found that the spike protein of Omicron variant showed the weakest infectivity and binding ability among all tested strains. Finally, we also proved this through virus infection experiments, and found that the cytotoxicity of Omicron seems to be not strong enough. The results in this study provide guidelines for prevention and control of COVID‐19.

Comirnaty (BNT162b2) and Spikevax (mRNA-1273) COVID-19 vaccines encode a full-length SARS-CoV-2 Spike (S) protein. To evaluate whether the S-protein expressed following treatment with the two vaccines differs in the real-world context, two cell lines were treated for 24 h with two concentrations of each vaccine, and the expression of the S-protein was evaluated using flow cytometry and ELISA. Vaccines were obtained from three vaccination centers in Perugia (Italy) that provided us with residual vaccines present in vials after administration. Interestingly, the S-protein was detected not only on the cell membrane but also in the supernatant. The expression was dose-dependent only in Spikevax-treated cells. Furthermore, the S-protein expression levels in both cells and supernatant were much higher in Spikewax-than in Comirnaty-treated cells. Differences in S-protein expression levels following vaccine treatment may be attributed to variations in the efficacy of lipid nanoparticles, differences in mRNA translation rates and/or loss of some lipid nanoparticles’ properties and mRNA integrity during transport, storage, or dilution, and may contribute to explaining the slight differences in the efficacy and safety observed between the Comirnaty and Spikevax vaccines.

The safety, inherent immunogenicity, stability, and low-cost production of bacteriophages make them an ideal platform for vaccine development. Most vaccination strategies against COVID-19 have targeted the spike protein of SARS-CoV-2 to generate neutralizing antibodies. P1, a truncated RBD-derived spike protein, has been shown to induce virus-neutralizing antibodies in preclinical studies. In this study, we first investigated whether recombinant phages displaying P1 on the M13 major protein could immunize mice against COVID-19, and second, whether inoculation with 50 µg of purified P1 in addition to the recombinant phages would stimulate the immune systems of the animals. The results showed that the mice that received recombinant phages were immunized against the phage particles, but did not have anti-P1 IgG. In contrast, compared with the negative control, the group that received a combination of P1 protein and recombinant phage was immunized against the P1 protein. In both groups, CD4+ and CD8+ T cells appeared in the lung tissue. These results suggest that the number of antigens on the phage body plays a crucial role in stimulating the immune system against the bacteriophage, although it is immunogenic enough to function as a phage vaccine.

Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.

Background: SARS-CoV-2 vaccines play an important role in reducing disease severity, hospitalization, and death, although they failed to prevent the transmission of SARS-CoV-2 variants. Therefore, an effective inhibitor of galectin-3 (Gal-3) could be used to treat and prevent the transmission of COVID-19. ProLectin-M (PL-M), a Gal-3 antagonist, was shown to interact with Gal-3 and thereby prevent cellular entry of SARS-CoV-2 in previous studies. Aim: The present study aimed to further evaluate the therapeutic effect of PL-M tablets in 34 subjects with COVID-19. Methods: The efficacy of PL-M was evaluated in a randomized, double-blind, placebo-controlled clinical study in patients with mild to moderately severe COVID-19. Primary endpoints included changes in the absolute RT-PCR Ct values of the nucleocapsid and open reading frame (ORF) genes from baseline to days 3 and 7. The incidence of adverse events, changes in blood biochemistry, inflammatory biomarkers, and levels of antibodies against COVID-19 were also evaluated as part of the safety evaluation. Results: PL-M treatment significantly (p = 0.001) increased RT-PCR cycle counts for N and ORF genes on days 3 (Ct values 32.09 ± 2.39 and 30.69 ± 3.38, respectively) and 7 (Ct values 34.91 ± 0.39 and 34.85 ± 0.61, respectively) compared to a placebo treatment. On day 3, 14 subjects in the PL-M group had cycle counts for the N gene above the cut-off value of 29 (target cycle count 29), whereas on day 7, all subjects had cycle counts above the cut-off value. Ct values in placebo subjects were consistently less than 29, and no placebo subjects were RT-PCR-negative until day 7. Most of the symptoms disappeared completely after receiving PL-M treatment for 7 days in more patients compared to the placebo group. Conclusion: PL-M is safe and effective for clinical use in reducing viral loads and promoting rapid viral clearance in COVID-19 patients by inhibiting SARS-CoV-2 entry into cells through the inhibition of Gal-3.

Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2, which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants’ proteins, especially in the spike protein (SP) used for viral entry. These mutations, especially in the SP, enable the virus to evade immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of a vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase its immunogenicity.

Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large motions of its receptor binding domains (RBDs) to enter the host cell. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations, and characterize the transition pathways via inter-domain interactions. Transient salt-bridges between RBDA and RBDC and the interaction with glycan at N343B support RBDA motions from Down to one-Up. Reduced interactions between RBDA and RBDB in one-Up induce RBDB motions toward two-Up. The simulations overall agree with cryo-electron microscopy structure distributions and FRET experiments and provide hidden functional structures, namely, intermediates along Down-to-one-Up transition with druggable cryptic pockets as well as one-Open with a maximum exposed RBD. The inherent flexibility of S-protein thus provides essential information for antiviral drug rational design or vaccine development.

Objectives The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has been unprecedentedly fast, spreading to more than 180 countries within 3 months with variable severity. One of the major reasons attributed to this variation is genetic mutation. Therefore, we aimed to predict the mutations in the spike protein (S) of the SARS-CoV-2 genomes available worldwide and analyze its impact on the antigenicity. Materials and Methods Several research groups have generated whole genome sequencing data which are available in the public repositories. A total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of SARS-CoV-2 available in NCBI till May 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (SNPs) in the genomic study. Further, the antigenicity of the predicted mutations inferred, and the epitopes were superimposed on the structure of the spike protein. Results The sequence analysis resulted in high SNPs frequency. The significant variations in the predicted epitopes showing high antigenicity were A348V, V367F and A419S in receptor binding domain (RBD). Other mutations observed within RBD exhibiting low antigenicity were T323I, A344S, R408I, G476S, V483A, H519Q, A520S, A522S and K529E. The RBD T323I, A344S, V367F, A419S, A522S and K529E are novel mutations reported first time in this study. Moreover, A930V and D936Y mutations were observed in the heptad repeat domain and one mutation D1168H was noted in heptad repeat domain 2. Conclusion S protein is the major target for vaccine development, but several mutations were predicted in the antigenic epitopes of S protein across all genomes available globally. The emergence of various mutations within a short period might result in the conformational changes of the protein structure, which suggests that developing a universal vaccine may be a challenging task.

Abstract Background Infectious bronchitis (IB) is a highly contagious respiratory disease in chickens and produces economic loss within the poultry industry. This disease is caused by a single stranded RNA virus belonging to Cronaviridae family. This study aimed to design a potential multi-epitopes vaccine against infectious bronchitis virus spike protein (S). Protein characterization was also performed for IBV spike protein. Methods The present study used various tools in Immune Epitope Database (IEDB) to predict conserved B and T cell epitopes against IBV spike (S) protein that may perform a significant role in provoking the resistance response to IBV infection. Results In B cell prediction methods, three epitopes ( 1139 KKSSYY 1144 , 1140 KSSYYT 1145 , 1141 SSYYT 1145 ) were selected as surface, linear and antigenic epitopes. Many MHCI and MHCII epitopes were predicted for IBV S protein. Among them 982YYITARDMY990 and 983 YITARDMYM 991 epitopes displayed high antigenicity, no allergenicity and no toxicity as well as great linkage with MHCI and MHCII alleles. Moreover, docking analysis of MHCI epitopes produced strong binding affinity with BF2 alleles. Conclusion Five conserved epitopes were expected from spike glycoprotein of IBV as the best B and T cell epitopes due to high antigenicity, no allergenicity and no toxicity. In addition, MHC epitopes showed great linkage with MHC alleles as well as strong interaction with BF2 alleles. These epitopes should be designed and incorporated and then tested as multi-epitope vaccine against IBV.

Introduction: Coronavirus family member SARS- CoV-2 is a current worldwide threat. It enters into the epithelium membrane of respiratory tract with the help of its antigenic spike proteins and cause Coronavirus disease 2019 (COVID -19). Methods: Considering SARS- CoV-2 a potent vaccine or diagnostic candidate, a bioinformatical study was done to determine its structure homology modeling, physiological properties and structure validation with presence of antigenic sites. Results: The surface glycoprotein of SARS-CoV-2 was found to be a stable protein with stereochemically good structure. It also contains 65 antigenic sites. Conclusion: The present study suggests further wet-lab research to develop a vaccine or diagnostic kit using this promising surface glycoprotein.

The spike protein of SARS-CoV-2 triggers macrophages and epithelial cells to produce excess levels of pro-inflammatory molecules, which can do more harm than good.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, pharmaceutical companies and research institutions have been actively working to develop vaccines, and the mass roll-out of vaccinations against COVID-19 began in January 2021. At the same time, during lockdowns, the consumption of alcoholic beverages increased. During the peak of vaccination, consumption remained at high levels around the world, despite the gradual relaxation of quarantine restrictions. Two of the popular queries on search engines were whether it is safe to drink alcohol after vaccination and whether this will affect the effectiveness of vaccines. Over the past two years, many studies have been published suggesting that excessive drinking not only worsens the course of an acute respiratory distress syndrome caused by the SARS-CoV-2 virus but can also exacerbate post-COVID-19 syndrome. Despite all sorts of online speculation, there is no specific scientific data on alcohol-induced complications after vaccination in the literature. Most of the published vaccine clinical trials do not include groups of patients with a history of alcohol-use disorders. This review analyzed the well-known and new mechanisms of action of COVID-19 vaccines on the immune system and the effects of alcohol and its metabolites on these mechanisms.

While most approved vaccines are based on the viral spike protein or its immunogenic regions, inactivated whole-virion vaccines (e.g., CoronaVac) contain additional antigens that may enhance protection. This study analyzes short-term humoral responses against the SARS-CoV-2 spike (S1) and nucleocapsid (NCP) protein in 50 Turkish adults without previous SARS-CoV-2 infection after CoronaVac immunization. Samples were collected before vaccination (t0), 28–29 days after the first vaccine dose and prior to the second dose (t1), as well as 14–15 days after the second dose (t2). Anti-S1 IgG and IgA as well as anti-NCP IgG were quantified using ELISA. At t1, seroconversion rates for anti-S1 IgG, anti-S1 IgA and anti-NCP IgG were 30.0%, 28.0% and 4.0%, respectively, increasing significantly to 98.0%, 78.0% and 40.0% at t2. The anti-NCP IgG median (t2) was below the positivity cut-off, while anti-S1 IgG and IgA medians were positive. Anti-S1 IgG levels strongly correlated with anti-S1 IgA (rs = 0.767, p < 0.001) and anti-NCP IgG (rs = 0.683, p < 0.001). In conclusion, two CoronaVac doses induced significant increases in antibodies against S1 and NCP. Despite strong correlations between the antibody concentrations, the median levels and seroconversion rates of S1-specific responses exceed those of NCP-specific responses as early as two weeks after the second vaccine dose.