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COVID-19, caused by the SARS-CoV-2 virus, is a highly pathogenic emerging infectious disease. Healthcare personnel (HCP) are presumably at higher risk of acquiring emerging infections because of occupational exposure. The prevalence of COVID-19 in HCP is unknown, particularly in low- to middle-income countries like El Salvador. The goal of this study was to determine the seroprevalence of anti-SARS-CoV-2 antibodies among HCP in El Salvador just prior to vaccine rollout in March 2021. We evaluated 2176 participants from a nationally representative sample of national healthcare institutions. We found 40.4% (n = 880) of the study participants were seropositive for anti-spike protein antibodies. Significant factors associated with infection included younger age; living within the central, more populated zone of the country; living in a larger household (≥7 members); household members with COVID-19 or compatible symptoms; and those who worked in auxiliary services (i.e., housekeeping and food services). These findings provide insight into opportunities to mitigate SARS-CoV-2 risk and other emerging respiratory pathogens in HCP in El Salvador.

Coronaviruses continue to disrupt health and economic productivity worldwide. Porcine epidemic diarrhea virus (PEDV) is a devastating swine disease and SARS-CoV-2 is the latest coronavirus to infect the human population. Both viruses display a similar spike protein on the surface that is a target of vaccine development. Despite the availability of commercial vaccines for both viruses, there is still a high occurrence of infections and a great need for enhanced efficacy and lower costs. We previously produced the PEDV spike protein (S) using transgenic maize, enabling a low-cost supply of the vaccine candidate. In this study, we (1) test orally delivered PEDV vaccine candidates in pigs to optimize the mucosal immune response; (2) generate the SARS-CoV-2 S1 protein in maize; and (3) perform structural characterization of the S1 protein for PEDV and SARS-CoV-2. We demonstrated high expression levels in maize of the S1 subunit of the SARS-CoV-2 spike protein, both with and without fusion to the heat-labile enterotoxin B (LTB) subunit. We found that the LTB fusion protein from both coronaviruses preferentially assembles into higher molecular weight multimers, consistent with the formation of trimers. For PEDV, administering the spike protein fused to LTB to young pigs elicited a higher level of mucosal IgAs compared to maize grain containing the S1 protein alone or controls. This suggests that fusing the coronavirus spike protein with LTB may provide better protection.

Background: Although treatments using thermal water have yielded beneficial effects in respiratory tract infections, the effects of thermal water under experimental conditions similar to those triggered by SARS-CoV-2 have yet to be evaluated. This study aimed to assess whether thermal water could interfere with the interaction between SARS-CoV-2 and host cells and influence inflammatory factors. Methods: Human nasal epithelial primary cells (HNEpCs) were stimulated with SARS-CoV-2 spike protein in the presence or absence of thermal water or tap water. Cell viability, cytokine concentration, ACE2 and TMPRSS2 levels, and ACE2 activity were determined in the cell cultures. Results: Exposure of HNEpCs to spike protein increased IL-6, IL-8, and IL-1β production, with decreased production observed in the presence of thermal water at an optimal dose. Treatment of cells with tap water did not affect cytokine release in unstimulated or spike-stimulated cells. Spike-protein-stimulated HNEpCs showed reduced levels of ACE2, which were partially restored only in the presence of thermal water. Spike protein did not affect the TMPRSS2 levels of the cell lysates. Stimulation with spike protein induced an increase in the concentration of both receptors in the supernatants, while treatment with thermal water reduced TMPRSS2 levels in both the cells and supernatants. Stimulation with spike protein increased ACE2 activity, which was reduced with thermal water. Conclusions: This study shows the regulatory effects of mineral-rich thermal water on spike-protein-induced pro-inflammatory cytokine production and the amount and activity of receptors mainly involved in viral entry, suggesting a potential use of this treatment as a support therapy for SARS-CoV-2 infection of the upper respiratory tract.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, known for its immune evasion and resistance to chemotherapy. Evidence indicates that the SARS-CoV-2 virus may worsen outcomes for RCC patients, as well as patients with diminished renal function. Evidence suggests that the SARS-CoV-2 virus may exacerbate outcomes in RCC patients and those with impaired renal function. This study explored the unidirectional effects of RCC cells and the SARS-CoV-2 spike protein (S protein) on human renal proximal tubule epithelial cells (RPTECs) using a microphysiological approach. We co-cultured RCC cells (Caki-1) with RPTEC and exposed them to the SARS-CoV-2 S protein under dynamic 3D conditions. The impact on metabolic activity, gene expression, immune secretions, and S protein internalization was evaluated. The SARS-CoV-2 S protein was internalized by RPTEC but poorly interacted with RCC cells. RPTECs exposed to RCC cells and the S protein exhibited upregulated expression of genes involved in immunogenic pathways, particularly those related to antigen processing and presentation via the major histocompatibility complex I (MHCI). Additionally, increased TNF-α secretion suggested a pro-inflammatory response. Metabolic shifts toward glycolysis were observed in RCC co-culture, while the presence of the S protein led to minor changes. The presence of RCC cells amplified the immune-modulatory effects of the SARS-CoV-2 S protein on the renal epithelium, potentially exacerbating renal inflammation and fostering tumor-supportive conditions. These findings suggest that COVID-19 infections can impact renal function in the presence of kidney cancer.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) virus and the resulting COVID-19 pandemic have had devastating and lasting impact on the global population. Although the main target of the disease is the respiratory tract, clinical outcomes, and research have also shown significant effects of infection on other organ systems. Of interest in this review is the effect of the virus on the cardiovascular system. Complications, including hyperinflammatory syndrome, myocarditis, and cardiac failure, have been documented in the context of COVID-19 infection. These complications ultimately contribute to worse patient outcomes, especially in patients with pre-existing conditions such as hypertension, diabetes, or cardiovascular disease (CVD). Importantly and interestingly, reports have demonstrated that COVID-19 also causes myocardial injury in adults without pre-existing conditions and contributes to systemic complications in pediatric populations, such as the development of multisystem inflammatory syndrome in children (MIS-C). Although there is still a debate over the exact mechanisms by which such complications arise, understanding the potential paths by which the virus can influence the cardiovascular system to create an inflammatory environment may clarify how SARS-CoV-2 interacts with human physiology. In addition to describing the mechanisms of disease propagation and patient presentation, this review discusses the diagnostic findings and treatment strategies and the evolution of management for patients presenting with cardiovascular complications, focusing on disease treatment and prevention.

SARS-CoV-2, the causative virus for the COVID-19 disease, uses its spike glycoprotein to bind to human ACE2 as a first step for viral entry into the cell. For this reason, great efforts have been made to find mechanisms that disrupt this interaction, avoiding the infection. Nitric oxide (NO) is a soluble endogenous gas with known antiviral and immunomodulatory properties. In this study, we aimed to test whether NO could inhibit the binding of the viral spike to ACE2 in human cells and its effects on ACE2 enzymatic activity. Our results show that ACE2 activity was decreased by the NO donors DETA-NONOate and GSNO and by the NO byproduct peroxynitrite. Furthermore, we found that DETA-NONOate could break the spike–ACE2 interaction using the spike from two different variants (Alpha and Gamma) and in two different human cell types. Moreover, the same result was obtained when using NO-producing murine macrophages, while no significant changes were observed in ACE2 expression or distribution within the cell. These results support that it is worth considering NO as a therapeutic agent for COVID-19, as previous reports have suggested.

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer’s dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells.

The emergence of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has sparked intense research on its spike protein, which is essential for viral entrance into host cells. Viral reproduction and transmission, host immune response regulation, receptor recognition and host cell entrance mechanisms, as well as structural and functional effects have all been linked to mutations in the spike protein. Spike protein mutations can also result in immune evasion mechanisms that impair vaccine effectiveness and escape, and they are linked to illness severity and clinical consequences. Numerous studies have been conducted to determine the effects of these mutations on the spike protein structure and how it interacts with host factors. These results have important implications for the design and development of medicines and vaccines based on spike proteins as well as for the assessment of those products’ efficiency against newly discovered spike protein mutations. This paper gives a general overview of how spike protein mutations are categorized and named. It further looks at the links between spike protein mutations and clinical outcomes, illness severity, unanswered problems, and future research prospects. Additionally, explored are the effects of these mutations on vaccine effectiveness as well as the possible therapeutic targeting of spike protein mutations.

Multidisciplinary research efforts on potential COVID-19 vaccine and therapeutic candidates have increased since the pandemic outbreak of SARS-CoV-2 in 2019. This search has become imperative due to the increasing emergences and limited widely available medicines. The presence of bioactive anti-SARS-CoV-2 molecules was examined from various plant sources. Among them is a group of proteins called lectins that can bind carbohydrate moieties. In this article, we present ten novel, chitin-specific Hevein-like lectins that were derived from Selaginella moellendorffii v1.0’s genome. The capacity of these lectin homologs to bind with the spike protein of SARS-CoV-2 was examined. Using the HDOCK server, 3D-modeled Hevein-domains were docked to the spike protein’s receptor binding domain (RBD). The Smo446851, Smo125663, and Smo99732 interacted with Asn343-located complex N-glycan and RBD residues, respectively, with binding free energies of −17.5, −13.0, and −26.5 Kcal/mol. The molecular dynamics simulation using Desmond and the normal-state analyses via torsional coordinate association for the Smo99732-RBD complex using iMODS is characterized by overall higher stability and minimum deformity than the other lectin complexes. The three lectins interacting with carbohydrates were docked against five individual mutations that frequently occur in major SARS-CoV-2 variants. These were in the spike protein’s receptor-binding motif (RBM), while Smo125663 and Smo99732 only interacted with the spike glycoprotein in a protein–protein manner. The precursors for the Hevein-like homologs underwent additional characterization, and their expressional profile in different tissues was studied. These in silico findings offered potential lectin candidates targeting key N-glycan sites crucial to the virus’s virulence and infection.

SARS-CoV-2, a single stranded positive RNA-virus, emerged in 2019 and caused a global pandemic. Some infected patients develop severe complications, such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. The mechanisms leading to lung fibrosis in the context of COVID-19 are still unclear. However, there are correlations between this condition and certain inflammatory mediators that are elevated in the plasma of patients. This study addressed the question of whether SARS-CoV-2 spike protein was able to directly activate immune cells to produce inflammatory and fibrogenic cytokines, independent of viral infection. By stimulating peripheral blood mononuclear cells (PBMC) from healthy blood donors through different formulations of SARS-CoV-2 spike protein, the expression of fibrogenic and inflammatory cytokines was measured. The spike protein induced a significantly increased expression of IL-1β and IL-6 mRNA in PBMC. Both cytokines are important players in the COVID-19 cytokine storm, in ARDS and in the development of pulmonary fibrosis. Different receptors with an affinity to the spike protein may be involved. However, the exact mechanism on how the spike protein leads to a higher cytokine expression in PBMC needs further investigation.

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in 2021 as a variant with heavy amino acid mutations in the spike protein, which is targeted by most vaccines, compared to previous variants. Amino acid substitutions in the spike proteins may alter their affinity for host viral receptors and the host interactome. Here, we found that the receptor-binding domain (RBD) of the omicron variant of SARS-CoV-2 exhibited an increased affinity for human angiotensin-converting enzyme 2, a viral cell receptor, compared to the prototype RBD. Moreover, we identified β- and γ-actin as omicron-specific binding partners of RBD. Protein complex predictions revealed that many omicron-specific amino acid substitutions affected the affinity between RBD of the omicron variant and actin. Our findings indicate that proteins localized to different cellular compartments exhibit strong binding to the omicron RBD.

Background: 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. Methods: 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. Results: 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. Conclusion: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner.

The rapid spread of SARS-COV-2 and the millions of worldwide deaths and hospitalizations have prompted an urgent need for the development of screening tests capable of rapidly and accurately detecting the virus, even in asymptomatic people. The easy collection and the biomarker content of saliva, together with the label-free and informative power of surface-enhanced Raman spectroscopy (SERS) analysis have driven the creation of point-of-care platforms capable of identifying people with COVID-19. Indeed, different salivary fingerprints were observed between uninfected and infected people. Hence, we performed a retrospective analysis of SERS spectra from salivary samples of COVID-19-infected and -vaccinated subjects to understand if viral components and/or the immune response are implicated in spectral variations. The high sensitivity of the proposed SERS-based method highlighted the persistence of molecular alterations in saliva up to one month after the first positive swab, even when the subject tested negative for the rapid antigenic test. Nevertheless, no specific spectral patterns attributable to some viral proteins and immunoglobulins involved in COVID-19 infection and its progression were found, even if differences in peak intensity, presence, and position were observed in the salivary SERS fingerprint.

The global effort to combat the COVID-19 pandemic faces ongoing uncertainty with the emergence of Variants of Concern featuring numerous mutations on the Spike (S) protein. In particular, the Omicron Variant is distinguished by 32 mutations, including 10 within its receptor-binding domain (RBD). These mutations significantly impact viral infectivity and the efficacy of vaccines and antibodies currently in use for therapeutic purposes. In our study, we employed structure-based computational saturation mutagenesis approaches to predict the effects of Omicron missense mutations on RBD stability and binding affinity, comparing them to the original Wuhan-Hu-1 strain. Our results predict that mutations such as G431W and P507W induce the most substantial destabilizations in the Wuhan-Hu-1-S/Omicron-S RBD. Notably, we postulate that mutations in the Omicron-S exhibit a higher percentage of enhancing binding affinity compared to Wuhan-S. We found that the mutations at residue positions G447, Y449, F456, F486, and S496 led to significant changes in binding affinity. In summary, our findings may shed light on the widespread prevalence of Omicron mutations in human populations. The Omicron mutations that potentially enhance their affinity for human receptors may facilitate increased viral binding and internalization in infected cells, thereby enhancing infectivity. This informs the development of new neutralizing antibodies capable of targeting Omicron’s immune-evading mutations, potentially aiding in the ongoing battle against the COVID-19 pandemic.

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.

The Omicron variant and its sub-lineages are the only current circulating SARS-CoV-2 viruses worldwide. In this study, the conformational stability of the isolated Receptor Binding Domain (RBD) of Omicron’s spike protein is examined in detail. The parent Omicron lineage has over ten mutations in the ACE2 binding region of the RBD that are specifically associated with its β hairpin loop domain. It is demonstrated through biophysical molecular computations that the mutations in the β hairpin loop domain significantly increase the intra-protein interaction energies of intra-loop and loop–RBD interactions. The interaction energy increases include the formation of new hydrogen bonds in the β hairpin loop domain that help stabilize this critical ACE2 binding region. Our results also agree with recent experiments on the stability of Omicron’s core β barrel domain, outside of its loop domain, and help demonstrate the overall conformational stability of the Omicron RBD. It is further shown here through dynamic simulations that the unbound state of the Omicron RBD remains closely aligned with the bound state configuration, which was not observed for the wild-type RBD. Overall, these studies demonstrate the significantly increased conformational stability of Omicron over its wild-type configuration and raise a number of questions on whether conformational stability could be a positive selection feature of SARS-CoV-2 viral mutational changes.

The continuous emergence of new variants of concern for SARS-CoV-2 has created a challenge for existing therapies. To address this, we developed a series of single-stranded DNA aptamers that not only bind specifically to the trimer S protein of SARS-CoV-2 but also block the interaction between the trimer S protein and ACE2 receptors. The systematic evolution of ligands by exponential enrichment (SELEX) was performed to select the aptamers for SARS-CoV-2 trimer S protein. ELISA-based assay and flow cytometry were performed to test the apatmers’ binding and inhibition of trimer S protein in vitro. Binding affinity was measured using surface plasmon resonance. Significance was determined in Prism 9.0 using the one-way ANOVA test (Dunnett’s multiple comparisons test) or two-way ANOVA test (Tukey’s multiple comparisons test) for comparisons. The p values < 0.05 were considered statistically significant. After 12 rounds of SELEX, eight highly enriched aptamers were able to bind to the trimer S protein of the SARS-CoV-2 Wuhan original strain as well as the trimer S proteins of the Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants, with affinities in the nM range, while also inhibiting their interaction with ACE2 receptors in Vero E6 cells. Modifications to our best aptamer were made by adding forward and reverse primer sequences and truncation. The modified aptamers AYA2012004_L and AYA2012004_L-M1 showed up to 70% inhibition of the binding of virus-like particles (VLPs) expressing S protein to the ACE2 receptor expressed in HEK293T cells. Our findings imply that the selected aptamers can prevent SARS-CoV-2 from entering host cells and hence suppress the viral infection. In addition, the findings suggest that the selected aptamers might be an innovative therapy for the treatment of COVID-19.

Pharmacovigilance databases are showing evidence of injury in the context of the modified COVID-19 mRNA products. According to recent publications, adverse event reports linked to the mRNA COVID-19 injections largely point to the spike protein as an aetiological agent of adverse events, but we propose that the platform itself may be culpable. To assess the safety of current and future mRNA vaccines, further analysis is needed on the risks due to the platform itself, and not specifically the expressed antigen. If harm can be exclusively and conclusively attributed to the spike protein, then it is possible that future mRNA vaccines expressing other antigens will be safe. If harms are attributable to the platform itself, then regardless of the toxicity, or lack thereof, of the antigen to be expressed, the platform may be inherently unsafe, pending modification. In this work, we examine previous studies of RNA-based delivery by a lipid nanoparticle (LNP) and break down the possible aetiological elements of harm.

Protein three-dimensional structural analysis using artificial intelligence is attracting attention in various fields, such as the estimation of vaccine structure and stability. In particular, when using the spike protein in vaccines, the major issues in the construction of SARS-CoV-2 vaccines are their weak abilities to attack the virus and elicit immunity for a short period. Structural information about new viruses is essential for understanding their properties and creating effective vaccines. However, determining the structure of a protein through experiments is a lengthy and laborious process. Therefore, a new computational approach accelerated the elucidation process and made predictions more accurate. Using advanced machine learning technology called deep neural networks, it has become possible to predict protein structures directly from protein and gene sequences. We summarize the advances in antiviral therapy with the SARS-CoV-2 vaccine and extracellular vesicles via computational analysis.

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.

Human coronaviruses (HCoVs) are seriously associated with respiratory diseases in humans and animals. The first human pathogenic SARS-CoV emerged in 2002–2003. The second was MERS-CoV, reported from Jeddah, the Kingdom of Saudi Arabia, in 2012, and the third one was SARS-CoV-2, identified from Wuhan City, China, in late December 2019. The HCoV-Spike (S) gene has the highest mutation/insertion/deletion rate and has been the most utilized target for vaccine/antiviral development. In this manuscript, we discuss the genetic diversity, phylogenetic relationships, and recombination patterns of selected HCoVs with emphasis on the S protein gene of MERS-CoV and SARS-CoV-2 to elucidate the possible emergence of new variants/strains of coronavirus in the near future. The findings showed that MERS-CoV and SARS-CoV-2 have significant sequence identity with the selected HCoVs. The phylogenetic tree analysis formed a separate cluster for each HCoV. The recombination pattern analysis showed that the HCoV-NL63-Japan was a probable recombinant. The HCoV-NL63-USA was identified as a major parent while the HCoV-NL63-Netherland was identified as a minor parent. The recombination breakpoints start in the viral genome at the 142 nucleotide position and end at the 1082 nucleotide position with a 99% CI and Bonferroni-corrected p-value of 0.05. The findings of this study provide insightful information about HCoV-S gene diversity, recombination, and evolutionary patterns. Based on these data, it can be concluded that the possible emergence of new strains/variants of HCoV is imminent.

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.

As SARS-CoV-2 continues to evolve and COVID-19 cases rapidly increase among children and adults, there is an urgent need for a safe and effective vaccine that can elicit systemic and mucosal humoral immunity to limit the emergence of new variants. Using the Chinese Hu191 measles virus (MeV-hu191) vaccine strain as a backbone, we developed MeV chimeras stably expressing the prefusion forms of either membrane-anchored, full-length spike (rMeV-preFS), or its soluble secreted spike trimers with the help of the SP-D trimerization tag (rMeV-S+SPD) of SARS-CoV-2 Omicron BA.2. The two vaccine candidates were administrated in golden Syrian hamsters through the intranasal or subcutaneous routes to determine the optimal immunization route for challenge. The intranasal delivery of rMeV-S+SPD induced a more robust mucosal IgA antibody response than the subcutaneous route. The mucosal IgA antibody induced by rMeV-preFS through the intranasal routine was slightly higher than the subcutaneous route, but there was no significant difference. The rMeV-preFS vaccine stimulated higher mucosal IgA than the rMeV-S+SPD vaccine through intranasal or subcutaneous administration. In hamsters, intranasal administration of the rMeV-preFS vaccine elicited high levels of NAbs, protecting against the SARS-CoV-2 Omicron BA.2 variant challenge by reducing virus loads and diminishing pathological changes in vaccinated animals. Encouragingly, sera collected from the rMeV-preFS group consistently showed robust and significantly high neutralizing titers against the latest variant XBB.1.16. These data suggest that rMeV-preFS is a highly promising COVID-19 candidate vaccine that has great potential to be developed into bivalent vaccines (MeV/SARS-CoV-2).

Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three—SARS, SARS-CoV-2 and MERS—are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them.

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.

The surface spike (S) glycoprotein mediates cell entry of SARS-CoV-2 into the host through fusion at the plasma membrane or endocytosis. Omicron lineages/sublineages have acquired extensive mutations in S to gain transmissibility advantages and altered antigenicity. The fusogenicity, antigenicity, and evasion of Omicron subvariants have been extensively investigated at unprecedented speed to align with the mutation rate of S. Cells that overexpress receptors/cofactors are mostly used as hosts to amplify infection sensitivity to tested variants. However, systematic cell entry comparisons of most prior dominant Omicron subvariants using human lung epithelium cells are yet to be well-studied. Here, with human bronchial epithelium BEAS-2B cells as the host, we compared single-round virus-to-cell entry and cell-to-cell fusion of Omicron BA.1, BA.5, BQ.1.1, CH.1.1, XBB.1.5, and XBB.1.16 based upon split NanoLuc fusion readout assays and the S-pseudotyped lentivirus system. Virus-to-cell entry of tested S variants exhibited cell-type dependence. The parental Omicron BA.1 required more time to develop full entry to HEK293T-ACE2-TMPRSS2 than BEAS-2B cells. Compared to unchanged P681, S-cleavage constructs of P681H/R did not have any noticeable advantages in cell entry. Omicron BA.1 and its descendants entered BEAS-2B cells more efficiently than D614G, and it was slightly less or comparable to that of Delta. Serine protease-pretreated Omicron subvariants enhanced virus-to-cell entry in a dose-dependent manner, suggesting fusion at the plasma membrane persists as a productive cell entry route. Spike-mediated cell-to-cell fusion and total S1/S2 processing of Omicron descendants were similar. Our results indicate no obvious entry or fusion advantages of recent Omicron descendants over preceding variants since Delta, thus supporting immune evasion conferred by antigenicity shifts due to altered S sequences as probably the primary viral fitness driver.

The emergence of COVID-19 in March 2020 challenged Zimbabwe to respond with limited medical facilities and therapeutic options. Based on early clinical indications of efficacy for the macrocyclic lactone, Ivermectin (IVM), against COVID-19, IVM-based combination treatments were deployed to treat it. Oxygen saturation (SpO2) data were retrospectively analyzed for 34 severe, hypoxic COVID-19 patients all on room air (without supplemental oxygen). The patients, median age 56.5, were treated at clinics or at home between August 2020 and May 2021. All but three of these 34 patients had significantly increased SpO2 values within 24 h after the first IVM dose. The mean increase in SpO2 as a percentage of full normalization to SpO2 = 97 was 55.1% at +12 h and 62.3% at +24 h after the first IVM dose (paired t-test, p < 0.0000001). These results parallel similar sharp, rapid increases in SpO2, all on room air, for 24 mostly severe COVID-19 patients in the USA (California) who were given an IVM-based combination treatment. All patients in both of these critical series recovered. These rapid increases in SpO2 values after IVM treatment stand in sharp contrast to declines in SpO2 and associated pulmonary function through the second week following the onset of moderate or severe COVID-19 symptoms under standard care.

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

The slow experimental acquisition of high-quality atomic structures of the rapidly changing proteins of the COVID-19 virus challenges vaccine and therapeutic drug development efforts. Fortunately, deep learning tools such as AlphaFold2 can quickly generate reliable models of atomic structure at experimental resolution. Current modeling studies have focused solely on definitions of mutant constellations of Variants of Concern (VOCs), leaving out the impact of haplotypes on protein structure. Here, we conduct a thorough comparative structural analysis of S-proteins belonging to major VOCs and corresponding latitude-delimited haplotypes that affect viral seasonal behavior. Our approach identified molecular regions of importance as well as patterns of structural recruitment. The S1 subunit hosted the majority of structural changes, especially those involving the N-terminal domain (NTD) and the receptor-binding domain (RBD). In particular, structural changes in the NTD were much greater than just translations in three-dimensional space, altering the sub-structures to greater extents. We also revealed a notable pattern of structural recruitment with the early VOCs Alpha and Delta behaving antagonistically by suppressing regions of structural change introduced by their corresponding haplotypes, and the current VOC Omicron behaving synergistically by amplifying or collecting structural change. Remarkably, haplotypes altering the galectin-like structure of the NTD were major contributors to seasonal behavior, supporting its putative environmental-sensing role. Our results provide an extensive view of the evolutionary landscape of the S-protein across the COVID-19 pandemic. This view will help predict important regions of structural change in future variants and haplotypes for more efficient vaccine and drug development.

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 world-wide COVID-19 pandemic has promoted a series of alternative vaccination strategies aiming to elicit neutralizing adaptive immunity in the human host. However, restricted efficacies of these vaccines targeting epitopes on the spike (S) protein that is involved in primary viral entry were observed and putatively assigned to viral glycosylation as an effective escape mechanism. Besides the well-recognized N-glycan shield covering SARS-CoV-2 spike (S) proteins, immunization strategies may be hampered by heavy O-glycosylation and variable O-glycosites fluctuating depending on the organ sites of primary infection and those involved in immunization. A further complication associated with viral glycosylation arises from the development of autoimmune antibodies to self-carbohydrates, including O-linked blood group antigens, as structural parts of viral proteins. This outline already emphasizes the importance of viral glycosylation in general and, in particular, highlights the impact of the site-specific O-glycosylation of virions, since this modification is independent of sequons and varies strongly in dependence on cell-specific repertoires of peptidyl-N-acetylgalactosaminyltransferases with their varying site preferences and of glycan core-specific glycosyltransferases. This review summarizes the current knowledge on the viral O-glycosylation of the SARS-CoV-2 spike protein and its impact on virulence and immune modulation in the host.

In its prefusion state, the SARS-CoV-2 spike protein (similarly to other class I viral fusion proteins) is metastable, which is considered to be an important feature for optimizing or regulating its functions. After the binding process of its S1 subunit (S1) with ACE2, the spike protein (S) undergoes a dramatic conformational change where S1 splits from the S2 subunit, which then penetrates the membrane of the host cell, promoting the fusion of the viral and cell membranes. This results in the infection of the host cell. In a previous work, we showed—using large-scale molecular dynamics simulations—that the application of external electric fields (EFs) induces drastic changes and damage in the receptor-binding domain (RBD) of the wild-type spike protein, as well of the Alpha, Beta, and Gamma variants, leaving a structure which cannot be recognized anymore by ACE2. In this work, we first extend the study to the Delta and Omicron variants and confirm the high sensitivity and extreme vulnerability of the RBD of the prefusion state of S to moderate EF (as weak as 104 V/m), but, more importantly, we also show that, in contrast, the S2 subunit of the postfusion state of the spike protein does not suffer structural damage even if electric field intensities four orders of magnitude higher are applied. These results provide a solid scientific basis to confirm the connection between the prefusion-state metastability of the SARS-CoV-2 spike protein and its susceptibility to be damaged by EF. After the virus docks to the ACE2 receptor, the stable and robust postfusion conformation develops, which exhibits a similar resistance to EF (damage threshold higher than 108 V/m) like most globular proteins.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging porcine intestinal coronavirus that can cause acute diarrhea, vomiting, rapid weight loss, and high mortality in newborn piglets. Cholesterol 25-hydroxylase (CH25H) is a molecular mediator of innate antiviral immunity and converts cholesterol to 25-hydroxycholesterol (25HC). Previous studies have reported that CH25H and 25HC have an antiviral effect against multiple viruses. However, the interplay between SADS-CoV infection and CH25H or 25HC is still uncertain. Here, we found that CH25H and its enzymatic product 25HC restrained SADS-CoV replication by blocking membrane fusion. Our results show that CH25H was upregulated by SADS-CoV infection in vitro and in vivo, and that it was an IFN-stimulated gene in porcine ileum epithelial cells. Moreover, CH25H and CH25H mutants lacking catalytic activity can inhibit SADS-CoV replication. Furthermore, 25HC significantly suppressed SADS-CoV infection by inhibiting virus entry. Notably, we confirmed that CH25H and 25HC blocked SADS-CoV spike protein-mediated membrane fusion. Our data provide a possible antiviral therapy against SADS-CoV and other conceivable emerging coronaviruses in the future.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)–NTD complexes, supported by molecular dynamics simulations and hydrogen–deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

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.

The Omicron variant of SARS-CoV-2 bears peptide sequence alterations that correlate with a higher infectivity than was observed in the original SARS-CoV-2 isolated from Wuhan, China. We analyzed the CendR motif of spike protein and performed in silico molecular docking with neuropilin-1 (Nrp1), a receptor–ligand interaction known to support infection by the original variant. Our analysis predicts conserved and slightly increased energetic favorability of binding for Omicron CendR:Nrp1. We propose that the viral spike:Nrp1 coreceptor pathway may contribute to the infectivity of the Omicron variant of SARS-CoV-2.

Background/Objectives : While post-acute COVID-19 syndrome is well known and extensively studied, the post-acute COVID vaccination syndrome (PACVS) is a more recent nosological entity that is poorly defined at the immunopathological level, although it shares many symptoms with the sequelae of viral infections. Methods : This single-center retrospective study reports a case series of 17 subjects vaccinated with mRNA or adenoviral vector vaccines who were healthy before vaccination and had never been infected with SARS-CoV-2 but who presented with symptoms similar to PACVS for a median time of 20 months (min 4, max 32). The medical records of all patients referred to our outpatient clinic over a one-year period were retrospectively analyzed. Results : In this group, serological tests showed that, in addition to positivity for anti-spike protein antibodies, a high percentage of subjects were positive for antibodies against G protein-coupled receptors and molecules involved in the response to SARS-CoV-2. In a panel of 16 autoantibodies tested, a few were positively associated with some of the symptoms reported by patients: anti-ATR1 with lymphadenopathy and/or tonsillitis; anti-ACE2 with skin symptoms such as ecchymosis, skin oedema, and rash; anti-MAS1 with widespread burning sensation; and anti-STAB1 with skin oedema and rash. Anti-ADRA2A were negatively associated with memory loss and/or mental fog. ACE2 correlated with the serum levels of anti-S antibodies, supporting the hypothesis of an anti-idiotype mechanism in the immunopathogenesis of PACVS. Conclusions : This exploratory analysis suggests that the levels of autoantibodies directed against ACE2, and probably also MAS1 and STAB1, may serve as biomarkers for PACVS.

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.

Cardiovascular complications are major clinical hallmarks of acute and post-acute coronavirus disease 2019 (COVID-19). However, the mechanistic details of SARS-CoV-2 infectivity of endothelial cells remain largely unknown. Here, we demonstrate that the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein shares a similarity with the proline-rich binding ena/VASP homology (EVH1) domain and identified the endoplasmic reticulum (ER) resident calreticulin (CALR) as an S-RBD interacting protein. Our biochemical analysis showed that CALR, via its proline-rich (P) domain, interacts with S-RBD and modulates proteostasis of the S protein. Treatment of cells with the proteasomal inhibitor bortezomib increased the expression of the S protein independent of CALR, whereas the lysosomal/autophagy inhibitor bafilomycin 1A, which interferes with the acidification of lysosome, selectively augmented the S protein levels in a CALR-dependent manner. More importantly, the shRNA-mediated knockdown of CALR increased SARS-CoV-2 infection and impaired calcium homeostasis of human endothelial cells. This study provides new insight into the infectivity of SARS-CoV-2, calcium hemostasis, and the role of CALR in the ER-lysosome-dependent proteolysis of the spike protein, which could be associated with cardiovascular complications in COVID-19 patients.

The spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on host cell surface glycans to facilitate interaction with the angiotensin-converting enzyme 2 (ACE-2) receptor. This interaction between ACE2 and the spike protein is a gateway for the virus to enter host cells and may be targeted by antiviral drugs to inhibit viral infection. Therefore, targeting the interaction between these two proteins is an interesting strategy to prevent SARS-CoV-2 infection. A library of glycan mimetics and derivatives was selected for a virtual screening performed against both ACE2 and spike proteins. Subsequently, in vitro assays were performed on eleven of the most promising in silico compounds to evaluate: (i) their efficacy in inhibiting cell infection by SARS-CoV-2 (using the Vero CCL-81 cell line as a model), (ii) their impact on ACE2 expression (in the Vero CCL-81 and MDA-MB-231 cell lines), and (iii) their cytotoxicity in a human lung cell line (A549). We identified five synthetic compounds with the potential to block SARS-CoV-2 infection, three of them without relevant toxicity in human lung cells. Xanthene 1 stood out as the most promising anti-SARS-CoV-2 agent, inhibiting viral infection and viral replication in Vero CCL-81 cells, without causing cytotoxicity to human lung cells.

TNX-1800 is a preclinical stage synthetic-derived live attenuated chimeric horsepox virus vaccine engineered to express the SARS-CoV-2 spike (S) gene. The objectives of this study were to assess the safety, tolerability, and immunogenicity of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated at three doses via percutaneous inoculation. The data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. At all dose levels, rabbits were more decerning regarding vaccine site reaction than hamsters. Lastly, no TNX-1800 genomes could be detected at the site of vaccination. Post-vaccination, all animals had anti-SARS-CoV-2 spike protein IgG specific antibody responses. These data demonstrate that TNX-1800 infection was limited, asymptomatic, and cleared by the end of this study, and a single dose was able to generate immune responses.

To contain the spread of the SARS-CoV-2 pandemic, rapid development of vaccines was required in 2020. Rational design, international efforts, and a lot of hard work yielded the market approval of novel SARS-CoV-2 vaccines based on diverse platforms such as mRNA or adenovirus vectors. The great success of these technologies, in fact, contributed significantly to control the pandemic. Consequently, most scientific literature available in the public domain discloses the results of clinical trials and reveals data of efficaciousness. However, a description of processes and rationales that led to specific vaccine design is only partially available, in particular for adenovirus vectors, even though it could prove helpful for future developments. Here, we disclose our insights from the endeavors to design compatible functional adenoviral vector platform expression cassettes for the SARS-CoV-2 spike protein. We observed that contextualizing genes from an ssRNA virus into a DNA virus provides significant challenges. Besides affecting physical titers, expression cassette design of adenoviral vaccine candidates can affect viral propagation and spike protein expression. Splicing of mRNAs was affected, and fusogenicity of the spike protein in ACE2-overexpressing cells was enhanced when the ER retention signal was deleted.

(1) Background: The COVID-19 pandemic left many intriguing mysteries. Retrospective vulnerability trends tie as strongly to odd demographics as to exposure profiles, genetics, health, or prior medical history. This article documents the importance of nasal microbiome profiles in distinguishing infection rate trends among differentially affected subgroups. (2) Hypothesis: From a detailed literature survey, microbiome profiling experiments, bioinformatics, and molecular simulations, we propose that specific commensal bacterial species in the Pseudomonadales genus confer protection against SARS-CoV-2 infections by expressing proteases that may interfere with the proteolytic priming of the Spike protein. (3) Evidence: Various reports have found elevated Moraxella fractions in the nasal microbiomes of subpopulations with higher resistance to COVID-19 (e.g., adolescents, COVID-19-resistant children, people with strong dietary diversity, and omnivorous canines) and less abundant ones in vulnerable subsets (the elderly, people with narrower diets, carnivorous cats and foxes), along with bioinformatic evidence that Moraxella bacteria express proteases with notable homology to human TMPRSS2. Simulations suggest that these proteases may proteolyze the SARS-CoV-2 spike protein in a manner that interferes with TMPRSS2 priming.

The SARS-CoV-2 virus rapidly spread worldwide, threatening public health. Since it emerged, the scientific community has been engaged in the development of effective therapeutics and vaccines. The subunit S1 in the spike protein of SARS-CoV-2 mediates the viral entry into the host and is therefore one of the major research targets. The S1 protein is extensively glycosylated, and there is compelling evidence that glycans protect the virus’ active site from the human defense system. Therefore, investigation of the S1 protein glycome alterations in the different virus variants will provide a view of the glycan evolution and its relationship with the virus pathogenesis. In this study, we explored the N-glycosylation expression of the S1 protein for eleven SARS-CoV-2 variants: five variants of concern (VOC), including alpha, beta, gamma, delta, and omicron, and six variants of interest (VOI), including epsilon, eta, iota, lambda, kappa, and mu. The results showed significant differences in the N-glycome abundance of all variants. The N-glycome of the VOC showed a large increase in the abundance of sialofucosylated glycans, with the greatest abundance in the omicron variant. In contrast, the results showed a large abundance of fucosylated glycans for most of the VOI. Two glycan compositions, GlcNAc4,Hex5,Fuc,NeuAc (4-5-1-1) and GlcNAc6,Hex8,Fuc,NeuAc (6-8-1-1), were the most abundant structures across all variants. We believe that our data will contribute to understanding the S1 protein’s structural differences between SARS-CoV-2 mutations.

The ongoing pandemic situation of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global threat to both the world economy and public health. Therefore, there is an urgent need to discover effective vaccines or drugs to fight against this virus. The flavonoids and their medicinal plant sources have already exhibited various biological effects, including antiviral, anti-inflammatory, antioxidant, etc. This study was designed to evaluate different flavonoids from medicinal plants as potential inhibitors against the spike protein (Sp) and main protease (Mpro) of SARS-CoV-2 using various computational approaches such as molecular docking, molecular dynamics. The binding affinity and inhibitory effects of all studied flavonoids were discussed and compared with some antiviral drugs that are currently being used in COVID-19 treatment namely favipiravir, lopinavir, and hydroxychloroquine, respectively. Among all studies flavonoids and proposed antiviral drugs, luteolin and mundulinol exhibited the highest binding affinity toward Mpro and Sp. Drug-likeness and ADMET studies revealed that the chosen flavonoids are safe and non-toxic. One hundred ns-MD simulations were implemented for luteolin-Mpro, mundulinol-Mpro, luteolin-Sp, and mundulinol-Sp complexes and the results revealed strong stability of these flavonoid-protein complexes. Furthermore, MM/PBSA confirms the stability of luteolin and mundulinol interactions within the active sites of this protein. In conclusion, our findings reveal that the promising activity of luteolin and mundulinol as inhibitors against COVID-19 via inhibiting the spike protein and major protease of SARS CoV-2, and we urge further research to achieve the clinical significance of our proposed molecular-based efficacy.