To generate antibodies (Abs) against SARS-CoV-2 emerging variants, we integrated multiple tools and engineered molecules with excellent neutralizing breadth and potency. Initially, the screening of an immune library identified a nanobody (Nb), termed Nb4, specific to the receptor-binding domain (RBD) of the Omicron BA.1 variant. A Nb4-derived heavy chain antibody (hcAb4) recognized the spike (S) of the Wuhan, Beta, Delta, Omicron BA.1, and BA.5 SARS-CoV-2 variants. A high-resolution crystal structure of the Nb4 variable (VHH) domain in complex with the SARS-CoV-2 RBD (Wuhan) defined the Nb4 binding mode and interface. The Nb4 VHH domain grasped the RBD and covered most of its outer face, including the core and the receptor-binding motif (RBM), which was consistent with hcAb4 blocking RBD binding to the SARS-CoV-2 receptor. In mouse models, a humanized hcAb4 showed therapeutic potential and prevented the replication of SARS-CoV-2 BA.1 virus in the lungs of the animals. In vitro, hcAb4 neutralized Wuhan, Beta, Delta, Omicron BA.1, and BA.5 viral variants, as well as the BQ.1.1 subvariant, but showed poor neutralization against the Omicron XBB.1.5. Structure-based computation of the RBD-Nb4 interface identified three Nb4 residues with a reduced contribution to the interaction with the XBB.1.5 RBD. Site-saturation mutagenesis of these residues resulted in two hcAb4 mutants with enhanced XBB.1.5 S binding and virus neutralization, further improved by mutant Nb4 trimers. This research highlights an approach that combines library screening, Nb engineering, and structure-based computational predictions for the generation of SARS-CoV-2 Omicron-specific Abs and their adaptation to emerging variants.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiated a global pandemic resulting in an estimated 775 million infections with over 7 million deaths, it has become evident that COVID-19 is not solely a pulmonary disease. Emerging evidence has shown that, in a subset of patients, certain symptoms - including chest pain, stroke, anosmia, dysgeusia, diarrhea and abdominal pain - all indicate a role of vascular, neurological and gastrointestinal (GI) pathology in the disease process. Many of these disease processes persist long after the acute disease has been resolved, resulting in 'long COVID' or post-acute sequelae of COVID-19 (PASC). The molecular mechanisms underlying the acute and systemic conditions associated with COVID-19 remain incompletely defined. Appropriate animal models provide a method of understanding underlying disease mechanisms at the system level through the study of disease progression, tissue pathology, immune system response to the pathogen and behavioral responses. However, very few studies have addressed PASC and whether existing models hold promise for studying this challenging problem. Here, we review the current literature on cardiovascular, neurological and GI pathobiology caused by COVID-19 in patients, along with established animal models of the acute disease manifestations and their prospects for use in PASC studies. Our aim is to provide guidance for the selection of appropriate models in order to recapitulate certain aspects of the disease to enhance the translatability of mechanistic studies.

The global pandemic of SARS-CoV-2 has highlighted the necessity for innovative therapeutic solutions. This research presents a new formulation utilising the metal-organic framework MIL-101(Al)-NH2, which is loaded with hypericin, aimed at addressing viral and bacterial challenges. Hypericin, recognised for its antiviral and antibacterial efficacy, was encapsulated to mitigate its hydrophobicity, improve bioavailability, and utilise its photodynamic characteristics. The MIL-101(Al)-NH2 Hyp complex was synthesised, characterised, and evaluated for its biological applications for the first time. The main objective of this study was to demonstrate the multimodal potential of such a construct, in particular the effect on SARS-CoV-2 protein levels and its interaction with cells. Both in vitro and in vivo experiments demonstrated the effective transport of hypericin to cells that express ACE2 receptors, thereby mimicking mechanisms of viral entry. In addition, hypericin found in the mitochondria showed selective phototoxicity when activated by light, leading to a decrease in the metabolic activity of glioblastoma cells. Importantly, the complex also showed antibacterial efficacy by selectively targeting Gram-positive Staphylococcus epidermidis compared to Gram-negative Escherichia coli under photodynamic therapy (PDT) conditions. To our knowledge, this study was the first to demonstrate the interaction between hypericin, MIL-101(Al)-NH2 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which inhibits cellular uptake and colocalises with ACE2-expressing cells. Therefore, the dual functionality of the complex - targeting the viral RBD and the antibacterial effect via PDT - emphasises its potential to mitigate complications of viral infections, such as secondary bacterial infections. In summary, these results suggest that MIL-101(Al)-NH2 Hyp is a promising multifunctional therapeutic agent for antiviral and antibacterial applications, potentially contributing to the improvement of COVID-19 treatment protocols and the treatment of co-infections.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a devastating global pandemic for 4 years and is now an endemic disease. With the emergence of new viral variants, COVID-19 is a continuing threat to public health despite the wide availability of vaccines. The virus-encoded trimeric spike protein (S protein) mediates SARS-CoV-2 entry into host cells and also induces strong immune responses, making it an important target for development of therapeutics and vaccines. In this Review, we summarize our latest understanding of the structure and function of the SARS-CoV-2 S protein, the molecular mechanism of viral entry and the emergence of new variants, and we discuss their implications for development of S protein-related intervention strategies.

Viral-bacterial co-infections pose significant global health challenges. Currently, there are limited vaccines that target both viral and bacterial pathogens simultaneously. In this study, we present HRBD, a novel fusion protein vaccine combining the receptor-binding domain (RBD) of SARS-CoV-2 and a detoxified α-hemolysin mutant (HlaH35A) from S.aureus. When tested in mice, HRBD induced robust IgG1-dominated antibody responses against both antigens, leading to neutralizing effects against SARS-CoV-2 pseudovirus (NT50 ∼ 103) and S. aureus hemolytic activity (NT50 ∼ 102). HRBD promoted a Th2 immune response with increased IL-4 levels, while also boosting IFN-γ production. Importantly, HRBD showed dose-sparing efficacy, achieving similar immune responses as high-dose single or mixed-antigen vaccines with half the antigen amount. Functional tests confirmed that HRBD sera could neutralize Hla-induced cell lysis and block SARS-CoV-2 entry and syncytium formation. HRBD vaccination protected hACE2 transgenic mice against the co-infection of SARS-CoV-2 pseudovirus and S. aureus. These results suggest that HRBD is a promising candidate for preventing SARS-CoV-2 and S. aureus co-infections.

The global pandemic caused by SARS-CoV-2 has underscored the critical necessity for effective antiviral therapies. The viral main protease (Mpro), crucial for viral replication, has emerged as a promising therapeutic target. In the present study, the inhibitory potential of ten drug-like compounds (KL1-KL10), designed as derivatives of the parent inhibitor K36, against Mpro, has been computationally investigated. To elucidate the binding affinities and interactions of the suggested drugs with the Mpro active site, molecular docking and molecular dynamics (MD) simulations till 500 nanoseconds have been applied. Our results revealed that many suggested inhibitors exhibited enhanced binding affinities compared to the parent inhibitor K36. Among these, KL7 displayed the most favourable binding characteristics, with a docking score of -13.54 and MM-PBSA binding energy of -34.57 kJ/mol, surpassing that of K36. Molecular dynamics simulations demonstrated persistent binding of these compounds to Mpro, with RMSD values ranging from 0.5 to 2.0 nm, suggesting their potential as effective inhibitors. These findings suggest that the proposed ligands hold promise as potential scaffolds for developing potent antiviral drugs against COVID-19.

Angiotensin-converting enzyme 2 (ACE2) receptor plays a pivotal role in the infection of several coronaviruses, including SARS-CoV and SARS-CoV-2. We combined computational and experimental protein engineering approaches to develop ACE2-YHA, a soluble, high-affinity ACE2 decoy with pan-coronavirus preventive and therapeutic potential. Leveraging native human ACE2-SARS-CoV/SARS-CoV-2 receptor binding domain (RBD) complex homology models, we employed in silico site-saturation mutagenesis to predict key ACE2-RBD interacting residues. Subsequent generation of ACE2 mutants and high-throughput screening identified specific ACE2 residue substitutions that enhanced binding to both SARS-CoV and SARS-CoV-2 RBDs. The triple mutant ACE2-YHA demonstrated significantly enhanced binding affinity to SARS-CoV, SARS-CoV-2, and bat SARSr-CoVs' RBDs. It effectively neutralized SARS-CoV and numerous SARS-CoV-2 variants with picomolar IC50s in pseudotyped virus assays. Notably, ACE2-YHA displayed potent neutralization against major variants of concern, including Delta and Omicron, in human airway epithelia, positioning it as a promising universal decoy for current and future ACE2-binding coronavirus outbreaks.

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

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

Emerging evidence suggests a potential association between periodontitis and adverse outcomes in COVID-19. Both conditions share risk factors and exhibit similar immune dysregulation, including elevated pro-inflammatory cytokines, altered myeloid compartments, and T-cell dysfunction. SARS-CoV-2 uses angiotensin-converting enzyme type 2 and transmembrane protease serine 2 membrane proteins, highly expressed in the oral cavity, for cellular entry. Periodontitis may exacerbate COVID-19 through mechanisms such as oral microbe aspiration, increased viral receptor expression, and systemic inflammation. The shared immunopathogenesis, characterized by cytokine storms and perturbed immune profiles, suggests periodontitis can predispose patients to more severe COVID-19 outcomes. This article aims to review the associations between periodontitis and the severity of COVID-19 and the possible immune mechanisms involved.

Background: Dynamic SUMO modifications play crucial roles in orchestrating cellular response to various stimuli, including viral infection, and hold significant therapeutic potential. The Spike (S) protein, a surface glycoprotein of SARS-CoV-2 (a global health threat), serves as the key mediator for viral entry and is a critical target for drug development. However, the function of SUMOylation in the Spike protein remains largely unclear. Methods: The SUMO modification of Spike was assessed by immunoprecipitation (IP), denatured IP and immunoblotting assays in lung epithelial cells or SUMO deficient cell line models. The effect of Spike SUMOylation on viral infection was explored by site-direct mutation, cell-to-cell transmission, cell-free infection, quantitative PCR and immunofluorescence staining experiments. The role of Spike SUMOylation-derived peptides was investigated using viral intranasally infection, immunohistochemistry assay in transgenic mouse model. Results: SARS-CoV-2 infection triggers the relocation of SUMO1 to the cytoplasm and SUMO2 to the perinuclear region. Notably, SUMO1 knockout increases Spike trimer formation and its co-localization with SUMO2 at perinuclear puncta, which facilitates virion particle release. SUMO2 knockout leads to enhanced Spike cleavage and promotes viral cell-to-cell transmission. Further bioinformatic and immuno-precipitation analyses reveal that the Spike protein contains highly conserve SUMO-interacting motifs (SIMs) and selectively promotes either SUMO1 (via SIM1) or SUMO2 (via SIM3/4) modifications on lysine residues 129 and 1269, respectively. Importantly, these modifications can be efficiently disrupted by the SIM2 motif. A cell-penetrating peptide (cpSIM2), derived from the SIM2 motif, exhibits robust and broad-spectrum inhibitory activity against SARS-CoV-2 variants infection in vitro and in hACE2-transgenic mice model. Conclusions: This study uncovers critical features of SUMOylation in regulating Spike-mediated viral spread and pathogenesis, providing a potential broad-spectrum therapeutic target for drug development against emerging SARS-CoV-2 infection.

Single-event completion times, such as are estimated in viral entry, offer both promise and challenge to kinetic interpretation. The promise is that they are able to constrain underlying kinetic models much more efficiently than bulk kinetics, but the challenge is that completion times alone can incompletely determine complex reaction topologies. Gamma distributions or mechanistic models have often been used to estimate kinetic parameters for such data, but the gamma distribution relies on homogenous processes contributing to the rate-limiting behavior of the system. Here, we introduce hypoexponential analysis to estimate heterogeneous kinetic processes. We demonstrate that hypoexponential fitting can indeed estimate rate constants separated by 2-3 orders of magnitude. We then apply this approach to measurements of SARS-CoV-2 entry, showing that ACE2 reduces the number of rate-limiting steps but does not change the rates of these kinetic processes. We propose a kinetic model whereby SARS-CoV-2 entry is driven by a mixture of ACE2-accelerated and ACE2-independent spike protein activation events. Inferring such models requires the capability to detect heterogeneous kinetic processes, provided by robust estimation of hypoexponential distributions.

Coronavirus disease 2019 (COVID-19) frequently presents with neurological symptoms in human patients and leads to long-lasting brain pathology in a hamster model. There is no overt SARS-CoV-2 virus replication in central neurons. Whether viral proteins are sufficient to cause this pathology requires further investigations. The SARS-CoV-2 Spike-protein S1-subunit (S1-protein) has recently gained interest for causing neuroinflammation and accelerating aggregation of alpha-synuclein (aSyn) in vitro. Here, we show the impact of S1-protein in a broad spectrum of brain regions after injection via three different application routes in C57/BL6 mice.

S1-protein was administered either intranasally, intravenously or intracerebrally. We quantified aSyn immunoreactivity and phosphorylated aSyn (pS129), microglia and astrocyte reactivity, ACE2/Neuropilin-1 receptor expression, and parvalbumin-positive interneurons in limbic system, basal ganglia, and cortical regions 14 days post-application. Plasma cytokine profiles were assessed 6 days post-injection.

While intracerebral injection resulted in decreased aSyn immunoreactivity with increased pS129 in males, intravenous injection led to increased levels of aSyn immunoreactivity and microglia cell density, predominantly in brain regions associated with Parkinson's disease pathology. Intranasal application of S1-protein induced microgliosis in some brain regions but resulted in sex-dependent alterations of aSyn levels, with increases in females and decreases in males. All routes showed sex-dependent alterations in astrocytic reactivity, receptor expression, and parvalbumin-positive interneurons.

Our results demonstrate that S1-protein itself leads to neuroinflammation, altered aSyn homeostasis, and disruption of inhibitory circuits in a route- and sex-dependent manner. These findings indicate the possibility of S1-protein being a crucial agent for both neuroinflammatory processes and altered protein regulation mechanisms. S1-protein trapped in tissue reservoirs could therefore explain symptoms occurring or persisting beyond viral clearance (Post COVID-19 condition).

The SARS-CoV-2 virus and its rapid spread have made it a global health concern. The aim of this was to investigate the microbial and metabolic faecal profiles of paediatric patients hospitalised for COVID-19 to try to identify biomarkers of predisposition to severity. The study included 16 patients (aged 4-14 years old) from six different Spanish hospitals and 20 age-matched healthy controls. The gut microbiota was characterised by sequencing of 16S rDNA amplicons and internal transcribed space amplicons, while the metabolic profile was analysed by liquid chromatography high resolution mass spectrometry. A different microbial profile was observed between patients and controls, with a significantly higher abundance of sequences belonging to the phyla Bacteroidota and Pseudomonadota in patients. A different metabolic profile was observed between the two groups. Non-infected children had higher faecal levels of vitamins such as niacin, thiamine, and vitamin D3 derivatives, which were negatively correlated with the abundance of pathogenic bacteria, such as members of Enterobacteriaceae. Hospitalisation due to SARS-CoV-2 infection in children was associated with changes in the gut microbiota and an altered metabolomic profile. For the first time, several relevant biological compounds were found to be reduced in the faeces of children hospitalised with COVID-19 compared to healthy controls.

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

Since its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, giving rise to multiple variants that have significantly altered the trajectory of the COVID-19 pandemic. These variants have resulted in multiple waves of the pandemic, exhibiting characteristic mutations in the spike (S) protein that may have affected receptor interaction, tissue tropism, and cell entry mechanisms. While the virus was shown to primarily utilize the angiotensin-converting enzyme 2 (ACE2) receptor and host proteases such as transmembrane serine protease 2 (TMPRSS2) for entry into host cells, alterations in the S protein have resulted in changes to receptor binding affinity and use of alternative receptors, potentially expanding the virus's ability to infect different cell types or tissues, contributing to shifts in clinical presentation. These changes have been linked to variations in disease severity, the emergence of new clinical manifestations, and altered transmission dynamics. In this paper, we overview the evolving receptor utilization strategies of SARS-CoV-2, focusing on how mutations in the S protein may have influenced viral entry mechanisms and clinical outcomes across the ongoing pandemic waves.

With the COVID-19 pandemic becoming endemic, vigilance for Long COVID-related cardiovascular issues remains essential, though their specific pathophysiology is largely unexplored.

Our study investigates the persistent cardiovascular symptoms observed in individuals long after contracting SARS-CoV-2, a condition commonly referred to as "Long COVID", which has significantly affected millions globally.

We meticulously describe the cardiovascular outcomes in five patients, encompassing a range of severe conditions such as sudden cardiac death during exercise, coronary atherosclerotic heart disease, palpitation, chest tightness, and acute myocarditis.

All five patients were diagnosed with myocarditis, confirmed through endomyocardial biopsy and histochemical staining, which identified inflammatory cell infiltration in their heart tissue. Crucially, electron microscopy revealed widespread mitochondrial vacuolations and the presence of myofilament degradation within the cardiomyocytes of these patients. These findings were mirrored in SARS-CoV-2-infected mice, suggesting a potential underlying cellular mechanism for the cardiac effects associated with Long COVID.

Our findings demonstrate a profound impact of SARS-CoV-2 on mitochondrial integrity, shedding light on the cardiovascular implications of Long COVID.

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

The UniProt REST API is a freely available, open-access resource that powers the UniProt.org website and gives users flexible programmatic interaction with protein knowledge data. It provides access to UniProtKB, UniRef, UniParc, Proteomes, GeneCentric, ARBA, UniRule, and the ID Mapping tool, along with supporting data and controlled vocabularies. Users can access the API with their favorite programming language and generate example code snippets to access the UniProt databases using the API documentation page (https://www.uniprot.org/api-documentation) in various languages. API results can be returned and downloaded in various formats. With an average of 303 million requests per month over the last year, the API enables structured search queries using logical operators and parentheses, allows users to specify fields of interest within results, and customize downloads for direct integration into workflows. The API is a powerful tool that empowers users to fully utilize UniProt data across multiple datasets, enabling download, analysis, and extraction of valuable research insights. This website is free and open to all users, and there is no login requirement.

Vaccines usually contain an adjuvant that activates innate immunity to promote the acquisition of adaptive immunity. Aluminum and lipid nanoparticles have been used for this purpose, but their accumulation or widespread circulation in the body can lead to adverse effects. In contrast, physical adjuvants, which use physical energy to transiently stress tissues, do not persist in exposed tissues or cause lasting adverse effects. Herein, we investigate the effects of intradermal injection of endotoxin-free ovalbumin (OVA) protein alone without additional adjuvants using a needle-free pyro-drive jet injector (PJI) on tumor vaccination efficacy. Intradermal injection of OVA protein alone using PJI significantly increased OVA-specific CD8+ T cell expansion in the lymph node, although lymph node swelling was much less than when aluminum hydroxide was used. The injection also induced OVA-specific killing activity and antibody production and showed strong CD8+ T cell-dependent prophylactic antitumor effects against transplanted E.G7-OVA tumors. In particular, intradermal injection of the fluorescent OVA protein significantly enhanced its uptake by XCR1+ dendritic cells, which have a strong ability to cross-present extracellular proteins in the skin and draining lymph nodes. In addition, the injection increased the expression of HMGB1, one of the potent danger signals whose expression has been reported to increase in response to shear stress. Thus, intradermal injection of OVA protein alone without any additional adjuvants using PJI induces potent CD8+ T cell-mediated antitumor immunity by enhancing its uptake into XCR1+ dendritic cells, which have a high cross-presentation capacity accompanied by an increased expression of shear stress-induced HMGB1.

mRNA vaccines have demonstrated considerable efficacy and safety against SARS-CoV-2, limiting the pandemic burden worldwide. The emergence of new variants of concern and the decline in neutralizing activity observed several weeks post-vaccination reinforced the call for repeated mRNA vaccination. We and others have shown that vaccine efficacy does not exclusively rely on antibody neutralizing activites; Fc-effector functions play an important role as well. However, it is well known that long-term exposure and repeated antigen stimulation elicit the IgG4 subclass of antibodies, which are inefficient at mediating Fc-effector functions. In this regard, recent studies highlighted concerns about IgG4 induction by mRNA vaccines. Here, we explored the impact of repeated mRNA vaccination on IgG4 induction and its impact on Fc-effector functions. We observed anti-Spike IgG4 elicitation after three doses of mRNA vaccine; the antibody levels further increased with additional doses. Vaccine-elicited IgG4 preferentially bound the ancestral D614G Spike. We also observed that Breakthrough Infection (BTI) after several doses of vaccine strongly increased IgG1 levels but had no impact on IgG4 levels, thereby improving Fc-effector functions. Finally, we observed that elderly donors vaccinated with Moderna mRNA vaccines elicited higher IgG4 levels and presented lower Fc-effector functions than donors vaccinated with the Pfizer mRNA vaccine. Altogether, our results highlight the importance of monitoring the IgG subclasses elicited by vaccination.

Coronaviruses have emerged as a significant challenge to human health. While earlier outbreaks of coronaviruses such as SARS-CoV and MERS-CoV posed serious threats, the recent SARS-CoV-2 pandemic has heightened interest in coronavirus research due to its pulmonary pathology, in addition to its neurological manifestations. In addition, the patients who have recovered from SARS-CoV-2 infection show long-term symptoms such as anosmia, brain fog and long COVID. A major hurdle in studying these viruses is the limited availability of specialized research facilities, emphasizing the need for prototype virus-based models to investigate the pathophysiology. The mouse hepatitis virus (MHV), a member of the β-coronavirus family, serves as an excellent model to unravel the mechanisms underlying virus-induced pathogenesis. This review highlights two decades of research efforts aimed at understanding the pathophysiological mechanism of coronavirus-induced diseases, focusing on the development of targeted recombinant strains to identify the minimal essential motif of the spike protein responsible for fusogenicity and neuropathogenicity. By synthesizing findings from these studies, the review identifies the most promising therapeutic targets against coronaviruses, paving the way for the development of pan-coronavirus antivirals.

The Angiotensin-converting enzyme 2 (ACE2) receptor, crucial for coronavirus recognition of host cells, is a key target for therapeutic intervention against SARS-CoV-2 and related coronaviruses. Therefore, thoroughly investigating the interaction mechanism between ACE2 and the Spike protein (S protein), as well as developing targeted inhibitors based on this mechanism, is vital for effectively controlling the spread of SARS-CoV-2 and preventing potential future pandemics caused by other coronaviruses.

This article comprehensively reviews the mechanisms underlying ACE2-S protein interaction that facilitate SARS-CoV-2 entry into host cells. It also analyzes the patent landscape regarding inhibitors targeting the ACE2-S interface since 2019.

In the 5 years since the outbreak of SARS-CoV-2, numerous methods and design strategies have been employed to develop innovative therapeutics against coronaviruses. Among these approaches, inhibitors targeting both the ACE2 receptor and the S protein have gained significant interest due to their potential in blocking various coronaviruses. Despite facing challenges similar to other protein-protein interaction inhibitors, progress has been made in developing these inhibitors through virtual screening, covalent protein binding, and peptide modification strategies. However, obstacles persist in clinical translation, necessitating a multidisciplinary strategy that integrates state-of-the-art methodologies to optimize S-ACE2 interface-targeted drug discovery.

In the life cycle of a virus, host cell entry represents the first step that a virus needs to undertake to gain access to the cell interior for replication. Once a virus attaches itself to its target cell receptor, it activates endogenous cellular responses and exploits host cell factors for its internalization, fusion, and genome release. Among the host factors that critically contribute to the viral entry processes are cathepsins, which are the most abundant endo/lysosomal proteases with diverse physiological functions. This review summarizes previous findings on how different cathepsins contribute to the host cell entry of human pathogenic viruses, focusing on their specific roles in the entry processes of both enveloped and non-enveloped RNA viruses. A comprehensive knowledge of the functions of different cathepsins in viral entry will provide valuable insights into the molecular mechanisms underlying viral infections and can be useful in the development of new antiviral strategies.

The ongoing COVID-19 has caused a global pandemic, resulting in millions of infections and deaths. While current vaccines target the SARS-CoV-2 spike (S) protein, its high mutation rate significantly compromises vaccine efficacy. We aimed to evaluate the potential of epitope-based nanoparticles (NPs) to induce broad cross-protection and durable immune responses against SARS-CoV-2.

Four conserved epitopes derived from the receptor-binding domain (RBD) and S2 subunit of the spike protein were integrated into Helicobacter pylori ferritin to create epitope-based NPs named S18-F, RBM-F, UH-F, and HR2-F. The immunogenicity of the epitope-based NPs was evaluated through animal experiments to measure epitope-specific antibody titers and assess neutralizing activity against SARS-CoV-2 pseudovirus. To characterize cellular immune responses, splenic lymphocyte proliferation following epitope stimulation was measured, and cytokine secretion profiles including IFN-γ, IL-2, IL-4, and IL-10 were analyzed to determine Th1/Th2 immune polarization. Antibody-dependent cellular cytotoxicity (ADCC) assays were performed to evaluate NP-enhanced recognition and elimination of infected target cells.

These NPs induced high titers of epitope-specific antibodies lasting three months post-immunization. Sera from the RBM-F, UH-F, and HR2-F groups exhibited neutralizing activity against the SARS-CoV-2 pseudovirus WH-1 in vitro. Splenic lymphocytes from the S18-F, RBM-F, and UH-F groups showed significantly increased proliferation. Lymphocytes from the RBM-F group demonstrated increased secretion of IFN-γ, IL-2, IL-4, and IL-10 cytokines, indicating a balanced Th1 and Th2 immune response. Immune sera from the S18-F and mixed-immunized groups exhibited antibody-dependent cellular cytotoxicity.

The results indicate that these NPs induce robust humoral and cellular immune responses, potentially offering a promising strategy for effective vaccine development against SARS-CoV-2.

SARS-CoV-2, first identified in December 2019, caused a global pandemic, resulting in over 6.8 million deaths by March 2023. The elderly, or individuals over 65, accounted for the majority of COVID-19 deaths, with 81% of fatalities in the US in 2020 occurring in this group. Beyond mortality, aging populations are also at higher risk of long-term cardiovascular complications and acute respiratory distress syndrome (ARDS). Although these outcomes may be influenced by comorbidities common in the elderly, age has been found to be a standalone risk factor for severe COVID-19 infection. Therefore, investigating age-related factors in COVID-19 outcomes is crucial in protecting this vulnerable group. Of particular interest is the cytokine storm phenomenon, an excessive inflammatory response that contributes to severe COVID-19 symptoms, including ARDS and cardiovascular damage. Elevated levels of multiple cytokines are common in severe cases of COVID-19. We propose that changes that occur to cytokine profiles as we age may contribute to these aberrant inflammatory responses. This review specifically explored the interleukin class cytokines IL-1, IL-6, IL-17, and IL-23 and considered the potential of biologics targeting these cytokines to alleviate severe outcomes in both COVID-19 and aging individuals.

The antibody-mediated immune response is crucial for the development of protective immunity against SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Understanding the interaction between SARS-CoV-2 and the immune system is critical because new variants emerge as a result of the virus's ongoing evolution. Understanding the function of B cells in the SARS-CoV-2 infection process is critical for developing effective and long-lasting vaccines against this virus. Triggered by the innate immune response, B cells transform into memory B cells (MBCs). It is fascinating to observe how MBCs provide enduring immune defence, not only eradicating the infection but also safeguarding against future reinfection. If there is a lack of B cell activation or if the B cells are not functioning properly, it can lead to a serious manifestation of the disease and make immunisation less effective. Individuals with disruptions in the B cells have shown increased production of cytokines and chemokines, resulting in a poor prognosis for the disease. Therefore, we have developed an updated review article to gain insight into the involvement of B cells in SARS-CoV-2 infection. The discussion has covered the generation, functioning, and dynamics of neutralising antibodies (nAbs). Furthermore, we have emphasised immunotherapeutics that rely on nAbs.

Evaluating long-term immunity after COVID-19 infection and vaccination is critical for managing potential outbreaks. The aim of this study was to develop a cost-effective in-house enzyme-linked immunosorbent assay (ELISA) based on Escherichia coli-expressed SARS-CoV-2 spike protein (E-S1) for antibody detection and to evaluate its performance. The system was validated by comparing the in-house ELISA results with those obtained using a commercial ELISA with HEK293-expressed spike protein (H-S1). Recombinant SARS-CoV-2 spike protein was produced in E. coli, purified, and validated for antigenicity via ELISA. Indirect ELISAs with both E-S1 and H-S1 antigens were performed on 386 serum samples from COVID-19 survivors, vaccinated individuals, and pre-pandemic controls collected at different time points. The E-S1 ELISA showed a statistically significant but weak correlation with H-S1 ELISA across all samples (r=0.205; p=0.0001). Stronger correlations were observed among vaccinated individuals with prior infection on day 90 (r=0.6017; p<0.001) and in naïve vaccine recipients on day 30 (r=0.5361; p=0.0003). Pre-pandemic sera from a rural population in Sumba Island exhibited high background reactivity in E-S1 ELISA, likely due to anti-E. coli antibodies, while urban pre-pandemic sera from Jakarta showed a stronger correlation with H-S1 ELISA. This suggests potential regional or immune background differences influencing assay performance. Although E-S1 retained antigenic properties, its diagnostic utility is limited by non-specific reactivity and reduced sensitivity compared to H-S1. In conclusion, E. coli expression systems may not be ideal for producing spike protein-based ELISA antigens specific to SARS-CoV-2. Alternative expression systems, such as human or baculovirus, could enhance diagnostic accuracy and specificity for COVID-19 antibody detection.

In May 2023 the World Health Organization (WHO) declared the end of COVID-19 as a public health emergency. Seroprevalence studies performed in African countries, such as Cameroon, depicted a much higher COVID-19 burden than reported by the WHO. To better understand humoral responses kinetics following infection, we enrolled 333 participants from Yaoundé, Cameroon between March 2020 and January 2022. We measured the levels of antibodies targeting the SARS-CoV-2 receptor-binding-domain (RBD) and the Spike glycoproteins of Delta, Omicron BA.1 and BA.4/5 and the common cold coronavirus HCoV-OC43. We also evaluated plasma capacity to neutralize authentic SARS-CoV-2 virus and to mediate Antibody-Dependent Cellular Cytotoxicity (ADCC). Most individuals mounted a strong antibody response against SARS-CoV-2 Spike. Plasma neutralization waned faster than anti-Spike binding and ADCC. We observed differences in humoral responses by age and circulating variants. Altogether, we show a global overview of antibody dynamics and functionality against SARS-CoV-2 in Cameroon.

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

After the emergence of the COVID-19 pandemic in late 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has undergone a dynamic evolution driven by the acquisition of genetic modifications, resulting in several variants that are further classified as variants of interest (VOIs), variants under monitoring (VUM) and variants of concern (VOC) by World Health Organization (WHO). Currently, there are five SARS-CoV-2 VOCs (Alpha, Beta, Delta, Gamma and Omicron), two VOIs (Lambda and Mu) and several other VOIs that have been reported globally. In this study, we report a natural compound, Curcumin, as the potential inhibitor to the interactions between receptor binding domain (RBD(S1)) and human angiotensin-converting enzyme 2 (hACE2) domains and showcased its inhibitory potential for the Delta and Omicron variants through a computational approach by implementing state of the art methods. The study for the first time revealed a higher efficiency of Curcumin, especially for hindering the interaction between RBD(S1) and hACE-2 domains of Delta and Omicron variants as compared to other lead compounds. We investigated that the mutations in the RBD(S1) of VOC especially Delta and Omicron variants affect its structure compared to that of the wild type and other variants and therefore altered its binding to the hACE2 receptor. Molecular docking and molecular dynamics (MD) simulation analyses substantially supported the findings in terms of the stability of the docked complexes. This study offers compelling evidence, warranting a more in-depth exploration into the impact of these alterations on the binding of identified drug molecules with the Spike protein. Further investigation into their potential therapeutic effects in vivo is highly recommended.

The growing societal impact of coronavirus disease 2019 (COVID-19) has underscored the urgent need for innovative strategies to address the ongoing challenges posed by the pandemic. While rapid therapeutic interventions remain critical for short-term mitigation, equally vital is the development of accessible and efficient diagnostic tools to curb viral transmission. In this context, optical sensing technologies have emerged as foundational tools for detection and diagnosis, owing to their rapid response, user-friendliness, and adaptability. These attributes strengthen their indispensable role in identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19. This review systematically outlines the structural components of SARS-CoV-2 virions and their respective biological functions, classifies optical biosensors according to their underlying principles and evaluates the advantages and limitations of each methodology in real-world diagnostic applications. By addressing current detection challenges, these optical platforms not only enhance our capacity to manage SARS-CoV-2 but also establish a framework for deploying optical sensing technologies in future pandemic scenarios.

Mucosal immunity plays a pivotal role in safeguarding against significant global infectious diseases caused by mucosal pathogens. The development of mucosal vaccines has been limited by the poor efficiency of antigen display and the risk of adjuvants. Here, we report an engineered yeast vaccine integrating a well-displayed antigen with an intrinsic adjuvant for the development of innate and adaptive immunity to the intestinal mucosa. Compared with antigen-secretory yeast, antigen-anchored yeast significantly activated gut dendritic cells (DCs) and promoted follicular helper T (Tfh) cell differentiation, thereby amplifying the immune response by the interaction with Tfh-B cells. Consequently, oral vaccination of SARS-CoV-2 receptor-binding domain (RBD)-anchored yeast triggered stronger RBD-specific IgA-neutralizing effects, providing potential adaptive protections. Given its corresponding impact on the functionality of both innate and adaptive mucosal responses, the proposed RBD-anchored yeast outperformed RBD-anchored bacteria and biomimetic nanovaccine in the production of RBD-specific IgA and IgG. Together, these results revealed how antigen-displaying patterns could be modulated to elicit intestinal mucosal immunity and demonstrated the translational potential of antigen-displayed yeast for effective mucosal protection.

Monoclonal antibody (mAb) drug treatments have proven effective in reducing COVID-19-related hospitalizations or fatalities, particularly among high-risk patients. Numerous experimental studies have explored the structures of spike proteins and their complexes with ACE2 or mAbs. These 3D structures provide crucial insights into the interactions between spike proteins and ACE2 or mAb, forming a basis for the development of diagnostic tools and therapeutics. However, the field of computational biology has faced substantial challenges due to the lack of methods for precise protein structural comparisons and accurate prediction of molecular interactions. In our previous studies, we introduced the Triangular Spatial Relationship (TSR)-based algorithm, which represents a protein's 3D structure using a vector of integers (keys). These earlier studies, however, were limited to individual proteins.

This study introduces new extensions of the TSR-based algorithm, enhancing its ability to study interactions between two molecules. We apply these extensions to gain a mechanistic understanding of spike - mAb interactions.

We expanded the basic TSR method in three novel ways: (1) TSR keys encompassing all atoms, (2) cross keys for interactions between two molecules, and (3) intra-residual keys for amino acids. This TSR-based representation of 3D structures offers a unique advantage by simplifying the search for similar substructures within structural datasets.

The study's key findings include: (i) The method effectively quantified and interpreted conformational changes and steric effects using the newly introduced TSR keys. (ii) Six clusters for CDRH3 and three clusters for CDRL3 were identified using all-atom keys. (iii) We constructed the TSR-STRSUM (TSR-STRucture SUbstitution Matrix), a matrix that represents pairwise similarities between amino acid structures, providing valuable applications in protein sequence and structure comparison. (iv) Intra-residual keys revealed two distinct Tyr clusters characterized by specific triangle geometries.

This study presents an advanced computational approach that not only quantifies and interprets conformational changes in protein backbones, entire structures, or individual amino acids, but also facilitates the search for substructures induced by molecular binding across protein datasets. In some instances, a direct correlation between structures and functions was successfully established.

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