Synthesis and Analysis of the SARS-CoV-2 ORF7a Accessory Protein Transmembrane Domain and Determination of the Oligomerization State

The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

SARS-CoV-2 Infection Increases the Gene Expression Profile for Alzheimer's Disease Risk

The COVID-19 pandemic is a global outbreak of coronavirus, an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One in five adults who have had COVID-19 in the past was still experiencing any one of the symptoms of long COVID like headache, brain fog, fatigue, and shortness of breath. Up to 30% of individuals with mild to severe infection show diverse neurological symptoms, including dementias. Hence, it is very much important to characterize the neurotropism and neurovirulence of the SARS-CoV-2 virus. This helps us understand the mechanisms involved in initiating inflammation in the brain, further leading to the development of earlyonset Alzheimer's disease and related dementias (ADRDs). In our brain gene expression analysis, we found that severe COVID-19 patients showed increased expression of innate immune response genes and genes that are implicated in AD pathogenesis. To study the infection-induced ADRDs, we used a mouse-adapted strain of the SARS-CoV-2 (MA10) virus to infect mice of different age groups (3, 6, and 20 Months). In this study, we found that aged mice showed evidence of viral neurotropism, prolonged viral infection, increased expression of tau aggregator FKBP51, interferoninducible gene Ifi204, and complement genes like C4 and C5AR1. Brain histopathology also showed the AD signature including tau-phosphorylation, tau-oligomerization, and alpha-synuclein expression in aged MA10-infected mice. The results from gene expression profiling of SARS-CoV-2 infected and AD brains and studies with MA10 aged mice show that COVID-19 infection increases the risk of AD in the aged population. Furthermore, this study helps us to understand the crucial molecular markers that are regulated during COVID infection that could act as major players in developing ADRDs. Future studies will be involved in understanding the molecular mechanisms of ADRD in response to COVID infection and developing novel therapies targeting AD.

Development of a spin-label model system for investigation of protein oligomerization states

The transmembrane domains of viral proteins are highly conserved and crucial to normal viral function. Oligomeric transmembrane domains present novel opportunities for drug development, as their disruption can prevent the assembly of the virus. The Reichart lab is particularly interested in developing retro-inverso peptide inhibitors. Retro-inverso peptides are peptides using D-amino acids mirroring a region of target protein, which allows the peptide to inhibit viral assembly, but they are also significantly less likely to be catabolized by natural metabolic or immunologic processes. The efficacy of these inhibitors is governed largely by the extent to which they mirror the target protein, making highly conserved regions, such as transmembrane domains, ideal target regions for these inhibitors. The primary technique in the literature for the investigation of oligomerization states uses fluorescence spectroscopy. We are now working on developing a novel alternative system to evaluate protein oligomerization using spin-labeled peptides that are directly incorporated into the peptide sequence. Direct incorporation of the spin-label into the peptide sequence is a more powerful technique than the standard procedures used in the literature. In particular, the ability to incorporate spin labels in various positions within the protein can give novel insights into the relative depth of the protein within a membrane, which is very difficult to study using other techniques and not possible using the fluorescence technique. The transmembrane domains of proteins with known and well-characterized monomer and trimer standard oligomerization states were synthesized using an Fmoc Solid- Phase Peptide Synthesis (SPPS) procedure incorporating an Fmoc-2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, (Fmoc-TOAC) instead of an alanine. Direct incorporation of stable N-oxide spin labels, which can be contrasted to labeling cysteine residues after the protein synthesis, has been used for the investigation of the secondary structure of proteins for decades, but the application of this spin labeling technique to study the oligomerization states of transmembrane domains of proteins is an understudied application. The products of SPPS were analyzed using a Liquid Chromatography Mass Spectroscopy instrument and purified using High Performance Liquid Chromatography. The spin-label was then deprotected and evaluated using Electron Spin Resonance (ESR) Spectroscopy. There are two primary future directions following this research project: first, the generation of viral proteins with spin labels incorporated in different positions to determine the relative depth of each position within the membrane;second, the incorporation of spin labels into SARS-CoV- 2 proteins to develop a model for in vitro evaluation of retro-inverso peptide assembly inhibitors. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

SARS-CoV-2 Nsp13 Catalytic Efficiency is Regulated by ATP: Mg2+ Stoichiometry and Functional Cooperativity Among Nsp13 Molecules

Coronavirus disease 19 (COVID-19) is a highly contagious and lethal disease caused by the SARS-CoV-2 positive-strand RNA virus. Nonstructural protein 13 (Nsp13) is the highly conserved ATPase/helicase required for replication of the SARS-CoV-2 genome which allows for the infection and transmission of COVID-19. We biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein expressed using a eukaryotic cell-based system and characterized its catalytic functions, focusing on optimization of its reaction conditions and assessment of functional cooperativity among Nsp13 molecules during unwinding of duplex RNA substrates. These studies allowed us to carefully determine the optimal reaction conditions for binding and unwinding various nucleic acid substrates. Previously, ATP concentration was suggested to be an important factor for optimal helicase activity by recombinant SARS-CoV-1 Nsp13. Apart from a single study conducted using fixed concentrations of ATP, the importance of the essential divalent cation for Nsp13 helicase activity had not been examined. Given the importance of the divalent metal ion cofactor for ATP hydrolysis and helicase activity, we assessed if the molar ratio of ATP to Mg2+ was important for optimal SARS-CoV-2 Nsp13 RNA helicase activity. We determined that Nsp13 RNA helicase activity was dependent on ATP and Mg2+ concentrations with an optimum of 1 mM Mg2+ and 2 mM ATP. Next, we examined Nsp13 helicase activity as a function of equimolar ATP:Mg2+ ratio and determined that helicase activity decreased as the equimolar concentration increased, especially above 5 mM. We determined that Nsp13 catalytic functions are sensitive to Mg2+ concentration suggesting a regulatory mechanism for ATP hydrolysis, duplex unwinding, and protein remodeling, processes that are implicated in SARS-CoV-2 replication and proofreading to ensure RNA synthesis fidelity. Evidence is presented that excess Mg2+ impairs Nsp13 helicase activity by dual mechanisms involving both allostery and ionic strength. In addition, using single-turnover reaction conditions, Nsp13 unwound partial duplex RNA substrates of increasing doublestranded regions (16-30 base pairs) with similar kinetic efficiency, suggesting the enzyme unwinds processively in this range under optimal reaction conditions. Furthermore, we determined that Nsp13 displayed sigmoidal behavior for helicase activity as a function of enzyme concentration, suggesting that functional cooperativity and oligomerization are important for optimal activity. The observed functional cooperativity of Nsp13 protomers suggests the essential coronavirus RNA helicase has roles in RNA processing events beyond its currently understood involvement in the SARS-CoV-2 replication-transcription complex (RTC), in which it was suggested that only one of the two Nsp13 subunits has a catalytic function, whereas the other has only a structural role in complex stability. Altogether, the intimate regulation of Nsp13 RNA helicase by divalent cation and protein oligomerization suggests drug targets for modulation of enzymatic activity that may prove useful for the development of novel anti-coronavirus therapeutic strategies. This work was supported by the Intramural Training Program, National Institute on Aging (NIA), NIH, and a Special COVID-19 Grant from the Office of the Scientific Director, NIA, NIH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis of the Transmembrane Domain of the Spike Protein from SARS-CoV-2 Using Solid Phase Peptide Synthesis and Determination of Its Oligomerization State

The spike protein in severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is directly responsible for the binding to ACE2 receptors in host cells. While the spike protein overall is known to form trimers, the oligomerization state of the transmembrane domain of the spike protein in SARS-CoV-2 is unknown. It is believed to be essential for the function of this protein. Since the transmembrane domain of the spike protein is highly conserved in SARS-CoV-2 it is important to investigate its character and determine its relationship to the function of the protein as awhole. The goal of this project was to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the spike protein in SARS-CoV-2. The most practical method to synthesize the TM domain of the S protein is through solid phase peptide synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will then need to be purified using high-performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography- mass spectrometry (LCMS) to confirm the identity of the synthesized peptides as well as any potential impurities. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS-CoV-2. The function of the S protein is believed to only present when the transmembrane domain forms a trimer. Therefore, the analysis of their oligomerization states will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and tetramethylrhodamine (TAMRA) in a fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

TARGETING THE HOST-VIRUS INTERFACE TO BLOCK SARS-CoV-2 ASSEMBLY IN AIRWAY CELLS

Background: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many therapies illustrates the need for antiviral treatments with high genetic barriers to resistance. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly with minimal host toxicity. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low susceptibility to evolutionary escape. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Method(s): Dose-dependent cytotoxicity of PAV-104 in Calu-3 cells was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 Gamma, Delta, and Omicron variants (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the N gene, immunofluorescence assay (IFA) of nucleocapsid (N) protein, and titration of supernatant (TCID50). Transient co-expression of four SARS-CoV-2 structural proteins (N, M, S, E) to produce virus-like particles (VLPs) was used to study the effect of PAV-104 on viral assembly. Drug resin affinity chromatography was performed to study the interaction between PAV-104 and N. Glycerol gradient sedimentation was used to assess N oligomerization. Total RNA-seq and the REACTOME database were used to evaluate PAV-104 effects on the host transcriptome. Result(s): PAV-104 reached 50% cytotoxicity in Calu-3 cells at 3732 nM (Fig.1A). 50 nM PAV-104 inhibited >99% of SARS-CoV-2 infection in Calu-3 cells (p< 0.01) and in primary AECs (p< 0.01) (Fig.1B-E). PAV-104 specifically inhibited SARS-CoV-2 post entry, and suppressed production of SARS-CoV-2 VLPs without affecting viral protein synthesis. PAV-104 interacted with SARS-CoV-2 N and interfered with N oligomerization. Transcriptome analysis revealed that PAV-104 treatment reversed SARS-CoV-2 induction of the interferon and maturation of nucleoprotein signaling pathways. Conclusion(s): PAV-104 is a pan-respiratory virus small molecule inhibitor with promising activity against SARS-CoV-2 in human airway epithelial cells that should be explored in animal models and clinical studies.

Current status of probiotic and related health benefits

This review is aimed to explore the health beneficial effects of probiotics which are live microorganisms that provide a positive health influence on humans when taken in sufficient quantity. Lactic acid bacteria, bifidobacteria, and yeast are frequently used as probiotics. These health-beneficial bacteria could compete with pathogens and modulate the gut microbiota, and exhibit immunomodulatory, anti-obesity, anti-diabetic, and anti-cancer activities which are discussed in this review. Moreover, recent studies showed that probiotics could neutralize COVID-19 infections. Hence, probiotics have become an alternative to several drugs including antibiotics. In addition, probiotic efficacy also depends on the delivery system as the delivery agents help the bacteria to survive in the harsh environment of the human gut. Considering these health benefits of probiotics, now it has been applied to different food materials which are designated as functional food. This review explored a portrait of the beneficial effects of probiotics on human health.Copyright © 2022 The Author(s)

D155Y substitution of SARS-CoV-2 ORF3a weakens binding with Caveolin-1.

The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

Shionone-Targeted Pneumolysin to Ameliorate Acute Lung Injury Induced by Streptococcus pneumoniae In Vivo and In Vitro.

Streptococcus pneumoniae (S. pneumoniae), as a Gram-positive bacterium, can cause severe bacterial pneumonia, and result in high morbidity and mortality in infected people. Meanwhile, isolated drug-resistant S. pneumoniae is growing, which raises concerns about strategies for combatting S. pneumoniae infection. To disturb S. pneumoniae pathogenicity and its drug-resistance, developing novel anti-infective strategies or compounds is urgent. In this study, the anti-infective effect of shionone was explored. A minimum inhibitory concentration (MIC) assay and growth curve determination were performed to evaluate the effect of the tetracyclic triterpenoid compound shionone against S. pneumoniae. Hemolysis tests, western blotting, oligomerization inhibition assays, and molecular docking were carried out to explore the anti-infective mechanism of shionone. Moreover, the protective effect of shionone was also confirmed in a mousepneumonia model. The results showed that the excellent hemolytic inhibitory activity of shionone was observed at less than 8 µg/mL. Meanwhile, shionone could disturb the oligomerization of pneumolysin (PLY) but did not interfere with PLY expression at less than 4 µg/mL. Molecular docking suggested that shionone targeted the ASP-59, ILE-60, THR-57, PHE-344, and ASN-346 amino acid sites to reduce S. pneumoniae pathogenicity. Furthermore, shionone alleviated lung histopathologic injury and decreased lung bacterial colonization in vivo. The above results showed that shionone could bind to the PLY active pocket under the concentrations of 8 µg/mL and neutralize PLY hemolysis activity to reduce S. pneumoniae pathogenicity in vitro and in vivo.

NLRP3 inflammasome: an important therapeutic target for SARS-CoV-2 infection

SARS-CoV-2, the pathogen of the COVID-19 pandemic, causes serious damage to human health and social stability.In severe COVID-19 cases, the infection triggers cytokine storm, resulting in multi-organ excessive inflammatory responses and even failure, which eventually leads to death.Recent studies have shown the activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome plays an essential role in the pathogenesis of COVID-19.SARS-CoV-2 can activate NLRP3 inflammasome through several pathways, thereby inducing the release of a large number of pro-inflammatory cytokines.This article reviews the activation of NLRP3 inflammasome caused by SARS-CoV-2 infection and the possible molecular mechanisms, and summarizes the progress in targeted inhibition of NLRP3 inflammation aiming to provide a new strategy for the treatment of SARS-CoV-2 infection.

THE DANGER OF HYPERGLYCEMIA DURING COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is an RNA beta-coronavirus responsible for the coronavirus disease 2019 (COVID-19). COVID-19 encompasses a large range of disease severity, from mild symptoms to severe forms with Intensive Care Unit admission and eventually death. The severe forms of COVID-19 are usually observed in high-risk patients, as those with type two diabetes mellitus. Acute hyperglycemia at hospital admission represents a risk factor for poor COVID-19 prognosis in patients with and without diabetes. Acute and chronic glycemic control are both emerging as major determinants of vaccination efficacy, disease severity, and mortality rate in COVID-19 patients. Mechanistically, it has been proposed that hyperglycemia might be a disease-modifier for COVID-19 through multiple mechanisms: 1- induction of glycation and oligomerization of ACE2, the main receptor of SARS-CoV-2;2- increased expression of the serine protease TMPRSS2, responsible for S protein priming;3- impairment of the function of innate and adaptive immunity despite the induction of higher pro-inflammatory responses, both local and systemic. Consistently, managing acute hyperglycemia through insulin infusion has been suggested to improve clinical outcomes while implementing chronic glycemic control positively affects the immune response following vaccination. Here, we review the available evidence linking acute and chronic hyperglycemia to COVID-19 outcomes, describing also the putative mediators of such interactions and proposing glycemic control as a potential route to optimize disease prevention and management.

Dysfunctional Heteroreceptor Complexes as Novel Targets for the Treatment of Major Depressive and Anxiety Disorders.

Among mental diseases, major depressive disorder (MDD) and anxiety deserve a special place due to their high prevalence and their negative impact both on society and patients suffering from these disorders. Consequently, the development of novel strategies designed to treat them quickly and efficiently, without or at least having limited side effects, is considered a highly important goal. Growing evidence indicates that emerging properties are developed on recognition, trafficking, and signaling of G-protein coupled receptors (GPCRs) upon their heteromerization with other types of GPCRs, receptor tyrosine kinases, and ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors. Therefore, to develop new treatments for MDD and anxiety, it will be important to identify the most vulnerable heteroreceptor complexes involved in MDD and anxiety. This review focuses on how GPCRs, especially serotonin, dopamine, galanin, and opioid heteroreceptor complexes, modulate synaptic and volume transmission in the limbic networks of the brain. We attempt to provide information showing how these emerging concepts can contribute to finding new ways to treat both MDD and anxiety disorders.

α-Hemolysin-Aided Oligomerization of the Spike Protein RBD Resulted in Improved Immunogenicity and Neutralization Against SARS-CoV-2 Variants.

The increase in confirmed COVID-19 cases and SARS-CoV-2 variants calls for the development of safe and broad cross-protective vaccines. The RBD of the spike protein was considered to be a safe and effective candidate antigen. However, the low immunogenicity limited its application in vaccine development. Herein, we designed and obtained an RBD heptamer (mHla-RBD) based on a carrier protein-aided assembly strategy. The molecular weight of mHla-RBD is up to 450 kDa, approximately 10 times higher than that of the RBD monomer. When formulated with alum adjuvant, mHla-RBD immunization significantly increased the immunogenicity of RBD, as indicated by increased titers of RBD-specific antibodies, neutralizing antibodies, Th2 cellular immune response, and pseudovirus neutralization activity, when compared to RBD monomer. Furthermore, we confirmed that RBD-specific antibodies predominantly target conformational epitopes, which was approximately 200 times that targeting linear epitopes. Finally, a pseudovirus neutralization assay revealed that neutralizing antibodies induced by mHla-RBD against different SARS-CoV-2 variants were comparable to those against the wild-type virus and showed broad-spectrum neutralizing activity toward different SARS-CoV-2 variants. Our results demonstrated that mHla-RBD is a promising candidate antigen for development of SARS-CoV-2 vaccines and the mHla could serve as a universal carrier protein for antigen design.

GeGen QinLian decoction alleviate influenza virus infectious pneumonia through intestinal flora.

Influenza in humans is often accompanied by gastroenteritis-like symptoms. GeGen QinLian decoction (GQD), a Chinese herb formula, has been widely used to treat infectious diarrhea for centuries and has the effect of restoring intestinal flora. Studies have also reported that GQD were used to treat patients with influenza. However, whether regulating the intestinal flora is one of the ways GQD treats influenza has not been confirmed. In present research, we conducted a systemic pharmacological study, and the results showed that GQD may acts through multiple targets and pathways. In influenza-infected mice, GQD treatment reduced mortality and lung inflammation. Most importantly, the mortality and lung inflammation were also reduced in influenza-infected mice that have undergone fecal microbiota transplantation (FMT) from GQD (FMT-GQD) treated mice. GQD treatment or FMT-GQD treatment restores the intestinal flora, resulting in an increase in Akkermansia_muciniphila, Desulfovibrio_C21_c20 and Lactobacillus_salivarius, and a decrease in Escherichia_coli. FMT-GQD treatment inhibited the NOD/RIP2/NF-κB signaling pathway in the intestine and affected the expression of downstream related inflammatory cytokines in mesenteric lymph nodes (mLNs) and serum. In addition, FMT-GQD treatment showed systemic protection by restraining the inflammatory differentiation of CD4+ T cells. In conclusion, our study shows that GQD can affect systemic immunity, at least in part, through the intestinal flora, thereby protect the mice against influenza virus infectious pneumonia.

Immunogenicity and Protective Efficacy of a Highly Thermotolerant, Trimeric SARS-CoV-2 Receptor Binding Domain Derivative.

The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of ∼80-100 mg/L in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of up to 100 °C and were stable to long-term storage of over 4 weeks at 37 °C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were ∼3-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1, and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

Antiviral Properties of the NSAID Drug Naproxen Targeting the Nucleoprotein of SARS-CoV-2 Coronavirus.

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.

High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2.

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.

Are Migraine Patients at Increased Risk for Symptomatic Coronavirus Disease 2019 Due to Shared Comorbidities?

The coronavirus disease 2019 (COVID-19) pandemic has rapidly transformed the whole world and forced us to look through comorbid diseases and risk factors from a different perspective. COVID-19 shows some inherent risk factors like cardiovascular comorbidities independent from age, gender, and geographic location. One of the most peculiar features of the COVID-19 pandemic is that severe acute respiratory syndrome coronavirus 2 respiratory infections disproportionately impact patients with hypertension, diabetes, and other cardiovascular comorbidities rather than those with allergic respiratory diseases and immune-compromised conditions. Migraine is a complex neuro-vasculo-inflammatory disorder that is also packed frequently with certain medical conditions including vascular disorders, hypertension, allergic diseases such as asthma and systemic inflammatory disorders. Accordingly, 2 different questions arise during the pandemic: (1) Do share comorbidities of cardiovascular diseases and hypertension increase the risk of symptomatic COVID-19 for migraine patients? (2) Do comorbid allergic and atopic diseases, including asthma act as opposite influencers alongside with female gender? This paper focuses on the co-existence of comorbidities of COVID-19, in comparison with migraine, based on a wide clinical dataset and available reports. Discussed mechanisms include potential strategic roles of angiotensin-converting enzyme 2, angiotensin-II, and nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 inflammasome, playing remarkable parts in the pathogenesis of COVID-19 and migraine. There are also some clues about the importance of endothelial and pericyte dysfunction and neuroinflammation in COVID-19 infection, related to complications and survival of the patients. The large epidemiological studies as well as basic research, focusing on migraine patients with COVID-19 will clarify these vital questions during the upcoming periods.

Enhancing innate immunity against virus in times of COVID-19: Trying to untangle facts from fictions.

INTRODUCTION: In light of the current COVID-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. METHODS: Without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. Some of these interventions can also stimulate the adaptive T- and B-cell responses, but our main focus is on the innate part of immunity. We divide the reviewed interventions into: 1) lifestyle related (exercise, >7 h sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) Non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod), and 3) specific vaccines with heterologous effect (BCG vaccine, mumps-measles-rubeola vaccine, etc). RESULTS: For each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections, and eventually a reduced mortality in severe respiratory infections in the intensive care unit. At the end, a summary table demonstrates the best trials supporting (or not) clinical evidence. CONCLUSION: Several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in COVID-19 should be conducted to support solid recommendations.