A Vaccine of SARS-CoV-2 S Protein RBD Induces Protective Immunity.

The pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed great threat to the world in many aspects. There is an urgent requirement for an effective preventive vaccine. The receptor binding domain (RBD), located on the spike (S) gene, is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. The RBD protein is an effective and safe antigen candidate. The six-helix bundle (6HB) "molecular clamp" is a novel thermally-stable trimerization domain derived from a human immunodeficiency virus (HIV) gp41 protein segment. We selected the baculovirus system to fuse and express the RBD protein and 6HB for imitating the natural trimeric structure of RBD, named RBD-6HB. Recombinant RBD-6HB was successfully obtained from the cell culture supernatant and purified to high homogeneity. The purity of the final protein preparation was more than 97%. The results showed that the protein was identified as a homogeneous polymer. Further studies showed that the RBD-6HB protein combined with AL/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges. Our findings highlight the importance of trimerized SARS-CoV-2 S protein RBD in designing SARS-CoV-2 vaccines and provide a rationale for developing a protective vaccine through the induction of antibodies against the RBD domain.

Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

An integrated approach of high-performance liquid chromatography-mass spectrometry-based chemical profiling, network pharmacology, and molecular docking to reveal the potential mechanisms of Qishen Gubiao granules for treating coronavirus disease 2019.

Qishen Gubiao granules, a traditional Chinese medicine preparation composed of nine herbs, have been widely used to prevent and treat coronavirus disease 2019 with good clinical efficacy. In the present study, an integrated strategy based on chemical profiling followed by network pharmacology and molecular docking was employed, to explore the active components and potential molecular mechanisms of Qishen Gubiao granules in the therapy of coronavirus disease 2019. Using the ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry technique, a total of 186 ingredients corresponding to eight structure types in Qishen Gubiao preparation were identified or structurally annotated with the elucidation of the fragmentation pathways in the typical compounds. The network pharmacology analysis screened 28 key compounds including quercetin, apigenin, scutellarein, luteolin and naringenin acting on 31 key targets, which possibly modulated signal pathways associated with immune and inflammatory responses in the treatment of coronavirus disease 2019. The molecular docking results observed that the top 5 core compounds had a high affinity for angiotensin-converting enzyme 2 and 3-chymotrypsin-like protease. This study proposed a reliable and feasible approach for elucidating the multi-components, multi-targets, and multi-pathways intervention mechanism of Qishen Gubiao granules against coronavirus disease 2019, providing a scientific basis for its further quality evaluation and clinical application.

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

A micro-sized vaccine based on recombinant Lactiplantibacillus plantarum fights against SARS-CoV-2 infection via intranasal immunization.

COVID-19 has globally spread to burden the medical system. Even with a massive vaccination, a mucosal vaccine offering more comprehensive and convenient protection is imminent. Here, a micro-sized vaccine based on recombinant Lactiplantibacillus plantarum (rLP) displaying spike or receptor-binding domain (RBD) was characterized as microparticles, and its safety and protective effects against SARS-CoV-2 were evaluated. We found a 66.7% mortality reduction and 100% protection with rLP against SARS-CoV-2 in a mouse model. The histological analysis showed decreased hemorrhage symptoms and increased leukocyte infiltration in the lung. Especially, rLP:RBD significantly decreased pulmonary viral loads. For the first time, our study provides a Lactiplantibacillus plantarum-vectored vaccine to prevent COVID-19 progress and transmission via intranasal vaccination.

Multi-robot task assignment for serving people quarantined in multiple hotels during COVID-19 pandemic.

Background: Efficiently combating with the coronavirus disease 2019 (COVID-19) has been challenging for medics, police and other service providers. To reduce human interaction, multi-robot systems are promising for performing various missions such as disinfection, monitoring, temperature measurement and delivering goods to people quarantined in prescribed homes and hotels. This paper studies the task assignment problem for multiple dispersed homogeneous robots to visit a set of prescribed hotels to perform tasks such as body temperature assessment or oropharyngeal swabs for people quarantined in the hotels while trying to minimize the robots' total operation time. Each robot can move to multiple hotels sequentially within its limited maximum operation time to provide the service. Methods: The task assignment problem generalizes the multiple traveling salesman problem, which is an NP-hard problem. The main contributions of the paper are twofold: (I) a lower bound on the robots' total operation time to serve all the people has been found based on graph theory, which can be used to approximately evaluate the optimality of an assignment solution; (II) several efficient marginal-cost-based task assignment algorithms are designed to assign the hotel-serving tasks to the robots. Results: In the Monte Carlo simulations where different numbers of robots need to serve the people quarantined in 30 and 90 hotels, the designed task assignment algorithms can quickly (around 30 ms) calculate near-optimal assignment solutions (within 1.15 times of the optimal value). Conclusions: Numerical simulations show that the algorithms can lead to solutions that are close to the optimal compared with the competitive genetic algorithm and greedy algorithm.

The E3 ligase RNF5 restricts SARS-CoV-2 replication by targeting its envelope protein for degradation.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe global health crisis; its structural protein envelope (E) is critical for viral entry, budding, production, and induction of pathology which makes it a potential target for therapeutics against COVID-19. Here, we find that the E3 ligase RNF5 interacts with and catalyzes ubiquitination of E on the 63rd lysine, leading to its degradation by the ubiquitin-proteasome system (UPS). Importantly, RNF5-induced degradation of E inhibits SARS-CoV-2 replication and the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model. We also found that RNF5 is distinctively expressed in different age groups and in patients displaying different disease severity, which may be exploited as a prognostic marker for COVID-19. Furthermore, RNF5 recognized the E protein from various SARS-CoV-2 strains and SARS-CoV, suggesting that targeting RNF5 is a broad-spectrum antiviral strategy. Our findings provide novel insights into the role of UPS in antagonizing SARS-CoV-2 replication, which opens new avenues for therapeutic intervention to combat the COVID-19 pandemic.

Dysregulation of glutamine/glutamate metabolism in COVID-19 patients: A metabolism study in African population and mini meta-analysis.

Coronavirus disease 2019 (COVID-19) remains a serious global threat. The metabolic analysis had been successfully applied in the efforts to uncover the pathological mechanisms and biomarkers of disease severity. Here we performed a quasi-targeted metabolomic analysis on 56 COVID-19 patients from Sierra Leone in western Africa, revealing the metabolomic profiles and the association with disease severity, which was confirmed by the targeted metabolomic analysis of 19 pairs of COVID-19 patients. A meta-analysis was performed on published metabolic data of COVID-19 to verify our findings. Of the 596 identified metabolites, 58 showed significant differences between severe and nonsevere groups. The pathway enrichment of these differential metabolites revealed glutamine and glutamate metabolism as the most significant metabolic pathway (Impact = 0.5; -log10P = 1.959). Further targeted metabolic analysis revealed six metabolites with significant intergroup differences, with glutamine/glutamate ratio significantly associated with severe disease, negatively correlated with 10 clinical parameters and positively correlated with SPO2 (rs = 0.442, p = 0.005). Mini meta-analysis indicated elevated glutamate was related to increased risk of COVID-19 infection (pooled odd ratio [OR] = 2.02; 95% confidence interval [CI]: 1.17-3.50) and severe COVID-19 (pooled OR = 2.28; 95% CI: 1.14-4.56). In contrast, elevated glutamine related to decreased risk of infection and severe COVID-19, the pooled OR were 0.30 (95% CI: 0.20-0.44), and 0.44 (95% CI: 0.19-0.98), respectively. Glutamine and glutamate metabolism are associated with COVID-19 severity in multiple populations, which might confer potential therapeutic target of COVID-19, especially for severe patients.

Effect of BMI on Proinflammatory Markers in Unvaccinated Adults Recently Diagnosed With COVID-19

Background: Obesity has arisen as a prominent risk factor for COVID-19 severity and mortality, potentially owed to an obesity-induced proinflammatory state impacting immune and thrombotic responses. This report characterized the associations between BMI and inflammatory biomarkers in an adult population recently diagnosed with COVID-19 and not hospitalized. Methods: These data were derived from a double-blind randomized placebo-controlled clinical trial conducted October 2020-March 2021 (NCT04912921). Participants (N=60) were unvaccinated and reported to the test site following diagnosis as allowed by the CDC's return to work policy. At study baseline and week four, anthropometrics and non-fasting blood samples were collected from participants. We examined the associations between BMI (continuous and categorical) and inflammatory markers via multiple regression models. Results: Our analyses revealed significant associations between baseline BMI and IL-6 (ß=0.036, p=0.0001), TNF-α (ß=0.015, p=0.0059), Ferritin (ß=0.018, p=0.0097), C-reactive protein (ß=0.057, p<0.0001), and WBC count (ß=0.104, p=0.0308) adjusting for age, sex, existing conditions, and interval between COVID-19 diagnosis and trial initiation. Analyses of BMI categories revealed similar significant associations between obesity (BMI>30 kg/m2) and elevations in IL-6, CRP, Ferritin, and WBC at baseline (p<0.05). For those individuals in the control group (n=30), after four weeks, an obese BMI was significantly associated with increases in CRP, TNF-α, soluble P-selectin, and ICAM-1 (p<0.05). Conclusions: The presence of a hyperinflammatory state is a main driver of the severity of COVID-19, and a suggested link between the increased risk of poor COVID-19 outcomes in individuals with obesity. The present study demonstrated significant associations between BMI and several proinflammatory markers associated with disease severity in an adult population recently diagnosed with COVID-19.

SARS-COV-2 as potential microRNA sponge in COVID-19 patients.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNA that can downregulate their targets by selectively binding to the 3' untranslated region (3'UTR) of most messenger RNAs (mRNAs) in the human genome. MiRNAs can interact with other molecules such as viruses and act as a mediator for viral infection. In this study, we examined whether, and to what extent, the SARS-CoV-2 virus can serve as a "sponge" for human miRNAs. RESULTS: We identified multiple potential miRNA/target pairs that may be disrupted during SARS-CoV-2 infection. Using miRNA expression profiles and RNA-seq from published studies, we further identified a highly confident list of 5 miRNA/target pairs that could be disrupted by the virus's miRNA sponge effect, namely hsa-miR-374a-5p/APOL6, hsa-let-7f-1-3p/EIF4A2, hsa-miR-374a-3p/PARP11, hsa-miR-548d-3p/PSMA2 and hsa-miR-23b-3p/ZNFX1 pairs. Using single-cell RNA-sequencing based data, we identified two important miRNAs, hsa-miR-302c-5p and hsa-miR-16-5p, to be potential virus targeting miRNAs across multiple cell types from bronchoalveolar lavage fluid samples. We further validated some of our findings using miRNA and gene enrichment analyses and the results confirmed with findings from previous studies that some of these identified miRNA/target pairs are involved in ACE2 receptor network, regulating pro-inflammatory cytokines and in immune cell maturation and differentiation. CONCLUSION: Using publicly available databases and patient-related expression data, we found that acting as a "miRNA sponge" could be one explanation for SARS-CoV-2-mediated pathophysiological changes. This study provides a novel way of utilizing SARS-CoV-2 related data, with bioinformatics approaches, to help us better understand the etiology of the disease and its differential manifestation across individuals.

Detection of SARS-CoV-2 Neutralizing Antibodies in Vaccinated Pregnant Women and Neonates by Using a Lateral Flow Immunoassay Coupled with a Spectrum-Based Reader.

The focus of this study was to investigate the detection of neutralizing antibodies (Nabs) in maternal serum and cord blood as the targeted samples by employing a lateral flow immunoassay combined with a spectrum reader (LFI-SR) and the correlation of Nab protection against different types of SARS-CoV-2. We enrolled 20 pregnant women who were vaccinated with the Moderna (mRNA-1273) vaccine during pregnancy and collected 40 samples during delivery. We used an LFI-SR for the level of spike protein receptor binding domain antibody (SRBD IgG) as Nabs and examined the correlation of the SRBD IgG concentration and Nab inhibition rates (NabIR) via enzyme-linked immunosorbent assays (ELISA). The LFI-SR had high confidence for the SRBD IgG level (p < 0.0001). Better NabIR were found in wild-type SARS-CoV-2 (WT) compared to Delta-type (DT) and Omicron-type (OT). Women with two-dose vaccinations demonstrated greater NabIR than those with a single dose. The cut-off value of the SRBD IgG level by the LFI-SR for NabIR to DT (≥30%; ≥70%) was 60.15 and 150.21 ng/mL for mothers (both p = 0.005), and 156.31 (p = 0.011) and 230.20 ng/mL (p = 0.006) for babies, respectively. An additional vaccine booster may be considered for those mothers with SRBD IgG levels < 60.15 ng/mL, and close protection should be given for those neonates with SRBD IgG levels < 150.21 ng/mL, since there is no available vaccine for them.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Antiviral Effect of pIFNLs against PEDV and VSV Infection in Different Cells.

Type III and type I interferon have similar mechanisms of action, and their different receptors lead to different distributions in tissue. On mucosal surfaces, type III interferon exhibits strong antiviral activity. Porcine epidemic diarrhea virus (PEDV) is an economically important enteropathogenic coronavirus, which can cause a high incidence rate and mortality in piglets. Here, we demonstrate that porcine interferon lambda 1 (pIFNL1) and porcine interferon lambda 3 (pIFNL3) can inhibit the proliferation of vesicular stomatitis virus with an enhanced green fluorescent protein (VSV-EGFP) in different cells, and also show strong antiviral activity when PEDV infects Vero cells. Both forms of pIFNLs were shown to be better than porcine interferon alpha (pIFNα), the antiviral activity of pIFNL1 is lower than that of pIFNL3. Therefore, our results provide experimental evidence for the inhibition of PEDV infection by pIFNLs, which may provide a promising treatment for the prevention and treatment of Porcine epidemic diarrhea (PED) in piglets.

Real-time detection of viruses in aerosols with weak light imaging based on Tesla discharge

The analysis and detection of nucleic acid and specific antigens and antibodies are the most basic technologies for virus monitoring. However, the potential window for applying these technologies exists within a late specific period in the early monitoring and control of unknown viruses, especially human and animal pathogenic viruses transmitted via aerosols, e.g., SARS-CoV-2 and its variants. This is because early, real-time, and convenient monitoring of unknown viruses in the air or exhaled gas cannot be directly achieved through existing technologies. Herein, we report a weak light spectral imaging technology based on Tesla discharge (termed T-DAI) that can quickly monitor for viruses in real time in simulated aerosols with 71% sensitivity and 76% specificity for aerosol virus concentrations exceeding approximately 2800 vp/μl. This technology realizes the rapid detection of low concentrations of viruses in aerosols and could provide an important means for predicting, screening, and monitoring unknown or pandemic pathogenic viruses in the air or exhaled breath of humans and animals. [ FROM AUTHOR] Copyright of Applied Physics Letters is the property of American Institute of Physics and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Epidemic and Evolutionary Characteristics of Swine Enteric Viruses in South-Central China from 2018 to 2021.

Swine enteric viruses are a major cause of piglet diarrhea, causing a devastating impact on the pork industry. To further understand the molecular epidemiology and evolutionary diversity of swine enteric viruses, we carried out a molecular epidemiological investigation of swine enteric viruses (PEDV, PDCoV, PoRVA, and TGEV) on 7107 samples collected from pig farms in south-central China. The results demonstrated that PEDV is the predominant pathogen causing piglet diarrhea, and its infection occurs mainly in relatively cold winter and spring in Hunan and Hubei provinces. The positive rate of PEDV showed an abnormal increase from 2020 to 2021, and that of PoRVA and PDCoV exhibited gradual increases from 2018 to 2021. PEDV-PoRVA and PEDV-PDCoV were the dominant co-infection modes. A genetic evolution analysis based on the PEDV S1 gene and ORF3 gene revealed that the PEDV GII-a is currently epidemic genotype, and the ORF3 gene of DY2020 belongs to a different clade relative to other GII-a strains isolated in this study. Overall, our results indicated that the variant PEDV GII-a is the main pathogen of piglet diarrhea with a trend of outbreak. G9 is the dominant PoRVA genotype and has the possibility of outbreak as well. It is therefore critical to strengthen the surveillance of PEDV and PoRVA, and to provide technical reserves for the prevention and control of piglet diarrhea.