A Novel Targeted RIG-I Receptor 5'Triphosphate Double Strain RNA-Based Adjuvant Significantly Improves the Immunogenicity of the SARS-CoV-2 Delta-Omicron Chimeric RBD-Dimer Recombinant Protein Vaccine.

The rapid mutation and spread of SARS-CoV-2 variants recently, especially through the emerging variants Omicron BA5, BF7, XBB and BQ1, necessitate the development of universal vaccines to provide broad spectrum protection against variants. For the SARS-CoV-2 universal recombinant protein vaccines, an effective approach is necessary to design broad-spectrum antigens and combine them with novel adjuvants that can induce high immunogenicity. In this study, we designed a novel targeted retinoic acid-inducible gene-I (RIG-I) receptor 5'triphosphate double strain RNA (5'PPP dsRNA)-based vaccine adjuvant (named AT149) and combined it with the SARS-CoV-2 Delta and Omicron chimeric RBD-dimer recombinant protein (D-O RBD) to immunize mice. The results showed that AT149 activated the P65 NF-κB signaling pathway, which subsequently activated the interferon signal pathway by targeting the RIG-I receptor. The D-O RBD + AT149 and D-O RBD + aluminum hydroxide adjuvant (Al) + AT149 groups showed elevated levels of neutralizing antibodies against the authentic Delta variant, and Omicron subvariants, BA1, BA5, and BF7, pseudovirus BQ1.1, and XBB compared with D-O RBD + Al and D-O RBD + Al + CpG7909/Poly (I:C) groups at 14 d after the second immunization, respectively. In addition, D-O RBD + AT149 and D-O RBD + Al + AT149 groups presented higher levels of the T-cell-secreted IFN-γ immune response. Overall, we designed a novel targeted RIG-I receptor 5'PPP dsRNA-based vaccine adjuvant to significantly improve the immunogenicity and broad spectrum of the SARS-CoV-2 recombinant protein vaccine.

Forecasting ward-level bed requirements to aid pandemic resource planning: Lessons learned and future directions.

During the COVID-19 pandemic, there has been considerable research on how regional and country-level forecasting can be used to anticipate required hospital resources. We add to and build on this work by focusing on ward-level forecasting and planning tools for hospital staff during the pandemic. We present an assessment, validation, and deployment of a working prototype forecasting tool used within a modified Traffic Control Bundling (TCB) protocol for resource planning during the pandemic. We compare statistical and machine learning forecasting methods and their accuracy at one of the largest hospitals (Vancouver General Hospital) in Canada against a medium-sized hospital (St. Paul's Hospital) in Vancouver, Canada through the first three waves of the COVID-19 pandemic in the province of British Columbia. Our results confirm that traditional statistical and machine learning (ML) forecasting methods can provide valuable ward-level forecasting to aid in decision-making for pandemic resource planning. Using point forecasts with upper 95% prediction intervals, such forecasting methods would have provided better accuracy in anticipating required beds on COVID-19 hospital units than ward-level capacity decisions made by hospital staff. We have integrated our methodology into a publicly available online tool that operationalizes ward-level forecasting to aid with capacity planning decisions. Importantly, hospital staff can use this tool to translate forecasts into better patient care, less burnout, and improved planning for all hospital resources during pandemics.

Rapid Single-Round Pool Testing of Infectious Disease Enabled by Multicolor Digital Melting PCR.

Pooled nucleic acid amplification test is a promising strategy to reduce cost and resources for screening large populations for infectious disease. However, the benefit of pooled testing is reversed when disease prevalence is high, because of the need to retest each sample to identify infected individual when a pool is positive. Split, Amplify, and Melt analysis of Pooled Assay (SAMPA) is presented, a multicolor digital melting PCR assay in nanoliter chambers that simultaneously identify infected individuals and quantify their viral loads in a single round of pooled testing. This is achieved by early sample tagging with unique barcodes and pooling, followed by single molecule barcode identification in a digital PCR platform using a highly multiplexed melt curve analysis strategy. The feasibility is demonstrated of SAMPA for quantitative unmixing and variant identification from pools of eight synthetic DNA and RNA samples corresponding to the N1 gene, as well as from heat-inactivated SARS-CoV-2 virus. Single round pooled testing of barcoded samples with SAMPA can be a valuable tool for rapid and scalable population testing of infectious disease.

Research Advances on the Stability of mRNA Vaccines.

Compared to other vaccines, the inherent properties of messenger RNA (mRNA) vaccines and their interaction with lipid nanoparticles make them considerably unstable throughout their life cycles, impacting their effectiveness and global accessibility. It is imperative to improve mRNA vaccine stability and investigate the factors influencing stability. Since mRNA structure, excipients, lipid nanoparticle (LNP) delivery systems, and manufacturing processes are the primary factors affecting mRNA vaccine stability, optimizing mRNA structure and screening excipients can effectively improve mRNA vaccine stability. Moreover, improving manufacturing processes could also prepare thermally stable mRNA vaccines with safety and efficacy. Here, we review the regulatory guidance associated with mRNA vaccine stability, summarize key factors affecting mRNA vaccine stability, and propose a possible research path to improve mRNA vaccine stability.

Predicting DNA-binding protein and coronavirus protein flexibility using protein dihedral angle and sequence feature.

The flexibility of protein structure is related to various biological processes, such as molecular recognition, allosteric regulation, catalytic activity, and protein stability. At the molecular level, protein dynamics and flexibility are important factors to understand protein function. DNA-binding proteins and Coronavirus proteins are of great concern and relatively unique proteins. However, exploring the flexibility of DNA-binding proteins and Coronavirus proteins through experiments or calculations is a difficult process. Since protein dihedral rotational motion can be used to predict protein structural changes, it provides key information about protein local conformation. Therefore, this paper introduces a method to improve the accuracy of protein flexibility prediction, DihProFle (Prediction of DNA-binding proteins and Coronavirus proteins flexibility introduces the calculated dihedral Angle information). Based on protein dihedral Angle information, protein evolution information, and amino acid physical and chemical properties, DihProFle realizes the prediction of protein flexibility in two cases on DNA-binding proteins and Coronavirus proteins, and assigns flexibility class to each protein sequence position. In this study, compared with the flexible prediction using sequence evolution information, and physicochemical properties of amino acids, the flexible prediction accuracy based on protein dihedral Angle information, sequence evolution information and physicochemical properties of amino acids improved by 2.2% and 3.1% in the nonstrict and strict conditions, respectively. And DihProFle achieves better performance than previous methods for protein flexibility analysis. In addition, we further analyzed the correlation of amino acid properties and protein dihedral angles with residues flexibility. The results show that the charged hydrophilic residues have higher proportion in the flexible region, and the rigid region tends to be in the angular range of the protein dihedral angle (such as the ψ angle of amino acid residues is more flexible than rigid in the range of 91°-120°). Therefore, the results indicate that hydrophilic residues and protein dihedral angle information play an important role in protein flexibility.

Assessing the spatial distribution of and inequality in 15-minute PCR test site accessibility in Beijing and Guangzhou, China.

China has been planning to construct SARS-CoV-2 antigen testing sites within a 15-min walk in most major cities to timely identify asymptomatic cases and stop the transmission of COVID-19. However, little is known about the spatial distribution of 15-min accessibility to PCR test sites. In this study, we analyze the spatial distribution of and inequality in 15-min accessibility to PCR test sites in two major Chinese cities (Beijing and Guangzhou) based on the cumulative-opportunity model. The results indicate that the current distribution of 15-min accessibility to PCR test sites is satisfactory when normal commuting is not disrupted. However, disruptions of normal commuting (e.g., due to work-from-home restrictions) can negatively influence 15-min accessibility to PCR test sites and increase its inequality. Our study provides policymakers with up-to-date knowledge about the spatial distribution of 15-min accessibility to PCR test sites, identifies the disadvantaged neighborhoods in terms of test site accessibility, and highlights the changes in accessibility and inequality because of travel disruptions.

Blood transcriptome and machine learning identified the crosstalk between COVID-19 and fibromyalgia: a preliminary study.

OBJECTIVES: The COVID-19 pandemic caused by SARS-CoV-2 has seriously threatened the human health. Growing evidence shows that COVID-19 patients who recovery will persist with symptoms of fibromyalgia (FM). However, the common molecular mechanism between COVID-19 and FM remains unclear. METHODS: We obtained blood transcriptome data of COVID-19 (GSE177477) and FM (GSE67311) patients from GEO database, respectively. Subsequently, we applied Limma, GSEA, Wikipathway, KEGG, GO, and machine learning analysis to confirm the common pathogenesis between COVID-19 and FM, and screened key genes for the diagnosis of COVID-19 related FM. RESULTS: A total of 2505 differentially expressed genes (DEGs) were identified in the FM dataset. Functional enrichment analysis revealed that the occurrence of FM was intimately associated with viral infection. Moreover, WGCNA analysis identified 243 genes firmly associated with the pathological process of COVID-19. Subsequently, 50 common genes were screened between COVID-19 and FM, and functional enrichment analysis of these common genes primarily involved in immunerelated pathways. Among these common genes, 3 key genes were recognised by machine learning for the diagnosis of COVID-19 related FM. We also developed a diagnostic nomogram to predict the risk of FM occurrence which showed excellent predictive performance. Finally, we found that these 3 key genes were closely relevant to immune cells and screened potential drugs that interacted with the key genes. CONCLUSIONS: Our study revealed the bridge role of immune dysregulation between COVID-19 and fibromyalgia, and screened underlying biomarkers to provide new clues for further clinical research.

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia <1220> and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7−9.8 ng/mL for pseudovirus and EC50: 9.6−127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369−379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70−143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.

Research progress on circular RNA vaccines.

Owing to the success of linear mRNA coronavirus disease 2019 (COVID-19) vaccines, biopharmaceutical companies and research teams worldwide have attempted to develop more stable circular RNA (circRNA) vaccines and have achieved some preliminary results. This review aims to summarize key findings and important progress made in circRNA research, the in vivo metabolism and biological functions of circRNAs, and research progress and production process of circRNA vaccines. Further, considerations regarding the quality control of circRNA vaccines are highlighted herein, and the main challenges and problem-solving strategies in circRNA vaccine development and quality control are outlined to provide a reference for circRNA vaccine-related research.

Heterologous Omicron-adapted vaccine as a secondary booster promotes neutralizing antibodies against Omicron and its sub-lineages in mice.

Over one billion people have received 2-3 dosages of an inactivated COVID-19 vaccine for basic immunization. Whether a booster dose should be delivered to protect against the Omicron variant and its sub-lineages, remains controversial. Here, we tested different vaccine platforms targeting the ancestral or Omicron strain as a secondary booster of the ancestral inactivated vaccine in mice. We found that the Omicron-adapted inactivated viral vaccine promoted a neutralizing antibody response against Omicron in mice. Furthermore, heterologous immunization with COVID-19 vaccines based on different platforms remarkably elevated the levels of cross- neutralizing antibody against Omicron and its sub-lineages. Omicron-adapted vaccines based on heterologous platforms should be prioritized in future vaccination strategies to control COVID-19.

Research progress on circular RNA vaccines

Owing to the success of linear mRNA coronavirus disease 2019 (COVID-19) vaccines, biopharmaceutical companies and research teams worldwide have attempted to develop more stable circular RNA (circRNA) vaccines and have achieved some preliminary results. This review aims to summarize key findings and important progress made in circRNA research, the in vivo metabolism and biological functions of circRNAs, and research progress and production process of circRNA vaccines. Further, considerations regarding the quality control of circRNA vaccines are highlighted herein, and the main challenges and problem-solving strategies in circRNA vaccine development and quality control are outlined to provide a reference for circRNA vaccine-related research.

Chest CT Features of Patients with COVID-19 Pneumonia Following Hospital Discharge.

OBJECTIVE: To evaluate chest computed tomographic (CT) findings in patients with coronavirus disease 2019 (COVID-19) pneumonia following hospital discharge. METHODS: 52 patients with confirmed COVID-19 pneumonia underwent follow-up chest CT. The scans were obtained on average 43.1 days after hospital admission and analyzed for parenchymal abnormality (e.g., ground-glass opacities, consolidation, or interstitial thickening) and evidence of fibrosis (e.g., assigned to one of three groups: Group 1 (normal lung), Group 2 (parenchymal abnormality but without evidence of fibrosis), and Group 3 (evidence of fibrosis)). Clinical data and CT manifestations of the patients were compared among the three groups. RESULTS: 30.8% (16/52) of patients with COVID-19 pneumonia showed normal lung and were designated as Group 1. 69.2% (36/52) of patients showed parenchymal abnormality ranging from residual ground-glass opacities, consolidation, or interstitial thickening in Group 2 (51.9%) to fibrosis in Group 3 (17.3%). All patients in Group3 had severe/critical COVID-19, while most patients in Group 2 and Group 1 had common COVID-19. Patients in Group 3 were older (60.9 vs. 40.8 and 36.8 years, p<0.001, there is a significant difference), had a longer hospitalization day (20.2 vs. 15.3 and 12.3 days, p<0.05, there is a significant difference), a higher ratio of patients with comorbidities (88.9%vs14.8% and 25%, p<0.001, there is a significant difference), and higher peak CT scores (13 vs. 6.2 and 3.2, p<0.001, there is a significant difference) than those patients in Group 2 and Group 1. CONCLUSIONS: Elderly severe/critical COVID-19 patients with comorbidities are more prone to develop fibrosis early on following hospital discharge. On the other hand, lung inflammation in younger patients with common COVID-19 can be resolved completely.

Exploring the Transmission Path, Influencing Factors and Risk of Aerosol Transmission of SARS-CoV-2 at Xi'an Xianyang International Airport.

SARS-CoV-2 genetic sequence results collected from native COVID-19 cases who waited or saw relatives off at Xi'an Xianyang International Airport were highly consistent with the imported cases. In order to explore the routes of transmission and influencing factors that may cause the transmission of SARS-CoV-2 at the airport, a field simulation experiment of aerosol diffusion was adopted based on epidemiological survey data and a detailed field investigation of airport structure and ventilation. The results showed that the inbound passengers waited for approximately 3 h in the rest area on the first level of the international arrival area (Zone E). During the period, masks were removed for eating and drinking, resulting in the viral aerosols rising from the first level to the second level with hot air. After deplaning, the inbound passengers handled the relevant procedures and passed through the corridor on the second floor. The local side wall of the corridor adopted fan coil air conditioning, combined with fresh air supply and personnel walking, resulting in airflow flowing to Zone E. After merging with diffused air containing virus aerosol from the first floor, it continued to spread upward to the connected third-layer area. There was a local suspended ceiling on the top of the third floor, but it was approximately 4 m high and connected to the corridor from Terminal 2 to Terminal 3. When the virus aerosol diffused above the Terminal 2-Terminal 3 corridor, where the temperature was low and the air diffused downward, it could cause an infection risk for people passing through the corridor. In addition, the investigation found that the exhaust pipes of the nucleic acid sampling rooms at the international arrival corridor were directly discharged outdoors without treatment. Only one exhaust pipe and poor ventilation in the bathroom in Zone E had a risk of viral aerosol diffusion. Therefore, the international arrival area should be set up alone or separated from the other areas by hard isolation to avoid the existence of communication between different areas that could cause viral aerosols to diffuse with airflow. The toilet ventilation should be increased to avoid the accumulation of viral aerosols at high concentrations. The exhaust pipes of the toilet and the nucleic acid sampling rooms should be equipped with disinfection and efficient filtration devices, and high-altitude emission should be adopted to reduce the risk of virus aerosol diffusion.

Toward a Healthy Urban Living Environment: Assessing 15-Minute Green-Blue Space Accessibility

Exposure to green-blue space has been shown to be associated with better physical and mental health outcomes. The advent of COVID-19 has underlined the importance for people to have access to green-blue spaces in proximity to their residences due to pandemic-related restrictions on activity space. The implementation of the 15-min concept, which advocates that people should be able to reach locations of essential functions like green-blue spaces within 15 min of active travel, can bring green-blue spaces nearer to where people live. Nonetheless, there is still a lack of understanding of the social and spatial (in)equality in 15-min green-blue space accessibility by active travel in cities seeking to embrace the concept, such as Hong Kong. This study explores 15-min green-blue space accessibility by walking and cycling in Hong Kong to reveal the distribution of disadvantaged neighborhoods. The results show that neighborhoods in Kowloon's districts are the most disadvantaged in accessing green-blue spaces within 15 min of active travel. Our study provides policymakers with valuable insights and knowledge conducive to formulating policies aimed at reducing inequality in 15-min accessibility.

Factors Influencing Consumer Satisfaction of Fresh Produce E-Commerce in the Background of COVID-19 - A Hybrid Approach Based on LDA-SEM-XGBoost

In order to clarify the influencing factors of fresh produce e-commerce consumer satisfaction in the context of COVID-19, a hybrid approach based on LDA-SEM-XGBoost was proposed by studying online reviews. Firstly, topic elements were extracted through the LDA topic model, PLS-SEM was established to explore the paths between variables, and XGBoost models were applied to rank the importance of each topic variable based on satisfaction. The results showed that epidemic factors had a significant impact on logistics factors, product factors, and platform factors, with the epidemic factors having the greatest impact on logistics factors. Logistics factors, product factors, platform factors, and epidemic factors had a significant impact on consumer satisfaction, with logistics factors having the greatest impact on satisfaction. The topic variables affecting fresh produce e-commerce consumer satisfaction were, in order: logistics time, shipping speed, product quality, delivery speed, after-sales strategy, logistics packaging, product price, the impact of COVID-19, marketing strategy, and product brand. Based on these findings, recommendations are made for the sustainable production and marketing of fresh produce.

COVID-19 vaccination boosts the potency and breadth of the immune response against SARS-CoV-2 among recovered patients in Wuhan.

The immunity of patients who recover from coronavirus disease 2019 (COVID-19) could be long lasting but persist at a lower level. Thus, recovered patients still need to be vaccinated to prevent reinfection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or its mutated variants. Here, we report that the inactivated COVID-19 vaccine can stimulate immunity in recovered patients to maintain high levels of anti-receptor-binding domain (RBD) and anti-nucleocapsid protein (NP) antibody titers within 9 months, and high neutralizing activity against the prototype, Delta, and Omicron strains was observed. Nevertheless, the antibody response decreased over time, and the Omicron variant exhibited more pronounced resistance to neutralization than the prototype and Delta strains. Moreover, the intensity of the SARS-CoV-2-specific CD4+ T cell response was also increased in recovered patients who received COVID-19 vaccines. Overall, the repeated antigen exposure provided by inactivated COVID-19 vaccination greatly boosted both the potency and breadth of the humoral and cellular immune responses against SARS-CoV-2, effectively protecting recovered individuals from reinfection by circulating SARS-CoV-2 and its variants.

A Short 5'triphosphate RNA nCoV-L Induces a Broad-Spectrum Antiviral Response by Activating RIG-I.

Small molecular nucleic acid drugs produce antiviral effects by activating pattern recognition receptors (PRRs). In this study, a small molecular nucleotide containing 5'triphosphoric acid (5'PPP) and possessing a double-stranded structure was designed and named nCoV-L. nCoV-L was found to specifically activate RIG-I, induce interferon responses, and inhibit duplication of four RNA viruses (Human enterovirus 71, Human poliovirus 1, Human coxsackievirus B5 and Influenza A virus) in cells. In vivo, nCoV-L quickly induced interferon responses and protected BALB/c suckling mice from a lethal dose of the enterovirus 71. Additionally, prophylactic administration of nCoV-L was found to reduce mouse death and relieve morbidity symptoms in a K18-hACE2 mouse lethal model of SARS-CoV-2. In summary, these findings indicate that nCoV-L activates RIG-I and quickly induces effective antiviral signals. Thus, it has potential as a broad-spectrum antiviral drug.

Clinical Characterizations and Radiological Findings of COVID-19: A 4 Case Report.

BACKGROUND: Coronavirus disease 2019 (COVID-19, previously known as novel coronavirus [2019-nCoV]), first reported in China, has now been declared a global health emergency by World Health Organization. The clinical severity ranges from asymptomatic individuals to death. Here, we report clinical features and radiological changes of a cured family cluster infected with COVID-19. CASE PRESENTATION: In this report, we enrolled a family of 4 members who were admitted to our hospital in January 2020. We performed a detailed analysis of each patient's records. All patients underwent chest computed tomography (CT) examination with 120 kilovolts peak and 150 kilovolt-ampere. Realtime polymerase chain reaction (RT-PCR) examinations for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid were done using nasopharyngeal swabs. CONCLUSION: In the family members infected with COVID-19 who were accompanied by other diseases or had low immunity, the pneumonia was prone to be aggravated.

Integrated analysis of doubly disadvantaged neighborhoods by considering both green space and blue space accessibility and COVID-19 infection risk.

The ongoing COVID-19 pandemic has taken a heavy toll on the physical and mental health of the public. Nevertheless, the presence of green and blue spaces has been shown to be able to encourage physical activities and alleviate the mental distress caused by COVID-19. However, just as the impact of COVID-19 varies by geographical region and area, the distribution of green and blue spaces is also different across different neighborhoods and areas. By using Hong Kong as the study area, we determine the local neighborhoods that suffer from both high COVID-19 infection risk as well as low green and blue space accessibility. The results show that some of the poorest neighborhoods in the territory such as Sham Shui Po, Kwun Tong and Wong Tai Sin are also among the most doubly disadvantaged in terms of COVID-19 infection risk as well as green and blue space accessibility.