High-throughput saturation mutagenesis generates a high-affinity antibody against SARS-CoV-2 variants using protein surface display assay on a human cell.

As new mutations continue to emerge, the ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus to evade the human immune system and neutralizing antibodies remains a huge challenge for vaccine development and antibody research. The majority of neutralizing antibodies have reduced or lost activity against SARS-CoV-2 variants. In this study, we reported a novel protein surface display system on a mammalian cell for obtaining a higher-affinity antibody in high-throughput manner. Using a saturation mutagenesis strategy through integrating microarray-based oligonucleotide synthesis and single-cell screening assay, we generated a group of new antibodies against diverse prevalent SARS-CoV-2 variants through high-throughput screening the human antibody REGN10987 within 2 weeks. The affinity of those optimized antibodies to seven prevalent mutants was greatly improved, and the EC50 values were no higher than 5 ng/mL. These results demonstrate the robustness of our screening system in the rapid generation of an antibody with higher affinity against a new SARS-CoV-2 variant, and provides a potential application to other protein molecular interactions.

Biparatopic antibody BA7208/7125 effectively neutralizes SARS-CoV-2 variants including Omicron BA.1-BA.5.

SARS-CoV-2 Omicron subvariants have demonstrated extensive evasion from monoclonal antibodies (mAbs) developed for clinical use, which raises an urgent need to develop new broad-spectrum mAbs. Here, we report the isolation and analysis of two anti-RBD neutralizing antibodies BA7208 and BA7125 from mice engineered to produce human antibodies. While BA7125 showed broadly neutralizing activity against all variants except the Omicron sublineages, BA7208 was potently neutralizing against all tested SARS-CoV-2 variants (including Omicron BA.1-BA.5) except Mu. By combining BA7208 and BA7125 through the knobs-into-holes technology, we generated a biparatopic antibody BA7208/7125 that was able to neutralize all tested circulating SARS-CoV-2 variants. Cryo-electron microscopy structure of these broad-spectrum antibodies in complex with trimeric Delta and Omicron spike indicated that the contact residues are highly conserved and had minimal interactions with mutational residues in RBD of current variants. In addition, we showed that administration of BA7208/7125 via the intraperitoneal, intranasal, or aerosol inhalation route showed potent therapeutic efficacy against Omicron BA.1 and BA.2 in hACE2-transgenic and wild-type mice and, separately, effective prophylaxis. BA7208/7125 thus has the potential to be an effective candidate as an intervention against COVID-19.

Do COVID-19 CT features vary between patients from within and outside mainland China? Findings from a meta-analysis

Background Chest computerized tomography (CT) plays an important role in detecting patients with suspected coronavirus disease 2019 (COVID-19), however, there are no systematic summaries on whether the chest CT findings of patients within mainland China are applicable to those found in patients outside. Methods Relevant studies were retrieved comprehensively by searching PubMed, Embase, and Cochrane Library databases before 15 April 2022. Quality assessment of diagnostic accuracy studies (QUADAS) was used to evaluate the quality of the included studies, which were divided into two groups according to whether they were in mainland China or outside. Data on diagnostic performance, unilateral or bilateral lung involvement, and typical chest CT imaging appearances were extracted, and then, meta-analyses were performed with R software to compare the CT features of COVID-19 pneumonia between patients from within and outside mainland China. Results Of the 8,258 studies screened, 19 studies with 3,400 patients in mainland China and 14 studies with 554 outside mainland China were included. Overall, the risk of quality assessment and publication bias was low. The diagnostic value of chest CT is similar between patients from within and outside mainland China (93, 91%). The pooled incidence of unilateral lung involvement (15, 7%), the crazy-paving sign (31, 21%), mixed ground-glass opacities (GGO) and consolidations (51, 35%), air bronchogram (44, 25%), vascular engorgement (59, 33%), bronchial wall thickening (19, 12%), and septal thickening (39, 26%) in patients from mainland China were significantly higher than those from outside;however, the incidence rates of bilateral lung involvement (75, 84%), GGO (78, 87%), consolidations (45, 58%), nodules (12, 17%), and pleural effusion (9, 15%) were significantly lower. Conclusion Considering that the chest CT features of patients in mainland China may not reflect those of the patients abroad, radiologists and clinicians should be familiar with various CT presentations suggestive of COVID-19 in different regions.

Heterologous booster with inhaled adenovirus vector COVID-19 vaccine generated more neutralizing antibodies against different SARS-CoV-2 variants.

The rapid widespread Omicron subvariant BA.5 of SARS-CoV-2 has become a potential imminent pandemic threat, but available vaccines lack high efficacy against this subvariant. Thus, it is urgent to find highly protective vaccination strategies within available SARS-CoV-2 vaccines. Here, by using a SARS-CoV-2 pseudovirus neutralization assay, we demonstrated that the aerosol inhalation of adenoviral vector COVID-19 vaccine after two dose of inactivated vaccine (I-I-Ad5) led to higher levels of neutralizing antibodies against D614G strain (2041.00[95% CI, 1243.00-3351.00] vs 249.00[149.10-415.70]), Omicron BA.2 (467.10[231.00-944.40] vs 72.21[39.31-132.70]), BA.2.12.1(348.5[180.3-673.4] vs 53.17[31.29-90.37]), BA.2.13 (410.40[190.70-883.3] vs 48.48[27.87-84.32]), and BA.5 (442.40 vs 56.08[35.14-89.51]) than three inactivated vaccine doses (I-I-I). Additionally, the level of neutralizing antibodies against BA.5 induced by I-I-Ad5 was 2.41-fold higher than those boosted by a third dose of RBD subunit vaccine (I-I-S) (p = 0.1308). The conventional virus neutralizing assay confirmed that I-I-Ad5 induced higher titre of neutralizing antibodies than I-I-I (116.80[84.51-161.5] vs 4.40[4.00-4.83]). In addition, I-I-Ad5 induced higher, but later, anti-RBD IgG and IgA in plasma than I-I-I. Our study verified that mucosal immunization with aerosol inhalation of adenoviral vector COVID-19 vaccine may be an effective strategy to control the probable wave of BA.5 pandemic in addition to two inactivated vaccines.

Capturing Time Dynamics From Speech Using Neural Networks for Surgical Mask Detection.

The importance of detecting whether a person wears a face mask while speaking has tremendously increased since the outbreak of SARS-CoV-2 (COVID-19), as wearing a mask can help to reduce the spread of the virus and mitigate the public health crisis. Besides affecting human speech characteristics related to frequency, face masks cause temporal interferences in speech, altering the pace, rhythm, and pronunciation speed. In this regard, this paper presents two effective neural network models to detect surgical masks from audio. The proposed architectures are both based on Convolutional Neural Networks (CNNs), chosen as an optimal approach for the spatial processing of the audio signals. One architecture applies a Long Short-Term Memory (LSTM) network to model the time-dependencies. Through an additional attention mechanism, the LSTM-based architecture enables the extraction of more salient temporal information. The other architecture (named ConvTx) retrieves the relative position of a sequence through the positional encoder of a transformer module. In order to assess to which extent both architectures can complement each other when modelling temporal dynamics, we also explore the combination of LSTM and Transformers in three hybrid models. Finally, we also investigate whether data augmentation techniques, such as, using transitions between audio frames and considering gender-dependent frameworks might impact the performance of the proposed architectures. Our experimental results show that one of the hybrid models achieves the best performance, surpassing existing state-of-the-art results for the task at hand.

Stringency of COVID-19 Containment Response Policies and Air Quality Changes: A Global Analysis across 1851 Cities.

The COVID-19 containment response policies (CRPs) had a major impact on air quality (AQ). These CRPs have been time-varying and location-specific. So far, despite having numerous studies on the effect of COVID-19 lockdown on AQ, a knowledge gap remains on the association between stringency of CRPs and AQ changes across the world, regions, nations, and cities. Here, we show that globally across 1851 cities (each more than 300 000 people) in 149 countries, after controlling for the impacts of relevant covariates (e.g., meteorology), Sentinel-5P satellite-observed nitrogen dioxide (NO2) levels decreased by 4.9% (95% CI: 2.2, 7.6%) during lockdowns following stringent CRPs compared to pre-CRPs. The NO2 levels did not change significantly during moderate CRPs and even increased during mild CRPs by 2.3% (95% CI: 0.7, 4.0%), which was 6.8% (95% CI: 2.0, 12.0%) across Europe and Central Asia, possibly due to population avoidance of public transportation in favor of private transportation. Among 1768 cities implementing stringent CRPs, we observed the most NO2 reduction in more populated and polluted cities. Our results demonstrate that AQ improved when and where stringent COVID-19 CRPs were implemented, changed less under moderate CRPs, and even deteriorated under mild CRPs. These changes were location-, region-, and CRP-specific.

Cross-reactivity of eight SARS-CoV-2 variants rationally predicts immunogenicity clustering in sarbecoviruses.

A steep rise in Omicron reinfection cases suggests that this variant has increased immune evasion ability. To evaluate its antigenicity relationship with other variants, antisera from guinea pigs immunized with spike protein of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) were cross-tested against pseudotyped variants. The neutralization activity against Omicron was markedly reduced when other VOCs or VOIs were used as immunogens, and Omicron (BA.1)-elicited sera did not efficiently neutralize the other variants. However, a Beta or Omicron booster, when administered as the 4th dose 3-months after the 3rd dose of any of the variants, could elicit broad neutralizing antibodies against all of the current variants including Omicron BA.1. Further analysis with 280 available antigen-antibody structures and quantification of immune escape from 715 reported neutralizing antibodies provide explanations for the observed differential immunogenicity. Three distinct clades predicted using an in silico algorithm for clustering of sarbecoviruses based on immune escape provide key information for rational design of vaccines.

Characteristics of the methane (CH4) mole fraction in a typical city and suburban site in the Yangtze River Delta, China

China is the largest emitter of greenhouse gases in the world. However, the atmospheric observation of greenhouse gases is relatively sparse. In this study, surface measurements of CH4 over 5 years at a typical city site (Hangzhou) in an economically developed region in China were conducted to study the temporal variations and the influence of meteorological factors and airmass transport. The CH4 observations from a suburban site (Lin'an station [LAN]) which is a World Meteorological Organization/Global Atmosphere Monitoring Program (WMO/GAW) regional site, were also compared. Our results showed that the atmospheric CH4 mole fraction in Hangzhou was not only affected by meteorological factors and topography, but also by strong local emissions. Although the distance between the two stations was only 50 km, there was a significant difference in the temporal CH4 variations. The strong anthropogenic emissions in the city were responsible for the urban-suburban site difference. The CH4 peaks in the diurnal cycles in Hangzhou corresponded to rush hours, and there were unique variations during special periods (i.e., the National Day holiday, coronavirus disease 2019 [COVID - 19] lock-down). It also led to an annual average CH4 mole fraction at the Hangzhou station (HZ) that was on average 111.1 ± 1.6 ppb higher than that at the LAN from 2016 to 2020. The lock-down measures caused by the outbreak of COVID - 19 decreased the atmospheric CH4 mole fractions by 6.8% in Hangzhou but only 1.9% in Lin'an in 2020 compared to those in 2019. Excluding the data in 2020, the annual growth rate of the CH4 mole fraction was 19.0 ppb yr−1 in Hangzhou. Our results indicated that the CH4 mole fraction in Hangzhou was mainly driven by local anthropogenic emissions, although they were influenced by emissions from surrounding cities such as Nanjing and Ningbo.

Novel cleavage sites identified in SARS-CoV-2 spike protein reveal mechanism for cathepsin L-facilitated viral infection and treatment strategies.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) "up" activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

ACE2 decoy receptor generated by high-throughput saturation mutagenesis efficiently neutralizes SARS-CoV-2 and its prevalent variants.

The recent global pandemic was a spillover from the SARS-CoV-2 virus. Viral entry involves the receptor binding domain (RBD) of the viral spike protein interacting with the protease domain (PD) of the cellular receptor, ACE2. We hereby present a comprehensive mutational landscape of the effects of ACE2-PD point mutations on RBD-ACE2 binding using a saturation mutagenesis approach based on microarray-based oligo synthesis and a single-cell screening assay. We observed that changes in glycosylation sites and directly interacting sites of ACE2-PD significantly influenced ACE2-RBD binding. We further engineered an ACE2 decoy receptor with critical point mutations, D30I, L79W, T92N, N322V, and K475F, named C4-1. C4-1 shows a 200-fold increase in neutralization for the SARS-CoV-2 D614G pseudotyped virus compared to wild-type soluble ACE2 and a sevenfold increase in binding affinity to wild-type spike compared to the C-terminal Ig-Fc fused wild-type soluble ACE2. Moreover, C4-1 efficiently neutralized prevalent variants, especially the omicron variant (EC50=16 ng/mL), and rescued monoclonal antibodies, vaccine, and convalescent sera neutralization from viral immune-escaping. We hope to next investigate translating the therapeutic potential of C4-1 for the treatment of SARS-CoV-2.

Electronic health literacy among older adults in the context of the COVID-19 pandemic: A mixed-methods study.

AIM: This study aimed to investigate eHealth literacy about coronavirus disease 2019 (COVID-19) among older adults during the pandemic. BACKGROUND: The COVID-19 pandemic promoted the development of online health care. Higher demand for accessing information from the Internet was seen. METHODS: This was a sequential explanatory mixed-method study, involving a survey of older adults to explore the status and influencing factors of eHealth literacy regarding COVID-19. Semi-structured interviews were used to understand experiences and challenges regarding information retrieval, judgment and utilization. RESULTS: A total of 337 older adults participated in the online questionnaire survey. Overall, older adults had slightly higher scores on eHealth literacy during the COVID-19 pandemic. Participants' location in the past month and current health issues were associated with eHealth literacy. Qualitative data were collected from nine older adults and included that some older adults retrieved health-related information during the pandemic. However, those who used non-smartphones described difficulties in information retrieval. A glut of misinformation has resulted in an 'infodemic', which has not only increased the difficulty of judging information but also posed challenges in information utilization for older adults. CONCLUSION: Improving older adults' eHealth literacy is essential in promoting an improved response to major public health events and in providing better health care for this group in the future. It is essential that government health agencies and health care providers provide evidence-based health information via social media platforms. Further efforts are needed to combine aspects of traditional and online health care services and provide reliable and updated online information and resources for older adults. IMPLICATIONS FOR NURSING MANAGEMENT: Providing evidence to eHealth literacy improvement and health management of older adults in the context of public health events.

eHealth Literacy of Chinese Residents During the Coronavirus Disease 2019 Pandemic: A Cross-sectional Survey.

The Coronavirus Disease 2019 (COVID-19) pandemic has become a leading societal concern. eHealth literacy is important in the prevention and control of this pandemic. The purpose of this study is to identify eHealth literacy of Chinese residents about the COVID-19 pandemic and factors influencing eHealth literacy. A total of 15 694 individuals clicked on the link to the questionnaire, and 15 000 agreed to participate and completed the questionnaire for a response rate of 95.58%. Descriptive statistics, χ2 test, and logistic regression analysis were conducted to analyze participants' level of eHealth literacy about COVID-19 and its influencing factors. The results showed 52.2% of participants had relatively lower eHealth literacy regarding COVID-19 (eHealth literacy score ≤ 48). The scores of the information judgment dimension (3.09 ± 0.71) and information utilization dimension (3.18 ± 0.67) of the eHealth literacy scale were relatively lower. The logistics regression showed that sex, age, education level, level of uncertainty, having people around the respondent diagnosed with COVID-19, relationship with family, and relationship with others were associated to eHealth literacy (χ2 = 969.135, P < .001). The public's eHealth literacy about COVID-19 needs to be improved, especially the ability to judge and utilize online information. Close collaboration among global health agencies, governments, healthcare institutions, and media is needed to provide reliable online information to the public. Interventions to improve eHealth literacy should take into account and accentuate the importance of sex, age, educational background, level of uncertainty, exposure to disease, and social support.

Antigenicity comparison of SARS-CoV-2 Omicron sublineages with other variants contained multiple mutations in RBD.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, particularly those with multiple mutations in receptor-binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID-19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster.

A high-throughput single cell-based antibody discovery approach against the full-length SARS-CoV-2 spike protein suggests a lack of neutralizing antibodies targeting the highly conserved S2 domain.

Coronavirus disease 2019 pandemic continues globally with a growing number of infections, but there are currently no effective antibody drugs against the virus. In addition, 90% amino acid sequence identity between the S2 subunit of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV S proteins attracts us to examine S2-targeted cross-neutralizing antibodies that are not yet well defined. We therefore immunized RenMab mice with the full-length S protein and constructed a high-throughput antibody discovery method based on single-cell sequencing technology to isolate SARS-CoV-2 S-targeted neutralizing antibodies and cross-neutralizing antibodies against the S2 region of SARS-CoV-2/SARS-CoV S. Diversity of antibody sequences in RenMab mice and consistency in B-cell immune responses between RenMab mice and humans enabled screening of fully human virus-neutralizing antibodies. From all the frequency >1 paired clonotypes obtained from single-cell V(D)J sequencing, 215 antibodies with binding affinities were identified and primarily bound S2. However, only two receptor-binding domain-targeted clonotypes had neutralizing activity against SARS-CoV-2. Moreover, 5' single-cell RNA sequencing indicated that these sorted splenic B cells are mainly plasmablasts, germinal center (GC)-dependent memory B-cells and GC B-cells. Among them, plasmablasts and GC-dependent memory B-cells were considered the most significant possibility of producing virus-specific antibodies. Altogether, using a high-throughput single cell-based antibody discovery approach, our study highlighted the challenges of developing S2-binding neutralizing antibodies against SARS-CoV-2 and provided a novel direction for the enrichment of antigen-specific B-cells.

Circular RNA vaccines against SARS-CoV-2 and emerging variants.

As the emerging variants of SARS-CoV-2 continue to drive the worldwide pandemic, there is a constant demand for vaccines that offer more effective and broad-spectrum protection. Here, we report a circular RNA (circRNA) vaccine that elicited potent neutralizing antibodies and T cell responses by expressing the trimeric RBD of the spike protein, providing robust protection against SARS-CoV-2 in both mice and rhesus macaques. Notably, the circRNA vaccine enabled higher and more durable antigen production than the 1mΨ-modified mRNA vaccine and elicited a higher proportion of neutralizing antibodies and distinct Th1-skewed immune responses. Importantly, we found that the circRNARBD-Omicron vaccine induced effective neutralizing antibodies against the Omicron but not the Delta variant. In contrast, the circRNARBD-Delta vaccine protected against both Delta and Omicron or functioned as a booster after two doses of either native- or Delta-specific vaccination, making it a favorable choice against the current variants of concern (VOCs) of SARS-CoV-2.

Cover Image, Volume 94, Number 5, May 2022

Front Cover Caption: The cover image is based on the Research Article Aggregation of high-frequency RBD mutations of SARS-CoV-2 with three VOCs did not cause significant antigenic drift by Tao Li et al., https://doi.org/10.1002/jmv.27596.

Analysis of SARS-CoV-2 variants B.1.617: host tropism, proteolytic activation, cell-cell fusion, and neutralization sensitivity.

SARS-CoV-2 has caused the COVID-19 pandemic. B.1.617 variants (including Kappa and Delta) have been transmitted rapidly in India. The transmissibility, pathogenicity, and neutralization characteristics of these variants have received considerable interest. In this study, 22 pseudotyped viruses were constructed for B.1.617 variants and their corresponding single amino acid mutations. B.1.617 variants did not exhibit significant enhanced infectivity in human cells, but mutations T478K and E484Q in the receptor binding domain led to enhanced infectivity in mouse ACE2-overexpressing cells. Furin activities were slightly increased against B.1.617 variants and cell-cell fusion after infection of B.1.617 variants were enhanced. Furthermore, B.1.617 variants escaped neutralization by several mAbs, mainly because of mutations L452R, T478K, and E484Q in the receptor binding domain. The neutralization activities of sera from convalescent patients, inactivated vaccine-immunized volunteers, adenovirus vaccine-immunized volunteers, and SARS-CoV-2 immunized animals against pseudotyped B.1.617 variants were reduced by approximately twofold, compared with the D614G variant.

Heterologous boosting with third dose of coronavirus disease recombinant subunit vaccine increases neutralizing antibodies and T cell immunity against different severe acute respiratory syndrome coronavirus 2 variants.

Waned vaccine-induced immunity and emerging severe acute respiratory syndrome coronavirus 2 variants with potential for immune escape pose a major threat to the coronavirus disease (COVID-19) pandemic. Here, we showed that humoral immunity components, including anti-S + N, anti-RBD IgG, and neutralizing antibodies (NAbs), gradually waned and decreased the neutralizing capacity against emerging Omicron variants at 3 and 6 months after two inactivated COVID-19 vaccinations. We evaluated two boosting strategies with either a third dose of inactivated vaccine (homologous, I-I-I) or a recombinant subunit vaccine (heterologous, I-I-S). Both strategies induced the production of high levels of NAbs with a broad neutralizing capacity and longer retention. Interestingly, I-I-S induced 3.5-fold to 6.8-fold higher NAb titres than I-I-I, with a broader neutralizing capacity against six variants of concern, including Omicron. Further immunological analysis revealed that the two immunization strategies differ considerably, not only in the magnitude of total NAbs produced, but also in the composite pattern of NAbs and the population of virus-specific CD4+ T cells produced. Additionally, in some cases, heterologous boosted immunity induced the production of more effective epitopes than natural infection. The level of I-I-S-induced NAbs decreased to 48% and 18% at 1 and 3 months after booster vaccination, respectively. Overall, our data provide important evidence for vaccination strategies based on available vaccines and may help guide future global vaccination plans.