Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

Mouse models susceptible to HCoV-229E and HCoV-NL63 and cross protection from challenge with SARS-CoV-2.

Human coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63) are endemic causes of upper respiratory infections such as the "common cold" but may occasionally cause severe lower respiratory tract disease in the elderly and immunocompromised patients. There are no approved antiviral drugs or vaccines for these common cold coronaviruses (CCCoV). The recent emergence of COVID-19 and the possible cross-reactive antibody and T cell responses between these CCCoV and SARS-CoV-2 emphasize the need to develop experimental animal models for CCCoV. Mice are an ideal experimental animal model for such studies, but are resistant to HCoV-229E and HCoV-NL63 infections. Here, we generated 229E and NL63 mouse models by exogenous delivery of their receptors, human hAPN and hACE2 using replication-deficient adenoviruses (Ad5-hAPN and Ad5-hACE2), respectively. Ad5-hAPN- and Ad5-hACE2-sensitized IFNAR-/- and STAT1-/- mice developed pneumonia characterized by inflammatory cell infiltration with virus clearance occurring 7 d post infection. Ad5-hAPN- and Ad5-hACE2-sensitized mice generated virus-specific T cells and neutralizing antibodies after 229E or NL63 infection, respectively. Remdesivir and a vaccine candidate targeting spike protein of 229E and NL63 accelerated viral clearance of virus in these mice. 229E- and NL63-infected mice were partially protected from SARS-CoV-2 infection, likely mediated by cross-reactive T cell responses. Ad5-hAPN- and Ad5-hACE2-transduced mice are useful for studying pathogenesis and immune responses induced by HCoV-229E and HCoV-NL63 infections and for validation of broadly protective vaccines, antibodies, and therapeutics against human respiratory coronaviruses including SARS-CoV-2.

Plasma cell-free RNA characteristics in COVID-19 patients.

The pathogenesis of COVID-19 is still elusive, which impedes disease progression prediction, differential diagnosis, and targeted therapy. Plasma cell-free RNAs (cfRNAs) carry unique information from human tissue and thus could point to resourceful solutions for pathogenesis and host-pathogen interactions. Here, we performed a comparative analysis of cfRNA profiles between COVID-19 patients and healthy donors using serial plasma. Analyses of the cfRNA landscape, potential gene regulatory mechanisms, dynamic changes in tRNA pools upon infection, and microbial communities were performed. A total of 380 cfRNA molecules were up-regulated in all COVID-19 patients, of which seven could serve as potential biomarkers (AUC > 0.85) with great sensitivity and specificity. Antiviral (NFKB1A, IFITM3, and IFI27) and neutrophil activation (S100A8, CD68, and CD63)-related genes exhibited decreased expression levels during treatment in COVID-19 patients, which is in accordance with the dynamically enhanced inflammatory response in COVID-19 patients. Noncoding RNAs, including some microRNAs (let 7 family) and long noncoding RNAs (GJA9-MYCBP) targeting interleukin (IL6/IL6R), were differentially expressed between COVID-19 patients and healthy donors, which accounts for the potential core mechanism of cytokine storm syndromes; the tRNA pools change significantly between the COVID-19 and healthy group, leading to the accumulation of SARS-CoV-2 biased codons, which facilitate SARS-CoV-2 replication. Finally, several pneumonia-related microorganisms were detected in the plasma of COVID-19 patients, raising the possibility of simultaneously monitoring immune response regulation and microbial communities using cfRNA analysis. This study fills the knowledge gap in the plasma cfRNA landscape of COVID-19 patients and offers insight into the potential mechanisms of cfRNAs to explain COVID-19 pathogenesis.

Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19.

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Occurrence and sources of microplastics in dust of the Ebinur lake Basin, northwest China.

Currently, there is a lack of studies on microplastic pollution in mountain terrains and foothills areas in Northwest China and Central Asia. Here, we collected monthly dusts samples for one year and we studied the distribution, pollution levels, and sources of microplastics in atmospheric dust fall in the Ebinur Lake Basin in Northwest China. Results showed that the average content of dust microplastic on construction land was 28.61 ± 1.13 mg/kg, followed by farmland (20.25 ± 1.56 mg/kg), forest (19.52 ± 1.06 mg/kg), and deserts (8.08 ± 0.56 mg/kg). Regarding different land use types, atmospheric dust reduction dominated on farmland (58.64%), followed by urban area (26.65%), forest (9.76%), and desert (4.95%). Regarding the shape of microplastics, the order of occurrence in dust was film (46.85%) > fiber (35.15%) > foam(12.35%) > fragment (5.65%). In this study, four colors of microplastics were found in dust, and white accounted for the largest proportion (52.15%), followed by transparent (18.65%), black (19.45%), and green (9.75%). The main components of film microplastics in atmospheric dustfall in the Ebinur Lake Basin were PE and PP, and their sources were mainly plastic products in daily life, plastic industrial packaging materials from urban enterprises, broken plastic woven bags, and PET mostly from fabric fragment emissions. The abundance of microplastics in dust was correlated with atmospheric dust pH, EC, and total salt content. The contents of seven heavy metals (Cu, Ni, Cd, Pb, Cr, Mn, and Co) adsorbed by microplastics were also correlated with pH, EC, and total salt content. Our results represent a reference for microplastics pollution prevention in mountain terrains and foothills areas in northwest China and Central Asia.

Tacrolimus alleviates LPS-induced AKI by inhibiting TLR4/MyD88/NF-κB signalling in mice.

Lipopolysaccharide (LPS)-induced sepsis-associated acute kidney injury (SA-AKI) is a model of clinical serious care syndrome, with high morbidity and mortality. Tacrolimus (TAC), a novel immunosuppressant that inhibits inflammatory response, plays a pivotal role in kidney diseases. In this study, LPS treated mice and cultured podocytes were used as the models of SA-AKI in vivo and in vitro, respectively. Medium- and high-dose TAC administration significantly attenuated renal function and renal pathological manifestations at 12, 24 and 48 h after LPS treatment in mice. Moreover, the Toll-like receptor 4 (TLR4)/myeloid differential protein-88 (MyD88)/nuclear factor-kappa (NF-κB) signalling pathway was also dramatically inhibited by medium- and high-dose TAC administration at 12, 24 and 48 h of LPS treatment mice. In addition, TAC reversed LPS-induced podocyte cytoskeletal injury and podocyte migratory capability. Our findings indicate that TAC has protective effects against LPS-induced AKI by inhibiting TLR4/MyD88/NF-κB signalling pathway and podocyte dysfunction, providing another potential therapeutic effects for the LPS-induced SA-AKI.

Increased Pathogenicity and Virulence of Middle East Respiratory Syndrome Coronavirus Clade B In Vitro and In Vivo.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory disease in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. We isolated the clade B MERS-CoV ChinaGD01 strain from a patient infected during the South Korean MERS outbreak in 2015 and compared the phylogenetics and pathogenicity of MERS-CoV EMC/2012 (clade A) and ChinaGD01 (clade B) in vitro and in vivo Genome alignment analysis showed that most clade-specific mutations occurred in the orf1ab gene, including mutations that were predicted to be potential glycosylation sites. Minor differences in viral growth but no significant differences in plaque size or sensitivity to beta interferon (IFN-ß) were detected between these two viruses in vitro ChinaGD01 virus infection induced more weight loss and inflammatory cytokine production in human DPP4-transduced mice. Viral titers were higher in the lungs of ChinaGD01-infected mice than with EMC/2012 infection. Decreased virus-specific CD4+ and CD8+ T cell numbers were detected in the lungs of ChinaGD01-infected mice. In conclusion, MERS-CoV evolution induced changes to reshape its pathogenicity and virulence in vitro and in vivo and to evade adaptive immune response to hinder viral clearance.IMPORTANCE MERS-CoV is an important emerging pathogen and causes severe respiratory infection in humans. MERS-CoV strains from early epidemic clade A and contemporary epidemic clade B have not been phenotypically characterized to compare their abilities to infect cells and mice. In this study, we showed that a clade B virus ChinaGD01 strain caused more severe disease in mice, with delayed viral clearance, increased inflammatory cytokines, and decreased antiviral T cell responses, than the early clade A virus EMC/2012. Given the differences in pathogenicity of different clades of MERS-CoV, periodic assessment of currently circulating MERS-CoV is needed to monitor potential severity of zoonotic disease.

Current research and perspective of microplastics (MPs) in soils (dusts), rivers (lakes), and marine environments in China.

In this study, we first reviewed the current research progress regarding the presence of environmental microplastics (MPs) in environment in China from 2010 to 2019. Results showed that: (1) current research has primarily focused on river and marine environments rather than soils and dusts, mainly located in eastern China, i.e., the Yangtze river, Poyang lake, Dongting lake, Yellow sea, and Bohai sea; (2) the abundance of MPs found in water bodies (sediments) of the rivers in China ranged from 3.9 to 7900 items·m-3 (19.0 × 103-13600.5 × 103 items·km-2), and 20-24300 items·kg-2 (170-5500 × 106 items·km-2) in the sediments, respectively; in lake water the range was 340-8900 items·m-3 (5 × 103-340 × 105 items·km-2) and 8 to 1200 items·m-2/25-300 items·kg-1 in the sediments, respectively; in marine water the range was 0.003-540 items·m-3 (0-380,100 item·km-2) and 1.3-14700 item·kg-1 in the sediments, respectively; in fish, shellfish, and natural planktons from ocean and freshwater, the range was 0-57 items·individuals-1 (0-168 items·g-1); (3) The absorption and toxicological effects of MPs in freshwater and oceans have mainly focused on polyethylene (PE), polypropylene (PP), and polystyrene (PS); (4) the sources of microplastics in soils and dusts primarily come from urban/town activities; for rivers and lakes (estuary), they primarily come from urban activities; for coastal waters, fishing gear and nets, and the maritime activities were the main sources.

Pollution assessment and health risk evaluation of eight (metalloid) heavy metals in farmland soil of 146 cities in China.

Heavy metals are the most important indicator for farmland soil; however, in China, few provincial and national scales of studies have been done on heavy metals. Herein, by retrieving published studies, we calculated the spatial distribution characters and evaluated the health risks of Cu, Zn, Cd, Ni, Pb, Cr, As, and Hg in the farmland soil of 146 cities in China. Results showed that (1) the range (mean) values of eight (metalloid) heavy metals were as follows in mg/kg: Cu 0.236-251.015 (44.604), Zn 0.151-1547.060 (154.203), Cd 0.014-39.100 (1.497), Ni 0.709-554.420 (41.968), Pb 0.327-495.400 (55.143), Cr 0.078-333.510 (70.093), As 0.836-60.000 (12.207), and Hg 0.008-12.190 (0.371). The coefficient of variation values of Cu, Cr, and As displayed moderate variation, and Zn, Cd, Ni, Pb, and Hg displayed high variation (142.148-364.960%). (2) the Igeo values of As, Cu, Cd, Ni, Zn, Cr, Pb, and Hg were - 4.329 to 1.837, - 7.166 to 2.888, - 3.378 to 8.070, - 5.831 to 3.780, - 9.527 to 3.797, - 10.120 to 1.866, - 6.899 to 3.667, and - 3.681 to 6.966, respectively; in many cities, there was some degree of heavy metal pollution of the farmland soil such as Zn in Pingdu, Pb in Huludao, and Hg in Tongguan, Funshun, Huludao, and Qinglong (Igeo > 3); there were no obvious spatial patterns of Cr, Ni, and As, and Zn, Cu, but Cd, Ni, Pb, and Hg mainly located in some cities in the southwest, central or eastern parts of China. (3) Health risk assessment showed that with the exception of Cd, Cr, and As by the respiration route and Ni, Cr, and As through skin exposure, the average amount of daily exposure of the eight (metalloid) heavy metals all showed values for children > adults, and the HQ and HI values were all lower than 1.0, indicating noncarcinogenic risks; calculation of carcinogenic risks showed there were no carcinogenic risks for As, Cr, Ni, and Cd; however, the value for Cr was the maximum and contributed 98.505% of the total.

Sources identification and pollution evaluation of heavy metals in the surface sediments of Bortala River, Northwest China.

The current study focused on the Bortala River - a typical inland river located in an oasis of arid area in northwestern China. The sediment and soil samples were collected from the river and drainage basin. Results showed that: (1) the particle size of the sand fraction of the sediments was 78-697 µm, accounting for 78.82% of the total samples; the average concentrations of eight heavy metals fell within the concentration ranges recommended by the Secondary National Standard of China, while the maximum concentrations of Pb, Cd, and Hg exceeded these standards; (2) results from multivariate statistical analysis indicated that Cu, Ni, As, and Zn originated primarily from natural geological background, while Cd, Pb, Hg and Cr in the sediments originated from human activities; (3) results of the enrichment factor analysis and the geo-accumulation index evaluation showed that Cd, Hg, and Pb were present in the surface sediments of the river at low or partial serious pollution levels, while Zn, Cr, As, Ni, and Cu existed at zero or low pollution levels; (4) calculation of the potential ecological hazards index showed that among the eight tested heavy metals, Cd, Pb, Hg, and Cr were the main potential ecological risk factors, with relative contributions of 25.43%, 22.23%, 21.16%, and 14.87%, respectively; (5) the spatial distribution of the enrichment factors (EF(S)), the Geo-accumulation index (I(geo)), and the potential ecological risk coefficient (E(r)(i)) for eight heavy metals showed that there was a greater accumulation of heavy metals Pb, Cd, and Hg in the sediments of the central and eastern parts of the river. Results of this research can be a reference for the heavy metals pollution prevention, the harmony development of the ecology protection and the economy development of the oases of inland river basin of arid regions of China, Central Asia and also other parts of the world.

Spatial distribution and potential sources of trace metals in insoluble particles of snow from Urumqi, China.

The purpose of this work is to characterize trace elements in snow in urban-suburb gradient over Urumqi city, China. The spatial distribution patterns of 11 trace metals in insoluble particulate matters of snow were revealed by using 102 snow samples collected in and around urban areas of Urumqi, a city suffering from severe wintertime air pollution in China. Similar spatial distribution for Mn, Cu, Zn, Ni, and Pb was found and their two significant high-value areas located in the west and east, respectively, and a high-value area in the south, which were correlated with factory emissions, traffic activities, and construction fugitive dust. The high-value areas of Cr, Ni, and V occurred in the northeast corner and along main traffic paths, which were linked to oil refinery and vehicular emissions. High value of Be presented in the west of the city. The high-value area of Co in the northeast could be related to local soil. Cd and U displayed relatively even spatial patterns in the urban area. In view of distance from the urban center, e.g., from the first circular belt to the fourth circular belt, except Be, V, Cd, and U, the contents of other metals generally decreased from the first circular belt to the forth circular belt, implying the effect of human activity clearly. Additionally, prevailing northwesterly winds and occasionally southeasterly winds in winter were associated with decreased, generally, concentrations of trace metal in snow from the urban center to the southern suburb along a northwest and southeast transect. The information on concentrations and spatial distributions of these metals in insoluble particles of snow in winter will be valuable for further environmental protection and planning.

Prognostic value of preoperative white blood cell to hemoglobin ratio and fibrinogen to albumin ratio in patients with colorectal cancer.

The prognostic value of preoperative white blood cell to hemoglobin ratio (WHR) and fibrinogen to albumin ratio (FAR) in colorectal cancer (CRC) is unknown. The purpose of this study was to analyze the correlation between preoperative WHR and FAR and the prognosis of CRC patients. The retrospective study analyzed the medical records of 207 patients with colorectal cancer who were admitted to Linyi People's Hospital between June 1, 2017 and June 1, 2021. The receiver operator curve was used to determine the cutoff value of 4.604 for WHR and 0.086 for FAR, and the patients were divided into high and low groups for comparative analysis of clinical data. Cox proportional hazards regression models were used to assess independent risk factors for disease-free survival (DFS) and overall survival (OS) in univariate and multifactorial analyses. Kaplan-Meier methods were used for survival analysis and logrank tests were used to assess survival differences. Multifactorial Cox analysis showed that tumor pathological stage (HR = 6.224, 95% CI:3.063-12.647, P < .001), and WHR (HR = 3.681, 95% CI:1.768-7.401, P < .001) were the independent risk factors for DFS in CRC patients. Tumor pathological stage (HR = 4.080, 95% CI:1.992-8.360, P < .001), and WHR (HR = 3.397, 95% CI:1.662-6.940, P = .001) were independent risk factors for OS. High levels of WHR and high levels of FAR were associated with lower DFS (P < .001) and OS (P < .001).CRC patients with both higher WHR and FAR had significantly lower DFS (P < .001) and OS (P < .001). DFS and OS may be shorter in CRC patients with high WHR and high FAR, perhaps associated with poor prognosis in CRC patients, and WHR and FAR may be potential CRC prognostic markers.

A bispecific antibody exhibits broad neutralization against SARS-CoV-2 Omicron variants XBB.1.16, BQ.1.1 and sarbecoviruses.

The Omicron subvariants BQ.1.1, XBB.1.5, and XBB.1.16 of SARS-CoV-2 are known for their adeptness at evading immune responses. Here, we isolate a neutralizing antibody, 7F3, with the capacity to neutralize all tested SARS-CoV-2 variants, including BQ.1.1, XBB.1.5, and XBB.1.16. 7F3 targets the receptor-binding motif (RBM) region and exhibits broad binding to a panel of 37 RBD mutant proteins. We develop the IgG-like bispecific antibody G7-Fc using 7F3 and the cross-neutralizing antibody GW01. G7-Fc demonstrates robust neutralizing activity against all 28 tested SARS-CoV-2 variants and sarbecoviruses, providing potent prophylaxis and therapeutic efficacy against XBB.1 infection in both K18-ACE and BALB/c female mice. Cryo-EM structure analysis of the G7-Fc in complex with the Omicron XBB spike (S) trimer reveals a trimer-dimer conformation, with G7-Fc synergistically targeting two distinct RBD epitopes and blocking ACE2 binding. Comparative analysis of 7F3 and LY-CoV1404 epitopes highlights a distinct and highly conserved epitope in the RBM region bound by 7F3, facilitating neutralization of the immune-evasive Omicron variant XBB.1.16. G7-Fc holds promise as a potential prophylactic countermeasure against SARS-CoV-2, particularly against circulating and emerging variants.

A potent neutralizing nanobody targeting a unique epitope on the receptor-binding domain of SARS-CoV-2 spike protein.

SARS-CoV-2 and its variants continue to threaten public health. Nanobodies that block the attachment of the RBD to host cell angiotensin-converting enzyme 2 (ACE2) represent promising drug candidates. In this study, we reported the identification and structural biological characterization of a nanobody from a RBD-immunized alpaca. The nanobody, termed as 2S-1-19, shows outstanding neutralizing activity against both pseudotyped and authentic SARS-CoV-2 viruses. The crystal structure of 2S-1-19 bound to SARS-CoV-2 RBD reveals an epitope that overlaps with the binding site for ACE2. We also showed that 2S-1-19 reserves promising, though compromised, neutralizing activity against the Delta variant and that the trivalent form of 2S-1-19 remarkably increases its neutralizing capacity. Despite this, neither the monomeric or trimeric 2S-1-19 could neutralize the Omicron BA.1.1 variant, possibility due to the E484A and Q493K mutations found within this virus variant. These data provide insights into immune evasion caused by SARS-CoV-2 variants.

Mosaic RBD Nanoparticles Elicit Protective Immunity Against Multiple Human Coronaviruses in Animal Models.

To combat SARS-CoV-2 variants and MERS-CoV, as well as the potential re-emergence of SARS-CoV and spillovers of sarbecoviruses, which pose a significant threat to global public health, vaccines that can confer broad-spectrum protection against betacoronaviruses (ß-CoVs) are urgently needed. A mosaic ferritin nanoparticle vaccine is developed that co-displays the spike receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 Wild-type (WT) strain and evaluated its immunogenicity and protective efficacy in mice and nonhuman primates. A low dose of 10 µg administered at a 21-day interval induced a Th1-biased immune response in mice and elicited robust cross-reactive neutralizing antibody responses against a variety of ß-CoVs, including a series of SARS-CoV-2 variants. It is also able to effectively protect against challenges of SARS-CoV, MERS-CoV, and SARS-CoV-2 variants in not only young mice but also the more vulnerable mice through induction of long-lived immunity. Together, these results suggest that this mosaic 3-RBD nanoparticle has the potential to be developed as a pan-ß-CoV vaccine.

An unconventional VH1-2 antibody tolerates escape mutations and shows an antigenic hotspot on SARS-CoV-2 spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein continues to evolve antigenically, impacting antibody immunity. D1F6, an affinity-matured non-stereotypic VH1-2 antibody isolated from a patient infected with the SARS-CoV-2 ancestral strain, effectively neutralizes most Omicron variants tested, including XBB.1.5. We identify that D1F6 in the immunoglobulin G (IgG) form is able to overcome the effect of most Omicron mutations through its avidity-enhanced multivalent S-trimer binding. Cryo-electron microscopy (cryo-EM) and biochemical analyses show that three simultaneous epitope mutations are generally needed to substantially disrupt the multivalent S-trimer binding by D1F6 IgG. Antigenic mutations at spike positions 346, 444, and 445, which appeared in the latest variants, have little effect on D1F6 binding individually. However, these mutations are able to act synergistically with earlier Omicron mutations to impair neutralization by affecting the interaction between D1F6 IgG and the S-trimer. These results provide insight into the mechanism by which accumulated antigenic mutations facilitate evasion of affinity-matured antibodies.

Characterization of humoral immune responses against SARS-CoV-2 accessory proteins in infected patients and mouse model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, encodes several accessory proteins that have been shown to play crucial roles in regulating the innate immune response. However, their expressions in infected cells and immunogenicity in infected humans and mice are still not fully understood. This study utilized various techniques such as luciferase immunoprecipitation system (LIPS), immunofluorescence â€‹assay (IFA), and western â€‹blot (WB) to detect accessory protein-specific antibodies in sera of COVID-19 patients. Specific antibodies to proteins 3a, 3b, 7b, 8 and 9c can be detected by LIPS, but only protein 3a antibody was detected by IFA or WB. Antibodies against proteins 3a and 7b were only detected in ICU patients, which may serve as a marker for predicting disease progression. Further, we investigated the expression of accessory proteins in SARS-CoV-2-infected cells and identified the expressions of proteins 3a, 6, 7a, 8, and 9b. We also analyzed their ability to induce antibodies in immunized mice and found that only proteins 3a, 6, 7a, 8, 9b and 9c were able to induce measurable antibody productions, but these antibodies lacked neutralizing activities and did not protect mice from SARS-CoV-2 infection. Our findings validate the expression of SARS-CoV-2 accessory proteins and elucidate their humoral immune response, providing a basis for protein detection assays and their role in pathogenesis.

Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants.

The constant emergence of SARS-CoV-2 variants continues to impair the efficacy of existing neutralizing antibodies, especially XBB.1.5 and EG.5, which showed exceptional immune evasion properties. Here, we identify a highly conserved neutralizing epitope targeted by a broad-spectrum neutralizing antibody BA7535, which demonstrates high neutralization potency against not only previous variants, such as Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5, but also more recently emerged Omicron subvariants, including BF.7, CH.1.1, XBB.1, XBB.1.5, XBB.1.9.1, EG.5. Structural analysis of the Omicron Spike trimer with BA7535-Fab using cryo-EM indicates that BA7535 recognizes a highly conserved cryptic receptor-binding domain (RBD) epitope, avoiding most of the mutational hot spots in RBD. Furthermore, structural simulation based on the interaction of BA7535-Fab/RBD complexes dissects the broadly neutralizing effect of BA7535 against latest variants. Therapeutic and prophylactic treatment with BA7535 alone or in combination with BA7208 protected female mice from the circulating Omicron BA.5 and XBB.1 variant infection, suggesting the highly conserved neutralizing epitope serves as a potential target for developing highly potent therapeutic antibodies and vaccines.

Identification of a broad sarbecovirus neutralizing antibody targeting a conserved epitope on the receptor-binding domain.

Omicron, as the emerging variant with enhanced vaccine tolerance, has sharply disrupted most therapeutic antibodies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subgenus Sarbecovirus, members of which share high sequence similarity. Herein, we report one sarbecovirus antibody, 5817, which has broad-spectrum neutralization capacity against SARS-CoV-2 variants of concern (VOCs) and SARS-CoV, as well as related bat and pangolin viruses. 5817 can hardly compete with six classes of receptor-binding-domain-targeted antibodies grouped by structural classifications. No obvious impairment in the potency is detected against SARS-CoV-2 Omicron and subvariants. The cryoelectron microscopy (cryo-EM) structure of neutralizing antibody 5817 in complex with Omicron spike reveals a highly conserved epitope, only existing at the receptor-binding domain (RBD) open state. Prophylactic and therapeutic administration of 5817 potently protects mice from SARS-CoV-2 Beta, Delta, Omicron, and SARS-CoV infection. This study reveals a highly conserved cryptic epitope targeted by a broad sarbecovirus neutralizing antibody, which would be beneficial to meet the potential threat of pre-emergent SARS-CoV-2 VOCs.