Publications by authors named "Youchun Wang"

149 Publications

Unmethylated CpG motif-containing genomic DNA fragments of bacillus calmette-guerin improves immune response towards a DNA vaccine for COVID-19.

Vaccine 2021 Sep 3. Epub 2021 Sep 3.

Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, No.31 Huatuo Street, Daxing District, Beijing 102629, China. Electronic address:

The development of an effective vaccine to control the global coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) is of utmost importance. In this study, a synthetic DNA-based vaccine candidate, known as pSV10-SARS-CoV-2, expressing the SARS-CoV-2 spike protein was designed and tested in 39 BALB/c mice with BC01, an adjuvant derived from unmethylated CpG motif-containing DNA fragments from the Bacillus Calmette-Guerin genome. Mice vaccinated with pSV10-SARS-CoV-2 with BC01 produced early neutralizing antibodies and developed stronger humoral and cellular immune responses compared to mice that received the DNA vaccine only. Moreover, sera from mice vaccinated with pSV10-SARS-CoV-2 with BC01 can neutralize certain variants, including 614G, 614G + 472 V, 452R, 483A, 501Y.V2, and B.1.1.7. The results of this study demonstrate that the addition of BC01 to a DNA-vaccine for COVID-19 could elicit more effective neutralizing antibody titers for disease prevention.
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http://dx.doi.org/10.1016/j.vaccine.2021.08.103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8413458PMC
September 2021

Methods to Identify Immunogenic Peptides in SARS-CoV-2 Spike and Protective Monoclonal Antibodies in COVID-19 Patients.

Small Methods 2021 Jul 19;5(7):2100058. Epub 2021 Jun 19.

Department of Microbiology Immunology & Molecular Genetics University of California Los Angeles Los Angeles CA 90095 USA.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the associated COVID-19 diseases are an emerging threat to global public health. Although considerable scientific research on the immune, especially antibody, responses to SARS-CoV-2 infection have been conducted, additional dominant epitopes and protective antibodies are needed for diagnosis and treatment of COVID-19 patients. Here, two different phage libraries are used to identify immunogenic epitopes across the spike protein and monoclonal antibodies from COVID-19 patients. Three peptides are further characterized in the receptor-binding motif (RBM) and measured their antibody levels in COVID-19 patients, from which one identifies one most immunodominant epitope with the highest antibody response in COVID-19 patients and in immunized mice. More importantly, monoclonal antibodies specifically binding to this peptide isolated from COVID-19 patients have therapeutic potential to neutralize SARS-CoV-2 infection. Thus, the approaches to systemically identify immunogenic peptides and directly identify human monoclonal antibodies from patients will provide useful diagnostic and therapeutic tools for COVID-19 and other emerging infectious diseases.
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http://dx.doi.org/10.1002/smtd.202100058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420616PMC
July 2021

A broadly neutralizing humanized ACE2-targeting antibody against SARS-CoV-2 variants.

Nat Commun 2021 08 17;12(1):5000. Epub 2021 Aug 17.

CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

The successive emergences and accelerating spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages and evolved resistance to some ongoing clinical therapeutics increase the risks associated with the coronavirus disease 2019 (COVID-19) pandemic. An urgent intervention for broadly effective therapies to limit the morbidity and mortality of COVID-19 and future transmission events from SARS-related coronaviruses (SARSr-CoVs) is needed. Here, we isolate and humanize an angiotensin-converting enzyme-2 (ACE2)-blocking monoclonal antibody (MAb), named h11B11, which exhibits potent inhibitory activity against SARS-CoV and circulating global SARS-CoV-2 lineages. When administered therapeutically or prophylactically in the hACE2 mouse model, h11B11 alleviates and prevents SARS-CoV-2 replication and virus-induced pathological syndromes. No significant changes in blood pressure and hematology chemistry toxicology were observed after injections of multiple high dosages of h11B11 in cynomolgus monkeys. Analysis of the structures of the h11B11/ACE2 and receptor-binding domain (RBD)/ACE2 complexes shows hindrance and epitope competition of the MAb and RBD for the receptor. Together, these results suggest h11B11 as a potential therapeutic countermeasure against SARS-CoV, SARS-CoV-2, and escape variants.
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http://dx.doi.org/10.1038/s41467-021-25331-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371079PMC
August 2021

Novel quinolone derivatives targeting human dihydroorotate dehydrogenase suppress Ebola virus infection in vitro.

Antiviral Res 2021 Oct 12;194:105161. Epub 2021 Aug 12.

School of Medicine, Tsinghua University, Beijing, 100084, China. Electronic address:

Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 outbreak in West Africa. Currently, no effective antiviral treatments have been approved for clinical use. Compound 1 RYL-634 is a quinolone-derived compound that can inhibit dihydroorotate dehydrogenase, a rate-limiting enzyme in the de novo pyrimidine synthesis pathway and it exhibited antiviral activity against multiple RNA virus infection. In this study, we evaluated the efficacy of a panel of newly developed compounds based on RYL-634 against EBOV infection. Our data showed that RYL-634 as well as its derivatives are effective against EBOV transcription- and replication-competent virus-like particle (trVLP) infection and authentic EBOV infection in vitro at low nanomolar IC values and relatively high CC. Of note, the new derivative RYL-687 had the lowest IC at approximately 7 nM and was almost 6 times more potent than remdesivir (GS-5734). Exogenous addition of different metabolites in the pyrimidine de novo synthesis pathway confirmed DHODH as the target of RYL-687. These data provide evidence that such quinolone-derived compounds are promising therapeutic candidates against EBOV infection.
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http://dx.doi.org/10.1016/j.antiviral.2021.105161DOI Listing
October 2021

Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat spike protein RBD protects mice against COVID-19.

Emerg Microbes Infect 2021 Dec;10(1):1574-1588

Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Tropical Medicine and Laboratory Medicine, The First Affiliated Hospital, Hainan Medical University, Haikou, People's Republic of China.

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus-vectored vaccines expressing spike (S) protein have been approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD), or tandem-repeat dimeric RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular immune responses. AdC7-RBD-tr2 showed higher antibody responses compared to either AdC7-S or AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. AdC7-RBD-tr2-elicted sera preserved the neutralizing activity against the circulating variants, especially the Delta variant. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.
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http://dx.doi.org/10.1080/22221751.2021.1959270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8366625PMC
December 2021

Three epitope-distinct human antibodies from RenMab mice neutralize SARS-CoV-2 and cooperatively minimize the escape of mutants.

Cell Discov 2021 Jul 20;7(1):53. Epub 2021 Jul 20.

Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

Coronavirus disease 2019 (COVID-19), a pandemic disease caused by the newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 3.8 million deaths to date. Neutralizing antibodies are effective therapeutic measures. However, many naturally occurring mutations at the receptor-binding domain (RBD) have emerged, and some of them can evade existing neutralizing antibodies. Here, we utilized RenMab, a novel mouse carrying the entire human antibody variable region, for neutralizing antibody discovery. We obtained several potent RBD-blocking antibodies and categorized them into four distinct groups by epitope mapping. We determined the involved residues of the epitope of three representative antibodies by cryo-electron microscopy (Cryo-EM) studies. Moreover, we performed neutralizing experiments with 50 variant strains with single or combined mutations and found that the mixing of three epitope-distinct antibodies almost eliminated the mutant escape. Our study provides a sound basis for the rational design of fully human antibody cocktails against SARS-CoV-2 and pre-emergent coronaviral threats.
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http://dx.doi.org/10.1038/s41421-021-00292-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290868PMC
July 2021

Lentil lectin derived from exhibit broad antiviral activities against SARS-CoV-2 variants.

Emerg Microbes Infect 2021 Dec;10(1):1519-1529

Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), Beijing, People's Republic of China.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutated continuously and newly emerging variants escape from antibody-mediated neutralization raised great concern. S protein is heavily glycosylated and the glycosylation sites are relatively conserved, thus glycans on S protein surface could be a target for the development of anti-SARS-CoV-2 strategies against variants. Here, we collected 12 plant-derived lectins with different carbohydrate specificity and evaluated their anti-SARS-CoV-2 activity against mutant strains and epidemic variants using a pseudovirus-based neutralization assay. The -derived lentil lectin which specifically bind to oligomannose-type glycans and GlcNAc at the non-reducing end terminus showed most potent and broad antiviral activity against a panel of mutant strains and variants, including the artificial mutants at N-/O-linked glycosylation site, natural existed amino acid mutants, as well as the epidemic variants B.1.1.7, B.1.351, and P.1. Lentil lectin also showed antiviral activity against SARS-CoV and MERS-CoV. We found lentil lectin could block the binding of ACE2 to S trimer and inhibit SARS-CoV-2 at the early steps of infection. Using structural information and determined N-glycan profile of S trimer, taking together with the carbohydrate specificity of lentil lectin, we provide a basis for the observed broad spectrum anti-SARS-CoV-2 activity. Lentil lectin showed weak haemagglutination activity at 1 mg/mL and no cytotoxicity activity, and no weight loss was found in single injection mouse experiment. This report provides the first evidence that lentil lectin strongly inhibit infection of SARS-COV-2 variants, which should provide valuable insights for developing future anti-SARS-CoV-2 strategies.
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http://dx.doi.org/10.1080/22221751.2021.1957720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330776PMC
December 2021

Distribution and origin of anomalously high permeability zones in Weizhou formation, Weizhou 12-X oilfield, Weixinan Sag, China.

Earth Sci Inform 2021 Jul 13:1-13. Epub 2021 Jul 13.

School of Geosciences, Northeast Petroleum University, Daqing, 163318 Heilongjiang China.

In order to study the dominant seepage channel of the third member of Weizhou formation (E ) in Weizhou 12-X oilfield, Weixinan Sag, Beibu Gulf Basin, and tap the potential of remaining oil. The distribution and causes of the anomalously high permeability zones in Weizhou Formation were studied by using conventional core physical property analysis, scanning electron microscope, laser particle size analysis, X-ray diffraction and thin section microscopic identification. As the results show, vertically,there are three anomalously high permeability zones in the A , A and A micro-stage of the middle diagenetic stage, with the depth range of 2300 m ~ 2400 m, 2400 m ~ 2600 m, 2600 m ~ 2900 m respectively. Grain size, sorting, dissolution and early emplacement of hydrocarbons are the main causes of anomalously high permeability zones. Although both grain size and sorting affect porosity and permeability, the effect of grain size on permeability is stronger than sorting, and sorting has a stronger effect on porosity than grain size. Magmatic hydrothermal and organic acid promote dissolution and concomitant porosity and permeability increase by dissolving unstable minerals. The early emplacement of hydrocarbons retard the cementation and accompanying porosity and permeability reduction by reducing the water-rock ratio. Finally, sandstone reservoirs in the E are characterized by anomalously high permeability zones.
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http://dx.doi.org/10.1007/s12145-021-00670-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276689PMC
July 2021

Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2.

Nat Commun 2021 07 9;12(1):4210. Epub 2021 Jul 9.

Institute for Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People's Hospital; The Second Affiliated Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province, China.

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-24514-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270942PMC
July 2021

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants.

Virol Sin 2021 Jul 5. Epub 2021 Jul 5.

State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high-affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446-S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351, and reacted with mutations of N501Y, E484K, and L452R, indicated that it may also neutralize the recent India endemic strain B.1.617. The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants, which mitigated the risk of viral escape. Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-CoV-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-CoV-2 variants.
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http://dx.doi.org/10.1007/s12250-021-00409-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255729PMC
July 2021

The Antigenicity of Epidemic SARS-CoV-2 Variants in the United Kingdom.

Front Immunol 2021 17;12:687869. Epub 2021 Jun 17.

Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

To determine whether the neutralization activity of monoclonal antibodies, convalescent sera and vaccine-elicited sera was affected by the top five epidemic SARS-CoV-2 variants in the UK, including D614G+L18F+A222V, D614G+A222V, D614G+S477N, VOC-202012/01(B.1.1.7) and D614G+69-70del+N439K, a pseudovirus-neutralization assay was performed to evaluate the relative neutralization titers against the five SARS-CoV-2 variants and 12 single deconvolution mutants based on the variants. In this study, 18 monoclonal antibodies, 10 sera from convalescent COVID-19 patients, 10 inactivated-virus vaccine-elicited sera, 14 mRNA vaccine-elicited sera, nine RBD-immunized mouse sera, four RBD-immunized horse sera, and four spike-encoding DNA-immunized guinea pig sera were tested and analyzed. The N501Y, N439K, and S477N mutations caused immune escape from nine of 18 mAbs. However, the convalescent sera, inactivated virus vaccine-elicited sera, mRNA vaccine-elicited sera, spike DNA-elicited sera, and recombinant RBD protein-elicited sera could still neutralize these variants (within three-fold changes compared to the reference D614G variant). The neutralizing antibody responses to different types of vaccines were different, whereby the response to inactivated-virus vaccine was similar to the convalescent sera.
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http://dx.doi.org/10.3389/fimmu.2021.687869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247764PMC
July 2021

The first Chinese national standards for SARS-CoV-2 neutralizing antibody.

Vaccine 2021 06 19;39(28):3724-3730. Epub 2021 May 19.

Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China. Electronic address:

In order to meet the domestic urgent needs of evaluating the immunogenicity of vaccines and the potency testing of therapeutic antibody products against coronavirus disease 2019 (COVID-19), the first Chinese national standards for SARS-CoV-2 neutralizing antibody were established. The potency and stability of the candidate standards were determined by neutralization assay and accelerated degradation study. The stability studies showed that the standards were stable in the short-term. The collaborative study showed that the candidate standards could reduce the variations in neutralization titers between labs and thus improve comparability of neutralizing antibody measurements. Sample 22 has been approved by the Biological Product Reference Standards Sub-Committee of the National Drug Reference Standards Committee as the first Chinese National Standard for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) neutralizing antibody, with an assigned potency of 1,000 units per milliliter (U/ml). This standard will contribute to the standardized assessment of the quality and efficacy of vaccines and therapeutics for COVID-19 in China.
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http://dx.doi.org/10.1016/j.vaccine.2021.05.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133612PMC
June 2021

A practical method for evaluating the efficacy of EVA-71 vaccine using a hSCARB2 knock-in mouse model.

Emerg Microbes Infect 2021 Dec;10(1):1180-1190

Division of Animal Model Research, Institute for Laboratory Animal Resources, National Institutes for Food and Drug Control (NIFDC), Beijing, People's Republic of China.

Hand-foot-and-mouth disease is a contagious disease common among children under 5 years old worldwide. It is caused by strains of enterovirus, especially EV-A71, which can lead to severe disease. Vaccines are the only way to fight this disease. Accordingly, it is necessary to establish an efficient and accurate methodology to evaluate vaccine efficacy . Here, we established a practical method using a hSCARB2 knock-in mouse model, which was susceptible to EV-A71 infection at 5-6 weeks of age, to directly determine the efficacy of vaccines. Unlike traditional approaches, one-week-old hSCARB2 mice were immunized twice with a licensed vaccine, with an interval of a week. The titre of antibodies was measured after 1 week. Mice at 4 weeks of age were challenged with EV-A71 intraperitoneally and intracranially, respectively. The unimmunized hSCARB2 mice displayed systemic clinical symptoms and succumbed to the disease at a rate of approximately 50%. High viral loads were detected in the lungs, brain, and muscles, accompanied by clear pathological changes. The expression of IL-1β, IL-13, IL-17, and TNF-α was significantly upregulated. By contrast, the immunized group was practically normal and indistinguishable from the control mice. These results indicate that the hSCARB2 knock-in mouse is susceptible to infection in adulthood, and the efficacy of EV-A71 vaccine could be directly evaluated in this mouse model. The method developed here may be used in the development of new vaccines against HFMD or quality control of licensed vaccines.
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http://dx.doi.org/10.1080/22221751.2021.1934558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8205003PMC
December 2021

A new nairo-like virus associated with human febrile illness in China.

Emerg Microbes Infect 2021 Dec;10(1):1200-1208

Department of Emerging Infectious Diseases, The First Hospital of Jilin University, Changchun, People's Republic of China.

Several nairo-like viruses have been discovered in ticks in recent years, but their relevance to public health remains unknown. Here, we found a patient who had a history of tick bite and suffered from a febrile illness was infected with a previously discovered RNA virus, Beiji nairovirus (BJNV), in the nairo-like virus group of the order . We isolated the virus by cell culture assay. BJNV could induce cytopathic effects in the baby hamster kidney and human hepatocellular carcinoma cells. Negative-stain electron microscopy revealed enveloped and spherical viral particles, morphologically similar to those of nairoviruses. We identified 67 patients as BJNV infection in 2017-2018. The median age of patients was 48 years (interquartile range 41-53 years); the median incubation period was 7 days (interquartile range 3-12 days). Most patients were men (70%), and a few (10%) had underlying diseases. Common symptoms of infected patients included fever (100%), headache (99%), depression (63%), coma (63%), and fatigue (54%), myalgia or arthralgia (45%); two (3%) patients became critically ill and one died. BJNV could cause growth retardation, viremia and histopathological changes in infected suckling mice. BJNV was also detected in sheep, cattle, and multiple tick species. These findings demonstrated that the newly discovered nairo-like virus may be associated with a febrile illness, with the potential vectors of ticks and reservoirs of sheep and cattle, highlighting its public health significance and necessity of further investigation in the tick-endemic areas worldwide.
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http://dx.doi.org/10.1080/22221751.2021.1936197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212832PMC
December 2021

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines.

Cell Res 2021 07 21;31(7):732-741. Epub 2021 May 21.

School of Pharmaceutical Sciences, IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, China.

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242-244 deletion (242-244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242-244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.
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http://dx.doi.org/10.1038/s41422-021-00514-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138844PMC
July 2021

Cellular tropism and antigenicity of mink-derived SARS-CoV-2 variants.

Signal Transduct Target Ther 2021 05 17;6(1):196. Epub 2021 May 17.

Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

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http://dx.doi.org/10.1038/s41392-021-00617-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127491PMC
May 2021

Functional comparison of SARS-CoV-2 with closely related pangolin and bat coronaviruses.

Cell Discov 2021 Apr 6;7(1):21. Epub 2021 Apr 6.

Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, No. 31 Huatuo Street, Daxing District, Beijing 102629, China.

The origin and intermediate host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be determined. Coronaviruses found to be closely related to SARS-CoV-2 include RaTG13 derived from bat and two clusters (PCoV-GD and PCoV-GX) of coronaviruses identified in pangolin. Here, we studied the infectivity and antigenicity patterns of SARS-CoV-2 and the three related coronaviruses. Compared with the other three viruses, RaTG13 showed almost no infectivity to a variety of cell lines. The two pangolin coronaviruses and SARS-CoV-2 showed similar infectious activity. However, in SARS-CoV-2-susceptible cell lines, the pangolin coronaviruses presented even higher infectivity. The striking difference between the SARS-CoV-2 and pangolin coronaviruses is that the latter can infect porcine cells, which could be partially attributed to an amino acid difference at the position of 498 of the spike protein. The infection by SARS-CoV-2 was mainly mediated by Furin and TMPRSS2, while PCoV-GD and PCoV-GX mainly depend on Cathepsin L. Extensive cross-neutralization was found between SARS-CoV-2 and PCoV-GD. However, almost no cross-neutralization was observed between PCoV-GX and SARS-CoV-2 or PCoV-GD. More attention should be paid to pangolin coronaviruses and to investigate the possibility of these coronaviruses spreading across species to become zoonoses among pigs or humans.
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http://dx.doi.org/10.1038/s41421-021-00256-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022302PMC
April 2021

Clofazimine: A Promising Inhibitor of Rabies Virus.

Front Pharmacol 2021 18;12:598241. Epub 2021 Mar 18.

Department of HIV/AIDS and Sex-transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China.

With an almost 100% mortality rate, rabies virus (RABV) infection is a global concern. Limited post-exposure prophylaxis and lack of an effective treatment necessitate novel antiviral therapies against RABV. Here, using a high-throughput screening (HTS) method developed in our lab, 11 candidates with anti-RABV activity were identified from a library of 767 clinical drugs. Clofazimine (CFZ), an anti-leprosy drug, displayed an EC of 2.28 μM, and SI over 967 against RABV. Investigations into the underlying mechanisms revealed that CFZ targeted viral membrane fusion at the early stages of virus replication. Moreover, CFZ and Clofazimine salicylates (CFZS) exhibited elevated survival rates , compared with the positive control T-705. Thus, this study revealed CFZ as a promising drug against RABV infection.
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http://dx.doi.org/10.3389/fphar.2021.598241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012719PMC
March 2021

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

Cell 2021 04 23;184(9):2362-2371.e9. Epub 2021 Feb 23.

Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, No. 31 Huatuo Street, Daxing District, Beijing 102629, China. Electronic address:

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.
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http://dx.doi.org/10.1016/j.cell.2021.02.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901273PMC
April 2021

Publisher Correction: A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Nature 2021 Feb;590(7844):E23

Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

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http://dx.doi.org/10.1038/s41586-020-03108-4DOI Listing
February 2021

[Challenges and considerations on quality control and evaluation of pathogen metagenomic next-generation sequencing].

Sheng Wu Gong Cheng Xue Bao 2020 Dec;36(12):2598-2609

National Institutes for Food and Drug Control, Beijing 100050, China.

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.
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http://dx.doi.org/10.13345/j.cjb.200377DOI Listing
December 2020

Spike-specific circulating T follicular helper cell and cross-neutralizing antibody responses in COVID-19-convalescent individuals.

Nat Microbiol 2021 01 16;6(1):51-58. Epub 2020 Nov 16.

The Baoqing Psychiatric Hospital, Shaoyang, China.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and individuals with COVID-19 have symptoms that can be asymptomatic, mild, moderate or severe. In the early phase of infection, T- and B-cell counts are substantially decreased; however, IgM and IgG are detectable within 14 d after symptom onset. In COVID-19-convalescent individuals, spike-specific neutralizing antibodies are variable. No specific drug or vaccine is available for COVID-19 at the time of writing; however, patients benefit from treatment with serum from COVID-19-convalescent individuals. Nevertheless, antibody responses and cross-reactivity with other coronaviruses in COVID-19-convalescent individuals are largely unknown. Here, we show that the majority of COVID-19-convalescent individuals maintained SARS-CoV-2 spike S1- and S2-specific antibodies with neutralizing activity against the SARS-CoV-2 pseudotyped virus, and that some of the antibodies cross-neutralized SARS-CoV, Middle East respiratory syndrome coronavirus or both pseudotyped viruses. Convalescent individuals who experienced severe COVID-19 showed higher neutralizing antibody titres, a faster increase in lymphocyte counts and a higher frequency of CXCR3 T follicular help (T) cells compared with COVID-19-convalescent individuals who experienced non-severe disease. Circulating T cells were spike specific and functional, and the frequencies of CXCR3 T cells were positively associated with neutralizing antibody titres in COVID-19-convalescent individuals. No individuals had detectable autoantibodies. These findings provide insights into neutralizing antibody responses in COVID-19-convalescent individuals and facilitate the treatment and vaccine development for SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41564-020-00824-5DOI Listing
January 2021

Alterations of Oral Microbiota in Chinese Patients With Esophageal Cancer.

Front Cell Infect Microbiol 2020 21;10:541144. Epub 2020 Oct 21.

Department of Gastroenterology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, China.

Emerging evidence supports that oral microbiota are associated with health and diseases of the esophagus. How oral microbiota change in Chinese patients with esophageal cancer (EC) is unknown, neither is their biomarker role. For an objective to understand alterations of oral microbiota in Chinese EC patients, we conducted a case-control study including saliva samples from 39 EC patients and 51 healthy volunteers. 16S rDNA genes of V3-V4 variable regions were sequenced to identify taxon. Relationship between oral flora and disease was analyzed according to alpha diversity and beta diversity. Resultantly, the Shannon index ( = 0.2) and the Simpson diversity index ( = 0.071) were not significant between the two groups. Yet we still found several species different in abundance between the two groups. For the EC group, the most significantly increased taxa were , and , while the most significantly decreased taxa were , and . In conclusion, there are significant alterations in abundance of some oral microbiomes between the EC patients and the healthy controls in the studied Chinese participants, which may be meaningful for predicting the development of EC, and the potential roles of these species in EC development deserve further studies.
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http://dx.doi.org/10.3389/fcimb.2020.541144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609410PMC
June 2021

Structural characterization of a neutralizing mAb H16.001, a potent candidate for a common potency assay for various HPV16 VLPs.

NPJ Vaccines 2020 23;5:89. Epub 2020 Sep 23.

Division of HIV/AIDS and Sexually Transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC), No.31 Huatuo Street, Daxing District, 102629 Beijing, China.

With more human papillomavirus (HPV) virus-like particle (VLP) vaccines to hit the market in future, a monoclonal antibody (mAb) with preferably comparable reactivity against vaccines from different expression systems and bioprocesses is urgently needed for the potency characterization. Among all mAbs against HPV16 collected, rabbit mAb H16.001 is potently neutralizing with the highest affinity, recognizes an immune-dominant epitope, and can comparably react with HPV16 vaccines from various sources. Cryo-electron microscopic (cryo-EM) structure demonstrated that 360 H16.001 Fabs could bind to HPV16 capsid in preferable binding manner without steric hindrance between neighboring Fabs, potentially supporting its identification for VLP structural integrity and utility in monitoring VLP structural probity. This structural analysis indicated that mAb H16.001 afforded unbiased potency characterization for various HPV16 vaccines and was potential for use in vaccine regulation practice. This study also showed a model process for selecting suitable mAbs for potency assays of other vaccines.
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http://dx.doi.org/10.1038/s41541-020-00236-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511963PMC
September 2020

Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.

Nat Protoc 2020 11 25;15(11):3699-3715. Epub 2020 Sep 25.

Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay could discriminate the protective agents from the antibody family. This protocol includes production and titration of the SARS-CoV-2 S pseudotyped virus and the neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies and fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience in handling cells is needed before implementing this protocol.
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http://dx.doi.org/10.1038/s41596-020-0394-5DOI Listing
November 2020

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Cell 2020 11 14;183(4):1013-1023.e13. Epub 2020 Sep 14.

Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing 102629, China.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.09.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489885PMC
November 2020

Dysregulation of Ephrin receptor and PPAR signaling pathways in neural progenitor cells infected by Zika virus.

Emerg Microbes Infect 2020 Dec;9(1):2046-2060

Centre for Biologics Evaluation, Biologics and Radiopharmaceutical Drugs Directorate, HPFB, Health Canada and WHO Collaborating Centre for Standardization and Evaluation of Biologicals, Ottawa, ON, Canada.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.
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http://dx.doi.org/10.1080/22221751.2020.1818631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534353PMC
December 2020

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.

Cell 2020 09 17;182(5):1284-1294.e9. Epub 2020 Jul 17.

Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC) and WHO Collaborating Center for Standardization and Evaluation of Biologicals, No. 31 Huatuo Street, Daxing District, Beijing 102629, China; Graduate School of Peking Union Medical College, No. 9 Dongdan Santiao, Dongcheng District, Beijing 100730, China. Electronic address:

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.
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http://dx.doi.org/10.1016/j.cell.2020.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366990PMC
September 2020

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Nature 2020 10 29;586(7830):572-577. Epub 2020 Jul 29.

Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.
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http://dx.doi.org/10.1038/s41586-020-2599-8DOI Listing
October 2020
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