Neutralizing monoclonal antibodies(mAb)are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection.The continuous emergence of new SARS-CoV-2 variants wor...Neutralizing monoclonal antibodies(mAb)are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection.The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs.In this study,we immunized mice with the receptor-binding domain(RBD)of the SARS-CoV-2 prototypic strain(WIV04)and screened 35 RBDspecific mAbs using hybridoma technology.Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection.The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results.A representative mAb was selected from each category(RD4,RD10,and RD14)to determine the binding kinetics and median inhibitory concentration(IC_(50))of WIV04 and two variants of concern(VOC):B.1.351(Beta)and B.1.617.2(Delta).RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ng/mL;however,it completely lost neutralizing activity against the B.1.351 variant.RD10 neutralized both variants with an IC50 exceeding 100 ng/mL;whereas RD14 neutralized two variants with a higher IC50(>1 mg/mL).Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections.RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 mg/kg,while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 mg/kg.These results highlight the potential for future modifications of the mAbs for practical use.展开更多
Background:With the ongoing worldwide coronavirus disease 2019(COVID-19)pandemic,an increasing number of viral variants are being identified,which poses a challenge for nucleic acid-based diagnostic tests.Rapid tests,...Background:With the ongoing worldwide coronavirus disease 2019(COVID-19)pandemic,an increasing number of viral variants are being identified,which poses a challenge for nucleic acid-based diagnostic tests.Rapid tests,such as real-time reverse transcription-polymerase chain reaction(rRT-PCR),play an important role in monitoring COVID-19 infection and controlling its spread.However,the changes in the genotypes of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARSCoV-2 detection over time.Methods:We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase(RdRp)and nucleocapsid(N)genes of SARS-CoV-2.We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern(VOCs).In addition,we also developed two rRT-PCR assays(S484K and S501Y)targeting the spike gene,which when combined with the open reading frames(ORF)1ab assay,respectively,to form duplex rRT-PCR assays,were able to detect SARS-CoV-2 VOCs(lineages B.1.351 and B.1.1.7).Results:Using a SARS-CoV-2 stock with predetermined genomic copies as a standard,the detection limit of both assays targeting RdRp and N was five copies/reaction.Furthermore,no cross-reactions with six others human CoVs(229E,OC43,NL63,HKU1,severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus)were observed using these assays.In addition,the S484K and S501Y assays were combined with the ORF1ab assay,respectively.Conclusions:Four rRT-PCR assays(RdRp,N,S484K,and S501Y)were used to detect SARS-CoV-2 variants,and these assays were shown to be effective in screening for multiple virus strains.展开更多
基金supported by grants from the National Key R&D Program of China(2021YFC2301700)National Natural Science Foundation of China(82061138021)+1 种基金the Key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001)Hubei Natural Science Foundation for Distinguished Young Scholars(2021CFA050).
文摘Neutralizing monoclonal antibodies(mAb)are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection.The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs.In this study,we immunized mice with the receptor-binding domain(RBD)of the SARS-CoV-2 prototypic strain(WIV04)and screened 35 RBDspecific mAbs using hybridoma technology.Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection.The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results.A representative mAb was selected from each category(RD4,RD10,and RD14)to determine the binding kinetics and median inhibitory concentration(IC_(50))of WIV04 and two variants of concern(VOC):B.1.351(Beta)and B.1.617.2(Delta).RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 ng/mL;however,it completely lost neutralizing activity against the B.1.351 variant.RD10 neutralized both variants with an IC50 exceeding 100 ng/mL;whereas RD14 neutralized two variants with a higher IC50(>1 mg/mL).Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections.RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 mg/kg,while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 mg/kg.These results highlight the potential for future modifications of the mAbs for practical use.
基金This work was supported by grants from the National Key Research and Development Program of China(Nos.2016YFD0500301,2021YFC0863300,and 2020YFC0840900).
文摘Background:With the ongoing worldwide coronavirus disease 2019(COVID-19)pandemic,an increasing number of viral variants are being identified,which poses a challenge for nucleic acid-based diagnostic tests.Rapid tests,such as real-time reverse transcription-polymerase chain reaction(rRT-PCR),play an important role in monitoring COVID-19 infection and controlling its spread.However,the changes in the genotypes of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARSCoV-2 detection over time.Methods:We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase(RdRp)and nucleocapsid(N)genes of SARS-CoV-2.We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern(VOCs).In addition,we also developed two rRT-PCR assays(S484K and S501Y)targeting the spike gene,which when combined with the open reading frames(ORF)1ab assay,respectively,to form duplex rRT-PCR assays,were able to detect SARS-CoV-2 VOCs(lineages B.1.351 and B.1.1.7).Results:Using a SARS-CoV-2 stock with predetermined genomic copies as a standard,the detection limit of both assays targeting RdRp and N was five copies/reaction.Furthermore,no cross-reactions with six others human CoVs(229E,OC43,NL63,HKU1,severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus)were observed using these assays.In addition,the S484K and S501Y assays were combined with the ORF1ab assay,respectively.Conclusions:Four rRT-PCR assays(RdRp,N,S484K,and S501Y)were used to detect SARS-CoV-2 variants,and these assays were shown to be effective in screening for multiple virus strains.
