Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—...Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—suchas animal immunization or B cell isolation from infected individuals—are often hindered by biosafety concerns,lengthy development timelines,and limited adaptability during outbreaks.In the present study,we aimed toestablish a robust and rapid in vitro platform for the efficient isolation of neutralizing antibodies targetingconserved viral epitopes.We developed an epitope-guided negative screening strategy that integrates phagedisplay technology with rational antigen mutagenesis to exclude antibodies against variable regions whileenriching for those that recognize functionally constrained epitopes.When applied to the receptor-binding domainof severe acute respiratory syndrome coronavirus 2,this method enabled the identification of six neutralizingantibodies(one IgG and five nanobodies)exhibiting broad-spectrum neutralizing activity across multiple viralvariants.Notably,antibodies recognizing distinct epitopes demonstrated significant synergistic neutralizationwhen used in combination(P<0.05).This screening approach facilitates the rapid discovery of potent andmutation-resistant antibodies and holds promise for application to other emerging pathogens.Our findingsunderscore the potential of epitope-guided,in vitro platforms in expediting therapeutic antibody developmentunder conditions of high biosafety requirements.展开更多
To explore the mechanisms underlying ocular infection by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),we conducted a comprehensive review of current literature,focusing on viral entry pathways,receptor ...To explore the mechanisms underlying ocular infection by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),we conducted a comprehensive review of current literature,focusing on viral entry pathways,receptor expression in ocular tissues,and associated clinical manifestations.This review encompasses studies published within the last five years with a focus on original research and systematic reviews that provide molecular,histological,or clinical evidence.The findings show that SARS-CoV-2 can infect ocular tissues through multiple receptors beyond angiotensin-converting enzyme 2(ACE2),including transmembrane serine protease 2(TMPRSS2),CD147,alanyl aminopeptidase N(ANPEP),dipeptidyl peptidase 4(DPP4),angiotensin II receptor type 2(AGTR2),and polymeric immunoglobulin receptor(PIGR),which are expressed in retinal,conjunctival,corneal,limbal,and photoreceptor cells.The virus may also reach ocular structures via neurovascular invasion.Clinically,patients with coronavirus disease 2019(COVID-19)may present with a broad spectrum of ophthalmic manifestations,including conjunctivitis,hyperreflective lesions in the inner retinal layers,flame-shaped hemorrhages,cottonwool spots,retinal pallor,hard exudates,and various forms of maculopathy,such as paracentral acute middle maculopathy and acute macular neuroretinopathy(AMN).These signs reflect both direct viral damage and secondary effects of systemic inflammation and microvascular injury.Understanding the molecular and clinical spectrum of ocular involvement is essential for early diagnosis,appropriate ophthalmologic care,and the prevention of long-term visual sequelae in patients affected by COVID-19.展开更多
目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫...目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫PBS作为对照,每组10只。初次免疫后第7、14、21、28、35和42天采集血清,间接ELISA法检测血清中针对Delta株病毒S和N蛋白的结合抗体效价,微量中和试验检测针对Delta株病毒的中和抗体效价。初次免疫后第42天,取小鼠脾脏,进行Elispot检测,评价细胞免疫水平。结果初次免疫后第7天即可检测到S蛋白结合抗体,加强免疫后抗体效价进一步升高,至第21天抗体几何平均滴度(geometric mean titer,GMT)为89144;而初次免疫后N蛋白结合抗体水平较低,加强免疫后迅速升高,与S蛋白抗体水平相当。初次免疫后第7、14天小鼠中和抗体阳转数为4/10和8/10,加强免疫后全部小鼠抗体阳转,中和抗体GMT达391。初次免疫后第42天,疫苗组IFNγ和IL-2平均斑点数均显著高于对照组(t分别为8.094和13.08,P均<0.0001)。结论SARS-CoV-2灭活疫苗2次免疫能够有效刺激小鼠产生针对Delta株病毒的体液免疫和细胞免疫。展开更多
基金supported by the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(Grant No.22KJB310001 to W.G.)the Special Project of the Jiangsu Provincial Department of Science and Technology(Grant No.BE2023603 to W.G.)the Nanjing Medical University Science and Technology Development Foundation(Grant No.NMUB20210006 to W.G.).
文摘Neutralizing antibodies are essential tools in antiviral therapy and epidemic preparedness,capable of directlyinhibiting viral entry and limiting disease progression.However,traditional antibody discovery strategies—suchas animal immunization or B cell isolation from infected individuals—are often hindered by biosafety concerns,lengthy development timelines,and limited adaptability during outbreaks.In the present study,we aimed toestablish a robust and rapid in vitro platform for the efficient isolation of neutralizing antibodies targetingconserved viral epitopes.We developed an epitope-guided negative screening strategy that integrates phagedisplay technology with rational antigen mutagenesis to exclude antibodies against variable regions whileenriching for those that recognize functionally constrained epitopes.When applied to the receptor-binding domainof severe acute respiratory syndrome coronavirus 2,this method enabled the identification of six neutralizingantibodies(one IgG and five nanobodies)exhibiting broad-spectrum neutralizing activity across multiple viralvariants.Notably,antibodies recognizing distinct epitopes demonstrated significant synergistic neutralizationwhen used in combination(P<0.05).This screening approach facilitates the rapid discovery of potent andmutation-resistant antibodies and holds promise for application to other emerging pathogens.Our findingsunderscore the potential of epitope-guided,in vitro platforms in expediting therapeutic antibody developmentunder conditions of high biosafety requirements.
文摘To explore the mechanisms underlying ocular infection by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),we conducted a comprehensive review of current literature,focusing on viral entry pathways,receptor expression in ocular tissues,and associated clinical manifestations.This review encompasses studies published within the last five years with a focus on original research and systematic reviews that provide molecular,histological,or clinical evidence.The findings show that SARS-CoV-2 can infect ocular tissues through multiple receptors beyond angiotensin-converting enzyme 2(ACE2),including transmembrane serine protease 2(TMPRSS2),CD147,alanyl aminopeptidase N(ANPEP),dipeptidyl peptidase 4(DPP4),angiotensin II receptor type 2(AGTR2),and polymeric immunoglobulin receptor(PIGR),which are expressed in retinal,conjunctival,corneal,limbal,and photoreceptor cells.The virus may also reach ocular structures via neurovascular invasion.Clinically,patients with coronavirus disease 2019(COVID-19)may present with a broad spectrum of ophthalmic manifestations,including conjunctivitis,hyperreflective lesions in the inner retinal layers,flame-shaped hemorrhages,cottonwool spots,retinal pallor,hard exudates,and various forms of maculopathy,such as paracentral acute middle maculopathy and acute macular neuroretinopathy(AMN).These signs reflect both direct viral damage and secondary effects of systemic inflammation and microvascular injury.Understanding the molecular and clinical spectrum of ocular involvement is essential for early diagnosis,appropriate ophthalmologic care,and the prevention of long-term visual sequelae in patients affected by COVID-19.
文摘目的评价原型株SARS-CoV-2灭活疫苗免疫BALB/c小鼠后对Delta株病毒的体液及细胞免疫效果,为现有疫苗对变异株的保护效果评价以及研发更加安全有效的疫苗提供参考。方法将SARS-CoV-2灭活疫苗经腹腔免疫雌性BALB/c小鼠2次,间隔14 d,以免疫PBS作为对照,每组10只。初次免疫后第7、14、21、28、35和42天采集血清,间接ELISA法检测血清中针对Delta株病毒S和N蛋白的结合抗体效价,微量中和试验检测针对Delta株病毒的中和抗体效价。初次免疫后第42天,取小鼠脾脏,进行Elispot检测,评价细胞免疫水平。结果初次免疫后第7天即可检测到S蛋白结合抗体,加强免疫后抗体效价进一步升高,至第21天抗体几何平均滴度(geometric mean titer,GMT)为89144;而初次免疫后N蛋白结合抗体水平较低,加强免疫后迅速升高,与S蛋白抗体水平相当。初次免疫后第7、14天小鼠中和抗体阳转数为4/10和8/10,加强免疫后全部小鼠抗体阳转,中和抗体GMT达391。初次免疫后第42天,疫苗组IFNγ和IL-2平均斑点数均显著高于对照组(t分别为8.094和13.08,P均<0.0001)。结论SARS-CoV-2灭活疫苗2次免疫能够有效刺激小鼠产生针对Delta株病毒的体液免疫和细胞免疫。
