| BackgroundZika virus disease(ZVD)is an acute disease caused by Zika Virus(ZIKV)infection.Since 2015,the outbreak of the ZVD in South America,North America,Europe,Asia and many other countries or regions,caused serious complications such as the Guillain-Barr syndrome and microcephaly,which attrated concern in public health.With the rapid spread of Zika virus(ZIKV)in the Americas in 2015-2016,and its association with fetal skull malformations and neurologic disorders in adults,WHO declared ZIKV a global emergency.A total of 24 imported cases were reported during the year 2016 in China.South China had sporadic cases of dengue virus(DENV)infection for decades with the serotype 1 of DENV(DENV1)predominant in circulation.A major DENV1 outbreak in 2014 in Guangdong had resulted in 13800 hospitalizations with an estimated 300 cases of severe dengue and five reported deaths.The preexisting DENV immune status in population combined the risk of endemic spread of ZIKV infection,have raised the question of how to diagnosis the specific infection,and whether disease severity will be altered due to antibody cross-reactivity because of ZIKV and DENV.ZIKV is a positive sense,single-strand ribonucleic acid(RNA)with a genome size of approximately 10.8 kilobases.The RNA si translated 3 structureal protein(capid;membrance;and envelope)as well as 7 nonstructrural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B and NS5).The structural proteins,as the name suggests,form the virus particles.The nonstructral proteins assist in replication and packaging of the genome as well as in subverting the host pathways in favor of the virus.Both E and NS1 proteins are main targets of human antibody response.The high cross-reactivity between ZIKV and DENV E-specific Abs was commonly known because of the similarity of their E proteins in sequence and structure,especially at an early time point during DENV and ZIKV infections.We reported that the peak of EDI/EDII binding Abs in sera from ZIKV infected individual peaked and waned earlier than EDIII binding Abs,consisting with the knowledge that ZIKV and DENV have a highly conserved fusion loop epitope(FLE)in EDII.The monoclonal antibodies(mAbs)isolated from the plasma cell or memory B cell appearing at early ZIKV infection also showed cross-reactivity with DENV and binding to the EDI/EDII.The non-structural protein 1(NS1)of flavivirus can be released from infected cell to the blood or on the cell surface.NS1 is also highly antigenic and contributes to the human antibody response repertoire against the virus.Recently the NS1-based serological tests have been developed for distinguishing ZIKV from DENV infection.But little is known about the NS1-specific Ab response during the course of the ZIKV infection with respect to its specificity,magnitude,and kinetics are of great relevance for diagnostics.ObjectivesThe characteristics of NS1 antibody response at both sera level and monoclonal antibody level were analyzed in samples from ZIKV infected individiuls.The kinetics and specificity of NS1 Ab revealed had important implications for NS1-based diagnostics and vaccine development.Also,the capture-ELISA method was established which could be used to identify ZIKV especially in DENV epidemic areas.The high specific monoclonal antibodies were isolated from B cells which can be applied to the development for early screening tools in ZIKV infecteds.Methods 1? Expression of recombinant NS1 protein and establishment of capture ELISA(1)The full-length Zika and Dengue virus NS1 genes were amplified from clinical samples and the homology of the amino acid sequence of dengue virus and Zika virus NS1 were analyzed.(2)NS1 proteins were expressed by transfecting the vectors into mammalian 293 T cell line.(3)NS1 capture-ELISA method was developed to detect antibodies response in serum.2?Cross-reactivity of NS1 antibody response during natural ZIKV infection and in DENV1 infected individuals(1)The characteristics of antibody response to NS1 were revealed during natural ZIKV infection by NS1-based capture ELISA.(2)Characteristics of NS1 antibody response reaction in serum were explored by analyzing the convalescent serum of DENV1 patients of Guangzhou in 2014.3?Binding and specificity of NS1 mAbs derived from memory B cells of ZIKV infected patients(1)B cells were isolated from PBMC samples of ZIKV individuals.The gene of variable regions for heavy and light chains were amplified and ligated into specific vector.Monoclone antibodies were expressed by co-transfecting both heavy chain and light chain vectors into 293 T cells.(2)The binding and specificity of NS1 mAbs were analyzed by capture-ELISA.4?The application of high specific NS1 monoclone antibodies The specific monoclonal antibodies were applied into the colloidal gold test strip for screening NS1 at early stage of ZIKV infected.Results 1?Expression of recombinant NS1 protein and establishment of capture ELISA(1)The complete NS1 gene was amplified from ZIKV infected samples,and cloned into modified vectors.(2)The full length NS1 proteins of ZIKV and DENV1-4 were expressed in mammalian 293 T cells and collected the culture supernatants containing NS1 s which were shown to be about 55 KD as detected in western blot.(3)These NS1 proteins were used to set up capture ELISA with D7 Ab pre-coated plates.The capture ELISA were tested by positive serum in dillution,which indicated the mothed can be used to detect antibody response in ZIKV infection.2?The characteristics of ZIKV-NS1 antibody response(1)Dynamics and cross-reactivity of NS1 antibody response during natural ZIKV infection To examine the dynamic changes of human NS1 antibody response during the course of ZIKV infection at polyclonal level,we measured the serum binding Abs in sequential samples from two ZIKV infected patients,with eight sequential plasma samples.Comparing to E specific Ab response reported previously in serum samples from the same patient,the NS1 specific antibodies appeared later,became detectable on day 15,also peaked later on day 106 and declined markedly during disease.Similar trends of E and NS1 specific Ab responses were observed in Pt2 with peaks on day 12 and day 66,respectively.In contrast to high cross-reactivity of serum antibodies to DENV1-4 E proteins,there were no detectable binding of serum antibodies to DENV1-4 NS1 from both patients at all time points.(2)Global analysis of NS1 cross-reactivity in DENV infected individuals To investigate whether the lack of significant cross-reactivity between anti-ZIKV NS1 antibodies and anti-DENV NS1 antibodies is not restricted to a few selected patients,we performed NS1 serum binding assay on a total of 108 convalescent sera from DENV1 infection including primary infection(n=35),secondary infection(n=20)and undetermined cases(n=53).Lack of cross-reactivity to ZIKV was observed in all DENV1 infected individuals except for the two cases.As expected,these DENV1 immune sera exhibited significant cross-reactivity to DENV2,DENV3,and DENV4.3?Binding and specificity of NS1 mAbs derived from memory B cells of ZIKV infected patients(1)Five NS1 mAbs named ZKns3G2?ZKns2E11?ZKns14G5?ZKns4B8?ZKns4F10 were isolated from two ZIKV infected patients by memory B cell culture method and characterized their CDR3 regions and pattern of reactivity were analyzed.(2)Most of mAbs(4/5)showed strong binding to ZIKV NS1 with EC50 values ranging from 9.8 to 277.4 ng/ml,and no reactivity to DENV 1-4 NS1 proteins.Only one mAb(1/5),ZKns4F10,cross-reacted with DENV 2 and DENV 4 NS1 with EC50 of 116.1ng/ml and 351.4ng/ml,respectively;10-30 fold less than that to ZIKV NS1(10.9ng/ml),which can be explained by the partial sequence and structure similarity shared between ZIKV and DENV.4?The application of high specific NS1 monoclone antibodiesThe specific NS1 monoclone antibodies were matched and used to develop the NS1 antigen rapid screening kit.The sensitivity for recombined NS1 protein was 10ng/mL.The kit worked well to detect as positive for urine samples from ZIKV infected individiuls.ConclusionBy analyzing sera obtained from patients with ZIKV infection,we had discovered anti-NS1 being high specific at polyclonal antibodies levels,a phenomenon which is distinct from the high cross-reactive antibody responses to ZIKV and DENV E proteins.This supports the development of NS1 based diagnostic tools for areas where DENV and ZIKV co-circulate.The monoclone antibodies were high specific with great binding activity.And the monoclonal antibodies were applied to develop kits of colloidal gold test strip.The results indicated that the high specific NS1 monoclonal antibodies could be used in the development of diagnostic kits. |
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