Production And Characterization Of Monoclonal Antibodies Specific For Dengue Serotype 3 Virus Nonstructural Protein NS1, And Establishment Of NS1 Antigen Capture ELISA

Dengue virus belongs to the Flavivirus genus of the Flaviviridae family and is transmitted to humans by Aedes aegypti or Aedes albopictus mosquitoes.Dengue virus include four antigenically related serotypes(DV 1～4),all of which cause disease.Infection with any of the four serotypes of dengue virus causes a spectrum of clinical features,ranging from asymptomatic infections,undifferentiated fever,and classical dengue fever(DF) to life-threatening manifestations,dengue hemorrhagic fever(DHF) and dengue shock syndrome(DSS).Dengue is an endemic viral disease affecting tropical and subtropical regions around the world.The disease is now endemic in more than 100 countries around the world and there may be 50 million cases of dengue virus infections each year.In areas where multiple dengue serotypes are transmitted concurrently,clinical cases caused by more than 1 serotype of dengue virus may be more common than previously thought.The high attack rates of cases that occur during epidemics would likely results in many infections with multiple virus serotypes in humans,and also provide opportunities for mosquitoes to become infected with two or more serotypes.Infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infections by the other three serotypes.Instead,it has generally been accepted that secondary infection or infection with secondary or multiple infections with various dengue virus serotypes is a major risk factor for DHF/DSS due to antibody-dependent enhancement.At present,however,there is no protective vaccine or specific treatment available for dengue virus infection.Thus,early clinical management can reduce the morbidity and mortality of DHF or DSS.Since symptoms of DV infections are insufficiently specific for accurate clinical differentiation from other febrile illnesses and hemorrhagic fever,the definitive diagnosis of dengue virus infection can only be made in the laboratory,and it depends on the isolation of these viruses,the detection of viral antigens or RNA in serum or tissues,or the detection of specific antibodies in the patients' serum.Currently, laboratory diagnosis of DV infections is based on viral isolation,serology,and RNA detection.Although the detection of antibodies using whole virus antigens based enzyme-linked immunosorbent assay(ELISA) is the most commonly used,the limitations of this assay are cross-reactivity with all serotypes of DV and also other members of the flavivirus family.Viral isolation is a gold standard for the diagnosis and the serotype of DV infections,but this method is time-consuming and requires a sophisticated laboratory.Viral nucleic acid detection typically provides more sensitive and rapid diagnosis than traditional virus isolation method does.However, the molecular diagnosis requires experienced technicians and specialized laboratory equipment and ease to bring out false-positive results due to contamination.Therefore, early diagnosis and determination of serotype still remains a problem,as it mainly depends on RT-PCR or virus isolation methods.Up to now,only our laboratory developed a specific antigen capture ELISA for early detection and serotyping of DV1 by using well-characterized monoclonal antibodies(MAbs) specific to NS1 of DV1.Dengue virus is a positive-stranded encapsulated RNA virus.The genomic RNA is approximately 11 kb in length and is composed of three structural protein genes that encode the nucleocapsid or core protein(C),a membrane associated protein(M), an envelope protein(E),and seven nonstructural(NS) protein genes.The gene order is 5'-CprM(/M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3',as for other flaviviruses.The organization of the lipid bilayer and of the envelope glycoprotein shell has been observed by cryoelectron microscopy and image reconstruction,which provided three-dimensional structure of DV.The NS proteins are assumed to be involved primarily in the replication of viral RNA as a part of the replication complex. The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells:an intracellular,membrane-associated form essential for viral replication,a cell surface-associated form that may be involved in signal transduction,and a secreted form(sNS1),the biological properties of which remain elusive.And we propose that NS1 may play a role in the immunopathogenesis of severe dengue virus infections.Glycoprotein NS1 appears to be essential for virus viability although no precise function has yet been ascribed to it.NS1 is a highly conserved 48-kDa glycoprotein with 12 invariant cysteine residues.It possesses not only group-specific but type-specific determinants and has been recognized as an important immunogen in DV infections.Therefore NS1 protein might be as an early diagnostic marker and a serotyping marker.Our laboratory have developed and characterized a panel of MAbs to serotype-specific epitopes of NS1 from DV1,by which a rapid and sensitive serotype-specific assay for the early diagnosis of DV infections was established.This laid the basis for us to produce and characterize a panel of monoclonal antibodies of dengue type 3 virus nonstructural protein NS1,and established of NS1 antigen capture ELISA.This two-site sandwich antigen-capture ELISA was tested and optimized for detection of DV3NS1.Ⅰ.Cloning and expression of DV3 nonstructural protein NS1 and identification of its antigenicityThe cDNA of nonstructural protein NS1 was amplified with RT-PCR from the C6/36 cells infected with DV3 and was cloned into the multi-cloning sites of pQE31 and expressed with induction of IPTG.After the purification under denature condition, the immunogenicity of the recombinant protein was identified by the monoclonal antibody 7E9A2 against DVNS1 through Western Blot and ELISA methods. Experimental results showed the recombinant NS1 protein was highly expressed in E.coli M15 and the purified NS1 could be recognized and combined by the monoclonal antibody against the DVNS1.The expressed NS1 protein has good antigenicity,which may provide a potential source for studying the function of NS1 and use as an antigen for dengue serum detection.Ⅱ.Preparation and characterization of monoclonal antibodies against DV3 nonstructural protein 116 Balb/c mice were immunized by recombinant DV3NS1 protein and inactive DV3 alternately.Then the splenocytes of the immunized mice were fused with myeloma cells to produce hybridoma cell line,which could secrete anti-DV3NS1 protein antibodies.ELISA,immnofluorescence assay(IFA) and Western blot analysis were applied to identify specificity of antibodies.Ultimately 26 strains of hybridoma cell lines steadily secrete antibodies of NS1 were obtained.These antibodies had characteristics of binding to DV and recombinant NS1 protein.Among them,two strains was identified as IgG2a isotype and the other twenty four were all IgG1.Six out of the twenty six strains were specific to DV3 without detectable cross-reactivity with the other three serotypes of dengue virus.Seven out of the twenty six strains were cross-reactive to with the other three serotypes of dengue virus.Competitive inhibition assay showed that these mAbs recognized at least seven distinct epitopes of NS1 antigen.These obtained antibodies with high activity and specificity will provide a potential value for vaccine researches and early diagnosis of dengue virus infection.Ⅲ.Establishment of the antigen capture enzyme-linked immunosorbent assay for detection dengue serotype 3 virus nonstructural protein NS1To develop the mAb-based antigen capture ELISA for DV3NS1,the antibodies were paired one by one based on their recognizing epitopes and antibody titers to choose the optimal coating mAb and detecting mAb through determination the detection sensitivity and specificity.The best coating antibody and Biotin-conjugate detection antibody selected ultimately to construct the antigen capture ELISA was 1D 14A2A10 and Biotin-5D32A17.The lowest limit of detection of the recombinant NS1 protein with this capture ELISA is 0.4μg/ml approximately.Furthermore,this mAb-based antigen-capture assay is specific to DV3NS1 and has no cross-reactivity with either other serotypes of DV or other closely related members of the flavivirus family(Japanese encephalitis virus and Yellow fever virus) when the virus-infected cell culture was used.All these results demonstrated that the established test is sensitive and specific which may lay the basis for integrate laboratory diagnosis tool for all serotypes of dengue virus infection.In conclusion,1.The DV3NS1 protein was expressed and purified successfully which has good antigenicity,and a panel of mAbs specific to the NS1 of DV3 and mAbs cross-reactivity to the NS1 of DV was successfully obtained.This may provide a potential source for studying the function of NS1 and for vaccine researches and early diagnosis of dengue virus infections.2.Among the twenty six strains of hybridoma cell lines steadily secrete antibodies of nonstructual protein 1,enzyme-linked immunosorbent assay(ELISA), immnofluorescence assay(IFA) and Western blot analysis were applied to identify specificity of antibodies.The result of ELISA and IFA is that six strains out of the twenty six strains were specific to DV3NS1,but the western bot result is nagetive. Furthermore,the detection sensitivity of denature NS1 protein of DV3-specific mAb-based antigen-capture ELISA is lower than nature DV3NS 1 protein.Therefore, we supposed that the capture antibody recognize a conformentional determinant of DV3NS1 protein;however the genuine reason was to be investigated.3.A mAb-based two-site sandwich antigen-capture ELISA was successfully established.The assay employs a mAb specific to the DV3NS1 and mAb cross-reactivity to the DVNS1 as the capture and detection antibodies,respectively.It has high detection specificity to DV3 without cross-reactions with either other three DV serotypes or other closely related members of the flavivirus.The characteristic will be helpful in rapid serotyping and differential diagnosis of dengue virus infections.