Production And Characterization Of Antibodies For Dengue Serotype 1 Virus Envelope Protein Domain Ⅲ, And Expression Of H5 Subtype Hemagglutinin Of Influenza A Virus In Insect Cells

Dengue virus is a member of the Flavivirus genus in the Flaviviridae family and has four distinct serotypes(DENV-1,DENV-2,DENV-3 and DENV-4).Infection with any of the four serotypes of DV causes a spectrum of clinical features ranging from asymptomatic infections,undifferentiated fever,and classical dengue fever to life-threatening manifestations such as dengue hemorrhagic fever(DHF) and dengue shock syndrome(DSS).The diease is endemic in more than 100 countries and is found virtually throughout the tropics and cause an estimated 50-100 million illnesses annually,including 250 000-500 000 cases of dengue haemorrhagic fever.After being infected by one serotype dengue virus,an individual develops long-lived immunity to homologous serotypes,but antibodies to one serotype are not sufficient to protect against other serotypes,subsequent heterologous infections may lead to the developing DHF or DSS.This is known as antibody dependent enhancement(ADE) for it is postulated that subneutralizing cross-reactive antibodies may facilitate viral infection through Fc receptor by binding with the virus. Antibodies induced by dengue virus infections can be roughly divided into type-specific neutralizing antibodies,cross-reactive nonneutralizing antibodies,and cross-neutralizing antibodies.It is thought that cross-reactive nonneutralizing antibodies and neutralizing antibodies in sub-neutralizing concentration will both lead to ADE.Yamanaka et al.reported that antibodies showed enhancing activities or neutralizing activities depended on the levels of complement in vitro and complement levels were considered an additional factor involved in the in vivo ADE phenomenon: neutralizing at a normal level of complement,but increaseing infetion at a low level.Envelope(E) protein is the the major protein on the surface of DENV virions,it can mediate receptor binding and membrane fusion.Antibodies against E protein long-lastingly exist in patients' sera,and most importantly,confers protective immunity.E protein contains three domains:a central domain(DⅠ),a dimerization domain(DⅡ),and an immunoglobulin(Ig)-like domain(DⅢ).It had been found many type- and subtype- specific neutralizing epitopes were mapped to domainⅢ,and antibodies to this domain were the most powerful blockers to virus infection.At present,however,there is no specific treatment available for DENV infections and no vaccine that is effective against all four DENV serotypes,so preparation of antibodies with neutralizing abilities is meaningful for the study of antigen epitopes and the production of vaccine.For the great importance of EDⅢin inducing neutralizing antibodies,we chose the recombinant DENV-1 EDⅢprotein as the antigen to immunize five BALB/c mice and one New Zealand rabbit for producing monoclonal antibodies and rabbit hyperimmune serum and tried to find out which epitopes were serotype specific and which were cross-reactive.We also tried to find out andibodies recoganized these epitopes,which had serotype specific neutralizing abilities and which had cross-neutralizing abilities.These will give guidelines for designing a vaccine without inducing ADE.This study consists of three parts,as follows:Ⅰ.Preparation and characterization of antibodies to DENV-1 EDⅢ A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivity with the rDENV1- EDⅢprotein.The immunoglobulin isotype determinations revealed that the 24 MAbs comprised IgG2a(n=2),IgG2b(n=1),and IgG1(n=21).The serotype specificity of the MAbs was further identified by testing their reactivity to the four rDENV EDⅢproteins by ELISA and the four serotypes DENV in the IFA.Of the 24 MAbs,8 MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.Ⅱ.To study the DENV-1 EDⅢepitopes recoganized by these antibodies and detect their neutralizing abilities to DENV with Plaque Reduction Neutralization Test.The distinct binding epitopes of the 24 DENV EDⅢMAbs were determined by competition ELISA using recombinant DENV1-EDⅢas the immobilized antigen. The results showed that the 24 MAbs bound at least to three different epitopes on the DENV-1 EDⅢprotein and 13 MAbs recognized eptiopeⅠshowed a cross-reactivity with other DENV serotypes in ELISA and IFA.Each of the 24 hybridoma cell lines were injected intraperitoneally in BALB/c mice to produce ascitic fluids. Neutralization ability to DENV-1～4 of these ascetic fluids were detected by Plaque Reduction Neutralization Test(PRNT).The results indicated that 23 MAbs had neutralizing activities to DENV,from which 13 cross-reactive MAbs recognized eptiopeⅠalso showed cross-neutralizing abilities to DENV,but there was no certain relationship between the two.Four MAbs of the 13 could neutralize all four dengue-serotypes;The 7 MAbs recoganized epitopeⅢshowed great neutralizing abilities to DENV-1,except 9E18A8 and 9F26A1.Three MAbs from the greatly neutralizing antibodies were serotype-specifc and could be developed as therapeutic antibodies for DENV-1;The rabbit hyperimmune serum neutralizd only DENV-1 and DENV-3,which indicated,that DENV-1 EDⅢand DENV-3 EDⅢshared crossly protective epitopes. Ⅲ.To evaluate the neutralizing abilities of anti-DENV antibodies with DENV-1 NS1 antigen capture ELISA.DENV-1 neutralized firstly by antibodies were used to infect C6/36 cell,the DENV-1 NS 1 antigen capture ELISA was used to evaluate the neutralizing abilities of these antibodies by detecting the NS1 secreted from the the infected cell.The results indicated that only 14 MAbs of the 23 neutralizing MAbs detected by PRNT had neutralizing abilities,while the rabbit hyperimmune serum had neutralizing ability detected by both methods.Neutralizing titers detected by NS1 capture ELISA were far lower than that detected by PRNT.Antibodies recoganized epitopeⅢshowed greater neutralizing titers than other antibodie in both methods.Conclusions:1.A total of 24 hybridoma cell lines that stably produced MAbs were initially established on the basis of their strong positive reactivities with the rDENV1- EDⅢprotein.Eight MAbs specifically reacted to DENV1 with no cross-reactivities to the other three DENV serotypes in either the ELISA or the IFA.The remaining 16 MAbs and the rabbit hyperimmune serum had cross-reactivities to the four DENV serotypes in different cross-reactivity patterns.These obtained antibodies can be used to study their neutralizing ablilities and are potential tools for studing the mechanism of reduced or enhanced virus infection.2.The 24 MAbs bound to at least three different epitopes on the DENV-1 EDⅢprotein and MAbs recognized eptiopeⅠshowed a cross-reactivities with other DENV serotypes in ELISA and IFA and cross-neutralizing abilities to DENV in PRNT.This could be used to study common epitopes of DENV,which is meaningful for developing DENV vaccine;Most of the MAbs recoganized epitopeⅢshowed serotype specificities and great neutralizing abilities to DENV-1.It was supposed that epitopeⅢplayed a important role in virus infection and cell binding,so this epitope could be used as one of the potential candidates of tetravalent vaccine.3.The rabbit hyperimmune serum to DENV-1 EDⅢcould protected cells against DENV-1 and only crossly protected cells from being infected by DENV-3,it indicated that DENV-1 EDⅢwould not generate sufficient protection for DENV-2 and DENV-4 as a subunit vaccine.4.DENV-1 NS1 antigen capture ELISA was not sensitive enough for evaluating antibodies' neutralizing abilities.But it could be used for screening the antibodies with high neutralizing abilities as a supplement to PRNT.