Draw Topography Of DENV NS1Protein And Establish A ELISPOT Micro-neutralization Test To Evaluate DENV Antibodies Neutralizing Activity Rapidly And High-throughput

Dengue virus (DENV) is a RNA virus of the family Flaviviridae; genus Flavivirus and primarily transmitted by Aedes mosquitoes among vertebrates. DENV is classified four serotypes according to their reaction to antigen, DENV1-4, each of which is capable of causing same clinical manifestations including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).The transmission of DENV is limited in early mainly in tropical and subtropical regions. But now DENV is endemic in over100countries and regiaons in Africa, America, Southeast Asia and the West Pacific region along with the world economics integration and internetional exchange. Evidence shows that each year around the world50-100million people infected with DENV and there are about0.5million patients would be emergence with DHF or DSS,20-25thousand death cases, mostly children, mortality is about5%. Therefore, DENV infection has become an vital public health problem worldwide, how to effectively control the infection is the key to deal with this problem.The people who infected by DENV would be asymptomatic or develop flu-like symptoms, self-healing or mild symptoms DF, which appeared mainly in the initial infection patients but most of children need hospital treatment. However, sever symptoms including DHF and DSS would emergence in second infection patients. There is no specific treatment, patients can only accept symptomatic treatment, and no an effective vaccine to prevent DENV infection. Most researchs suggest that antibody-dependent inhancment (ADE) is the main cause of DHF and DSS because there are four different dengue virus serotypes. Studies show that there are various antibodies in sera of DENV infection patients and they have a short-term cross-reactive with all four serotypes. But the cross-reactive antibodies do not have long-term protection function. Instead, they would potentially cause the DHF/DSS. Their binding to virus particle can’t block but to enhance virus enter cells through Fc receptor-mediated entry pathway if the cells (such as monocytes and dendritic cells) express Fc receptor, which cotributes to the uptaking of virus and viral replication in cells, leading to infection enhancement and severe dengue disease occurrence. Vaccine is the most effective way to prevent dengue disease transmission, but if the vaccine can only protect one of serotypes of virus, the ADE would occurrence when the patient is infected with the other different serotype virus, which affects the life of the patient. Therefore, the key point in the development of various types of vaccines is to avoid the ADE.First stain of DENV had been isolated more than sixty years ago, but there are not any effective methods to prevent and cure the diseases caused by DENV, Which is the biggest obstacle to dengue virus infection of immune mechanism is unknown and there is not a focus to study. Dengue virus genomic structure is a single stranded positive-sense RNA molecules,11000nt, encoding three structural proteins, capsid protein (C), membrane protein (M) and envelope protein (E), and seven nonstructural proteins (non-structure protein, NS), NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The E protein contributes to viral infection through binding the surface of the target cells with the virus receptor, which causes the fusion between viral and cellular membrane. Although the nonstructural proteins are not the composition of mature virus particles, but newly researchs show that they have important function during virus infection and immune process. NS1is the main non-structural protein whichs have three forms. First is the secreted NSl (sNS1), secreted out of infected cells and contributes to the early diagnosis of DENV infection. Second is intracellular form which mainly presents in the intracellular biological membranes. The last form is membrane asociated NS1(mNS1) which existences on the infected cell membrane. The function of mNS1is not clear now, researchs show it maybe involved in viral clearance through cytotoxicity and contributed to viral RNA replication. And because the structure of NS1and the binding with antibody is unknow, so many further researchs would be performed in the futrue. Various technical analysis to predict secondary structure of protein before the crystal structrue accomplishment, such as electron cryo-microscopy, yeast display, point mutation and the peptide binding site analysis. This study will use a panel of148monoantibodies araised from NS1protein to bind the overlapping peptides of NS1and analysis the linear binding sites of mAbs. If an antibody has two binding sites in different domains, the sites would be near in physical location, which would contribute to draw the topography of DENV NS1protein. At the same time, in order to have some knowledge if our panel mAbs can binding the mNS1, we also screen all mAbs binding patterm using flow cytometry, which would produce important foundation data to explained the mNS1fuction in the immune mechanisms of DENV infection. Vaccine is the most effective way to prevent the infectious disease. So as in DENV transmittion, but DENV have four different serotypes, which would cause ADE effect in secondary infection and severe diseases would occurance. Thus the value of vaccine against DENV is not only the protection but also no ADE effect. Unfortunately, up to day, there is no a satisfied vaccine development, either a single valence or multivalent vaccince. Neutralization test is most important technology during vaccine research. The traditional method of neutralization is plaque reduction neutralization test (PRNT), but it is time-consuming, laborious and lack of objective judgment. Many researchers want to develop a method to replace it including based on flow cytometric method, the based on ELISA method. Although these methods can save time and labor, but still exist some problems, such as need more virus dosage than PRNT, don’t be available for some clinical isolation virus and have poor repeatability. Recently, researchs develop a new microneutralization to detecte neutralization of antibody beaed on enzyme-linked immunospot-based microneutralization assay (ELISPOT). Our study would evaluation of this method and establish a platform to detecte the neutralization activity of the antibodies againsts DENV and a panel of29Convalescent sera originate the patients who infected by DENV-1over three years ago.This research is divided into three parts as flowing:Part I:Draw the topography of DENV NS1protein according to analysis the binding sites of their monoantibodiesMore and more researchs about DENV infectionimmune mechanism focus on NS1protein because it can avoid the ADE. NS1has three species and the mNS1maybe induce the cy to toxic function and contribute to the viral clearance. Some researchs’ results showed that NS1protein had three regions, RI, RII and RIII, but the exact function of each domain is still not proved because the structure don’t be developed yet. Our research focues on the binding site of a panel of148mAbs to the NS1according to detection the binding between mAbs and overlapping peptides of NS1(total35peptides and every peptide overlap5aa with upper peptide except the last overlap8aa). The results show that a total of82mAbs can bind to peptides of NS1,7of82mAbs have two sites in different domain,1A11A9and2E8A5binds to NO.1peptide in RⅠ domain and NO.22peptide in RⅡ domain,6D14A4binds to NO.1and14peptide in RⅠ domain and NO.22peptide in RⅡ domain,1F32A1binds to NO.1peptide in RⅠ domain and NO.31peptide in RⅡ,7E1A4and7E9A2binds to NO.5peptide in RⅠ domain and NO.27peptide in RⅢ domain,1B7A1binds to NO.22peptide in RⅡ and NO.32peptide in RⅢ domain, which indicates that there is a close connection among three domain. We draw the secondary structure simulation diagram of DENV NS1protein according to the binding sites because the different binding sites of an antibody would be near in physical location. Some researchs indicated that antibodies to WNV NS1protein can clear infected cell through antibody dependent cell cytotoxicity, which is based on the binding mNS1antigen. We used flow cytometry to analysis the binding patterns of the panel of148mAbs. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RⅡ domain and60%(29/48) mAbs to RⅠ can bind to mNS1. Our study results are similar to the former research on the NS1protein of Flavivirus. These data would contribute to the study of NS1function and the mAbs application research.Part Ⅱ:Establish and Evaluation of a Micro-neutralization Test Based on ELISPOTPRNT is the gold standard method for neutralization test; this method is performed in6-well format and required for7-10days incubation to form visible plaques. If there are a large number of neutralization test specimens to perform, it would be quite time-consuming and labor-intensive using PRNT. Micro neutralization test is performed in96-well format and widely used in high throughput screening. This research evaluates a newly developed microneutralization test based on ELISPOT (ELISPOT-MNT). For estabilish the methodology, we used VERO E6cell as the target cell of DEVN infection and optimize the virus dosage and the incubation time after DENV infection. After the methodology being established, the neutralization activity of33mAbs, arised from the domain III of the DENV envelope protein (EDIII) against four serotype of DENV, was performed using the method. A panel of10from33mAbs was selected to evaluate this method using PRNT. The same virus stain and mAbs dilution gradient were used in two methods and compare the results. The results show that the spots are clear with the virus dosage200pfus per well of96-well plate when the infected cell incubate for2-4days. The incubation time is4days for DENV-1, DENV-2and DENV-4for2days, DENV-3for3days. The comparison of two methods show that there is a good relationship between them(r=0.863, P<0.001). McNemar’s association test was used to measure the disagreement between the two assay methods. There was no significant (P=1.000) disagreement between them.. The method showed a sensitivity of95.6%(22/23) and a specificity of88.24%(15/17) when these10MAbs were tested against four DENV serotype strains and used the PRNT as the reference. Therefore, ELISPOT-MNT shows potential as a primary method for screening neutralization activities of large numbers of antibodies.Part Ⅲ The Pattern of Neutralization in Convalescent Sera from the Patients with DENV-1InfectionThere are not any specific and efficient treatments or drugs for the diseases caused by DENV. Presently, the complaints of dengue can not be well replicated in animal model, which hamper the vaccine research. We can learn some about the antivital mechanism from the immune status of human body after naturally infected with DENY We used the ELISPOT-MNT to detecte the neutralization activity to four DENV serotypes of29sera from the patients infected with DENV-1in2006and compared the results. The results showed that most of sera have cross-neutralization activity to four serotype DENVs. The mean values(IC50, the recip of serum dilution) of DENV-1and DENV-2,-3and-4were analysised by kruska-Wallis H test with SPSS13.0, the result showed that there is significant difference among four DENV serotypes (X2=21.134, P<0.001), the value of DENV-1is the highest and DENV-4is the lowest. Our study indicates that there is cross-neutralization activity to four serotype DENVs in patient primary infection of one serotype DENV, but the strength to heterotype is lower than homeotype. The weak cross neutralization antibodies maybe contribute to the ADE effect when the second infection of different serotype DENV occurrence. Summarization:1, We sythesize35overlapping peptides of DENV-1NS1that be used to bind to a panel of148mAbs against NS1and anslysis the binding site. There are seven mAbs binding to different domain of NS1, which is the base to draw a secondary structure simulation diagram of DENV NS1protein. The results are similar to to the former research on the NS1protein of Flavivirus. This research would contribute to learning the profile of NS1protein before the crystal constructure developed. We also have screened the binding mode to mNS1of148mAbs and obtained the profile of them using flow cytometric. The results show that92,87,75and91mAbs can bind to mNS1of hemeotype DENV-1, DENV-2, DENV-3and DENV-4respectively infection cells. And82%(18/22) mAbs to RII domain and60%(29/48) mAbs to RI can bind to mNS1. 2, The microneutralization test based on the ELISPOT was developed to detecte the neutralization activity of antibody or immune sera against DENV. It is a time-saving and high-throughput method, which would be the support of the virus nerutralization research. We also evaluated this method compared with PRNT. The results show that it has high specificity(95.6%), high sensitivity(88.24%), good correlation with PRNT(r=0.863, P<0.001) and more objective judgement of result than PRNT. ELISPOT-MNT is availabe for batch processing.3, We obtain the neutralization titer profiles against homeotypic and heterotypic DENV of29convalescent sera from patients with DENV-1infection three years ago.The statistical analysis indicates that the neutralization activity against homeotypic virus is far stronger than heterotypic virus, which verifies the theory that antibodies arise primary infection can strongly protecte the challenge of hemeotypic virus but weak to heterotypic virus.