| Dengue virus (DENV), a member of the Flavivirus genus of the Flaviviridae family. It was principally transmitted to humans by the bite of infected Aedes mosquitoes. There are four antigenically distinct serotypes of DENV (DENV-1, DENV-2, DENV-3and DENV-4), infection with one serotype can cause a broad spectrum of clinical symptoms ranging from asymptomatic infection or mild dengue fever (DF) to severelly fatal dengue hemorrhagic fever (DHF) and dengue shock symdrome (DSS). Dengue fever is endemic mainly in tropical and subtropical regions, almost two-fifths of the world’s population is at risk of dengue infection, In attribute to the increment of international exchange, greenhouse effect, urbanization, the endemic area is widen to more than100countries. The WHO reports that the sharp increase of reported cases of infection after2000has been maintained at an annual level of100million. The life threatening DHF/DSS cases caused22,000deaths every500,000to1million, mainly in children. Dengue virus now becomes to be a global concern of the public health.Although the DENV has been isolated more than60years, To date, no safe and effective vaccine or specific therapy is available, mostly attribute to the ambiguous knowledge of the pathogenic mechanism of DHF/DSS. Primary infection with anyone of the four serotypes produces cross-neutralizing antibody against all four serotypes. However, the cross-protection is usually last for a few months and wanes afterwards. Unfortunately, the secondary infection of a heterologous DENV, these cross-reactive or sub-neutralizing antibodies can promote the intake of the heterologous DENV into target cells, which is supposed to be antibody-dependent enhancement (ADE). ADE is indicated to be major risk of the development of DHF/DSS.DENV is an enveloped icosahedral virus with a single-stranded, positive-sense RNA genome. The diameter of virion is～50nm,10.7-kb genome encodes three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The E glycoprotein is dynamic protein, which is responsible for receptor attachment, entry, viral fusion, and viral assembly during the virus life cycle, and thus, required to rearrangement on the virus particle to acquire several distinct conformations. E protein is the major surface-exposed protein of the DENV virion, and the neutralizing antibodies immunity to the virus is mediated primarily by E protein. The E protein is composed of three distinct domains, domain Ⅰ (ED Ⅰ) is located in the centre, which is a eight-stranded β-barrel, participates in conformational changes required for viral entry and nucleocapsid escape from the endosomal compartment. Domain Ⅱ (ED Ⅱ) is the site of dimerization and contains a highly conserved fusion loop relevant to membrane fusion. Domain Ⅲ (EDⅢ) has an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites. All the three domains can elicit antibody response to DENV, however, antibodies recognized ED Ⅰ are predominately non-neutralizing; the immunodominant epitopes surrounding ED Ⅱ fusion peptide raise broadly cross-reactive antibodies among the flaviviruses, lacks the corresponding cross-neutralizing activity; many of the most potent monoclonal antibodies are virus-type specific and bind epitopes within EDⅢ.The E proteins of the four distinct serotypes are72%～80%identical at the amino acid level. In addition to the well characterized type-specific epitopes, EDⅢ also contains sub-type and complex cross-reactive neutralizing antibodies, thus, dengue vaccine target to EDⅢ may display a broadly anti-virus effect as stimulating neutralizing antibodies against all four serotypes. Due to the lack of a approved dengue vaccine or antiviral therapy, protection against DENV and other related flaviviruses is mainly associated with the development of neutralizing antibodies. The studies of murine antibodies in vivo indicated that EDⅢ neutralizing antibodies is strongly protective both in prophylactic vaccination and post-exposure treatment. Administration of the strongly neutralizing EDⅢ antibodies rapidly reduces the virus load, which is important to control the disease. EDⅢ protein and EDⅢ antibodies with strong neutralization activity is significant in antiviral research, provide useful tool to the study of broadly cross-reactive epitopes, development of new dengue vaccine and antiviral therapy.The definitude of interaction between dengue virus and antibodies is crucial to clear pathopoiesis and protection immunity of DENV. Currently, most of the knowledge to antiviral antibody is based on murine monoclonal antibodies. It is demonstrated that the most potent neutralizing antibody recognise epitopes in ED Ⅲ,and the epitope is well characterized, which is located in A strand and lateral ridge. Although previous study reported that the neutralization is closely correlate to the level of EDⅢ antibody in human immune serum, recently, researches focus on natural infection found human immune response fo DENV is dominated by cross-reactive antibodies, EDⅢ specific antibody occupied only a small portion of human immune serum. EDⅢ-depleted human immune serum retained the potent protection against homologous DENV infection, the neutralizing amtibodies may target other epitopes outside EDⅢ. The significance of EDⅢ specific antibodies in natural antiviral immunity is controversial. Clarification of neutralizing antibody response and the protective efficacy of EDⅢ has important implication in anti-DENV researches, which needs further validation in a larger number of clinical samples.The research is divided into two parts as follow:Part Ⅰ The neutralizing activity of an monoclonal antibody against envelope protein domain Ⅲ and analysis of the neutralization mechanismEnvelope (E) protein, the major structure protein of DENV, is compose of three distinct structural domain (ED Ⅰ, ED Ⅱ and EDⅢ). EDⅢ participates in cellar receptor attachment, which elicits the most potent neutralizing antibody. Thus, EDⅢ plays an crucial role in the study of pathogenesis and specific therapy of DENV and becomes the target of dengue DNA or recombined protein vaccine. A batch of EDⅢ-reactive monoclonal antibodies (mAbs) were prepared in the our previous study, using recombinant DENV1～4EDⅢ separately or mixed together to immunize BALB/c mice. The reactivity of each antibody was determined using indirect immunofluorescence assay (IFA) on C6/36cells infected with DENV and indirect ELISA of EDⅢ using recombinant EDⅢ protein from the four DENV serotypes. One of the monoclonal antibodies cross-react with all four serotypes was screened, To identify if the cross-reactive mAb2D73possess the cross-neutralization against all four serotypes, Enzyme-linked immunospot micro-neutralizing test (ELISPOT-MNT) was performed to identify its neutralizing activities in vitro, the data showed mAb2D73displayed strong neutralizing activities against DENV1～4with50%inhibitory concentration(IC50) of0.28,0.16,0.18and18.82μg/ml, respectively. A stringent intracranial challenge one-day old suckling mice model was established to evaluated the protective capacity of mAb2D73against lethal DENV1-4infection. The2D73treatment showed protection to DENV1～4in a dose-dependent manner, as the increased dose of antibody the onset of pathogenesis was delayed and the survival rate was improved obviously (P<0.05), the protective efficacy is correlated with the neutralization activity in vitro. EDⅢ has been argued to contain a cell surface receptor recognition site, antibodies raise against EDⅢ may play an important role in blocking the binding between viruses and target cells. To define the neutralization mechanism mediated by mAb2D73, pre-and post-adsorption assay were employed. Infection was effectively inhibited in pre-adsorption assay, while the inhibition was less effective in post-adsorption assay, which indicates that2D73mediated neutralization is mainly by blocking the virus attaches to the cellar receptor. Structural analysis of crystallization of complexes between EDⅢ and Fab fragments from2D73reveals the epitope recognized by2D73is highly conserved between the DENVs (a job by another teammate). The data suggest that neutralizing antibody recognizes epitope highly conserved on EDⅢ among all4DENV serotypes has important implications in the development of dengue vaccine.Part Ⅱ The neutralization pattern of convalescent serum from patients primary infected DENV-1and the relation with the titer of EDⅢ specific antibodyA clear understanding of the characteristic of neutralizing antibodies response in human DENV immune serum plays an important role in the validation of dengue vaccine, the epidemiological survey and dengue fever diagnosis. Currently, most study on neutralizing antibody are baesd on murine mAbs. To define the specificity of neutralizing antibodies raise in natural DENV infection and the relationship between antibody mediated neutralization and the titer of EDⅢ specific antibody, a panel of convalescent sera samples from the patients infected with DENV-13years ago was collected. Enzyme-linked immunospot micro-neutralizing test (ELISPOT-MNT) was performed to identify the neutralizing activities to four DENV serotypes of each serum and a reliable double antigen sandwich ELISA was established to detect the EDⅢ specific antibodies, which used recombinant EDⅢ as double antigen expressed in pichia pastoris. Analyse the relation between neutralization activity to DENV-1and the titer of EDⅢ type-specific antibodies. Our experiment showed neutralization activity of DENV-1convalescent sera was the most potent against DENV-1among four serotypes, which indicated the neutralizing activity of heterologous antibody declined over time, whereas the neutralizing activity of homologous antibody enhanced, and to be the dominant neutralizing antibody in convalescent phase. The titers of30sera samples varied from1:80to below the cut-off value. The neutralization against DENV-1and the titer of EDⅢ specific antibody were uncorrelated (R=0.194, P=0.305), which is consistent with the theory that type-specific EDⅢ antibodies are not the dominant antibodies mediated neutralization of homologous DENV.Summarization:In summary, three major findings and conclusions were achieved, which were depicted as follows:1. A murine monoclonal antibody targeted to EDⅢ was obtained, which is cross-reactive with all four DENV serotypes. The mAb displayed potent neutralization against DENV1～4in vitro, as well as a corresponding protective capacity in vivo. Infection was efficiently inhibited by the mAb in early step of the virus life cycle, mainly by blocking the attachment between the virus and target cell. The mAb may provide useful tool to the study of broadly cross-reactive epitopes, development of new dengue vaccine and antiviral therapy.2. We established a double antigen sandwich ELISA method specific for detecting the EDⅢ reacting antibody. For the recombinant EDⅢs antigen were expressed in the yeast expression system, the recombinant protein was well folded and it was believed to be closed to the native conformation. Thus, using the recombinant DENV-1EDⅢ with and without HRP label as double antigen could be more specific and reliable to detect the EDⅢ specific antibody in human immune serum from natural infection.3. We obtained the neutralization titer profiles against homologous and heterologous dengue viruses of30convalescent sera samples from the patients primary infected with DENV-13years ago. Statistical analysis of the data indicates that the neutralization activity is more potent to homologous serotype, whereas, those to heterologous serotypes are far weaker, which supports the theory that primary infection DENV will elicit life-long neutralizing antibody against the homologous serotype. However, we also verified the potent neutralization activity to DENV-1is unrelated to the level of type-specific EDⅢ antibody. |
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