Function And Structure Analysis Of Neutralizing Antibodies Against Envelope Protein E Domain Ⅲ Of Dengue Virus

Dengue virus (DENV) is a member of the family Flaviviridae, genus Flavivirus, which is closely related to other important human pathogens such as yellow fever (YF), West Nile (WN), Japanese encephalitis (JE) and tick-borne encephalitis (TBE). There are four circulating serotypes of dengue (DENV1, DENV2, DENV3, DENV4) that share approximately60-70%sequence identity. Human infection with each serotype of DENV may result in a spectrum of clinical disease, ranging from an acute, debilitating, self-limited febrile illness (DF) to a life-threatening hemorrhagic (DHF) and capillary leak syndrome (DSS). Worldwide, an estimated50million infections with dengue occur annually, with approximately500,000cases of severe dengue and20,000deaths. Considerable research efforts for over60years have sought to develop an effective vaccine strategy, however neither effective nor antiviral treatment is approved for human use, and only supportive therapy is available. Infection results in life-long protective immunity against the serotype responsible, however risk of severe disease is increased in the case of secondary infection with a heterologous serotype in a process known as antibody dependent enhancement (ADE) of disease. ADE is thought to occur due to the presence of weakly neutralizing cross-reactive antibodies from a primary response to one serotype, which dominate the human immune response and facilitate viral entry into Fcγ-receptor-positive cells during a secondary infection with a different serotype. ADE is thought to be the major obstacle for the development of dengue vaccine. A clearer understanding of the mechanisms of antibody-mediated neutralization and the key antigenic sites the antibodies recognizewillassist in the design of new vaccine strategies and in assessing the efficacy and safety of those already in clinical trials.DENV is an enveloped virus with a single-stranded, positive-sense RNA genome that encodes three structural proteins (C, prM/M, and E) and seven non-structural proteins (NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5). The E protein is the major component of the mature virion surface and along with prM, found on immature particles, is the primary antigen targeted by the host’s antibody response. During cell entry, the conformational change in the surface-arrayed dimeric E subunits that drives virus and host-cell membrane fusion is triggered by the low pH of endosomal compartments, allowing release of the RNA genome into the cytoplasm to initiate infection. The virion surface ectodomain (sE) comprises three structural domains, a centrally-located domain I (EDI), an extended EDII containing the fusion loop (fusion peptide, FP), and EDⅢ with an immunoglobulin-like fold comprising ten β-strands (A-G and AXCXDX), which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (MAb).In our previous work, a panel of monoclonal antibodies (MAbs) raised against dengue envelope protein domain III were produced by hybridoma technique and characterized by using enzyme linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) and western blot (WB). Then the neutralizing activity of these MAbs were determined by enzyme-linked immunospot based micro-neutralization test (ELISPOT-MNT), which is established previously. In this study, two MAbs3E31and2D73that cross-react with the four serotypes of dengue and neutralize all four dengue serotypes have been characterized by further analysis of function and structure.Thus, there are three major objectives for our current study:first of all, to develop a new method for accurate titration of dengue virus; secondly, to characterize the anti-EDIII cross-reactive neutralizing MAbs by functional assays; finally, to obtain structural insight into the neutralization mechanisms of our MAbs by X-ray crystallography and thereby to lay the foundation for the development of dengue vaccine and therapeutic drugs.Thereby, this study is divided into three parts:I. Development of ELISA-based TCID50assay (TCID50-ELISA) for titrating DENVResearch on DENV is often hindered by inefficient and inaccurate or costly viral titration methods. Hence, a simple and efficient assay for accurate titration of DENV in infected cultures would greatly facilitate dengue research, vaccine development, and laboratory detection. To date, a variety of methods for titrating DENV have been developed, including classical assays, the plaque assay and the tissue culture infectious dose-50assay (TCID50), and immunofluorescence-based assays such as fluorescence-activated cell sorting (FACS) assay and fluorescent focus assay. As a standard method for titrating DENV, however, the plaque or TCID50assays have their disadvantages, as they are limited to some strains and passages of the virus, and a few cell lines. Most primary clinical isolates do not form clear plaques or have a visible cytopathic effect (CPE) on cell monolayers. Furthermore, both of these assays require manual microscope examination daily, which is time consuming and labour intensive. FACS and fluorescent focus assays can provide more rapid and accurate quantitation of DENV than the traditional plaque assay. However, each of these techniques requires experienced technicians and sophisticated laboratories, hindering its application in most laboratories lacking sophisticated equipment. Therefore, at the beginning of our research, we set to establish a new method for titrating dengue virus to overcome the disadvantages of classical methods. As we know, nonstructural protein1(NS1), a multifunctional glycoprotein in dengue virus, is highly conserved for all serotypes of DENV and is strongly immunogenic. Some of the NS1protein is expressed as a soluble secreted form, which has been implicated to contribute to dengue viral propagation and the amount secreted is closely related to dengue viral titer. In our previous study, we established a dengue NS1antigen capture enzyme-linked immunosorbent assay (ELISA). In the present study, a novel TCID50assay was developed, which employs this dengue NS1antigen capture ELISA instead of observation of CPE in the classical assay. The novel TCID50-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID50-CPE for titration of dengue virus.Ⅱ. Function analysis of cross-reactive MAbs against envelope protein domain Ⅲ of dengue virusIn our previous work, we expressed DENV1-4EDⅢ recombinant protein by using yeast system. Then, a panel of anti-EDⅢ MAbs were produced from BALB/c mice immunized with recombinant EDⅢ from DENV1-4. Furthermore, the serotype specificity and cross-reactivity of these MAbs were determined by indirect ELISA and indirect immunofluorescent assay (IFA). As the aim of this study is to develop antibodies against all four DENV serotypes,3E31and2D73were selected for further studies because they were found to neutralize the infection of all four dengue serotypes. In this study, MAb3E31and2D73were further characterized by a serial of functional assays such as surface plasmon resonance (SPR), membrane fusion inhibition assay, temperature-dependent assay and ADE assay. SPR can not only provide the affinity data (equilibrium dissociation constant, KD), but also with the association constant (Ka) and dissociation constant (Kd) of the binding of EDⅢ MAbs to EDⅢ protein, as well as the percentage activitiy, which represent the accessibility of the protein. In addition, the SPR data also provide insight into the next co-crystallization of antibody and antigen. The SPR results revealed that the affinity of two MAbs could reach nM and there is no significant difference between the binding affinities for DENV1, DENV2, DENV3and DENV4. Additionally, the data did not show any relationship with the neutralization capacity. For the fusion assay, to our surprise, while3E31inhibited fusion,2D73enhanced fusion in a dose-dependent manner. To our knowledge, this is the first time fusion enhancing antibodies have been described for any virus. Finally, the ADE activity of MAb3E31and2D73were eveluated. We have observed that3E31does not induce ADE in Fcy-receptor-expressing cells. By contrast, binding by2D73led to ADE in DENV2, DENV3and DENV4but not DENV1. On the basis of the functional analysis data, we proposed that the neutralization mechanism of MAb3E31and MAb2D73might be different.Ⅲ. Structure analysis of the neutralization mechanism of anti-EDⅢ cross-reactive MAbs using X-ray crystallography.To understand the structural basis of neutralization by our mAbs, crystal structures of the complexes between recombinant DENV4EDⅢ and Fab fragments from3E31and2D73were determined at2.2A and2.0A resolution, respectively. The crystal structures were analyzed by software and the3E31epitope and2D73epitope were finally defined. The MAbs recognize two distinct, slightly overlapping epitopes on EDⅢ. For3E31, the epitope comprises the AB loop and β-strand E. Residues that form hydrogen bonds with the Fab and are strictly conserved in DENV1-4, consistent with the neutralization assay, which showed that MAb blocked virus infection of all four serotypes. For2D73, the epitope cluster on β-strands A and β-strand G. The key epitope residues are again highly conserved. In addition, antibody:EDⅢ structures that show binding to this region have been reported with the3E31epitope being similar to that recognized by mAb2H12and the2D73epitope being similar to those described for the A-strand-specific mAbs1A1D-2and4E11. Moreover, to investigate the accessibility of these anti-EDIII mAb epitopes on native forms of the E protein, we superimposed the crystal structures of our Fab:EDⅢ complexes onto both dimeric pre-fusion and trimeric post-fusion sE structures, as well as E proteins in the context of immature and mature whole virions. The results revealed that both epitopes are partially hidden in the mature virion dimer, which raises the question of how the MAbs could bind to any of the E-DⅢ epitopes. Location of the epitope on the post-fusion trimer structure suggests that fusion inhibition observed for3E31is most likely due to a steric blockade of E trimer formation. By contrast, the availability of the2D73epitope in the post-fusion trimer suggests that it could remain bound during the conformational transition from dimer to trimer and possibly shifting the equilibrium to trimer formation and this way activate fusion. In combination with the functional data and crystal structures, we finally determine the neutralization mechanisms and reveal stark differences between the two antibodies3E31and2D73, a finding that has far reaching consequences in vaccine and therapeutic drugs development and understanding the role of antibodies in ADE.Summarization:1. Using the DENV group NS1antigen capture ELISA established previously, we developed a novel TCID50-ELISA based on the classical TCID50. With this new method, DENV1-4could be accurately titrated at day6. Virus titers obtained by TCID50-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID50-cytopathic effect (CPE) test (TCID50-CPE), with a better reproducibility. Because the observation of the CPE was replaced by determination of NS1protein, which can eliminate subjective variations between operators and laboratories. Moreover, this TCID50-ELISA assay overcomes the flaws inherent in the plaque assay and the TCID50-CPE assay. For instance, the TCID50-ELISA assay showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. Finally, the TCID50-ELISA assay has been used successfully for clinically DENV isolates, which might not be assessable by other titration methods. Based on its reliability and ease of execution, the TCID50-ELISA test represents a promising assay for titration of DENV, and will facilitate dengue research.2. In this study, some functional approaches were carried out to characterize two cross-reactive neutralizing MAbs raised against DENV EDIII. Both MAbs could block the infection of all four dengue serotypes. Additionally, the affinity and neutralization capacity against dengue viral particle are temperature sensitive. Furthermore, the activity of3E31and2D73in membrane fusion and ADE are totally different, indicating that the neutralization mechanism of them might be different. We found that2D73enhanced the membrane fusion, to our knowledge this is the first time fusion enhancing antibodies have been described for any virus. This new finding provides insight into the functional study of anti-viral neutralization antibodies. Finally, MAb2D73showed no ADE activity, highlighting its potential as immunotherapeutics.3. To understand the structural basis of neutralization by3E31and2D73, crystal structures of the complexes between recombinant DENV4EDIII and Fab fragments from3E31and2D73were determined by crystallography technique. The crystals were diffracted by X-ray diffraction and then two data sets with high resolution were collected. According to the solved3D crystal structures, the epitopes recognized by MAb3E31and2D73were defined accurately. Moreover, we superimposed the crystal structures of our Fab:EDIII complexes onto both dimeric pre-fusion and trimeric post-fusion sE structures, as well as E proteins in the context of immature and mature whole virions to investigate the accessibility of these anti-EDIII mAb epitopes on native forms of the E protein. In conculsion, the results of our combined structural and functional studies uncover two distinct mechanisms of neutralization used by our MAbs and possibly other recently reported MAbs targeting EDIII, and may further people’s understanding of cross-neutralization as well as the structural basis for ADE, critical for the design of effective vaccines in the future.