Identification And Functional Analysis Of Neutralizing Epitope On The Envelope Protein Of Dengue 2 Virus

In three structural proteins of dengue viruses, the E glycoprotein is the major structure protein on the DENV virion. It has three distinct domain (domains I, II and III) in the N-terminal 400 amino acids of the E-protein and plays a central role in viral assembly, receptor attachment, entry, and viral fusion during the flavivirus life cycle. E protein also is the principal protective antigen that induces neutralizing antibodies to elicit protective immune response, thereby is crucial for control of severe DENV infection. However, subtle antigenic structure and function of the E protein, especially region outside of domain III still remain poorly understood. Variation on antibody binding site between different and identical DENV strains and their involvement in viral tissue tropism, replication efficacy and pathognesis need to be more investigated. In addition, only a relatively few epitope that relate with ADE have been characterized. These unresolved questions severely impede the elucidating the mechanism of viral pathogensis and inmmune. Therefore, determining neutralizing and ADE epitopes of DENV E protein, elucidating the mechanism of antibody-mediated neutralization and enhancement, and further investigating some key amino acids related with function of E protein, not only are first step to understand mechanism of pathogenesis and immune, but give important information for specific control and treatment.In this study, using a panel of anti-DENV2 E monoclonal antibodies, we analyzed the neutralizing epitopes of DENV E protein, identified key amino acids which antibody recognizes and mechanism of antibody-mediated neutralization, and initially investigated influence of amino acid variance to viral biology characteristics.1. Establishment of anti-DENV2 E protein mAbsIn this study, we employed two different methods, heat-inactivated virus or plasmid DNA encoding the DENV2 prME proteins to produce and deliver DENV2 immunogens. Eventually, we generated a panel of mAbs (2B8, 2H11, 2D7, 2F2, 2F10, 2A10G6, 2B4, 2D5, 4C10, and 6B4) against DENV2 E protein.To determine which viral surface glycoprotein these Mabs recognized, we examined the abilities of Mabs to react the DENV2 prME protein expressed in eukaryotic systems by using indirect immunofluorescence assays (IFA). As expected, all mAbs recognized an intracellular reticular pattern in BHK21 cells transfected with the recombinant plasmid expressing the prME protein, suggesting that these mAbs are directed against the DENV2 prME protein. Subsequently, we next determined if the mAbs recognized the recombinant DENV2 DI-DII or DIII protein by ELISA and western blot techniques. 2A10G6 and 2F10 react with the DENV2 DI-DII protein, suggesting that two mAbs may recognize epitopes within of DI-DII protein. Another eight mAbs reacted with the DIII protein, suggesting that these mAbs are directed against DIII. To further determine cross-reactivities of mAbs with dengue virus other serotypes and distantly related ?aviviruses, we infected BHK21 cells with the specific viruses and stained with mAbs by using IFA. The result indicated that four mAbs (2B8, 2H11, 2D7, and 2F2) against DIII protein were DENV2-specific antibodies. Another six mAbs cross-reacted with DENV1–4, TBEV, JEV, WNV and YFV were broadly flavivirus cross-reactive antibodies. These results are helpful for study on the specific epitopes of envelope glycoprotein and for developing novel diagnosis reagent of dengue.2. The molecular basis of antibody-mediated neutralization of DENV2We observed neutralizing and protective activities of anti-DENV2 E protein mAbs by using a standard plaque reduction neutralization assay and an established mouse model. These results indicated that we obtained three neutralizing Mabs (2A10G6, 2F10, and 2B8), and DI-II-specific mAb 2A10G6 has more potent neutralization and protection against DENV2 than DIII-specific mAb 2B8, 2A10G6 also could afford 66.7% and 40% protection of mice at 4 and 24h after challenge with DENV2. Meanwhile, 2A10G6 potently neutralized DENV1, DENV3, DENV4, YFV and WNV, suggesting 2A10G6 is a more broad-spectrum flavivirus cross-neutralizing mAb in comparison with other reported mAbs.To further define the molecular basis of antibody-mediated neutralization of flavivirus infection, we tested on where mAb could inhibit DENV2 infection during virus life cycle by using real-time RT-PCR and plaque assay. Importantly, neutralizing DIII-specific mAb 2B8 inhibited virus binding by 3.1-fold, whereas DI-II-specific Mab 2A10G6 potently neutralizes infection by blocking membrane fusion after cellular attachment. These results indicated that those two neutralizing antibodies inhibit DENV2 infection at different step of virus life cycle by binding different epitopes on the E protein. On the other hand, we also evaluated whether subneutralizing concentrations of 2A10G6 enhance infection in macrophages and found that 2A10G6 augment DENV1-4 infection on K562 cells beyond 5 to 10 fold.3. Identification of neutralizing epitopes of DENV2 E proteinTo map the epitope determinants of 2A10G6, a standard phage-display technique was performed. The result found that 2A10G6 specifically recognizes the 98DRXW101 motif on the fusion loop peptide of DENV2 E protein. Then the reactivity of 2A10G6 to the synthetic peptide corresponding to the amino acids of the DENV2 E protein was determined by ELISA. The results also showed that this region containing the three common residues constitutes a mimotope of the 2A10G6 epitope. We compared this peptide with the amino acid sequences of E proteins of DENV1–4, YFV, WNV, JEV and TBEV and found that all flavivirus strains shared the same amino acids at positions 98 (D), 99 (R), and 101 (W) within the highly conserved N-terminal fusion loop peptide of the E protein DII. Furthermore, three-dimensional structural analysis showed that the 98DRXW101 motif was located at the tip of the fusion loop of the DENV2 E protein. The structure of the 2A10G6 epitope was similar in the three known E protein structures.On the base of neutralizing epitope of 2A10G6 were identified by the phage display technique, to further determine whether D98, R99, and W101 are critical epitope residues on mAb 2A10G6 binding, site-directed mutagenesis of the E protein was used to investigate the specific amino acid residues important for binding of antibodies. Western blot assay showed that binding of D98R or R99A mutant alone with mAb 2A10G6 was no significant difference compared with wild type DENV E protein. However, W101R mutation had a strong effect on MAb binding, suggesting W101 were considered to be critical epitope residues, which was identical with the function of W101 on membrane fusion. Furthermore, D98R and R99A combinant mutation also significantly reduced mAb binding, this would imply that residues D98 and R99 are not critical for the interaction of these mAbs with E protein but are still likely to be located on the periphery of this epitope. In addition, we have made use of 2A10G6 to select neutralization-escape mutants in C6/36 cells and purified an escape mutant in BHK21 cells. Enventually, we obtained a mutant that can be neutralization resistant to the selecting mAb 2A10G6. Sequence analysis of the escape mutant E protein genes defined a single nucleotide substitution of A to G at position 376, resulting in the change of Glu to Lys at amino acid position 126, suggesting E126 is also an animo acid residue that 2A10G6 recognizes, which can constitutes the 2A10G6 epitope with D98,R99 and W101 during virus life cycle. Meanwhile, the growth rates of the escape mutant was similar to parent DENV2, but exhibited a significantly reduced neurovirulent, indicating that E126 is also an important virulence-related residue.4. Construction of chimeric antibody and its biological functionsTo analysis antibody Fc-mediated effector function and lay a good foundation for its further humanization into clinical therapy, we firstly amplified and sequenced the genes encoding VH and VL of 2A10G6 were by 5'RACE assay, we also predicted the Framework (FR) and Complementarity determining region (CDR). Our results indicated that three CDR regions of heavy chain were EYTMH, GIDPNNGGTNYNQKFKG, and RDYYALDY; these of light chain were KASQHVGSAVA, SASNRYT, and QQYNSYPT. To construct the chimeric antibody light and heavy chain expression vectors, the cloned antibody light and heavy chain variable region cDNAs (VH) were respectively fused to the human antibody light and heavy chain constant region genes (CH). The chimeric light and heavy chain genes were respectively inserted into the expression vectors, resulting in the chimeric light and heavy chain expression vectors. Then appropriate light and heavy chain expression vector was co-transfected into CHO cells and the clones producing the highest amount of chimeric antibody were selected. Indirect ELISA and IFA assays demonstrated that chimeric antibody was shown to bind well to dengue virus antigen, suggesting that the chimeric antibody was correctly constructed and produced. In neutralizing activity in vitro, 2A10G6 could effectively block with dengue virus infection on BHK21 cells, which indicated that the chimeric antibody possessed affinity and specificity similar to that of the original murine antibody.In summary, these findings provide the theoretical foundation and the microbiological tools for further analyzing structure and function of DENV E protein, improving flaviviral serodiagnosis as well as developing novel ?avivirus vaccines and antiviral therapeutic drugs.