Construction And Charaterization Of A Broadly Neutralizing Antibody Against Four Dengue Virus Serotypes Without Antibody-Dependent Enhancement

Dengue viruses (DENV) are composed of four genetically and antigenically related viruses termed DENV1, DENV2, DENV3 and DENV4. Dengue is the most important arbovirus disease in tropical and subtropical countries. Clinical symptoms range from self-limited, acute, febrile disease called dengue fever (DF) to severe dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Over 2.5 billion people in the world are at risk of contracting dengue, and 50-100 million people develop symptomatic infections of which up to 500,000 develop DHF/DSS each year. However, little is known about the mechanisms of DHF/DSS and no efficient preventative and therapeutic drugs are available now. Antibody-based therapy is an important way for treatment of virus diseases. With thegreat progress obtained in the last few years, therapeutic antibodies have cought more and more attentions now. Compared with other therapeutics, mAbs are more effective, more specific with less side effects, and have the ability of elimiting viruses by CDC and ADCC. Previous studies have shown that neutralizing mAbs could show therapeutic effects both before and after virus infection.DENV have a relatively simple enveloped virion which is 50 nm in diameter and consist of three structural proteins (a capsid protein C, a membrane protein M, and a major envelope glycoprotein E) and seven non-structural proteins(NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Of the three structural proteins, E glycoprotein is the major structural protein on the DENV virion. It has three distinct domains designated domain I, domain II, and domain III (ED1,ED2 and ED3) and plays a critical role in viral assembly, receptor attachment, entry and viral fusion in the flavivirus life cycle. It is also the most important protective antigen inducing neutralizing antibodies against DENV. ED1 is a central, eight strandedβ-barrel, which contains a single N-linked glycan in most DENV strains. ED2 is a long, finger-like protrusion from ED1 with a highly conserved fusion peptide (residues 98–110) at its distal end and mediates post-entry endosomal fusion; it contains the major flavivirus group and subgroup cross-reactive epitopes. ED3 adopts an immunoglobulin-like fold and is characteristic of many cell receptors. In addition, ED3 contains the critical and dominant virus subcomplex and type-specific neutralization sites. ED3 protein could block adsorption of dengue virus competitively, and mAbs that target ED3 are the most potent neutralizing antibodies. ED3 is a potential protein target both in vaccine development and antibody-based therapy.However, relative few neutralizing eipitopes have been fine mapped yet and the relationshiop between amino acids variations among and between different DENV serotypes and neutralizing antibody efficacies need to be further investigated. Mechanisms of antibody neutralization for DENV still remain poorly understood. All these problems have prevented the development of effective vaccines and therapeutic mAbs. A more thorough analysis of DENV ED3 neutralizing epitopes will provide a better understanding of the molecular mechanism of DENV neutralization and aid in the development of candidate DENV vaccines and antibody therapy.Antibody-dependent enhancement (ADE) has been found to be an underlying pathogenic mechanism of DHF/DSS. Epidemiological data suggests that a subsequent infection with a different DENV serotype is more frequently associated with severe dengue illness and DHF occurs during infection with a second serotype. This observation has led to the hypothesis of antibody-dependant enhancement (ADE) contributing to dengue severity. It's presumed that preexisting cross-reactive and subneutralizing antibodies form complexes with the infecting DENV which could facilitate the binding to Fc receptor-bearing cells, leading to increased virus infection. This phenomenon has greatly hampered the development of dengue vaccines and the application of antibody therapy. Concurrent or sequential infections with different DENV serotypes are common, and an effective neutralizing antibody should possess the ability to neutralize four DENV serotypes. There are also some cross-neutralizing mAbs, but its neutralizing titres are variable between different DENV serotypes, and the risk of ADE still exists. One possible strategy is to develop several mAbs as combination therapies, which is costly and subject to safety considerations. The technology of dual-variable-domain immunoglobulin (DVD-Ig) could construct a novel antibody molecule engineered from two mAbs of distinct specificities and efficacies. Furthermore, we could eliminate the ADE effect by introducing Fc mutations which could abrogate the binding of Fc and Fc receptors. In the case of DENV, we could obtain a broadly neutralizing DVD-Ig by combining two potent neutralizing mAbs that could only neutralize several serotypes each.In this study, we intended to obtain neutralizing mAbs against DENV using hybridoma technology, analyze neutralizing epitopes and identify identical amino acids. Finally, we would construct chimeric antibodies and broadly neutralizing antibodies against four DENV serotypes. This work contains four individual parts: 1. Generation and characterization of DENV ED3-specific mAbsIn this study, we employed the method of immuning mice with tandem ED3 protein to produce ED3 specific mAbs against four DENV. Finally, we generated 14 mAbs (1B12, 1E12, 1G6, 1H8, 2F9, 2G9, 2H12, 3A10, 3H12, 4H10, 5C10, 6E1, 6H7 and 7G5) to aid in our development of serologic diagnostic reagents and neutralizing antibodies.To determine whether these mAbs that reacted with recombinant ED3 protein would certainly bind with whole dengue virus, we examined the abilities of mAbs to react with the DENV-infected cells by indirect immunofluorescence assays (IFA) analysis. As expected, all mAbs recognized BHK21 cells infected with DENV. Subsequently, we determined if the mAbs cross-react with other flavivirus, and the results showed that none of these mAbs cross-reacted with other flaviviruses including WNV, JEV, YFV and TBEV by IFA, suggesting that all these mAbs are DENV-specific. Of these mAbs, 1B12, 1E12, 2H12, 3A10, 5C10, 6H7 and 7G5 are DENV1 type-specific; 1H8 and 2G9 are DENV3 type-specific; 1G6 is DENV4 type-specific; 2F9 cross-reacts with DENV1 and DENV3; 3H12 and 4H10 cross-react with DENV1-3; 6E1 could cross-react with four dengue serotypes. All these mAbs reacted with the expressed respective recombinant ED3 protein, suggesting that they are directed against ED3. These mAbs might be helpful for both studying epitopes of ED3 protein and developing novel diagnosis reagent of dengue.The neutralizing ability of mAbs were then determined by a standard plaque reduction neutralization test (PRNT) in vitro. The results indicated that a DENV4-specific antibody 1G6 has potent neutralizing ability in vitro. In an established suckling mouse model, at the concentration of 100μg/ml, mAb 1G6 could protect 60% suckling mive from lethal virus challenge. In the sukling mice therapeutic model, a single 50μg dose of mAb could afford 90% and 30% protection at 4h and 24h after infection, indicating that it had therapeutic effect and could be a potential candidate for antibody-based therapeutics. In pre- and post-adsorption assays, mAb 1G6 neutralized DENV4 infection primarilly through blocking the step of viral adsorption.2. Screening and identification of neutralizing epitopesTo map the epitope of mAb 1G6, a standard phage-display technique was firstly carried out. Through analysis of phage-displayed peptide and sequence alignment, we found that mAb 1G6 specifically recognizes the 387LTLH390 motif on the DENV4 envelope protein. Furthermore, the dependency of the phage-displayed epitope was confirmed by sequential truncations of the DENV4 rED3 from the C termini by 5 aa intervals. To further identify the critical residues involved in the epitope recognized by mAb 1G6, a total of 5 single amino acid substitutions covering 386-390aa and a combination of substitutions (T388 and H390) were introduced by site-directed mutagenesis, based on the above results and the crystallization of DENV4 ED3. The results showed that when amino acids T388 and H390 were substituted by glacine, the affinity were reduced by 11.7 and 10.5 times, respectively, while the L389G mutation resulted in only 7.7 times reduction of affinity. These results indicated that T388 and H390 were critical residues and L389 was a peripheral residue for the epitope recognized by mAb 1G6. However, when the amino acids T388 and H390 were mutated simutaneously, the binding of mAb 1G6 was abrogated, indicating that T388 and H390 had some synergistic effect.In the result of DENV1-4 ED3 amino acid sequences alignment, at the mAb 1G6 epitope region, although residues L387 and L389 are highly conserved among four DENV serotypes, the charges of critical residues T388 and H390 are unique compared with the corresponding residues of other three serotypes, which is consistent with the inability of mAb 1G6 to bind DENV1-3. MAb 1G6 might interact primarily with residues in the surface-exposed loops of ED3 protein, and the binding and specificity of its epitope might be associated with variations in charge of the surface of ED3 protein.In 3-D structures, the mAb 1G6 epitope region is exposed completely on the surface of DENV4 ED3 and constitute a linear epitope. The overlay result of crystal structures of DENV1-4 ED3 showed that the backbones of ED3 are highly similar among four DENV serotypes; however, the critical residue H390, with an imidazole ring exposed, has the most different side chain from its neighboring residues. The result indicated that besides variations in charge, the subtle structure differences might also play an important role.3. Construction of chimeric antibodies and characterization of its biological functionsTo reduce the antigenicity of mouse antibody and lay a good foundation for further construction of DVD-Ig, we firstly amplified and sequenced the genes encoding variable regions of both light chain and heavy chain of 1G6 by 5'RACE system,and then synthesised the sequences of genes encoding both light and chain variable regions of a cross-neutralizing antibody 1A1D-2. The cloned variable genes encoding both light chains and heavy chains of the two antibodies were fused with human antibody light and heavy chain constant region genes, respectively. The genes encoding the whole chimeric light chain and heavy chain were inserted into pcDNA3.1(+) expression vector. Then the light chain and heavy chain expression vectors were co-transfected into CHO cells, and the clones producing the highest amount of chimeric antibodies were selected. Indirect ELISA and IFA showed that the chimeric antibodies had similar affinity with parental antibodies and bound well to inactivaed whole dengue virus, and plaque reduction neutralizaiton test (PRNT) also showed similar neutralizing activities, suggesting that the chimeric antibodies were successfully constructed and secreted.4. Construction of a DVD-Ig without ADE and characterization of its biological functionsThe VH and VL of mAb 1A1D-2 and 1G6 were genetically fused by overlap PCR technology in orientations of either 1A1D-1G6 or 1G6-1A1D by nine amino acids linkers from N-terminus of heavy chain and light chain constant regions of human IgG 1 respectively. The constructed 1A1D-1G6-VH, 1A1D-1G6-VL or 1G6-1A1D-VH, 1G6-1A1D-VL were connected with constant regions of human IgG 1 and then inserted into the expression vector pcDNA3.1(+).CHO cells were then co-transfected with heavy chain and light chain expression vectors using lipofectamine 2000 reagent. The cell clones with higher expression levels were selected and expended in serum-free medium. The DVD-Ig was purified from the culture medium by proteinA affinity column.The resulting DVD1A1D-1G6 was demonstrated to retain better affinity with ED3 proteins of four dengue virus serotypes than DVD1G6-1A1D. DVD1A1D-1G6 was able to bind and neutralize four dengue virus serotypes both in vitro and in vivo with the same neutralizing titres with the two chimeric antibodies. After introducing the deletion of nine amino acids which mediate Fc-FcR interaction, ADE was abrogated in vitro while did not show any influence on its neutralizing ability.In summary, this study generated a new DENV4 type-specific neutralizing antibody and mapped a novel neutralizing epitope, these findings help to elucidate the relationship between structure and functions of envelope protein, and provide theoretical foundations for further investigation of pathogenesis of DENV. The construction of DVD-Ig identify a novel strategy for the design of broadly neutralizing antbodies against DENV. The chimeric mAbs and the DVD-1A1D-1G6 without ADE might be potential candidates for anti-DENV therapeutics.