Recombinant dengue type 2 viruses with altered e protein domain III epitopes are efficiently neutralized by human immune sera

Humans develop polyclonal, serotype-specific neutralizing antibody responses after dengue virus (DENV) infection. Many mouse antibodies that neutralize DENV bind to the lateral ridge or A strand epitopes on domain III of the viral envelope (EDIII) protein. It has been assumed that these epitopes are also the main target of human neutralizing antibodies. Using recombinant dengue serotype 2 viruses with altered EDIII epitopes, we demonstrate that EDIII epitopes are not the main target of human neutralizing antibody.     

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.     

Identification and characterization of a 43 kDa actin protein involved in the DENV-2 binding and infection of ECV304 cells

Characterization of the primary host factors associated with host–virus interaction is critical for understanding how a virus infects its host cell. In this study, a modified virus overlay protein binding assay was developed. Host factors with 34, 43, and 55 kDa proteins, which could interact with EDIII, a cell receptor-binding domain of Dengue virus (DENV)-enveloped E protein, were isolated from ECV304 cells. Mass spectrometry identified peptide masses of 43 kDa protein matched to actin, a cytoskeleton protein in eukaryotic cells. The interaction between 43 kDa actin and DENV-2 EDIII was further confirmed by competitive blocking and co-immunoprecipitation assays. Actin cytoskeleton rearrangement was observed within 1 h p.i. of DENV-2-infected ECV304 cells in the confocal immunofluorescent assay. The co-localization of DENV-2 E protein with the actin filaments occurred in the late stage of the DENV replication cycle. Finally, a docking complex was constructed, and the functional residues involved in the interaction of actin and DENV-2 EDIII protein were predicted. Our findings suggest that the direct contact of DENV E protein with 43 kDa actin protein may have a crucial function in DENV infection of ECV304 cells.     

Development of a humanized antibody with high therapeutic potential against dengue virus type 2

Background

Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.

Methodology/Principal Findings

We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.

Conclusions/Significance

We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.     

Functional Analysis of Antibodies against Dengue Virus Type 4 Reveals Strain-Dependent Epitope Exposure That Impacts Neutralization and Protection

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.     

Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8?M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124?mg of highly purified and biologically active EDIII protein was obtained from 100?g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.     

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.     

登革病毒四型联合重组包膜蛋白III区的表达和免疫原性鉴定

登革热在全球范围内广泛流行,但是目前为止却仍然没有疫苗上市,疫苗的开发迫在眉睫。抗体依赖增强感染效应是登革病毒疫苗开发中遇到的一个瓶颈问题。研究表明登革病毒的包膜蛋白III区能够介导中和抗体产生,且诱导产生较少的交叉抗体或无交叉抗体,能够大大减弱抗体依赖增强感染效应,因而是登革热重组蛋白疫苗的首选靶标。通过酵母密码子优化后合成同时包含4种血清型登革病毒包膜蛋白III区的四价联合DV EDIII蛋白序列,随后构建酵母表达质粒,并获得酵母表达菌株,经诱导后四联DV EDIII蛋白获得高效表达。通过Western blot、ELISA检测及蛋白质免疫原性鉴定,结果表明登革病毒四联DV EDIII蛋白表达质粒构建成功,重组蛋白在毕赤酵母获得高效表达,免疫小鼠后能够介导产生较高水平的血清效价。这表明已获得了能引起有效免疫反应的四型登革病毒EDIII蛋白,为登革病毒疫苗的研究提供了良好的基础。     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

Background

Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.

Methods

In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.

Results

Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.

Conclusions

The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.     

Induction of Neutralizing Antibodies against Four Serotypes of Dengue Viruses by MixBiEDIII,a Tetravalent Dengue Vaccine

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1–2 and type 3–4) of DENV connected by a Gly-Ser linker ((Gly₄Ser)₃) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.     

A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion

Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.     

High-Avidity and Potently Neutralizing Cross-Reactive Human Monoclonal Antibodies Derived from Secondary Dengue Virus Infection

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.     

Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein

Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus

The four serotypes of dengue virus (DENV-1 to -4) cause the most important emerging viral disease. Protein E, the principal viral envelope glycoprotein, mediates fusion of the viral and endosomal membranes during virus entry and is the target of neutralizing antibodies. However, the epitopes of strongly neutralizing human antibodies have not been described despite their importance to vaccine development. The chimpanzee Mab 5H2 potently neutralizes DENV-4 by binding to domain I of E. The crystal structure of Fab 5H2 bound to E from DENV-4 shows that antibody binding prevents formation of the fusogenic hairpin conformation of E, which together with in-vitro assays, demonstrates that 5H2 neutralizes by blocking membrane fusion in the endosome. Furthermore, we show that human sera from patients recovering from DENV-4 infection contain antibodies that bind to the 5H2 epitope region on domain I. This study, thus, provides new information and tools for effective vaccine design to prevent dengue disease.     

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.