EDIII Protein Research Articles

Development of Colloidal Gold-Based Immunochromatographic Strips for Rapid Detection and Surveillance of Japanese Encephalitis Virus in Dogs across Shanghai, China.

Japanese encephalitis virus (JEV) causes acute encephalitis in humans and is of major public health concern in most Asian regions. Dogs are suitable sentinels for assessing the risk of JEV infection in humans. A neutralization test (NT) or an enzyme-linked immunosorbent assay (ELISA) is used for the serological detection of JEV in dogs; however, these tests have several limitations, and, thus, a more convenient and reliable alternative test is needed. In this study, a colloidal gold immunochromatographic strip (ICS), using a purified recombinant EDIII protein, was established for the serological survey of JEV infection in dogs. The results show that the ICSs could specifically detect JEV antibodies within 10 min without cross-reactions with antibodies against other canine viruses. The test strips could detect anti-JEV in serum with dilution up to 640 times, showing high sensitivity. The coincidence rate with the NT test was higher than 96.6%. Among 586 serum samples from dogs in Shanghai examined using the ICS test, 179 (29.98%) were found to be positive for JEV antibodies, and the high seropositivity of JEV in dogs in China was significantly correlated with the season and living environment. In summary, we developed an accurate and economical ICS for the rapid detection of anti-JEV in dog serum samples with great potential for the surveillance of JEV in dogs.

Self-assembled nanoparticle with E protein domain III of DTMUV based on ferritin as carrier can induce a more comprehensive immune response and against DTMUV challenge in duck

Duck Tembusu virus (DTMUV) causes severe reduction in egg production and neurological symptoms in ducklings. Vaccination is the primary measure used to prevent DTMUV infections. In this study, self-assembled nanoparticles with the E protein domain III of DTMUV, using ferritin as a carrier (EDⅢ-RFNp), were prepared using a prokaryotic expression system. Ducks were intramuscularly vaccinated with EDⅢ-RFNp, EDⅢ protein, an inactivated vaccine HB strain (InV-HB), and PBS. At 0, 4, and 6 weeks post-primary vaccination, the EDIII protein-specific antibody titre, IL-4, and IFN-γ concentrations in serum were determined by ELISA, and neutralising antibodies titres in sera were determined by virus neutralising assay. Peripheral blood lymphocytes proliferation was determined by CCK-8 kit. Following challenge with the virulent DTMUV strain, the clinical signals and survival rate of the vaccinated ducks were recorded, and DTMUV RNA levels in the blood and tissues of the surviving ducks were determined by real-time quantitative RT-PCR. The near-spherical EDⅢ-RFNp nanoparticles with 13.29 ± 1.43 nm diameter were observed by transmission electron microscope. At 4 and 6 weeks post-primary vaccination, special and Virus neutralisation (VN) antibodies, lymphocyte proliferation (stimulator index, SI), and concentrations of IL-4 and IFN-γ in the EDⅢ-RFNp group were significantly higher than in the EDⅢ and PBS groups. In the DTMUV virulent strain challenge test, the EDⅢ-RFNp-vaccinated ducks showed milder clinical signs and higher survival rates than EDⅢ- and PBS-vaccinated ducks. The DTMUV RNA levels in the blood and tissues of EDⅢ-RFNp-vaccinated ducks were significantly lower than those in EDⅢ- and PBS-vaccinated ducks. Additionally, the EDⅢ protein-special and VN antibodies, SI value, and concentration of IL-4 and IFN-γ in the InV-HB group was significantly higher than that of the PBS group at 4 and 6 weeks post-primary vaccination. InV-HB provided more efficient protection than PBS based on a higher survival rate, milder signals, and lower levels of the DTMUV virus in the blood and tissues. These results indicated that EDⅢ-RFNp effectively protected ducks against DTMUV challenge and could be a vaccine candidate to prevent DTMUV infection.

Tetravalent formulation of polymeric nanoparticle-based vaccine induces a potent immune response against dengue virus.

Dengue is a mosquito-borne disease caused by the four serotypes of the dengue virus (DENV 1-4). It is growing at an alarming rate globally, which could be partly attributed to the lack of an effective therapeutic regimen. Therefore, strategies for developing an effective vaccine have gained more significance in the given scenario. Failure of the existing live attenuated vaccine candidates to mount effective and broader protection against all the four serotypes of DENV has generated a new interest in exploring novel strategies for augmenting the efficacy of non-infectious, non-replicating subunit vaccines. In the current study, we employed a new strategy of encapsulating the immunodominant EDIII domain of Envelop protein of all the serotypes of DENV (1-4) into PLGA nanoparticles separately. All four nano formulations were physically mixed to develop a tetravalent nano formulation in combination with TLR agonists. Further, we examined its immunological efficacy using a mouse and in vitro infection model system. Interestingly, our results demonstrate that majority of EDIII protein loaded PLGA nanoparticles were polydispersed and less than 1 μm in size with optimal encapsulation efficacy. Tetravalent nanoformulation along with TLR agonists (MPLA + R837) enhanced the magnitude of antigen-specific polyfunctional T cell response. It triggered robust antibody responses in mice concurrent with the increased level of genes involved in the programming of memory B-cell formation and the maintenance and maturation of GCs, leading to the formation of long-lived plasma cells secreting antigen-specific antibodies. Further assessment revealed that tetravalent nanoformulation in combination with TLR ligands upon immunization in mice aids in the enhanced production of serotype-specific neutralizing antibodies, which can effectively neutralize all the four serotypes of DENV (DENV 1-4). The findings of this study reveal a new strategy for enhancing the immunogenicity of vaccine candidates and might pave the way for the development of a tetravalent vaccine against all the serotypes of Dengue Virus.

A dose-response study in mice of a tetravalent recombinant dengue envelope domain III protein secreted from insect cells

BackgroundDENV is the most globally prevalent mosquito-transmitted virus. Induction of a broadly and potently immune response is desirable for dengue vaccine development. MethodsSeveral formulations of secreted tetravalent EDIII protein containing different amounts of antigen from eukaryotic cells were used to evaluate the immune responses in mice. ResultsWe demonstrated that the tetravalent protein induced humoral immunity against all four serotypes of DENV, even at the lowest dose assayed. Besides, cellular immunities against DENV-1 and DENV-2 were elicited by medium dose group. Importantly, the immune responses induced by the tetravalent protein were functional in clearing DENV-2 in circulation of mice. ConclusionsWe believe that the tetravalent secreted EDIII protein is a potential vaccine candidate against DENV and suggest further detailed studies of this formulation in nonhuman primates.

Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

Zika Virus-Derived E-DIII Protein Displayed on Immunologically Optimized VLPs Induces Neutralizing Antibodies without Causing Enhancement of Dengue Virus Infection.

Zika virus (ZIKV) is a flavivirus similar to Dengue virus (DENV) in terms of transmission and clinical manifestations, and usually both viruses are found to co-circulate. ZIKV is usually transmitted by mosquitoes bites, but may also be transmitted by blood transfusion, via the maternal–foetal route, and sexually. After 2015, when the most extensive outbreak of ZIKV had occurred in Brazil and subsequently spread throughout the rest of South America, it became evident that ZIKV infection during the first trimester of pregnancy was associated with microcephaly and other neurological complications in newborns. As a result, the development of a vaccine against ZIKV became an urgent goal. A major issue with DENV vaccines, and therefore likely also with ZIKV vaccines, is the induction of antibodies that fail to neutralize the virus properly and cause antibody-dependent enhancement (ADE) of the infection instead. It has previously been shown that antibodies against the third domain of the envelope protein (EDIII) induces optimally neutralizing antibodies with no evidence for ADE for other viral strains. Therefore, we generated a ZIKV vaccine based on the EDIII domain displayed on the immunologically optimized Cucumber mosaic virus (CuMVtt) derived virus-like particles (VLPs) formulated in dioleoyl phosphatidylserine (DOPS) as adjuvant. The vaccine induced high levels of specific IgG after a single injection. The antibodies were able to neutralise ZIKV without enhancing infection by DENV in vitro. Thus, the here described vaccine based on EDIII displayed on VLPs was able to stimulate production of antibodies specifically neutralizing ZIKV without potentially enhancing disease caused by DENV.

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication

BackgroundThe rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated.MethodsGeraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays’. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay.ResultsCytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin’s interaction with rE-DIII with high affinity.ConclusionsGeraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Accuracy estimation of an indirect ELISA for the detection of West Nile Virus antibodies in wild birds using a latent class model

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus, are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies (Pica pica) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73–99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68–99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe=91% (95%PCI: 79–99%), and DSp=77% (95%PCI: 59–98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52–99%), and the DSp rose to 94% (95%PCI: 83–100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds infected by other Flaviviruses, such as USUV.

Scaling Up of Recombinant Dengue Virus Type 3 Envelope Domain III Protein Production from Escherichia coli

Abstract This study describes an efficient production and purification system developed for large-scale production of biologically active recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein. Process optimization was carried out at small scale for enhancement of recombinant protein production. Under the optimized conditions, 100L pilot-scale fermentation was carried out to produce a large amount of biomass for purification. The recombinant protein was renatured using an on-column refolding process. This protein was purified using affinity and cation exchange chromatography processes. Approximately 29 mg of pure and biologically active rDen 3 EDIII protein were obtained per gram of dry cell weight. The purified protein was used for immunization in mice. Biochemical characterization of purified rDen 3 EDIII protein was performed by Western blotting and enzyme-linked immunosorbent assays (ELISA). The results demonstrated that the purified rDen 3 EDIII protein could react with anti-Den 3...

Use of Envelope Domain III Protein for Detection and Differentiation of Flaviviruses in the Free State Province, South Africa

The presence of the mosquito-borne flavivirus species West Nile virus (WNV) and Wesselsbron virus (WESSV) in southern Africa is well established; however, their true prevalence remains unknown. To date, the presence of tick-borne flaviviruses has not been confirmed in this region. Serological assays using reagents that can be handled in a biosafety level 2 or lower facility were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle, and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect immunoglobulin G (IgG) enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on the KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV, and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive using the KUNV assay, of which 71, 457, and 431 were positive using the r-LGTVEDIII, r-WNVEDIII, and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive using the KUNV assay but not using any of the other assays, suggesting that there are possibly other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses, but it has yet to be confirmed. The EDIII protein is a useful tool that can be used in the detection and differentiation of flaviviruses in resource-limited laboratories, but virus neutralization assays are suggested for accurate confirmation of results.

Fine mapping of a linear epitope on EDIII of Japanese encephalitis virus using a novel neutralizing monoclonal antibody

The domain III (EDIII) of the envelope protein of Japanese encephalitis virus (JEV) is proposed to play an essential role in JEV replication and infection; it is involved in binding to host receptors and contains specific epitopes that elicit neutralizing antibodies. However, most previous studies have not provided detailed molecular information about the functional epitopes on JEV EDIII protein. In this study, we described a monoclonal antibody (mAb 2B4) we produced and characterized by IFA, PRNT, ELISA and Western blot analyses. The results showed that mAb 2B4 was specific to JEV EDIII protein and possessed high neutralization activity against JEV in vitro. Furthermore, we found that the motif, 394HHWH397, was the minimal unit of the linear epitope recognized by mAb 2B4 through screening a phage-displayed random 12-mer peptide library. Using sequence alignment analysis it was found that this motif was highly conserved among JEV strains and was present in West Nile Virus (WNV). Indeed, ELISA data showed that this epitope could be recognized by both JEV-positive swine serum and WNV-positive swine serum. Notably, this linear epitope was highly hydrophilic and was located within the terminal end of a β-pleated sheet of EDIII. An analysis of the spatial conformation supported the possibility of inducing specific antibodies to this epitope. Taken together, we identified 394HHWH397 as an EDIII-specific linear epitope recognized by mAb 2B4, which would be beneficial for studying the pathogenic mechanism of JEV; and mAb 2B4 was also a potential diagnostic and therapeutic reagent.

Domain Ⅲ of Dengue Virus Serotype 2 Envelope: Expression at High Levels in Escherichia coli and Competitive Inhibition of Virus Entry

Abstract Obejective The domain Ⅲ of dengue virus type 2 envelope was cloned and expressed in Escherichia coli and the recombinant protein inhibited virus effect was tested. Methods In this study, the domain Ⅲ (DⅢ) protein of the dengue virus type-2 (DENV-2) envelope (E) antigen was expressed in Escherichia coli by fusion with a carrier protein. The protein was purified using enzymatic cleavage and affinity purification. Rabbit immunization and antibody detection was carried out. Inhibition of DENV-2 infection was observed by DENV-2 EDⅢ protein and its immunity rabbits serum. Results The recombinant expression DENV-2 EDⅢ protein plasmid was constructed successfully. After isopropyl thiogalactoside induction, a specific soluble 29 kD protein was obtained, and the expression product accounted for 68.87% of the total protein of the cell lysate. Western blotting demonstrated the reactivity of the recombinant protein with his-tag and DENV (Ⅰ-Ⅳ) monoclonal antibodies. The protein was purified using enzymatic cleavage and affinity purification. The purified recombinant EDⅢ protein inhibited the entry of DENV-2 into BHK-21 cells. DENV-2 plaque neutralization assays were carried out using serially diluted antibodies against EDⅢ protein. At a 1:16 dilution, the antibodies produced at least 90% neutralization of the DENV-2 virus. Furthermore, the antibodies continued to exhibit high neutralization effects (approximately 80%) until the anti-EDⅢ antibody titer reached 1:1 024. Conclusions DENV-2 EDⅢ was cloned and expressed successfully. DENV-2 EDⅢ protein could be useful in the development of inexpensive dengue vaccine. The data also suggested that DENV-2 employed an attachment molecule or receptor for its entry into C6/36 mosquito cells.

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 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 8M 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 ~124mg of highly purified and biologically active EDIII protein was obtained from 100g 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.

Eliciting cross-neutralizing antibodies in mice challenged with a dengue virus envelope domain III expressed inEscherichia coli

Dengue viruses (DENVs) are mosquito-borne infectious pathogens that pose a serious global public health threat, and at present, no therapy or effective vaccines are available. Choosing suitable units as candidates is fundamental for the development of a dengue subunit vaccine. Domain III of the DENV-2 E protein (EDIII) was chosen in the present study and expressed in Escherichia coli by N-terminal fusion to a bacterial leader (pelB), and C-terminal fusion with a 6×His tag based on the functions of DENV structure proteins, especially the neutralizing epitopes on the envelope E protein. After two-step purification using Ni-NTA affinity and cation-exchange chromatography, the His-tagged EDIII was purified up to 98% homogenicity. This recombinant EDIII was able to trigger high levels of neutralizing antibodies in both BALB/c and C57BL/6 mice. Both the recombinant EDIII and its murine antibodies protected Vero cells from DENV-2 infection. Interestingly, the recombinant EDIII provides at least partial cross-protection against DENV-1 infection. In addition, the EDIII antibodies were able to protect suckling mice from virus challenge in vivo. These data suggest that a candidate molecule based on the small EDIII protein, which has neutralizing epitopes conserved among all 4 DENV serotypes, has important implications.

Recombinant dengue virus type 3 envelope domain III protein from Escherichia coli

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. The envelope protein of dengue virus is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. Optimization of culture media was carried out for enhanced production of recombinant dengue virus type 3 envelope domain III (rDen 3 EDIII) protein in E. coli. Further, batch and fed-batch cultivation process were also developed in optimized medium. After fed-batch cultivation, the dry cell weight was about 22.80 g/L of culture. The rDen 3 EDIII protein was purified using immobilized metal affinity chromatography. This process produced ∼649 mg of purified rDen 3 EDIII protein per liter of culture. The purity of the protein was determined by SDS-PAGE analysis and the reactivity was checked by Western blotting as well as ELISA. These results show that the purified protein may be used for the dengue diagnosis or further prophylactic studies for dengue infection.

Production and characterization of recombinant dengue virus type 4 envelope domain III protein

Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Cultivation process for production of recombinant dengue virus type 4 envelope domain III (rDen 4 EDIII) protein in Escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. The dissolved oxygen level was maintained at 20–30% of air saturation. The culture was induced with 1 mM of isopropyl β- d-thiogalactoside when dry cell weight was 13.78 g l −1 and cells were further grown for 4 h to reach 17.31 g l −1 of culture. The protein was overexpressed in the form of insoluble inclusion bodies. The rDen 4 EDIII protein was purified by affinity chromatography and analyzed by SDS-PAGE. The final yield of purified rDen 4 EDIII protein in this method was approximately 196 mg l −1 of culture. The purified protein was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue infected human serum samples. These results show that the product has the potential to be used for the diagnosis of dengue infection or for further studies in vaccine development. This production system may also be suitable for the high yield of other recombinant dengue proteins.