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.     

Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate

An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.     

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.     

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 1mM of isopropyl beta-d-thiogalactoside when dry cell weight was 13.78 g l(-1) and cells were further grown for 4h 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.     

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

High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli

Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine. The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity. We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli. A 6x His tag was inserted at the N terminus to facilitate its one-step purification. The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol. We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of approximately 30 mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 neutralizing epitope known to reside in domain III. The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope. In addition, murine and human polyclonal sera also recognized the recombinant protein. The in vitro refolded recombinant protein preparation was biologically functional. It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells. This expression/purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts.     

Synthesis and assembly of dengue virus envelope protein fused to cholera toxin B subunit into biologically active oligomers in transgenic tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

Dengue is the fastest growing mosquito-borne disease worldwide, causing nearly 400 million infections annually. A universally applicable dengue virus vaccine is required to arrest its spread. Here, we generated an edible dengue vaccine by expressing the dengue fusion protein in tomatoes, which is a desirable expression system owing to the inherent adjuvanticity of alpha tomatine and immunogenicity of the tomato lectin/microbial antigen complex. The B subunit of Vibrio cholera toxin (CTB) was genetically fused to dengue envelope antigen for improved delivery to antigen-presenting cells and enhanced immunogenicity, while avoiding immunological tolerance. We utilized domain III of the dengue envelope protein (EDIII), as it has been shown to induce serotype-specific neutralizing antibodies. The CTB–EDIII fusion gene construct containing an endoplasmic reticulum target sequence was introduced into tomato plants by Agrobacterium tumefaciens-mediated gene transformation, and the expression of CTB–EDIII in transgenic plants was confirmed by DNA, RNA and protein analyses. Accumulated fusion protein accounted for up to 0.015 % of total soluble protein, and it assembled into fully functional pentamers as demonstrated by binding to GM1 ganglioside. Future work will involve testing of transgenic tomatoes for immunogenicity in mice following oral delivery.     

A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity

A novel dengue vaccine candidate comprised of a consensus dengue virus envelope protein domain III (cED III) was developed to fight against dengue virus infection. The amino acid sequence of this novel cED III was obtained by alignment of amino acid sequences from different isolates of the four serotypes of dengue viruses. A proof-of-concept study demonstrated that BALB/c mice immunized with the recombinant cED III developed neutralizing antibodies against all serotypes of dengue virus. Moreover, formulation of recombinant cED III with aluminum phosphate could induce long-lasting antibody responses and anamnestic neutralizing antibody responses following challenge with dengue virus at week 28 after priming. These results demonstrate the possibility of developing a single tetravalent vaccine against dengue viral infections.     

EDIII‐DENV3 nanospheres drive immature dendritic cells into a mature phenotype in an in vitro model

Domain III of E protein of dengue virus (DENV) is a target for vaccine development. Unfortunately, this protein based platform has low general immunogenicity. To circumvent this problem, the use of an adjuvant‐nanoparticle delivery system to facilitate immunogenicity of soluble DENV‐EDIII protein was investigated. One of the key features of this delivery system is its ability to simultaneously deliver antigens and exert adjuvanticity on specialized immune cells. In this study, N‐trimethyl chitosan (TMC) nanoparticles (NPs) were generated to be used as adjuvant and carrier for soluble E‐domain III of dengue virus serotype 3 (sEDIII‐D3). Using ionotropic gelation, purified sEDIII‐D3 was encapsulated into TMC NPs to form EDIII‐D3 TMC NPs. After optimization, EDIII‐D3 TMC particles exhibited a loading efficiency of 81% and a loading capacity of 41%. The immunogenicity of EDIII‐D3 TMC NPs was tested using monocyte‐derived dendritic cells (MoDCs). It was found that EDIII‐D3 TMC NPs were well taken up by MoDCs. In addition, EDIII‐D3 TMC NP treated MoDCs significantly upregulated maturation markers (CD80, CD83, CD86 and HLA‐DR) and induced secretion of various cytokines and chemokines (IFN‐α, IL‐1β, IL‐6, IL‐2, IL‐12p70, IFN‐γ, IL‐4, IL‐10, IL‐8, MCP‐1, macrophage inflammatory protein‐1β, granulocyte‐colony stimulating factor, granulocyte–macrophage colony‐stimulating factor and IL‐7). These results indicate that EDIII‐D3 TMC NPs are potent immunogens, at least in vitro , with the ability to induce maturation of DCs and highlight the potential use of TMC NPs for enhancing immunogenicity of a non‐replicating dengue vaccine.

     

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.     

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。     

Lipidated Dengue-2 Envelope Protein Domain III Independently Stimulates Long-Lasting Neutralizing Antibodies and Reduces the Risk of Antibody-Dependent Enhancement

Background

Dengue virus is a mosquito-transmitted virus that can cause self-limiting dengue fever, severe life-threatening dengue hemorrhagic fever and dengue shock syndrome. The existence of four serotypes of dengue virus has complicated the development of an effective and safe dengue vaccine. Recently, a clinical phase 2b trial of Sanofi Pasteur''s CYD tetravalent dengue vaccine revealed that the vaccine did not confer full protection against dengue-2 virus. New approaches to dengue vaccine development are urgently needed. Our approach represents a promising method of dengue vaccine development and may even complement the deficiencies of the CYD tetravalent dengue vaccine.

Methodology/Principal Findings

Two important components of a vaccine, the immunogen and immunopotentiator, were combined into a single construct to generate a new generation of vaccines. We selected dengue-2 envelope protein domain III (D2ED III) as the immunogen and expressed this protein in lipidated form in Escherichia coli, yielding an immunogen with intrinsic immunopotentiation activity. The formulation containing lipidated D2ED III (LD2ED III) in the absence of exogenous adjuvant elicited higher D2ED III-specific antibody responses than those obtained from its nonlipidated counterpart, D2ED III, and dengue-2 virus. In addition, the avidity and neutralizing capacity of the antibodies induced by LD2ED III were higher than those elicited by D2ED III and dengue-2 virus. Importantly, we showed that after lipidation, the subunit candidate LD2ED III exhibited increased immunogenicity while reducing the potential risk of antibody-dependent enhancement of infection in mice.

Conclusions/Significance

Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus and other pathogens.     

A novel single-dose dengue subunit vaccine induces memory immune responses

To protect against dengue viral infection, a novel lipidated dengue subunit vaccine was rationally designed to contain the consensus amino acid sequences derived from four serotypes of dengue viruses. We found that the lipidated consensus dengue virus envelope protein domain III (LcED III) is capable of activating antigen-presenting cells and enhancing cellular and humoral immune responses. A single-dose of LcED III immunization in mice without extra adjuvant formulation is sufficient to elicit neutralizing antibodies against all four serotypes of dengue viruses. In addition, strong memory responses were elicited in mice immunized with a single-dose of LcED III. Quick, anamnestic neutralizing antibody responses to a live dengue virus challenge were elicited at week 28 post-immunization. These results demonstrate the promising possibility of a future successful tetravalent vaccine against dengue viral infections that utilizes one-dose vaccination with LcED III.     

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.     

Expression of a consensus dengue virus envelope protein domain III in transgenic callus of rice

An effective dengue vaccine should elicit immune responses against all four different dengue virus serotypes. This study optimized the codon usage of a gene encoding consensus dengue virus envelope protein domain III (cEDIII) with cross-neutralizing activity against four dengue virus serotypes for plant expression. Then, a plant expression vector was constructed with this gene under the control of the rice amylase 3D promoter (RAmy3D), which is a strong inducible promoter under sugar starvation conditions. The synthetic cEDIII gene was fused with the RAmy3D signal peptide and ER retention signal, SEKDEL, and was introduced into rice callus by particle bombardment-mediated transformation. The integration and expression of cEDIII gene in transgenic rice callus was confirmed by genomic DNA PCR amplification, Northern blot analysis, and western blot analysis, respectively. Densitometric analysis determined that the highest expression level of the cEDIII protein in lyophilized rice callus was approximately 0.45 mg g⁻¹. These results suggest that it is feasible to use transgenic rice callus to produce the consensus dengue virus envelop protein domain III for edible vaccine purposes.     

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.     

Pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.     

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.     

Production of IgM specific recombinant dengue multiepitope protein for early diagnosis of dengue infection

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.