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。     

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.     

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.     

Expression of a cholera toxin B subunit and consensus dengue virus envelope protein domain III fusion gene in transgenic rice callus

Dengue (DEN) is one of the most important emerging mosquito-borne viral human diseases. Therefore, an effective dengue vaccine with immune responses against all four dengue virus serotypes is highly needed. A fusion gene encoding a synthetic consensus envelope protein domain III (scEDIII) of dengue virus with neutralizing activity against the four dengue virus serotypes and with the B subunit of cholera toxin (CTB) to increase its mucosal immunogenicity was constructed and was introduced into rice callus under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of the CTB-scEDIII fusion gene in transgenic rice callus were confirmed by genomic DNA PCR amplification, Northern, and Western blot analyses, respectively. The biological binding activity of the CTB-cEDIII fusion protein to its G_M1-ganglioside receptor was confirmed via G_M1-ELISA with anti-CT and anti-dengue virus antibodies. Delivery of the CTB-cEDIII fusion protein into mucosal immune inductive sites (including M cells) in BALB/c mice was confirmed by in vitro and in vivo antigen uptake assays. These results showed that the CTB-cEDIII fusion protein was produced in the transgenic rice callus, and that plant-produced ligand fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of the overall immune responses.     

C5a receptor-targeting ligand-mediated delivery of dengue virus antigen to M cells evokes antigen-specific systemic and mucosal immune responses in oral immunization

Oral mucosal immunization is a feasible and economic vaccination strategy. In order to achieve a successful oral mucosal vaccination, antigen delivery to gut immune inductive site and avoidance of oral tolerance induction should be secured. One promising approach is exploring the specific molecules expressed on the apical surfaces of M cells that have potential for antigen uptake and immune stimulation. We previously identified complement 5a receptor (C5aR) expression on human M-like cells and mouse M cells and confirmed its non-redundant role as a target receptor for antigen delivery to M cells using a model antigen. Here, we applied the OmpH ligand, which is capable of targeting the ligand-conjugated antigen to M cells to induce specific mucosal and systemic immunities against the EDIII of dengue virus (DENV). Oral immunization with the EDIII–OmpH efficiently targeted the EDIII to M cells and induced EDIII-specific immune responses comparable to those induced by co-administration of EDIII with cholera toxin (CT). Also, the enhanced responses by OmpH were characterized as Th2-skewed responses. Moreover, oral immunization using EDIII–OmpH did not induce systemic tolerance against EDIII. Collectively, we suggest that OmpH-mediated targeting of antigens to M cells could be used for an efficient oral vaccination against DENV infection.     

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.     

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.     

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.     

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.     

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 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.     

M Cell-Targeting Ligand and Consensus Dengue Virus Envelope Protein Domain III Fusion Protein Production in Transgenic Rice Calli

The spread of dengue (DEN) virus is becoming a major concern due to the possibility of primary infection with one of the four dengue serotypes (DEN 1–4) and secondary infection with other heterotypes, which can further aggravate clinical manifestations. A gene encoding consensus envelope protein domain III (cEDIII) of dengue virus with neutralizing activity against four dengue virus serotypes was fused to M cell-targeting peptide ligand (Co1) to increase its mucosal immunogenicity and was introduced into rice calli under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of scEDIII–Co1 fusion gene in transgenic rice calli were confirmed by genomic DNA PCR amplification, Northern and Western blot analyses, respectively. The deliveries of cEDIII–Co1 fusion proteins into mucosal immune inductive site (including M cells) were confirmed by in vitro and in vivo antigen uptake assays. These results showed that plant-produced M cell-targeting peptide ligand, Co1, fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of immune responses.     

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.     

Induction of tetravalent protective immunity against four dengue serotypes by the tandem domain III of the envelope protein

In the present study, the domain IIIs of all four dengue virus (DENV) serotypes were connected sequentially to construct the tandem domain III. The resulting DNA fragment was then cloned into pBAD/Topo ThioFusion plasmid. After induction, the Escherichia coli expression protein was purified and used to immunize BALB/c mice by subcutaneous route. The sera from mice immunized with the purified protein were confirmed to contain specific high antibody titers against DEN1, DEN2, and DEN4, and moderate antibody titer against DEN3. In suckling mouse model, 70% of the mice challenged with DEN1, DEN2, and DEN4 in combination with sera from mice immunized with the purified protein were protected, and 18% of the mice challenged with DEN3 in combination with the same sera were protected. Our data suggest that the tandem domain III of the envelope protein can be used as a potential tetravalent dengue vaccine based on a single antigen.     

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.

     

An Alphavirus Vector-Based Tetravalent Dengue Vaccine Induces a Rapid and Protective Immune Response in Macaques That Differs Qualitatively from Immunity Induced by Live Virus Infection

Despite many years of research, a dengue vaccine is not available, and the more advanced live attenuated vaccine candidate in clinical trials requires multiple immunizations with long interdose periods and provides low protective efficacy. Here, we report important contributions to the development of a second-generation dengue vaccine. First, we demonstrate that a nonpropagating vaccine vector based on Venezuelan equine encephalitis virus replicon particles (VRP) expressing two configurations of dengue virus E antigen (subviral particles [prME] and soluble E dimers [E85]) successfully immunized and protected macaques against dengue virus, while antivector antibodies did not interfere with a booster immunization. Second, compared to prME-VRP, E85-VRP induced neutralizing antibodies faster, to higher titers, and with improved protective efficacy. Third, this study is the first to map antigenic domains and specificities targeted by vaccination versus natural infection, revealing that, unlike prME-VRP and live virus, E85-VRP induced only serotype-specific antibodies, which predominantly targeted EDIII, suggesting a protective mechanism different from that induced by live virus and possibly live attenuated vaccines. Fourth, a tetravalent E85-VRP dengue vaccine induced a simultaneous and protective response to all 4 serotypes after 2 doses given 6 weeks apart. Balanced responses and protection in macaques provided further support for exploring the immunogenicity and safety of this vaccine candidate in humans.     

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.     

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.     

菠菜乙醇酸氧化酶同工酶的亚基组成

我们首次报告 ,菠菜有ＧＯⅠ (ｐＩ≈ 7.4 )、ＧＯⅡ(ｐＩ≈ 9.4 )和ＧＯⅢ (ｐＩ≈ 8.3 ) 3种乙醇酸氧化酶(ＧＯ)同工酶 ;ＧＯⅠ只含 4 0± 2ｋＤ一种亚基 ,而ＧＯⅡ和ＧＯⅢ的亚基组成未被研究 ;3种同工酶之间均有免疫同源性[1-4 ] .水稻也存在 3种ＧＯ同工酶 ,其中ＧＯⅡ (ｐＩ >8.3 )能被乙醇酸所诱导 用柱层析法纯化可获得经ＳＤＳ ＰＡＧＥ后为 4 3± 2ｋＤ单带的水稻ＧＯⅠ[5～ 7] .以上初步解释了前人报告ＧＯ电荷不均一的原因[5] .最近从菠菜分离得到含ＧＯⅡ的蛋白和含ＧＯⅢ的蛋白 ,其ＳＤＳ ＰＡＧＥ分别为 67± 2ｋＤ和 4 0±…     

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.