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.

     

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.     

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.     

Strategies for the plant-based expression of dengue subunit vaccines

Despite significant efforts in many countries, there is still no commercially viable dengue vaccine. Currently, attention is focused on the development of either live attenuated vaccines or live attenuated chimaeric vaccines using a variety of backbones. Alternate vaccine approaches, such as whole inactivated virus and subunit vaccines are in the early stages of development, and are each associated with different problems. Subunit vaccines offer the advantage of providing a uniform antigen of well-defined nature, without the added risk of introducing any genetic material into the person being inoculated. Preliminary trials of subunit vaccines (using dengue E protein) in rhesus monkeys have shown promising results. However, the primary disadvantages of dengue subunit vaccines are the low levels of expression of dengue proteins in mammalian or insect cells, as well as the added unknown risks of antigens produced from mammalian cells containing other potential sources of contamination. In the past two decades, plants have emerged as an alternative platform for expression of biopharmaceutical products, including antigens of bacterial, fungal or viral origin. In the present minireview, we highlight the current plant expression technologies used for expression of biopharmaceutical products, with an emphasis on plants as a production system for dengue subunit vaccines.     

Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2?N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2?N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development.     

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.     

Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis

Dengue virus causes about 100 million cases of dengue disease per year in the world. Laboratory diagnosis is done mainly by serological techniques, which in many cases use crude virus extracts that may cause cross-reactions to other flaviviruses. These undesirable cross-reactions can be reduced or eliminated by using recombinant proteins based on restricted epitopes. Aiming to decrease flaviviral cross-reactions and non-specific interactions in dengue serological assays, a plant expression system was chosen for recombinant antigen production as a reliable and inexpensive dengue diagnostic tool. In the present report, the lettuce plastid transformation system was applied to achieve efficient and stable tetra-epitope peptide antigen production, and its reactivity was evaluated. For this purpose, one putative epitope at positions 34 to 57 of E protein within the junction site of domains I and II of dengue virus (DENV) 1 to 4 serotypes linked by glycine linkers was expressed in lettuce chloroplasts. The potential immunoreactivity for the four DENV serotypes was evaluated using sera from patients of positive and negative dengue cases. Results indicated an overall sensitivity of 71.7 % and specificity of 100 %. No cross-reactions with the sera of yellow fever-positive or healthy individuals vaccinated against yellow fever were observed. This novel approach may provide an alternative system for the large-scale production of dengue recombinant antigens useful for serodiagnosis.     

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.     

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.     

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.     

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。     

Chloroplast‐derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin‐B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen‐1 (AMA1) and merozoite surface protein‐1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB‐AMA1 and CTB‐MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen‐specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross‐reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB‐specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin‐10⁺ T cell but not Foxp3⁺ regulatory T cells, suppression of interferon‐γ and absence of interleukin‐17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast‐derived vaccine antigens for long‐term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.     

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.     

Lactic acid bacteria — promising vaccine vectors: possibilities,limitations, doubts

Gram‐positive, nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of novel, safe production and delivery systems of heterologous proteins. Recombinant LAB strains were shown to elicit specific systemic and mucosal immune responses against selected antigens. For this reason, this group of bacteria is considered as a potential replacement of classical, often pathogenic, attenuated microbial carriers. Mucosal administration of recombinant LAB, especially via the best explored and universal oral route, offers many advantages in comparison to systemic inoculation, and is attractive from the immunological and practical point of view. Research aimed at designing efficient, mucosally applied vaccines in combination with improved immunization efficiency, monitoring of in vivo antigen production, determination of optimal dose for vaccination, strain selection and characterization is a priority in modern vaccinology. This paper summarizes and organizes the available knowledge on the application of LAB as live oral vaccine vectors. It constitutes a valuable source of general information for researchers interested in mucosal vaccine development and constructing LAB strains with vaccine potential.     

Direct and indirect interactions in the recognition between a cross‐neutralizing antibody and the four serotypes of dengue virus

Dengue fever is the most important vector‐borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Secondary infection by a different serotype is a risk factor for severe dengue. Monoclonal antibody mAb4E11 neutralizes the four serotypes of DENV with varying efficacies by recognizing an epitope located within domain‐III (ED3) of the viral envelope (E) protein. To better understand the cross‐reactivities between mAb4E11 and the four serotypes of DENV, we constructed mutations in both Fab4E11 fragment and ED3, and we searched for indirect interactions in the crystal structures of the four complexes. According to the serotype, 7 to 12 interactions are mediated by one water molecule, 1 to 10 by two water molecules, and several of these interactions are conserved between serotypes. Most interfacial water molecules make hydrogen bonds with both antibody and antigen. Some residues or atomic groups are engaged in both direct and water‐mediated interactions. The doubly‐indirect interactions are more numerous in the complex of lowest affinity. The third complementarity determining region of the light chain (L‐CDR3) of mAb4E11 does not contact ED3. The structures and double‐mutant thermodynamic cycles showed that the effects of (hyper)‐mutations in L‐CDR3 on affinity were caused by conformational changes and indirect interactions with ED3 through other CDRs. Exchanges of residues between ED3 serotypes showed that their effects on affinity were context dependent. Thus, conformational changes, structural context, and indirect interactions should be included when studying cross‐reactivity between antibodies and different serotypes of viral antigens for a better design of diagnostics, vaccine, and therapeutic tools against DENV and other Flaviviruses. Copyright © 2014 John Wiley & Sons, Ltd.     

肺炎链球菌糖疫苗的研究进展

肺炎链球菌(Streptococcus pneumoniae)是引起多种疾病的主要病原体,包括侵袭性感染(如败血症和脑膜炎菌血症),以及更常见的粘膜部位感染(如肺炎、中耳炎和鼻窦炎).根据肺炎链球菌表面荚膜多糖结构的不同可以分成不同的血清型,至今已经鉴定出98种,其中有20种具有高毒力.为了预防肺炎链球菌感染,已研制出...     

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.