An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8 T cells and antibody when expressed from modified vaccinia Ankara

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8⁺ T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8⁺ T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8⁺ T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8⁺ T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8⁺ T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.     

Immune correlates of protection for dengue: State of the art and research agenda

Dengue viruses (DENV1-4) are mosquito-borne flaviviruses estimated to cause up to ∼400 million infections and ∼100 million dengue cases each year. Factors that contribute to protection from and risk of dengue and severe dengue disease have been studied extensively but are still not fully understood. Results from Phase 3 vaccine efficacy trials have recently become available for one vaccine candidate, now licensed for use in several countries, and more Phase 2 and 3 studies of additional vaccine candidates are ongoing, making these issues all the more urgent and timely. At the “Summit on Dengue Immune Correlates of Protection”, held in Annecy, France, on March 8–9, 2016, dengue experts from diverse fields came together to discuss the current understanding of the immune response to and protection from DENV infection and disease, identify key unanswered questions, discuss data on immune correlates and plans for comparison of results across assays/consortia, and propose a research agenda for investigation of dengue immune correlates, all in the context of both natural infection studies and vaccine trials.     

Targeting of envelope domain III protein of DENV type 2 to DEC-205 receptor elicits neutralizing antibodies in mice

Dengue virus (DENV) is the causal agent of severe disease and, in some cases, mortality in humans, but no licensed vaccines against dengue are available. An effective vaccine against dengue requires long-term humoral and cellular immune responses. Several viral proteins have been the subjects of intense research, especially the envelope (E) protein, aimed at developing a vaccine. Domain III of the envelope protein (EDIII) has been identified as a potential candidate because it is involved in binding to host cell receptors and contains epitopes that elicit virus neutralizing antibodies. However, this domain is not sufficiently antigenic when is expressed and administered as antigen to elicit a strong immune response. One alternative to enhance immunogenicity is to target the antigen to dendritic cells to induce T-cells for broad antibody responses. In this work, a single chain antibody fragment (scFv) raised against the DEC-205 receptor fused with the EDIII was successfully expressed in Nicotiana benthamiana. The recombinant protein was expressed and purified from the plant and evaluated in BALB/c mice to test its immunogenicity and ability to induce neutralizing antibodies against DENV. The mice immunized with the recombinant protein produced specific and strong humoral immune responses to DENV. Only two immunizations were required to generate a memory response to DENV without the presence of adjuvants. Also, recognition of the recombinant protein with sera from DENV-infected patients was observed. These findings suggest that this strategy has potential for development of an effective vaccine against DENV.     

ADE and dengue vaccination

The vaccine against Dengue virus (DENV), Dengvaxia® (CYD), produced by Sanofi-Pasteur, has been registered by several national regulatory agencies; nevertheless, the performance and security of this vaccine have been challenged in a series of recent papers. In this work, we intend to contribute to the debate by analyzing the concept of an enhancing vaccine, presenting objections to the epidemiological model base of the concept and, likewise, presenting data that contradict that concept.     

Identifying protective dengue vaccines: Guide to mastering an empirical process

A recent clinical trial of a live-attenuated tetravalent chimeric yellow fever-dengue vaccine afforded no protection against disease caused by dengue 2 (DENV-2). This outcome was unexpected as two or more doses of this vaccine had raised broad neutralizing antibody responses. Data from pre-clinical subhuman primate studies revealed that vaccination with the monotypic DENV-2 component failed to meet established criteria for solid protection to homotypic live virus challenge. Accordingly, it is suggested that preclinical testing adopt more rigorous criteria for protection and that Phase I testing be extended to require evidence of solid monotypic protective immunity for each component of a dengue vaccine by direct challenge with live-attenuated DENV. Because live-attenuated tetravalent DENV vaccines exhibit evidence of immunological interference phenomena, during Phase II, volunteers given mixtures of DENV 1–4 vaccines should be separately challenged with monotypic live-attenuated DENV. Immune responses to live-attenuated challenge viruses and vaccine strains should be studied in an attempt to develop useful in vitro correlates of in vivo protection. Finally, it will be important to learn if DENV non-structural protein 1 (NS1) contributes to pathogenesis of the vascular permeability syndrome in humans. If so, immunity to dengue 1–4 NS1 may be crucial to prevent severe disease.     

Detection of Dengue viruses among febrile patients in Lagos,Nigeria and phylogenetics of circulating Dengue serotypes in Africa

Dengue fever, a mosquito borne viral disease, is caused by Dengue virus. This virus and its vector is endemic in most tropical countries including Nigeria. Dengue presents with febrile symptoms and is a major cause of morbidity and mortality in affected countries. The infection presently has no licensed drugs and vaccine is only available for previously exposed individuals. Despite the endemicity of Dengue in Nigeria, very few studies have identified circulating Dengue genotypes in the country. There is also sparse information on the occurrence, distribution and temporal patterns of circulating dengue virus serotypes as well as genotypes in Africa. This situation creates barriers to effective control of the infection in the continent.This study identified Dengue serotypes and genotypes among febrile patients in two health centers in Lagos, Nigeria. Phylogenetic analysis of Dengue sequences previously collected from African countries and submitted to GenBank database from 1944 till date was also performed. One hundred and thirty febrile persons were recruited for the study between April and August 2018. Eleven (8.5%) persons were Dengue virus positive. Dengue virus serotypes 1 (genotype I) and 3 (genotype I) were identified as actively circulating in Lagos, Nigeria. DENV 1 genotype V, DENV 2 cosmopolitan genotype and DENV 3 genotype III has over the years been the predominant circulating Dengue strains in Africa. Relative genotypic stability of circulating Dengue serotypes in Africa occurred over the past five decades. This may be due to limited investigations on circulating Dengue serotypes among asymptomatic individuals in the region as most studies focused on disease outbreaks and imported cases.There is the need to describe circulating Dengue genotypes in northern Africa, southern Africa as well as among asymptomatic individuals in other parts of Africa as this will provide further information on the diversity of Dengue genotypes circulating in the region.     

A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titers met the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection.     

DNA vaccine against the non-structural 1 protein (NS1) of dengue 2 virus

Dengue is one of the most important mosquito-borne viral disease causing dengue fever and/or dengue shock syndrome/haemorrhagic fever. In some reports, the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. In order to investigate the potential of a DNA vaccine based on the NS1 protein against DENV, we used the plasmid pcTPANS1, which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. All Balb/c mice intramuscularly inoculated with the pcTPANS1 presented high levels of NS1-specifc antibodies. Vaccinated animals were challenged with intracerebral DENV-2 virus inoculations and a 100% survival was observed. In general, results demonstrate that the pcTPANS1 plasmid is able to induce protection in mice, and then may be used as a vaccination approach against DENV in further assays.     

Changing Pattern of Dengue Virus Serotypes in Thailand between 2004 and 2010

Dengue virus infection is a major concern in several countries, and more than 50 million people are infected worldwide each year. Thailand is one of the countries where people are susceptible to infection due to favourable geographical and environmental conditions. In this retrospective study, we reported the changing pattern of dengue virus serotypes during the period between 2004 and 2010. The following percentage prevalence showed different serotypes of dengue virus (DENV) predominant in respective years: DENV1 in 2004 (56.41%), DENV4 in 2007 (50%), DENV1 in 2008 (57.41%), and DENV3 in 2010 (38.7%). Moreover, the major serotypes were not stable as they showed a shift from one serotype to another. We also found co-infection with two different serotypes and reported the clinical manifestations, which were not different from infection with a single serotype. Co-infection with various serotypes may not necessarily cause more severe disease.Key words: Co-infection, Dengue virus, Prevalence, Serotype, Thailand     

2015-2017年汕头市登革病毒分子流行病学研究

目的为了解汕头市登革病毒的分子特征与病毒的来源和传播途径,对汕头市登革病毒进行分子监测,并对登革病毒进行分子流行病学研究。方法利用2015-2017年间采集的174例登革热疑似患者的血清样本;对样本进行登革病毒IgM和IgG、NS1抗原检测;采用实时rt-PCR进行登革病毒RNA检测和血清分型;PCR扩增病毒的包膜(E)基因并进行序列测定;对序列进行系统发育分析,以推测病毒的基因型、起源和传播。结果 174例登革热疑似病例中,59例(33.9%)检出登革病毒感染。血清型以DENV-2为主(15例,占51.7%),其次为DENV-1(12例,占41.4%),DENV-3和DENV-4各1例;DENV-1型毒株的基因型有I型和IV型,DENV-2型毒株均属于cosmopolitan基因型;序列分析表明,汕头菌株与东南亚国家分离的菌株关系密切。结论本研究揭示了汕头市登革病毒的血清型和基因型的分布,血清型以DENV-2为主,基因型以cosmopolitan为主;推测汕头登革热流行的病毒株多来源于市外,而非本地流行;病毒毒株的主要起源地为东南亚国家。     

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.     

Genotype replacement of dengue virus type 3 and clade replacement of dengue virus type 2 genotype Cosmopolitan in Dhaka,Bangladesh in 2017

Dengue is a mosquito-borne disease that has spread to >100 countries and is caused by the dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to -4), and each serotype is further divided into distinct genotypes. In India, it is reported that all 4 serotypes of DENV co-circulate. Although Bangladesh is a neighboring country of India, very few reports have published DENV sequence data for the country, especially after 2012. To understand the current distribution of DENV genotypes in Bangladesh, we determined the nucleotide sequences of envelope regions obtained from 58 DENV-positive patients diagnosed at Apollo Hospitals Dhaka during the period between September 2017 and February 2018. We found 5 DENV-1, 47 DENV-2, and 6 DENV-3 serotypes. A phylogenetic analysis of the obtained viral sequences revealed that DENV-3 genotype I was present instead of DENV-3 genotype II, which was predominant in Bangladesh between 2000 and 2009. Furthermore, we found two distinct lineages of the Cosmopolitan genotype of DENV-2, one of which was closely related to strains from Southeast Asia and has never been reported previously in Bangladesh. These results indicated that DENVs in Bangladesh have increased in genotypic diversity and suggest that the DENV genotypic shift observed in other Asian countries also might have been taking place in Bangladesh.     

Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue,West Nile and Japanese encephalitis viruses

Dengue viruses (DENV), West Nile virus (WNV) and Japanese encephalitis virus (JEV) are major global health and growing medical problems. While a live-attenuated vaccine exists since decades against the prototype flavivirus, yellow fever virus (YFV), there is an urgent need for vaccines against dengue or West Nile diseases, and for improved vaccines against Japanese encephalitis. Live-attenuated chimeric viruses were constructed by replacing the genes coding for Premembrane (prM) and Envelope (E) proteins from YFV 17D vaccine strain with those of heterologous flaviviruses (ChimeriVax™ technology). This technology has been used to produce vaccine candidates for humans, for construction of a horse vaccine for West Nile fever, and as diagnostic reagents for dengue, Japanese encephalitis, West Nile and St. Louis encephalitis infections. This review focuses on human vaccines and their characterization from the early stages of research through to clinical development. Phenotypic and genetic properties and stability were examined, preclinical evaluation through in vitro or animal models, and clinical testing were carried out. Theoretical environmental concerns linked to the live and genetically modified nature of these vaccines have been carefully addressed. Results of the extensive characterizations are in accordance with the immunogenicity and excellent safety profile of the ChimeriVax™-based vaccine candidates, and support their development towards large-scale efficacy trials and registration.     

An adenovirus type 5 (AdV5) vector encoding an envelope domain III-based tetravalent antigen elicits immune responses against all four dengue viruses in the presence of prior AdV5 immunity

Dengue is a mosquito-borne viral disease caused by four antigenically distinct serotypes of dengue viruses (DENVs). This disease, which is prevalent in over a hundred tropical and sub-tropical countries of the world, represents a significant global public health problem. A tetravalent dengue vaccine capable of protecting against all four DENV serotypes has been elusive so far. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we have utilized a discrete carboxy-terminal region of the major DENV envelope (E) protein, known as domain III (EDIII), which mediates virus entry into target cells and contains multiple serotype-specific neutralizing epitopes, to create a chimeric tetravalent antigen. This antigen derived by in-frame fusion of the EDIII-encoding sequences of the four DENV serotypes was expressed using a replication-defective recombinant human adenovirus type 5 (rAdV5) vaccine vector. This rAdV5 vector induced cell-mediated immune responses and virus-neutralizing antibodies specific to each of the four DENVs in mice. Interestingly, anti-AdV5 antibodies did not suppress the induction of DENV-specific neutralizing antibodies. We observed that anti-AdV5 antibodies in the sera of immunized mice could promote uptake of a rAdV5-derived reporter vector into U937 cells, suggesting that pre-existing immunity to AdV5 may in fact facilitate the uptake of rAdV5 vectored vaccines into antigen presenting cells. This work presents an alternative approach to developing a single component tetravalent vaccine that bypasses the complexities inherent in the currently adopted four-in-one physical mixture approach.     

Elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence

Dengue viruses (DENVs) are re-emerging pathogens transmitted by mosquitoes mainly in tropical and subtropical regions. Each year, they are estimated to infect 390 million people globally. The major challenge confronting dengue vaccine development is the need to induce balanced, long lasting tetravalent immune responses against four co-circulating virus serotypes (DENV-I, -II, -III, -IV), because primary infection by any one of which may predispose infected individuals to more severe diseases during a heterotypic secondary infection. Another difficulty is to select representative strains in vaccine design to provide cross-protection against most circulating virus strains. In this study, aimed at developing a tetravalent subunit vaccine with a representative single protein, we designed two vaccines (named cE80(D4) and cE80(max)) based on the consensus sequences of the ectodomain of envelope protein of 3127 DENV strains, and then expressed them in the baculovirus expression system. Both vaccines were capable of eliciting specific antibodies against all four DENV serotypes, and the predominant IgG subtype elicited by the two vaccines was IgG1. Moreover, these vaccines activated both type I and type II antigen-specific helper T cells that secreted IFN-γ and IL-4, respectively. This proof-of-concept study has set foundation for further optimization of a single protein-based tetravalent DENV vaccine.     

African genetic ancestry is associated with a protective effect on Dengue severity in colombian populations

The wide variation in severity displayed during Dengue Virus (DENV) infection may be influenced by host susceptibility. In several epidemiological approaches, differences in disease outcomes have been found between some ethnic groups, suggesting that human genetic background has an important role in disease severity. In the Caribbean, It has been reported that populations of African descent present considerable less frequency of severe forms compared with Mestizo and White self-reported groups. Admixed populations offer advantages for genetic epidemiology studies due to variation and distribution of alleles, such as those involved in disease susceptibility, as well to provide explanations of individual variability in clinical outcomes. The current study analysed three Colombian populations, which like most of Latin American populations, are made up of the product of complex admixture processes between European, Native American and African ancestors; having as a main goal to assess the effect of genetic ancestry, estimated with 30 Ancestry Informative Markers (AIMs), on DENV infection severity. We found that African ancestry has a protective effect against severe outcomes under several systems of clinical classification: Severe Dengue (OR: 0.963 for every 1% increase in African ancestry, 95% confidence interval (0.934–0.993), p-value: 0.016), Dengue Haemorrhagic Fever (OR: 0.969, 95% CI (0.947–0.991), p-value: 0.006), and occurrence of haemorrhages (OR: 0.971, 95% CI (0.952–0.989), p-value: 0.002). Conversely, decrease from 100% to 0% African ancestry significantly increases the chance of severe outcomes: OR is 44-fold for Severe Dengue, 24-fold for Dengue Haemorrhagic Fever, and 20-fold for occurrence of haemorrhages. Furthermore, several warning signs also showed statistically significant association given more evidences in specific stages of DENV infection. These results provide consistent evidence in order to infer statistical models providing a framework for future genetic epidemiology and clinical studies.     

A novel chimeric dengue vaccine candidate composed of consensus envelope protein domain III fused to C-terminal-modified NS1 protein

There is an urgent need for a safe and effective vaccine against dengue virus (DENV) which infects about 390 million humans per year. In the present study we combined modifications of two DENV proteins, the nonstructural protein 1 (NS1) and the envelope (E) protein, to produce a DENV vaccine candidate with enhanced features. One of these modified proteins was a C-terminal-deleted fragment of NS1 called ΔC NS1 which we have shown previously to be protective without the potentially harmful effects of cross-reactive epitopes common to surface antigens on platelets and endothelial cells. The other modified protein was an envelope protein domain III (cEDIII) containing a consensus amino acid sequence among the four serotypes of DENV, which induces neutralizing antibody against all four DENV serotypes. The cEDIII and ΔC NS1 were expressed as a fusion protein cEDIII-ΔC NS1 and its protective effects against DENV were evaluated in a mouse model. C3H/HeN mice were immunized three times with cEDIII-ΔC NS1 fusion protein mixed with alum as adjuvant. Sera collected from cEDIII-ΔC NS1-immunized mice neutralized four serotypes of DENV and also caused complement-mediated cytolysis of HMEC-1 cells infected with each of the four different DENV serotypes. Mice immunized with cEDIII-ΔC NS1 and challenged with DENV showed reduced serum virus titer, soluble NS1 and bleeding time, compared with mice infected with DENV alone. The results reveal that antibodies induced by cEDIII-ΔC NS1 not only show anti-viral efficacy by in vitro assays but also provide protective effects against DENV infection in a mouse model. The cEDIII-ΔC NS1 thus represents a novel, effective DENV vaccine candidate.     

Dengue human infection models to advance dengue vaccine development

Dengue viruses (DENV) currently infect approximately 400 million people each year causing millions to seek care and overwhelming the health care infrastructure in endemic areas. Vaccines to prevent dengue and therapeutics to treat dengue are not currently available. The efficacy of the most advanced candidate vaccine against symptomatic dengue in general and DENV-2 in particular was much lower than expected, despite the ability of the vaccine to induce neutralizing antibody against all four DENV serotypes. Because seroconversion to the DENV serotypes following vaccination was thought to be indicative of induced protection, these results have made it more difficult to assess which candidate vaccines should or should not be evaluated in large studies in endemic areas. A dengue human infection model (DHIM) could be extremely valuable to down-select candidate vaccines or therapeutics prior to engaging in efficacy trials in endemic areas. Two DHIM have been developed to assess the efficacy of live attenuated tetravalent (LATV) dengue vaccines. The first model, developed by the Laboratory of Infectious Diseases at the U. S. National Institutes of Health, utilizes a modified DENV-2 strain DEN2Δ30. This virus was derived from the DENV-2 Tonga/74 that caused only very mild clinical infection during the outbreak from which it was recovered. DEN2Δ30 induced viremia in 100%, rash in 80%, and neutropenia in 27% of the 30 subjects to whom it was given. The Walter Reed Army Institute of Research (WRAIR) is developing a DHIM the goal of which is to identify DENV that cause symptomatic dengue fever. WRAIR has evaluated seven viruses and has identified two that meet dengue fever criteria. Both of these models may be very useful in the evaluation and down-selection of candidate dengue vaccines and therapeutics.     

Design and production of dengue virus chimeric proteins useful for developing tetravalent vaccines

Dengue virus (DENV) is a Flavivirus estimated to cause 390 million infections/year. Currently, there is no anti-viral specific treatment for dengue, and efficient DENV vector control is still unfeasible. Here, we designed and produced chimeric proteins containing potential immunogenic epitopes from the four DENV serotypes in an attempt to further compose safer, balanced tetravalent dengue vaccines.For this, South American DENV isolate sequences were downloaded from the NCBI/Virus Variation/Dengue virus databases and intraserotype-aligned to generate four consensuses. Four homologous DENV sequences were retrieved using BLAST and then interserotype-aligned. In parallel, sequences were subjected to linear B epitope prediction analysis. Regions of the envelope and NS1 proteins that are highly homologous among the four DENV serotypes, non-conserved antigenic regions and the most antigenic epitopes found in the C, prM, E and NS1 DENV proteins were used to construct 11 chimeric peptides. Genes encoding the chimeric proteins were commercially synthesized, and proteins were expressed, purified by affinity chromatography and further subjected to ELISA assays using sera from individuals infected with DENVs 1, 2, 3 or 4. As a proof-of-concept, the chimeric EnvEpII protein was selected to immunize BALB/c and C57BL/6 mice strains. The immunization with EnvEpII protein associated with aluminum induced an increased number of T CD4⁺ and CD8⁺ cells, high production of IgG₁ and IgG₂ antibodies, and increased levels of IL-2 and IL-17 cytokines, in both mouse strains. Because the EnvEpII protein associated with aluminum induced an efficient cellular response by stimulating the production of IL-2, IL-4, IL-17 and induced a robust humoral response in mice, we conclude that it resembles an efficient specific response against DENV infection. Although further experiments are required, our results indicate that epitope selection by bioinformatic tools is efficient to create recombinant proteins that can be used as candidates for the development of vaccines against infectious diseases.     

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1–4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.