Dengue Virus Serotype 1 Conformational Dynamics Confers Virus Strain-Dependent Patterns of Neutralization by Polyclonal Sera.

Dengue virus cocirculates globally as four serotypes (DENV1 to -4) that vary up to 40% at the amino acid level. Viral strains within a serotype further cluster into multiple genotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of vaccine design, and efforts to characterize epitopes targeted by polyclonal mixtures of antibodies are ongoing. Previously, we identified two E protein residues (126 and 157) that defined the serotype-specific antibody response to DENV1 genotype 4 strain West Pac-74. DENV1 and DENV2 human vaccine sera neutralized DENV1 viruses incorporating these substitutions equivalently. In this study, we explored the contribution of these residues to the neutralization of DENV1 strains representing distinct genotypes. While neutralization of the genotype 1 strain TVP2130 was similarly impacted by mutation at E residues 126 and 157, mutation of these residues in the genotype 2 strain 16007 did not markedly change neutralization sensitivity, indicating the existence of additional DENV1 type-specific antibody targets. The accessibility of antibody epitopes can be strongly influenced by the conformational dynamics of virions and modified allosterically by amino acid variation. We found that changes at E domain II residue 204, shown previously to impact access to a poorly accessible E domain III epitope, impacted sensitivity of DENV1 16007 to neutralization by vaccine immune sera. Our data identify a role for minor sequence variation in changes to the antigenic structure that impacts antibody recognition by polyclonal immune sera. Understanding how the many structures sampled by flaviviruses influence antibody recognition will inform the design and evaluation of DENV immunogens. IMPORTANCE Dengue virus (DENV) is an important human pathogen that cocirculates globally as four serotypes. Because sequential infection by different DENV serotypes is associated with more severe disease, eliciting a protective neutralizing antibody response against all four serotypes is a major goal of vaccine efforts. Here, we report that neutralization of DENV serotype 1 by polyclonal antibody is impacted by minor sequence variation among virus strains. Our data suggest that mechanisms that control neutralization sensitivity extend beyond variation within antibody epitopes but also include the influence of single amino acids on the ensemble of structural states sampled by structurally dynamic virions. A more detailed understanding of the antibody targets of DENV-specific polyclonal sera and factors that govern their access to antibody has important implications for flavivirus antigen design and evaluation.

Selection-based design of in silico dengue epitope ensemble vaccines.

Dengue virus affects approximately 130 countries. Twenty-five percentage of infections result in febrile, self-limiting illness; heterotypic infection results in potentially fatal dengue haemorrhagic fever or dengue shock syndrome. Only one vaccine is currently available. Its efficacy is very variable. Thus, to target dengue, we used an innovative immunoinformatics protocol to design a putative epitope ensemble vaccine by selecting an optimal set of highly conserved epitopes with experimentally verified immunogenicity. From 1597 CD4+ and MHC II epitopes, six MHC Class I epitopes (RAVHADMGYW, GPWHLGKLEM, GLYGNGVVTK, NMIIMDEAHF, KTWAYHGSY and WAYHGSYEV) and nine MHC Class II epitopes (LAKAIFKLTYQNKVV, GKIVGLYGNGVVTTS, AAIFMTATPPGSVEA, AAIFMTATPPGTADA, GKTVWFVPSIKAGND, KFWNTTIAVSMANIF, RAIWYMWLGARYLEF, VGTYGLNTFTNMEVQ and WTLMYFHRRDLRLAA) were selected; this candidate vaccine achieved a world population coverage of 92.49%.

Chitosan stabilized nasal emulsion delivery system for effective humoral and cellular response against recombinant tetravalent dengue antigen.

Nasal vaccine delivery systems are emerging alternatives to the conventional sub unit vaccine delivery systems owing to their ability to stimulate potent antigen specific humoral and cellular immune responses. Additional virtue of nasal delivery is its close proximity of immune cells to external epithelial layer which is the route of entry to pathogens. Toxicity of emulsion based vaccine delivery systems may be attributed to the presence of high quantities of surfactants used for stabilizing the emulsions. A safer approach would be to reduce physiologically unwanted surfactant burden in the emulsion to the bare limit to necessity. Oleic acid was used as oil phase due to its ability to enhance penetration of system in nasal mucosa. This emulsion was designed with the purpose that it activates the innate (TLR 4) and adaptive immune systems apart from performing its antigen delivery function. Proving the hypothesis, emulsion when immunized along with recombinant tetravalent dengue antigen has elicited a profound antigen specific humoral and cellular response. Antigen cross presenting and sustained release of antigen by emulsion is the key factor in shaping this immune response. Moreover, the dose sparing effect of emulsion has also been proven which has a crucial role in modern day vaccine delivery. This significant humoral and cellular response elicited proves the suitability of this emulsion system for enhancing the protective effect of vaccines against various intracellular pathogens.

Optimization of Surface Display of DENV2 E Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses.

The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally. An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV. The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials. DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines. Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation. Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation. Highly neutralizing epitopes are displayed by DENV-E quaternary structures. To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed. Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology. We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display. Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice. This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design.

Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.

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-1ß, IL-6, IL-2, IL-12p70, IFN-γ, IL-4, IL-10, IL-8, MCP-1, macrophage inflammatory protein-1ß, 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.

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2. CTB-EDIII fused to an endoplasmic reticulum (ER) retention signal, SEKDEL, was introduced into potatoes by A. tumefaciens-mediated gene transformation. The integration of the CTB-EDIII fusion gene into the nuclear genome of transgenic plants was confirmed by genomic DNA polymerase chain reaction (PCR), and mRNA transcripts of CTB-EDIII were detected. CTB-EDIII fusion protein was expressed in potato tubers and assembled into a pentameric form capable of binding monosialotetrahexosylganglioside (GM1). The level of expression was determined to be ∼0.005% of total soluble protein in potato tubers. These results suggest that dengue virus antigen could be produced in potatoes, raising the possibility that edible plants are employed in mucosal vaccines for protection against dengue infection.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

Structural basis of potent Zika-dengue virus antibody cross-neutralization.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

Early Transcriptional Signatures of the Immune Response to a Live Attenuated Tetravalent Dengue Vaccine Candidate in Non-human Primates.

BACKGROUND: The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV), suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN) response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal) vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30. CONCLUSIONS/SIGNIFICANCE: These results suggest that early transcriptional responses may be predictive of development of adaptive immunity to TDV vaccination in cynomolgus macaques, and will inform studies of human responses to dengue vaccines.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

The Interplay of Dengue Virus Morphological Diversity and Human Antibodies.

Dengue virus (DENV) infects ∼400 million people annually, and there is no available vaccine or therapeutics. It is not clear why candidate vaccines provide only modest protection. In addition to the presence of four different dengue serotypes, there is also structural heterogeneity in DENV infectious particles, even within a strain. This severely complicates the development of vaccines and therapeutics. The currently known different morphologies of DENV are: immature, partially mature, compact mature, and expanded mature forms of the virus. In this review I describe these forms of the virus, their infectivity, and how antibodies could recognize these morphologies. I also discuss possible vaccine and antibody therapeutic formulations to protect against all morphologies.

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

Development of Global Consensus of Dengue Virus Envelope Glycoprotein for Epitopes Based Vaccine Design.

Dengue virus (DENV) is the member of Flaviviridae and causative agent of Dengue Haemorrhagic Fever and Dengue Shock Syndrome. Every year, around 70% of the world population is at risk, due to epidemic episodes orchestrated by one or more of its serotypes. So, a tetravalent DENV vaccine is needed which may induce the immune response against all four DENV serotypes. In this study, B-cell and T-cell epitopes have been predicted from the DENV envelope glycoprotein (Eg) using a consensus based approach in complement with the physico-chemical property (PCP) conservancy analysis. Through DENV-Eg analysis, a total of 7 PCP conserved, water soluble, in vitro and in vivo stable epitopes were predicted which may induce the B-cell and T-cell mediated anti-viral immune response.

Computational prediction and analysis of envelop glycoprotein epitopes of DENV-2 and DENV-3 Pakistani isolates: a first step towards Dengue vaccine development.

Dengue fever of tropics is a mosquito transmitted devastating disease caused by dengue virus (DENV). There is no effective vaccine available, so far, against any of its four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). There is a need for the development of preventive and therapeutic vaccines against DENV to decrease the prevalence of dengue fever, especially in Pakistan. In this research, linear and conformational B-cell epitopes of envelope glycoprotein of DENV-2 and DENV-3 (the most prevalent serotypes in Pakistan) were predicted. We used Kolaskar and Tongaonkar method for linear epitope prediction, Emini's method for surface accessibility prediction and Karplus and Schulz's algorithm for flexibility determination. To propose three dimensional epitopes, the E proteins for both serotypes were homology modeled by using Phyre2 V 2.0 server, and ElliPro was used for the prediction of surface epitopes on their globular structure. Total 21 and 19 linear epitopes were predicted for DENV-2 and DENV-3 Pakistani isolates respectively. Whereas, 5 and 4 discontinuous epitopes were proposed for DENV-2 and DENV-3 Pakistani isolates respectively. Moreover, the values of surface accessibility, flexibility and solvent-accessibility can be helpful in analyzing vaccines against DENV-2 and DENV-3. In conclusion, the proposed continuous and discontinuous antigenic peptides can be valuable candidates for diagnostic and therapeutics of DENV.

Site-specific characterization of envelope protein N-glycosylation on Sanofi Pasteur's tetravalent CYD dengue vaccine.

Recently, several virus studies have shown that protein glycosylation play a fundamental role in the virus-host cell interaction. Glycosylation characterization of the envelope proteins in both insect and mammalian cell-derived dengue virus (DENV) has established that two potential glycosylation residues, the asparagine 67 and 153 can potentially be glycosylated. Moreover, it appears that the glycosylation of these two residues can influence dramatically the virus production and the infection spreading in either mosquito or mammalian cells. The Sanofi Pasteur tetravalent dengue vaccine (CYD) consists of four chimeric viruses produced in mammalian vero cells. As DENV, the CYDs are able to infect human monocyte-derived dendritic cells in vitro via C-type lectins cell-surface molecules. Despite the importance of this interaction, the specific glycosylation pattern of the DENV has not been clearly documented so far. In this paper, we investigated the structure of the N-linked glycans in the four CYD serotypes. Using MALDI-TOF analysis, the N-linked glycans of CYDs were found to be a mix of high-mannose, hybrid and complex glycans. Site-specific N-glycosylation analysis of CYDs using nanoLC-ESI-MS/MS demonstrates that both asparagine residues 67 and 153 are glycosylated. Predominant glycoforms at asparagine 67 are high mannose-type structures while mainly complex- and hybrid-type structures are detected at asparagine 153. In vitro studies have shown that the immunological consequences of infection by the CYD dengue viruses 1-4 versus the wild type parents are comparable in human monocyte-derived dendritic cells. Our E-protein glycan characterizations of CYD are consistent with those observations from the wild type parents and thus support in vitro studies. In addition, these data provide new insights for the role of glycans in the dengue virus-host cell interactions.

Investigating the efficacy of monovalent and tetravalent dengue vaccine formulations against DENV-4 challenge in AG129 mice.

Dengue (DEN) is the most important mosquito-borne viral disease, with a major impact on global health and economics, caused by four serologically and distinct viruses termed DENV-1 to DENV-4. Currently, there is no licensed vaccine to prevent DEN. We have developed a live attenuated tetravalent DENV vaccine candidate (TDV) (formally known as DENVax) that has shown promise in preclinical and clinical studies and elicits neutralizing antibody responses to all four DENVs. As these responses are lowest to DENV-4 we have used the AG129 mouse model to investigate the immunogenicity of monovalent TDV-4 or tetravalent TDV vaccines, and their efficacy against lethal DENV-4 challenge. Since the common backbone of TDV is based on an attenuated DENV-2 strain (TDV-2) we also tested the efficacy of TDV-2 against DENV-4 challenge. Single doses of the tetravalent or monovalent vaccines elicited neutralizing antibodies, anti-NS1 antibodies, and cellular responses to both envelope and nonstructural proteins. All vaccinated animals were protected against challenge at 60 days post-immunization, whereas all control animals died. Investigation of DENV-4 viremias post-challenge showed that only the control animals had high viremias on day 3 post-challenge, whereas vaccinated mice had no detectable viremia. Overall, these data highlight the excellent immunogenicity and efficacy profile of our candidate dengue vaccine in AG129 mice.

[Research progress in the structure and function of dengue virus non-structural 1 protein].

Dengue virus (DENV) is a re-emerging disease transmitted by the Aedes mosquitoes and has become a major public health problem in southern China. Currently, no antiviral drug or effective vaccine exist to control this disease. The chimeric DENV structural protein vaccine cannot elicit balanced levels of protective immunity to each of the four viral serotypes; therefore, non-structural protein components may be required to construct an effective DENV vaccine. The Dengue virus non-structural 1 (DENV NS1) protein plays a critical role in viral pathogenesis and protective immunity. Therefore, immunity to Dengue 1-4 NS1 subtypes may be crucial for the prevention of severe disease. This review attempts to provide an overview about the structure and function of DENV NS1.

Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design.

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime-boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309-320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime-boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.