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BackgroundDengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.Methodology/principal findingsWe report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Conclusions/significanceDirecting the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate.

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BackgroundFour antigenically distinct serotypes (1–4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype.ResultsWe cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies.ConclusionsThis work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0280-y) contains supplementary material, which is available to authorized users.

Dengue is a viral pandemic caused by four dengue virus serotypes (DENV-1, 2, 3, and 4) transmitted by Aedes mosquitoes. Reportedly, there has been a 2-fold increase in dengue cases every decade. An efficacious tetravalent vaccine, which can provide long-term immunity against all four serotypes in all target populations, is still unavailable. Despite the progress being made in the live virus-based dengue vaccines, the World Health Organization strongly recommends the development of alternative approaches for safe, affordable, and efficacious dengue vaccine candidates. We have explored virus-like particles (VLPs)-based nonreplicating subunit vaccine approach and have developed recombinant envelope ectodomains of DENV-1, 2, and 3 expressed in Pichia pastoris These self-assembled into VLPs without pre-membrane (prM) protein, which limits the generation of enhancing antibodies, and elicited type-specific neutralizing antibodies against the respective serotype. Encouraged by these results, we have extended this work further by developing P. pastoris-expressed DENV-4 ectodomain (DENV-4 E) in this study, which was found to be glycosylated and assembled into spherical VLPs without prM, and displayed critical neutralizing epitopes on its surface. These VLPs were found to be immunogenic in mice and elicited DENV-4-specific neutralizing antibodies, which were predominantly directed against envelope domain III, implicated in host-receptor recognition and virus entry. These observations underscore the potential of VLP-based nonreplicative vaccine approach as a means to develop a safe, efficacious, and tetravalent dengue subunit vaccine. This work paves the way for the evaluation of a DENV E-based tetravalent dengue vaccine candidate, as an alternative to live virus-based dengue vaccines.

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Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors. Following a three-dose immunisation schedule, the T-mVLPs elicited EDIII-directed antibodies in mice which could neutralise all four DENV serotypes. Importantly, anti-T-mVLP antibodies did not augment sub-lethal DENV-2 infection of dengue-sensitive AG129 mice, based on multiple parameters. The ‘four-in-one’ tetravalent T-mVLPs possess multiple desirable features which may potentially contribute to safety (non-viral, prM-lacking and ADE potential-lacking), immunogenicity (induction of virus-neutralising antibodies), and low cost (single tetravalent immunogen produced using P. pastoris, an expression system known for its high productivity using simple inexpensive media). These results strongly warrant further exploration of this vaccine candidate.

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO(4)GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO(4)GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (-19.8 ± 0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2 ± 0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283 ± 7 and 134 ± 5 nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO(4)GalNAc assures for targeting resident macrophages.