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The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1–4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy., Graphical Abstract, Developing a successful vaccine against dengue has been challenging. Arun Sankaradoss and colleagues developed a genotype-specific DNA DENV vaccine candidate that elicits robust dengue immune responses. The low cost and thermostability of DNA vaccines should allow countries to implement large-scale vaccination programs, which could greatly reduce dengue cases.

Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. OurIn silicostructural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.

A transient increase in trans-endothelial cell permeability in dengue patients leads to vascular leakage and shock syndrome. Here, we analysed the molecular mechanisms that cause permeability changes in human dermal microvascular endothelial cells (HMEC-1) using a direct dengue virus (DENV) infection model or treatment with NS1, a secreted DENV non-structural protein. In HMEC-1 cells, both treatments increase permeability with a concordant increase in the secretion of angiopoietin-2 (Ang-2). There is phosphorylation and loss of the junction protein VE-Cadherin from the inter-endothelial cell junctions and phosphorylation of RhoA. Direct virus infection results in activation of Src by phosphorylation, whereas NS1 treatment alone does not lead to Src activation. Furthermore, treatment with recombinant Ang-1, a physiological antagonist of Ang-2, prevents Ang-2 release, VE-Cadherin phosphorylation and internalization, and phosphorylation of RhoA and Src, resulting in restoration of barrier function. The permeability increase could also be prevented by blocking the Ang1/2 signalling receptor, Tie-2, or using a Rho/ROCK-specific inhibitor. Dasatinib, a Src-family kinase (SFK) inhibitor that inhibits Src phosphorylation, prevents enhanced permeability induced by direct DENV infection whereas in NS1 protein-treated cells its effect is less significant. The results provide important insights on the mechanisms of increased trans-endothelial permeability in DENV infection, and suggest the therapeutic potential of using recombinant Ang-1 or targeting these key molecules to prevent vascular leakage in dengue.