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The lack of an immunocompetent animal model for dengue mimicking the disease in humans is a limitation for advances in this field. Inoculation by intracerebral route of neuroadapted dengue strains in mice is normally lethal and provides a straightforward readout parameter for vaccine testing. However, systemic effects of infection and the immune response elicited in this model remain poorly described. In the present work, BALB/c mice infected by the intracerebral route with neuroadapted DENV2 exhibited several evidences of systemic involvement. DENV-inoculated mice presented virus infective particles in the brain followed by viremia, especially in late stages of infection. Infection induced cellular and humoral responses, with presence of activated T cells in spleen and blood, lymphocyte infiltration and tissue damages in brain and liver, and an increase in serum levels of some pro-inflammatory cytokines. Data highlighted an interplay between the central nervous system commitment and peripheral effects under this experimental condition.

We analyzed four DNA vaccines based on DENV-2 NS1: pcENS1, encoding the C-terminal from E protein plus the NS1 region; pcENS1ANC, similar to pcENS1 plus the N-terminal sequence from NS2a (ANC); pcTPANS1, coding the t-PA signal sequence fused to NS1; and pcTPANS1ANC, similar to pcTPANS1 plus the ANC sequence. The NS1 was detected in lysates and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected cells and not in cells with pcTPANS1ANC. Only the pcENS1ANC leads the expression of NS1 in plasma membrane, confirming the importance of ANC sequence for targeting NS1 to cell surface. High levels of antibodies recognizing conformational epitopes of NS1 were induced in mice immunized with pcTPANS1 and pcENS1, while only few pcENS1ANC-inoculated animals presented detectable anti-NS1 IgG. Protection against DENV-2 was verified in pcTPANS1- and pcENS1-immunized mice, although the plasmid pcTPANS1 induced slight higher protective immunity. These plasmids seem to activate distinct patterns of the immune system.

Dengue is one of the most important arboviral diseases in humans, and although efforts over the last decades have dealt with the development of a vaccine, this vaccine is not available yet. In order to evaluate the potential of a DNA vaccine based on the non-structural 1 (NS1) protein against dengue virus (DENV), we constructed the pcTPANS1 plasmid 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. Results indicate that pcTPANS1 promotes correct expression of NS1 in eukaryotic cells and drives secretion of the recombinant protein to the surrounding medium in a dimeric form. Balb/c mice, intramuscularly inoculated with this plasmid, presented high levels of antibodies, recognizing mainly surface-exposed conformational epitopes present in the NS1 protein expressed by insect cells. Long-term antibody response was observed in animals 56 weeks after the first plasmid inoculation, and a rapid, efficient secondary response was observed after a DNA boost. Vaccinated animals were challenged against DENV-2 in two murine models, based on intracerebral (i.c.) and intraperitoneal (i.p.) virus inoculations, and in both cases, pcTPANS1-immunized mice were protected. Overall, these results provide further support for the use of such a plasmid in a possible approach for the development of a vaccine against DENV.

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.