a–e, Active immunization with DENV2 ΔNS1 prevented DENV2 acquisition by mosquitoes. a, Schematic representation of the study design. b,c, DENV2 infection in immunized AG6 mice. b, Detection of DENV2 viremia. Blood was collected from the tail veins of infected mice from 0 to 5 days post-infection. The presence of infectious viral particles in the blood plasma was assessed using a plaque assay. c, Detection of DENV2 sNS1 concentration. Mouse sera were used to determine DENV2 sNS1 quantities by ELISA. d,e, NS1 immunization reduced DENV acquisition by A. aegypti. In d, the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. In e, data are represented as the percentage of mosquito infection. Differences in mosquito infective ratios were compared using Fisher's exact test. f–h, Vaccination with DENV2 ΔNS1 protected mice from lethal DENV-induced vascular leakage. In f, AG6 mice were immunized and infected following the same procedure as shown in a. n, number of mice in each group. Survival rates of the infected mice were statistically analysed using the log-rank (Mantel–Cox) test. In g and h, Evans blue dye was intravenously injected into the mice at 18 days post-infection. The dye was extracted from various tissues using formamide and OD610 was measured (g). Infected mice were used to visualize DENV-induced vascular leakage in various tissues (h). In b–g, the red P values represent a comparison between full-length DENV2 NS1-immunized and control mice, and the blue P values represent a comparison between DENV ΔNS1-immunized and control mice. P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if P < 0.025. *P < 0.025, **P < 0.005, ***P < 0.0005. In b, c and g, data are representative of at least five infected AG6 mice. Values in the graph represent the mean ± s.e.m. A non-parametric Mann–Whitney test was used to determine significant differences. In b–h, experiments were biologically reproduced at least three times. d.p.i., days post inoculation; NS, not significant.