a, Schematic representation of DENV2 ΔNS1. DENV2 ΔNS1 lacks the following antigenic regions from full-length DENV2 NS1 that potentially elicit the production of cross-reactive antibodies: amino acids (AA) 116–119 (ref. 14), 221–266 (ref. 15) and 311–330 (ref. 16). b,c, DENV NS1 antibody cross-reactivity with HUVEC cells. Purified antibodies against DENV2 full-length NS1 or DENV2 ΔNS1 were incubated with HUVECs. A pre-immune antibody served as a mock control. Antibody attachment to the HUVECs was quantified by flow cytometry (b) and ELISA (c). DENV2 NS1 antibodies were stained using anti-mouse Alexa 488-conjugated IgG for fluorescence-activated cell sorting (FACS) assay and anti-mouse HRP-conjugated IgG for ELISA. The values in the graph represent the mean ± s.e.m. A non-parametric Mann–Whitney test was used to determine significant differences. P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if P < 0.025. d,e, An antibody generated against DENV2 ΔNS1 prevents DENV acquisition by mosquitoes. Murine antisera (1:100 dilution) against DENV2 full-length NS1 or DENV2 ΔNS1 were incubated with supernatant from DENV-infected Vero cells (50% vol/vol) and fresh human blood (50% vol/vol). The mixture was used for in vitro membrane feeding of A. aegypti. The same dilution of pre-immune serum served as a mock control. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. 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. P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if P < 0.025. In b–e, experiments were biologically repeated at least three times with similar results. NS, not significant.