Adaptation and characterization of D2-vFLM D2-FLM.

A) Immunofluorescent staining of DV2 and D2-FLM infected Vero-81 cells with a-NS3 antibody at 24 hours post-infection. B) Combined staining of prM and E of DV-2 and D2-FLM infected Vero-81 cells at 32°C and 37°C at 5 days post-infection. C) Schematic of D2-vFLM mammalian cell adaptation. Multiple-step growth curves of DV2, D2-FLM, and D2-vFLM in D) Vero-81 and E) C6/36 cells. F) Western blot of DV2 and D2-vFLM with a-E and a-prM antibodies. Right: over-exposure to visualize prM in D2-vFLM. G) Quantification of prM:E ratio of D2-vFLM, normalized to DV2. Growth curves were analyzed by two-way ANOVA with multiple comparison to DV2 control at different time points. Western blot quantification was analyzed by Students t-test.

Compensatory mutations in structural proteins are important for Vero-81 adaptation.

A) Virus yield of D2-vFLM-strucutral and -nonstructural infectious clones grown on C6/36 cells. Multiple-cycle growth curves of B) Vero grown or C) C6/36 grown virus D2-vFLM-strucutral and -nonstructural viruses on Vero-81 cells. Growth curves were analyzed by two-way ANOVA with multiple comparison to D2-vFLM control at different time points.

Engineered and acquired mutations for different D2-vFLM mutants in Vero-81 cells.

Grafting and adaptation of FLM mutations on DENV4.

A) Growth curves of DV4, D4-FLM and D4-vFLM on Vero-81 cells. B) Acquired mutations of D4-vFLM after serial passage in Vero-81 cells. C) Structural model of DENV-Envelope-dimer with color coded FL mutations and Vero adaptation mutations from DENV2 and DENV4. Growth curves were analyzed by two-way ANOVA with multiple comparison to DV4 control at different time points.

Antigenicity of DENV and DENV-vFLM.

FRNT50 of (A) DV2 and D2-vFLM or (B) DV4 and D4-vFLM against mAbs on Vero-81 cells. EC50 of (C) DV2-nLuc and D2-vFLM-nLuc or (D) DV4-nLuc and D4-vFLM-nLuc against mAbs on Vero-81 cells. Statistics were performed by Students t-test.

Immunogenicity of DVs and D2-vFLMs in vivo.

A) Homotypic (against DV2-nLuc) and heterotypic (against DV4-nLuc) EC50 of pooled serum of DV2 and D2-vFLM from pre-immunization, 2wk after prime, 2wk after 1°boost, and 2 wk after 2°boost. B) Homotypic and heterotypic EC50 of individual DV2 and D2-vFLM serum from immunized C57BL/6 mice after 2°Boost. EC50 of pooled serum were analyzed by two-way ANOVA with multiple comparisons compared to pre-immunization. Mean EC50 of the 2°boost was compared by Students t-test.

High-throughput nLuc-based ADE assay shows no heterotypic ADE from serum isolated from D2-vFLM immunized mice.

Monoclonal Ab control and fold-increase in infectivity of DV4-nLuc and D4-vFLM-nLuc against A) 4G2, B) 2H2, and C) B7 in K562 cells. D) Heterotypic ADE against pooled serum from DV2 and D2-vFLM immunized mice that are positive against DV4. Error bars denote +1 S.D. Data was analyzed by two-way ANOVA; statistical significance shown corresponds to the interaction between groups.

D2-vFLM immunization protects AG129 mice from lethal challenge.

AG129 mice were immunized with DV2 (n=4), D2-vFLM (n=5), or mock (n=6) immunized and challenged with D220 virus. A) Percent weight loss, analyzed by linear mixed-effects model with repeated measures. Pairwise group comparisons were performed using estimated marginal means with Tukey adjustment. B) Survival curves up to 10 days post challenge, assessed with Log-rank (Mantel–Cox) test, and pairwise comparisons performed with Bonferroni correction for multiple testing. C) Serum viral titer from d1pi - d4pi. Individual mice are only bled twice per week for humane treatment. P < 0.05 was considered statistically significant. Θ represents unexpected mouse death on d1pi in the DV2 immunization group.

Temperature and pH stability of DV2 and D2-vFLM assessed using a nLuc reporter assay.

A) Infectivity of DV2-nLuc and D2-vFLM-nLuc following incubation at temperatures ranging from 4°C to 55°C, normalized to the control 4°C condition. B) Infectivity of DV2-nLuc and D2-vFLM-nLuc following exposure to buffers at pH 8.4, 7.4, 6.4, or 5.4, normalized to the untreated control. Statistics were performed by Students t-test.

Neutralization curves of pooled serum samples from A) DV2 and B) D2-vFLM immunized mice against homotypic and heterotypic DENV-nLuc.

Individual neutralization curves of A) DV2 and B) D2-vFLM serum after 2°boost against homotypic (DV2-nLuc) and heterotypic (DV4-nLuc).

Individual heterotypic ADE curves of DV4-nLuc and D4-vFLM-nLuc against DV2 and D2-vFLM serum from immunized mice.

Vertical dotted lines denote the EC50 of the serum against DV4-nLuc.