 mice produced DENV-2 specific neutralizing antibody with mean reciprocal 50% endpoint geometric mean titers ranging from 173 to 361. None of the mean neutralizing antibody titers from the cross-reactivity reduced immunized mice differed significantly from that of WT immunized mice. There was however, a significant effect of vaccine treatment on DENV-2 neutralizing antibody titer as determined by a non-parametric Kruskal-Wallis test (p = 0.03), and a Dunn’s post test revealed RD immunized mouse sera, that had the highest mean neutralizing antibody titer, was significantly different from RDERR vaccinated sera that had the lowest titer (p = 0.02). In mice immunized with cross-reactivity reduced vaccines containing substitutions in EDIII, ERR and RDERR, there was a trend toward a decrease in homologous DENV-2 neutralization. The apparent reduction in neutralization induced by EDIII substitution containing vaccines could be due to reductions in EDIII cross-reactive neutralizing antibody, some of which can neutralize potently, e.g., MAb 1A1D-2 or 9D12 [16, 19, 31]. There were no significant differences in mean 50% FRμNT titers as determined by a non-parametric Kruskal-Wallis test between WT and any of the cross-reactivity reduced vaccine immunized mouse sera when tested against DENV-1 (p = 0.08), DENV-3 (p = 0.18), and DENV-4 (p = 0.39) (Figure 1B-D).
Cross-reactivity reduced vaccines elicit DENV specific neutralizing IgG. (A) DENV-2 16681 neutralizing-antibody (Nt-Ab) titers elicited by WT and cross-reactivity reduced vaccines as determined by 50% FRμNT. (B) DENV-1 56BC94/95 Nt-Ab titers. (C) DENV-3 116RC1396 Nt-Ab titers. (D) DENV-4 H241 Nt-Ab titers. Data are expressed as GMT +/− 95%CI. for n = 10. Kruskal-Wallis test and Dunn’s post test was used to analyze data. p < 0.05 were considered significant; single asterisk, p < 0.05.
We utilized an epitope-specific IgG ELISA [19] to determine the quantity and quality of the cross-reactivity reduced vaccine DENV-2 antibody response (Table 2). Both murine and human monoclonal antibodies directed against the EDIIFP have been shown to enhance DENV infection leading to lethal disease in the AG129 mouse model [23–25]. Similar to studies examining DENV infected human sera [17, 19], the vaccines containing an unmodified EDIIFP, WT and ERR vaccine, elicited a large proportion of the total E response directed against EDIIFP epitopes (average 66.8% and 81.5%, respectively), which was highly variable between individual outbred mouse sera. This data agrees with the monoclonal antibody mapping of Table 1 where WT and ERR VLPs maintain full reactivity toward group cross-reactive antibodies. Cross-reactivity reduced plasmids containing EDIIFP substitutions (RD and RDERR) elicited almost no antibodies recognizing the WT EDIIFP epitope (Table 1; means of 0.3% and <1.0%, and p = 0.005 and p = 0.002 compared to WT for RD and RDERR respectively). Despite this altered antibody profile, total DENV-2 IgG endpoint mean titers elicited by WT and all cross-reactivity reduced plasmids were similar and ranged from 4.6 × 104 to 1.6 × 105 (n = 10 each, p = 0.22).
We next compared the three different cross-reactivity reduced vaccine constructs to determine whether altered cross-reactive antibody profiles can reduce the potential for ADE in vitro. FcRII bearing human K562 cells were infected with DENV alone, with DENV-immune complexes formed in the presence of mouse sera from WT or cross-reactivity reduced vaccine immunized mice, or with DENV-immune complexes formed in the presence of EDIIFP recognizing MAb 4G2. The K562 cell line has previously been shown to support ADE of infection as a relevant model for DHF [32–37]. Sera from WT immunized mice significantly enhanced the infection of homologous DENV-2 at relatively high serum dilutions whereas cross-reactivity reduced vaccine immune mouse sera did not enhance DENV-2 ant any serum concentration tested. WT vaccinated sera enhanced at a peak titer of 1:1,250 compared to RD (p = 0.01), ERR (p = 0.021), and RDERR (p = 0.01) (Figure 2A).
Cross-reactivity reduced vaccines have reduced potential to participate in ADE in vitro compared to WT vaccine. (A) DENV-2 16681 enhancement reported as percent infection compared to the virus input control (dashed line) (B) DENV-1 56BC94/95 percent infection. (C) DENV-3 116RC1396 percent infection. (D) DENV-4 H241 percent infection. All data represent the mean+/−s.e.m. of two independent experiments. Two-way ANOVA were performed on square-root transformed data with a Bonfferoni post test; p < 0.05 were considered significant; single asterisk, p < 0.05; two asterisks, p < 0.01.
In heterologous ADE assays, sera from WT vaccinated mice typically enhanced at the lowest serum dilutions in the assay, RD and ERR vaccinated sera enhanced DENV-1 and −3 at some serum dilutions, and only RDERR vaccinated sera lacked statistically significant enhancement of any DENV serotype at any dilution (Figure 2B-D). 4G2 control antibody enhanced DENV-1, -3, and −4 strongly between 1:50 and 1:1,250 dilutions but did not enhance DENV-2 at the dilutions we tested. This observation is consistent with both in vitro and in vivo studies demonstrating that this DENV-2 raised antibody enhanced infection in vitro at dilutions greater than 200,000, beyond the dilutions of this high titer ascites fluid tested here [38, 39]. 4G2 has also been demonstrated to neutralize and even protect from DENV-2 challenge at higher concentrations [23, 25, 38, 39]. WT vaccinated sera significantly enhanced DENV-1 infection at the lowest dilution tested (1:2) compared to RD (p = 0.05), ERR (p = 0.01), and RDERR (p = 0.001), and also enhanced DENV-1 infection at a 1:10 dilution compared to ERR (p = 0.04) and RDERR (p = 0.03) (Figure 2B). WT vaccinated sera significantly enhanced DENV-3 infection at a dilution of 1:2 compared to only RDERR (p = 0.029) and at a dilution of 1:10 compared to RD (p = 0.026) and RDERR (p = 0.006) (Figure 2C), whereas ERR vaccinated sera enhancement was not significantly different from that of WT vaccinated sera. This suggests an important role of the immunodominant EDIIFP targeting antibody response in the enhancement of severe disease because RD and RDERR vaccines do not produce antibodies which recognize WT EDIIFP while ERR immunized mice produced similar proportions of EDIIFP recognizing antibody as WT (Table 2). None of the serum from vaccinated mice significantly enhanced DENV-4 infection (Figure 2D). Thus, the in vitro enhancement analysis indicated that the combination of substitutions in EDIIFP and EDIIICR incorporated into the RDERR plasmid elicited the highest quality antibody response as only RDERR immune sera lacked DENV enhancing capabilities.
Cross-reactivity reduced vaccine increases survival from ADE in vivo. Survival of AG129 mice passively transferred 100 or 50 μL of pooled WT or RDERR immune sera from homologous ADE with 4.2 × 104 ffu of DENV-2 S221. Kaplan-Meyer survival analysis (n = 10). p < 0.008 were considered significant due to Bonferonni adjusted alpha for multiple comparisons; three asterisk p < 0.0001.
Because of the high DENV-2 neutralizing antibody titer, the combination of reduced cross-reactive antibodies with the lack of any in vitro enhancement, we selected RDERR as the best cross-reactivity reduced vaccine candidate to examine potential improvements in the quality of anti-DENV antibody response by the in vivo ADE assay using the published AG129 mouse model [23, 40].
Unfortunately, there are no published DENV-1, -3 or −4 mouse adapted dengue strains available to us that can cause vascular leak-associated enhanced disease in AG129 mice, making heterologous in vivo ADE difficult to examine. Previous studies have described the capability of mouse-adapted DENV-2 S221 strain to produce DHF-like disease via ADE in AG129 mice [23] allowing us to utilize this virus to test if reductions in cross-reactive antibody populations of passively transferred RDERR vaccinated Swiss Webster mouse sera can reduce homologous ADE in vivo. We passively transferred 100 μl or 50 μl of pooled sera from WT immunized, RDERR immunized, or naïve Swiss mice into AG129 mice (n = 10 each treatment). Sixteen hours post serum transfer, 2 randomly selected mice per group were tail bled and their circulating neutralizing antibody titers were determined. Eighteen hours post transfer, all mice