Characterization of recent and minimally passaged Brazilian dengue viruses inducing robust infection in rhesus macaques

Abstract

The macaque is widely accepted as a suitable model for preclinical characterization of dengue vaccine candidates. However, the only vaccine for which both preclinical and clinical efficacy results were reported so far showed efficacy levels that were substantially different between macaques and humans. We hypothesized that this model's predictive capacity may be improved using recent and minimally passaged dengue virus isolates, and by assessing vaccine efficacy by characterizing not only the post-dengue virus challenge viremia/RNAemia but also the associated-cytokine profile. Ten recent and minimally passaged Brazilian clinical isolates from the four dengue virus serotypes were tested for their infectivity in rhesus macaques. For the strains showing robust replication capacity, the associated-changes in soluble mediator levels, and the elicited dengue virus-neutralizing antibody responses, were also characterized. Three isolates from dengue virus serotypes 1, 2 and 4 induced viremia of high magnitude and longer duration relative to previously reported viremia kinetics in this model, and robust dengue virus-neutralizing antibody responses. Consistent with observations in humans, increased MCP-1, IFN-γ and VEGF-A levels, and transiently decreased IL-8 levels were detected after infection with the selected isolates. These results may contribute to establishing a dengue macaque model showing a higher predictability for vaccine efficacy in humans.

Fig 1. Viremia and RNAemia detected after inoculation with DENV-1 0111/2011, DENV-2 0126/2010 or DENV-4 BEL 83791.

Rhesus macaques were subcutaneously inoculated with ~10⁵ plaque- or focus-forming units (PFU and FFU, respectively) of DENV-1 0111/2011 (n = 5), DENV-2 0126/2010 (n = 5) or DENV-4 BEL 83791 (n = 6). Sera were collected daily during the 14 days post-inoculation and tested, in duplicate, by plaque or focus assay for their infectious virus content, i.e. viremia, expressed as PFU or FFU/mL (A) and by real-time RT-PCR for their DENV genome equivalent (ge) content, i.e. RNAemia, expressed as ge/mL (B). The individual viremia and RNAemia curves are shown. Horizontal dashed lines indicate the threshold of detection for the plaque/focus assay and the real-time RT-PCR assay, i.e. 2.5 PFU or FFU/mL and 10² ge/mL, respectively. In the absence of viremia and/or RNAemia detection, the corresponding sample was assigned an arbitrary titer corresponding to half the limit of detection.

Fig 2. DENV-neutralizing antibody responses detected after infection with DENV-1 0111/2011, DENV-2 0126/2010 or DENV-4 BEL 83791.

Sera collected 2 weeks after DENV inoculation were tested by plaque-reduction neutralization test (PRNT) for their neutralizing activity against DENV-1 60305, DENV-2 44/2, DENV-3 16562 and DENV-4 TVP360. The individual serum titers associated with 50% reduction in plaque counts (PRNT50), geometric mean titers (GMT) and 95% confidence intervals (CI) are shown. Dashed lines indicate the limit of detection. In the absence of detection of neutralizing activity, the corresponding sample was assigned an arbitrary titer corresponding to half the limit of detection. P-values were calculated using the Tukey’s multiple comparisons test: **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig 3. Serum cytokine profiles observed after infection with DENV-1 0111/2011, DENV-2 0126/2010 or DENV-4 BEL 83791.

Sera collected before (baseline) and at days 1, 4, 6, 8 and 14 after DENV inoculation were tested, in duplicate, for their concentration in the indicated cytokines. Results were expressed as pg/mL. When no signal was detected, the corresponding sample was assigned the arbitrary value of half the limit of detection for the corresponding cytokine. Shown are the mean changes from baseline and SEM from 5 (DENV-1 0111/2011 and DENV-2 0126/2010) and 6 (DENV-4 BEL 83791) animals.