Detection of post-vaccination enhanced dengue virus infection in macaques: An improved model for early assessment of dengue vaccines

Abstract

The need for improved dengue vaccines remains since the only licensed vaccine, Dengvaxia, shows variable efficacy depending on the infecting dengue virus (DENV) type, and increases the risk of hospitalization for severe dengue in children not exposed to DENV before vaccination. Here, we developed a tetravalent dengue purified and inactivated vaccine (DPIV) candidate and characterized, in rhesus macaques, its immunogenicity and efficacy to control DENV infection by analyzing, after challenge, both viral replication and changes in biological markers associated with dengue in humans. Although DPIV elicited cross-type and long-lasting DENV-neutralizing antibody responses, it failed to control DENV infection. Increased levels of viremia/RNAemia (correlating with serum capacity at enhancing DENV infection in vitro), AST, IL-10, IL-18 and IFN-γ, and decreased levels of IL-12 were detected in some vaccinated compared to non-vaccinated monkeys, indicating the vaccination may have triggered antibody-dependent enhancement of DENV infection. The dengue macaque model has been considered imperfect due to the lack of DENV-associated clinical signs. However, here we show that post-vaccination enhanced DENV infection can be detected in this model when integrating several parameters, including characterization of DENV-enhancing antibodies, viremia/RNAemia, and biomarkers relevant to dengue in humans. This improved dengue macaque model may be crucial for early assessment of efficacy and safety of future dengue vaccines.

Fig 1. DPIV elicited broad and long-lasting DENV-nAb responses.

Three groups of rhesus macaques received two intra-muscular administrations, 28 days apart, of the indicated formulations. Sera collected before and after immunization were tested, in duplicate, using a plaque reduction neutralization test (PRNT) for their neutralizing activity against each of the four DENV types. The individual reciprocal serum dilutions associated with 50% reduction in plaque counts (PRNT50 titers) were determined. Shown are the geometric mean titers (GMT) and 95% confidence intervals (CI) (n = 10/group except for Gr.3 at day 254, n = 9). Dotted lines indicate the limit of detection. ^§Sera were collected in Gr.3 at day 168 instead of 173 in Gr.1-2.

Fig 2. Viremia and RNAemia detected after challenge of Gr.1, 2 and 4 with either DENV-1 0111/2011 or DENV-2 0126/2010.

At month 8 post-second immunization, Gr.1, 2 and 4 were divided into two subgroups each (n = 5) which were subcutaneously inoculated with approximately 10⁵ plaque-forming units (PFU) of either DENV-1 0111/2011 or DENV-2 0126/2010. (A) Shown are the individual viremia (expressed as plaque-forming units (PFU)/mL) and RNAemia (expressed as genome equivalent (ge)/mL) determined, using frozen-thawed sera, after inoculation with DENV-1 0111/2011 or DENV-2 0126/2010. Horizontal black and grey dotted lines indicate the threshold of detection for viremia and RNAemia, respectively. Horizontal green and red dashed lines indicate the lowest and highest RNAemia peaks detected in the corresponding non-vaccinated subgroup. Animals with RNAemia peaks higher than the highest peaks detected in non-vaccinated groups are indicated in red font. (B) Fresh sera collected at days 2 and 7 post-challenge were also tested for their viremia content, in parallel. Shown are the individual viremia titers determined using either fresh or frozen-thawed sera from days 2 and 7 post-challenge. Circle, square and triangle symbols correspond to values obtained with Gr.1, 2 and 4, respectively. Open and black symbols correspond to values obtained after challenge with DENV-1 0111/2011 and DENV-2 0126/2010, respectively. The horizontal dashed line indicates the threshold of detection for the plaque assay. (C) Shown are geometric mean ratio (GMR) and 95% confidence intervals (CI) for RNAemia area under the curves (AUC) and peak levels between each of the vaccinated groups and the non-vaccinated Gr.4. RNAemia AUC and peak levels were compared between vaccinated and non-vaccinated groups using an ANOVA model and a non-parametric analysis (ANOVA on ranks), respectively.

Fig 3. Viremia and RNAemia detected after challenge of Gr.3 and 5 with either DENV-2 0126/2010 or DENV-2 S16803.

At month 8.5 post-second immunization, Gr.3 and 5 were divided into two subgroups each (n = 5 but Gr.3/DENV-2 S16803, n = 4) which were subcutaneously inoculated with approximately 4x10⁴ plaque-forming units (PFU) of DENV-2 S16803 or approximately 10⁵ PFU of DENV-2 0126/2010. (A) Shown are the individual viremia (expressed as plaque-forming units (PFU)/mL) and RNAemia (expressed as genome equivalent (ge)/mL) curves determined using either fresh (solid symbols) or frozen-thawed (open symbols) sera. Horizontal black and grey dotted lines indicate the threshold of detection for viremia and RNAemia, respectively. Horizontal green and red dashed lines indicate the lowest and highest RNAemia peaks detected in the corresponding non-vaccinated subgroup. Animals with RNAemia peaks higher than the highest peaks detected in non-vaccinated groups are indicated in red font. (B) Shown are the geometric mean ratio (GMR) and 95% confidence intervals (CI) for viremia and RNAemia area under the curves (AUC) determined using fresh versus frozen-thawed sera. Statistical comparisons were based on a paired t-test (*, p<0.05). (C) Shown are the GMR and 95% CI for viremia (determined using fresh sera) and RNAemia (determined using frozen-thawed sera) AUC and peak levels between the vaccinated Gr.3 and the non-vaccinated Gr.5. RNAemia AUC and peak levels were compared between vaccinated and non-vaccinated groups using an ANOVA model and a non-parametric analysis (ANOVA on ranks), respectively (both without adjusting for multiplicity; *, p<0.05).

Fig 4. Correlation between pre-challenge DENV-nAb titers or serum DENV-enhancing capacities and post-challenge RNAemia peaks.

Spearman correlations were performed to compare individual RNAemia peaks with pre-challenge (day 254) PRNT50 titers (A) or serum DENV-enhancing capacities (B), as measured, in a U937 cells-based antibody-dependent enhancement assay, by the serum dilution associated with infection enhancement peak, with the lowest and highest dilutions corresponding to the highest and lowest enhancing capacities, respectively (**, p<0.01; ***, p<0.001).

Fig 5. Post-challenge immune mediator profiles among vaccinated versus non-vaccinated macaques.

Sera collected before and after DENV challenge were tested for their concentration in the indicated immune mediators. (A) Shown are the mean changes from baseline and SEM (n = 5 but Gr.3/DENV-2 S16803, n = 4). The log₁₀-transformed changes from baseline were analyzed using an ANCOVA model. The p-values compare vaccinated to their corresponding non-vaccinated control subgroups with color codes referring to the vaccinated groups (*, p<0.05; **, p<0.01; ***, p<0.001). (B) Heat map representation of normalized scores of individual maximum changes from baseline in immune mediator levels. Monkeys were grouped by DENV challenge strain/wave, further divided based on their vaccination status, and ranked, within each subgroup, based on their maximum RNAemia level, monkeys with the lowest and the highest RNAemia peaks being on the left and the right sides, respectively. The immune mediators for which the maximum levels were further shown to significantly differ between vaccinated and non-vaccinated macaques are shown in red font. Also indicated are the macaques having shown more than 1-log higher RNAemia peaks compared to the highest RNAemia peaks detected in the corresponding non-vaccinated control groups (AF125 and AG179) as well as the only vaccinated macaque who was protected from post-challenge DENV replication (AH85). (C) A stratified non-parametric test was used to compare, across the DENV challenge strains/waves and between vaccinated and non-vaccinated macaques, the maximum immune mediator levels detected after DENV challenge. Shown are the individual values for the immune mediators for which the maximum levels significantly differed between vaccinated and non-vaccinated macaques (*, p<0.05; **, p<0.01; ***, p<0.001).