Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques

Abstract

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.

Fig. 1. Vaccination schedule and serum collection by study day.

Injections are indicated by a syringe. Serum and PBMC collections are indicated by their respective blood tube symbols.

Fig. 2. In vitro enhancement of DENV infection by sera from day 28 post-PIV vaccination.

Each of the four DENV types was mixed with a 1/8 dilution of sera from each group, and the infectivity of each was measured relative to the virus alone (fold-ADE). Bar heights are the means for each group. Data were analyzed using a one-way ANOVA model with Tukey’s multiple comparisons test. *p < 0.05.

Fig. 3. 50% neutralization titers against DENV-1–4 of sera from day 28 post-PIV vaccination.

Log₁₀-transformed data are shown. The lowest dilution tested was 1/8. Bar heights are the geometric means for each group. Data were analyzed using a one-way ANOVA model with Tukey’s multiple comparisons test. *p < 0.05.

Fig. 4. Viremia of DENV-2 live-attenuated vaccine virus.

Sera from days 0–9 post-LAV boost were tested by qRT-PCR for replication of the DENV-1–4 vaccine viruses. Only replication of DENV-2 was detectable. Data are presented as log₁₀-transformed genome equivalents (GE)/mL. Both group geometric means and data for individual animals within each group are shown. Data were analyzed using a mixed-effects model with Geisser–Greenhouse correction and Dunnett’s multiple comparisons test. Error bars show the standard error of the means.

Fig. 5. Peak viremia for DENV-2 live-attenuated vaccine virus and wild-type challenge virus.

Shown are the peak viremia values from each animal following LAV vaccination or the following wild-type DENV-2 infection of unvaccinated control animals. Data are presented as log₁₀-transformed genome equivalents (GE)/mL. Horizontal bars are the geometric means for each group. Data were analyzed by Welch’s ANOVA model and Dunnett’s T3 multiple comparisons test. *p < 0.05.

Fig. 6. Relationships between in vitro ADE and viremia.

Spearman correlations and non-linear regression analyses between in vitro fold-ADE of day 28 sera at a 1/8 dilution and either peak LAV viremia (a) or duration of LAV viremia (b) were performed. The upper panels show the data fit with an exponential, one-phase association curve with unconstrained parameters. The lower panels show the data fit similarly, but using an initial plateau region constrained to a fold-ADE value of 3. Use of the initial plateau region increased the Goodness of Fit r² value by >11%. Shaded regions represent 95% confidence intervals.

Fig. 7. Neutralizing antibody response following LAV vaccination.

Blood samples collected on day 28 post LAV vaccination (study day 56) were assessed for the neutralizing antibody titer of each group against each DENV type. Neutralizing antibody titers are presented as log₁₀-transformed NT50. The lowest dilution tested was 1/40. Bar heights are the geometric means for each group. Data were analyzed using a one-way ANOVA model with Tukey’s multiple comparisons test. *p < 0.05.

Fig. 8. Type-specific neutralizing antibody responses of representative animals in each PIV primed/LAV boosted group.

Sera were depleted of antibody by sequential rounds of incubation with polystyrene magnetic particles coated with BSA (control depleted), DENV-2 virions (D2 depleted), or DENV-1, -3, and -4 virions (D1 + D3 + D4 depleted). Neutralizing activity of depleted sera was tested by FlowNT. Data are presented as log₁₀-transformed NT50 values against each DENV type.

Fig. 9. Viremia resulting from the challenge of vaccinated and unvaccinated control animals with wild-type DENV-2.

All vaccinated animals were protected from detectable viremia upon challenge. All unvaccinated control animals had detectable DENV-2 viremia lasting from day 2 until day 8 or beyond. Data are presented as log₁₀-transformed genome-equivalents (GE)/mL. a Geometric mean viremia by the group; error bars show the standard error of the means. b Viremia curve of each animal in the unvaccinated control group shown in panel (a).