Zika virus pathogenesis in rhesus macaques is unaffected by pre-existing immunity to dengue virus

Abstract

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis.

Figure 1. In vitro ADE of ZIKV infection by macaque DENV-immune antisera in K-562 cells.

Sera from four DENV-immune (two per serotype) and four naïve macaques were serially diluted and incubated with ZIKV strain H/PF/2013 at a multiplicity of infection (MOI) of 1. K-562 cells were added and incubated for 22 h, and the percentage of ZIKV-infected cells were determined by flow cytometry after intracellular staining with E-specific mAb 4G2 conjugated to Alexa-488. DENV-1 immune macaques are in blue, DENV-2 immune ones in magenta, and naïve ones in black. Data are representative of the average of triplicate samples per concentration in one experiment. Statistically significant differences among groups were calculated by two-way ANOVA using Bonferroni's multiple comparisons test.

Figure 2. Experimental design of ZIKV infection in DENV-immune and naïve macaques.

Two cohorts of rhesus macaques (Macaca mulatta) were used. Cohort 1 was composed of four macaques previously infected with DENV (5 × 10⁶ p.f.u., s.c.) in 2013. Two macaques were from a group (n=4) infected with DENV-1 Western Pacific 74 (WP74) strain (BS14 and BP71), and the other two macaques from a group (n=4) infected with DENV-2 New Guinea 44 (NGC44) strain (MA071 and BS02). Cohort 1 in 2013 was sequentially bled at baseline, 30, 60 d.p.i., 1.5 and 2.5 years post DENV infection. Macaques in cohort 1 include one with high and one with low NAb titres against DENV-1 or DENV-2 (Supplementary Fig. 3). Cohort 2 contains four flavivirus-naïve macaques as control (5K6, CB52, 2K2 and 6N1). Macaques in both cohorts matched in age and sex. Cohorts were bled 30 days (Baseline) prior to infecting with ZIKV, and in August 1, 2016 they were infected with ZIKV (1 × 10⁶ p.f.u., s.c.) H/PF/2013 strain. Afterwards, all macaques were first bled from 1 to 10 d.p.i., and then on 15, 30 and 60 d.p.i. Furthermore, saliva and urine samples were also collected at the same timepoints. ZIKV infection was performed 2.8 years after DENV infection.

Figure 3. Vital signs and clinical status of macaques before and after ZIKV infection.

(a) Weight expressed in kilograms (kg). (b) Corporal temperature (in Celsius) measured using an infrared device. (c) Rectal temperature (in Celsius) was also measured. (d–g) Cell subsets: white blood cell (WBC), lymphocyte (LYM), neutrophil (NEU) and monocyte (MON) kinetics obtained from complete blood cell (CBC) counts (10⁶ cells per ml) at baseline and on days 7 and 15 p.i. in absolute numbers. Comparison of absolute NEU numbers within cohorts at day 7 related to their own baseline values was performed using a two-tailed unpaired t-test with Sidak–Bonferroni correction (P<0.01). (h–j) Levels of platelets (10⁶ PLT per ml), Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) at baseline, 7 and 15 d.p.i. In all panels, the DENV-1 immune macaques are in blue, DENV-2 immune ones in magenta, and naïve ones in black.

Figure 4. DENV-immune serum cross-reacts with ZIKV.

(a) Binding of baseline samples from the naïve (black circles) and the DENV-immune macaques (blue and magenta symbols for DENV-1 and DENV-2 immune macaques, respectively) assayed in six concentrations of serum and expressed as OD values at 492 nm. (b) Binding of samples collected 30 d.p.i. is represented as in a. In the first two-endpoints, serum from previous immunity to DENV resulted in significantly higher OD values (P<0.01 and P<0.05). Data are representative of duplicate samples per concentration in one experiment. Comparison of ZIKV IgG titres, expressed as OD values, within cohorts related to their own baseline values was performed using a two-tailed unpaired t-test with Sidak–Bonferroni correction (*P<0.05).

Figure 5. ZIKV RNA kinetics in serum.

ZIKV replication detected in serum in the first 10 days p.i. and on days 15 and 30 p.i. Genome copies per ml in blood serum samples from individual macaques are shown logarithmically. Orange and black lines represent mean values for the DENV-immune and naïve macaques, respectively. The limit of detection, 500 genome copies per ml, of our assay is indicated in black dotted line.

Figure 6. Pre-exposure to DENV results in lower activation of B and T cells.

(a) Frequency of activated B cells (CD20⁺CD3⁻CD14⁻CD69⁺) were statistically higher compared to their basal level in the naïve cohort at 24 and 48 h.p.i. (b,c) T cells were measured by flow cytometry 0, 1, 2, 7, 10 and 15 d.p.i of ZIKV. (b) CD4 T cells (CD3⁺CD4⁺CD69⁺) showed statistically higher activation at 24 and 48 h and on days 10 and 15 p.i. in the naïve compared to their own baseline values while DENV-immune macaques had higher frequency only at 24 and 48 h p.i. Comparison of frequency of activation of B and CD4⁺ T cells inside cohorts related to their own baseline values was performed using a two-tailed unpaired t-test with Sidak–Bonferroni correction. (c) CD8 T cell (CD3⁺CD8⁺CD69⁺) subsets showing significant higher frequency in naïve cohort at 24 and 48 h and on days 7 and 10 p.i. Comparison of frequency of activation of CD8⁺ T cells between two cohorts was performed using a two-tailed unpaired t-test with Sidak–Bonferroni correction. Median values are shown in orange and black lines for DENV-immune and naïve macaques, respectively. Black circles represent individual naïve macaques, and blue and magenta squares represent individuals previously exposed to either DENV-1 or DENV-2, respectively.

Figure 7. Antigen-specific CD8⁺ and CD4⁺ T cell responses in naïve and DENV-immune macaques.

(a–l) All percentages shown are subtracted from the unstimulated background. (a–f) Analysis of the CD8⁺ T cell from the PBMCs of ZIKV-infected naïve and DENV-immune macaques (n=4 per cohort). PBMCs were harvested on day 30 and 60 p.i., and intracellular cytokine staining of IFN-γ (a), TNF-α (b) and CD107a (c) were analysed in CD8⁺ T cells after ex vivo re-stimulation with the listed stimuli. Intracellular cytokine staining of IFN-γ (d), TNF-α (e) and CD107a (f) were analysed for CD8⁺ T cells after ex vivo re-stimulation with the listed stimuli (for naïve group 30 d.p.i. n=3). (g–l) Analysis of the CD4⁺ T cell from the PBMCs of ZIKV-infected naïve and DENV-immune macaques (n=4 per group). PBMCs were harvested on day 30 and 60 p.i., and intracellular cytokine staining of IFN-γ (g), TNF-α (h) and CD107a (i) were analysed in CD4⁺ T cells after ex vivo re-stimulation with the listed stimuli. Intracellular cytokine staining of IFN-γ (j), TNF-α (k) and CD107a (l) were analysed in CD4⁺ T cells after ex vivo re-stimulation with the listed stimuli (for naïve group 30 d.p.i n=3).

Figure 8. Previous exposure to DENV modulates the cytokine and chemokine profiles after ZIKV infection.

(a–h) Profiles of marked cytokines and chemokines after ZIKV infection of DENV-immune and naïve cohorts are depicted logarithmically in pg ml⁻¹. Mean values are shown in orange and black lines for DENV-immune and naïve cohorts, respectively. Comparison of the levels of cytokines and chemokines within cohorts related to their own baseline values or among cohorts was performed using a two-tailed unpaired t-test with Sidak–Bonferroni correction. Black circles represent individual naïve macaques, and blue and magenta squares represent macaques previously exposed to either DENV-1 or DENV-2, respectively. Dotted lines indicate the threshold of detection for each assay.