Human T cell responses to Dengue and Zika virus infection compared to Dengue/Zika coinfection

Abstract

Introduction: Zika virus (ZIKV) and dengue virus (DENV) co-circulated during latest outbreaks in Brazil, hence, it is important to evaluate the host cross-reactive immune responses to these viruses. So far, little is known about human T cell responses to ZIKV and no reports detail adaptive immune responses during DENV/ZIKV coinfection.

Methods: Here, we studied T cells responses in well-characterized groups of DENV, ZIKV, or DENV/ZIKV infected patients and DENV-exposed healthy donors. We evaluated chemokine receptors expression and single/multifunctional frequencies of IFNγ, TNF, and IL2-producing T cells during these infections. Even without antigenic stimulation, it was possible to detect chemokine receptors and IFNγ, TNF, and IL2-producing T cells from all individuals by flow cytometry. Additionally, PBMCs' IFNγ response to DENV NS1 protein and to polyclonal stimuli was evaluated by ELISPOT.

Results: DENV and ZIKV infections and DENV/ZIKV coinfections similarly induced expression of CCR5, CX3CR1, and CXCR3 on CD4 and CD8 T cells. DENV/ZIKV coinfection decreased the ability of CD4⁺ T cells to produce IFNγ⁺ , TNF⁺ , TNF ⁺ IFNγ⁺ , and TNF ⁺ IL2⁺ , compared to DENV and ZIKV infections. A higher magnitude of IFNγ response to DENV NS1 was found in donors with a history of dengue infection, however, a hyporesponsiveness was found in acute DENV, ZIKV, or DENV/ZIKV infected patients, even previously infected with DENV.

Conclusion: Therefore, we emphasize the potential impact of coinfection on the immune response from human hosts, mainly in areas where DENV and ZIKV cocirculate.

Keywords: Dengue; Zika: T lymphocytes.

Figure 1

Chemokine receptor expression on CD4⁺ and CD8⁺ T cell populations in acute DENV, ZIKV, and DENV/ZIKV patients. Peripheral blood mononuclear cells (2 × 10⁵) were stained with mAb against surface markers CD3, CD8, CD4, CCR5, CX3CR1, and CXCR3. The expression of CCR5, CX3CR1, and CXCR3 on CD4⁺ (a) and CD8⁺ T cells (e) was showed in contour plots from one representative exposed donor, DENV‐, ZIKV, and DENV/ZIKV‐patients by flow cytometry. Frequency, median, 25th and 75th percentile of CCR5⁺ (b), CX3CR1⁺ (c), and CXCR3⁺ (d) CD4⁺ T cells from acute viral patients were compared between them and with those in exposed healthy controls. The same strategy was used for CD8⁺ T cells (f–h). Patient 2 in late acute phase (22 days of illness) was show as open squares. Statistical significance of differences between groups was determined by using two‐tailed Mann–Whitney test, where p < 0.05 were considered significant (*p < 0.05; **p < 0.01).

Figure 2

Frequency of IFNγ‐, TNF‐, and IL2‐producing CD4⁺ and CD8⁺ T cells in acute DENV, ZIKV, and DENV/ZIKV‐patients and exposed donors. Cultures of 2 × 10⁵ PBMCs were stimulated with PMA/Ionomycin or unstimulated (medium) for 6 h in presence of brefeldin in the last 4 h. Then, cells were stained with mAb against surface markers CD3, CD8, CD4, and mAb against intracellular IFNγ, TNF, and IL2. Frequency of IFNγ, TNF, and IL2 on CD4⁺ (a) and CD8⁺ T cells (f) was exhibited in counter plots from one representative exposed donor, DENV‐, ZIKV, and DENV/ZIKV‐patients by flow cytometry for both conditions. Frequency, median, 25th and 75th percentile of IFNγ⁺ (b), TNF⁺ (c), and IL2⁺ (d) CD4⁺ T cells in unstimulated condition from acute viral patients were compared between them and with those in exposed healthy controls. The same strategy was used (g–i). Patient 2 in late‐acute phase (22 days of illness) was show as open squares. Bars represent the median of frequency of CD4⁺ T cells (e) and CD8⁺ T cells (j) expressing each of the seven possible combinations of IFNγ, TNF, and IL2 among the studied groups in unstimulated condition. Statistical significance of differences between groups and comparisons among the multifunctional populations were determined by using two‐tailed Mann–Whitney test and represented by lines. Values of p < 0.05 were considered significant (*p < 0.05; **p < 0.01).

Figure 3

DENV‐specific cells targeting DENV 1–4 NS1 protein in acute DENV, ZIKV, and DENV/ZIKV‐patients, and donors experiencing dengue. Peripheral blood mononuclear cells (2 × 10⁵ in 0.1 mL) were incubated with recombinant mammalian (rm) NS1 (0.1 μg/mL) derived from four different DENV serotypes or PHA for 20 h and IFNγ production was measured by ELISPOT assay. (a) Representative IFNγ production from one representative exposed donor, DENV, ZIKV, and DENV/ZIKV‐patients are shown against rmNS1. (b) The values obtained of IFNγ production, expressed as spot‐forming cells (SFC) relative to 10⁶ PBMC, is shown for the rmNS1 and PHA were compared between them and with those in exposed healthy controls. Graphs show the median, 25th and 75th percentile from acute‐patients and DENV‐exposed ZIKV naïve donors. Statistical significance was determined by using the two‐tailed Mann–Whitney test, where p < 0.05 were considered significant (*p < 0.05; **p < 0.01).