Figure 5.

Protection against heterologous challenge with a Puerto Rican ZIKV isolate. (a) Six animals (three females and three males) were re-challenged subcutaneously with 1 × 10⁵ TCID₅₀ of a PR ZIKV isolate 45 d after primary infection. Viral RNA levels in blood plasma were monitored between days 1 and 21 after the secondary challenge. ZIKV RNA copies in blood plasma are indicated. A single positive sample, detected in one male monkey 1 d after infection, is indicated (blue solid line). Lymphocyte activation and frequencies were measured during re-challenge. Activation and numbers of T cells, B cells, NK cells and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5 and 7 after ZIKV infection of three male and three female monkeys. (b,c) Percentage of activated (CD69⁺) and total numbers of naive (CD28⁺CD95⁻) (left), central memory (CD28⁺CD95⁺) (middle), and effector and effector memory (CD28⁻CD95⁺) (right) CD4⁺ (b) and CD8⁺ (c) T cell subsets in whole blood. (d) Percentage of activated (CD69⁺) and total numbers of CD16⁺ (left), CD16⁻CD56⁺ (middle) and CD16⁻CD56⁻ (right) NK cells in whole blood. (e) CD38 expression (geometric mean fluorescence of CD38) and total numbers of naive (CD27⁻) (left) and memory (CD27⁺) (right) B cells in whole blood. (f) Percentage of activated and total numbers of monocytes in whole blood. Black line indicates median. Individual values are shown for males (blue) and females (red). (g) ZIKV-specific T cell responses were measured during ZIKV re-challenge. T cell degranulation and cytokine production (in PBMCs) was measured by flow cytometry after the stimulation of cells on days 0, 1, 2, 3, 5 and 7 after re-challenge with overlapping peptide pools spanning the entire ZIKV capsid and envelope proteins. Percentage of cells expressing CD107a, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-2 after a 6-h peptide stimulation are shown. The threshold for determining positive values, after background subtraction of unstimulated corresponding samples, was applied as previously described⁴³. Values below this threshold were set to a value of 0. Values greater than 1.5-fold above that of the day of re-challenge were considered to be anamnestic. Each monkey is indicated by a different color symbol. Triangles indicate males, and circles indicate females.