In-utero exposure to maternal HIV infection alters T-cell immune responses to vaccination in HIV-uninfected infants

Abstract

Objective: In sub-Saharan Africa, HIV-exposed uninfected (HEU) infants have higher morbidity and mortality than HIV-unexposed infants. To evaluate whether immune dysfunction contributes to this vulnerability of HEU infants, we conducted a longitudinal, observational cohort study to assess T-cell immune responses to infant vaccines (Mycobacterium bovis BCG and acellular pertussis) and staphylococcal enterotoxin B (SEB). In total, 46 HEU and 46 HIV-unexposed infants were recruited from Khayelitsha, Cape Town.

Methods: Vaccine-specific T-cell proliferation (Ki67 expression) and intracellular expression of four cytokines [interferon-γ, interleukin (IL)-2, IL-13 and IL-17] were measured after whole blood stimulation with antigens at 6 and 14 weeks of age.

Results: HEU infants demonstrated elevated BCG-specific CD4 and CD8 T-cell proliferative responses at 14 weeks (P = 0.041 and 0.002, respectively). These responses were significantly increased even after adjusting for birth weight, feeding mode and gestational age. Similar to BCG, increased CD4 and CD8 T-cell proliferation was evident in response to SEB stimulation (P = 0.004 and 0.002, respectively), although pertussis-specific T cells proliferated comparably between the two groups. Within HEU infants, maternal CD4 cell count and length of antenatal antiretroviral exposure had no effect on T-cell proliferation to BCG or SEB. HIV exposure significantly diminished measurable cytokine polyfunctionality in response to BCG, Bordetella pertussis and SEB stimulation.

Conclusion: These data show for the first time, when adjusting for confounders, that exposure to HIV in utero is associated with significant alterations to CD4 and CD8T-cell immune responses in infants to vaccines and nonspecific antigens.

Fig. 1. T-cell proliferative responses to BCG at 6 (a) and 14 (b) weeks: frequency of CD4⁺ (filled ovals and triangles on the left side of each graph) and CD8⁺ (open ovals and triangles on the right side of each graph) proliferating (Ki67+) T cells in whole blood of HIV-exposed uninfected infants (HEU: ovals) compared to HIV-unexposed infants (triangles)

(a) T-cell proliferative responses to BCG at 6 weeks; (b) T-cell proliferative responses to BCG at 14 weeks. Frequencies reported are the frequencies of proliferating cytokine producing T cells out of total CD4⁺ or CD8⁺ T cells. Bars indicate medians; alpha = 0.05; statistical analysis was performed using Mann–Whitney U test.

Fig. 2. Cytokine production by BCG-specific T cells

(a–d) proportions of BCG-specific proliferating CD4⁺ and CD8⁺ T cells producing no cytokine, one, or a combination of cytokine(s) at 6 and 14 weeks of life in HIV-exposed uninfected (HEU) versus HIV-unexposed infants. (a) Comparison of the proportion of proliferating CD4⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s), based on expression of IL-2, IFN-γ, IL-13 and/or IL-17, at 6 weeks by HEU versus HIV-unexposed infants. (b) Comparison of the proportion of proliferating CD8⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) at 6 weeks by HEU versus HIV-unexposed infants. (c) Comparison of the proportion of proliferating CD4⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) at 14 weeks. (d) Comparison of the proportion of proliferating CD8⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) at 14 weeks by HEU versus HIV-unexposed infants. Statistical analysis was performed using Wilcoxon signed-rank test. (e–h) Frequency of total CD4⁺ (a and c) and CD8⁺ (b and d) T cells expressing ki67 and no cytokine, each one, or each combination of IL-2, IFN-γ, IL-13 and/or IL-17, at 6 and 14 weeks of life in HEU versus HIV-unexposed infants. Each possible single cytokine or combinations thereof within the responding CD4⁺ and CD8⁺ T cells at 6 weeks (a and b) and 14 weeks (c and d). Frequency of HEU (blue) versus HIV-unexposed (red) T cells within the total CD4⁺ (left) and CD8⁺ (right) T cells that are proliferating and producing each specific cytokine or cytokine combination. Error bars and whiskers correspond to medians and interquartile ranges (IQRs), respectively. IFN, interferon; IL, interleukin; (^*) HU significantly higher; (^*) HEU significantly higher. ^*P <0.05; ^**P <0.05 after adjustment for multiple comparisons; statistical analysis was performed using Wilcoxon signed-rank test and multiple comparison adjustment using the Holm step-down procedure.

Fig. 3. Cytokine production by Bordetella pertussis-specific T cells

(a, b) Proportions of Bordetella pertussis-specific proliferating CD4⁺ and CD8⁺ T cells producing no cytokine, one, or a combination of cytokine(s) at 14 weeks of life in HEU versus HIV-unexposed infants. (a) Comparison of the proportion of cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) by proliferating CD4⁺ (left) and CD8⁺ (right) T cells in HEU versus HIV-unexposed infants in response to B. pertussis antigens at 14 weeks of life. (c, d) Frequency of total CD4⁺ (a) and CD8⁺ (b) T cells expressing ki67 and no cytokine, each one, or each combination of IL-2, IFN-γ, IL-13 and/or IL-17, at 6 and 14 weeks of life in HEU versus HIV-unexposed. Each possible single cytokine or combinations thereof of the cytokines IFN-γ, IL-2, IL-17, and IL-13 within the total CD4⁺ and CD8⁺ T cells. Frequency of HEU (blue) versus HIV-unexposed (red) T cells within the total CD4⁺ (left) and CD8⁺ (right) T cells expressing Ki67 and each specific cytokine or cytokine combination. Error bars and whiskers correspond to medians and interquartile ranges (IQRs), respectively. HEU, HIV-exposed uninfected; IFN, interferon; IL, interleukin. (^*) HIV-unexposed significantly higher; (^*) HEU significantly higher. ^*P <0.05; ^**P <0.05 after adjustment for multiple comparisons; statistical analysis was performed using Wilcoxon signed-rank test and multiple comparison adjustment using the Holm step-down procedure.

Fig. 4. T-cell proliferative responses to SEB at 6 and 14 weeks

Frequency of CD4⁺ (filled ovals and triangles on the left side of each graph) and CD8⁺ (open ovals and triangles on the right side of each graph) proliferating T cells of HIV-exposed uninfected infants (HEU: ovals) presented on the left of each side of the plot compared to HIV-unexposed infants (triangles) presented on the right of each side of the plot. Frequencies reported are the frequencies of proliferating cytokine producing cells out of total CD4⁺ or CD8⁺ T cells. Bars indicate medians; alpha = 0.05; statistical analysis was performed using Mann–Whitney U-test. SEB, staphylococcal enterotoxin B.

Fig. 5. Proportions of proliferating CD4⁺ and CD8⁺ T cells producing no cytokine, one, or a combination of cytokine(s) in response to SEB at 14 weeks of life in HIV-exposed uninfected (HEU) versus HIV-unexposed infants

(a) Comparison of the proportion of proliferating CD4⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s), based on expression of IL-2, IFN-γ, IL-13 and/or IL-17, at 14 weeks by HEU versus HIV-unexposed. (b) Comparison of the proportion of proliferating CD8⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) at 14 weeks by HEU versus HIV-unexposed infants. (c) Comparison of the proportion of proliferating CD4⁺ T cells expressing no cytokine (pink), any one (green), any combination of two (yellow), three (blue) or all four (red) intracellular cytokine(s) at 14 weeks. Statistical analysis was performed using Wilcoxon signed-rank test. (d) T cells that are SEB-specific and producing no cytokine, each one, or each combination of IL-2, IFN-γ, IL-13 and/or IL-17, at 14 weeks of life in HEU versus HIV-unexposed infants. Each possible single cytokine or combinations thereof within the responding CD4⁺ and CD8⁺ T cells at 14 weeks. Frequency of HEU (blue) versus HIV-unexposed (red) proliferating T cells within the total CD4⁺ (left) and CD8⁺ (right) T cells producing each specific cytokine or cytokine combination. Error bars and whiskers correspond to medians and interquartile ranges (IQRs), respectively. IFN, interferon; IL, interleukin; SEB, staphylococcal enterotoxin B. ^*P <0.05; ^**P <0.05 after adjustment for multiple comparisons; statistical analysis was performed using Wilcoxon signed-rank test and multiple comparison adjustment using the Holm step-down procedure. ^* HIV-unexposed significantly higher, ^*HEU significantly higher.