Incomplete recovery of pneumococcal CD4 T cell immunity after initiation of antiretroviral therapy in HIV-infected malawian adults

Abstract

HIV-infected African adults are at a considerably increased risk of life-threatening invasive pneumococcal disease (IPD) which persists despite antiretroviral therapy (ART). Defects in naturally acquired pneumococcal-specific T-cell immunity have been identified in HIV-infected adults. We have therefore determined the extent and nature of pneumococcal antigen-specific immune recovery following ART. HIV-infected adults were followed up at 3, 6 and 12 months after initiating ART. Nasopharyngeal swabs were cultured to determine carriage rates. Pneumococcal-specific CD4 T-cell immunity was assessed by IFN-γ ELISpot, proliferation assay, CD154 expression and intracellular cytokine assay. S. pneumoniae colonization was detected in 27% (13/48) of HIV-infected patients prior to ART. The rates remained elevated after 12 months ART, 41% (16/39) (p = 0.17) and significantly higher than in HIV-uninfected individuals (HIVneg 14%(4/29); p = 0.0147). CD4+ T-cell proliferative responses to pneumococcal antigens increased significantly to levels comparable with HIV-negative individuals at 12 months ART (p = 0.0799). However, recovery of the pneumococcal-specific CD154 expression was incomplete (p = 0.0015) as were IFN-γ ELISpot responses (p = 0.0040) and polyfunctional CD4+ T-cell responses (TNF-α, IL-2 and IFN-γ expression) (p = 0.0040) to a pneumolysin-deficient mutant strain. Impaired control of pneumococcal colonisation and incomplete restoration of pneumococcal-specific immunity may explain the persistently higher risk of IPD amongst HIV-infected adults on ART. Whether vaccination and prolonged ART can overcome this immunological defect and reduce the high levels of pneumococcal colonisation requires further evaluation.

Figure 1. CD4+ T-cell phenotypes in peripheral blood of adults before and after antiretroviral therapy at 3, 6 and 12 months ART and in healthy HIV-uninfected individuals.

(A) expansion of CD4 T- cell counts (0mth, n = 43; 3mths, n = 43; 6mths, n = 41; 12mths, n = 34; HIV-, 23) (B) naive CD4+ T cells (T_N: CD45RA+CCR7+), central memory CD4+ T cells (T_CM: CD45RA-CCR7+) and effector memory CD4+ T cells (T_EM: CD45RA-CCR7-) (0mth, n = 35; 3mths, n = 34; 6mths, n = 34; 12mths, n = 26; HIV-, 29) (C) CD28-CD57+ senescent cells (0mth, n = 34; 3mths, n = 34; 6mths, n = 39; 12mths, n = 20; HIV-, 10) (D) CD4+CD25^hiFoxP3+ T regulatory cells (0mth, n = 33; 3mths, n = 30; 6mths, n = 30; 12mths, n = 22; HIV-,13) Black horizontal bars represent median and IQRs. Wilcoxon matched pairs were used to compare T-cell characteristics of HIV-infected persons on ART over time, and the Mann Whitney U test in the HIV-infected and HIV-uninfected comparisons. Representative flow cytometric data demonstrating gating strategy are shown in Figure S1.

Figure 2. Pneumococcal-specific CD4+ T-cell responses in peripheral blood during ART.

Patients were analysed prior to initiation of ART and followed-up at 3, 6 and 12 mths ART for CD4⁺ T-cell responses. Responses from HIV-infected persons at 12 months ART were compared with HIV-negative persons recruited from the same community (A) ex vivo IFN-γ ELISpot responses to wild-type Streptococcus pneumoniae strain concentrated culture supernatant (CCS) (D39WT CCS) (0mth, n = 36; 3mths, n = 36; 6mths, n = 26; 12mths, n = 10; HIV-, 21) (B) ex vivo IFN-γ ELISpot responses to an isogenic pneumolysin (ply)-deficient mutant (D39Ply- CCS) (0mth, n = 36; 3mths, n = 36; 6mths, n = 26; 12mths, n = 10; HIV-, 21) (C) proliferative responses (8 day CFSE dilution assay) to D39WT CCS (0mth, n = 16; 6mths, n = 13; 12mths, n = 13; HIV-, 28) (D) proliferative responses to D39Ply- CCS (0mth, n = 17; 6mths, n = 14; 12mths, n = 13; HIV-, 28) (E) CD154 expression on activated CD4⁺ T cells in response to D39WT CCS (0mth, n = 33; 3mths, n = 30; 6mths, n = 30; 12mths, n = 16; HIV-, 29) (F) CD154 expression on activated CD4⁺ T cells in response to D39Ply- CCS (0mth, n = 30; 3mths, n = 29; 6mths, n = 28; 12mths, n = 14; HIV-, 29). Black horizontal bars represent median and IQR after background responses were substrated from all antigen-specific CD4⁺ T cell responses. Wilcoxon matched pairs were used to compare T-cell characteristics of HIV-infected persons on ART over time, and the Mann Whitney U test in the HIV-infected and HIV-uninfected comparisons. Representative flow cytometric data demonstrating CD4⁺ T-cell proliferative responses and CD154 expression and gating strategy are shown in Figure S1.

Figure 3. Antigen (PPD and Flu)-specific CD4+ T-cell responses in peripheral blood during ART.

Patients were analysed prior to initiation of ART and followed-up at 3, 6 and 12 mths ART for CD4⁺ T-cell responses. Responses from HIV-infected persons at 12 months ART were compared with HIV-negative persons recruited from the same community (A) ex vivo IFN-γ ELISpot responses to M.tuberculosis PPD (0mth, n = 36; 3mths, n = 36; 6mths, n = 26; 12mths, n = 10; HIV-, 24) (B) ex vivo IFN-γ ELISpot responses to influenza antigens (0mth, n = 36; 3mths, n = 36; 6mths, n = 26; 12mths, n = 10; HIV-, 24) (C) proliferative responses (8 day CFSE dilution assay) to M.tuberculosis PPD (0mth, n = 12; 6mths, n = 12; 12mths, n = 12; HIV-, 28) (D) proliferative responses to influenza antigens (0mth, n = 14; 6mths, n = 12; 12mths, n = 13; HIV-, 28) (E) CD154 expression on activated CD4⁺ T cells in response to M.tuberculosis PPD (0mth, n = 33; 3mths, n = 30; 6mths, n = 30; 12mths, n = 16; HIV-, 29) (F) CD154 expression on activated CD4⁺ T cells in response to influenza antigens (0mth, n = 30; 3mths, n = 28; 6mths, n = 30; 12mths, n = 16; HIV-, 29). Black horizontal bars represent median and IQR after background responses were substrated from all antigen-specific CD4⁺ T-cell responses. Wilcoxon matched pairs were used to compare T cell characteristics of HIV-infected persons on ART over time, and the Mann Whitney U test in the HIV-infected and HIV-uninfected comparisons. Representative flow cytometric data demonstrating CD4⁺ T cell proliferative responses and CD154 expression and gating strategy are shown in Figure S1.

Figure 4. Functionality of CD4+ T cell during ART.

Patients were analysed prior to initiation of ART and followed-up at 3, 6 and 12 mths ART for different combinations of IFN-γ, TNF-α and IL-2 using flow cytometry. Polyfunctional CD4⁺ T-cell profiles of HIV-infected persons on ART were compared with HIV-negative persons recruited from the same community. CD4⁺ T-cell responses to (A) wild-type Streptococcus pneumoniae strain concentrated culture supernatant (CCS) (D39WT CCS) (B) an isogenic pneumolysin (ply)-deficient mutant (D39Ply- CCS) (C) Influenza vaccine (D) M. tuberculosis PPD. Collated data (background responses in the negative control substrated and threshold set at 0.01%) from HIV negative (n = 9) and those infected with HIV (n = 18). Pies were analysed according to slice colour using SPICE software and p values are indicated. The frequency of CD4⁺ T cells producing one, two or three cytokines specific for D39WT CCS, D39Ply- CCS, influenza vaccine and PPD are shown in Figure S2.