Incomplete Recovery of Pneumococcal CD4 T Cell Immunity after Initiation of Antiretroviral Therapy in HIV-Infected Malawian Adults

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

Evaluation of Cynomolgus Macaque (Macaca fascicularis) Endogenous Retrovirus Expression Following Simian Immunodeficiency Virus Infection

Human endogenous retrovirus type K (HERV-K) transcripts are upregulated in the plasma of HIV-infected individuals and have been considered as targets for an HIV vaccine. We evaluated cynomolgus macaque endogenous retrovirus (CyERV) mRNA expression by RT-qPCR in PBMCs isolated from a cohort of animals previously utilized in a live attenuated SIV vaccine trial. CyERV env transcript levels decreased following vaccination (control and vaccine groups) and CyERV env and gag mRNA expression was decreased following acute SIV-infection, whereas during chronic SIV infection, CyERV transcript levels were indistinguishable from baseline. Reduced susceptibility to initial SIV infection, as measured by the number of SIV challenges required for infection, was associated with increased CyERV transcript levels in PBMCs. In vitro analysis revealed that SIV infection of purified CD4+ T-cells did not alter CyERV gene expression. This study represents the first evaluation of ERV expression in cynomolgus macaques following SIV infection, in an effort to assess the utility of cynomolgus macaques as an animal model to evaluate ERVs as a target for an HIV/SIV vaccine. This non-human primate model system does not recapitulate what has been observed to date in the plasma of HIV-infected humans suggesting that further investigation at the cellular level is required to elucidate the impact of HIV/SIV infection on endogenous retrovirus expression

Impairment of pneumococcal antigen specific isotype-switched Igg memory B-cell immunity in HIV infected Malawian adults

Pneumococcal disease is associated with a particularly high morbidity and mortality amongst adults in HIV endemic countries. Our previous findings implicating a B-cell defect in HIV-infected children from the same population led us to comprehensively characterize B-cell subsets in minimally symptomatic HIV-infected Malawian adults and investigate the isotype-switched IgG memory B-cell immune response to the pneumococcus. We show that similar to vertically acquired HIV-infected Malawian children, horizontally acquired HIV infection in these adults is associated with IgM memory B-cell (CD19(+) CD27(+) IgM(+) IgD(+)) depletion, B-cell activation and impairment of specific IgG B-cell memory to a range of pneumococcal proteins. Our data suggest that HIV infection affects both T-cell independent and T-cell dependent B-cell maturation, potentially leading to impairment of humoral responses to extracellular pathogens such as the pneumococcus, and thus leaving this population susceptible to invasive disease

Delayed reconstitution of B cell immunity to pneumococcus in HIV-infected Malawian children on antiretroviral therapy

SummaryObjectiveDespite CD4+ count restoration and viral load suppression with antiretroviral therapy (ART), HIV-infected children remain at increased risk of life-threatening infections including invasive pneumococcal disease (IPD). We therefore investigated whether persistent susceptibility to IPD following ART is associated with incomplete recovery of B-cell function.Methods41 HIV-infected Malawian children commencing ART were followed-up for a 1 year period during which time blood samples were collected at 0, 3, 6 and 12 months for comprehensive immunophenotyping and pneumomococcal-specific Memory B-cell Enzyme-Linked Immunospot assays. In addition, nasopharyngeal swab samples were cultured to determine pneumococcal carriage rates.ResultsNormalization of major lymphocyte subsets such as CD4+ percentages was evident following 3 months of ART. The proportions of mature naïve B cells (CD19+ CD10− CD27− CD21hi) and resting memory B cells (CD19+ CD27+ CD21hi) increased and apoptosis-prone mature activated B cells (CD19+ CD21lo CD10−) decreased markedly by 12 months. However, in the context of high nasopharyngeal pneumococcal carriage rates (83%), restoration of pneumococcal protein antigen-specific B-cell memory was more delayed.ConclusionsThese data show that, in chronically HIV-infected children receiving ART, improvement in B-cell memory profiles and function is slower than CD4+ T-cells. This supports early initiation of ART and informs research into optimal timing of immunization with pneumococcal vaccines

IgM memory B-cells are depleted while isotype-switched memory B-cells are maintained in HIV-infected Malawian adults.

<p>(A) IgM memory B-cells (CD19⁺ CD27⁺ IgD⁺) absolute numbers, (B) percentages and (C) isotype-switched memory B-cell (CD19⁺ CD27⁺ IgD⁻) numbers, (D) percentages in the circulation of HIV⁻ controls (n = 44), HIV⁺ >350 (n = 16) and HIV⁺ ≤350 (n = 24) adult. Horizontal bars represent medium values. Statistical significance of differences between groups was assessed using the Mann Whitney <i>U</i> test.</p

Correlation between circulating IgM memory B-cells and CD4⁺ T-cells.

<p>(<b>A</b>) Scatter plots showing absolute IgM memory B-cell numbers & CD4⁺ T-cell counts and (<b>B</b>) % IgM memory B-cells & CD4⁺ T-cells measured using flow cytometry. The association between the two parameters (IgM memory B-cells and CD4 counts) was determined by Spearman’s correlation using Stata (Version 10). Each point represents the result from one subject. r_s is the Spearman rank correlation coefficient.</p

Baseline demographic and immune parameters of HIV-infected and uninfected Malawian adult study participants.

<p>Median and interquartile ranges (IQR) for age and immunological data are shown. Statistical significance of differences between the HIV negative group and all HIV-infected adults was tested using Fisher’s exact test (for categorical variables) or Mann Whitney <i>U</i> test (for continuous variables). † Data on <i>S. pneumoniae</i> carriage are missing for some patients for technical reasons.</p

CyERV gene expression levels in CD4⁺ T-cells following <i>in vitro</i> SIV infection.

<p>CyERV gene expression was quantitated by RT-qPCR using RNA isolated from mock- and SIV-infected CD4⁺ T-cells. CyERV envelope (A) and gag (B) gene expression levels relative to GAPDH are compared between <i>in vitro</i> mock- and SIV-infected CD4⁺ T-cells. Paired-samples t-tests (2-tailed) were performed to determine statistical significance.</p

<b>RT-qPCR Primers.</b>

<p>The slope and correlation represent the mean value.</p

CyERV gene expression negatively associated with CD4⁺ T-cell counts and SIV viral load.

<p>During acute SIV infection, CyERV envelope (n = 11) and gag (n = 10) gene expression levels negatively correlated with CD4⁺ T-cell counts (CD4⁺ T-cells/ul of PBMCs) (A-B) and negatively associated with acute SIV viral load for CyERV envelope (n = 9) (C). CyERV gag (n = 8) expression showed a slight trend towards a positive association with acute SIV viral load (D). Bivariate correlations were performed using Pearson’s correlation coefficient (r), statistical significance values (p) are shown.</p