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Immune Correlates of LTBI in HIV-exposed Infants
About 2 billion people worldwide harbor asymptomatic latent TB infection (LTBI), activation of which results in progression to TB disease. In areas of high tuberculosis (TB) prevalence, infants and young children have a 3-fold higher risk of progression and death from TB at age < 5 years compared to older children(1, 2). The only approved TB vaccine, Mycobacterium bovis Bacillus Calmette–Guérin (BCG) has been in use for almost 100 years for newborns in endemic areas. BCG is effective in 60% individuals for preventing progression of infection to pulmonary TB, but it is highly effective in preventing meningeal and miliary TB and thus is a critically important vaccine.
Why BCG fails in some cases and why immunity wanes with time has been elusive (3-5). Identifying people at the greatest risk of activation of TB is not clear-cut. HIV infection is known to increase the risk of TB infection and reactivation. In infants born to HIV+ women, immunity to BCG appears to be differentially altered in HIV-exposed infected (HEI) and HIV-exposed uninfected (HEU), but the data are controversial and inconclusive (6-8). Strategies to prevent progression or reactivation of latent TB are being explored, and include different drug options including Isoniazid (INH) prevention therapy (IPT).
IMPAACT protocol P1078 (TB Apprise) was conducted to assess safety of IPT given antepartum or postpartum for HIV+ pregnant women in high TB incidence settings. It enrolled 956 women from eight countries during pregnancy and followed mother-infant pairs for one-year post-partum. Infants were breast-fed and most were given BCG within 48hr of birth. At study entry, prevalence of latent TB infection (LTBI) in the women was 30%, as determined by the Quantiferon-TB Gold In-Tube (QGIT) diagnostic test, an IFNg release assay (IGRA). 34 infants developed LTBI with a positive IGRA at ages 12 wk. and 44wk. In a random subset of mothers and infants, blood was collected for immunology studies, creating a rich repository of samples from participants whose LTBI status was disclosed at study end.
This proposal represents a collaboration between maternal-pediatric investigators with clinical and laboratory expertise. We will utilize infant/maternal repository samples of PBMC and plasma to test the hypotheses that infants born to women with LTBI can become sensitized to mycobacterial antigens, which, together with maternal factors, can affect their immune response to BCG vaccination. Furthermore, infants diagnosed with LTBI have distinct gene transcriptomic profiles in monocytes and antigen specific CD4+ T cells.
Aim 1: To determine if infants born to women with LTBI develop immunologic memory due to sensitization to mycobacterial antigens and have an altered immune response to BCG vaccine. PBMC from 100 infants without LTBI born to mothers with (n=50) or without (n=50) and 10 infants with LTBI will be evaluated by 17 color flow cytometry (FC) assays for phenotype and cytokine responses to Mtb antigens ESAT-6 and CFP 10, DosR latency antigens, and purified protein antigen (PPD). CD4 and CD8 T cell maturation subsets (Naive, Central Memory, Effector Memory and Effector), regulatory T cells (Tregs), peripheral T follicular helper cells (pTfh) and checkpoint Inhibitor (CPI) molecules PD-1, LAG3, and Tim3 will be analyzed. Antigen induced CD40L in memory CD4 T cell subsets, their proliferation and intracellular cytokines TNF, IL-21, IFNg, IL-17, IL-22 and IL-2 as well as cytokines in culture supernatants will be measured as functional readouts. Week 12 and week 44 infant age samples will be tested to determine time sensitive characteristics.
Aim 2: To investigate role of maternal factors in influencing infant immunity to Mtb and BCG and development of LTBI. A systems serology approach will be performed in plasma of IGRA+/- mothers of infants tested in Aim 1 plus samples of 32/34 mothers whose infants developed LTBI to identify distinctive Mtb Ab with Fc-mediated effector functions. Plasma of infants matched with mothers will also be tested for systems serology for passively transferred maternal IgG Ab and development of infant Ab based on IgM and IgA subclasses or increase of IgG. Antibody results will be correlated with the best identifiable antigen specific infant immune responses for Mtb antigens, DosR, and PPD. Plasma cytokine profile and maternal intake of INH during pregnancy will be taken into consideration in data analysis. Maternal PBMC, matched to infants in Aim 1 will be tested for immune cell phenotypes and antigen specific CD4 T cell characteristics.
Aim 3: To identify immunologic risk factors for infant acquisition of TBI within one year of life, by profiling CD4 T cells and monocytes. PBMC samples are available from 10/34 infants who tested IGRA+ whose mothers were IGRA+ or IGRA negative. Paired Infant/Maternal samples (assembled based on IGRA+ or – status) as Infant/Mother +/+ (7), +/- (3), -/+ (5) and -/- (5) will be analyzed using a novel technology for single cell RNA sequencing. PBMC will be stimulated with Mtb antigens followed by sorting of monocytes and CD40L+ CD4 T cells for whole transcriptome analysis. Index sorting will integrate cell subset characteristics of monocytes and CD4 T cells with gene transcript expression. Transcriptional profiles will be correlated with immune profiling data in Aims 1 and 2.