Does the BCG vaccine protect infants from severe forms of childhood tuberculosis by trained innate and humoral immune mechanisms?

结核病（TB）是一个重大的全球健康负担，根据世界卫生组织的报告，2015年有180万例TB相关死亡（20万例儿童）。最近的研究表明，婴儿的免疫反应可能是次优的，使他们容易患结核病及其并发症。然而，目前的抗结核疫苗，牛分枝杆菌BCG，保护他们免受结核病并发症的影响，并可能产生非特异性的影响，降低其他无关感染的婴儿死亡率。调节这些特性的机制尚不清楚;然而，一个候选者可能是最近发现的单核细胞免疫记忆（或训练），通过表观遗传和代谢修饰来调节。这种现象被证明可以改善卡介苗接种的成年单核细胞中的单核细胞活化和炎症细胞因子的产生，直至接种后3个月，以响应BCG以外的刺激。这种机制可能有助于保护婴儿免受结核病的侵袭，可能改善单核细胞功能，从而增强T细胞（抗结核免疫的主要效应子）活化或促炎反应。最近的研究将抗分枝杆菌抗体与降低婴儿结核病风险联系起来，早期的研究表明，这种抗体可以改善BCG的吞噬作用，控制其生长，甚至是T细胞反应。由于先天免疫细胞表达可识别抗体结构域的受体，它们可能协同保护婴儿免受结核病并发症的影响。由于BCG训练的先天免疫和抗分枝杆菌抗体应答在婴儿中尚未得到广泛研究，本研究旨在填补目前对这些机制在婴儿结核病和BCG免疫生物学中的作用的理解中的差距。可能有助于新的结核病疫苗设计策略。为此，该研究将结合免疫学、细胞和分子生物学、代谢和定量2方法，表征卡介苗体液免疫和单核细胞训练的多层效应。首先，婴儿单核细胞表面活化标志物和细胞因子谱将通过流式细胞术/Luminex/细胞内染色，以将BCG接种前后婴儿的单核细胞表面和功能表型与其训练状态相关联。表观遗传组蛋白修饰和DNA甲基化分析将用于确定哪些基因调控变化与接种疫苗后BCG训练诱导的单核细胞表型相关。此外，还将测定婴儿卡介苗接种前后的单核细胞代谢表型。通过R/Matlab软件进行计算分析，检测表面、功能、表观遗传和代谢标志物与BCG诱导的单核细胞训练之间的关系，并检测抗BCG抗体在婴儿结核保护中的作用。卡介苗接种前和接种后婴儿血样抗分枝杆菌抗体滴度将通过ELISA/Luminex进行测量。为了测试它们对抗TB免疫的功能贡献，将使用来自接种前或接种后样品的细胞婴儿吞噬细胞和血清测定来确定它们的活化标志物和细胞因子谱。此外，它们激活抗分枝杆菌T细胞的能力将通过流式细胞术和ELISA/Luminex测定来测量。为了研究它们在抗结核免疫中的临床意义，将测试卡介苗接种前后血清对分枝杆菌生长的抗菌作用。计算R/Matlab分析将用于确定血清学标志物与其他免疫区室组分活化之间的相关性。

Tuberculosis (TB) is a significant global health burden, with 1.8 million TB-related deaths(0.2 million in children) in 2015, as reported by the WHO. Recent research suggested thatinfant immune responses may be suboptimal, predisposing them to TB and itscomplications. However, the current anti-TB vaccine, Mycobacterium bovis BCG, protectsthem from TB complications and may exert non-specific effects, reducing overall infantmortality from other, unrelated infections.The mechanisms mediating these properties are yet unknown; however, one candidate maybe a recently discovered monocyte immune memory (or training), regulated throughepigenetic and metabolic modifications. This phenomenon was shown to improve monocyteactivation and the inflammatory cytokine production in BCG vaccinated adult monocytes forup to 3 months post-vaccination in response to stimuli other than BCG. This mechanismmay contribute to infant protection from TB, possibly improving monocyte function and soenhancing T-cell (a major effector of anti-TB immunity) activation or proinflammatoryresponses.Anti-BCG antibodies may also contribute to protecting infants from severe TB forms. Recentresearch associated anti-mycobacterial antibodies with reduced infant TB risk, and theearlier studies suggested that such antibodies could improve BCG phagocytosis, control ofits growth and even T-cell responses. As the innate immune cells express receptors for thecrystallisable antibody domains, they may possibly act synergistically protecting infants fromTB complications.As neither BCG-trained innate immunity, nor the anti-mycobacterial antibody responseshave been extensively studied in infants, this study aims to fill the gap in the currentunderstanding on the role of these mechanisms in infant TB and BCG immunobiology,potentially contributing to novel TB vaccine design strategies. To do so, the study willcombine a mixture of immunology, cell and molecular biology, metabolic and quantitative 2approaches characterising the multi-layered effects of BCG humoral immunity and trainingin monocytes.First, infant monocyte surface activation marker and cytokine profiles will be determined byflow cytometry/Luminex/intracellular staining to associate monocyte surface and functionalphenotypes with their training status in infants prior to and post the BCG vaccination. Theepigenetic histone modification and DNA methylation analyses will then be applied todetermine which gene regulation changes correlate with monocyte phenotypes induced byBCG training upon vaccination. In addition, monocyte metabolic phenotypes prior to andpost the infant BCG vaccination will be determined. Computational analysis via R/Matlabwill be used to test the relationship between the surface, functional, epigenetic andmetabolic markers detected and BCG-induced monocyte training.This study will also test the anti-BCG antibody role in infant protection from TB. Pre- andpost-BCG vaccination infant blood sample anti-mycobacterial antibody titres will bemeasured by ELISA/Luminex. To test their functional contribution to anti-TB immunity,cellular infant phagocyte and sera assays from the pre- or post-vaccination samples will beused to characterise their activation marker and cytokine profiles. In addition, their ability toactivate anti-mycobacterial T-cells will be measured by flow cytometry and ELISA/Luminexassays. To study their clinical implications in anti-TB immunity, the pre- and post-BCGvaccination sera antimicrobial effect on mycobacterial growth will be tested. ComputationalR/Matlab analysis will be used to determine the correlation between serological markersand other immune compartment component activation.