Newton Sandra, Martineau Adrian, Kampmann Beate
Department of Paediatrics, Imperial College London, UK.
J Vis Exp. 2011 Sep 14(55):3332. doi: 10.3791/3332.
Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/Abbreviations BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).
功能测定长期以来在衡量特定疫苗的免疫原性方面发挥着关键作用。这通常以血清杀菌效价来表示。针对儿童疫苗的血清杀菌效价研究使我们能够制定并验证保护性免疫反应的临界水平，且此类临界水平已在常规使用。对于主要诱导以效应T细胞反应形式存在的细胞介导免疫的疫苗，如结核病疫苗，尚未有此类测定方法被纳入其常规评估。在动物模型中，给定候选疫苗的性能通常在标准化杀菌测定中进行评估，并且所有当前新型结核病候选疫苗在进入1期人体试验之前的评估中都经历了这一步骤。理想情况下，新型抗结核疫苗的免疫原性评估以及因此其具有保护效力的可能性评估现在应在人体模型中进行类似的逐步评估，包括在杀菌测定中的测量。在结核病疫苗研究背景下的杀菌测定在动物模型中已经确立，在那里它们被用于筛选潜在有前景的候选疫苗。各种器官中细菌载量的降低作为免疫原性的主要读数。然而，迄今为止，此类测定尚未纳入新型抗结核疫苗的临床试验。尽管导致人类巨噬细胞内分枝杆菌被杀灭的确切机制仍存在不确定性，但巨噬细胞和T细胞与分枝杆菌的相互作用显然是必需的。本文所述的测定代表了新一代杀菌测定，它能够在全血中存在所有其他细胞和体液因子的情况下研究此类关键细胞成分，而无需对它们各自的相对贡献进行假设。我们团队描述的测定使用少量全血，并且已经用于结核病流行地区成人和儿童的研究。我们已经展示了卡介苗的免疫原性、HIV阳性患者中分枝杆菌生长的增加，以及抗逆转录病毒疗法和维生素D对体外分枝杆菌存活的影响。在此，我们总结该方法，并展示使用这种相对简单、低成本且适合现场使用的模型获得的数据的可重复性。注意：定义/缩写BCG lux = 牛分枝杆菌卡介苗，蒙特利尔菌株，用携带来自哈维氏弧菌luxAB基因的穿梭质粒pSMT1转化，在分枝杆菌GroEL（hsp60）启动子的控制下。CFU = 菌落形成单位（分枝杆菌活力的一种度量）。
