Development of a novel TB vaccine safer and more effective than BCG based on a precisely controlled replication-limited Mycobacterium tuberculosis engineered for optimal in vivo growth and clearance

摘要 结核病(TB)由结核分枝杆菌(Mtb)引起，是世界上主要的致病原因之一。 死亡。卡介苗是唯一获得许可的结核病疫苗，是一种与结核分枝杆菌高度同源的减毒细菌，但 在具有免疫能力的个体中是安全的，因为它已经失去了几个赋予毒力的基因。卡介苗有好的 对儿童结核病有效，但对青少年和成人结核病效果不佳。因此，疫苗要多得多 显然需要比BCG更强大的力量。然而，几乎可以肯定，任何替代疫苗都需要 基于改良(如重组)卡介苗或减毒结核分枝杆菌，以保持卡介苗的实质益处。 该项目的目标是开发一种比卡介苗更安全、更有效的减毒结核分枝杆菌突变体。 我们的新策略涉及操纵活疫苗的两个关键特征：(1)它们的初始阶段 寄主的生长和(2)它们的淘汰率。卡介苗和卡介苗诱导的保护作用不足 非复制型结核分枝杆菌突变体至少部分可以归因于它们在宿主中缺乏复制。延长了 在宿主中的持久性也是一个负面因素，导致产生主要的效应器和效应器 记忆T细胞而不是中央记忆T细胞，对长期免疫很重要。我们假设 在免疫后的短时间内有限复制结核分枝杆菌突变体，模仿自然的 感染结核分枝杆菌后，迅速清除会引发强烈的免疫反应，但又能避免出现阴性反应 长期感染结核分枝杆菌引起的炎症反应。 为了实现我们的目标，我们首先将设计一个在两种铁获得方面都有缺陷的减毒Mtb突变体。 铁途径-铁载体介导的铁获得(SMIA)和血红素铁获得(HIA)。这样一个变种人 将无法从宿主获得铁，但可以在体外预先加载准确的铁含量，以允许 在主机中实现最佳复制。因此，Mtb∆smia∆hia突变体将使我们能够解决第一个重要的问题 因素-控制主机中的复制范围。而结核分枝杆菌∆SmiA∆Hia在宿主中的生长将停止 一旦它耗尽了铁的供应，这种有机体可能会持续很长一段时间。因此，要解决 第二个重要因素，从主机的清除速度，我们将进一步修改Mtb∆SMIA∆HIA，通过两个 方法-1)敲除持久性基因，2)有条件地沉默必要的基因。虽然两者都 如果能改善清除，有条件的沉默可能会导致更快的清除。我们应该接种疫苗- 吃具有持久性和条件性沉默突变的小鼠，并执行清除和保护效果 研究以确定最佳复制和清除。我们预计复制和持久性受到限制 快速清除的结核分枝杆菌突变株将比卡介苗有效得多，而且与卡介苗相比，即使在 免疫功能受损的宿主。一旦我们优化了疫苗对小鼠的保护性免疫，我们将 在小鼠体内检测其免疫原性，以评估初步的保护相关性，评估其在免疫中的安全性。 在第二个结核病豚鼠动物模型中检测其安全性和有效性。

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the world's leading causes of death. BCG, the only licensed vaccine against TB, is an attenuated bacterium highly homologous to Mtb, yet safe in immunocompetent individuals because it has lost several genes that confer virulence. BCG has good efficacy against TB in children, but poor efficacy against TB in adolescents and adults. Hence, a vaccine much more potent than BCG is clearly needed. However, any replacement vaccine will almost certainly need to be based on modified (e.g. recombinant) BCG or attenuated Mtb to preserve the substantial benefits of BCG. The goal of this project is to develop an attenuated Mtb mutant that is safer and more potent than BCG. Our novel strategy involves manipulating two key characteristics of live vaccines: (1) their initial period of growth in the host and (2) their rate of elimination. The inadequate protective efficacy induced by BCG and non-replicating Mtb mutants can be attributed, at least in part, to their lack of replication in the host. Prolonged persistence in the host is also a negative factor, resulting in the generation of primarily effector and effector memory T cells rather than central memory T cells, important for long-term immunity. We hypothesize that limited replication of an Mtb mutant for a brief period after immunization, mimicking the early stage of a natural Mtb infection, followed by rapid clearance will induce a potent immune response and yet avoid the negative inflammatory responses induced by prolonged Mtb infection. To achieve our goal, we first shall engineer an attenuated Mtb mutant defective in both of its iron acquisi- tion pathways - siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA). Such a mutant will be unable to obtain iron from the host but can be pre-loaded in vitro with the precise amount of iron to allow optimal replication in the host. Thus, an Mtb ∆SMIA ∆HIA mutant will allow us to address the first important factor - controlling the extent of replication in the host. While growth of Mtb ∆SMIA ∆HIA in the host will cease once it exhausts its supply of iron, the organism may persist for a prolonged period. Thus, to address the second important factor, the rate of clearance from the host, we shall further modify Mtb ∆SMIA ∆HIA, via two approaches – 1) knocking out persistence genes and 2) conditional silencing of essential genes. While both should result in improved clearance, conditional silencing likely will result in faster clearance. We shall vaccin- ate mice with persistence and conditional silencing mutants and perform clearance and protective efficacy studies to determine the optimal replication and clearance. We expect a replication- and persistence-limited Mtb mutant with rapid clearance will be much more efficacious than BCG and, in contrast to BCG, safe even in an immunocompromised host. Once we have optimized the vaccine for protective immunity in mice, we shall examine its immunogenicity in mice to assess preliminary correlates of protection, assess its safety in immuno- compromised SCID mice, and examine its safety and efficacy in a second animal model of TB - guinea pigs.