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 由结核分枝杆菌（Mtb）引起的结核病（TB）是世界上引起结核病的主要原因之一。 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 在免疫功能正常的个体中是安全的，因为它已经失去了几个赋予毒力的基因。BCG有很好的 对儿童结核病的疗效，但对青少年和成人结核病的疗效较差。因此，疫苗 显然需要比卡介苗更有效的疫苗然而，任何替代疫苗几乎肯定需要 基于修饰的（例如重组的）BCG或减毒的Mtb，以保留BCG的实质性益处。 该项目的目标是开发一种比BCG更安全和更有效的减毒Mtb突变体。 我们的新策略涉及操纵活疫苗的两个关键特征：（1）它们的初始期， 在宿主中的生长和（2）它们的消除速率。卡介苗和卡介苗接种后保护效果不佳， 非复制型Mtb突变体可至少部分归因于它们在宿主中缺乏复制。延长 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子和效应子 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 免疫后短时间内Mtb突变体的有限复制，模仿自然免疫的早期阶段。 结核杆菌感染后，迅速清除，将诱导强有力的免疫反应，但避免阴性 由延长的Mtb感染诱导的炎症反应。 为了实现我们的目标，我们首先将设计一种减毒的Mtb突变体，其铁获得和铁代谢都有缺陷。 铁载体介导的铁获得（SMIA）和血红素铁获得（HIA）。这种突变体 将不能从宿主获得铁，但可以在体外预先装载精确量的铁， 主机中的最佳复制。因此，Mtb BmSMIA BmHIA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。虽然宿主中结核分枝杆菌的生长将停止， 一旦它耗尽了铁的供应，有机体可以持续很长一段时间。因此，为了解决 第二个重要的因素，从主机清除率，我们将进一步修改Mtb的MtbSMIA MtbHIA，通过两个 方法- 1）敲除持久性基因和2）必需基因的条件性沉默。虽然这两 应该导致更好的清除，条件性沉默可能会导致更快的清除。我们将接种疫苗- 吃小鼠与持久性和条件沉默突变体和执行清除和保护效力 研究，以确定最佳的复制和清除。我们期望一个复制和持久性受限的 具有快速清除的Mtb突变体将比BCG有效得多，并且与BCG相反，即使在体内也是安全的。 免疫力低下的宿主一旦我们优化了小鼠保护性免疫的疫苗，我们将 检查其在小鼠中的免疫原性以评估保护的初步相关性，评估其在免疫中的安全性， 受损的SCID小鼠，并在第二种TB动物模型-豚鼠中检查其安全性和有效性。

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.