Characterization of an M. tuberculosis vaccine

由于耐药性、艾滋病、贫困和流动性，结核病在世界大部分地区死灰复燃。 该项目的目标是生产一种改进的疫苗。 基于我们的观察，MTB株H37 Rv的抗原85 A缺失突变体（Ag 85 A-）显著减毒并且具有增加的抗原呈递能力。 我们的理论基础有三个组成部分：首先，已知最有效的抗结核免疫是由先前感染MTB本身提供的。 因此，我们建议适当减毒的MTB将接近结核病疫苗效力的自然极限。 其次，MTB抑制吞噬体-溶酶体（P-L）融合。 初步数据表明，MTB的Ag 85 A基因的缺失恢复了P-L融合，增强了抗原呈递，并且可能增加了MTB的免疫原性。 第三，由于MTB是一种克隆生物体，没有证明具有水平转移基因的能力，因此可以确保减毒MTB的安全性。 具体目的是：1）评价Ag 85 A突变体作为结核病疫苗的有效性和安全性。 在用毒性MTB进行气溶胶激发之前，将通过对C57 BL/6小鼠、远系繁殖小鼠和豚鼠进行单次或多次免疫来研究有效性（限制原发感染和传播的能力）。将通过感染一系列动物评价安全性，包括天然更易患病的豚鼠、遗传异质性远系小鼠、免疫同源性SCID小鼠和类固醇治疗小鼠。 将根据疫苗微生物产生疾病的能力及其对攻毒后野生型MTB的持久性和致病性的影响评价安全性。2)在Ag 85 A-菌株中引入额外的缺失突变，以提高安全性，同时保持免疫原性。 我们预期任何在组织中存活时间延长的疫苗都可能在免疫抑制的人群中产生疾病。 因此，将尝试产生保留Ag 85 A的免疫原性并且不能在组织中存活的双敲除突变MTB。 将靶向16 kDa α晶状体蛋白基因和硝酸还原酶基因。 我们预期，一种安全的增强免疫原性的MTB减毒活疫苗将被证明在防治成人肺结核以及原发病方面有价值。

Tuberculosis is resurgent in most of the world fueled by drug resistance, AIDS, poverty and mobility. The goal of this project is to produce an improved vaccine. It is based on our observations that an antigen 85A deletion mutant of MTB strain H37Rv (Ag85A-) is markedly attenuated and has increased antigen presenting capability. Our rationale has three components: First, the most effective immunity known against tuberculosis is provided by prior infection with MTB itself. Consequently, we propose that an appropriately attenuated MTB will approach the natural limit of efficacy of vaccines for tuberculosis. Second, MTB inhibits phagosome-lysosome (P-L) fusion. Preliminary data demonstrates that deletion of the Ag85A gene of MTB restores P-L fusion, enhances antigen presentation and probably increases the immunogenicity of MTB. Third, since MTB is a clonal organism with no demonstrated ability for horizontal transfer of genes, the safety of attenuated MTB can be assured. The specific aims are: 1) Evaluate the efficacy and safety of the Ag85A-mutant as a vaccine against tuberculosis. The efficacy (ability to limit primary infection and dissemination) will be investigated by single or multiple immunizations of C57BL/6 mice, outbred mice and guinea pigs prior to aerosol challenge with virulent MTB. Safety will be evaluated by infection of a spectrum of animals including guinea pigs that are naturally more susceptible to disease, genetically heterogeneous outbred mice, immunocomormized SCID mice and steroid treated mice. Safety will be evaluated in terms the capacity of the vaccine organism to produce disease and its effect on persistence and pathogenicity of wild type MTB following challenge. 2) Introduce additional deletion mutations into the Ag85A- strain to improve safety while maintaining immunogenicity. We anticipate that any vaccine with prolonged survival in tissue may produce disease in immunosuppressed people. Consequently, attempts will be made to produce a double knockout mutant MTB that retains the immunogenicity of Ag85A- and is unable to survive in tissue. The 16 kDa alpha crystallin protein gene and nitrate reductase gene will be targeted. We anticipate that a safe live attenuated MTB vaccine with enhanced immunogenicity will prove valuable in combating adult pulmonary tuberculosis as well primary disease.