Characterization of an M. tuberculosis vaccine

结核病在世界大部分地区死灰复燃，原因是耐药性、艾滋病、贫困和流动性。这个项目的目标是生产一种改良的疫苗。根据我们的观察，MTB菌株H37Rv的抗原85A缺失突变体（Ag85A-）明显减弱，抗原提呈能力增强。我们的理由有三个组成部分：首先，已知对结核病最有效的免疫是由先前感染结核分枝杆菌本身提供的。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。第二，结核分枝杆菌抑制吞噬体-溶酶体（P-L）融合。初步数据表明，MTB Ag85A基因的缺失恢复了P-L融合，增强了抗原呈递，可能增加了MTB的免疫原性。第三，由于MTB是一种克隆生物，没有证明基因水平转移的能力，因此可以保证减毒MTB的安全性。具体目的是：1)评价ag85a突变体作为结核病疫苗的有效性和安全性。将通过对C57BL/6小鼠、近交小鼠和豚鼠进行单次或多次免疫，在毒力MTB气溶胶攻击之前，对其效力（限制原发感染和传播的能力）进行调查。安全性将通过对一系列动物的感染来评估，包括对疾病自然更敏感的豚鼠、基因异种的远交种小鼠、免疫一致的SCID小鼠和类固醇治疗小鼠。安全性将根据疫苗生物体产生疾病的能力及其对野生型MTB在攻击后的持久性和致病性的影响进行评估。2)在Ag85A-菌株中引入额外的缺失突变，在保持免疫原性的同时提高安全性。我们预计，任何在组织中存活时间较长的疫苗都可能在免疫抑制的人群中产生疾病。因此，将尝试产生一种双敲除突变MTB，它保留Ag85A-的免疫原性，并且不能在组织中存活。16 kDa α结晶蛋白基因和硝酸还原酶基因将成为目标。我们预计，一种安全的具有增强免疫原性的MTB减毒活疫苗将被证明在对抗成人肺结核和原发性疾病方面有价值。

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.