Characterization of an M. tuberculosis vaccine

由于抗药性、艾滋病、贫困和人口流动，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。根据我们的观察，结核分枝杆菌H37Rv株(Ag85A-)的抗原85A缺失突变株(Ag85A-)显著减弱并增强了抗原提呈能力。我们的理由有三个部分：第一，已知的对结核病最有效的免疫力是通过先前感染结核杆菌本身来提供的。因此，我们建议，适当减毒的结核分枝杆菌将接近结核病疫苗的自然效力极限。第二，MTB抑制吞噬小体-溶酶小体(P-L)的融合。初步资料表明，结核分枝杆菌Ag85A基因缺失恢复了P-L融合，增强了抗原提呈，可能增强了结核分枝杆菌的免疫原性。第三，由于结核分枝杆菌是克隆生物，没有证明有水平转移基因的能力，因此可以保证减毒结核分枝杆菌的安全性。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。在用毒力结核杆菌气雾剂攻击之前，将通过对C57BL/6小鼠、杂交小鼠和豚鼠进行单次或多次免疫来考察其有效性(限制原发感染和传播的能力)。安全性将通过感染一系列动物进行评估，包括自然更容易感染疾病的豚鼠、遗传异质性的近交系小鼠、免疫同化的SCID小鼠和类固醇治疗的小鼠。安全性将根据疫苗机体的致病能力及其对野生型结核分枝杆菌在挑战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，以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人群中产生疾病。因此，将尝试生产一种双基因敲除突变体MTB，它保留了Ag85A的免疫原性，并且无法在组织中存活。将以16 kDaα晶体蛋白基因和硝酸还原酶基因为靶点。我们预计，一种具有增强免疫原性的安全减毒结核分枝杆菌活疫苗将在抗击成人肺结核和原发疾病方面证明是有价值的。

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.