CHOLERA--PATHOGENESIS AND IMMUNOLOGY

这项研究的目的是为了帮助理解 霍乱--一种由弧菌引起的流行性腹泻病的发病机制 01霍乱弧菌，在生物和分子水平上都有 期望这些信息将导致Rational的发展 和有效的免疫预防手段。霍乱是霍乱的原型 腹泻疾病的数量不断增加，这些疾病由 结构、功能和免疫学上的肠毒素 与霍乱肠毒素(S)有关，观察结果与 数十亿病例和数百万人死亡的更大的全球问题 每年因腹泻病死亡，尤其是第三世界的儿童。 霍乱肠毒素(CT)于1959年被发现，并经纯化和鉴定 1969年出现了部分特征。在接下来的一段时间里，人们期望， 就像白喉和破伤风一样，类毒素疫苗可以预防这种疾病。 尽管早期鼓舞人心的实验仍未实现 观察。现在人们知道霍乱是一种有效的 一种免疫过程，在这一过程中，人类宿主被呈递给一个联合体 由在体内生长的霍乱弧菌精制的产品 小肠。除肠道毒素外，还包括定植 毒素共调节菌毛(TCP)、其他表面成分等因素 包括脂多糖、外膜蛋白、透明质酸/蛋白酶 和其他可溶性因子，细胞相关的甘露糖敏感 E1Tor生物型的血凝素，鞭毛，可能还有其他 各种因素。这种疾病在人身上引发的固体免疫力还没有 由非生物制剂复制，非肠道给药或 口服，或通过活的减毒突变体，口服， 尽管已经获得了某种程度的保护--即使在没有 毒素抗原。对毒素抗原本身的研究结果 更令人失望。这项建议解决了一些原因 对于以前的毒素疫苗的失败；它解决了一种 新型霍乱疫苗将由无毒的脱脂内毒素结合物组成 和霍乱毒素；此外，它还将继续处理其他 可能导致野生-霍乱弧菌毒力和免疫原性的因素- 活体霍乱弧菌分型。该技术包括合成和合成 序列连续重叠多肽的分析 CT免疫优势B亚单位蛋白及其结合位点的应用 产生CT的霍乱弧菌的特异性诱变 它无毒，但表达全毒素的主要表位。这 CT还将与内毒素低聚糖交联，形成偶联物 既能激发抗菌免疫又能抗毒素免疫的疫苗。我们 将进一步研究和定义HA/蛋白酶和甘露糖的作用- 霍乱弧菌附着和/或脱离中的敏感血凝素 并检测霍乱弧菌浑浊程度中位相变化的意义 殖民地。

The goals of this research are to contribute to the understanding of the pathogenesis of cholera, an epidemic diarrheal disease caused by Vibrio cholerae 01, at both the organismal and the molecular levels, with the expectation that this information will lead to the development of rational and effective means of immunoprophylaxis. As cholera is the prototype of an expanding number of diarrheal diseases which are mediated by enterotoxins which are structurally, functionally and immunologically related to the cholera enterotoxin(s), the observations are pertinent to the larger global problem of the billions of cases and millions of deaths annually from diarrheal diseases especially in children in the Third World. Cholera enterotoxin (CT) was discovered in 1959 and it was purified and partially characterized in 1969. In the ensuing period, expectations that, as with diptheria and tetanus, a toxoid vaccine would prevent the disease have not been fulfilled despite early encouraging experimental observations. It is now known that the disease, cholera, is an effective immunizing process in which the human host is presented with a consortium of products elaborated by the cholera vibrios growing in vivo in/on the small bowel. In addition to the enterotoxin, these include colonization factors, such as toxin-coregulated pili (TCP), other surface components including lipopolysaccharide (LPS) and outer membrane proteins, HA/protease and other soluble factors, the cell-associated mannose-sensitive hemagglutinin of the E1 Tor biotype, the flagellum and, possibly, other factors. The solid immunity evoked in people by the disease has not yet been duplicated by non-living preparations, administered parenterally or perorally, or by living attenuated mutants, administered perorally, although some degree of protection has been attained -- even in the absence of toxin antigen. Results of studies with toxin antigen by itself have been even more disappointing. This proposal addresses some of the reasons for previous failures of toxoid vaccines; it addresses the development of a new cholera vaccine to consist of a non-toxic conjugate of de-lipidated LPS and cholera toxin; and, in addition, it will continue to address other factors which may contribute to the virulence and immunogenicity of wild- type cholera vibrios in vivo. The technology includes the synthesis and analysis of sequential continuous overlapping peptides comprising the immunologically dominant B-subunit protein of CT and the use of site- specific mutagenesis to create a V. cholerae strain which produces a CT which is not toxic but expresses the major epitopes of the holotoxin. This CT will also be cross-linked to LPS oligosaccharide to form a conjugate vaccine which will stimulate both antibacterial and antitoxic immunity. We will further examine and define the role of HA/protease and the mannose- sensitive hemagglutinin in attachment and/or detachment of cholera vibrios and examine the significance of phase variation in opacity of V. cholerae colonies.