COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT III

少数民族健康合作项目--项目三

• 批准号: 2228533
• 负责人: David E Briles
• 金额: $ 10.16万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2228533
• 关键词: Streptococcus pneumoniae vaccine; bacterial disease; child (0-11); diagnostic tests; human tissue; immunological substance; laboratory mouse; polymerase chain reaction; recombinant proteins; sickle cell anemia; surface antigens

Sepsis with Streptococcus pneumoniae is a leading cause of death among sickle cell disease (SCD) patients. While immunization with a capsular polysaccharide vaccine can increase resistance to pneumococcal infection in older SCD patients, it is not effective in SCD patients less than 2 years of age because young children do not make adequate responses to most polysaccharide antigens. However, they do make good immune responses to most proteins. We have identified a pneumococcal surface protein (PspA) that has the potential of being a good pneumococcal vaccine for children. In mice, it is highly immunogenic and elicits antibodies that are protective against fatal infections with S. pneumoniae. PspA is a virulence factor that slows the clearance of S. pneumoniae from the blood. Although PspA is serologically variable, different PspAs are sufficiently cross-reactive that antibodies against an individual PspA can elicit protection against strains of different PspA types. Thus, a mixture of only a few PspAs (or a recombinant protein containing specific epitopes from only a few PspAs) would be needed for a vaccine. We have sequenced the pspA gene of strain Rx1. The N-terminal 49% of the inferred protein sequence is highly charged and consistent with an alpha-helix capable of forming linear coiled-coil fibers. The alpha-helical region contains the epitopes recognized by all protective monoclonal antibodies. Just C-terminal to the alpha-helical domain is a proline-rich domain followed by an anchoring domain containing ten 20 amino acid repeats. Most of the protective MAbs to Rx1 PspA recognize epitopes in the C-terminal third of the alpha-helical region. These epitopes are more cross-reactive than others in the alpha- helical region. Southern blots and the ability of specific pspA primers to amplify diverse pspAs by polymerase chain reaction (PCR) demonstrate that the portion of pspA encoding the alpha-helical domain is more variable than that encoding the C-terminal half of PspA. In the proposed studies we will sequence several pspAs and determine the portions of their alpha-helical sequences that contain epitopes capable of eliciting protective antibody in normal and SCD mice. We plan to determine whether humans make protective responses to the same epitopes by using affinity purified antibody from convalescent SCD and non-SCD patients. These studies could lead to the development of an inexpensive recombinant PspA fusion protein to be used as a vaccine against pneumococcal infection in children. We will also use the pspA sequence information to identify conserved regions for use as PCR primers to aid in the diagnosis of pneumococcal infection. This type of diagnostic capability is needed so that new vaccines and treatments can be accurately evaluated. Better diagnosis would also enable physicians to use more appropriate antibiotic treatments. An advantage of using PspA as the target of diagnostic PCR amplification is that RFLPs of the amplified products could facilitate epidemiologic studies of strains of S. pneumoniae that infect normal and SCD patients. The proposed studies will use isolates and convalescent serum from SCD patients.

肺炎链球菌败血症是导致死亡的主要原因 镰状细胞病（SCD）患者。 当用胶囊免疫时 多糖疫苗可增强对肺炎球菌感染的抵抗力 对于老年 SCD 患者，对于小于 2 岁的 SCD 患者无效 年龄，因为年幼的孩子没有做出足够的反应 大多数多糖抗原。 然而，它们确实具有良好的免疫力 对大多数蛋白质的反应。 我们已经确定了肺炎球菌表面 具有成为良好肺炎球菌潜力的蛋白质（PspA） 儿童疫苗。 在小鼠中，它具有高度免疫原性并引发 抗体可预防致命的金黄色葡萄球菌感染。 肺炎杆菌。 PspA 是一种毒力因子，可减缓 S 的清除。 肺炎来自血液。 尽管 PspA 在血清学上存在差异， 不同的 PspA 具有足够的交叉反应性，因此针对 单个 PspA 可以引发针对不同菌株的保护 PspA 类型。 因此，只有少数 PspA 的混合物（或重组体） 含有仅来自少数 PspAs 的特定表位的蛋白质）将是 需要疫苗。 我们对菌株 Rx1 的 pspA 基因进行了测序。 推断蛋白质序列的 N 端 49% 带高电荷， 与能够形成线性卷曲线圈的α螺旋一致 纤维。 α-螺旋区域包含所有生物识别的表位 保护性单克隆抗体。 仅 C 端至 α 螺旋 结构域是富含脯氨酸的结构域，后跟锚定结构域 含有10个20个氨基酸重复序列。 大多数 Rx1 的保护性单克隆抗体 PspA 识别 α 螺旋 C 端三分之一的表位 地区。 这些表位比α-中的其他表位更具交叉反应性 螺旋区域。 Southern 印迹和特定 pspA 引物的能力 通过聚合酶链式反应 (PCR) 扩增不同的 pspAs 证明 pspA 编码 α 螺旋结构域的部分更多 比编码 PspA C 端一半的变量更可变。 在拟议的研究中，我们将对几个 pspA 进行测序并确定 它们的α螺旋序列的部分包含能够 在正常和 SCD 小鼠中引发保护性抗体。 我们计划 确定人类是否对相同表位产生保护性反应 使用来自恢复期 SCD 和非 SCD 的亲和纯化抗体 患者。 这些研究可能会导致开发一种廉价的 重组 PspA 融合蛋白可用作疫苗 儿童肺炎球菌感染。 我们还将使用 pspA 序列 确定用作 PCR 引物的保守区域的信息，以帮助 在肺炎球菌感染的诊断中。 这种类型的诊断 需要能力，以便能够开发出新的疫苗和治疗方法 准确评估。 更好的诊断也将使医生能够 使用更合适的抗生素治疗。 使用 PspA 的优势 诊断性 PCR 扩增的目标是 RFLP 扩增产物可以促进菌株的流行病学研究 感染正常人和 SCD 患者的肺炎链球菌。 拟议的研究 将使用来自 SCD 患者的分离株和恢复期血清。