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COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT III

少数民族健康合作项目--项目三

基本信息

批准号：
2029047
负责人：
David E Briles
金额：
$ 11.54万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-01-15 至 1998-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2029047
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 vaccine development

项目摘要

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镰状细胞病(SCD)患者。同时用胶囊进行免疫多糖疫苗可增强对肺炎球菌感染的抵抗力对于年龄较大的SCD患者，小于2岁的SCD患者无效 5岁，因为年幼的孩子对大多数多糖类抗原。然而，它们确实具有很好的免疫力。对大多数蛋白质的反应。我们发现了一种肺炎球菌表面有可能成为一种良好肺炎球菌的蛋白质(PSPA) 儿童疫苗。在小鼠身上，它具有高度的免疫原性并引发对致命性感染S。肺炎。PSPA是一种毒力因子，可减缓S。血液中的肺炎。尽管PSPA在血清学上是可变的，不同的PspA具有足够的交叉反应，抗体可以对抗单个PSPA可以诱导对不同菌株的保护作用 PSPA类型。因此，只有几个PSPA的混合物(或重组只包含几个PspA特定表位的蛋白质)将是需要一种疫苗。我们已经对RX1菌株的PSPA基因进行了测序。推测的蛋白质序列的N-末端49%是高电荷的，并且与能够形成线性螺旋的阿尔法螺旋一致纤维。α-螺旋区包含所有人识别的表位。保护性的单抗。从C-末端到α-螺旋结构域是富含脯氨酸的结构域，后跟一个锚定结构域包含10个20个氨基酸重复。大多数对RX1具有保护性的单抗 PSPA识别α-螺旋C-末端三分之一的表位区域。这些表位比阿尔法中的其他表位具有更强的交叉反应。螺旋区。Southern杂交与PSPA特异引物的能力聚合酶链式反应扩增多种多样的PspA PSPA编码α-螺旋结构域的部分更多比编码PSPA的C-末端一半的变量。在拟议的研究中，我们将对几个PSPA进行排序，并确定其包含表位的α-螺旋序列的一部分在正常和SCD小鼠中诱导保护性抗体。我们计划确定人类是否对相同的表位做出保护性反应应用康复期SCD和非SCD亲和纯化抗体病人。这些研究可能会导致开发一种廉价的重组PSPA融合蛋白用作猪瘟疫苗儿童肺炎球菌感染。我们还将使用PSPA序列用于识别保守区的信息，用作聚合酶链式反应引物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，以便新的疫苗和治疗方法能够准确评估。更好的诊断也将使医生能够使用更适当的抗生素治疗。使用PSPA的优势作为诊断性聚合酶链式反应扩增的目标是扩增产物可促进对猪瘟病毒株的流行病学研究感染正常人和SCD患者的肺炎链球菌。建议进行的研究将使用SCD患者的分离株和恢复期血清。