COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT III

脓毒症与肺炎链球菌是一个主要的死亡原因， 镰状细胞病（SCD）患者。 当用一种胶囊 多糖疫苗可增加对肺炎球菌感染的抵抗力 在年龄较大的SCD患者中，对于小于2岁的SCD患者， 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，它们确实使良好的免疫力 对大多数蛋白质的反应。 我们发现了一个肺炎球菌表面 蛋白质（PspA），有潜力成为一个良好的肺炎球菌 儿童疫苗。 在小鼠中，它具有高度的免疫原性和免疫原性， 抗体是保护免受致命的感染与S。 肺炎。 PspA是一种毒力因子，可减缓S. 血液中的肺炎 虽然PspA在血清学上是可变的， 不同的PspA具有足够的交叉反应性， 单独的PspA可以引起针对不同菌株的保护， PspA类型。 因此，仅几种PspA的混合物（或重组体）可以用于制备PspA。 含有仅来自少数PspA的特异性表位的蛋白质）将被 需要疫苗。 我们测定了Rx 1菌株的pspA基因。 推测的蛋白质序列的N-末端49%是高电荷的， 与能够形成线性卷曲螺旋的α-螺旋一致 纤维 α-螺旋区含有所有抗原识别的表位， 保护性单克隆抗体。 就在阿尔法螺旋的C末端 结构域是富含脯氨酸的结构域，后面是锚定结构域 含有10个20个氨基酸的重复序列。 大多数针对Rx 1的保护性单克隆抗体 PspA识别α-螺旋多肽C-末端三分之一中的表位。 地区 这些表位比α-淀粉样蛋白中的其他表位更具交叉反应性。 螺旋区 Southern印迹和特异性pspA引物的能力 通过聚合酶链反应（PCR）扩增不同的pspA， 编码α-螺旋结构域的pspA部分比编码α-螺旋结构域的pspA部分更多， 与编码PspA的C-末端一半的蛋白相比， 在拟议的研究中，我们将对几种pspA进行测序，并确定 它们的α-螺旋序列的部分含有能够 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的表位产生保护性反应， 通过使用来自恢复期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融合蛋白用作抗 儿童肺炎球菌感染。 我们还将使用pspA序列 用于鉴定用作PCR引物的保守区域的信息， 肺炎球菌感染的诊断。 这种诊断 需要有能力使新的疫苗和治疗方法能够 准确评价。 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 作为诊断性PCR扩增的靶点， 扩增产物可以促进菌株的流行病学研究， S.肺炎，感染正常和SCD患者。 拟议的研究 将使用SCD患者的分离物和恢复期血清。

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.