Vaccine potential of the proline-rich domain of pneumococcal surface protein A

 说明(由申请人提供)：针对中耳炎、非复杂性肺炎和脑膜炎的保护需要一种非囊型特定的疫苗溶液。肺炎球菌表面蛋白PSPA对小鼠的肺炎、菌血症、定植和致死性败血症有保护作用。PSPA处于FDA批准的安全性(第一阶段)试验(1998、2002、2010和2012-14)。这些试验没有发现安全问题，PSPA诱导的人类抗体(Ab)保护小鼠免受致命的脓毒症。纯化的rPspA的人体试验(1998、2002和2010)检查了PSPA N-末端的~300aa-螺旋结构域(AHD)引起的保护作用。AHD是可变的；一种疫苗可能需要多达三种不同的AHD，以涵盖所有Sp中PSPA的多样性。我们最近发现，~80aa的富含脯氨酸的结构域(PRD)对脓毒症也有保护作用。所有PSPA中都有PRD域。每个PRD包含1到5个不同的6-10个氨基酸重复序列。我们有针对其中一个短重复序列的保护性单抗(MAb)，以及针对保守的22aa非脯氨酸阻断(NBP)序列的单抗，该序列存在于~50%的PRD中。然而，缺乏这两个表位之一的珠江三角洲仍可能引起交叉保护，这表明存在额外的保护性表位。我们已经在人血清中发现了针对四个PRD重复序列的抗体。我们将通过包含其所有常见的保护诱导表位来创建广泛交叉反应的rPRD。开发一种高度交叉保护的PRD免疫原是很重要的，因为PSPA进入第三阶段疗效试验的一些延迟是由于担心在AHD中的变异性 结构可能允许Sp围绕PSPA和疫苗进化。珠江三角洲将提供第二套表位，用于覆盖所有PSPA。我们将确定rPRD可以在多大程度上取代或增强AHD对所有相关衣壳类型的Sp菌株所产生的交叉保护。目的：1.确定在小鼠、兔子和人类中具有免疫原性的PRD中主要的保护诱导重复序列，并利用这些信息构建预期具有广泛交叉保护作用的候选rPRD。AIM2.评估在AIM 1中构建的rPRD免疫原在小鼠败血症、局灶性肺炎和定植模型中所产生的交叉保护作用。比较PRD、AHD和PRD+AHD针对不同Sp的保护能力。PSPA结构的变异与被膜类型没有遗传或功能上的关系。因此，长期以来，人们一直认为，抗PSPA抗体对少数小鼠毒力包膜类型的保护作用适用于非小鼠毒力包膜类型的Sp。为了检测对所有相关包膜类型侵袭性菌株的保护作用，我们将：1)使用我们的体外功能试验，2)检测由包膜类型比败血症更多的包膜类型引起的小鼠局灶性肺炎，以及3)使用宿主炎症相关分子暂时增强通常不具有小鼠毒力的包膜类型侵袭性临床IPD分离株的毒力。Aim3.确定抗PRD抗体是否能促进C‘在Sp上的沉积，并增强杀菌肽对Sp的杀灭作用。AB通过这两种机制发挥PSPA的作用。

 DESCRIPTION (provided by applicant): Protection against otitis media, non-complicated pneumonia, and meningitis requires a vaccine solution that is not capsular type-specific. The pneumococcal surface protein PspA elicits protection in mice against pneumonia, bacteremia, colonization, and fatal sepsis. PspA was in FDA-approved safety (phase 1) trials (1998, 2002, 2010, and 2012-14). These trials revealed no safety problems, and the human antibody (Ab) elicited to PspA protected mice from fatal sepsis. The human trials of purified rPspA (1998, 2002, and 2010) examined protection elicited by the ~300 aa a-helical domain (aHD) at the N-terminal end of PspA. The aHD is variable; up to three different aHD may be required in a vaccine to cover the diversity of PspA in all Sp. We have recently shown that the ~80 aa proline-rich domain (PRD) also elicits protection against sepsis. The PRD domain is found in all PspAs. Each PRD contains 1 to 5 different 6-10 AA repeat sequences. We have protective monoclonal antibody (mAb) to one of these short repeat sequences and to a conserved 22 aa non-proline block (NBP) sequence, present in ~50% of PRD. However, a PRD lacking either of these epitopes could still elicit cross-protection, indicating the existence of additional protective epitopes. We have already found Ab to four of the PRD repeat sequences in human sera. We will create broadly cross-reactive rPRD by including all of its common protection-eliciting epitopes. Developing a highly cross-protective PRD immunogen is important because some of the delay in moving PspA into phase 3 efficacy trials is due to concern that variability in the aHD structure may allow Sp to evolve around a PspA aHD vaccine. The PRD will provide a second set of epitopes with which to cover all PspAs. We will determine the degree to which rPRD can replace, or augment, the cross-protection elicited by the aHD against Sp strains of all relevant capsular types. Aim1. Identify major protection-eliciting repeats in PRD that are immunogenic in mice, rabbits, and humans and use this information to construct candidate rPRD expected to be broadly cross-protective. Aim2. Evaluate cross- protection elicited by the rPRD immunogens constructed in Aim 1 in mouse sepsis, focal pneumonia, and colonization models. Compare the ability of PRD, aHD, and PRD+aHD to elicit protection against diverse Sp. Variations in PspA structure are not genetically or functionally related to capsule type. Thus, it has long been assumed that the protection observed against the few mouse-virulent capsule types with Ab to PspA would hold for Sp of non-mouse virulent capsular types. To examine protection against invasive strains of all relevant capsular types we will: 1) use our in vitro functional assay, 2) ue focal pneumonia which is caused in the mouse by many more capsular types than sepsis, and 3) use host inflammation-associated molecules to temporarily enhance the virulence of invasive clinical IPD isolates of capsular types that are not normally mouse-virulent. Aim3. Determine whether Ab to PRD can enhance C' deposition on Sp and enhance the killing of Sp by bactericidal peptides. Ab to the aHD of PspA functions by both of these mechanisms.