背景:肺炎球菌的致病性高,死亡率,多药耐药模式一直在增加。许可的基于多糖的疫苗的有限覆盖和非疫苗血清型的替代是产生成功的血清型非依赖性疫苗的主要原因。肺炎球菌表面蛋白A(PspA)是一种极其重要的毒力因子,是一种有趣的基于保守蛋白的肺炎球菌疫苗的候选疫苗,分为两个主要家族,包含五个进化枝。PspA家族引起的免疫是进化枝依赖性的,并且PspA交叉反应性的水平限于同一家族。
方法:为了涵盖和克服本研究中PspA的进化枝依赖性免疫,我们设计并测试了一种PspA1-5c+p候选疫苗,该疫苗由每个进化枝的B-和T-细胞表位截短结构域的免疫显性覆盖率最高组成,重点关注PspA的两个交叉反应性B和C区.抗原性,毒性,物理化学性质,三维结构预测,使用分子动力学(MD)模拟设计的蛋白质的稳定性和灵活性,构建体与HLADRB1*(01:01)和人乳铁蛋白N-lop的分子对接,和免疫模拟使用免疫信息学工具进行评估。在实验部分,在用明矾佐剂化的重组PspA1-5c+p对小鼠进行腹膜内免疫后,我们评估了免疫反应,交叉反应性,和使用ELISA的抗PspA1-5c+p抗体的功能,视吞噬杀伤活性,和血清杀菌试验。
结果:第一次,这项工作使用免疫信息学工具提出了一种基于PspA的新型疫苗候选物.预计设计的PspA1-5c+p蛋白具有高度抗原性,无毒,可溶性,在MD模拟中稳定且灵活性低,能够刺激体液和细胞免疫反应。在对接研究中,设计的蛋白质还可以与HLADRB1*(01:01)和人乳铁蛋白N-lop强烈相互作用。我们的免疫信息学预测使用实验数据进行了验证。结果表明,抗PspA1-5cpIgG不仅具有高滴度,对所用的所有肺炎球菌血清型具有强且相同的交叉反应性,而且使用补体系统和吞噬细胞对肺炎球菌清除具有高且有效的生物活性。
结论:我们的研究结果阐明了PspA1-5c+p候选疫苗作为具有强交叉反应性特征的血清型非依赖性肺炎球菌疫苗的潜在应用。应进行针对其他PspA进化枝的进一步体外和体内研究,以确认PspA1-5c+p疫苗候选物的完全保护。
The pathogenicity of pneumococcus with high morbidity, mortality, and multi-drug resistance patterns has been increasing. The limited coverage of the licensed polysaccharide-based vaccines and the replacement of the non-vaccine serotypes are the main reasons for producing a successful serotype-independent vaccine. Pneumococcal surface protein A (
PspA) is an extremely important virulence factor and an interesting candidate for conserved protein-based pneumococcal vaccine classified into two prominent families containing five clades.
PspA family-elicited immunity is clade-dependent, and the level of the
PspA cross-reactivity is restricted to the same family.
To cover and overcome the clade-dependent immunity of the PspAs in this study, we designed and tested a PspA1-5c+p vaccine candidate composed of the highest immunodominant coverage of B- and T-cell epitope truncated domain of each clade focusing on two cross-reactive B and C regions of the PspAs. The antigenicity, toxicity, physicochemical properties, 3D structure prediction, stability and flexibility of the designed protein using molecular dynamic (MD) simulation, molecular docking of the construct withHLADRB1*(01:01) and human lactoferrin N-lop, and immune simulation were assessed using immunoinformatics tools. In the experimental section, after intraperitoneal immunization of the mice with Alum adjuvanted recombinant PspA1-5c+p, we evaluated the immune response, cross-reactivity, and functionality of the Anti-PspA1-5c+p antibody using ELISA, Opsonophagocytic killing activity, and serum bactericidal assay.
For the first time, this work suggested a novel
PspA-based vaccine candidate using immunoinformatics tools. The designed PspA1-5c+p protein is predicted to be highly antigenic, non-toxic, soluble, stable with low flexibility in MD simulation, and able to stimulate both humoral and cellular immune responses. The designed protein also could interact strongly with HLADRB1*(01:01) and human lactoferrin N-lop in the docking study. Our immunoinformatics predictions were validated using experimental data. Results showed that the anti-PspA1-5c+p IgG not only had a high titer with strong and same cross-reactivity coverage against all pneumococcal serotypes used but also had high and effective bioactivity for pneumococcal clearance using complement system and phagocytic cells.
Our findings elucidated the potential application of the PspA1-5c+p vaccine candidate as a serotype-independent pneumococcal vaccine with a strong cross-reactivity feature. Further in-vitro and in-vivo investigations against other
PspA clades should be performed to confirm the full protection of the PspA1-5c+p vaccine candidate.