Utilization of the adjuvant effect of CRM197 protein to develop a trivalent protein-vaccine against Streptococcus pneumoniae infections

摘要 肺炎链球菌(Spn)是引起一系列非侵袭性(中耳炎)的主要呼吸道病原体 媒体、鼻窦炎、肺炎)以及侵袭性(败血症、脑膜炎)疾病。尽管总体上出现了下降 现有疫苗(Spn/肺炎球菌结合疫苗：PCV)造成的SPN疾病负担， SPN疾病继续发生，并仍然是一个重大的医学问题。由于新技术的出现， 替换血清型(STS)，以Spn蛋白为基础的疫苗，旨在预防Spn疾病 不依赖血清型的方法被认为是最好的解决方案。然而，迫切需要 开发针对Spn的增强抗体应答及其生物学功能的新型佐剂 蛋白质抗原，考虑到这些疫苗的目标人群是儿童、老年人和免疫低下者 个人。 我们开发了一种三价蛋白质疫苗，涉及两种著名的Spn候选疫苗，N-末端 肺炎球菌表面蛋白A(PSPA)和肺炎溶血素结构域4(PlyD4)，与白喉基因融合 选择N-PSPA和PlyD4的目的是基于 它们在免疫动物模型中的先验特征，证明了它们对Spn定植的有效性 还有败血症。因此，CRM197与已证实的Spn候选基因的基因融合将有助于比较CRM197驱动的 通过已发表的工作增强免疫原性和保护效力，并将平台扩展到 随后包括其他有希望的候选疫苗。 解毒抗原CRM197(CRM197-N-PSPA-PlyD4)。 我们的初步数据显示，基因融合 CRM197显著提高了抗PSPA N-末端抗体效价(数倍)。此外， 基因融合(三价疫苗)导致抗血清与Spn细菌的结合显著增加，这是一项措施 抗体的功能。此外，Spn细菌与三价抗血清的孵育显著减少。 SPN对A549细胞的粘附性。 根据我们的初步数据，我们假设CRM197的基因融合增强了记忆T细胞 对Spn抗原的反应，导致增强抗体功能和防止Spn定植 和疾病。“这一假设将在两个结构化目标中进行测试，这两个目标将确定CRM197在 增强Spn抗原、N-PSPA和PlyD4的免疫原性和保护性。调查结果： 拟议的工作可能导致CRM197在增强免疫方面的佐剂作用得到更广泛的利用 对Spn和非Spn蛋白抗原的反应，导致更强大的T细胞依赖免疫 反应和保护作用。

Abstract Streptococcus pneumoniae (Spn) is a major respiratory pathogen that causes a spectrum of non-invasive (otitis media, sinusitis, pneumonia) as well as invasive (sepsis, meningitis) diseases. Despite a decline in the overall Spn disease burden caused by currently available vaccines (Spn/pneumococcal conjugate vaccines: PCVs), Spn diseases continue to occur and remain a significant medical problem. Due to the emergence of new, replacement serotypes (STs), Spn protein-based vaccines that aim to protect against Spn diseases in a serotype-independent manner are considered to be the best solution. However, there is a pressing need to develop novel adjuvants that can enhance antibody response and its biological function directed against Spn proteins antigens, given the target populations of these vaccines are children, elderly, and immunocompromised individuals. We developed a trivalent protein vaccine involving two well-known Spn vaccine candidates, N-terminal pneumococcal surface protein A (PspA) and domain 4 of pneumolysin (PlyD4), genetically fused with diphtheria The purpose of choosing N-PspA and PlyD4 is based on their prior characterization in animal models of vaccination with demonstratable efficacy against Spn colonization and sepsis. Therefore, genetic fusion of CRM197 to proven Spn candidates will help compare the CRM197 driven enhancement of immunogenicity and protective efficacy with published work and to expand the platform to include other promising vaccine candidates subsequently. detoxified antigen CRM197 (CRM197-N-PspA-PlyD4). Our preliminary data shows that the genetic fusion of CRM197 significantly enhanced (several folds) the antibody titers against N-terminal PspA. Additionally, the genetic fusion (trivalent vaccine) led to a significant increase in the binding of antisera to Spn bacteria, a measure of antibody functionality. Furthermore, the incubation of Spn bacteria with trivalent antisera significantly reduced Spn adherence to A549 cells. Based on our preliminary data, we hypothesize that “Genetic fusion of CRM197 enhances the memory T-cell response to Spn antigens, leading to enhanced antibody functionality and protection against Spn colonization and disease.” This hypothesis will be tested in two structured aims that will establish the role of CRM197 in enhancing the immunogenic and protective efficacy of Spn antigens, N-PspA and PlyD4. The findings of the proposed work could lead to a broader utilization of the adjuvant effect of CRM197 in enhancing the immune response to Spn as well as non-Spn protein antigens, leading to a more robust T-cell dependent immune response and protective effect.