“分域隔离”纳米疫苗抗泛耐药肺炎克雷伯菌的作用机制研究

Extensively drug-resistant klebsiella pneumoniae (XDR-Kp) has become a challenging problem in the field of critical care medicine worldwide because of the increasing incidence and the high mortality. Nanovaccines newly developed in recent years can hopefully become an alternative treatment for XDR-Kp cases, which have the functions of both prevention and treatment, vaccinating in high-risk patients in advance to clear off bacteria spontaneously. From a variety of XDR-Kp subtypes and drug-resistant strains collected and isolated in recent years in China, we have preliminarily isolated and screened out five O antigen and two K antigen epitopes as candidate antigen epitopes of nanovaccine. But the antigen epitopes are non T cell-dependent antigens, they can only produce primary immune response with low antibody abundance and short acting time, and therefore cannot clear bacteria out of the body of the patient completely. In order to enhance and amplify the immunogenicity of the antigen epitope, it is necessary to connect with carrier protein. But the carrier protein-induced epitopic suppression is common in research of nanovaccines against XDR-Kp, which is a key and bottleneck technique that has not been well dissolved. The essence of the carrier protein-induced epitopic suppression is epitope competition. As carrier protein is absolutely superior to the bacterial antigen epitope in identifying B cells, the vaccine produces both high-titer carrier protein antibodies and low-titer bacterial antibodies. To solve this technical crux, We are trying to use the self-assembly, microfluidics and chemical modification technology, to established a “dual isolated” nanovaccine on the basis of their previous successful establishment of hollow Nanospheres of the nuclear-hull structure, in which they expose the XDR-Kp antigen epitope to the surface of the Nanosphere and isolate the carrier protein within the Nanosphere. When the nanovaccine enters the body, the bacterial pathogen will be identified by B cell-specific priorities and presented to T helper cells in priority. As the carrier protein is isolated within the Nanosphere, it loses the possibility of contacting with and being identified by B cells. As a result, the side effect of carrier protein-induced epitopic suppression can be eliminated or inhibited. When the outer shell of the Naosphere engulfed by B cells is decomposed in B cells, the carrier protein inside is released and begins its action of enhancing the bacterial antigen immunogenicity, which further promotes B cells to secrete large numbers of bacteria-specific antibodies. Using the previously established mouse model of XDR-Kp pulmonary infection, the next work is to verify the efficacy and safety of the nanovaccine, and further explore the immunological mechanism of the nanovaccine within the organism, including how it triggers the specific response, breaks the immune tolerance effectively, and finally achieves the goal of clearing bacteria out of the body. The prospect of this research project is to develop a novel nanovaccine with independent intellectual property rights, thus providing a new drug and strategy for the clinical treatment of XDR-Kp.

高致死率的泛耐药肺炎克雷伯菌日益增多，成为重症医学的棘手难题。近年新兴的纳米疫苗兼有预防和治疗双重作用，通过对特定高危人群提前接种，有望成为抗菌药无效的挽救手段。前期我们从泛耐药肺炎克雷伯菌株中筛选出7个抗原的多糖表位作为疫苗候选。多糖表位需链接载体蛋白才能增强免疫原性，但载体蛋白诱导的表位抑制是目前疫苗研发中的技术瓶颈。因载体蛋白比细菌多糖表位在B细胞识别上占优势，导致机体产生高滴度的载体蛋白抗体和低滴度的细菌抗体。为突破此瓶颈，我们拟通过自组装、微流控和化学修饰等技术，构建一种分域隔离的新型纳米疫苗，将细菌表位置于纳米表面，被B细胞优先识别和递呈；将载体蛋白隔离至纳米内部，消除其表位抑制作用，仅发挥增强细菌抗原的免疫原性作用。在小鼠肺部感染模型上验证该疫苗的疗效和安全性，阐明该疫苗增强免疫原性、激发特异性应答、防治细菌的免疫学机制。本项目有望为防治泛耐药肺炎克雷伯菌提供新的药物和策略。

高致死率的泛耐药肺炎克雷伯杆菌日益增多，细菌疫苗的制备成为几年来新兴的研究领域。通过对蛋白的表位筛选来制备多肽疫苗是最常见的方法，多肽需偶联载体蛋白才能产生免疫原性，由载体蛋白引发的载体蛋白表位抑制现象是此类疫苗研发中的棘手难题。我们通过“氧化-热聚法”构建粒径可控的纳米载体，通过粒径调控、投料比来优化载体蛋白，从粒径、电位、形貌、稳定性、形成机理等方面对其理化性质进行表征。利用细菌外膜囊泡多表位的先天优势，辅以纳米载体内核组装成抗菌疫苗，在提高囊泡稳定性同时也大大增强了免疫原性。通过生物信息学技术手段，对肺炎克雷伯杆菌的MrkD菌毛蛋白的理化性质进行了研究，并筛选了B细胞表位，合成抗原多肽，结合文献已经验证的PCSK9蛋白多肽，对纳米抗菌疫苗和多肽纳米疫苗的各种理化性质、与免疫细胞的体内外相互作用进行考察。.我们证明了纳米载体的形成机理主要为二硫键，在胞内具有还原敏感性，通过优化载体粒径和不同蛋白的投料比，最大限度地减少载体特异性抗体滴度，并提高了抗菌抗体滴度，而且该抗体特异性高、亲和力高、持续时间久。 “分域隔离”纳米疫苗具有良好的生物相容性，100纳米左右的类病毒结构使得它对免疫细胞有强大的促吞噬作用。纳米疫苗可以有效地促进巨噬细胞、树突状细胞等免疫细胞的成熟和活化。经过优化的“分域隔离”纳米疫苗几乎可以抑制载体蛋白抗体的产生，使得目的抗体滴度稳健上升，对肺炎克雷伯杆菌诱导的脓毒症小鼠具有保护作用。. 在本研究中，我们拟通过合成具有抑制干扰性载体蛋白抗体产生的纳米载体，提高外膜囊泡抗菌疫苗和多肽抗菌疫苗的免疫效果，为重症领域棘手的细菌耐药难题提供新的治疗思路，同时也为多肽疫苗制备中遇到的载体蛋白表位抑制现象的克服提供了新的策略。