Development of a Subunit Vaccine Against Q Fever

甲型Q热疫苗仍然是一个重要的公共卫生和国家安全目标。这样做的目的是 应用克隆伯氏杆菌脂多糖的O抗原表位 异源脂蛋白A和核心或N连接的糖蛋白，并与保护性T细胞刺激相结合 重组减毒沙门氏菌疫苗中的重组蛋白。疫苗 候选的重组抗原的保护能力将赋予强大的T细胞 BL6和MHC完全人源化(DR1或DR4)疫苗诱导和感染衍生免疫应答 老鼠和繁殖的豚鼠。我们将通过以下具体目标实现这一目标：(一) 鉴定和鉴定亚单位LPSO抗原表达所需的基因。保护性碳水化合物 伯氏弧菌内毒素O侧链上的表位可在异源脂质A核心受体上表达 沙门氏菌通过克隆编码合成O抗原的生物合成途径的必要基因。 重组O抗原碳水化合物合成的选择将包括噬菌体抗性、内毒素特异性 抗体反应性和O抗原合成预测基因的直接克隆。(2)异构性 以N-连接糖蛋白的形式表达O多糖。O-端的保护性碳水化合物表位 伯氏杆菌内毒素链将利用转移酶(PgIB)以N-连接的糖蛋白的形式表达 空肠弯曲菌受体(AcrA)蛋白。偶联O侧链碳水化合物将是 纯化，结构和抗原性表征。一种伯氏杆菌抗原蛋白(Com-1)将是 用受体基序修饰以取代AcrA，并评估其作为抗原性N-连锁的能力 载体蛋白。(3)鉴定和鉴定模拟O-多糖的模拟表位多肽。 将筛选组合多肽库以寻找与伯氏梭菌O抗体发生交叉反应的靶点 多糖类。这些多肽将在疫苗接种后通过识别识别来测试保护性抗体反应 伯氏华支睾吸虫LPS.候选多肽将与KLH或TT载体蛋白偶联并用于刺激 小鼠的抗体反应。(4)评价异源表达的巴氏梭菌的保护作用。 小鼠和豚鼠挑战模型中的碳水化合物和蛋白质链。实况RASV， 从RASV制备的微囊和纯化的与异源内毒素结合的重组蛋白将 被比较。

A Q fever vaccine remains an important public health and national security goal. The objective of this application is to clone the O antigen epitopes from C. burnetii lipopolysaccharide (LPS) expressed on a heterologous LPS lipid A and core or N-linked glycoprotein, and combine this with protective T cellstimulating recombinant proteins in Recombinant Attenuated Salmonella Vaccines (RASV). The vaccine candidates will be characterized for the protective capacity of recombinant antigens that confer strong T-cell responses in vaccine-induced and infection-derived immunity in BL6 and MHC ll-humanized (DR1 or DR4) mice and outbred guinea pigs. We will accomplish the objective by pursuing the following specific aims: (1) Identify and characterize genes required forsubunit LPS O-antigen expression. Protective carbohydrate epitopes on the O-side chain of C. burnetii LPS can be expressed on a heterologous lipid A-core acceptor in Salmonella by cloning essential genes encoding the biosynthetic pathways for O antigen synthesis. Selection for recombinant O antigen carbohydrate synthesis will include phage resistance, LPS-specific antibody reactivity, and direct cloning of predicted genes for O antigen synthesis. (2) Heterologously express O polysaccharide as an N-linked glycoprotein. Protective carbohydrate epitopes on the O-side chain of C. burnetii LPS will be expressed as an N-linked glycoprotein using the transferase (PgIB) and acceptor (AcrA) proteins from Campylobacterjejuni. The coupled O side chain carbohydrates will be purified, and structurally and antigenically characterized. A C. burnetii antigenic protein (Com-1) will be modified with an acceptor motif to replace AcrA and evaluated for the ability to act as an antigenic N-linked carrier protein. (3) Identify and characterize mimetope peptides that antigenically model O-polysaccharide. Combinatorial peptide libraries will be screened for targets that cross react with antibody specific for C. burnetii O polysaccharide. These peptides will be tested for protective antibody responses upon vaccination by recognizing C. burnetii LPS. Candidate peptides will be conjugated to KLH or TT carrier protein and used to stimulate antibody responses in mice. (4) Evaluate the protective capacity of heterologously expressed C. burnetii Oside chain carbohydrates and proteins in mouse and guinea pig challenge models. Live RASV, microvesicles prepared from RASV, and purified recombinant proteins conjugated to heterologous LPS will be compared.