Development of a Subunit Vaccine Against Q Fever

Q热疫苗仍然是一个重要的公共卫生和国家安全目标。的目的 应用是从C.贝氏脂多糖（LPS）表达在 异源LPS脂质A和核心或N-连接糖蛋白，并将其与保护性T细胞刺激联合收割机结合 重组减毒沙门氏菌疫苗（RASV）中的重组蛋白。疫苗 候选者将被表征重组抗原的保护能力，所述重组抗原赋予强的T细胞免疫应答。 在BL 6和MHC II人源化（DR 1或DR 4）中疫苗诱导的和感染衍生的免疫应答 小鼠和远系豚鼠。我们将通过以下具体目标来实现这一目标：（1） 鉴定和表征亚单位LPS O-抗原表达所需的基因。保护性碳水化合物 C. O-侧链上的表位贝氏菌LPS可以在异源脂质A-核心受体上表达， 通过克隆编码O抗原合成的生物合成途径的必需基因来研究沙门氏菌。 用于重组0抗原碳水化合物合成的选择将包括噬菌体抗性、LPS特异性 抗体反应性，以及直接克隆用于O抗原合成的预测基因。(2)异源 将O多糖表达为N-连接的糖蛋白。O侧的保护性糖表位 链C。贝氏菌LPS将使用转移酶（PgIB）表达为N-连接的糖蛋白， 受体（AcrA）蛋白。偶联的O侧链碳水化合物将是 纯化，并在结构上和抗原性上表征。梭贝氏体抗原蛋白（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-连接的 载体蛋白(3)鉴定和表征抗原性模拟O-多糖的模拟肽。 将筛选组合肽文库中与C特异性抗体交叉反应的靶标。伯内特岛 多糖这些肽将在接种疫苗后通过识别 C.贝氏菌候选肽将与KLH或TT载体蛋白缀合，并用于刺激KLH或TT的表达。 小鼠的抗体反应。(4)评估异源表达的C.白藓 链碳水化合物和蛋白质的小鼠和豚鼠攻击模型。实时RASV， 从RASV制备的微泡和与异源LPS缀合的纯化重组蛋白将 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.