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以豬胸膜肺炎放射桿菌之 ApxII A 蛋白開發新型疫苗佐劑
指導教授:楊文仁 博士 國立高雄大學生物科技研究所 學生:蔣庭彬 國立高雄大學生物科技研究所 摘要 佐劑是一種用以增加抗原免疫原性的免疫促進劑，藉此增加疫苗保護效力或改變疫 苗免疫反應類型。許多研究主要以大腸桿菌毒素及霍亂毒素次單位蛋白進行黏膜佐劑之 開發，本研究嘗試以豬胸膜肺炎放線桿菌產生的呼吸道毒素結構蛋白為標的，開發新型 佐劑。ApxII 在已知豬胸膜肺炎放線桿菌的毒素中，具有較弱的溶血能力及細胞毒性， ApxIIA 為其結構蛋白。依據已知 RTX 家族毒素的不同功能性區域，將 ApxIIA 基因分 為全長(F)、N 端(N)及 C 端(C)片段進行選殖、表達與純化。此等重組蛋白質對巨噬細胞 的進行毒性分析，其中以 ApxIIA-N 的毒性最弱，在 100 μg/ml 仍有 45 %細胞存活率， 而 ApxIIA-F 及 ApxIIA-C 二者於 50 μg/ml 時，造成所有細胞死亡，其毒性低於大腸桿菌 腸毒素 LTB 約 10 倍，低於豬胸膜肺炎放線桿菌脂多醣約 200 倍。以此等蛋白開發為疫 苗佐劑，在安全性的考量上更具有優勢。依據此等重組蛋白誘導細胞激素表現情形，推 測pxIIA-N 能誘導 Th1 路徑，而 ApxIIA-F 與 ApxIIA-C 能同時誘導 Th1 與 Th2 路徑。 以卵白蛋白為抗原，搭配上述重組蛋白免疫小鼠進行佐劑功能測試，其中含有 ApxIIA 之佐劑免疫組抗體效價可達 51200-102400，為僅施打卵白蛋白抗原的 8-16 倍。進一步 分析其誘發之 IgG1 與 IgG2a 抗體亞型表現，結果顯示 ApxIIA-F、ApxIIA-N 及 ApxIIA-C 組皆能顯著提升 IgG1 含量及部份提升 IgG2a 含量，推測主要能誘導 Th2 免疫途徑。分 析肺沖洗液 IgA 抗體效價，顯示添加 ApxIIA 蛋白組的 IgA 效價可高於僅施打卵白蛋白 抗原的 10 倍以上。在佐劑對體重影響試驗方面，在第三次免疫後，ApxIIA-F 及 ApxIIA-C 組體重沒有如無佐劑組正常地增加，由先前細胞實驗結果推測可能是由於 ApxIIA-F 及 ApxIIA-C 蛋白較具有細胞毒性。綜合上述結果，證實 ApxIIA 蛋白確實有發展為黏膜佐 劑之潛力，能有效增強抗體效價。但由於 ApxIIA-F 與 ApxIIA-C 較具有毒性，因此以 ApxIIA-N 最適合發展為新型的黏膜佐劑。 關鍵字：黏膜佐劑、ApxIIA、豬胸膜肺炎放線桿菌、卵白蛋白2
Development of novel vaccine adjuvant using
ApxIIA protein of Actinobacillus pleuropneumoniae
Advisor: Dr. Wen-Jen Yang Institute of Biotechnology National University of Kaohsiung
Student: Ting-Bin Jiang Institute of Biotechnology National University of Kaohsiung
ABSTRACT
Adjuvant is an immune stimulator which can enhance the immunogenicity of vaccine. The function of adjuvant is to enhance the protective immunity of vaccine or to change the type of immune response. The development of mucosal adjuvant was major focused in the subunit protein of E. coli heat labile toxin (LT) and cholera toxin (CT). In this study, a novel mucosal adjuvant was developed using the structural protein of respiratory track toxin produced by Actinobacillus pleuropneumoniae. It was known that ApxII has the lower hemolytic and cytotoxic activity in all Apx toxins of A. pleuropneumoniae, and ApxIIA is the structural protein of ApxII. According to the functional domain of RTX (repeats in toxin) family toxin, the full length, N terminal and C terminal fragment of ApxIIA gene was cloned, expressed and purified, respectively. In the MTT assay for the toxicity analysis of these recombinant proteins to macrophage, revealed that ApxIIA-N has lowest cytotoxic activity. The viability of 100 μg/ml ApxIIA-N treatment is still about 45 %. In the ApxIIA-F or ApxIIA-C treatment, all cells could not survive in the concentration of 50 μg/ml. The toxicity is 10-fold less than E. coli heat labile toxin B subunit (LTB) and 200-fold less than LPS of A. pleuropneumoniae. These recombinant proteins have the superiority to develop as adjuvant in safety concerned issue. According to the cytokine expression profile induced by these recombinant proteins, the data indicated that ApxIIA-N could induce Th1 pathway, and both Th1 and Th2 pathway could be induced by ApxIIA-F and ApxIIA-C. Using ovalbumin as antigen and combined with these recombinant proteins to evaluate their adjuvant effect in mice. The antibody titers specific to ovalbumin of ApxIIA adjuvant-containing groups could reach 51200-102400, which are 8- to 16-fold than adjuvant-free group. Analysis the expression of IgG1 and IgG2a subclass revealed that all ApxIIA adjuvant-containing groups could significantly enhance IgG1 and limited IgG2a titer. It indicates that the major induced pathway was Th2. The analysis of IgA titer in bronchoalveolar lavages fluid (BALF) of all ApxIIA adjuvant-containing groups showed that was 10-fold than adjuvant-free group. The body
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weight of mice was measured to evaluate the influence of adjuvant administered. After third immunization, the mice body weight of ApxIIA-F and ApxIIA-C groups did not increase normally as adjuvant-free group. It indicates that ApxIIA-F and ApxIIA-C may have higher cytotoxic activity as previously in vitro results shown. In summary, ApxIIA protein could enhance the antibody titer and has the potential for the development of mucosal adjuvant. However, ApxIIA-F and ApxIIA-C have higher cytotoxic activity. Therefore, ApxIIA-N is the best candidate for the development of novel mucosal adjuvant.
Keywords: Mucosal adjuvant, ApxIIA protein, Actinobacillus pleuropneumoniae, Ovalbumin
