抗鼠傷寒沙門氏菌之去脂多醣-鞭毛蛋白共軛自佐劑疫苗：製備與效果評估

Preparation and Evaluation of Lipid-A Free Lipopolysaccharide-Flagellin Conjugate Self-adjuvanting Vaccine against Salmonella typhimurium

彭啟鈞(Chi-Jiun Peng)

指導教授 ： 方俊民

國立臺灣大學/理學院/化學研究所/博士(2019年)

https://doi.org/10.6342/NTU201900705

摘要

細菌鞭毛蛋白 (flagellin, FliC) 可以作為共軛疫苗 (conjugate vaccine) 載運抗原的載體蛋白，亦為類鐸受體 (toll-like receptor 5, TLR5) 之配位體，可以透過其自佐劑 (self-adjuvnat) 活性有效提高疫苗效力。鞭毛蛋白的佐劑效力來自其 D1 結構域與 TLR5 辨識結合後，再由 D0 結構域負責活化後續的訊息傳遞鏈。若以傳統不具位置選擇性的化學方法連結標的抗原與鞭毛載體生產共軛疫苗，結果將導致 D0 與 D1 結構域的過度修飾，造成鞭毛共軛產物 (conjugates) 喪失其佐劑活性。為使鞭毛蛋白能夠有效作為自佐劑載體，本研究首先利用鞭毛單體聚合鞭毛絲時產生的立體自我保護效應 (steric self-protection effect)，開發一個具有位置選擇性的化學修飾方法 (site-selective modification, SSM)，選擇性地於鞭毛蛋白中的 D2 與 D3 結構域建構生物正交官能基 (bioorthogonal groups) 以供後續抗原結合之用，有效避免 D0 和 D1 結構域的過度修飾，保留了鞭毛蛋白之自佐劑活性。
革蘭氏陰性菌表面具有獨特的脂多醣 (lipopolysaccharides, LPS)。這些具有輕度免疫原性的多醣是疫苗的合適抗原。本研究的第二個目的是利用去脂多醣 (lipid-A free LPS, LFPS) 結合前述細菌鞭毛蛋白，製備並評估一個新型抗傷寒沙門氏菌共軛疫苗。我們透過使用碘促進去羧基醯胺化反應 (iodine-promoted decarboxylative amidation) 與無痕跡 Staudinger 連接 (traceless Staudinger ligation)，運用不同的連結分子製備了三種保留了自佐劑活性的 stLFPS-stFliC 共軛疫苗(11)、(12)與(13)。動物實驗結果表明，以三種不同的共軛疫苗免疫的小鼠均能產生對脂多醣和鞭毛蛋白的特異性免疫反應。而用來連接去脂多醣和鞭毛蛋白的連結分子則不具有明顯的免疫原性。施打疫苗顯著延長了小鼠的存活時間並提升了存活率，這可能說明了 stLFPS-stFliC 共軛產物的自佐劑活性和多抗原表位特性確實如預期提高了疫苗效力。本研究的結果為開發具有自佐劑活性的 LFPS-FliC 共軛疫苗提供了可行的方法，並對疫苗發展提供了另外一個具有潛力的方向。

關鍵字

鞭毛 ； 疫苗 ； 佐劑 ； 脂多醣 ； 傷寒沙門氏菌

並列摘要

Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines and as an intrinsic adjuvant to activate the toll-like receptor 5 (TLR5) to enhance vaccine potency. The adjuvant activity of flagellin is derived from the binding of the D1 domain to TLR5 recognition site along with the subsequent signaling cascades activated by D0 domain. If a conventional conjugation method is used to link antigens to flagellin to produce a conjugate vaccine, over-modification of the D0 and D1 domains will result in the loss of adjuvant activity of the conjugate. To enable the use of FliC as a self-adjuvanting carrier, a site-selective modification (SSM) method based on the steric self-protection effect produced by the assembling of flagellin monomers to flagellar filaments is explored. This method selectively constructs bioorthogonal groups on the pertinent amino-acid residues in the D2 and D3 domains of FliC without excessive modification of the D0 and D1 domains, effectively retains the self-adjuvant activity of the flagellin.
Gram-negative bacteria exhibit unique lipopolysaccharide (LPS) on their surface. These accessible polysaccharides with mild immunogenicity are an appealing target for vaccination. Therefore, the second aim of this research is to prepare and evaluate an advanced conjugate vaccine against Salmonella typhimurium. Three self-adjuvanting stLFPS-stFliC conjugates (11), (12) and (13) were prepared through iodine-promoted decarboxylative amidation and traceless Staudinger ligation. Animal experiments showed that mice immunized with three different stLFPS-stFliC vaccines produced a significant immune response specific for lipopolysaccharide and flagellin of S. typhimurium. Meanwhile, the linker molecules are not significantly immunogenic. The vaccines significantly prolonged the survival time of mice and increased the survival rate, which may indicate that the self-adjuvanting ability and multiple epitopes of stLFPS-stFliC conjugates both provide immunity, as expected to improve the vaccine efficacy. The results of this research provide a viable approach to develop LFPS-FliC conjugate vaccines with self-adjuvant activity and offer a potential option for vaccine development.

並列關鍵字

Flagellin ； vaccine ； adjuvant ； lipopolysaccharide ； Salmonella typhimurium

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