困難梭狀芽孢桿菌被認為是全世界醫療系統中感染性腹瀉的主要原因之一，其致病機制和所分泌的兩個外毒－毒素A及毒素B素有關。在本篇研究中，我們建構一個移除了毒素活性域的重組蛋白rTcdB作為疫苗的抗原候選，此抗原蛋白由毒素B的C端受體結合域(1852-2363殘基)所構成，包覆於聚谷氨酸/幾丁聚醣(γ-PGA / chitosan)奈米微粒中，此奈米微粒來自自然材質，具有生物可分解性、無毒性且能夠引發高程度的免疫反應，此外，聚谷氨酸/幾丁聚醣奈米微粒在近年來被報導能夠應用於口服疫苗載體，其可在腸道誘發強烈的黏膜免疫反應。我們比較了注射和口服接種兩種途徑，發現無論是注射還是口服皆可引發足夠的免疫反應，達到保護小鼠免於腹瀉及死亡的效果，此保護效果是來自疫苗所誘發的大量毒素中和抗體，並且由奈米顆粒所包覆的抗原能夠產生比佐以傳統佐劑氫氧化鋁者更加長效的抗體反應；這些結果證實了rTcdB具有高免疫原的特性，而聚谷氨酸/幾丁聚醣奈米微粒包覆蛋白質抗原確實能夠保護小鼠免於感染，顯示此形式的奈米微粒能夠作為安全且強效的疫苗佐劑。總結以上，此研究顯示預防性注射或口服接種包裹rTcdB的奈米微粒能夠提供足夠的保護，達到預防困難梭狀芽孢桿菌感染的效果。

Clostridium difficile is now considered to be one of the major causes of infectious diarrhea in healthcare systems worldwide. C. difficile infection is believed to be a toxin-mediated intestinal disease caused mainly by two large exotoxins, toxins A and B. In this study, we constructed a non-toxic recombinant protein, rTcdB, which consists of residues 1852-2363 of Toxin B receptor binding domain as a potential vaccine candidate. rTcdB was encased in nanoparticles (NPs) composed of γ-PGA and chitosan, which are made of natural materials, biodegradable, non-toxic and able to induce a high degree of immune response. Moreover, the NPs were recently reported as a mucosal adjuvant; it could induce a strong mucosal immunity in the gastrointestinal tract. We compared intraperitoneal injection and mucosal vaccination regimens and found that both methods provided mice full protection from lethal dose of C. difficile spore challenge. Protection was associated with high levels of toxin-neutralizing antibodies, and the rTcdB-encapsulating NPs elicited longer-lasting antibody responses than rTcdB with the conventional adjuvant, aluminum hydroxide. These results suggest that rTcdB is highly immunogenic when encapsulated by the safe and potent vaccine adjuvant NPs. In conclusion, this study demonstrates that prophylactic parenteral or oral vaccination with rTcdB-encapsulating NPs can provide protection from C. difficile infection.

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