以Lactococcus lactis GEM particles作為鼻腔免疫黏膜佐劑之效果

黏膜免疫系統是身體對抗大多數外來病原體的第一道防線，主要是利用分泌性的IgA 來防止微生物的附著，也可藉由中和細菌外毒素的作用避免黏膜組織遭受破壞。藉由黏膜途徑接種能夠引發良好的黏膜及全身性的免疫反應，但是可溶性抗原本身所能引起的免疫性較弱，因此我們使用一非基因改造且屬GRAS (generally regarded as safe)級的乳酸球菌(Lactococcus lactis)，藉由其低免疫原性及佐劑功能當作抗原攜帶系統，增強局部的免疫反應。首先我們將抗原建構在帶有nisA 啟動子和訊息胜肽Usp45 的載體中，經由乳酸鏈球菌素的誘導後產生分泌型的蛋白質，這些分泌型的蛋白藉由羧基端具有與細胞壁胜肽聚醣結合能力的 protein anchor domain 結合到用酸和加熱處裡過的Lactococcus lactis Gram-positive enhancer matrix (GEM) particles 上。實驗中成功的將轉糖鏈球菌 (Streptococcus mutans)的Immunodominant glycoprotein 60 (IDG-60)建構到上述載體中，分別使用抗IDG-60、protein anchor 和6×His 的抗體執行西方墨點法，發現不管是細菌體內或是培養液中的IDG-60 重組蛋白質，都可以被這三種抗體辨認到。進一步，將培養液中的IDG-60 重組蛋白質或純化出的IDG-60 與GEM particles 混合後發現，兩者都可以黏附到GEM particles 上。採用以鼻腔內免疫途徑給予純化出的IDG-60 或IDG-60 與GEM particles 的混合物的方式接種BALB/c 小鼠，探討其調節小鼠黏膜及全身特異性免疫反應之影響，接種三次後，發現 GEM particles 可增加小鼠鼻咽相關淋巴組織、頸部淋巴結、脾臟與腸繫膜淋巴結中對IDG-60 特異性分泌細胞激素IL-2、IL-4、IL-10 及IFN-γ的細胞數，特別是在鼻咽相關淋巴組織的部位，並可顯著提升小鼠專一性抗IDG-60 唾液及糞便分泌型IgA 及血清IgG 的產量。

關鍵字

黏膜免疫；佐劑； GEM particles ；鼻腔內免疫；分泌型IgA

並列摘要

Mucosal immune system is the first line of defense against foreign pathogens that prevents adherence of microorganisms and neutralizes bacterial toxins through secretory IgA to maintain the integrity of the mucosal lining. Mucosal immunization can induce both mucosal and systemic immunity. To improve the immunogenecity of soluble antigens, we used generally regarded as safe (GRAS) bacteria, Lactococcus lactis to generate gram-positive enhancer matrix (GEM) particles with adjuvant properties as antigen display and delivery system. Firstly, we successfully constructed the expression system for immunodominant glycoprotein 60 (IDG-60) of Streptococcus mutans, under the control of a nisin-inducible promoter, with the lactococcal Usp45 signal sequence to drive secretion, and a protein anchor to direct surface localization on the GEM particles. Recombinant IDG-60 could be detected in cell lysates or cell-free culture supernatant and secreted IDG-60 could bind to GEM particles. To investigate the adjuvant effect of GEM particles for mucosal and systemic immune responses, mice were immunized intra-nasally with IDG-60 in soluble or GEM-bound form three times at weekly intervals. GEM particles enhanced IDG-60 specific serum antibody responses, including IgG1, IgG2a, IgG2b and IgG3. GEM particles also increased IDG-60 specific IgA levels in saliva and fecal samples. Although IDG-60 specific Th1- and Th2-type cytokine producing cells could be detected in different lymphoid tissues such as NALT, CLN, spleen or MLN, the incorporation of GEM enhanced significantly an increase in the numbers of cytokine producing cells preferentially in NALT.

並列關鍵字

mucosal immune ； adjuvant ； GEM particles ； intralnasal immunization ； secretory IgA

參考文獻

Steidler, L., Robinson, K., Chamberlain, L., Schofield, K.M., Remaut, E., Le Page, R.W. and Wells, J.M. (1998) Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect Immun. 66: 3183-3189.

Armstrong, M.E., Lavelle, E.C., Loscher, C.E., Lynch, M.A. and Mills, K.H. (2005) Proinflammatory responses in the murine brain after intranasal delivery of cholera toxin: implications for the use of AB toxins as adjuvants in intranasal vaccines. J Infect Dis. 192: 1628-1633.

Arnau, J., Hjerl-Hansen, E. and Israelsen, H. (1997) Heterologous gene expression of bovine plasmin in Lactococcus lactis. Appl Microbiol Biotechnol. 48: 331-338.

Audouy, S.A., van Roosmalen, M.L., Neef, J., Kanninga, R., Post, E., van Deemter, M., et al (2006) Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization. Vaccine. 24: 5434-5441.

Audouy, S.A., van Selm, S., van Roosmalen, M.L., Post, E., Kanninga, R., Neef, J., et al (2007) Development of lactococcal GEM-based pneumococcal vaccines. Vaccine. 25: 2497-2506.

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以Lactococcus lactis GEM particles作為鼻腔免疫黏膜佐劑之效果

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