TWI565474B - 一種用於增進一抗原之免疫原性的組合物 - Google Patents
一種用於增進一抗原之免疫原性的組合物 Download PDFInfo
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- TWI565474B TWI565474B TW103130443A TW103130443A TWI565474B TW I565474 B TWI565474 B TW I565474B TW 103130443 A TW103130443 A TW 103130443A TW 103130443 A TW103130443 A TW 103130443A TW I565474 B TWI565474 B TW I565474B
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Description
本發明提供一種用於增進一抗原之免疫原性的組合物，其包含：(i)該抗原；(ii)第一免疫輔助劑(adjuvant)，其包含LZ-8蛋白；以及(iii)第二免疫輔助劑，且該第二免疫輔助劑不含有LZ-8蛋白。本發明另提供一種疫苗組合物，其包含：(i)一抗原；(ii)第一免疫輔助劑(adjuvant)，其包含LZ-8蛋白；以及(iii)第二免疫輔助劑，且該第二免疫輔助劑不含有LZ-8蛋白。
樹突狀細胞(DCs)是專職的抗原呈現細胞(antigen-presenting cell)，作為連接天然和獲得性免疫的橋樑(Steinman,R.M.Nat Med 2007；13：1155-1159.)。DCs是異源性的，但其所有的亞群具有固有的協同免疫調節功能(Wu,L.et al,Immunity 2007；26：741-750；Iwasaki,A.Ann Rev Immunol 2007；25：381-418.)。當暴露於特定的抗原或環境時，它們能夠引導天然T細胞朝向免疫原性或免疫耐受性的方向(Abbas,A.K.et al.,Nat Immunol 2005；6：227-228.)。當通過發炎物質或微生物致病體刺激時，DCs在遷移到淋巴的過程中變成熟，這顯著地增強了DCs活化抗原專一性T細胞(antigen-specific T cells)的能力(Reis e Sousa,C.Nat Rev Immunol 2006；6：476-483.)。Toll樣受體(Toll-like receptors，TLRs)在天然識別病原相關分子模式(pathogen-associated molecular patterns，PAMPs)和活化DCs中之免疫反應上扮演著主要的角色(Lee,M.S.et al,Ann Rev Biochem 2007；76：13.1-13.34.)，一旦受TLR刺激，成熟DCs中之II類MHC(MHC class II)和協同刺激分子的表現量便上升，尤其是CD40、CD80和CD86，所有這些分子對活化T細胞都很重要(Kawai,T.et al,Semin Immunol 2007；19：24-32.)。因DCs在免疫反應中的關鍵調節作用，目前正在開發DCs來
治療癌症、過敏反應和病毒感染，並作為有效新疫苗的輔助劑來預防或治療癌症和感染性疾病(Banchereau,J.et al,Nat Rev Immunol 2005；5：296-306.；Steinman,R.M.et al,Nature 2007；449：419-426.)。促進DC活化的物質有潛力應用於免疫療法和疫苗接種。
從非褶菌目(Aphyllophorales)中已經分離出大量具生物活性的天然物質，這些非褶菌目為所謂的多孔菌。多孔菌是屬於擔子菌亞門(phylum Basdiomycota)(擔子菌)的一大類陸生真菌，它們與子囊菌門(Ascomycota)的一些種一起形成了藥理活性物質的主要來源(Zjawiony,J.K.et al,J Nat Prod 2004；67：300-310.)，靈芝(Ganoderma lucidum(Leyss.ex Fr.)Karst.(Ling-Zhi or Reishi))是一種有名的藥用真菌，屬於多孔菌科、菌褶少的擔子菌類真菌。在亞洲，這些菇類的醫藥性質，包括許多促進健康和治療的效用，被人們所熟知已有數個世紀。至目前已經積累了許多關於靈芝(G.lucidum)在許多疾病中其醫藥應用上的證據，如：腫瘤、癌轉移、高血壓、肝炎、胃炎、關節炎、支氣管炎、哮喘、厭食和免疫紊亂(Lin,Z.,Acta Pharmacol Sin 2004；25：1387-1395；Lin,Z.J Pharmacol Sci 2005；99：144-153.)。近來，研究人員已從子實體、純培養菌體和培養濾液(培養液)中萃取或純化出生物活性組分，並研究其生物作用(Shiao,M.S.,The Chem Record 2003；3：172-180.)；然而，靈芝的醫藥用途仍需要更另人信服的證據來支持。
從靈芝的擔子果和菌絲體中分離出的藥用活性化合物包括多糖、三萜類化合物(triterpenoids)、蛋白質、凝集素(lectins)、固醇類(sterols)、生物鹼(alkaloids)、核苷酸、內酯(lactones)和脂肪酸(Zhou,X.et al,Phytochemistry 2006；67：1985-2001.)。許多研究已顯示多糖是靈芝的主要活性成分(Hsu,H.Y.et al,J Immunol 2004；173：5989-5999.)；此外，一些研究已經證實靈芝中的天然三萜類化合物具有抗炎、抗肝炎和抗癌活性(Wang,G.et al,Int Immunopharmacol 2007；7：864-870.)。在靈芝的研究中，與多糖和三萜類化合物相比，少有著重於蛋白功能的研究(Jeurink,P.V.et al,Int Immunopharmacol 2008；8：1124-1133.)。一種分子量為12.4kDa之蛋白已從靈芝菌絲體中分離出來，命名為LZ-8的免疫調節蛋白(Kino,K.et al,J Biol
Chem 1989；264：472-478.)。從另一個種松杉靈芝(G.tsugae)中也分離到了該蛋白，命名為FIP-gts(Lin,W.H.et al,J Biol Chem 1997；272：20044-20048.)。一些研究已顯示LZ-8對自身免疫性和移植具有免疫調節作用，並且LZ-8作為促細胞***劑來活化T細胞(Hsu,H.Y.et al.,J Cell Physiol 2008；215：15-26.)。由於DCs在免疫系統中起著重要的作用，因此靈芝對DCs的作用已被研究過，但研究僅限靈芝中之多糖(Lin,Y.L.et al,Mol Pharmacol 2006；70：637-644.)，而尚未有相關之研究揭示LZ-8蛋白對DCs的作用。
樹突狀細胞(DCs)在活化和調節免疫反應中扮演著重要角色，並作為連接天然和獲得性免疫的橋樑。成熟的DCs能夠吸引、干擾和活化天然T細胞從而活化初級免疫反應。DCs還能夠直接活化自然殺手細胞(Natural killer cell,NK cell)，並能在遭遇病毒病原體時生成大量的干擾素。本發明提供了一種增加天然和獲得性免疫的方法，其透過活化DCs和巨噬細胞，經LZ-8蛋白之投予，DCs被活化並產生細胞介質(cytokine)和趨化因子(chemokine)。
之前的研究已經證實了LZ-8的免疫調節活性。然而，還不清楚LZ-8蛋白是否對DCs有任何作用。本發明揭示LZ-8蛋白可活化DCs和巨噬細胞。在本發明中，透過LZ-8之投予增強天然和獲得性免疫。
LZ-8蛋白透過促進DCs活化和成熟來調節獲得性免疫，因為促發炎因子(pro-inflammatory cytokine)的生成是DC活化的主要證據，所以檢測LZ-8在骨髓源性樹突狀細胞DC(bone marrow-derived DC,BMDCs)中腫瘤壞死因子-α(TNF-α)的生成。用LZ-8處理BMDCs後，生成之TNF與劑量之關係顯示LZ-8可活化DCs(圖1A)，除TNF之外，細胞內的IL-6和IL-12 p40也可偵測到；LZ-8處理後，其他之細胞激素(cytokine)也進行檢測，如圖1B所示，LZ-8處理的BMDCs顯著地分泌TNFα、介白素-1α(Interleukin 1α,IL-1α)、介白素-1β(IL-1β)、介白素-2(IL-2)、介白素-6(IL-6)和介白素-12(IL-12)。LZ-8刺激後之BMDCs亦刺激趨化因子之生成，如：單核細胞趨化蛋白1(monocyte chemoattractant protein 1，MCP-1)、巨噬細
胞發炎蛋白1α(macrophage inflammatory protein 1α，MIP-1α)、巨噬細胞發炎蛋白1β(macrophage inflammatory protein-1β，MIP-1β)與活化調節正常T細胞的表現和分泌(regulated upon activation,normal T-cell expressed and secreted,RANTES)(圖1C)。本發明中之LZ-8蛋白活化DCs從而分泌細胞激素和趨化因子，並增強了獲得性免疫反應。
DCs之成熟是其調節功能的一個關鍵步驟；偵測LZ-8刺激後BMDCs的成熟狀態，LZ-8促進BMDC的成熟，其係通過增加第II類MHC(MHC class II)、CD40、CD54(ICAM-1)、CD80與CD86的表現，以及降低CD119(IFN-γ受體)的表現(圖3A)，DC的活化還伴隨大分子吞噬作用(endocytosis)的降低，故本發明中，LZ-8的處理降低了BMDC對FITC-標記的右旋糖酐(FITC-labeled dextran)的吞噬(圖3B)。這些結果證明本發明中的LZ-8能夠活化DCs並促進DC成熟。
本發明中，以LZ-8刺激的BMDCs在體內體外均能誘導抗原特異性T細胞的活化，同時也揭示LZ-8能相對增強DC成熟的結果。誘導抗原特異性T細胞的活化是成熟DCs的主要功能。LZ-8活化的BMDCs促進了T細胞增殖(圖4A)。所有T細胞被以LZ-8刺激的BMDCs活化，並在其刺激下生成更多的IFN-γ(圖四)。本發明還揭示，在亞單位元免疫模型中，從LZ-8免疫處理之小鼠中分離的T細胞，其在LZ-8的處理後，其增殖活性增加(圖4B)。因此，基於此發現，本發明進一步提供了用於增強抗原免疫原性的方法，包括給予受試者LZ-8蛋白融合抗原(LZ-8 protein-fused antigen)。本發明使用的LZ-8蛋白係做為輔助劑增強受試者的免疫反應。在較佳實施例中，LZ-8蛋白融合抗原之投予係經注射。
為進一步了解LZ-8之生物機制，本發明亦揭露與BMDC被LZ-8活化之相關路徑，其中促***活化蛋白激酶(mitogen-activated protein kinase，MAPKs)訊息傳輸路徑與nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)路徑係涉及DCs的活化與成熟。LZ-8蛋白刺激促***活化蛋白激酶(MAPKs)的活化(圖5)，其中，MAPKs訊息傳輸路徑包括細胞外訊息調節激酶(extracellular signal-regulated kinases，ERK)、JUN N-端激酶(JUN N-terminal kinases，JNK)
或壓力活化蛋白激酶2A(stress-activated protein kinase 2A)(p38)。
本發明的方法不僅是透過促進DCs的活化與成熟增加天然和獲得性免疫，而且還透過誘導巨噬細胞的活化增加天然免疫。將巨噬細胞RAW264.7(RAW)細胞以LZ-8和脂多醣體(LPS)培養，用ELISA法偵測TNFα的生成。如圖六所示，LZ-8促進了RAW細胞中TNFα的分泌，此結果顯示LZ-8能夠活化巨噬細胞並增強天然免疫。
本發明已證實LZ-8對小鼠DCs的影響，此外，由於人類DCs與小鼠DCs相比表現出一些不同的特徵，所以亦以LZ-8處理人類單核細胞源性DCs(MoDCs)，並觀察細胞集落。本發明揭示LZ-8增加以LZ-8處理過的MoDCs中之CD80、CD83與CD86表現(圖7A)。以LZ-8刺激MoDCs，可誘導其細胞激素的生成(圖7B)。其中，細胞激素包括以下數種：TNFα、IFN-γ、IL-2和IL-6。本發明揭示LZ-8可活化人類之DCs，其與在小鼠DCs上的作用一致。另，本發明亦提供LZ-8在哺乳動物(如：人)中以DC做免疫治療上的應用。
本發明的LZ-8蛋白分離自靈芝，或者通過重組蛋白技術在宿主細胞中製得。宿主細胞係酵母菌或細菌系統。
所述的宿主細胞是釀酒酵母(Saccharomyces cerevisiae)、畢赤酵母(Pichia pastoris)、漢遜酵母(Hansenula polymorpha)、產阮假絲酵母(Candida utilis)、波氏假絲酵母(Candida boidinii)、麥芽糖假絲酵母(Candida maltosa)、乳酸克魯維斯酵母(Kluyveromyces lactis)、耶羅維亞酵母(Yarrowia lipolytica)、酵母菌(Schwanniomyces occidentalis)、粟酒裂殖酵母(Schizosaccaromyces pombe)、球擬酵母(Torulopsis)、Arxula adeninivorans)、曲黴菌(Aspergillus,A.nidulans,A.niger,A.awamori,A.oryzae)或瑞氏木黴(Tricoderma reesei)。
本發明中的LZ-8還能在體外活化人之MoDCs；本發明亦提供了一種LZ-8體外促進BMDC之成熟和功能的方法。本發明能以DC疫苗之形式應用於腫瘤治療和感染性疾病。
本發明亦提供一種增加抗原之免疫原性的組合物，此組合物包含LZ8融合蛋白抗體。
本發明亦提供一種用於增進一抗原之免疫原性的組合物，其包含：(i)該抗原；(ii)第一免疫輔助劑(adjuvant)，其包含LZ-8蛋白；以及(iii)第二免疫輔助劑，且該第二免疫輔助劑不含有LZ-8蛋白。
在本發明較佳實施例中，該第二免疫輔助劑為一乳化佐劑，其係選自於由佛朗氏不完全佐劑(Incomplete Freund's Adjuvant)和佛朗氏完全佐劑(Complete Freund's Adjuvant)所組成之群組。
在本發明較佳實施例中，該增進抗原之免疫原性的組合物係被調配成一注射投藥劑型，而該抗原為一蛋白質抗原。
本發明亦提供一種疫苗組合物，其包含：(i)一抗原；(ii)第一免疫輔助劑(adjuvant)，其包含LZ-8蛋白；以及(iii)第二免疫輔助劑，且該第二免疫輔助劑不含有LZ-8蛋白。
在本發明較佳實施例中，該第二免疫輔助劑為一乳化佐劑，其係選自於由佛朗氏不完全佐劑(Incomplete Freund's Adjuvant)和佛朗氏完全佐劑(Complete Freund's Adjuvant)所組成之群組。
在本發明較佳實施例中，該疫苗組合物被調配成一注射投藥劑型，而該抗原為一蛋白質抗原。
圖1為骨髓源性樹突狀細胞(BMDCs)在LZ-8刺激後細胞激素(cytokine)和趨化因子(chemokine)的生成。(A)劑量反應曲線。BMDCs以不同劑量的LZ-8培養6小時，最後在佈雷菲德菌素A的存在下培養4小時。通過流式細胞儀測定生成的TNFα之CD11c+細胞的百分含量；(B和C)BMDCs用不同劑量的LZ-8培養24小時(6小時用於TNFα)，收集上清液，用酵素聯結免疫吸附法(ELISA)測定；(B)TNFα、IL-1α、IL-1β、IL-2、IL-6和IL-12；(C)MCP-1、MIP-1α、MIP-1β和RANTES。誤差線表示三個樣品之±標準差(SD)，此數據為三次獨立的實驗。
圖2表示LZ-8蛋白誘導的BMDC活性並非由污染所造成。BMDCs用LPS(20毫微克/毫升)或LZ-8(5微克/毫升)培養6小時，最後在佈雷菲德菌素A的存在下培養4小時。通過流式細胞儀測定生成TNFα之CD11c+細胞的百分含量，並示於區域標記上方。(A)檢驗LPS、LZ-8和背
景對照組；(B)在將LPS和LZ-8加入BMDCs之前，用多粘菌素(polymyxin,PMB)B(5微克/毫升，Sigma-Aldrich)處理30分鐘；(C)處理之前用蛋白酶K處理LZ-8和LPS 1小時與(D)處理前將LZ-8和LPS煮沸20和50分鐘，資料表示三次獨立的實驗。
圖3表示LZ-8對促進BMDC成熟的作用。(A)以LZ-8(5微克/毫升)(黑色實線)處理或未處理(虛線)BMDCs 16小時。淺灰線表示用同類型匹配的對照抗體染色，用流式細胞儀測定DC的成熟，將細胞用抗I-Ab、CD86、CD80、CD40、CD54和CD119的單株抗體染色與；(B)用LZ-8(5微克/毫升)、對照溶液(LZ-8溶液的背景)和LPS(20毫微克/毫升)對BMDCs培養或不處理16小時，透過在4℃(虛線)和37℃(實線)下右旋糖苷-FITC的吞噬情形來測定DCs的吞噬作用。區域標記上方表示右旋糖酐-FITC+(Dextran-FITC+)細胞的百分比，所示數據為在CD11c+細胞上的篩選結果，此為三次獨立實驗之結果。
圖4表示LZ-8處理的BMDCs對T細胞的活化。(A)從OT-I/OT-II小鼠中分離出CD8+/CD4+ T細胞，並與LZ-8(5微克/毫升)和LPS(20毫微克/毫升)活化處理的BMDCs以DC：T細胞數為2：1一同培養，且在OVA257-264/OVA323-339肽(1微克/毫升)的存在下一起培養72小時，利用[3H]胸腺嘧啶核苷的結合測定T細胞的增殖(如上圖)；以及利用酵素聯結免疫吸附法(ELISA)測定IFN-γ的生成(如下圖)與(B)經足墊注射僅混有IFA或混有IFA+LZ-8(10微克)的OVA323-339肽(10微克)對C57BL/6小鼠進行免疫反應，10天後收集淋巴結細胞，在96孔盤中以不同濃度的OVA323-339肽培養3天，再利用[3H]胸腺嘧啶核苷的結合測定T細胞的增生。誤差線表示三個樣本±標準差(SD)，此為三次獨立的實驗結果。
圖5表示通過LZ-8對BMDCs的刺激而誘導的MAPKs和NF-κB活化。收集BMDCs、使其饑餓、再以LZ-8(10微克/毫升)處理，然後在不同時間點收集細胞，並將其打破。將樣品分離在SDS-PAGE凝膠上，轉移到硝酸纖維素薄膜，並以西方點墨法(western blotting)分析。分別用抗磷酸化特異抗體和抗蛋白抗體檢測磷酸化和未磷酸化的JNK、ERK和p38 MAPK蛋白；以及，以抗IκB抗體測定IκB的降解。所示資料表示三次獨
立的實驗。
圖6表示LZ-8對巨噬細胞的活化。將RAW264.7細胞用LZ-8(5微克/毫升)和LPS(100毫微克/毫升)培養16小時，收集上清液，用酵素聯結免疫吸附法(ELISA)測定TNFα的生成。此為兩次獨立實驗之的結果。
圖7表示LZ-8對人單核細胞源性樹突狀細胞(monocyte-derived DCs,MoDCs)的活化；將未成熟的MoDCs用脂多醣體(LPS 1微克/毫升)、poly(I：C)(pIC,25微克/毫升)或各種濃度的LZ-8處理48小時。(A)將細胞用CD80、CD86(Immunotech)和CD83(BD PharMingen)的抗體染色，然後用流式細胞儀分析，資料表示三次獨立之實驗與與(B)收集細胞培養之上清液，利用人類CBA試劑盒(cytometric bead array)分析細胞激素的生成，誤差線表示三次獨立實驗的標準差。
實施例1
為了測定LZ-8的作用，檢測了骨髓源性樹突狀細胞(BMDCs)中TNF的誘導。LZ-8處理後BMDCs之TNFα生成與劑量相關(圖1A)表明LZ-8具活化DCs之潛力。除了TNFα外，細胞內的IL-6和IL-12 p40也是可檢測(資料未示)。我們利用ELISA法定量檢測了由LZ-8處理過的BMDCs分泌的細胞激素。如圖1B所示，LZ-8處理過的BMDCs顯著分泌TNFα、IL-1α、IL-1β、IL-2、IL-6和IL-12。我們還檢測了趨化因數的生成，LZ-8刺激的BMDCs生成MCP-1、MIP-1α、MIP-1β和RANTES(圖1C)。
實施例2
本文使用的LZ-8蛋白來自酵母表達的重組蛋白。為了排除在LZ-8製備過程中酵母成分污染的可能性，從表達空載體的酵母中製備背景溶液作為對照。如圖2A所示，對照溶液對骨髓源性樹突狀細胞(BMDCs)TNF的生成沒有影響，排除了污染的影響。此外，多粘菌素B(Polymyxin B)未顯著地抑制LZ-8的活性，顯示DC的活化不是因為內毒素的污染(圖2B)。
此外，在LZ-8蛋白處理BMDCs之前，將LZ-8以蛋白酶K(proteinase K)在37℃下作用1小時或100℃下加熱25和50分鐘使其失活。如圖2C和2D所示，用蛋白酶K分解和加熱去活性的LZ-8喪失刺激BMDCs的能力，顯示該活性源自LZ-8蛋白本身。這些資料證明LZ-8對DCs的刺激活性不是由酵母成分的污染所引起的。
實施例3
通過流式細胞儀檢測了LZ-8刺激後BMDCs的成熟狀態。將培養6天的BMDCs用LZ-8(5微克/毫升)處理16小時。然後，將細胞先用抗CD16/CD32之單株抗體(mAb)2.4G2(BD Pharmingen)阻斷，再用針對CD11c、CD40、CD54、CD80、CD86、CD119和I-Ab(Biolegend)的抗體染色，並用流式細胞儀分析。如圖3A所示，LZ-8增加II類MHC、CD40、CD54(ICAM-1)、CD80和CD86的表現，並減少CD119(IFN-γ受體)的表現。
此外，已知DC的活化伴有大分子吞噬作用的降低；因此，亦檢測了BMDCs的吞噬作用。將未以LZ-8處理或已處理的BMDCs以200毫克/毫升右旋糖酐-FITC(Dextran-FITC,M.W.~77kD,Sigma-Aldrich)在4℃或37℃下培養1小時。用冷的磷酸鹽緩衝溶液(PBS)洗滌細胞，再CD11c單株抗體染色，然後通過流式細胞儀分析；如圖3B所示，LZ-8的處理減少了BMDCs對FITC-標記的右旋糖酐的攝取，且控制組溶液未改變BMDCs的吞噬作用，故從這些結果顯示LZ-8確實活化DCs並促進DC之成熟。
實施例4
誘導抗原特異性T細胞的活化是成熟樹突狀細胞(DCs)的主要功能。從OT-I或OT-II TCR轉基因小鼠中分離T細胞，將細胞與LZ-8-處理過的OVA257-264(OVAP1)或OVA257-264(OVAP2)加BMDCs一起培養72小時。然後，通過[3H]胸腺嘧啶核苷的結合測定T細胞的增殖。如前所述測定BMDCs的抗原呈現。透過使用EasySep陽性選擇試劑盒(EasySep Positive Selection Kit)(StemCell Technology)純化BMDCs，將細胞接種於96孔平底盤(Costar Corning)，並加入1微克/毫升含或不含LZ-8的OVAP1或OVAP2，培養3小
時。用EasySep陽性選擇試劑盒從OT-I或OT-II TCR轉基因小鼠中分離出T細胞，並以DC/T細胞數=1/2之比率，將T細胞加至DC培養液中，培養細胞72小時，再利用[3H]胸腺嘧啶核苷的結合測定T細胞的增生，如圖4A所示，LZ-8活化的BMDCs在體外刺激T細胞之增生較對照組細胞多；以及，被以LZ-8刺激之BMDCs活化的所有T細胞，其生成的IFN-γ亦比對照組多(圖4A)。
還進一步進行了體內T細胞活化的檢測。為了測定體內LZ-8對T細胞活化的誘導，採用亞單位元疫苗模型(subunit vaccine model)來評價LZ-8對T細胞活化的影響。為了進行檢驗，用10微克僅混有不完全佐劑(incomplete Freund’s Adjuvant，IFA)(Sigma-Aldrich)或混有IFA+LZ-8(10微克)的OVAP2經足墊注射對C57BL/6小鼠進行免疫，10天後從免疫的小鼠中分離出淋巴結細胞，再將細胞以OVAP2培養3天。利用[3H]胸腺嘧啶核苷的結合測量T細胞的增殖，結果顯示，在對OVAp2之反應，從LZ-8免疫的小鼠中分離的細胞比對照組小鼠的細胞增生更明顯(圖4B)。這些資料揭示LZ-8刺激的BMDCs在體內和體外均能誘導抗原特異性T細胞的活化，並且也支持LZ-8對DC成熟有相對增強作用的結論。
實施例5
為了探討LZ-8誘導細胞活化的分子機制，檢測LZ-8處理過的骨髓源性樹突狀細胞(BMDCs)中MAPKs和NF-κB的活化，先以LZ-8處理BMDCs，並利用西方點墨法分析JNK、ERK和p38 MAPK的非活化和活化狀態。過程如下；收集BMDCs細胞，使其饑餓3小時，然後用LZ-8處理(10微克/毫升)，處理完後，將細胞打破、高溫加熱，最後以SDS-PAGE凝膠分離，然後轉移到Immunobilon NC薄膜(Millipore)。阻斷後，將薄膜與抗p38 MAPK(Thr180/Tyr182)、ERK(Thr202/Tyr204)和JNK(Thr183/Tyr185)(Cell Signaling Technology)之抗磷酸特異性抗體或抗p38 MAPK(Millipore)、ERK(BD Transduction Laboratories)、JNK和IκB(Santa Cruz Biotechnology)抗體反應，接著再與結合HRP之二級抗體反應(Chemicon)。使用ECL檢測試劑
(Pierce)檢測免疫反應性。
結果顯示LZ-8誘導了這些MAPKs的活化(圖5)，以及刺激IκB的降解，顯示該蛋白能夠活化NF-κB路徑，這些結果顯示LZ-8透過活化MAPKs和NF-κB路徑促進DC的成熟和發揮作用。
實施例6
將RAW264.7細胞係培養在含10%胎牛血清(fetal bovine serum，FBS)、2毫莫耳濃度麩醯胺酸(glutamine)、1%非必需氨基酸和1毫莫耳濃度丙酮酸鈉的RPMI中。將細胞置於37℃、5% CO2的濕潤培養箱中。為了刺激活化細胞，將細胞用LZ-8(5毫克/毫升)或LPS(100毫微克/毫升)處理16小時，收集上清液，用酵素聯結免疫吸附法(Enzyme-linked immunosorbant assay,ELISA)測定TNFα的生成。如圖6所示，LZ-8促進了RAW細胞中TNFα的分泌，這些結果顯示LZ-8能夠活化巨噬細胞，進而增加天然性免疫力。
實施例7
為了探討LZ-8對人類樹突狀細胞(DCs)的影響，用LZ-8對人類單核源性DCs(MoDCs)進行處理，在培養物中表現出細胞集落(數據未示出)。然後通過流式細胞儀檢測LZ-8處理的MoDCs的成熟狀態。將細胞用CD80、CD86(Immunotech)和CD83(BD PharMingen)的抗體染色，然後通過流式細胞儀分析。如圖7A所示，LZ-8誘導了MoDCs中CD80、CD83和CD86的表達，這些結果顯示與在小鼠BMDCs中的相同。
此外，還檢測了LZ-8刺激後MoDCs的介質的生成。為了檢測介質的生成，培養24小時後，收集用TLR配體或有效LZ-8培養的MoDC培養物的上清液。通過使用人流式微球分析(cytometric beadarray)試劑盒(BD PharMingen)檢測介質。如圖7B所示，檢測了TNFα、IFN-γ、IL-2和IL-6。令人意想不到的是，當與TLR-活化的細胞相比較時，還發現LZ-8誘導了單核細胞源性樹突狀細胞(monocyte-derived DCs,MoDCs)的IFN-γ和IL-2之生成；這些結果顯示LZ-8能活化人類之DCs，且與其在小鼠DCs上的作用一致。
雖然已經足夠詳細地描述和舉例說明瞭本發明，使本領域的技術人員能夠製造和使用它，但是在不脫離本發明的宗旨和範圍的情形下，各種替換、修改和改進是顯而易見的。
本領域的技術人員很容易理解本發明能夠充分實現本發明的目的，並獲得所提到的結果和優點，以及其中固有的優點。本領域的技術人員容易想到其中的修改以及其他用途。這些修改也包含在本發明的精神內並受到申請專利範圍第範圍的限定。
Claims (4)
- 一種用於增進一抗原之免疫原性的組合物，其包含：(i)該抗原；(ii)第一免疫輔助劑(adjuvant)，其包含LZ-8蛋白；以及(iii)第二免疫輔助劑，且該第二免疫輔助劑不含有LZ-8蛋白，其中該第二免疫輔助劑為一乳化佐劑，其中該抗原為一蛋白質抗原。
- 如申請專利範圍第1項之組合物，其中該乳化佐劑係選自於由佛朗氏不完全佐劑(Incomplete Freund's Adjuvant)和佛朗氏完全佐劑(Complete Freund's Adiuvant)所組成之群組。
- 如申請專利範圍第1項之組合物，其被調配成一注射投藥劑型。
- 如申請專利範圍第1項之組合物，其係用於製備疫苗。
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