TW201311718A - 類鐸受體-2 增效劑及其用於刺激免疫反應之用途 - Google Patents
類鐸受體-2 增效劑及其用於刺激免疫反應之用途 Download PDFInfo
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Abstract
本發明揭露兩種新穎的類鐸受體-2增效劑,VP1及VP3,其為口蹄疫病毒(foot-and-mouth disease virus,FMDV)之結構蛋白。此外,VP3之第91-150胺基酸是其可以活化類鐸受體-2的主要區域。VP1或VP3或其複合蛋白質因此可被用以促進個體之免疫反應。特定而言,本文所提供之活體內試驗顯示VP3-4xM2e具活性而可作為疫苗佐劑。
Description
本發明係關於類鐸受體-2(Toll-like receptor,TLR)增效劑,及其用於刺激免疫反應之用途。
口蹄疫病毒(FMDV)屬於口瘡病毒(Aphthovirus)屬,為小核醣核酸病毒科家族中之一員(Sáiz,2002)。FMDV不具封套之殼體係為二十面體對稱,其為小核醣核酸病毒科家族之典型的結構特徵。FMDV殼體係由四個結構蛋白所組成,分別為VP1、VP2、VP3、及VP4,其中VP1、VP2、及VP3為表面朝外,然而VP4則朝向內部(Sobrino,2001)。FMDV可使感染或接種疫苗的動物誘發急性體液性抗體反應,其被認為係對抗FMD感染之最重要的保護因子。近年來,越來越多的研究探討FMDV感染或免疫接種期間的先天免疫反應(Grubman,2004)。FMDV可以硫酸乙醯肝素結合依賴之方式,與豬的單核細胞所衍生的樹突細胞進行交互作用,且可被該細胞所吞噬(Harwood.L.J.,2008)。此外,巨噬細胞透過未知的機制,進行FMDV的吞噬作用(Rigden,2002)。許多種類的促發炎細胞激素及趨化激素,例如,IL-6、IL-8及IL-12,可在經高效力的去活化FMDV免疫後的豬隻中被測得,因此,推測去活化的FMDV可誘發單核細胞的活性(Barnett,2002)。
類鐸受體(TLRs)為模式識別受體(pattern recognition receptors,PPRs),其可辨識微生物成分及內源性配體。迄今,已描述有13種小鼠類鐸受體,11種人類類鐸受體。當其與專一的配體結合時,類鐸受體誘發快速的細胞內訊號傳遞路徑,該路徑涉及轉錄因子NFκB、MAP激酶及干擾素調控因子的活化,其將導致先天免疫的活化,包含促發炎細胞激素、趨化激素、干擾素及免疫球蛋白、及共激分子的生成(Garantziotis,
2008;Ishii,2005)。在類鐸受體中,類鐸受體-2可辨識最廣範圍的微生物衍生增效劑,包含源自不同細菌株的脂多醣體、脂胜肽、脂阿拉伯甘露聚糖、脂化甘露聚醣、糖基化磷脂醯肌醇、壁脂酸、各種不同的蛋白包含脂蛋白及醣蛋白、酵母聚糖及肽聚糖(Zähringer,2008)。類鐸受體-2亦涉及病毒或病毒蛋白所誘發的訊號傳遞路徑及細胞激素生成,包含Epstein-Barr(EB)病毒、麻疹病毒、水痘帶狀疱疹病毒、B型及C型肝炎病毒、人類及鼠的巨細胞病毒、單純皰疹病毒、牛痘病毒及淋巴球性脈絡叢腦膜炎病毒(Barton,2007;Bieback,2002;Boehme,2006;Bowie,2005;Compton,2003;Cooper,2005;Dolganiuc,2004;Gaudreault,2007;Wang,2005;Zhu,2007)。類鐸受體-2可以與類鐸受體-1或類鐸受體-6形成異二聚體,且藉此以辨識不同的配體。例如,三酸脂胜肽及脂阿拉伯甘露聚糖可被類鐸受體-1/2所辨識,而二酸脂胜肽、酵母聚糖及壁脂酸可被類鐸受體-2/6所辨識。此種配體專一性的擴增可以使得對於微生物的辨識範圍變得更廣泛(Ishii,2005;West,2006)。
由配體及類鐸受體之交互作用所媒介的訊號傳遞路徑,係由類鐸受體家族中大多數成員所共用,且與toll/IL-1受體(TIR區域)、髓分化標記88(MyD88)、IL-1R-相關的激酶(IRAK)、干擾素調控因子(IRF)、TNF-受體-相關因子(TRAF)、TGF-β-活化激酶、IκB激酶、IκB、及NF-κB有關連(請參閱示例:Akira,S.(2003)J.Biol.Chem.278:38105及Geller at al.(2008)Curr.Drug Dev.Tech.5:29-38)。更具體而言,對於類鐸受體1、2、4、5、6、7、8、9、及11而言,此訊號傳遞起始於當PAMP配體與膜結合的類鐸受體產生交互作用而使其活化時;該類鐸受體係以同型二聚體存在於內體膜或細胞表面。隨後的活化,該受體進行構形變化,以招集含有MyD88蛋白的TIR區域;該MyD88蛋白係為一銜接蛋白,其常見於所有的類鐸受體之訊號傳遞路徑中,除了類鐸受體-3之外。MyD88招引IRAK4,此可將IRAK1磷酸化及活化。該活化的IRAK1會與TRAF6
結合,此可催化多聚泛蛋白加入TRAF6之反應。泛蛋白之加入可活化TAK/TAB複合物,其可將IRFs磷酸化,導致NF-κB的釋放並將NF-κB輸送至細胞核。NF-κB於細胞核中誘發促發炎基因的表現(參閱如:Trinchieri and Sher(2007)Nat.Rev.Immunol.7:179-190)。
基於該等因子參與調控發炎反應之結果,類鐸受體已顯示於許多疾病的致病機制中扮演角色,包含自體免疫、感染疾病及發炎(Papadimitraki et al.(2007)J.Autoimmun.29:310-318;Sun et al.(2007)Inflam.Allergy Drug Targets 6:223-235;Diebold(2008)Adv.Drug Deliv.Rev.60:813-823;Cook,D.N.et al.(2004)Nature Immunol.5:975-979;Tse and Horner(2008)Semin.Immunopathol.30:53-62;Tobias & Curtiss(2008)Semin.Immunopathol.30:23-27;Ropert et al.(2008)Semin.Immunopathol.30:41-51;Lee et al.(2008)Semin.Immunopathol.30:3-9;Gao et al.(2008)Semin.Immunopathol.30:29-40;Vijay-Kumar et al.(2008)Semin)。
用以調控類鐸受體活性之組合物可依所欲用於多種不同之目的,包含作為佐劑。本發明即著重於此主題。
本發明提供用於刺激免疫反應之組合物。本發明係發現口蹄疫病毒(FMDV)的殼體蛋白VP1及VP3可作為哺乳動物的類鐸受體-2的活化劑,且可被用於刺激個體之免疫反應。例如,VP1及VP3多胜肽,作為類鐸受體-2增效劑,可誘發骨髓衍生之樹突細胞的成熟及細胞激素的生成。具體而言,VP3的91-150殘基負責類鐸受體-2的活化。
本發明之部分具體實施例中,VP1或VP3多胜肽係作為佐劑以提升疫苗的免疫反應。本發明之VP1或VP3多胜肽,作為佐劑時,可能與疫苗抗原共同調配,如:將兩者共同配製於懸浮液或溶液中、與所欲之多胜肽抗原或其類似物融合。例
如,於老鼠攻毒模式中,將VP3與流感病毒基質蛋白融合,可有效作為佐劑以對抗流感病毒。
因此,本發明提供一種刺激個體免疫反應之組合物,其包含類鐸受體-2增效劑VP1或VP3多胜肽。於一具體實施例中,該VP3多胜肽包含FMDV VP3的1-220殘基(全長,SEQ ID NO:2)、91-150殘基(SEQ ID NO:3)、1-150殘基(SEQ ID NO:4)、131-149殘基(SEQ ID NO:7)、131-170殘基(SEQ ID NO:8)、131-190殘基(SEQ ID NO:9)、111-150殘基(SEQ ID NO:10)、或91-111殘基(SEQ ID NO:11)。於另一具體實施例中,該組合物可進一步包含抗原,其為多胜肽,可與VP1或VP3多胜肽融合。
本發明亦提供VP1或VP3多胜肽用於製備刺激個體免疫反應之佐劑的用途。在部分具體實施例中,該VP3多胜肽包含FMDV VP3的1-220殘基(全長,SEQ ID NO:2)、91-150殘基(SEQ ID NO:3)、1-150殘基(SEQ ID NO:4)、131-149殘基(SEQ ID NO:7)、131-170殘基(SEQ ID NO:8)、131-190殘基(SEQ ID NO:9)、111-150殘基(SEQ ID NO:10)、或91-111殘基(SEQ ID NO:11)。
進一步提供一單離的多胜肽,包含FMDV VP3的1-220殘基(全長,SEQ ID NO:2)、91-150殘基(SEQ ID NO:3)、1-150殘基(SEQ ID NO:4)、131-149殘基(SEQ ID NO:7)、131-170殘基(SEQ ID NO:8)、131-190殘基(SEQ ID NO:9)、111-150殘基(SEQ ID NO:10)、或91-111殘基(SEQ ID NO:11)。於一具體實施例中,本發明之單離的多胜肽係與一抗原融合。
另一方面,本發明提供刺激個體之免疫反應的組合物,其包含編碼本發明之類鐸受體-2增效劑VP1或VP3多胜肽之核酸。
本發明亦提供一種編碼本發明VP1或VP3多胜肽之核酸的用途,其係用於製備刺激個體免疫反應之佐劑。
本發明之各種不同具體實施例係於之後詳述。本發明之其他特徵將由以下各種不同的具體實施例及申請專利範圍之詳細說明,以清楚呈現。
我們相信屬於本發明領域中之具有通常技藝者,可基於本文之說明,且不需進一步闡述,即可利用本發明至其最廣泛之範圍。因此,以下說明應被理解為示例性目的而非以任何方式來侷限本發明之範圍。
本發明係關於調控發炎及免疫反應之組合物及方法,其係透過個體(如人類、非人類靈長類、囓齒類等)中增效劑多胜肽與類鐸受體-2結合所致。在本文所述之研究中,顯示VP1及VP3為類鐸受體-2之增效劑,且可透過與類鐸受體-2結合以刺激免疫反應。其亦顯示類鐸受體-2增效劑VP1或VP3多胜肽於免疫刺激中具有佐劑活性。
在一方面中,本發明提供刺激個體免疫反應之組合物,包含VP1或VP3多胜肽的類鐸受體-2增效劑。該組合物包含類鐸受體-2增效劑VP1或VP3多胜肽,當將其投予一個體(如人類、非人類靈長類、囓齒類等)時,將使得該個體之免疫反應受到刺激,例如,於目標細胞或組織中之至少一種細胞激素的表現。此外,該組合物可作為佐劑,並與抗原併同使用以投予個體(如人類、非人類靈長類、囓齒類)。本發明之組合物可提升該個體對於該抗原之免疫反應。該組合物可進一步包含醫藥可接受之載劑。
本文所使用的「類鐸受體-2增效劑」乙詞是指一試劑可透過類鐸受體-2以活化細胞訊號傳遞。增效劑可為自然存在之類鐸受體-2的活化劑,或增效劑可為非自然存在之類鐸受體-2的活化劑。增效劑亦包含本文所述之多胜肽,特別係指殼體蛋白VP1及VP3,以及包含,但不限於,如含有FMDV VP3之91-150殘基胺基酸的多胜肽。該多胜肽可由FMDV VP3之
91-150殘基所組成、或基本上由FMDV VP3之91-150殘基所組成、或該多胜肽可包含FMDV VP3之91-150殘基融合至一自然的或非自然的序列。例如,FMDV VP3之91-150殘基可能是於VP3蛋白內提供,或可能與具抗原性的多胜肽、細胞激素、或其他佐劑序列及其類似物融合。
本文所使用的「拮抗劑」乙詞是指一試劑可抑制增效劑之效用。例如,類鐸受體-2拮抗劑抑制VP1或VP3多胜肽之類鐸受體-2的活化。拮抗劑可為小分子、巨分子(如抗體)、胜肽及非胜肽。小分子、巨分子(如抗體)、胜肽及非胜肽可為自然存在者或合成的。
本文所使用的「有效量」乙詞是指本文所述之組合物的量,當其投予一個體時,足以達到療效(如提升發炎或免疫反應、誘發細胞激素的表現、或增加保護效果以對抗疾病或狀況)。
本文所使用的「免疫有效量」乙詞是指當投予該劑量給一個體時,無論是單劑量或一系列中之部分,對於治療是有效的,如刺激個體之先天或後天免疫反應。
本文所使用的「基因」乙詞是指一核酸分子,其編碼特定蛋白、或於特定例子中,編碼功能性或結構性RNA分子。例如,VP3基因編碼VP3蛋白。VP3核酸(如基因)係為此領域已知者。
本文所使用的「核酸」或「核酸分子」乙詞是指兩個或多個核苷酸鏈,例如,RNA(核糖核酸)及DNA(去氧核糖核酸)。「純化的」核酸分子係指一核酸分子從細胞中或生物體中,將之與其他自然存在的核酸序列實質上分開或單離。該術語包含,如嵌入載體、質體、病毒、或原核生物或真核生物之基因體內之重組的核酸分子。
本文所使用的「多胜肽」乙詞是指任何胜肽連結的胺基酸鏈,無論其長度或轉譯後修飾,如醣化或磷酸化。「VP3多胜肽」乙詞是指對應於FMDV之VP3殼體蛋白的蛋白、或其片
段。相似地,「VP1」係指FMDV之VP1殼體蛋白。本文所使用的「單離的」多胜肽或胜肽片段乙詞是指一多胜肽或一胜肽片段,其不具有自然存在的對應部分、或已從自然來源中分離或純化者。本發明之單離的多胜肽(或胜肽片段)可藉由下列方式取得,例如:藉由編碼該胜肽的重組核酸之表現;或藉由化學合成。一胜肽於細胞系統中生成或以化學合成,與自然原始來源有所不同者,稱為「單離的」。
當第一個核酸序列與第二個核酸序列以功能性關係放置時,第一個核酸序列可被「操作地」與第二個核酸序列連結在一起。例如,若啟動子影響編碼序列的轉錄或表現,則稱啟動子操作地連結於編碼序列。一般而言,操作地連結的核酸序列係為連續的,且其必須能將兩個蛋白編碼區域以可讀的框架方式連結在一起。
「表現控制序列」乙詞是指一核酸,其於一接收細胞中,可調控一編碼序列之複製、轉錄及轉譯。表現控制序列的例子包含啟動子序列、聚腺苷酸化(polyadenylation,pA)訊號、內含子、轉錄終止序列、加強子、上游調控區域、複製起始區、及內部的核醣體進入位。本文所使用的「啟動子」乙詞是指一DNA調控序列,其可被RNA聚合酶所結合,以起動下游(往3’端的方向)編碼序列的轉錄。
本文所述之方法包含一含有試劑(如VP3類鐸受體-2增效劑多胜肽、或編碼VP3多胜肽之核酸)之組合物,當將其投予一個體時,可活化類鐸受體-2,並刺激該個體之免疫反應。該試劑可藉由,例如,與類鐸受體-2進行交互作用(如VP3多胜肽結合至類鐸受體-2)而活化類鐸受體-2。活化類鐸受體-2之試劑可改變基因之轉錄、增加mRNA之轉譯、或增加蛋白之活性,其涉及調節類鐸受體-2之細胞反應過程。例如,活化類鐸受體-2之試劑可誘發IL-8、IL-12p40、及IL-23的表現。
根據本發明,VP1或VP3多胜肽可為全長的VP1或VP3
蛋白、或其具有類鐸受體-2增效劑活性之片段。於本發明之一具體實施例中,該全長的VP1或VP3蛋白具有SEQ ID NO:1或SEQ ID NO:2之胺基酸序列。
於本發明之另一具體實施例中,作為類鐸受體-2增效劑的VP3片段係為91-150殘基之多胜肽,其具有SEQ ID NO:3之胺基酸序列、或為1-150殘基之多胜肽,其具有SEQ ID NO:4之胺基酸序列。
本發明之另一具體實施例中,作為類鐸受體-2增效劑的VP3片段係為131-149殘基之多胜肽,其具有SEQ ID NO:7之胺基酸序列、為131-170殘基之多胜肽,其具有SEQ ID NO:8之胺基酸序列、為131-190殘基之多胜肽,其具有SEQ ID NO:9之胺基酸序列、為111-150殘基之多胜肽,其具有SEQ ID NO:10之胺基酸序列、為91-111殘基之多胜肽,其具有SEQ ID NO:11之胺基酸序列、或其組合。
組合物包含可活化類鐸受體-2之試劑(如VP3多胜肽、VP3多胜肽模仿物等),可被投予至個體(如齧齒類、人類、非人類靈長類)以刺激極有需要之個體(如疑似暴露於感染性疾病之個體、具有癌症之個體等)的免疫反應。
將包含可活化類鐸受體-2之試劑之組合物投予至個體,可誘發免疫調節細胞激素的表現,如IL-12p40、IL-8、及IL-23。於部分具體實施例中,包含可活化類鐸受體-2之試劑之組合物係為一佐劑,將之與特定抗原投予而可能達到致免疫之效用,以對抗感染性因子或不正常之細胞,例如,癌細胞。該組合物包含免疫有效量的試劑,其可活化類鐸受體-2。
另一方面,本發明亦提供一組合物,以刺激個體之免疫反應,其包含酸編碼本發明之類鐸受體-2增效劑VP1或VP3多胜肽之核酸。編碼本發明之VP1或VP3多胜肽之核酸可用於製備佐劑,以刺激個體之免疫反應。
本文所述之組合物可以任何適用方法於任何適用配方中以投予個體,包括人類。例如,包含可活化類鐸受體-2之試劑
(如VP3多胜肽或編碼VP3多胜肽的核酸)之組合物,可被直接地導入個體,包含藉由靜脈(intravenous,IV)注射、腹腔(intraperitoneal,IP)注射、或原位(in situ)注射進入目標組織,例如,肌肉注射。為了改善病患的依從性,可將藥物製備成口服形式。當作為疫苗佐劑時,其將會伴隨接種方法或接種路徑使用。例如,使用常規的注射器及注射針,以將包含可調節類鐸受體-2的活化之試劑之組合物注射進入個體。藉由注射之非經腸投予可由,例如,彈丸式注射或連續灌流注射,而予以實施。注射的配方可以單位劑量形式呈現,例如,於安瓿或於多劑量容器中,其中具有添加的保存劑。組合物亦可能採用如同懸浮液、溶液或於油性或水性載體中之乳化液形式,且可能含有配方劑,例如,懸浮劑、穩定劑及/或分散劑。或者,組合物可能為粉狀(如凍乾的),在使用前以適用的載體還原,例如,無菌且無致熱原的水。
本發明之組合物可根據習知方法以製備成醫藥上可用的組合物,藉此,該等物質、或其功能性衍生物,可以與醫藥上可接受之載劑結合成一混合物。適用的載劑及其配方,包括其他人類蛋白,如人類血清白蛋白,係描述於,例如,Remington的藥物科學(16.sup.th ed.,Osol,A.ed.,Mack Easton Pa.(1980))。為了形成適於有效投予之醫藥上可接受組合物,該組合物將含有有效量的前述化合物,並伴隨適當含量之載劑。
於部分具體實施例中,包含免疫有效量之可活化類鐸受體-2之試劑之組合物,係投予至個體以刺激該個體之免疫反應。免疫有效量會依受治療個體之健康及身體狀況、受治療個體的種類(如人類、非人類靈長類等)、個體免疫系統合成抗體的能力、所欲達到之保護程度、疫苗之配方、治療醫師對於醫療狀況的評估、要治療或預防的狀況、及其他相關因子而有所不同。可預期的是該量將可能是一個相對廣泛的範圍,其可藉由例行試驗決定。
本發明現在將伴隨以下具體實施例以更特定地描述,所提
供之具體實施例僅用於示例之目的而非侷限之目的。
不同類鐸受體(TLRs)及其相關的配體的發現,以及類鐸受體的結合後產生的細胞激素的生成及樹突細胞的成熟,已加速了疫苗研究及配方調製。於本研究中,我們發現口蹄疫病毒的殼體蛋白VP3可活化類鐸受體-2。我們進一步藉由使用不同的VP3缺失的突變,發現VP3的91-150殘基對於類鐸受體-2的活化很重要。VP3可誘導骨髓衍生之樹突細胞的成熟以及細胞激素的生成。再者,將VP3與四個保留的流感病毒基質蛋白M2之重複串連的膜外區域融合(VP3-4xM2e),發現於老鼠攻毒模式中,可有效作為佐劑以對抗流感病毒。VP3-4xM2e亦可提供交叉保護,以對抗A型流感病毒之致死攻毒,包含California/07/2009(H1N1)、NIBRG-14(H5N1)、及PR8(H1N1)流感病毒。綜合考量,該等發現指出VP3係為新穎的類鐸受體-2增效劑,具有發展成佐劑之潛力。
Pam3CSK4係購自Invivogen(CA,USA)。用於功能阻斷試驗之抗鼠/人類的類鐸受體-2之鼠的單株抗體,及抗鼠CD14之鼠的單株抗體,係購自eBioscience(CA,USA);鼠的同型控制IgG係購自PIERCE(IL,USA)。氯丙嗪(chlorpromazine,CPZ)或金雀異黃酮(genistein)係購自Simga-Aldrich(St.Louis,MO)。
本發明使用FMDV O/TWN/97病毒株,其生長於BHK-21細胞。收集感染後40小時後之樣本,並以4,000 rpm進行離心
以分離澄清。將0.1 M的溴乙胺氫溴酸鹽加入冰冷的0.2N氫氧化鈉溶液中,並置於室溫以進行環化,藉此以製備BEI。將終濃度為1 mM之BEI加入澄清的病毒培養物中,並於37℃培養,伴隨連續攪拌,達24小時。病毒的殼體顆粒經過BEI去活化處理後,係藉由蔗糖梯度離心收集,並以Braford試驗(PIERCE,IL,USA)測量蛋白濃度以量化。
細胞培養
野生型之C57BL/6小鼠係購自國家實驗動物中心(台北,台灣)。類鐸受體-2 -/-小鼠係購自Jackson Lab(Maine,USA)。巨噬細胞係從腹膜滲出液中所分離出,其中該腹膜滲出液係自以2ml之3% wt/vol的硫乙醇酸溶液腹腔注射小鼠,經注射3天後從小鼠取得。該分離的巨噬細胞係於添加10%熱去活化的胎牛血清、50 μg/ml青黴素、50 μg/ml硫酸鏈黴素、及100 μg/ml硫酸新黴素(Invitrogen,CA,USA)的RPMI-1640培養基培養。
將HEK293T細胞(1.5×104個細胞/孔)種於96孔盤上,並進行整夜培養。根據製造商之使用說明,以Lipofectamine2000(Invitrogen,CA,USA)將細胞以0.05 μg類鐸受體表現質體、0.03 μg p5xNF-κB-luc質體(Stratagene,TX,USA)、及0.02 μg EGFP-N1(Clontech,Palo Alto,CA,USA)進行轉染24小時。將細胞與類鐸受體的配體培養6小時,之後以PBS洗滌兩次,接著將之溶解。NF-κB螢光酵素活性係根據製造商之使用說明,以螢光酵素分析系統(Promega,WI,USA)測量。螢火蟲螢光酵素表現程度係藉由EGFP之強度作為轉染效率之對照組而予以正常化,並以相較於未受刺激的pcDNA3.1空載體對照組之刺激倍數表示之。
將RAW264.7細胞(8×104個細胞/孔)種於24孔盤上,並培養隔夜。將該等細胞以刺激物處理24小時。再根據製造商之使用說明,以ELISA試驗套組(Invitrogen,CA,USA)測量於培養基中細胞激素之生成量。
使用改良的SUMO融合蛋白表現系統,以產生水溶的重組Sumo-VP1、VP3、如圖4所示之不同的VP3缺失突變(dN150(151-220殘基))、131-170、131-190、111-150、91-150殘基及dC70(1-150殘基))、或如先前研究所述之VP42蛋白(Lee et al,2009)。以大腸桿菌生產之重組蛋白首先以鈷珠進行純化,接著以一自製之SUMO蛋白酶His6-Ulp1403-621-His6進行蛋白水解消化。該SUMO-融合的標記係藉由鈷珠移除。最後,該重組的VP3或VP42蛋白即可從上清液中取得。
為了分離樹突細胞,從野生型C57BL/6及類鐸受體-2 -/-小鼠之腿骨中分離全部的骨髓,並將細胞培養於含有重組的鼠GM-CSF(10 ng/mL)的完全RPMI1640中。於第2天及第4天,加入含有GM-CSF之新培養基。於培養第6天時,收集未成熟的DCs,並重種於6孔盤中。以1 μM VP3或1 μg/ml Pam3Csk4處理該等DCs 48小時。以流式細胞分析BMDCs表面標誌(CD80及CD86)之表現,以判定DC之成熟狀況。
將BMDCs培養於不含(假處理)或含有1 μM VP3或1 μg/ml Pam3Csk4中48小時。將該等處理後之BMDCs以CD86-PE、CD80-FITC及其同型控制抗體,於冰上進行30分鐘之免疫染色。經過PBS洗滌後,係以流式細胞儀分析該等樣本。藉由以FITC-及PE-連結的同型配對控制抗體染色的BMDCs,決定象限的位置。右上角之數字係指成熟DC之百分比。
代表VP3之140-149或131-149殘基的胜肽係藉由中央研究院生物化學研究所之研究設施所合成。
取自攻毒前4週之免疫小鼠的血清,以56℃作用1小時使之失去活性。新鮮配製的California/07/2009(H1N1)、NIBRG-14(H5N1)、及PR8(H1N1)病毒(National Institute for Biological Standards and Control,Potters Bar,U.K.)係以半數組織培養感染量(TCID50)予以量化。將100倍TCID50之病毒與同體積之2倍系列稀釋之去活化血清原液於96孔盤混合,並於37℃作用1小時。將混合物加入盤上之MDCK細
胞中(每孔1.5x104細胞),接著於37℃作用3天。將細胞以冰冷的1xPBS洗滌,並固定於甲醇溶液中,並加入200μl 0.5%的結晶紫。經過3次的ddH2O洗滌後,將盤子風乾整夜。若一血清樣本含有抗體,其可阻斷病毒感染,則大部分的細胞將會存活且呈現紫色;若病毒不能被血清所阻斷,則細胞將會被感染,圓化,然後從細胞培養盤脫離,因此,被感染的細胞不會有紫色呈現。該MN力價係以可阻斷病毒感染之最高血清稀釋倍數表示。
將6到8週之BALB/c公鼠於第0週及第2週以肌肉注射進行免疫,以5 μg的血球凝集素(hemagglutinin,HA)蛋白,分別併同50 μg之氫氧化鋁(Alum;Sigma)、30 μg的VP3-4xM2e、或對照組PBS進行注射。將免疫後之小鼠以致死劑量的California/07/2009(H1N1)、NIBRG-14(H5N1)、及PR8(H1N1)病毒於第4週進行鼻內攻毒。攻毒後每天觀察14天,監測存活的小鼠數目。此動物試驗係經中央研究院之動物管理及使用委員會評估及同意。
VP1全長胺基酸序列(SEQ ID NO:1)(全長213個胺基酸):TTSAGESADP VTATVENYGG ETQVQRRQHT DIAFILDRFV KVKPKEQVNV LDLMQIPAHT LVGALLRTAT YYFSDLELAV KHEGDLTWVP NGAPETALDN TTNPTAYHKE PLTRLALPYT APHRVLATVY NGSSKYGDTS TNNVRGDLQV LAQKAERTLP TSFNFGAIKA TRVTELLYRM KRAETYCPRP LLAIQPSDAR HKQRIVAPAK QLL
VP3全長胺基酸序列(SEQ ID NO:2)(全長220個胺基酸):GIFPVACSDG YGGLVTTDPK TADPVYGKVF NPPRNLLPGR FTNLLDVAEA CPTFLHFDGD VPYVTTKTDS DRVLAQFDLS LAAKHMSNTF LAGLAQYYTQ
YSGTINLHFM FTGPTDAKAR YMVAYAPPGM EPPKTPEAAA HCIHAEWDTG LNSKFTFSIP YLSAADYAYT ASDVAETTNV QGWVCLFQIT HGKADGDALV VLASAGKDFD LRLPVDARTQ
VP3片段(91-150殘基)胺基酸序列(SEQ ID NO:3):LAGLAQYYTQ YSGTINLHFM FTGPTDAKAR YMVAYAPPGM EPPKTPEAAA HCIHAEWDTG
VP3片段(dC70;1-150殘基)胺基酸序列(SEQ ID NO:4):GIFPVACSDG YGGLVTTDPK TADPVYGKVF NPPRNLLPGR FTNLLDVAEA CPTFLHFDGD VPYVTTKTDS DRVLAQFDLS LAAKHMSNTF LAGLAQYYTQ YSGTINLHFM FTGPTDAKAR YMVAYAPPGM EPPKTPEAAA HCIHAEWDTG
VP3片段(dN150;151-220殘基)胺基酸序列(SEQ ID NO:5):LNSKFTFSIP YLSAADYAYT ASDVAETTNV QGWVCLFQIT HGKADGDALV VLASAGKDFD LRLPVDARTQ
VP3片段(140-149殘基)胺基酸序列(SEQ ID NO:6):AHCIHAEWDT
VP3片段(131-149殘基)胺基酸序列(SEQ ID NO:7):EPPKTPEAAA HCIHAEWDT
VP3片段(131-170殘基)胺基酸序列(SEQ ID NO:8):EPPKTPEAAA HCIHAEWDTG LNSKFTFSIP YLSAADYAYT
VP3片段(131-190殘基)胺基酸序列(SEQ ID NO:9):EPPKTPEAAA HCIHAEWDTG LNSKFTFSIP YLSAADYAYT ASDVAETTNV QGWVCLFQIT
VP3片段(111-150殘基)胺基酸序列(SEQ ID NO:10):FTGPTDAKAR YMVAYAPPGM EPPKTPEAAA HCIHAEWDTG
VP3片段(91-111殘基)胺基酸序列(SEQ ID NO:11):LAG LAQYYTQ YSGTINLHFMF
先天性免疫識別係由模式識別受體(pattern recognition receptors,PPRs)所媒介,以偵測微生物物質及進一步活化免疫反應。於眾多的PPRs中,類鐸受體(TLRs)被瞭解的最為透徹(Medzhitov,2007)。我們分析類鐸受體是否能辨識I-FMDV以活化免疫細胞。以專一性類鐸受體表現及NF-κB報導質體,暫時性地轉染缺乏類鐸受體之HEK293T細胞中,以評估I-FMDV是否可經由類鐸受體活化NF-κB。我們發現I-FMDV經由類鐸受體-2以誘發NF-κB的活化,而非經由其他類鐸受體(圖1)。為了進一步確認類鐸受體-2為I-FMDV之受體,於添加I-FMDV之前,先使用類鐸受體-2專一性抗體以阻斷類鐸受體-2之細胞外區域,其係位於可穩定表現類鐸受體-2之HEK293T(HEK-TLR2)細胞的細胞膜上。當細胞以類鐸受體-2專一性抗體預處理後,I-FMDV所誘發的NF-κB活化或IL-8之生成皆顯著地減少;但是以同型控制IgG處理者則不會有此現象(圖2A)。此結果顯示由I-FMDV所誘發之NF-κB活化或促發炎因子生成,可被抗類鐸受體-2抗體所阻斷。
我們進一步研究從野生型或類鐸受體-2 -/-小鼠所分離之腹腔巨噬細胞所產生之細胞激素;該等老鼠係經I-FMDV處理(圖2B)。從類鐸受體-2 -/-小鼠所分離之腹腔巨噬細胞不會對I-FMDV刺激產生反應(圖2B)。該等數據指出I-FMDV可經由類鐸受體-2以誘發巨噬細胞中之細胞激素的生成。整體而
言,此等結果顯示I-FMDV之專一性受體係為類鐸受體-2,而非其他的類鐸受體。
FMDV殼體係由四個結構蛋白所組成,分別為VP1、VP2、VP3、及VP4。為了判別是哪一個次單位負責類鐸受體-2的活化,我們以大腸桿菌製備重組的VP1、VP3、及VP42蛋白。於本研究中,使用SUMO融合技術以得到可溶的殼體次單位(Lee et al,2009)。於本試驗中,採用重組的VP1及SUMO-tag(Sumo-VP1),而非無SUMO-tag之VP1,原因是若將Sumo-tag移除會使得蛋白沈澱。於HEK-TLR2細胞中研究NF-κB的活化對於sumo-VP1、VP3或VP42的反應。圖3顯示Sumo-VP1及VP3可以劑量依賴方式以誘發NF-κB的活化。相較於VP1及VP3,VP42或SUMO蛋白則不能活化NF-κB,即便是使用高濃度之VP42或SUMO。此結果顯示VP1及VP3係為FMDV用以誘發細胞之NF-κB活化的組分。
為了找尋VP3中負責類鐸受體-2之活化的重要區域,係以大腸桿菌表現不同之VP3缺失的突變,該等突變係帶有Sumo-tag。僅有代表VP3之140-149或131-149殘基之兩個胜肽係以化學合成的,因為該兩個胜肽之分子量太小,以致無法純化。NF-κB的活化係於HEK-TLR2細胞分析其對於不同的VP3缺失蛋白質的反應。雖然VP3之131-149殘基所組成者為短小胜肽足以活化類鐸受體-2,但其活化倍數相較於全長VP3低了許多。代表91-150殘基之VP3片段蛋白,其含有VP3之131-149殘基及其他的胺基酸,與全長的VP3相同一樣可誘發類鐸受體-2的活化(圖4)。此結果顯示VP3之91-150殘基負責類鐸受體-2的活化。此外,藉由比較91-150及111-150殘基的活性,我們相信涵蓋91-111殘基之胜肽對於類鐸受體-2
的活化亦相當重要。
類鐸受體增效劑已被發展為佐劑以預防癌症及感染性疾病(Kanzler et al,2007)。我們接著研究VP3於免疫細胞中之角色,以及探討使用VP3作為疫苗佐劑的潛在可能性。由於於樹突細胞(dendritic cell,DC)上之類鐸受體係為一重要的佐劑受體,且提供佐劑活性的分子基礎(Takeda et al,2003),我們分析經VP3處理,DC之成熟或於DC中所生成之細胞激素。結果顯示VP3誘發骨髓衍生的樹突細胞(bone marrow-drived dendritic cell,BDMC)的成熟(圖5),及細胞激素的生成(圖6)。為了進一步分析VP3作為疫苗佐劑的可能性,將重組的VP3蛋白與四個保留的流感病毒基質蛋白M2之重複串連的膜外區域融合(VP3-4xM2e),並將其與血球凝集素抗原(HA)一起免疫。於一攻毒研究中(圖7),以HA及VP3-4xM2e免疫之小鼠顯示較高的微中和(MN)力價,且較常用之疫苗佐劑氫氧化鋁(Alum)之保護效果。此外,我們亦發現VP3-4xM2e可提供交叉保護以對抗不同亞型之流感病毒的致死攻毒,包含California/07/2009(H1N1)、NIBRG-14(H5N1)、及PR8(H1N1)流感病毒(圖7)。綜合考量,此等結果證明VP3可被發展為一疫苗佐劑。
發展安全且有效的新穎的類鐸受體-2增效劑,以作為疫苗佐劑是一項長期不間斷的工作。於本研究中,我們發現兩種新穎的類鐸受體-2增效劑,VP1及VP3,其均為FMDV之結構蛋白。進一步的研究指出VP3之91-150殘基負責類鐸受體-2的活化。再者,活體內試驗亦證明VP3-4xM2e可提升HA疫苗之保護效力以對抗不同亞型的流感病毒。此等結果指出VP1及VP3可作為新穎的類鐸受體-2增效劑。
圖1:I-FMDV活化類鐸受體-2而非其他的類鐸受體。將HEK293T細胞以p5xNF-κB-luc、EGFP-N1(供正常化所用)及如圖所示之表現不同類鐸受體的質體或pcDNA3.1(空載體)予以暫時性轉染。於轉染後24小時,將細胞以90 μg/ml I-FMDV處理或不以其處理6小時。接著將細胞溶解,並分析NF-κB的活化。資料係以至少兩獨立試驗中一者的三重複之平均值±SD呈現。
圖2:I-FMDV活化類鐸受體-2。(A)將HEK-TLR2細胞以10 μg/ml類鐸受體-2抗體、10 μg/ml同型控制IgG或不處理,達1小時,接著以90 μg/ml I-FMDV處理。經6小時處理後,使用報導基因試驗及ELISA分別分析HEK-TLR2細胞的NF-κB(上圖)活化及IL-8生成(下圖)。(B)從野生型或類鐸受體-2-/-小鼠中分離出腹腔巨噬細胞,並以所示濃度之I-FMDV處理24小時。使用ELISA測量於培養基中之IL-6的生成。資料係以至少兩獨立試驗中一者的三重複之平均值±SD呈現。
圖3:Sumo-VP1及VP3透過類鐸受體-2活化NF-κB。將HEK-TLR2細胞以p5xNF-κB-luc、EGFP-N1轉染。經轉染後24小時,將細胞以不同劑量的I-FMDV、VP3、VP42、Sumo-VP1或Sumo蛋白處理,或不處理。經過6小時處理後,使用報導基因試驗以測量該等細胞的NF-κB活化。資料係以至少兩獨立試驗中一者的三重複之平均值±SD呈現。(unt=未處理之細胞)。
圖4:VP3的中心區域負責類鐸受體-2的活化。將HEK-TLR2細胞以p5xNF-κB-luc、EGFP-N1轉染。經轉染後24小時,將細胞以不同VP3缺失突變處理。經過6小時處理後,使用報導基因試驗以測量該等細胞的NF-κB活化。資料係以至少兩獨立試驗中一者的三重複之平均值±SD呈現。(unt=未處理之細胞)。
圖5:VP3誘發骨髓衍生之樹突細胞(BMDCs)的成熟。以流式細胞分析BMDCs表面標記(CD80及CD86)的表現情形。於免疫染色及流式細胞分析前,先將BMDCs培養於不含
(假處理)或含有1 μM VP3或1 μg/ml Pam3Csk4中48小時。藉由以FITC-及PE-連結的同型配對控制抗體染色的BMDCs,以決定象限的位置。右上角之數字係指成熟DC之百分比。
圖6:為因應VP3,BMDCs生成細胞激素。於上清液收穫前,先將BMDCs培養於不含或含有1 μg/ml Pam3Csk4或於特定濃度之VP3中48小時,再將收穫之上清液以ELSIA試驗測量(A)IL-6、(B)TNF-α、及(C)IL-12之程度。
圖7:VP3-4xM2e顯示佐劑功效。將BALB/c小鼠(n=6)於第0週及第2週以肌肉注射進行免疫,分別以5 μg的HA蛋白,及與50 μg之氫氧化鋁(Alum)、30 μg的VP3-4xM2e、或對照組PBS搭配注射。將免疫後之小鼠以致死劑量的H1N1(California/07/2009)病毒於第4週進行經鼻攻毒。攻毒後每天觀察,長達14天以記錄存活的小鼠數目。(A)於第四週從免疫小鼠所取得之血清,進行微中和(microneutralization,MN)力價測量。為了要評估疫苗之保護效果,於第4週將免疫小鼠以致死劑量的(B)California/07/2009(H1N1)、(C)NIBRG-14(H5N1)、及(D)PR8(H1N1)流感病毒進行點鼻攻毒。攻毒後每天觀察,長達14天以記錄存活的小鼠數目。
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Claims (11)
- 一種用於刺激個體免疫反應之組合物,其包含口蹄疫病毒(FMDV)類鐸受體(TLR)-2的VP1或VP3增效劑多胜肽。
- 如申請專利範圍第1項之組合物,其中該VP3類鐸受體-2增效劑多胜肽包含選自由SEQ ID NOS:2-4及7-11所組成群組之胺基酸序列。
- 如申請專利範圍第1或2項之組合物,其進一步包含抗原。
- 如申請專利範圍第3項之組合物,其中該抗原係為與VP3類鐸受體-2增效劑多胜肽融合之多胜肽。
- 一種VP1或VP3類鐸受體-2增效劑多胜肽或編碼該VP1或VP3類鐸受體-2增效劑多胜肽之核酸的用途,其係用於製備可併同疫苗抗原刺激個體之免疫反應之佐劑。
- 如申請專利範圍第5項之用途,其中該VP3類鐸受體-2增效劑多胜肽包含選自由SEQ ID NOS:2-4及7-11所組成群組之胺基酸序列。
- 如申請專利範圍第5或6項之用途,其中該VP3類鐸受體-2增效劑多胜肽係與疫苗抗原融合。
- 一種單離的多胜肽,其包含選自由SEQ ID NOS:2-4、7-11、及其組合所組成群組之胺基酸序列。
- 如申請專利範圍第8項之單離的多胜肽,係與疫苗抗原融合。
- 一種用以刺激個體免疫反應之組合物,其包含編碼如申請專利範圍第1或2項所述之VP1或VP3類鐸受體-2增效劑多胜肽之核酸。
- 一種編碼如申請專利範圍第1或2項所述之VP1或VP3類鐸受體-2增效劑多胜肽的核酸之用途,其係用於製備可併同疫苗抗原刺激個體之免疫反應之佐劑。
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US20140031250A1 (en) | 2010-10-07 | 2014-01-30 | David Tsai Ting | Biomarkers of Cancer |
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US10632151B2 (en) | 2015-01-22 | 2020-04-28 | University Of Massachusetts | Cancer immunotherapy |
WO2016146143A1 (en) * | 2015-03-16 | 2016-09-22 | Amal Therapeutics Sa | Cell penetrating peptides and complexes comprising the same |
JP2018524588A (ja) | 2015-06-26 | 2018-08-30 | ベス・イスラエル・ディーコネス・メディカル・センター,インコーポレイテッド | 骨髄由来サプレッサー細胞中のテトラスパニン33(Tspan33)を標的化するがん療法 |
EP3347044B1 (en) * | 2015-09-10 | 2023-04-19 | Academia Sinica | Bird flu vaccine combination comprising virus-like particles and novel adjuvants |
KR20230131498A (ko) | 2016-09-21 | 2023-09-13 | 아말 테라퓨틱스 에스에이 | 암 치료를 위한, 세포 투과 펩타이드, 멀티 에피토프 및 tlr 펩타이드 작용제를 포함하는 융합체 |
EP3532638A4 (en) | 2016-10-31 | 2020-07-29 | University of Massachusetts | TARGETING MICROARN-101-3 P IN CARCINOTHERAPY |
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US11426566B2 (en) | 2016-12-14 | 2022-08-30 | Biora Therapeutics, Inc. | Treatment of a disease of the gastrointestinal tract with a TLR modulator |
US11814623B2 (en) | 2018-01-30 | 2023-11-14 | University Of Massachusetts | Methods of treating a wound using epigenetic regulation |
AU2019217875A1 (en) | 2018-02-06 | 2020-08-20 | Icahn School Of Medicine At Mount Sinai | Repeat RNA as biomarkers of tumor immune response |
US20230107927A1 (en) | 2020-02-28 | 2023-04-06 | First Wave Bio, Inc. | Methods of treating iatrogenic autoimmune colitis |
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