結(jié)核分枝桿菌蛋白亞單位疫苗與疫苗誘導(dǎo)的T細(xì)胞免疫記憶研究進(jìn)展

祝秉東，馬瀾，劉勛，牛紅霞，白春香，李菲

蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)研究所，蘭州 730000

·綜述·

結(jié)核分枝桿菌蛋白亞單位疫苗與疫苗誘導(dǎo)的T細(xì)胞免疫記憶研究進(jìn)展

祝秉東，馬瀾，劉勛，牛紅霞，白春香，李菲

蘭州大學(xué)基礎(chǔ)醫(yī)學(xué)院病原生物學(xué)研究所，蘭州 730000

牛分枝桿菌減毒活疫苗——卡介苗(bacillus Calmette-Guérin，BCG)對(duì)預(yù)防嚴(yán)重的兒童結(jié)核病有效，但其免疫保護(hù)效率隨兒童年齡增長(zhǎng)而降低。BCG不能提供終身免疫保護(hù)可能與其誘導(dǎo)的記憶性T細(xì)胞主要是壽命較短的效應(yīng)記憶性T細(xì)胞有關(guān)。新型結(jié)核分枝桿菌蛋白亞單位疫苗將有效的抗原有機(jī)組合起來(lái)，在適宜的疫苗佐劑輔助下誘導(dǎo)Th1型免疫應(yīng)答。動(dòng)物實(shí)驗(yàn)表明，增加抗原譜可有效提高亞單位疫苗的保護(hù)效率。更重要的是，亞單位疫苗在體內(nèi)持續(xù)時(shí)間較短，可誘導(dǎo)壽命較長(zhǎng)的中央記憶性T細(xì)胞，提供比BCG更持久的免疫保護(hù)力。記憶性T細(xì)胞的分化受抗原特性與劑量、細(xì)胞因子、轉(zhuǎn)錄因子及雷帕霉素等的調(diào)控。對(duì)亞單位疫苗及其誘導(dǎo)的免疫記憶進(jìn)行研究將對(duì)新型結(jié)核分枝桿菌疫苗的設(shè)計(jì)與評(píng)價(jià)產(chǎn)生積極影響。

結(jié)核分枝桿菌；疫苗；亞單位疫苗；免疫記憶；T細(xì)胞

結(jié)核病(tuberculosis)是由結(jié)核分枝桿菌(Mycobacteriumtuberculosis)感染引起的慢性傳染性疾病，其患病人數(shù)和死亡人數(shù)長(zhǎng)期居高不下。2014年全球新增結(jié)核病約960萬(wàn)例，其中約150萬(wàn)例死亡[1]?？ń槊?bacillus Calmette-Guérin，BCG)是目前許多國(guó)家批準(zhǔn)應(yīng)用于人體預(yù)防結(jié)核病的唯一疫苗。研究表明，BCG接種雖可預(yù)防嚴(yán)重的兒童結(jié)核病，但對(duì)成人結(jié)核病的預(yù)防效果不佳[2]。因此，研發(fā)旨在增強(qiáng)機(jī)體長(zhǎng)期抗結(jié)核免疫保護(hù)的新型疫苗和免疫策略迫在眉睫。現(xiàn)階段國(guó)內(nèi)外研發(fā)的結(jié)核分枝桿菌疫苗主要包括：①活菌疫苗，包括改良BCG和結(jié)核分枝桿菌減毒活疫苗。②全菌滅活疫苗，如滅活的母牛分枝桿菌和草分枝桿菌疫苗。③亞單位疫苗，包括DNA疫苗、蛋白/多肽疫苗和病毒載體疫苗等。本文所述亞單位疫苗特指在佐劑輔助下的蛋白抗原疫苗。

1 結(jié)核分枝桿菌蛋白亞單位疫苗研究進(jìn)展

1.1 保護(hù)性抗原的篩選與融合蛋白疫苗的構(gòu)建

1.1.1 生長(zhǎng)期抗原的篩選與疫苗構(gòu)建 早在1995年，Andersen等在小鼠實(shí)驗(yàn)中發(fā)現(xiàn)結(jié)核分枝桿菌的培養(yǎng)濾液蛋白可識(shí)別結(jié)核分枝桿菌感染活化的T細(xì)胞，其中以Ag85B和早期分泌抗原靶6 kDa蛋白(early secretory antigenic target of 6 kDa，ESAT-6)最強(qiáng)。此外，人們發(fā)現(xiàn)BCG缺失的ESAT-6和MPT64抗原可誘導(dǎo)豚鼠皮膚發(fā)生明顯的遲發(fā)型超敏反應(yīng)[3]。ESAT-6為早期分泌蛋白，能誘導(dǎo)強(qiáng)烈的T細(xì)胞免疫應(yīng)答，并提供一定的抗結(jié)核免疫保護(hù)[2]。Ag85復(fù)合物包括相對(duì)分子質(zhì)量為30 000～32 000的3種蛋白(Ag85A、Ag85B和Ag85C)，是結(jié)核分枝桿菌和BCG的主要分泌性蛋白，在結(jié)核分枝桿菌H37Rv毒株中占分泌蛋白總量的30%。由于Ag85復(fù)合物可誘導(dǎo)很強(qiáng)的Th1型免疫應(yīng)答，現(xiàn)階段很多臨床前和臨床試驗(yàn)中的新型結(jié)核分枝桿菌疫苗均包含Ag85組分[2,4]。其他一些抗原如Mtb10.4、Mtb8.4、Rv1789、Rv2220和Rv3478可誘導(dǎo)較強(qiáng)的免疫保護(hù)力，后三者與ESAT-6-Ag85B聯(lián)合誘導(dǎo)出的免疫保護(hù)水平近似于BCG[5]。通過(guò)研究ESAT-6家族蛋白的免疫原性，研究人員構(gòu)建了早期分泌抗原ESX二聚體EsxD-EsxC、ExsG-EsxH和ExsW-EsxV，三者聯(lián)合(H65疫苗)也可誘導(dǎo)出與BCG近似的保護(hù)效果[6]。

1.1.2 潛伏期抗原的應(yīng)用與多期抗原疫苗的研究 結(jié)核分枝桿菌在體內(nèi)進(jìn)入潛伏期后，抗原表達(dá)與增殖期有所不同。由于被感染的細(xì)菌包含增殖期和休眠期，且可相互轉(zhuǎn)化[7]，所以理想的結(jié)核分枝桿菌疫苗不但要針對(duì)增殖期細(xì)菌，還要能針對(duì)休眠期結(jié)核分枝桿菌產(chǎn)生免疫應(yīng)答。已報(bào)道的免疫原性較強(qiáng)的潛伏期抗原有Rv2660c和HspX。將潛伏相關(guān)抗原Rv2660c與Ag85B-ESAT-6蛋白H1[8]融合，構(gòu)建新疫苗H56[9]，可達(dá)到甚至超過(guò)BCG的免疫保護(hù)效果，還可應(yīng)用于結(jié)核潛伏感染的治療，協(xié)助機(jī)體清除潛伏感染細(xì)菌。另一項(xiàng)研究將潛伏相關(guān)抗原Rv1813與其他3個(gè)結(jié)核分枝桿菌抗原(Rv2608、Rv3619、Rv3620)聯(lián)合構(gòu)建亞單位疫苗ID93，也達(dá)到與BCG相近的保護(hù)效果[10]。本課題組將融合蛋白疫苗EAMM(融合結(jié)核分枝桿菌生長(zhǎng)期抗原ESAT-6、Ag85B、Mtb8.4)與MH(融合結(jié)核分枝桿菌潛伏相關(guān)抗原HspX和生長(zhǎng)期抗原Mtb10.4)聯(lián)合應(yīng)用，可顯著降低結(jié)核分枝桿菌攻擊后的脾和肺組織的細(xì)菌載量，達(dá)到與BCG相同的保護(hù)效果[11]。進(jìn)一步分別將EAMM與潛伏感染相關(guān)抗原HspX及RV2626c融合，構(gòu)建融合蛋白疫苗LT69[12]和LT70[13]，小鼠毒力株攻擊保護(hù)實(shí)驗(yàn)表明，該疫苗長(zhǎng)期(免疫30周后)保護(hù)效率強(qiáng)于BCG。除蛋白抗原，結(jié)核分枝桿菌細(xì)胞壁糖脂組分也可激活CD1限制性T細(xì)胞及γδT細(xì)胞，具有免疫保護(hù)作用。限于篇幅，在此不展開討論。

1.2 結(jié)核分枝桿菌疫苗佐劑的研究

結(jié)核分枝桿菌感染人體后主要寄生在巨噬細(xì)胞內(nèi)。與抗胞外菌感染需誘導(dǎo)體液免疫不同，抗胞內(nèi)菌感染疫苗需誘導(dǎo)有效的T細(xì)胞免疫應(yīng)答[14]。鋁佐劑是目前唯一用于臨床的疫苗佐劑，主要介導(dǎo)體液免疫，對(duì)以細(xì)胞免疫為主的疾病效果不佳，因此亟需研發(fā)可誘導(dǎo)細(xì)胞免疫的新型佐劑。新型佐劑常由載體和免疫激活劑兩部分組成。免疫激活劑多為Toll樣受體(Toll-like receptor，TLR)激動(dòng)劑，可通過(guò)激活天然免疫影響獲得性免疫應(yīng)答的種類和方向，從而增強(qiáng)疫苗效應(yīng)。對(duì)于結(jié)核分枝桿菌亞單位疫苗，現(xiàn)階段進(jìn)入臨床試驗(yàn)的佐劑有4個(gè)：①AS01E，是含有單磷脂A(monophosphoryl lipid A，MPL)和皂角苷(QS21)的油包水乳劑，其中MPL是TLR4的激動(dòng)劑[15]。②IC31，由陽(yáng)離子肽KLKL(5)KLK和合成的寡聚脫氧核苷酸1(oligodeoxynucleotide 1，ODN1)構(gòu)成，其中ODN1為TLR9的激動(dòng)劑[16-17]。③GLA-SE，是TLR4的合成激動(dòng)劑GLA在角鯊烯水溶液中配制成的乳液[18]。④CAF01，由陽(yáng)離子脂質(zhì)體二甲基雙十八烷基銨(dimethyldioctadecyl ammonium，DDA)和6，6′-二十二酸酯海藻糖(trehalose-6,6′-dibehenate，TDB)構(gòu)成，其中TDB為分枝桿菌細(xì)胞壁成分6，6′-雙分枝菌酸海藻糖(trehalose-6-6′-dimycolate，TDM)的合成類似物，具有激活TLR和T細(xì)胞的作用[19]。陽(yáng)離子脂質(zhì)體DDA是有效的疫苗載體，可自組裝成雙層脂質(zhì)體囊泡。利用其表面為正電荷的特性，可吸附至帶負(fù)電荷的細(xì)胞表面，從而將包裹或結(jié)合的蛋白和DNA抗原呈遞給抗原呈遞細(xì)胞(antigen-presenting cell，APC)。結(jié)果DDA可直接呈遞內(nèi)吞抗原進(jìn)入細(xì)胞質(zhì)，并通過(guò)融合胞膜或交叉呈遞，誘導(dǎo)CD8+T細(xì)胞免疫應(yīng)答。表1列出目前進(jìn)入臨床試驗(yàn)的結(jié)核分枝桿菌亞單位疫苗抗原及其佐劑。

表1 進(jìn)入臨床試驗(yàn)的結(jié)核分枝桿菌蛋白亞單位疫苗

Tab.1Mycobacteriumtuberculosissubunit vaccine candidates in clinical trails

VaccineTargetAntigen CharacterofantigenDeliverysystemPhaseReferencesID93/GLA-SEPreventive/Postexposure/TherapeuticRv2608Rv3619Rv3620Rv1813PPEfamilyproteinVirulencefactorLatency-associatedproteinGLA-SEI[10,18]H1/IC31Preventive/PostexposureAg85BESAT-6MycolyltransferaseVirulencefactorIC31IIa[8,16-17]H1/CAF01Preventive/PostexposureAg85BESAT-6MycolyltransferaseVirulencefactorCAF01IIa[8-9,19]H4/IC31PreventiveAg85BTB10.4Mycolyltransferase,VirulencefactorIC31IIa[16]M72/AS01EPreventive/PostexposureRv1196Rv0125PPEfamilyproteinPeptidaseAS01EIIa[15,20]H56/IC31Preventive/Postexposure/TherapeuticAg85BESAT-6Rv2660cMycolyltransferaseVirulencefactorLatency-associatedproteinIC31IIa[9,16]

2 結(jié)核分枝桿菌疫苗誘導(dǎo)的T細(xì)胞免疫記憶

2.1 記憶性CD8+/ CD4+T細(xì)胞的發(fā)育和分化

由于記憶性T細(xì)胞(memory T cell，TM)研究以CD8+T細(xì)胞的發(fā)育和分化較多，以下有關(guān)TM的發(fā)育調(diào)控以CD8+T細(xì)胞為主。病原體感染和抗原刺激后，T細(xì)胞擴(kuò)增，分化為不同的亞群，主要分為效應(yīng)性T細(xì)胞(effector T cell，Teff)和TM。Teff主要分布在感染部位和外周，具有較強(qiáng)的免疫學(xué)活性，但維持時(shí)間較短。機(jī)體建立免疫記憶，產(chǎn)生再次免疫應(yīng)答主要依靠TM。TM根據(jù)分化程度、存在部位和存活時(shí)間，分為中央記憶性T細(xì)胞(central memory T cell，TCM)和效應(yīng)記憶性T細(xì)胞(effector memory T cell，TEM)。TCM具有淋巴結(jié)歸巢受體(CCR7和CD62L)，主要存在于淋巴結(jié)，存活時(shí)間長(zhǎng)，受抗原再次刺激時(shí)產(chǎn)生白細(xì)胞介素2(interleukin 2，IL-2)能力強(qiáng)，能大量增殖，增殖的T細(xì)胞轉(zhuǎn)化為TEM和Teff。TEM主要存在于外周，受抗原再次刺激時(shí)主要分泌γ干擾素(interferon γ，IFN-γ)等細(xì)胞因子。與TCM相比，TEM增殖能力較弱，存活時(shí)間較短。TEM和TCM的表面分子標(biāo)記分別為CD44hi/CD62Llo/CCR7lo和CD44hi/CD62Lhi/CCR7hi[21]。近年來(lái)發(fā)現(xiàn)TM有新的亞型，如有人提出了具有自我更新能力的干細(xì)胞樣記憶性 T 細(xì)胞(stem cell memory T cell，TSCM)，其分子表型為CD44loCD62LhiSca-1hi，再次受抗原刺激時(shí)可分化為TEM和TCM[22]。在外周組織器官中也發(fā)現(xiàn)組織定居記憶性T細(xì)胞(tissue-resident memory T cell，TRM)[23]。近期研究報(bào)道小鼠TEM和TCM的表面分子標(biāo)記分別為CD44+CD62L-CCR7-KLRG1+和CD44+CD62L+CCR7+KLRG1-；TSCM和TRM的分子表型分別為CD44-CD62L+KLRG1-CD103-CD69-Sca-1+[24-25]和CD44+CD62L-KLRG1-CD103+CD69+CCR7-[26]。

CD4+T細(xì)胞功能較CD8+T細(xì)胞更為復(fù)雜，其免疫記憶形成過(guò)程與CD8+T細(xì)胞有相似性。研究發(fā)現(xiàn)，受感染刺激后幾乎所有CD4+Teff均會(huì)產(chǎn)生相應(yīng)的TM，再次感染后TM又可分化為同一類型的Teff[27]。

2.2 影響TM發(fā)育和分化的因素

2.2.1 抗原呈遞特性決定TM的分化方向 抗原特性、抗原劑量和刺激持續(xù)時(shí)間被認(rèn)為是影響CD8+TM發(fā)育和分化的首要因素。在流感病毒感染模型中，低劑量短期抗原刺激有利于CD8+T細(xì)胞向TCM轉(zhuǎn)化；反之，高劑量持續(xù)抗原刺激促使T細(xì)胞向TEM甚至Teff轉(zhuǎn)化[28]。結(jié)核分枝桿菌融合蛋白H56疫苗研究發(fā)現(xiàn)，一定范圍內(nèi)低劑量抗原免疫反而誘導(dǎo)持久的免疫保護(hù)力[9]。本課題組在結(jié)核分枝桿菌亞單位疫苗LT69的研究中也觀察到同樣現(xiàn)象，即低劑量抗原誘導(dǎo)的免疫記憶更持久，表現(xiàn)出更高的保護(hù)效力[12]。

2.2.2 CD4+T細(xì)胞和細(xì)胞因子對(duì)CD8+TM發(fā)育和分化的調(diào)節(jié)作用 CD4+T細(xì)胞被認(rèn)為對(duì)CD8+TM的發(fā)育和分化發(fā)揮重要作用，CD4+T細(xì)胞缺陷小鼠和靈長(zhǎng)類動(dòng)物再次受病原體感染后CD8+T細(xì)胞擴(kuò)增明顯減少[29-30]。CD4+T細(xì)胞可能通過(guò)天然免疫作用[29]和分泌IL-2等細(xì)胞因子調(diào)節(jié)CD8+T細(xì)胞的分化。TM的分化和維持還需細(xì)胞因子IL-7、IL-15的參與。在IL-7和IL-15受體缺失的情況下，流感病毒感染不能誘導(dǎo)抗原特異的CD8+T細(xì)胞分化，表明IL-7和IL-15是TM分化所必需的細(xì)胞因子[31]。IL-2和IL-15通過(guò)其受體CD122發(fā)揮作用。研究表明，弱的CD122信號(hào)為T細(xì)胞存活提供信號(hào)，有利于TCM的分化；中等強(qiáng)度的CD122信號(hào)使T細(xì)胞向TEM分化；而強(qiáng)的CD122信號(hào)則促使T細(xì)胞向末端Teff分化[32]。

2.2.3 轉(zhuǎn)錄因子對(duì)TM分化的影響 外因和內(nèi)因?qū)細(xì)胞的分化調(diào)控最終大多通過(guò)轉(zhuǎn)錄因子的作用實(shí)現(xiàn)。通過(guò)生物信息學(xué)分析，人們發(fā)現(xiàn)了許多可調(diào)控T細(xì)胞發(fā)育和分化的轉(zhuǎn)錄因子，并證實(shí)了一些轉(zhuǎn)錄因子的作用[33]。已發(fā)現(xiàn)和證實(shí)調(diào)節(jié)CD8+TM分化的轉(zhuǎn)錄因子有Tcf7、kif2、Bach2、Bcl-6、Blimp-1、c-Myc、Id2、Id3、NFAT、NF-κB、Notch 1、Notch 2、Tbet、STAT3等。其中，Blimp-1與存活期短的Teff分化有關(guān)。Blimp-1通過(guò)抑制Id3啟動(dòng)子下調(diào)Id3表達(dá)而發(fā)揮調(diào)節(jié)作用；增強(qiáng)Id3表達(dá)可恢復(fù)短期存活效應(yīng)細(xì)胞的生存時(shí)間，增強(qiáng)再次免疫應(yīng)答[34]。Id2/Id3缺失會(huì)導(dǎo)致CD8+Teff和TM喪失，Id3高表達(dá)預(yù)示T細(xì)胞向長(zhǎng)期存活的TM分化[35]。在STAT3突變患者中，TM相關(guān)轉(zhuǎn)錄因子Bcl-6降低，導(dǎo)致CD4+和CD8+TCM增殖和分化活性降低，從而易被各種病毒、細(xì)菌和真菌感染。因此，STAT3也是調(diào)節(jié)T細(xì)胞免疫記憶的重要轉(zhuǎn)錄因子[36]。Tcf7是Wnt信號(hào)通路的下游轉(zhuǎn)錄因子，激活Wnt/β-catenin信號(hào)通路，可促進(jìn)CD8+T細(xì)胞向多能記憶干細(xì)胞(multipotent memory stem cell)(CD44loCD62LhiSca-1hiCD122hiBcl-2hi)分化[22]。生物信息學(xué)分析發(fā)現(xiàn)，Id3、Bach2和Tcf7等10個(gè)轉(zhuǎn)錄因子與其他轉(zhuǎn)錄因子有廣泛的相互作用，為較關(guān)鍵的轉(zhuǎn)錄調(diào)控因子。

此外，哺乳動(dòng)物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)和磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)/AKT信號(hào)通路活化會(huì)促進(jìn)T細(xì)胞向末端Teff分化[37]。傳統(tǒng)的免疫抑制劑雷帕霉素可抑制mTOR信號(hào)通路，使T細(xì)胞向TM分化。在腫瘤疫苗和BCG免疫過(guò)程中加入雷帕霉素可促進(jìn)TCM分化[38]。mTOR信號(hào)的調(diào)節(jié)分子Tsc1在單核細(xì)胞增多性李斯特菌感染引起的CD8+TM分化中發(fā)揮重要作用。Tsc1參與免疫功能和細(xì)胞代謝的轉(zhuǎn)錄調(diào)節(jié)，是機(jī)體建立再次免疫應(yīng)答和促進(jìn)CD8+T細(xì)胞向TM分化的關(guān)鍵因子[39-40]。

2.2.4 其他 其他因素也會(huì)影響TM的分化，如炎癥反應(yīng)、趨化因子和調(diào)節(jié)性T細(xì)胞等。疫苗佐劑對(duì)TM的分化也有重要影響。例如，佐劑通過(guò)延緩抗原在體內(nèi)的降解釋放可誘導(dǎo)TM形成。細(xì)胞因子、雷帕霉素等新型佐劑更可使人們有目的地對(duì)T細(xì)胞的分化加以調(diào)控。但TM分化是一個(gè)復(fù)雜的過(guò)程，對(duì)其調(diào)控的認(rèn)識(shí)需不斷研究和完善。本課題組應(yīng)用細(xì)胞因子IL-28B下調(diào)調(diào)節(jié)性T細(xì)胞后，雖然短期抗感染效應(yīng)增強(qiáng)，但疫苗誘導(dǎo)的長(zhǎng)期免疫記憶水平下降[41]，提示TM的發(fā)育調(diào)節(jié)較為復(fù)雜，在誘導(dǎo)更多Teff和TEM分化的同時(shí)，反而減弱了TCM的產(chǎn)生。

值得注意的是，對(duì)T細(xì)胞免疫記憶尤其是TEM和TCM的研究主要在急性病毒感染中獲得。在結(jié)核分枝桿菌等慢性感染中，TM的分化和抗感染作用可能與急性感染不盡相同。例如，在急性感染中IL-7、IL-2、IL-15能促進(jìn)T細(xì)胞增殖和免疫記憶的維持；在瘧疾引起的慢性感染中，IL-2被發(fā)現(xiàn)具有刺激細(xì)胞增殖和增強(qiáng)再次免疫應(yīng)答的作用，IL-7雖能增強(qiáng)CD4+TM增殖，但不能增強(qiáng)再次免疫應(yīng)答水平[42]。

2.3 結(jié)核分枝桿菌亞單位疫苗誘導(dǎo)的免疫記憶特征

Th1和Th17型CD4+T細(xì)胞、CD8+T細(xì)胞、γδT細(xì)胞及CD1限制性T細(xì)胞均參與機(jī)體抗結(jié)核免疫應(yīng)答，本文側(cè)重描述CD4+Th1細(xì)胞和CD8+T細(xì)胞的免疫記憶。結(jié)核分枝桿菌蛋白亞單位疫苗Ag85B-ESAT-6/CAF01[9]、M72[20]、H56[9]和H65[6]等可誘導(dǎo)多功能CD4+TM，這些多功能T細(xì)胞受抗原刺激可分泌IFN-γ、腫瘤壞死因子α(tumor necrosis factor α，TNF-α)和IL-2等細(xì)胞因子，與疫苗誘導(dǎo)的長(zhǎng)期免疫保護(hù)作用相關(guān)。蛋白亞單位疫苗誘導(dǎo)的CD4+T細(xì)胞是一種以分泌IL-2為特征的TCM[43]，而BCG免疫后持續(xù)存在的活菌誘導(dǎo)CD4+T細(xì)胞分化為TEM，主要分泌TNF-α和IFN-γ[11]。動(dòng)物實(shí)驗(yàn)發(fā)現(xiàn)，結(jié)核分枝桿菌亞單位疫苗Ag85B-ESAT-6/CAF01和H56誘導(dǎo)的免疫保護(hù)時(shí)間較BCG長(zhǎng)[9]。本課題組也發(fā)現(xiàn)亞單位疫苗LT69和LT70誘導(dǎo)的長(zhǎng)期免疫保護(hù)力要高于BCG[12-13]，可能與其誘導(dǎo)的TM亞型有關(guān)。在牛結(jié)核模型中也發(fā)現(xiàn)BCG誘導(dǎo)的TM維持時(shí)間短，免疫保護(hù)時(shí)間也短[44]。敲除BCG菌株尿素酶C基因(ureC)，并重組李斯特菌溶胞素基因(hly)，構(gòu)建rBCG ΔureC∷hly，與傳統(tǒng)的BCG相比，該重組BCG在體內(nèi)持續(xù)時(shí)間短，能促使更多TM尤其是CD4+TCM產(chǎn)生。疫苗免疫后分選出小鼠體內(nèi)抗原特異性CD4+TCM，過(guò)繼轉(zhuǎn)移給未免疫小鼠，攻毒實(shí)驗(yàn)證實(shí)CD4+TCM在抗結(jié)核免疫中具有非常重要的作用[45]。

BCG初始免疫后，應(yīng)用亞單位疫苗加強(qiáng)免疫被認(rèn)為可延長(zhǎng)和增強(qiáng)機(jī)體抗結(jié)核分枝桿菌感染的能力。然而，近期以痘苗病毒為載體的MVA85A臨床試驗(yàn)表明，MVA85A并未明顯增強(qiáng)BCG的免疫保護(hù)力[46]。MVA85A “失敗”的原因可能與以下因素有關(guān)：①抗原譜窄，僅Ag85A一個(gè)抗原不足以誘導(dǎo)全面的免疫保護(hù)。②未激活足夠的TM。對(duì)T細(xì)胞表型和功能的研究表明，MVA85A強(qiáng)化BCG免疫后，Teff數(shù)量明顯增加，但TCM數(shù)量未見增加[47]。③強(qiáng)化免疫方案有待改進(jìn)。亞單位疫苗強(qiáng)化免疫方案關(guān)系到疫苗激活T細(xì)胞的類型，不合理的方案可能會(huì)誘導(dǎo)更多Teff，而TCM并未增多，反而可能減少。因此，相關(guān)方案還需進(jìn)一步深入研究。此外，有研究比較了蛋白亞單位疫苗與病毒載體疫苗誘導(dǎo)免疫記憶的差異，發(fā)現(xiàn)蛋白疫苗H28(Ag85B-TB10.4-Rv2660c)可激活分泌TNF-α和IL-2的CD4+T細(xì)胞，而痘苗病毒載體疫苗MVA28主要誘導(dǎo)分泌IFN-γ的T細(xì)胞。BCG初免后分別用H28和MVA28強(qiáng)化免疫，H28較MVA28疫苗誘導(dǎo)的保護(hù)時(shí)間更長(zhǎng)，毒株攻擊后病理?yè)p傷也較輕[48]，提示結(jié)核分枝桿菌融合蛋白疫苗較病毒載體疫苗能誘導(dǎo)更長(zhǎng)的免疫記憶，其機(jī)制可能與誘導(dǎo)更多的TCM有關(guān)。

不同類型TM在抗結(jié)核免疫保護(hù)中所起的作用還有待細(xì)致研究。TCM和TEM誘導(dǎo)的保護(hù)作用差異可能與所采用的動(dòng)物模型、檢測(cè)時(shí)間、攻毒劑量和方式等有關(guān)?？乖掷m(xù)刺激可激活更多的TEM而表現(xiàn)出較高的免疫保護(hù)效應(yīng)，但TEM維持時(shí)間不夠長(zhǎng)，其長(zhǎng)期保護(hù)效率往往有所下降。維持時(shí)間較長(zhǎng)的TCM是否一定具有有效的抗結(jié)核保護(hù)作用?不同研究結(jié)論也不完全相同。例如，有研究報(bào)道雷帕霉素可促進(jìn)BCG免疫小鼠TM的分化，并顯著提高抗結(jié)核水平[38]；也有報(bào)道指出IL-15處理增加了BCG誘導(dǎo)的CD8+TM，但保護(hù)效率未見提高[49]。因此，如何延長(zhǎng)免疫保護(hù)力是結(jié)核分枝桿菌疫苗的關(guān)鍵問(wèn)題，仍需進(jìn)行深入研究。

3 結(jié)語(yǔ)

卡介苗和結(jié)核分枝桿菌亞單位疫苗誘導(dǎo)不同類型的TM。從已有的研究報(bào)道看，結(jié)核分枝桿菌亞單位疫苗傾向于誘導(dǎo)TCM。疫苗免疫過(guò)程中，疫苗劑量與免疫策略、細(xì)胞因子和轉(zhuǎn)錄因子等均對(duì)TM分化具有調(diào)節(jié)作用。如何調(diào)控免疫應(yīng)答向TM分化，從而提高結(jié)核分枝桿菌疫苗的免疫保護(hù)作用有待深入研究。結(jié)核分枝桿菌亞單位疫苗及其免疫記憶特征的研究，對(duì)新型結(jié)核分枝桿菌疫苗的設(shè)計(jì)和評(píng)價(jià)具有重要意義。

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. ZHU Bingdong, E-mail: bdzhu@lzu.edu.cn

Progress on tuberculosis subunit vaccine and memory T cells

ZHU Bingdong, MA Lan, LIU Xun, NIU Hongxia, BAI Chunxiang, LI Fei

InstituteofPathogenBiology,SchoolofBasicMedicalSciences,LanzhouUniversity,Lanzhou730000,China

AttenuatedMycobacteriumbovisbacillus Calmette-Guérin (BCG) is effective for the prevention of severe tuberculosis infection in childhood, but its protective efficiency shrinks along with children growing up. BCG persisted for a long time after vaccination, therefore it mainly induces short-lived effector memory T cells. This may be the reason why BCG can’t provide long-term protection. Novel tuberculosis subunit vaccine composed of effective antigens with suitable adjuvants could induce Th1 type cell-mediated immune responses and provide protection against tuberculosis. Animal experiments showed that expanding the spectrum of antigens could improve the protective efficacy of subunit vaccine effectively. Moreover, subunit vaccine has been proven to induce long-lived central memory T cells, which helps to provide a longer-term protective immunity compared with BCG. The differentiation of memory T cells is regulated by antigen characteristic and dose, cytokines, transcription factors, and drugs like rapamycin,etc. The study on the subunit vaccine and vaccine-induced immune memory will be helpful to the design and evaluation of novel tuberculosis vaccines.

Mycobacteriumtuberculosis; Vaccine; Subunit vaccine; Immune memory; T lymphocyte

國(guó)家自然科學(xué)基金(31470895)

祝秉東

2016-06-24)