CN103908668B - 蝇蛆抗菌肽佐剂及其制备方法和含该佐剂的疫苗制剂及用途 - Google Patents
蝇蛆抗菌肽佐剂及其制备方法和含该佐剂的疫苗制剂及用途 Download PDFInfo
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Abstract
本发明涉及具有免疫佐剂作用的蝇蛆抗菌肽及其制备方法和含该抗菌肽佐剂的疫苗制剂，以及它们在制备疫苗制剂中的应用。该抗菌肽是从蝇蛆中提取分离的多肽组分，可促进免疫小鼠的细胞免疫和体液免疫应答，诱生机体同时产生平衡的Th1型和Th2型免疫反应，显示出比现有技术中已知的铝胶佐剂更佳的佐剂特性，可以作为多种疫苗的免疫佐剂，发挥理想的免疫效果。以该抗菌肽为佐剂的疫苗制备工艺简单、方法简便、质量容易控制、使用方便、可冰冻保存，安全性好。
Description
技术领域
本发明涉及从蝇蛆提取分离的具有免疫佐剂作用的蝇蛆抗菌肽，及其制备方法和含该抗菌肽佐剂的疫苗制剂，以及它们在制备疫苗制剂中的应用。
背景技术
疫苗接种是预防和控制传染性疾病最经济、有效的手段。DNA疫苗、重组疫苗和合成肽疫苗等新型疫苗是近年来发展迅速的一类生物制剂，比常规疫苗具有抗原纯度高、特异性强、安全性好等优势。然而，这些新型疫苗的免疫原性弱，免疫效力低下[OystonP,RobinsoK.Thecurrentchallengesforvaccinedevelopment.JMedMicrobiol2012;61(7):889–894]。同此，当前疫苗研究正面临一些复杂病原如疟疾、结核、人免疫缺陷病毒等的重要挑战[Leroux–RoelsG.Unmetneedsinmodernvaccinology:Adjuvantstoimprovetheimmuneresponse.Vaccine2010;28(Supp.3):C25–36]。佐剂是传统疫苗和当前新型疫苗必不可少的组成成分，不仅能影响机体对疫苗免疫应答的强度，而且能针对特定病原体诱导最有效的免疫应答类型[MbowML,DeGregorioE,ValianteNM,etal.Newadjuvantsforhumanvaccines.CurrentOpinioninImmunology2010;22(3):411–416]。目前研究的免疫佐剂种类很多，如油佐剂、弗氏佐剂、微生物及其代谢产物、核酸及其类似物、细胞因子、脂质体等[DeyAK,SrivastavaIK.Noveladjuvantsanddeliverysystemsforenhancingimmuneresponsesinducedbyimmunogens.ExpertRevVaccines2011;10:227–51]，但由于存在不同程度的毒副作用或安全隐患等一些不可避免的缺陷而难以实际应用[MbowML,DeGregorioE,ValianteNM,RappuoliR.Newadjuvantsforhumanvaccines.CurrOpinImmunol2010,22(3):411–6;Batista–DuharteA,LindbladEB,Oviedo–OrtaE.Progressinunderstandingadjuvantimmunotoxicitymechanisms.ToxicolLett2011;203(2):97–105]。铝胶佐剂自1920s使用以来，直至最近由铝盐和TLR4激动剂3–O–去酰基–4’–单磷酰脂质A组成的AS04被批准之前一直是唯一通过FDA批准的人用疫苗佐剂[MarrackP,McKeeAS,MunksMW.Towardsanunderstandingoftheadjuvantactionofaluminium.NatRevImmunol2009;9:287–293]。然而，铝胶佐剂存在一些弊端：①主要通过诱导Th2型免疫应答产生抗体起保护作用，不能诱生Th1型免疫应答和细胞介导的免疫反应，只适用于以抗体为主要保护性免疫的疫苗(ExleyC,SiesjoP,ErikssonH.Theimmunobiologyofaluminiumadjuvants:howdotheyreallywork?TrendsImmunol2010;31:103–109)；②对人免疫缺陷病毒、丙型肝炎病毒、单纯疱疹病毒、流感病毒以及血吸虫病、百日咳和伤寒等多种抗原无佐剂作用[O’HaganDT,DeGregorioE.Thepathtoasuccessfulvaccineadjuvant-‘thelongandwindingroad’.DrugDiscovToday2009;14:541–551]；③可促进IgE抗体产生，容易诱导机体产生过敏反应；④在局部形成肉芽肿，甚至发生局部无菌性脓肿；⑤因其理化性质特点，含铝胶的疫苗怕冻，冻后铝胶容易变性；⑥对人、畜神经系统可能有影响。因此，铝胶佐剂已远远不能满足新型疫苗发展的要求[KoolM,FierensK,LambrechtBN.Alumadjuvant:someofthetricksoftheoldestadjuvant.JMedMicrobiol2012;61(7):927–934]。寻找安全、有效、新型的免疫佐剂是当前疫苗研究领域的一个热点[SchijnsVEJC,LavelleEC.Trendsinvaccineadjuvants.ExpertRevVaccines2011;10(4):539–550]。免疫佐剂研究已被列为疫苗研究的优先领域[HarandiAM,MedagliniD,ShattockRJ.Vaccineadjuvants:Apriorityforvaccineresearch.Vaccine2010;28(12):2363–2366;HarandAM?,BreweJ,SchijnV.ConferenceScene:Recentadvancementsinimmunopotentiatorsformodernvaccines.Immunotherapy2011;3(11):1297–301]。
天然药物用于防治疾病具有悠久的历史。当今，FDA批准的药物约30%来源于天然药物。新的具有免疫活性的天然产物的发现日益成为研究重点，尤其在寻找新一代疫苗佐剂方面[LicciardiPV,UnderwoodJR.Plant–derivedmedicines:Anovelclassofimmunologicaladjuvants.IntImmunopharmacol2011;11(3):390–398]。
蝇蛆为昆虫纲双翅目环裂亚目家蝇科家蝇MuscadomesticaL.的幼虫。蝇蛆在我国食用和药用历史悠久，主治臁烂、唇疔、毒痢作呕、小儿疳积诸症。蝇蛆的主要化学成分包括蛋白质、抗菌肽、不饱和脂肪酸、多糖、溶菌酶、凝集素、维生素和矿物质等[FengX,ChengG,ChenSY,YangH,HuangW.Evaluationoftheburnhealingpropertiesofoilextractionfromhouseflylarvainmice.JEthnopharmacol2010;130(3):586–92]。蝇蛆抗菌肽具有抗氧化[AiH,WangF,LeiC.Antioxidantactivitiesofprotein–enrichedfractionfromthelarvaeofhousefly,Muscadomestica.NatProdRes2008;22(6):507–15]、抗肿瘤[HouL,ShiY,ZhaiP,LeG.Antibacterialactivityandinvitroanti–tumoractivityoftheextractofthelarvaeofthehousefly(Muscadomestica).JEthnopharmacol2007;111(2):227–31;JinXB,MeiHF,LiXB,MaY,ZengAH,WangY,etal.Apoptosis–inducingactivityoftheantimicrobialpeptidececropinofMuscadomesticainhumanhepatocellularcarcinomacelllineBEL–7402andthepossiblemechanism.ActaBiochimBiophysSin2010;42(4):259–65;ZhuL,WangP,QinQL,ZhangH,WuYJ.Protectiveeffectofpolypeptidesfromlarvaofhousefly(Muscadomestica)onhydrogenperoxide–inducedoxidativedamageinHepG2cells.FoodChemToxicol2013;60:385–90]、抗真菌[FuP,WuJ,GuoG.PurificationandmolecularidentificationofanantifungalpeptidefromthehemolymphofMuscadomestica(housefly).CellMolImmunol2009;6(4):245–51.]、抗炎[ChuFJ,JinXB,XuYY,MaY,LiXB,LuXM,etal.Inflammatoryregulationeffectandactionmechanismofanti–inflammatoryeffectivepartsofhousefly(Muscadomestica)larvaeonatherosclerosis.Evid–basedComplementAlternMed2013;2013:ArticleID340267,10pages]、抗动脉粥样硬化[ChuFJ,JinXB,ZhuJY.Houseflymaggots(Muscadomestica)protein–enrichedfraction/extracts(PE)inhibitlipopolysaccharide–inducedatherosclerosispro–inflammatoryresponses.JAtherosclerThromb2011;18(4):282–90]、保肝[WangFR,AiH,ChenXM,LeiCL.Hepatoprotectiveeffectofaprotein–fractionfromthemaggots(Muscadomestica)againstCCl4–inducedhepaticdamage.BiotechnolLetter2007;29:853–8]、抗病毒以及免疫调节[AiH,WangF,ZhangN,ZhangL,LeiC.Antiviral,immunomodulatory,andfreeradicalscavengingactivitiesofaprotein–enrichedfractionfromthelarvaeofthehousefly,Muscadomestica.JInsectSci2013;13:112.]等活性。然而，迄今蝇蛆抗菌肽作为疫苗佐剂的研究尚未见报道。
发明内容
本发明的目的是提供一类用于疫苗、具有免疫佐剂作用的蝇蛆抗菌肽佐剂。
本发明的另一个目的是提供从蝇蛆中提取分离蝇蛆抗菌肽佐剂的方法。
本发明的进一步目的是提供一种包含所述蝇蛆抗菌肽佐剂的疫苗制剂。
本发明的还有一个目的是提供所述蝇蛆抗菌肽佐剂在制备疫苗制剂中的应用。
本发明的蝇蛆抗菌肽佐剂是从蝇蛆中提取、分离的蝇蛆多肽组分。蝇蛆抗菌肽中蛋白质的质量百分含量为56.24%±3.9%，分子量约10KDa。
本发明提供的蝇蛆抗菌肽是从蝇蛆中提取分离的，制备方法包括以下步骤：
a.将蝇蛆以质量浓度3%醋酸提取；
b.醋酸提取液100℃加热1h，4℃静置，滤过；
c.滤液浓缩，冷冻干燥，得蝇蛆提取物；
d.蝇蛆提取物上大孔吸附树脂柱进行色谱纯化，以水和55%乙醇依次洗脱，收集55%乙醇洗脱液，浓缩，冷冻干燥，得蝇蛆抗菌肽。
上述步骤d中所说的大孔吸附树脂为D101大孔吸附树脂。
本发明的含蝇蛆抗菌肽佐剂的疫苗制剂，包含有免疫有效量的蝇蛆抗菌肽佐剂以及含有一种或多种药物学上可接受的载体。
本发明的蝇蛆抗菌肽佐剂可在制备疫苗制剂中应用。
上文所述药学上可接受的载体是指药学领域的药物载体。例如：稀释剂、赋形剂如水、生理盐水、葡萄糖、甘露醇、甘油、乙醇及其混合物等；填充剂如淀粉、蔗糖等；粘合剂如纤维素衍生物、海藻酸盐、明胶和聚乙烯吡咯烷酮；湿润剂如甘油；崩解剂如碳酸钙和碳酸氢钠；吸收促进剂如季铵化合物；表面活性剂如吐温–80；润滑剂如滑石、硬脂酸钙、硬脂酸镁和聚乙二醇等。另外还可以在组合物中加入其它辅料如香味剂、甜味剂等。
本发明的疫苗制剂可以是溶液、混悬液或乳浊液形式，可通过口服、鼻吸入、直肠、肠胃外或经皮给药的方式施用于需要接种的对象。本发明的疫苗制剂用于口服时，可将其制成常规的固体制剂如片剂、粉剂、颗粒剂、胶囊剂、丸剂、缓释微丸、固体分散体、包含物等，制成的液体制剂如混悬剂、乳剂、溶胶剂、糖浆剂、合剂、溶液剂等；用于肠胃外给药时，可将其制成注射用的溶液、水或油性混悬剂、乳剂、冻干粉、脂质体、微囊、微球、纳米囊、纳米球等。优先的形式是注射用冻干粉及溶液。
本发明的疫苗制剂可按照药学和疫苗领域的任何常规生产方法制备。
疫苗制剂的典型单剂量可以根据制剂的形式而变化，并根据抗原种类、所需的抗体水平、接种对象的特异性及所需的免疫程序等各种因素进行确定。本发明的疫苗制剂中蝇蛆抗菌肽佐剂的量可为0.01μg～1g/单剂量，优选为0.1～100μg/单剂量。可以一次或多次施用。
本发明的优点
本发明具有免疫佐剂作用的蝇蛆抗菌肽可促进免疫小鼠的细胞免疫和体液免疫应答，诱生机体同时产生平衡的Th1型和Th2型免疫反应，显示出比现有技术中已知的铝胶佐剂更佳的佐剂特性，可以作为多种疫苗的免疫佐剂，发挥理想的免疫效果。以该抗菌肽为佐剂的疫苗制备工艺简单、方法简便、质量容易控制、使用方便、可冰冻保存，安全性好。。所以，本发明的蝇蛆抗菌肽有望开发成疫苗佐剂。
附图说明
图1为蝇蛆抗菌肽（MDPF）对卵清蛋白（OVA）受免小鼠血清中特异性IgG、IgG1、IgG2a和IgG2b抗体效价的影响。QuilA：佐剂对照，系从南美皂树（QuillajasaponariaMolina）树皮中分离制备的佐剂活性总皂苷。aP<0.05、bP<0.01和cP<0.001vsOVA对照组。
图2为蝇蛆抗菌肽（MDPF）对ConA刺激的卵清蛋白（OVA）受免小鼠脾细胞增殖反应的影响。QuilA：佐剂对照。aP<0.05和bP<0.01vsOVA对照组。
图3为蝇蛆抗菌肽（MDPF）对LPS刺激的卵清蛋白（OVA）受免小鼠脾细胞增殖反应的影响。QuilA：佐剂对照。aP<0.和、bP<0.01vsOVA对照组组。
图4为蝇蛆抗菌肽（MDPF）对卵清蛋白（OVA）受免小鼠脾细胞分泌IL-2能力的影响。QuilA：佐剂对照。cP<0.001vsOVA对照组。
图5为蝇蛆抗菌肽（MDPF）对卵清蛋白（OVA）受免小鼠脾细胞分泌INF-γ能力的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsOVA对照组。
图6为蝇蛆抗菌肽（MDPF）对卵清蛋白（OVA）受免小鼠脾细胞分泌IL-10能力的影响。QuilA：佐剂对照。cP<0.001vsOVA对照组。
图7为蝇蛆抗菌肽（MDPF）对禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠血清中特异性IgG、IgG1、IgG2a和IgG2b抗体效价的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsrL-H5对照组。
图8为蝇蛆抗菌肽（MDPF）对ConA刺激的禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠脾细胞增殖反应的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsrL-H5对照组。
图9为蝇蛆抗菌肽（MDPF）对LPS刺激的禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠脾细胞增殖反应的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsrL-H5对照组。
图10为蝇蛆抗菌肽（MDPF）对H5抗原刺激的禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠脾细胞增殖反应的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsrL-H5对照组。
图11为蝇蛆抗菌肽（MDPF）对禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠自然杀伤（NK）细胞活性的影响。QuilA：佐剂对照。aP<0.05、bP<0.01和cP<0.001vsrL-H5对照组。
图12为蝇蛆抗菌肽（MDPF）对禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠脾细胞分泌IL-2能力的影响。QuilA：佐剂对照。bP<0.01和cP<0.001vsrL-H5对照组。
图13为蝇蛆抗菌肽（MDPF）对禽流感–新城疫重组二联活疫苗（rL-H5）受免小鼠脾细胞分泌INF-γ能力的影响。QuilA：佐剂对照。cP<0.001vsrL-H5对照组。
图14为蝇蛆抗菌肽（MDPF）对禽流感–新城疫重组二联活疫苗（rL–H5）受免小鼠脾细胞分泌IL-10能力的影响。QuilA：佐剂对照。cP<0.001vsrL-H5对照组。
具体实施方式
以下通过实例进一步说明本发明，而非限制其范围。
以下百分比浓度均指质量百分比浓度。
实施例1：蝇蛆抗菌肽制备
取蝇蛆1kg，以3%醋酸水溶液于高速组织捣碎机中匀浆5min，置4℃冰箱中放置过夜，离心，收集上清液；沉淀再以3%醋酸水溶液同法处理。合并两次上清液，100℃加热1h，4℃冰箱中静置，减压抽滤。滤液减压浓缩，冷冻干燥、得到蝇蛆提取物。蝇蛆提取物上D101大孔吸附树脂色谱柱，用水和55%乙醇依次进行洗脱，收集55%乙醇洗脱液，减压回收乙醇、浓缩，冷冻干燥，得蝇蛆抗菌肽。蝇蛆抗菌肽为浅黄白色粉末；茚三酮反应呈阳性；蛋白质质量百分含量为56.24%±3.9%；分子量约10KDa。
实施例2含蝇蛆抗菌肽佐剂的乙肝疫苗制剂
称取蝇蛆抗菌肽500μg和重组乙型肝炎病毒表面抗原（rHBsAg）100μg，以生理盐水10ml溶解，以0.22μm微孔滤膜滤过，无菌分装，每支1ml。每ml含抗菌肽50μg和rHBsAg10μg。
实施例3含铝胶和蝇蛆抗菌肽佐剂的乙肝疫苗制剂
称取蝇蛆抗菌肽适量，以生理盐水溶解，制成浓度为100μg/ml的溶液，以0.22μm微孔滤膜滤过。将该抗菌肽溶液与等体积的含铝佐剂乙型肝炎病毒表面抗原疫苗原液(每ml含rHBsAg20μg和氢氧化铝500μg)，混合均匀，无菌分装，每支1ml。每ml含蝇蛆抗菌肽50μg、rHBsAg10μg和氢氧化铝250μg。
实施例4蝇蛆抗菌肽对卵清蛋白受免小鼠免疫反应的佐剂作用
实验方法：清洁级ICR小鼠随机分6组，每组6只。生理盐水对照组：每鼠注射生理盐水（saline）0.2ml；卵清蛋白（OVA）对照组：每鼠注射含OVA25μg的生理盐水0.2ml；QuilA对照组：每鼠注射含10μgQuilA和25μgOVA的生理盐水0.2ml；蝇蛆抗菌肽实验组：每鼠注射含蝇蛆抗菌肽(5、10、25、50μg)和25μgOVA的生理盐水0.2ml。各组皮下注射免疫2次，第一次免疫和第二次免疫间隔14天。二免后14天，采血，分离血清，检测特异性抗体效价；将小鼠断颈处死，无菌条件下取脾，制备脾细胞悬液，进行脾细胞增殖反应和细胞因子分析。
结果表明：蝇蛆抗菌肽能显著提高OVA免疫小鼠血清中OVA特异性IgG、IgG1、IgG2a和IgG2b抗体效价（图1）；增强ConA和LPS诱导的OVA受免小鼠脾细胞增殖反应（图2～图3）；促进免疫小鼠脾细胞分泌细胞因子IL-2、INF-γ和IL-10的能力（图4～图6）。说明蝇蛆抗菌肽不仅能促进免疫小鼠对OVA的体液免疫和细胞免疫应答，而且可诱导平衡的Th1/Th2免疫反应，对模式抗原OVA具有显著的佐剂作用。
实施例5蝇蛆总抗菌肽对禽流感–新城疫重组二联活疫苗的佐剂作用
实验方法：将蝇蛆抗菌肽以生理盐水溶解制成浓度分别为0.5、1.0、2.0和3.0mg/ml的稀释液，以0.22μm微孔滤膜滤过，备用。将QuilA以生理盐水溶解制成0.1mg/ml的稀释液。取商品禽流感–新城疫重组二联活疫苗（rL-H5，500羽份/瓶）1瓶加入生理盐水50ml，混匀，制成疫苗稀释液。取疫苗稀释液与等量的生理盐水、QuilA稀释液、蝇蛆抗菌肽（MDPF）稀释液，混匀，分别于第1和15天皮下注射免疫小鼠。二免后14天，采血，分离血清，进行H5抗原特异性抗体效价检测；将小鼠断颈处死，无菌条件下取脾，制备脾细胞悬液，进行脾细胞增殖反应、自然杀伤（NK）细胞活性和细胞因子分析。
结果表明：蝇蛆抗菌肽（MDPF）能显著提高禽流感–新城疫重组二联活疫苗（rL-H5）免疫小鼠血清中H5抗原特异性IgG、IgG1、IgG2a和IgG2b抗体滴度（图7）；增强ConA、LPS和H5抗原诱导的受免小鼠脾细胞增殖反应（图8～10）以及NK细胞对K562细胞的杀伤活性（图11）；促进免疫小鼠脾细胞分泌细胞因子IL-2、INF-γ和IL-10的能力（图12～14）。说明蝇蛆抗菌肽不仅能促进免疫小鼠对禽流感–新城疫重组二联活疫苗（rL-H5）的体液免疫和细胞免疫应答，而且可同时诱导免疫小鼠对rL-H5疫苗的Th1型和Th2型免疫反应，对禽流感–新城疫重组二联活疫苗具有显著的佐剂作用。
综上所述，本发明的蝇蛆抗菌肽具有显著的免疫佐剂作用，可促进免疫小鼠的细胞免疫和体液免疫应答，诱生机体同时产生平衡的Th1型和Th2型免疫反应，显示出比现有技术中已知的铝胶佐剂更佳的佐剂特性。同时，以该抗菌肽为佐剂的疫苗制备工艺简单、方法简便、质量容易控制、使用方便、可冰冻保存、安全性好。因此，本发明的蝇蛆抗菌肽有望开发成疫苗佐剂。
Claims (2)
1.含蝇蛆抗菌肽佐剂的疫苗制剂，其特征在于它是包含有免疫有效量的从蝇蛆中提取、分离的蝇蛆多肽组分的蝇蛆抗菌肽佐剂和药物学上可接受的载体，该蝇蛆抗菌肽佐剂按以下步骤制得：
a.将蝇蛆以质量浓度3%醋酸提取；
b.醋酸提取液100℃加热1h，4℃静置，滤过；
c.滤液浓缩，冷冻干燥，得蝇蛆提取物；
d.蝇蛆提取物上大孔吸附树脂柱进行色谱纯化，以水和55%乙醇依次洗脱，收集55%乙醇洗脱液，浓缩，冷冻干燥。
2.根据权利要求1所述的含蝇蛆抗菌肽佐剂的疫苗制剂，其特征在于蝇蛆抗菌肽佐剂的量为0.01μg～1g/单剂量。
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