CN105400753A - 重组神经氨酸酶蛋白及其应用 - Google Patents
重组神经氨酸酶蛋白及其应用 Download PDFInfo
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- CN105400753A CN105400753A CN201510555504.4A CN201510555504A CN105400753A CN 105400753 A CN105400753 A CN 105400753A CN 201510555504 A CN201510555504 A CN 201510555504A CN 105400753 A CN105400753 A CN 105400753A
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Abstract
一种重组神经氨酸酶蛋白,其以人类新兴流感野生种病毒pH1N1-NA(A/Texas/05/2009)的氨基酸序列(SEQ?ID?NO:1)为基础。本发明的重组神经氨酸酶蛋白具有一细胞膜胞外域(ectodomain),该胞外域与SEQ?ID?NO:1的氨基酸序列具有至少95%的相同性,并在特定位置149、344、365及366被其它流感病毒的相对应氨基酸取代。本发明的重组神经氨酸酶蛋白具有交叉保护性免疫,并可作为广谱流感疫苗。
Description
技术领域
本发明涉及一种重组神经氨酸酶蛋白及其应用,特别涉及一种可作为广谱流感疫苗的重组神经氨酸酶蛋白及其应用。
背景技术
流行性感冒病毒为正粘液病毒科(Orthomyxoviridae)的成员,A型流感病毒为包膜病毒,其含有单股、8段反义(anti-sense)RNA基因组,通常用以编码11至12个病毒蛋白。A型流感病毒的亚型已根据血凝素(hemagglutinin,HA)及神经氨酸酶(neuraminidase,NA)糖蛋白的抗原特性进行分类,分别命名为H1-H16及N1-N9。最近的一篇报导叙述有关被流感病毒感染的果蝠体内,鉴定出H17N10及H18N11这两个新种。根据系统发育分析,N1-N9可以被分类为属于第一群组(包括N1、N4、N5及N8)或第二群组(包括N2、N3、N6、N7及N9)。迄今,只有N1、N2、N7及N9亚型已知可引起人类流行病。
NA为具有四聚体复合体结构的酶蛋白,又称唾液酸酶(sialidase)。
其可切割细胞表面的唾液酸(sialicacid)连接,从而有利于病毒从受感染细胞释放。NA还可藉由摧毁纤毛的诱饵受体(decoyreceptors)、粘蛋白(mucins)、及细胞醣质包被(glycocalyx)因而利于病毒传播及感染。NA的免疫原性(immunogenicity)最早见于以NA特异性去活化疫苗所接种的人类受试者。使用表达于酵母菌或昆虫细胞的重组NA(rNA)蛋白质在接种小鼠引起对抗致命病毒攻击的保护作用。以rNA蛋白质所接种的雪貂除了HA免疫本身所提供的保护力外,亦会产生抑制NA(NA-inhibiting,NI)抗体,以展现出独特类型的保护。NI抗体已知可限制病毒传播并减轻A型流感病毒感染的临床症状。以反向遗传工程技术生产含有季节性流感病毒NA的重组H1N1病毒(reverse-geneticreassortantH1N1virus)接种的小鼠展现交叉反应性NI抗体以及在人类新兴流感(pH1N1)病毒攻击时降低死亡率。季节性H1N1、H3N2及pH1N1不同品系的减毒活流感疫苗(Liveattenuatedinfluenzavaccines,LAIVs),已被报导在雪貂中可诱导对抗H5N1病毒的交叉反应性NI抗体,以及季节性的三价流感疫苗亦在雪貂中被报导可提供对抗致命的H5N1攻击的交叉保护性免疫。此外,含有NA、M1及M2的NA类病毒颗粒(VLP)已显示在小鼠中能引起更有效的NI抗体,并赋予对抗H5N1及pH1N1病毒攻击的交叉保护性免疫力。NI抗体也已在以含有H5N1的去活化疫苗接种的人类中检测出,以及暴露于自然感染的人类中亦可检测得到NI抗体。但NA的免疫原性及交叉保护机制目前仍不清楚。
综合上述,开发含有新颖性NA抗原的广谱流感疫苗,是目前极需努力的目标。
发明内容
本发明的目的为提供一种重组神经氨酸酶蛋白,其可具有交叉保护性免疫,并作为广谱流感疫苗。
依据本发明一个实施例,一种重组神经氨酸酶(NA)蛋白,其具有一胞外域(Ectodomain),该胞外域与SEQIDNO:1所示的氨基酸序列具有至少95%的相同性,其中氨基酸序列具有下列之一或其任意组合:(i)在位置149,I被取代为V;(ii)在位置344,N被取代为Y或H;以及(iii)在位置365、366,IS被取代为TA、TN或ED。
依据本发明的另一个实施例,一种多核苷酸,其编码上述重组神经氨酸酶蛋白。
依据本发明的另一个实施例,一种重组流感病毒,其组成包含上述重组神经氨酸酶,或编码上述重组神经氨酸酶蛋白的多核苷酸。
依据本发明的再另一实施例,一种流感疫苗,其包含上述重组神经氨酸酶蛋白,或编码上述重组神经氨酸酶蛋白的多核苷酸。其中流感疫苗可为灭活流感病毒疫苗、减毒流感病毒疫苗、类病毒颗粒疫苗或重组亚单位蛋白疫苗。
以下藉由具体实施例配合所附的图式详加说明,当更容易了解本发明的目的、技术内容、特点及其所达成的功效。
附图说明
图1A至1D说明水溶性重组NA(rNA)蛋白质的建构、表达及定性;
图2A至2E说明由接种rNA所诱导的对抗H5N1、pH1N1、H3N2及H7N9病毒的NA特异性IgG抗体;
图3A至3H说明由H5N1-rNA及pH1N1-rNA引起的对抗H5N1、pH1N1、H3N2及H7N9病毒的NA抑制性抗体;
图4说明由接种H5N1-rNA及pH1N1-rNA对同源及异源病毒所引起的相对应IC50值;
图5说明在脾脏中所检测到可分泌H5N1-rNA及pH1N1-rNA特异性抗体的B细胞;
图6A至6E说明H5N1-rNA及pH1N1-rNA对抗不同病毒攻毒攻击的保护性免疫反应;
图7A到7E说明H5N1-rNA及pH1N1-rNA对抗不同的病毒攻毒攻击后的体重恢复情形;
图8说明pH1N1及H5N1的突变位点及比较结果;
图9说明pH1N1NA的顶视图及侧视图;
图10A至10D说明由WT及第1组突变pH1N1-rNA对抗不同病毒株所产生的NA抑制性抗体增加曲线;
图11说明对抗同源病毒pH1N1及异亚型H5N1、H3N2及H7N9病毒的相对应IC50值;
图12A及12B说明不同群组的神经氨酸酶(NA)的氨基酸比对;
图13A至13D说明由WT及第2组突变pH1N1-rNA接种对抗不同病毒攻毒攻击所产生的NA抑制性抗体增加曲线;
图14说明对抗同源病毒pH1N1及异亚型H5N1、H3N2及H7N9病毒的相对应IC50值;
图15A至15C说明PR8病毒、pH1N1/PR8病毒、及具有I365T/S366N突变的pH1N1/PR8病毒的蚀斑块形态、病毒效价及NA酶活性。
具体实施方式
本发明提供了一种重组神经氨酸酶,其系基于新兴的野生型流感病毒pH1N1-NA(A/Texas/05/2009)序列(SEQIDNO:1)。本发明的重组神经氨酸酶包含胞外域(ectodomain),其具有与SEQIDNO:1实质相同的氨基酸序列。
参照表1,其显示pH1N1、H5N1、H3N2、H7N9在特定位置149、344、365及366的相对应氨基酸。本发明的目的为提供在特定位置以H5N1、H3N2、及H7N9的相对应氨基酸进行替代,以便产生交叉保护性免疫力。具体而言,上述氨基酸序列包含以下氨基酸之一或其任意组合:(i)在位置149,I被取代为V;(ii)在位置344,N被取代为Y或H;以及(iii)在位置365、366,IS被取代为TA、TN或ED。表1为pH1N1、H5N1、H3N2、H7N9在特定位置149、344、365及366的相对应氨基酸。
表1,pH1N1、H5N1、H3N2、H7N9在特定位置149、344、365及366的相对应氨基酸
菌株\位置 | 149 | 344 | 365、366 |
A/Texas/05/2009(H1N1) | I | N | IS |
A/Viet Nam/1203/2004(H5N1) | V | Y | TN |
A/Udorn/307/1972(H3N2) | I | H | ED |
A/Shanghai/02/2013(H7N9) | I | N | TA |
换言之,本发明的重组神经氨酸酶蛋白,可使用编码野生型pH1N1基因(SEQIDNO:1)的质粒作为模版,并利用定点突变(site-directedmutagenesis)在NA基因中导入突变氨基酸制备所得。
本发明所用的H5N1、H3N2及H7N9的序列如SEQIDNO:9、SEQIDNO:10、SEQIDNO:11所列。
目前已知流感病毒NA的胞外域的氨基酸中94.6%为保守(Conserved)序列。本领域人士可了解流感病毒NA的胞外域的一些氨基酸序列可以加以变换而不明显影响蛋白质的结构或功能。因此本发明神经氨酸酶蛋白的胞外域与SEQIDNO:1具有至少95%的序列相同性,较佳地,可具有至少97%或至少99%的序列相同性。
参照表2,在一具体实施例中,胞外域的氨基酸序列选自由SEQIDNO:2至8所组成的群组,较佳选自由SEQIDNO:2至5所组成的群组。
表2、重组神经氨酸酶蛋白的胞外域序列
SEQ ID NO. | 突变 | 简称 |
2 | I149V | MutA |
3 | N344Y | MutB |
4 | I365T/S366N | MutC |
6 | N344H | MutD |
7 | I365E/S366D | MutE |
8 | I365T/S366A | MutF |
本发明的主要目标之一是提供一种流感病毒疫苗,其能诱导广谱的不同亚型交叉反应的免疫反应。本发明的流感病毒疫苗可为灭活流感病毒疫苗、减毒流感病毒疫苗、类病毒颗粒疫苗或重组亚单位蛋白疫苗,亦即重组NA、编码重组NA的体内表达载体、类病毒颗粒和重组流感病毒都可以被用于疫苗制备。
在一具体实施例中,将本发明疫苗投递到个体,在该个体中引发针对人类新兴流感、人类季节性流感或各禽流感病毒亚型的广谱免疫反应。本发明的药理学活性化合物可以根据制剂学的常规方法进行加工,以产生用于投递至患者例如哺乳动物(包括人)的药剂。在另一具体实施例中,投递可经由例如但不限于皮下注射、肌肉注射、口腔投予、喷洒或基因枪注射的方式所达成。
以下通过具体实施例配合附图详加说明,可更容易了解本发明的目的、技术内容、特点及所达成的功效,并据以实施,但不能以此限定本发明的保护范围。
材料及方法
重组NA蛋白质表达及纯化
A/Vietnam/1203/2004(H5N1)(GI:145284408)及A/Texas/05/2009(pH1N1)(GI:255602223)NA基因的cDNA分别以昆虫细胞最佳化的密码子序列合成。具有额外N端序列(包含gp67信号肽、6-His残基、四聚体人血管扩张剂刺激磷蛋白(tetramerichumanvasodilator-stimulatedphosphoprotein,hvsp)结构域及凝血酶切割位点的H5N1与pH1N1的NA胞外域的编码序列被克隆到pFastBac表达载体。接下来,根据制造商说明书,使用Bac-to-Bac昆虫细胞表达系统生产rNA蛋白质。简言之,以表达H5N1及pH1N1的NA胞外域的重组杆状病毒感染Sf9细胞48小时,之后收集上清液以使用镍螯合树脂的亲和层析管柱进行rNA蛋白质纯化。H5N1-rNA及pH1N1-rNA的纯度是以考马斯蓝(Coomassieblue)染色证实。抗His的HRP共轭结合抗体被用于蛋白质印迹中定性。
H5N1及H7N9VLP的生产及纯化
H5N1及H7N9的VLP是用先前描述的方法所制备。简言之,将A/Thailand/1(KAN-1)/2004(H5N1)的H5HA基因、A/Shanghai/2/2013(H7N9)的H7HA基因、以及A/WSN/1933(H1N1)的M1基因克隆到pFastBacDual载体中。A/Vietnam/1203/2004(H5N1)的N1NA基因、A/Shanghai/2/2013(H7N9)的N9NA基因、以及A/WSN/1933(H1N1)的M2基因则克隆到另一个pFastBacDual载体中。有关生产H5N1VLP,Sf9细胞以BAC-H5HA-M1及BAC-N1NA-M2重组杆状病毒共感染,其MOI分别为3及1。在感染72小时后收集及浓缩培养上清液。使用20%蔗糖溶液及33000转离心3小时进一步纯化获得VLP。所得的H5N1类病毒样颗粒存放于4℃,直到用于NI测定。有关H7N9VLP,Sf9细胞则以Bac-H7HA-M1及Bac-N9NA-M2重组杆状病毒共感染72小时,其MOI分别为3及1。后续步骤均与生产H5N1VLP相同。
免疫接种老鼠
BALB/c小鼠(6-8周龄)购自台湾国家实验动物中心,如同先前报告所述,以2或20μgrNA蛋白质加10μgCpG及10%PEGb-PLACL、角鲨烯及(PELC)乳液佐剂以三周之间隔进行两次肌肉注射进行接种。在第二次接种后2周收集血清样品;再间隔一周后收集及分离脾脏细胞。
病毒攻击(Viralchallenges)
为达成两剂免疫接种策略,BALB/c小鼠(6-8周龄)共分为五组,其中每组由5只小鼠所组成,其以2或20μg的H5N1-rNA或pH1N1-rNA蛋白质加上CpG/PELC或PBS以三周之间隔进行接种。在第二次接种的三周后,所有的小鼠以10MLD50的H5N1(NIBRG-14,RG-14)、pH1N1(A/California/07/2009,CA/09)、或H7N9(A/Taiwan/01/2013,TW/13)病毒进行鼻内攻毒。以PBS接种的小鼠用以作为空白控制组。在14天中每日记录存活率及体重。根据IACUC准则,体重减轻25%以上被确立为终点。
酶联免疫吸附测定法(Enzyme-linkedimmunosorbentassays,ELISA)
96孔盘的每个孔以纯化的rNA蛋白质(2μg/ml浓度,100μl)涂布并在4℃保持过夜,以PBST(0.05%Tween-20溶于PBS中)洗涤3次,及以阻绝缓冲液(1%BSA溶于PBS中)阻绝至少1小时。接着,加入100μl的两倍连续稀释的血清样品并在室温下保持1小时,随后以PBST进行3次的额外洗涤。将HRP共轭结合的山羊抗小鼠IgG抗体加入到每个孔,静置1小时,并以PBST洗涤3次。测定抗NA的IgG抗体效价,其系藉由添加TMB受质,于室温下保持15分钟,并以2N硫酸停止反应而得到。终点效价被测定为在450nm提供0.2以上平均光密度(opticaldensity,OD)的最高稀释倍率血清浓度的倒数。
神经氨酸酶抑制(Neuraminidase-inhibiting,NI)测定法
NA抑制(NI)抗体的测量是使用先前所述的以胎球蛋白(Fetuin)为基础的检测方法所得到。简言之,将96孔盘以50μg/ml胎球蛋白涂布,并在4℃保持过夜,接着以PBST洗涤3次,并以阻绝缓冲液阻绝2小时。溶于阻绝缓冲液中的两倍连续稀释血清样品以等体积的1μg的VLP(H5N1或H7N9)或105p.f.u.病毒(pH1N1或H3N2)在37℃温育1小时,加入到已在4℃保持过夜的胎球蛋白涂布的孔盘并在37℃再保持另外1小时,然后用PBST洗涤3次。将过氧化物酶标记的花生凝集素(浓度2.5μg/mL,100μl)加入到每个孔,于室温下置放1小时,以及用PBST洗涤3次。病毒(pH1N1或H3N2)及VLPs(H5N1或H7N9)的NA活性水平的测定是通过加入TMB受质、于室温下保持15分钟,并以2N硫酸停止反应。96孔盘以ELISA读数器在450nm读取OD值。相对应的IC50值被定义为抑制50%病毒NA酶活性的血清稀释值的倒数。
分泌NA特异性抗体的B细胞
对每组rNA免疫或PBS免疫小鼠在第二次接种之后3周收集其脾细胞。多重筛选96孔盘以rNA蛋白质(每孔1μg)涂布并在4℃过夜。孔盘以200μl/孔的完全RPMI-1640(含有10%FBS、1xP/S、1x丙酮酸钠,1xNEAA及100μMβ-ME)阻绝并于室温保持1小时。稀释于完全RPMI-1640的脾细胞(2×105)加入到各个孔盘并在37℃下温育48小时。在用PBST洗涤3次后,HRP共轭结合的抗小鼠IgG抗体被加到每个孔及于室温保持2小时。在以PBST及洗涤3次及以PBS洗涤2次之后,加入AEC受质至每一孔盘,使在RT下保持10~60分钟并以ddH2O停止反应,以ELISPOT孔盘读数器测定每个免疫接种组别的免疫斑点。
制造pH1N1/PR8嵌合流感病毒
293T(3.5×105)及MDCK细胞(5×105)的混合物于6孔盘各孔播种于2mLOPTI-MEM培养基并于CO2培养箱中温育过夜。本发明使用八个不同的成份质体,以产生pH1N1/PR8嵌合病毒,包括来自A/Texas/05/2009(pH1N1)或PR8病毒株的WTHA、来自A/Texas/05/2009病毒株的WT或突变(I365T和S366N)NA成份、以及全部来自PR8病毒株的其它6个成份。这8个DNA质体利用TransIT-LT1转染试剂同时共转染到293T/MDCK细胞混合物。TransIT-LT1试剂/DNA复合体的混合物在37℃温育过夜。培养基替换为含有0.5μg/mlTPCK胰蛋白酶(Trypsin-TPCK)的3mL新鲜OPTI-MEM并再培养2~3天。收集上清液、滴定病毒滴度并保持于-80℃,直到在实验中使用。
pH1N1/PR8嵌合流感病毒蚀斑测试
MDCK细胞(6×105/孔),在37℃保持过夜。单层MDCK细胞以连续稀释的PR8、PR8xTexasHA(THA)xTexasNA(TNA)或PR8xTHAxTNA(I365T/S366N)RG病毒在37℃感染1小时。除去上清液并用PBS洗涤两次。被感染的MDCK细胞以MEM-α加上0.5%琼脂覆盖。于37℃再温育48小时后,被感染的细胞以4%多聚甲醛固定并以1%结晶紫溶液染色。观察及计算蚀斑数量。
统计分析
所有结果用Student’sttest分析。每个图中的星号表示统计显着(*,p<0.05;**,p<0.01;***,p<0.001)。
使用杆状病毒-昆虫细胞表达系统表达并纯化H5N1-rNA及pH1N1-rNA蛋白质。
如图1A呈现的水溶性H5N1-rNA及pH1N1-rNA的蛋白质建构图。上述两种重组蛋白是由以重组杆状病毒感染的Sf9细胞的培养上清液所产生,并使用镍螯合亲和层析法纯化。水溶性H5N1-rNA及pH1NA-rNA产量分别约为0.5mg/L及0.25mg/L,纯度为80-90%(图1B)。根据考马斯蓝染色(图1B)及蛋白质印迹(图1C)的SDS-PAGE凝胶结果,纯化的H5N1-rNA及pH1N1-rNA蛋白质分子量各自为53kDa。基于Eadie-Hofstee测量,H5N1-rNA及pH1N1-rNA的Km值分别为2.80及1.90(图1D),其说明表达H5N1-rNA及pH1N1-rNA的酶活性。
NA特异性IgG抗体是由H5N1-rNA及pH1N1-rNA免疫接种所诱导产生。
各组五只母BALB/c小鼠以三周间隔、肌肉注射(i.m.)方式,免疫接种两剂H5N1-rNA或pH1N1-rNA蛋白质(每剂2或20μg)。在第二次免疫接种后2周收集血清。每个组中对于相同的免疫原(H5N1-rNA或pH1N1-rNA的蛋白质)的NA特异性IgG效价数据显示于图2A。本发明使用抗小鼠N1NA特异性抗体作为阳性对照及以PBS免疫接种的血清作为阴性对照(分别为103.5-4及未测出的NA特异性IgG效价)。如图2A所示,相较以2μgH5N1-rNA或pH1N1-rNA免疫接种的小鼠,以20μgH5N1-rNA或pH1N1-rNA免疫接种的小鼠展现略高的NA-特异性总IgG抗体效价。本发明亦利用H5N1VLPs(图2B)及pH1N1(A/California/04/2009)(图2C)病毒进行ELISA试验。我们的数据显示,在2或20μg部分,无论H5N1-rNA及pH1N1-rNA皆与H5N1类病毒颗粒及pH1N1病毒交叉反应,而诱导类似数量的总IgG抗体效价(图2B-2C)。相似的交叉反应性结果亦发现于H3N2病毒(A/Udorn/307/1972)及H7N9VLPs(图2D-2E)。在相同的免疫接种群组中IgG1或IgG2a子类别被发现并无显着差异。唯一的例外是以20μgH5N1-rNA免疫接种的小鼠具有显着较高的IgG2a分泌(数据未显示)。
NI抗体是由H5N1-rNA及pH1N1-rNA免疫接种所引起。
为了测量NI抗体效价,将各免疫接种组的2倍连续稀释血清样品与1μgH5N1-VLP、105p.f.u.pH1N1(A/California/04/2009)病毒、105p.f.u.H3N2(A/Udorn/307/1972)或1μgH7N9-VLP混合,接着使用胎球蛋白分析,每个免疫接种组中对同组的两种病毒,H5N1及pH1N1,NA-抑制百分比及NI效价显示于图3A-3B及3E-3F。这些结果说明剂量相关性,其中所有的rNA免疫组的NI曲线显着高于PBS免疫组的曲线。NA-抑制百分比及NI效价也表现于H3N2病毒及H7N9VLPs,其具有相似的结果,但NI效价较低(图3C-3D及3G-3H)。关于IC50值,可观察到对同源病毒的类似NI效价范围,其中H5N1-rNA免疫接种对抗H5N1为3.7-3.8,以及pH1N1-rNA免疫接种对抗pH1N1为4.3-4.4(图4)。然而,以NI效价表示时,已被发现对H5N1、pH1N1、H3N2及H7N9异源病毒具有显着差异。相较由两个pH1N1-rNA免疫接种组对抗H5N1病毒所引起的效价,无论H5N1-rNA免疫组(2及20μg)皆表现较高的抗pH1N1异亚型NI效价。相反地,H5N1-rNA及pH1N1-rNA免疫接种对抗H3N2及H7N9病毒,则引起相对较低的NI效价,所有组别的IC50值皆小于2.32(图4)。我们的数据显示,H5N1-rNA免疫效果可产生更多对抗pH1N1病毒的有效异亚型NI抗体。
脾细胞中所检测的抗体分泌B细胞
为了测量由H5N1-rNA或pH1N1-rNA所引起的分泌抗NAIgG抗体的B细胞,在小鼠第二次免疫接种后3周,从免疫接种小鼠收集脾细胞,分别与1μgH5N1-rNA或pH1N1-rNA蛋白质反应,并使用ELISPOT试验测试。图5结果显示,H5N1-rNA免疫接种的对抗同源病毒的斑点数量略高于由pH1N1-rNA免疫接种所得的斑点数量。对抗H5N1及pH1N1的异源病毒仅在20μgH5N1-rNA免疫接种组别发现具有显着较高的斑点数量,其亦显示脾细胞对接同源及异源H5N1及pH1N1病毒展现较高数量的分泌抗体的B细胞(ASC)。
对抗H5N1、pH1N1及H7N9病毒攻击的保护性免疫力
为评估由rNA接种所引起的保护性免疫力,以2或20μgH5N1-rNA或pH1N1-rNA蛋白质进行接种的小鼠,在其第二次免疫接种后的3周以10MLD50的H5N1(RG-14)、pH1N1(CA/09)或H7N9(TW/13)病毒进行攻击。根据图6A-6B所示结果,其中除了以2μgpH1N1-rNA免疫接种的小鼠具有80%的存活率及PBS小鼠对照组存活率为0%外,所有的免疫接种组别对于同源H5N1或pH1N1病毒攻击具有100%的存活率。相较于2μgH5N1-rNA及2μgpH1N1-rNA免疫接种组别,以同源H5N1或pH1N1病毒攻击的20μgH5N1-rNA及20μgpH1N1-rNA免疫接种组别的小鼠观察到显着较少的体重减轻(图7A-7B)。就交叉保护程度而言,接受2或20μgH5N1-rNA接种的小鼠展现对抗病毒pH1N1攻击的完整的保护(图6C),其中在20μg组别中发现具有显着的较少体重减轻(图7C)。在20μgpH1N1-rNA免疫接种组别在以异源H5N1病毒攻击之后观察到具有60%的存活率(图6D)及较快的体重回复(图7D)。在20μgH5N1-rNA及20μgpH1N1-rNA免疫接种组别以H7N9病毒攻击之后观察到保护力为零(图6E及7E)。
由pH1N1-rNA突变蛋白质所引起的交叉反应性NI抗体
为了详述我们对交叉反应性NI抗原表位(epitopes)研究,本发明将A/Vietnam/1203/2004(H5N1)、A/Texas/05/2009(pH1N1)、A/Udorn/307/1972(H3N2)及A/Shanghai/02/2013(H7N9)的氨基酸序列加以排列。如图8所示,本发明识别出两个序列的NA胞外域(ectodomain)中具有34个不同氨基酸(如图中所标示黑色字体处),(94.6%为相同)。先前报告已识别流感NA酶的催(活)化位点位于残基118-119、151-152、198、224、227、243、274、276-277、292、330、350或425;因此,本发明针对残基149、344、365及366,其皆靠近NA酶的活性位点并促进引发NI抗体。在这四个残基的位点上设计点突变产生三个pH1N1-rNA蛋白质的突变体:I149V、N344Y及I365T/S366N,如图9所示。依据野生型及三个突变pH1N1-rNA的血清分析结果显示,I149V及I365T/S366N突变蛋白质引起更多对抗同源pH1N1菌株(图10A)的NI抗体,其中I365T/S366N蛋白质引发对抗H5N1(图10B)、H3N2(图10C)及H7N9病毒(图10D)的更有效的交叉反应性NI抗体。由NI抑制曲线计算的相对应IC50值说明,I149V及I365T/S366N突变蛋白质导致对抗同源pH1N1病毒的NI效价增加,以及这三个突变蛋白质皆导致对抗异亚型H5N1、H3N2及H7N9病毒的NI效价增加(图11)。pH1N1-rNA的I365T/S366N突变能引起对抗同源及异亚型株的最高数量的NI抗体效价。本发明亦建构并表达改变自pH1N1到H3N2或H7N9的其它突变pH1N1-rNA蛋白质,如图12所述。依据野生型及三个突变pH1N1-rNA的血清分析结果显示,pH1N1-N344H(N2)及pH1N1-I365T/S366A(N9)突变蛋白质引起更多对抗同源pH1N1菌株(图13A)及异源H5N1(图13B)、H7N9病毒(图13D)的有效的交叉反应性NI抗体,对抗H3N2病毒(图13C)的交叉反应性NI抗体则和野生型相似。由NI响应曲线计算的相对应IC50值说明,pH1N1-N344H(N2)及pH1N1-I365T/S366A(N9)突变蛋白质导致对抗同源pH1N1病毒的NI效价增加,以及pH1N1-N344H(N2)突变蛋白质导致对抗异亚型H7N9病毒的NI效价增加(图14)。
建构嵌合pH1N1/PR8(I365T/S366N)的突变病毒株
本发明利用PR8(A/PuertoRico/8/1934(H1N1))反向遗传系统,将HA及NA更换成A/Texas/05/2009(pH1N1)的HA及NA基因以建构含有野生型TexasHA和NA基因的嵌合pH1N1/PR8病毒((PR8xTHAxTNA)及NA基因的I365T/S366N病毒突变株(PR8xTHAxTNA-I365T/S366N)。具有I365T/S366N突变的所得嵌合pH1N1/PR8病毒具有类似的病毒蚀斑形态(图15A),其效价(titers)约为6x106PFU/ml。本发明亦量测三种病毒(PR8RG、PR8xTHAxTNA及PR8xTHAxTNA-I365T/S366N)在MDCK细胞中的复制生长曲线,所选的MOI为0.01。相较于PR8野生型及嵌合pH1N1/PR8病毒,PR8xTHAxTNA-I365T/S366N突变病毒株并未观察到在复制动力曲线方面(图15B)或酶活性方面(图15C)具有显着差异。
结论
相较于HA抗原,NA抗原在宿主免疫系统中变化量较小,因此NA流感疫苗具有其吸引力。在这项研究中,本发明建构并从Sf9昆虫细胞中纯化H5N1-rNA及pH1N1-rNA蛋白质,并且发现以H5N1-rNA及pH1N1-rNA蛋白质免疫接种的小鼠展现较高量的NA特异性总IgG、IgG1、IgG2a亚型及NI抗体效价、脾细胞中的ASC数量增加,以及在对抗活病毒攻击时具有较佳的保护性免疫力。相较以pH1N1-rNA对抗H5N1,发现以H5N1-rNA免疫接种能诱发更有效的交叉反应性NI抗体并诱导对抗pH1N1病毒的保护性免疫。交叉反应性的NI抗原表位进一步由具有I149V、N344Y及I365T/S366N的NA突变的pH1N1-rNA蛋白质进行分析。pH1N1-rNA的I365T/S366N突变被发现可增加对抗H5N1、H3N2及病毒H7N9的交叉反应性NI抗体。
以上所述的实施例仅是为说明本发明的技术思想及特点,其目的在使熟习此项技艺之人士能够了解本发明的内容并据以实施,当不能以之限定本发明的专利范围,即大凡依本发明所揭示的精神所作的均等变化或修饰,仍应涵盖在本发明的专利范围内。
Claims (16)
1.一种重组神经氨酸酶蛋白,其具有一胞外域,该胞外域与SEQIDNO:1所示的氨基酸序列具有至少95%的相同性,其中该氨基酸序列具有下列之一或其任意组合:
(i)在位置149,I被取代为V;
(ii)在位置344,N被取代为Y或H;以及
(iii)在位置365、366,IS被取代为TA、TN或ED。
2.如权利要求1所述的重组神经氨酸酶蛋白,其中该氨基酸序列与SEQIDNO:1具有至少97%的相同性。
3.如权利要求1所述的重组神经氨酸酶蛋白,其中该氨基酸序列与SEQIDNO:1具有至少99%的相同性。
4.如权利要求1所述的重组神经氨酸酶蛋白,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:8。
5.如权利要求1所述的重组神经氨酸酶蛋白,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:5。
6.一种重组流感病毒,包含一重组神经氨酸酶蛋白,其具有一胞外域,该胞外域与SEQIDNO:1所示的氨基酸序列具有至少95%的相同性,其中该氨基酸序列具有下列之一或其任意组合:
(i)在位置149,I被取代为V;
(ii)在位置344,N被取代为Y或H;以及
(iii)在位置365、366,IS被取代为TA、TN或ED。
7.如权利要求6所述的重组流感病毒,其中该氨基酸序列与SEQIDNO:1具有至少97%的相同性。
8.如权利要求6所述的重组流感病毒,其中该氨基酸序列与SEQIDNO:1具有至少99%的相同性。
9.如权利要求6所述的重组流感病毒,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:8。
10.如权利要求6所述的重组流感病毒,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:5。
11.一种流感疫苗,包含重组神经氨酸酶蛋白,该重组神经氨酸酶蛋白具有一胞外域,该胞外域与SEQIDNO:1所示的氨基酸序列具有至少95%的相同性,其中该氨基酸序列具有下列之一或其任意组合:
(i)在位置149,I被取代为V;
(ii)在位置344,N被取代为Y或H;以及
(iii)在位置365、366,IS被取代为TA、TN或ED。
12.如如权利要求11所述的流感疫苗,其中该氨基酸序列与SEQIDNO:1具有至少97%的相同性。
13.如如权利要求11所述的流感疫苗,其中该氨基酸序列与SEQIDNO:1具有至少99%的相同性。
14.如如权利要求11所述的流感疫苗,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:8。
15.如如权利要求11所述的流感疫苗,其中该氨基酸序列选自SEQIDNO:2至SEQIDNO:5。
16.如如权利要求11所述的流感疫苗,其中该流感疫苗为灭活流感病毒疫苗、减毒流感病毒疫苗、类病毒颗粒疫苗或重组亚单位蛋白疫苗。
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CN110551187A (zh) * | 2019-09-23 | 2019-12-10 | 新乡学院 | 化学合成的h7n9禽流感病毒na蛋白胞外区抗原片段及制备方法和应用 |
WO2024078631A1 (zh) * | 2022-10-14 | 2024-04-18 | 吴夙钦 | 流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途 |
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US20180128545A1 (en) * | 2016-11-08 | 2018-05-10 | Berry Metal Company | Modular furnace cooling wall |
CN108384808A (zh) * | 2018-04-24 | 2018-08-10 | 上海市静安区疾病预防控制中心 | H7n9禽流感病毒ha蛋白重组杆状病毒表达载体及其构建方法 |
MX2022004711A (es) * | 2019-10-22 | 2022-06-14 | Icahn School Med Mount Sinai | Neuraminidasa recombinante y usos de la misma. |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4029763A (en) * | 1974-08-05 | 1977-06-14 | Mount Sinai School Of Medicine Of The City University Of New York | Influenza vaccine containing purified neuraminidase antigen and method of using the same |
WO2007118284A1 (en) * | 2006-04-19 | 2007-10-25 | Biodiem Ltd | Influenza virus vaccine |
-
2014
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2015
- 2015-04-13 US US14/685,286 patent/US9688965B2/en active Active
- 2015-09-02 CN CN201510555504.4A patent/CN105400753B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4029763A (en) * | 1974-08-05 | 1977-06-14 | Mount Sinai School Of Medicine Of The City University Of New York | Influenza vaccine containing purified neuraminidase antigen and method of using the same |
WO2007118284A1 (en) * | 2006-04-19 | 2007-10-25 | Biodiem Ltd | Influenza virus vaccine |
Non-Patent Citations (4)
Title |
---|
HONGQUAN WAN ET AL.: "Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses", 《JOURNAL OF VIROLOGY》 * |
XIAOJIN XU ET AL.: "Structural Characterization of the 1918 Influenza Virus H1N1 Neuraminidase", 《JOURNAL OF VIROLOGY》 * |
XIUHUA LU ET AL.: "Evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza A (H1N1) virus and highly pathogenic avian influenza A (H5N1) virus", 《VIROLOGY》 * |
陈则: "A型流病毒血凝素、神经氨酸酶A疫苗研究", 《微生物学杂志》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN110551187A (zh) * | 2019-09-23 | 2019-12-10 | 新乡学院 | 化学合成的h7n9禽流感病毒na蛋白胞外区抗原片段及制备方法和应用 |
CN110551187B (zh) * | 2019-09-23 | 2022-09-16 | 新乡学院 | 化学合成的h7n9禽流感病毒na蛋白胞外区抗原片段及制备方法和应用 |
WO2024078631A1 (zh) * | 2022-10-14 | 2024-04-18 | 吴夙钦 | 流感病毒神经氨酸酶突变体、编码流感病毒神经氨酸酶突变体的核酸分子、包含流感病毒神经氨酸酶突变体的疫苗组合物及流感病毒神经氨酸酶突变体用于制备流感病毒疫苗组合物的用途 |
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US20160067328A1 (en) | 2016-03-10 |
TWI588260B (zh) | 2017-06-21 |
TW201610162A (zh) | 2016-03-16 |
US9688965B2 (en) | 2017-06-27 |
CN105400753B (zh) | 2019-06-07 |
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