CN117701511A - 一株h9亚型禽流感病毒分离株及其制备的疫苗组合物和应用 - Google Patents
一株h9亚型禽流感病毒分离株及其制备的疫苗组合物和应用 Download PDFInfo
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Abstract
本发明公开了一株H9亚型禽流感病毒分离株及其制备的疫苗组合物和应用,属于兽用医药技术领域。本发明分离得到的禽流感病毒JS121株,命名为H9亚型禽流感病毒JS121株(Avian influenza virus JS121),已于2023年11月12日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V2023102,保藏地址为中国武汉大学。本发明获得的H9亚型禽流感病毒JS121株在细胞上高滴度增殖,病毒效价达1011.5TCID50/ml,为低致病力毒株,同时具有良好的免疫原性,可用于制备H9亚型禽流感疫苗。制备的H9亚型禽流感疫苗免疫后,可产生高效价的抗体,对H9亚型禽流感流行株具有良好的免疫保护作用。
Description
技术领域
本发明属于兽用医药技术领域,尤其涉及一株H9亚型禽流感病毒分离株及其制备的疫苗组合物和应用。
背景技术
禽流感(Avian influenza,AI)是由禽流感病毒(Avian influenzavirus,AIV)引起的一种高度传染性的急性呼吸道疾病,AIV是一种单股负链分节段RNA病毒,属于正黏病毒科,A型流感病毒属。根据病毒致病性和毒力不同,在临床上可分为高致病性禽流感病毒和低致病性禽流感病毒。在我国鸡群中流行的有H5亚型、H7亚型和H9亚型。H5亚型和H7亚型由于致病性比较强,可引起禽类大批死亡并有感染人的风险,因此H5亚型和H7亚型禽流感病毒的分离鉴定及疫苗研制受国家管控,具备一定资质的研究院所和企业才可以研究和生产疫苗。H9亚型为低致病性禽流感,感染后不会导致群体大批死亡,但是感染率比较高,会导致产蛋率大幅下降,且由于易与其他致病微生物发生协同作用,造成继发感染,从而引起了禽群的高致死率,给养殖业也会带来巨大的经济损失。
目前疫苗接种是防控H9亚型禽流感的主要手段之一,但由于病毒可以通过抗原漂移和抗原转变两种途径产生变异株,导致抗原性发生变化,引起免疫失败。特别是近年来禽群感染H9亚型禽流感出现在免疫禽场,主要原因在于流行株与疫苗株存在较大的遗传距离及抗原性差异,导致商品化疫苗的免疫保护失败。另外,目前也有一些关于H9亚型禽流感疫苗的相关报道,但其只能对局部地域流行株提供有限保护,随着禽流感毒株在免疫压力下的出现变异,现有的疫苗已不能提供有效交叉保护,因此,急需开发一种可以针对现有流行株提供有效交叉保护的H9亚型禽流感疫苗。
发明内容
为解决上述技术问题,本发明针对病毒的变异情况开展大量的流行病学调查研究,最终成功从某鸡场发病鸡群中分离出一株H9亚型禽流感病毒,该病毒分离株对鸡胚的半数感染量EID50高达1010.5/ml,采用该病毒株制得的疫苗能够产生H9亚型特异性HI抗体,抗体效价的平均值达到9.6log2,且能完全抵御多种禽流感流行毒株的攻击。
本发明的第一个目的是提供一种禽流感病毒JS121株,所述H9亚型禽流感病毒JS121株命名为H9亚型禽流感病毒JS121株(Avian influenza virus JS121),于2023年11月12日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V2023102。
本发明的第二个目的是提供一种H9亚型(H9N2)禽流感疫苗,含有灭活的上述禽流感病毒JS121株。
进一步地,疫苗中禽流感病毒JS121株的含量不低于106.5TCID50/ml,优选为106.5TCID50/ml~108.5TCID50/ml。
本发明的第三个目的是提供一种H9亚型禽流感疫苗的制备方法,使用上述禽流感病毒JS121株,通过细胞悬浮培养技术制备得到H9亚型禽流感疫苗。采用细胞悬浮培养技术,克服了传统鸡胚、鸭胚生产制备工艺的缺点,降低了疫苗的批间差,提高疫苗质量,易于规模化生产,降低生产成本,获得市场优势。
进一步地,制备方法包括以下步骤:
S1、将悬浮MDCK细胞接种于生物反应器中,设置温度35~38℃,溶氧40%~50%,pH 6.8~7.2培养,当细胞密度达到4.0×106~6.0×106个/ml,接种上述禽流感病毒JS121株并加入终浓度为4~6μg/ml的TPCK-胰酶,设置温度32~34℃、溶氧40%~50%、pH 7.0~7.4悬浮培养48~72小时,当细胞密度低于2.0×106个/ml时收获病毒液;
S2、向病毒液中加入灭活剂使病毒灭活;
S3、将灭活后的病毒液与佐剂混合,乳化,得到所述H9亚型禽流感疫苗。
进一步地,禽流感病毒JS121株的接种量为0.001%~0.01%(v/v)。
进一步地,所述灭活剂选自甲醛、β-丙内酯中的一种或多种。
进一步地,灭活剂的终浓度为0.05~0.2%(v/v)。
进一步地,灭活操作包括:与灭活剂混合均匀后,转入无菌容器中,密封,35~38℃灭活12~36小时。
进一步地,灭活后的病毒液与佐剂的体积比为1:1.5~2。
本发明的第三个目的是提供包含灭活的禽流感病毒JS121株的疫苗组合物。
进一步地,所述疫苗组合物中含有灭活的新城疫病毒,制备得到的疫苗为新城疫、禽流感(H9亚型)二联灭活疫苗。
进一步地,疫苗组合物中,新城疫病毒的含量不低于106.0TCID50/ml,优选为106.0TCID50/ml~108.0TCID50/ml。
进一步地,灭活的新城疫病毒的制备方法同上,均采用悬浮细胞培养技术进行制备。
本发明的第四个目的是提供含有上述禽流感病毒JS121株在制备禽流感预防或治疗药物或禽流感诊断产品中的应用。
进一步地,所述禽流感诊断产品包括但不限于抗原试剂、阳性血清试剂等。
本发明的上述技术方案相比现有技术具有以下优点:
本发明通过血清中和实验,发现分离得到的禽流感病毒JS121株具有良好的纯净度,血凝实验的HA效价为1:512,且只能被H9亚型禽流感病毒阳性血清所抑制,证明该病毒株具有良好的特异性和免疫原性。利用上述病毒株JS121可成功制备灭活疫苗,测定静脉接种致病指数(IVPI)为0,证明该禽流感病毒为低致病性毒株,在鸡胚感染实验中表现良好,EID50为1010.5/ml,在细胞上高滴度增殖,病毒效价达1011.5TCID50/ml,H9亚型特异性HI抗体效价的平均值达到9.6log2。以上,说明本发明提供的禽流感病毒株可用于预防H9亚型禽流感,对禽流感病毒的防治具有重要意义。
生物材料保藏
禽流感病毒JS121株,命名为H9亚型禽流感病毒JS121株(Avian influenzavirusH9- JS121),于2023年11月12日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V2023102,保藏地址为中国武汉大学。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1病毒的鉴定
1、血凝试验
制备1%浓度的鸡红细胞,以收集的尿囊液为检测样品,通过血凝试验(HA)检测尿囊液的HA效价,试验设PBS阴性对照。HA效价大于1∶16判定为阳性。结果显示,鸡胚尿囊液对1%鸡红细胞的凝集效价为1∶512。
2、血凝抑制试验(HI)
根据HA试验的结果,用PBS(pH7.0~7.2)配制4个工作单位的病毒。以4单位病毒为待检抗原,与标准的鸡新城疫病毒血凝抑制试验阳性血清、鸡减蛋综合征病毒血凝抑制试验阳性血清、H9亚型禽流感病毒血凝抑制试验阳性血清、H5亚型禽流感病毒血凝抑制试验阳性血清和H7亚型禽流感病毒血凝抑制试验阳性血清进行血凝抑制试验(HI),鉴定病毒的类型。结果显示,分离株仅与禽流感病毒H9亚型血凝抑制试验阳性血清有交叉反应,与其他阳性血清均没有交叉反应,表明该尿囊液中只含有H9亚型禽流感病毒,没有新城疫病毒、鸡减蛋综合征病毒、H5亚型禽流感病毒和H7亚型禽流感病毒。
3、鸡胚半数感染量(EID50)的测定
将分离的病毒用灭菌生理盐水作10倍系列稀释,经尿囊腔接种10~11日龄SPF鸡胚,0.1ml/枚,每个稀释度接种5枚鸡胚,弃24小时死亡胚,于接种后120小时4℃过夜冻死鸡胚,逐胚测定其HA效价,HA效价大于1∶16判定为阳性,参考Reed-Muench方法计算出病毒EID50为1010.5/ml。
4、对鸡的毒力
为测定静脉接种致病指数(IVPI),将JS121株E1代病毒液10倍稀释后,静脉注射6周龄SPF鸡10只,每只0.1ml,攻毒后观察10日,未见任何异常反应,计算IVPI指数为0,确定该毒株为低致病力毒株。同时在攻毒后第5日时,采集所有攻毒鸡喉头及泄殖腔棉拭子放置无菌PBS中,经尿囊腔接种于10~11日龄SPF鸡胚分离病毒,结果10/10病毒分离阳性。
实施例2H9亚型禽流感灭活疫苗的制备
1、病毒液的制备
将悬浮型MDCK细胞接种于生物反应器中,使罐内细胞密度为1.0×106~1.3×106个/ml。设置温度37℃,溶氧(DO)值40%~50%,pH值7.0,以适宜转速培养。当细胞密度达4.0×106~6.0×106个/ml,按0.001%~0.01%(v/v)比例接种H9亚型禽流感病毒JS121株,同时加入终浓度为5μg/mlTPCK-胰酶,设置温度33℃、溶氧40%~50%、pH值7.2,以适宜转速悬浮培养48~72小时,当悬浮细胞出现死亡、崩解、破碎,细胞密度低于2.0×106个/ml时收获病毒液。
所收获病毒液取样,用含终浓度为5μg/mlTPCK-胰酶的无血清培养基作10倍系列稀释,分别接种长有单层MDCK细胞的96孔培养板,每孔0.1ml,每个稀释度接种5孔,同时设正常细胞对照,置33℃、含5%CO2培养箱中培养4日。逐孔取样测定红细胞凝集价,凝集价不低于1:16(微量法)者判为感染,记录各稀释度感染孔数,并按ReedMuench法计算TCID50。测定病毒含量,为1011.5TCID50/ml。
2、抗原灭活
将分析纯甲醛溶液用纯化水10倍稀释后,加入上述步骤的病毒液中,边加边摇,使其终浓度为0.1%(V/V)。充分摇震混匀后,转入另一无菌容器中,密封后37℃灭活24小时,间隔4~6小时震摇1次。
3、乳化
取注射用白油94容积份和司本-806容积份混合后,加硬脂酸铝2质量份,随加随搅拌至透明为止,高压灭菌冷却后备用,即为油相。
取禽流感抗原液,置于灭菌容器中,按抗原液体积的4%(V/V)加入灭菌的吐温-80,充分振摇,使吐温-80完全溶解为止,即为水相。
取油相2份导入乳化罐内,开动电机慢速搅拌,同时徐徐导入上述水相1份,以3600r/min乳化40分钟。具体配比见表1。
表1H9亚型禽流感病毒灭活疫苗配比
实施例3新流二联灭活疫苗的制备
1、新城疫病毒液的制备
将悬浮型BHK-21细胞接种于生物反应器中,使罐内细胞密度为0.8×106~1.0×106个/ml。设置温度37℃,溶氧(DO)值40%~50%,pH值7.0,以适宜转速培养。当细胞密度达4.0×106~5.0×106个/ml,按0.001%~0.01%(v/v)比例接种鸡新城疫病毒La Sota株,同时加入终浓度为5μg/mlTPCK-胰酶,设置温度37℃、溶氧40%~50%、pH值7.2,以适宜转速悬浮培养72~96小时,当悬浮细胞出现死亡、崩解、破碎,细胞密度低于2.0×106个/ml时收获病毒液。
所收获病毒液取样,用含终浓度为5μg/mlTPCK-胰酶的无血清培养基作10倍系列稀释,分别接种长有单层BHK-21细胞的96孔培养板,每孔0.1ml,每个稀释度接种5孔,同时设正常细胞对照,置37℃、含5%CO2培养箱中培养4日。逐孔取样测定红细胞凝集价,凝集价不低于1:16(微量法)者判为感染,记录各稀释度感染孔数,并按ReedMuench法计算TCID50。测定病毒含量,为1010TCID50/ml。
2、抗原灭活
将分析纯甲醛溶液用纯化水10倍稀释后,加入上述步骤的病毒液中,边加边摇,使其终浓度为0.1%(V/V)。充分摇震混匀后,转入另一无菌容器中,密封后37℃灭活24小时,间隔4~6小时震摇1次。
3、乳化
取注射用白油94容积份和司本-806容积份混合后,加硬脂酸铝2质量份,随加随搅拌至透明为止,高压灭菌冷却后备用,即为油相。
取鸡新城疫抗原液、禽流感抗原液,置于灭菌容器中,按比例混合均匀,再按抗原液体积的4%(V/V)加入灭菌的吐温-80,充分振摇,使吐温-80完全溶解为止,即为水相。
取油相2份导入乳化罐内,开动电机慢速搅拌,同时徐徐导入上述水相1份,以3600r/min乳化40分钟。具体配比见表2。
表2新流二联灭活疫苗配比
实施例4JS121株免疫保护试验
1、血清学方法
用3周龄SPF鸡35只,每组10只,分3组,各皮下注射H9亚型禽流感病毒JS121株制作的灭活疫苗0.3ml,另5只不免疫作对照。接种后21日,每只鸡分别采血,分离血清,按现行《中国兽药典》进行HI抗体效价测定。免疫组HI抗体效价的几何平均值可达1:776(微量法),未免疫对照组HI抗体效价的几何平均值均不高于1:2(微量法),检测结果见表3。
表3HI抗体效价结果
注:“/”表示此项无内容,“GMT”表示几何平均值。
结果显示,本发明禽流感灭活疫苗HI抗体效价远高于几何平均值1:16(微量法)疫苗标准。
2、免疫攻毒法
使用上述35只鸡,在免疫21天后,每只鸡各静脉注射H9亚型禽流感病毒JS121株病毒液,0.2ml/只(含2×106.0EID50)。攻毒后第5日,分别采集每只鸡的喉头及泄殖腔拭子,经尿囊腔接种10日龄SPF鸡胚5枚,每胚0.2ml,孵育观察120小时,逐胚测定鸡胚液的HA效价。每个拭子样品接种的5枚鸡胚中只要有1枚鸡胚液的HA效价不低于1∶16(微量法),即可判为病毒分离阳性。对病毒分离阴性的样品,应盲传1代后再进行判定。
结果详见表4。
表4攻毒后病毒分离结果
组别 | 病毒分离阳性率 |
疫苗1 | 0/10 |
疫苗2 | 0/10 |
疫苗3 | 0/10 |
对照组 | 5/5 |
结果显示,本发明禽流感灭活疫苗通过免疫攻毒法效力检验,病毒分离率为0,说明免疫本发明禽流感灭活疫苗后,鸡只不排毒,完全抵抗了H9亚型禽流感的感染,具有很好的免疫保护效果。
实施例5JS121株免疫攻毒交叉保护试验
用3周龄的SPF鸡75只,每组10只,分6组,其中3组各皮下免疫H9亚型禽流感病毒JS121株制备的灭活疫苗2,0.3ml/只;另外3组皮下免疫0.3ml/羽的商品化疫苗鸡新城疫、禽流感(H9亚型)二联灭活疫苗;另15只分为5只/组,作为对照组。免疫21日后,每只鸡分别采血,分离血清,进行HI抗体效价测定,结果显示,免疫组均达到较高抗体水平,然后分别用流行毒株A22-2020、A17-2021、A29-2022攻毒,分别采集每只鸡的喉头及泄殖腔拭子,经尿囊腔接种10日龄SPF鸡胚5枚,每胚0.2ml,孵育观察120小时,逐胚测定鸡胚液的HA效价。每个拭子样品接种的5枚鸡胚中只要有1枚鸡胚液的HA效价不低于1∶16(微量法),即可判为病毒分离阳性。对病毒分离阴性的样品,应盲传1代后再进行判定。结果详见表5。
其中流行毒株A22-2020、A17-2021、A29-2022分别从2020年、2021年、2022年来源于山东、河南和江苏商品蛋鸡养殖场的发病鸡病料中分离得到。
具体病毒分离步骤为:
1、采集疑似发病鸡群病料及肛咽拭子,无菌处理后,根据病毒RNA提取试剂盒操作步骤提取核酸。
2、按照GB/T 19438.4-2004、GB/T 19438.2-2004和GB/T 19438.3-2004合成相应的引物和探针,对1中提取的核酸分别进行H9亚型禽流感病毒核酸、H5亚型禽流感病毒核酸和H7亚型禽流感病毒核酸的检测。
3、将步骤2中H9亚型禽流感病毒核酸检测阳性、且H5亚型禽流感病毒核酸和H7亚型禽流感病毒核酸的检测均为阴性的组织病料研磨后,反复冻融3次,10000rpm离心5分钟后取上清,经0.45μm滤器过滤,按照0.2ml/胚的剂量经尿囊腔接种9~10日龄SPF鸡胚,置36℃~37℃恒温培养箱培养。每日照蛋,弃去24h内死亡的鸡胚,将培养24~72h的死亡及存活的鸡胚放于4℃过夜或放于-20℃2h后无菌收集尿囊液。
4、对3中收集的尿囊液取样测定红细胞凝集价(HA)、鸡胚半数感染量(EID50)和对鸡的毒力(IVPI)。
表5JS121株免疫攻毒交叉保护试验结果
预防H9亚型禽流感的商品化疫苗鸡新城疫、禽流感(H9亚型)二联灭活疫苗免疫SPF鸡后,使用2020~2022年分离到的H9亚型禽流感A22-2020、A17-2021、A29-2022毒株攻毒,攻毒5天后检测排毒,发现攻毒鸡的病毒检出率均在40%以上,即试验中早期的H9亚型禽流感商品化疫苗可以在免疫后产生高水平抗体,但不能抵御2020~2022年流行毒株的攻击。而本发明的灭活苗则能完全保护。
实施例6新流二联灭活疫苗免疫保护试验
1、新城疫部分免疫原性试验
取3周龄的SPF鸡35只,每组10只,分3组,各皮下注射免疫新流二联灭活疫苗,20μl/只;另5只不免疫作对照。所有试验鸡均隔离饲养,免疫后21日,每只鸡分别采血并分离血清。检测新城疫病毒HI抗体。结果见表6。
表6新流二联灭活疫苗新城疫部分免疫原性试验结果
注:HI抗体测定为免疫鸡抗体的几何平均数。
结果显示,本发明新流二联灭活疫苗在免疫21日后均能产生较高的新城疫抗体,可以完全保护强毒的攻击,可对鸡群提供完全的保护。
2、禽流感部分免疫原性试验
用3周龄SPF鸡35只,每组10只,分3组,各皮下注射新流二联灭活疫苗0.3ml,另5只不免疫作对照。接种后21日,每只鸡分别采血,分离血清,检测禽流感病毒HI抗体,同时每只鸡各静脉注射H9亚型禽流感病毒JS121株病毒液,0.2ml/只(含2×106.0EID50)。攻毒后第5日,分别采集每只鸡的喉头及泄殖腔拭子,经尿囊腔接种10日龄SPF鸡胚5枚,每胚0.2ml,孵育观察120小时,逐胚测定鸡胚液的HA效价。每个拭子样品接种的5枚鸡胚中只要有1枚鸡胚液的HA效价不低于1∶16(微量法),即可判为病毒分离阳性。对病毒分离阴性的样品,应盲传1代后再进行判定。结果详见表7。
表7新流二联灭活疫苗禽流感部分免疫原性试验结果
注:HI抗体测定为免疫鸡抗体的几何平均数。
结果显示,本发明新流二联灭活疫苗在免疫21日后均能产生较高的禽流感抗体,可以完全保护强毒的攻击,可对鸡群提供完全的保护。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种禽流感病毒JS121株,其特征在于,所述禽流感病毒JS121株的保藏编号为CCTCCNO:V2023102。
2.一种H9亚型禽流感疫苗,其特征在于,含有灭活的权利要求1所述禽流感病毒JS121株。
3.一种H9亚型禽流感疫苗的制备方法,其特征在于,使用权利要求1所述禽流感病毒JS121株,通过细胞悬浮培养技术得到病毒液制备得到H9亚型禽流感疫苗。
4.根据权利要求3所述的制备方法,其特征在于,包括以下步骤:
S1、将悬浮MDCK细胞接种于生物反应器中,设置温度35~38℃,溶氧40%~50%,pH6.8~7.2培养,当细胞密度达到4.0×106~6.0×106个/ml,接种上述禽流感病毒JS121株并加入终浓度为4~6μg/ml的TPCK-胰酶,设置温度32~34℃、溶氧40%~50%、pH 7.0~7.4悬浮培养48~72小时,当细胞密度低于2.0×106个/ml时收获病毒液;
S2、向病毒液中加入灭活剂使病毒灭活;
S3、将灭活后的病毒液与佐剂混合,乳化,得到所述H9亚型禽流感疫苗。
5.根据权利要求4所述的制备方法,其特征在于,禽流感病毒JS121株的接种量为0.001%~0.01%(v/v)。
6.根据权利要求4所述的制备方法,其特征在于,灭活后的病毒液与佐剂的体积比为1:1.5~2。
7.包含灭活的权利要求1所述禽流感病毒JS121株的疫苗组合物。
8.根据权利要求7所述的疫苗组合物,其特征在于,所述疫苗组合物中含有灭活的新城疫病毒。
9.权利要求1所述禽流感病毒JS121株在制备禽流感预防或治疗药物或禽流感诊断产品中的应用。
10.根据权利要求9所述的应用,其特征在于,所述禽流感诊断产品为抗原试剂或阳性血清试剂。
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