CN103908662A - 可生物分解的高效率登革疫苗、其制备方法及医药组合物 - Google Patents
可生物分解的高效率登革疫苗、其制备方法及医药组合物 Download PDFInfo
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- CN103908662A CN103908662A CN201310308184.3A CN201310308184A CN103908662A CN 103908662 A CN103908662 A CN 103908662A CN 201310308184 A CN201310308184 A CN 201310308184A CN 103908662 A CN103908662 A CN 103908662A
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Abstract
本发明是有关于一种可生物分解的高效率登革疫苗、其制备方法及医药组合物,其中该可生物分解的高效率登革疫苗包括一具生物分解性且带电性的纳米复合物结构,而在纳米复合物结构中包含一登革病毒蛋白,使纳米复合物结构在两次接种后,即具有抗体表达量;由此,与现有的用于登革疫苗的铝盐佐剂以及Ribi佐剂相比较,可减少所需接种疫苗的次数,进而降低免疫所需费用,更有利成为具商业等级品质的混合疫苗。
Description
技术领域
本发明有关于一种可生物分解的高效率登革疫苗,尤其是指一种将登革病毒蛋白包裹于一具生物分解性且带电性的纳米复合物结构,使其在两次的接种后,即具有抗体表达量,并同时具有大幅增加抗体生成量,使疫苗能更有效地引起免疫反应的技术。
背景技术
据报道,登革热(dengue fever),又称断骨热,是一种由埃及斑蚊(Aedes aegypti)或白线斑蚊(Aedes albopictus)所传播的急性病毒性热疾,其会发生发烧(39℃至40℃)或恶寒、皮肤出疹、并有四肢酸痛、肌肉痛、前额头痛、眼窝痛、腹部痛、背痛(断骨热名称的来源)、喉咙痛,或可能有呕吐和昏晕等症状;一般所称登革热是指典型登革热(classicdengue fever),其又称为原发性登革热(primary dengue fever),另外也可能发展成严重且可能致命的出血征候或休克为其特征的登革热,称之为出血性登革热(denguehemorrhagic fever,DHF)或登革休克症候群(dengue shock syndrome,DSS),也可称为续发性登革热(secondary dengue);据估计,全球每年约有五千万至一亿个登革热病例产生,而其中更有约二十五万至五十万人会引发出血性登革热,也因此登革热的预防与治疗,已成为各国政府相当重视的议题,而由于登革病毒是登革热的主要致病原,若能对其早期检测或以疫苗早期预防,将能有效控制登革热的致病率与致死率。
请参阅本申请发明人的一中国台湾发明专利第201210614公开号所揭露的“登革疫苗、包含该登革疫苗的医药组合物、以及核苷酸序列”,以及第201210615公开号所揭露的“登革疫苗、包含该登革疫苗的医药组合物、核苷酸序列、以及抗体组成物”,上述两个案例揭露了一种可去除自体免疫作用,达到降低与内皮细胞和血小板的交叉反应的效果,并缩短出血时间的登革疫苗,这两篇文献在此全部以引用的方式并入本文中;然而,上述的登革疫苗在实际实施时虽可达到预期效果,但仍有不足之处,是因为上述疫苗在实际实施时需要加入一般最广泛使用的氢氧化铝胶作为佐剂(又称为免疫刺激剂),其增进活体内免疫反应的能力还不是很理想。
一般而言,佐剂的作用机制一般包括有:(a)增加疫苗抗原的生物或免疫性储存生命;(b)帮助抗原被输送至抗原呈现细胞;(c)改善抗原呈现细胞的抗原处理;以及(d)诱导免疫调节细胞活素的产生等;而矿物质佐剂为最常被使用的佐剂之一,此类佐剂包括例如,金属盐如锌、钙、铈、铬、铁和铍,而最常使用特别指磷酸盐及氢氧化盐的铝盐(该佐剂常被通称为铝佐剂[Alum]),其作用原理是抗原吸附在作为免疫刺激剂的相同铝盐颗粒上,当抗原被提供抗原的细胞吸收时,共同吸附的免疫刺激剂便直接对同样提供抗原的细胞发挥其刺激活性;请一并参阅美国发明专利第7357936公告号,其揭露一种制造疫苗组合物的方法,包括将含有吸附于金属盐颗粒的免疫刺激剂与抗原混合,其特征在于该金属盐颗粒实质上不含其他抗原的佐剂组合物,其中抗原也吸附在金属盐颗粒上,且抗原可选自包括:衍生自人类免疫不全病毒、水痘病毒、人类细胞巨大病毒、登革热病毒、A、B、C或E型肝炎病毒等;事实上,铝佐剂经统计已被应用于超过50%以上的商用疫苗产品内,其包括B型肝炎疫苗(Alum-HBsAg)以及白喉和破伤风类毒素疫苗(Alum-DT)等;然而,上述的抗原/金属盐复合疫苗虽已经实行了很多年,而且已证实可更容易地被提供抗原的细胞吸收,但其重金属成分仍然具有安全性的疑虑;因此,目前的登革疫苗极需要开发出安全的佐剂,不仅需避免铝佐剂所引起的安全性问题,也需能够增加抗体生成量,以具有更佳的免疫反应,进而减少所需接种的次数及其所需费用。
发明内容
本发明人即是鉴于上述现有的登革疫苗在实际实施时仍具有多处的缺失,于是本着孜孜不倦的精神,并通过其丰富的专业知识及多年的实务经验所辅佐,而加以改善,并据此研创出本发明。
本发明主要目的为提供一种将登革病毒蛋白包裹于一具生物分解性且带电性的纳米复合物结构,使其在两次的接种后,即具有抗体表达量,并同时具有大幅增加抗体生成量,使疫苗能更有效地引起免疫反应。
为了达到上述实施目的,本发明人提出一种可生物分解的高效率登革疫苗,其包括一具生物分解性且带电性的纳米复合物结构,而在纳米复合物结构中包含一登革病毒蛋白,使纳米复合物结构在两次接种后,即具有抗体表达量。由此,与现有的用于登革疫苗的铝盐佐剂以及Ribi佐剂相比较下,可减少所需接种疫苗的次数,进而降低免疫所需费用,更有利成为具商业等级质量的混合疫苗;此外,由于纳米复合物结构具有生物分解性,使得登革疫苗进入人体后可以容易、自然的被人体分解、吸收与代谢并排出体外,不仅解决现有技术中使用铝佐剂所引起的重金属安全性问题,也使得被包裹于纳米复合物结构内的登革病毒蛋白可缓慢释放,进而达到持续、长效释放的功效。
在本发明的一实施例中,登革病毒蛋白包括一非结构性嵌合蛋白DJ NS1,该非结构性嵌合蛋白DJ NS1包括:一登革病毒非结构性蛋白1(DV NS1)的N端序列1-270;以及一日本脑炎病毒非结构性蛋白1(JEV NS1)的C端序列271-352;再者,非结构性嵌合蛋白DJ NS1的氨基酸序列与SEQ.ID.NO.1的序列相似度较佳为90%以上,更佳可为95%以上。
在本发明的一实施例中,其中一次接种的纳米复合物结构含量不超过25微克(μg);再者,当纳米复合物结构以带正电的几丁聚糖与带负电的聚谷氨酸混合所形成时,登革疫苗于第2次接种后,即具有至少32000倍的抗体效价(titer);而当纳米复合物结构以带正电的几丁聚糖与带负电的肝素混合所形成时,登革疫苗于第2次接种后,即具有至少32000倍的抗体效价;因此,与现有技术相较下,可大幅增加抗体生成量,使疫苗能更有效的引起免疫反应,以达成增加疫苗保护效力的功效。
此外,本发明再提供一种制备可生物分解的高效率登革疫苗方法,其步骤包括:首先,准备一可生物分解的第一高分子(可例如为肝素或聚谷氨酸中的一种)溶液,其中第一高分子带有第一电性;接着,将与第一电性同性的登革病毒蛋白溶于第一高分子溶液中,以形成一具第一电性的混合溶液;然后,准备一可生物分解的第二高分子(可例如为几丁聚糖或胶原蛋白中的一种)溶液,其中第二高分子带有第二电性,且第一电性与第二电性为电荷相反;最后,将混合溶液加入第二高分子溶液中,通过电荷异性相吸的作用力而形成一纳米复合物结构,且纳米复合物结构即可包覆登革病毒蛋白。
本发明提供的一种可生物分解的高效率登革疫苗的医药组合物,优选的,其包含至少一种所述的可生物分解的高效率登革疫苗或其与医药学上可接受的碱形成的加成盐以及一或多种医药学上可接受的赋形剂。
再者,本发明再提供一种包含具上述生物分解的高效率登革疫苗的医药组合物,其是用于制造治疗或预防出血性登革热或登革休克症候群疾病的疫苗或药剂。
本发明与现有技术相较之下,本发明具有以下优点:
1、本发明具纳米复合物的登革疫苗在两次接种后,即可引起小鼠抗登革蛋白的特异性抗体反应,相对于现有的用于登革疫苗的铝盐佐剂以及Ribi佐剂,本发明的高效率登革疫苗可减少所需接种疫苗的次数,进而降低免疫所需费用,更有利成为具商业等级质量的混合疫苗。
2、本发明的登革疫苗于第二次接种后即能具有32000倍的抗体效价,与现有技术相比较,大幅增加抗体生成量,使疫苗能更有效的引起免疫反应,以达成增加疫苗保护效力的功效。
3、本发明具纳米复合物的登革疫苗是以具可生物分解性的聚谷氨酸(或肝素)与几丁聚糖混合来包裹登革病毒蛋白,使得登革疫苗进入人体后可以容易、自然的被人体分解、吸收与代谢并排出体外,不仅可解决现有的使用铝佐剂所引起的重金属安全性问题,也使得被包裹于纳米复合物结构内的登革病毒蛋白可缓慢释放,进而达到持续、长效释放的功效。
附图说明
图1为本发明包覆登革病毒蛋白后的纳米复合物结构的电子显微镜影像。
具体实施方式
本发明的目的及其结构功能上的优点,将依据以下图所示的结构,配合具体实施例予以说明,以使人们能对本发明有更深入且具体的了解。
首先,本发明的可生物分解的高效率登革疫苗,其包括一具生物分解性且带电性的纳米复合物结构,而在纳米复合物结构中包含一登革病毒蛋白,使一生物体在纳米复合物结构两次的疫苗接种后,即具有抗体表达量(antibody response),其中一次疫苗接种的纳米复合物结构含量不超过25微克(μg);值得注意的,上述的登革病毒蛋白可为针对登革套膜蛋白(envelope protein)或登革非结构性蛋白发展策略所制备,较佳的特别适用于本发明人先前的中国台湾发明专利第201210614公开号“登革疫苗、包含该登革疫苗的医药组合物、以及核苷酸序列”所揭露的登革病毒蛋白,该申请的内容也被引证作为本申请的参考数据;其中,登革病毒蛋白包括一非结构性嵌合蛋白DJ NS1,该非结构性嵌合蛋白DJ NS1包括:一登革病毒非结构性蛋白1(DV NS1)的N端序列1-270;以及一日本脑炎病毒非结构性蛋白1(JEV NS1)的C端序列271-352;再者,非结构性嵌合蛋白DJ NS1的氨基酸序列与SEQ.ID.NO.1的序列相似度(sequence similarity)较佳为90%以上,更佳可为95%以上。
此外,通过下述步骤制备在第2次接种后,即具有至少256000倍抗体效价的纳米复合物结构实际实施例,可进一步证明本发明的工艺可实际应用的范围,但不以任何形式限制本发明的范围:
步骤一:准备一可生物分解的第一高分子溶液,其中第一高分子带有第一电性,且第一高分子可例如为肝素(heparin)或聚谷氨酸(γ-PGA)中的一种,且第一电性为负电,在本具体实施例中,第一高分子选自聚谷氨酸,其具体的制备方式取适当重量的聚谷氨酸粉末加入去离子水后,以电磁搅拌器搅拌至完全溶解;接着,将完全溶解的聚谷氨酸溶液装至透析膜中,置于去离子水中透析,使得聚谷氨酸中的钠离子被透析至去离子水中并随着去离子水被移除,其透析过程将去离子水及聚谷氨酸溶液保存于4℃冰箱中以避免聚谷氨酸长菌;然后,将透析完成的聚谷氨酸溶液置于-20℃冰箱中,待其完全结成冰晶以利进行冷冻干燥;接着,将结为冰晶的聚谷氨酸放入冷冻干燥机中进行冷冻干燥,以将溶液中的水份完全去除,此时聚谷氨酸呈结晶粉末状;再将此聚谷氨酸的结晶粉末保存于灭过菌的离心管中,并储藏于防潮箱中;最后,取出适当重量的聚谷氨酸结晶粉末加入去离子水,使聚谷氨酸溶液浓度达到所需的设定值,即完成一可生物分解且带负电的第一高分子溶液;然而必需注意的是,应了解上述第一高分子溶液的制备方法是为了方便说明一具体实施例,而非以本例所举为限,在阅读及了解本发明的教导后,相关的变化实施属于业界技能,因此,并不限定上述实施例第一高分子溶液的制备方式;
步骤二:将与第一电性同性的登革病毒蛋白溶于第一高分子溶液中,以形成一具负电的混合溶液;其中,登革病毒蛋白是以本发明人先前的中国台湾发明专利第201210614公开号所揭露的“登革疫苗、包含该登革疫苗的医药组合物、以及核苷酸序列”的登革病毒蛋白,且其剂量可例如为100、200、400微克,在此并不限定;
步骤三:准备一可生物分解的第二高分子溶液,其中第二高分子带有第二电性,且第一电性与第二电性为电荷相反;而第二高分子可例如为几丁聚糖(chitosan)或胶原蛋白(collagen)中的一种,且第二电性为正电;于本具体实施例中,第二高分子选自几丁聚糖;其具体的制备方式秤取5g的低黏性(low-viscous)几丁聚糖,并加入495ml的去离子水以及5ml的冰醋酸(Acetic acid glacial),以电磁加热搅拌器搅拌直至几丁聚糖溶液呈现黄色澄清透明的状态;接着,将上述溶液装至透析膜中,并置于去离子水中,以将几丁聚糖溶液中的冰醋酸透析至去离子水中,使得几丁聚糖溶液在透析后的pH值约在6.5左右;然后,将透析完成的几丁聚糖溶液以抽气过滤装置将其中的杂质滤掉;最后,置于加热搅拌器上,温度设置为135℃,以磁搅拌子搅拌直至几丁聚糖溶液浓度达到20~30mg/ml,即完成一可生物分解且带正电的第二高分子溶液;同样地,应了解上述第二高分子溶液的制备方法是为了方便说明一具体实施例,在阅读及了解本发明的教导后,相关的变化实施属于业界技能,在此并不限定上述实施例第二高分子溶液的制备方式;值得注意的,上述的第一、二高分子可例如分别为肝素或聚谷氨酸,以及几丁聚糖或胶原蛋白等天然高分子,也可为人工合成的可生物分解高分子;以及
步骤四:将混合溶液加入第二高分子溶液中,通过电荷异性相吸的作用力而形成一纳米复合物结构,且纳米复合物结构包覆登革病毒蛋白;其结果请参阅图1,为本发明包覆登革病毒蛋白后的纳米复合物结构的电子显微镜影像;值得注意的,上述带负电的登革病毒蛋白先与同样带负电的聚谷氨酸溶液混合后,再将混合液与带正电的几丁聚糖溶液混合以得到具纳米复合物结构溶液,其中的纳米复合物结构可呈现较佳的稳定性,但也可将带负电的登革病毒蛋白先与带正电的几丁聚糖溶液混合后,再将混合液与带负电的聚谷氨酸溶液混合;而由于第一、二高分子皆具可生物分解性,因此以第一、二高分子混合形成的纳米复合物结构也具有生物可分解性,其中生物可分解性是指该结构进入人体后可以容易、自然的被人体分解、吸收与代谢并排出体外,也因此被包裹于纳米复合物结构内的登革病毒蛋白可缓慢释放,进而达到持续、长效释放的功效;请参阅下表所示,为本实际实施例未包覆以及已包覆登革病毒蛋白的纳米复合物结构其粒径与接口电荷(Zeta potential)的分布比较表;其中,带正电的几丁聚糖溶液与带负电的聚谷氨酸溶液其电荷比较佳为4:1,因其所合成出来的纳米复合物结构可呈现最为稳定的状态,且下表1所述的实验数据的平均值数据是取3颗纳米复合物结构所得。
表1
未包覆 | 已包覆 | |
平均粒径(nm) | 126.4±5.1 | 124.5±1.8 |
界面电荷(mV) | 83.5±6.2 | 15.4±0.7 |
接者,使用包裹登革病毒蛋白的纳米复合物作为模式分子(model compound)接种小鼠,其是由自美国杰克森实验室(The Jackson Laboratory)取得C3H∕HeN鼠,并于成功大学医学院的实验动物中心以标准饲料及水饲养,动物实验是取用该鼠的八周大的后代来进行,而关于实验动物的居住环境、饲养、以及实验条件皆受到成功大学医学院的实验动物中心严密的管控;请参阅下表2所示,是本发明包覆登革病毒蛋白后的纳米复合物与现有的铝盐佐剂(Alum)以及Ribi佐剂产生具中和性抗体的能力比较表,由表中可看出在两次接种后,本发明具纳米复合物的登革疫苗即可引起小鼠抗登革蛋白的特异性抗体反应,且每次接种的纳米复合物结构含量仅为25微克(μg),即可具有256000倍的抗体效价,使得本发明可生物分解的高效率登革疫苗可减少所需接种疫苗的次数,更有利成为具商业等级质量的混合疫苗,表中的ND表示无法侦测(non-detectable);其中上述的抗体效价可通过例如诸如ELISA的标准方法来适当地量测;再者,所引起抗体反应的速度明显快于传统的铝盐佐剂以及Ribi佐剂,上述两佐剂皆必须于第三次接种才能引起小鼠抗登革蛋白的特异性抗体反应;此外,本发明纳米复合物的登革疫苗在第三次接种后,所产生的抗体数量也优于上述两者;这样的结果暗示着包裹登革病毒蛋白的纳米复合物对于登革病毒疫苗展现了增强的佐剂效果;其中Ribi佐剂是由Ribi ImmunochemResearch Inc.于1985年开发成功一种无毒性,无免疫性,属于Ribi adjuvant system(RAS)的油包水乳化佐剂;值得注意的,于第一高分子选自肝素而第二高分子选自几丁聚糖所形成的纳米复合物的登革疫苗另一实施例中,也可于第二次接种即引起小鼠抗登革蛋白的特异性抗体反应,且每次接种的纳米复合物结构含量仅为25微克(μg),且于第二次接种后具有32000倍的抗体效价。
表2
此外,可预见上述纳米复合物的登革疫苗将可被包含为一种医药组合物,是用于制造治疗或预防出血性登革热(dengue hemorrhagic fever)或登革休克症候群(dengue shocksyndrome)疾病的疫苗或药剂,而该医药组合物是可生物分解的高效率登革疫苗或其与医药学上可接受的碱形成的加成盐以及一或多种医药学上可接受的赋形剂;再者,该医药组合物可由已知途径投予动物,包括经口、经鼻、经黏膜、局部、透皮、和非经肠(如,静脉内、腹膜内、皮内、皮下或肌肉内)等途径,也可以使用途径组合来达到接种,例如使用非经肠途径第一次接种及随后使用黏膜途径接种;此外,该医药组合物所需的精确用量是随着个体不同,依据个体的物种、年龄、体重及一般状态,被治疗或预防的疾病、感染或病况的严重性,所使用的特定化合物及它的给予形式等而有所变化,本领域技术人员得知本说明书的公开的内容后,可仅仅利用例行实验而决定适当的用量,在初次给予后,个体可再接受一或多次适当间隔的追加免疫。
由上述的实施说明可知,本发明与现有技术相较之下,本发明具有以下优点:
1、本发明具纳米复合物的登革疫苗在两次接种后,即可引起小鼠抗登革蛋白的特异性抗体反应,相对于现有的用于登革疫苗的铝盐佐剂以及Ribi佐剂,本发明的高效率登革疫苗可减少所需接种疫苗的次数,进而降低免疫所需费用,更有利成为具商业等级质量的混合疫苗。
2、本发明的登革疫苗于第二次接种后即能具有32000倍的抗体效价,与现有技术相比较,大幅增加抗体生成量,使疫苗能更有效的引起免疫反应,以达成增加疫苗保护效力的功效。
3、本发明具纳米复合物的登革疫苗是以具可生物分解性的聚谷氨酸(或肝素)与几丁聚糖混合来包裹登革病毒蛋白,使得登革疫苗进入人体后可以容易、自然的被人体分解、吸收与代谢并排出体外,不仅可解决现有的使用铝佐剂所引起的重金属安全性问题,也使得被包裹于纳米复合物结构内的登革病毒蛋白可缓慢释放,进而达到持续、长效释放的功效。
Claims (14)
1.一种可生物分解的高效率登革疫苗,其特征在于,所述的可分解的高效率登革疫苗包括一具生物分解性且带电性的纳米复合物结构,在该纳米复合物结构中包含一登革病毒蛋白,使该纳米复合物结构在两次接种后,即具有抗体表达量。
2.根据权利要求1所述的可生物分解的高效率登革疫苗,其中一次接种的纳米复合物结构含量不超过25微克。
3.根据权利要求1所述的可生物分解的高效率登革疫苗,其中该纳米复合物结构在第2次接种,即具有至少32000倍的抗体效价。
4.根据权利要求3所述的可生物分解的高效率登革疫苗,其中该纳米复合物结构于第2次接种,即具有至少256000倍的抗体效价。
5.根据权利要求1所述的可生物分解的高效率登革疫苗,其中该登革病毒蛋白包括一非结构性嵌合蛋白DJ NS1,该非结构性嵌合蛋白DJ NS1包括:一登革病毒非结构性蛋白1(DV NS1)的N端序列1-270;以及一日本脑炎病毒非结构性蛋白1(JEV NS1)的C端序列271-352。
6.根据权利要求5所述的可生物分解的高效率登革疫苗,其中该非结构性嵌合蛋白DJ NS1的氨基酸序列与SEQ.ID.NO.1的序列相似度为90%以上。
7.根据权利要求1所述的可生物分解的高效率登革疫苗,其中该纳米复合物结构以带正电的几丁聚糖与带负电的肝素或聚谷氨酸混合所形成。
8.一种制备可生物分解的高效率登革疫苗的方法,其特征在于,所述的制备可分解的高效率登革疫苗的方法包括如下步骤:
步骤一:准备一可生物分解的第一高分子溶液,其中,该第一高分子带有第一电性;
步骤二:将与该第一电性同性的登革病毒蛋白溶于第一高分子溶液中,以形成一具第一电性的混合溶液;
步骤三:准备一可生物分解的第二高分子溶液,其中,该第二高分子带有第二电性,该第二电性与前述的第一电性的电荷相反;以及
步骤四:将该混合溶液加入该第二高分子溶液中,通过电荷异性相吸的作用力而形成一纳米复合物结构,且该纳米复合物结构包覆该登革病毒蛋白。
9.根据权利要求8所述的制备可生物分解的高效率登革疫苗的方法,其中该第一高分子为肝素或聚谷氨酸中的一种,且该第一电性为负电。
10.根据权利要求8所述的制备可生物分解的高效率登革疫苗的方法,其中该第二高分子为几丁聚糖或胶原蛋白中的一种,且该第二电性为正电。
11.根据权利要求8所述的制备可生物分解的高效率登革疫苗的方法,其中该登革病毒蛋白包括一非结构性嵌合蛋白DJ NS1,该非结构性嵌合蛋白DJ NS1包括:一登革病毒非结构性蛋白1(DV NS1)的N端序列1-270;以及一日本脑炎病毒非结构性蛋白1(JEV NS1)的C端序列271-352。
12.根据权利要求11所述的制备可生物分解的高效率登革疫苗的方法,其中该非结构性嵌合蛋白DJ NS1的氨基酸序列与SEQ.ID.NO.1的序列相似度为90%以上。
13.一种包含可生物分解的高效率登革疫苗的医药组合物,其包含至少一种如权利要求1至7中任一项所述的可生物分解的高效率登革疫苗或其与医药学上可接受的碱形成的加成盐以及一或多种医药学上可接受的赋形剂。
14.根据权利要求13所述的包含可生物分解的高效率登革疫苗的医药组合物,其是用于制造治疗或预防出血性登革热或登革休克症候群疾病的疫苗或药剂。
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