CN113939313A - 以肽核酸为基础的佐剂 - Google Patents
以肽核酸为基础的佐剂 Download PDFInfo
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- CN113939313A CN113939313A CN202080041821.3A CN202080041821A CN113939313A CN 113939313 A CN113939313 A CN 113939313A CN 202080041821 A CN202080041821 A CN 202080041821A CN 113939313 A CN113939313 A CN 113939313A
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Abstract
本发明提供一种对有效性高且安全性高的疫苗的制备有用的佐剂和包含该佐剂的疫苗组合物。本发明的佐剂由结合有细胞膜穿透性肽的肽核酸构成。
Description
技术领域
本发明涉及一种具有免疫活化能力的对细胞膜穿透性肽进行修饰而得的肽核酸。
背景技术
后天免疫系统具有针对经历过一次的异物形成在第2次以后的曝露中能够迅速应对的免疫记忆的能力。利用该免疫记忆的是疫苗，通过接种疫苗，能够在病原体曝露之前得到能够中和该病原体的免疫。
已获批准的疫苗有减毒活疫苗、灭活疫苗和纯化重组蛋白质等亚单位疫苗，特别是减毒活疫苗是有效性高的疫苗，但由于具有感染性，因此难以确保安全性，有产生未预期的副反应的顾虑。另一方面，灭活疫苗、亚单位疫苗是与减毒活疫苗相比在安全性方面优异的疫苗，但由于丧失感染性，因此缺乏引起自然免疫的能力，因此仅抗原的情况下无法赋予充分的后天免疫。因此，已批准的灭活疫苗和亚单位疫苗多为向抗原加入具有免疫活化能力的佐剂而成的制剂。因此，在安全性高的疫苗的开发中，应当使用安全性高的灭活疫苗、亚单位疫苗抗原，但为了获得充分的有效性，需要添加具有免疫活化能力的佐剂。
另外，为了提高病原体的感染防御效果，成为病原体的入侵门户的粘膜中的免疫诱导也较为重要。粘膜中的免疫诱导通过给药粘膜疫苗而实现，已批准的粘膜疫苗为减毒活疫苗、来自具有粘膜亲和性的毒素的疫苗，不具有粘膜亲和性的灭活疫苗和亚单位疫苗难以引起对感染防御而言充分的免疫。因此，这样的抗原的粘膜给药中也需要增强粘膜免疫的佐剂。
已批准的疫苗中使用的佐剂可举出铝盐或基于角鲨烯的乳液，但被批准的佐剂较少，已知铝盐会促进IgE诱导(非专利文献1)。另外，上述佐剂为注射剂用佐剂，难以与粘膜疫苗组合。
另一方面，作为将核酸的糖磷酸骨架转化为N-(2-氨基乙基)甘氨酸骨架的修饰核酸的肽核酸(PNA)具有较高的双链形成能力，不会被生物体内核酸酶分解，因此可期待应用为反义分子等。
另外，在以药物递送为代表的领域中，作为将蛋白质、肽或低分子化合物导入至细胞内使其发挥功能的技术，在这些分子上结合穿膜肽(Cell Penetrating Peptide，CPP)。
但是，将PNA、结合有CPP的PNA用作佐剂尚属未知。
现有技术文献
非专利文献
[非专利文献1]Marichal T,Ohata K,Bedoret D,Mesnil C,Sabatel C,KobiyamaK,Lekeux P,Coban C,Akira S,Ishii KJ,Bureau F,Desmet CJ.DNAreleased from dyinghost cells mediates aluminum adjuvant activity.Nat Med.2011Jul 17；17(8):996-1002.
发明内容
本发明涉及一种对有效性高且安全性高的疫苗的制备有用的佐剂和包含该佐剂的疫苗组合物。
本申请发明人等进行了深入研究，结果发现在肽核酸修饰HIV(HumanImmunodeficiency Virus，人类免疫缺陷病毒)的TAT(Trans-Activator ofTranscription Protein，转录反式激活蛋白)的细胞膜穿透性肽(CPP)，该结合有CPP的肽核酸在注射和经粘膜给药中均具有佐剂活性。
即，本发明涉及以下的1)～6)。
1)一种佐剂，由结合有细胞膜穿透性肽的肽核酸构成。
2)一种佐剂组合物，含有结合有细胞膜穿透性肽的肽核酸和药学上允许的载体。
3)一种疫苗组合物，其含有如1)的佐剂和抗原。
4)一种结合有细胞膜穿透性肽的肽核酸的用途，用于制造佐剂。
5)一种结合有细胞膜穿透性肽的肽核酸的作为佐剂的用途。
6)一种方法，使用结合有细胞膜穿透性肽的肽核酸作为佐剂。
根据本发明，能够提高安全性高的灭活疫苗的有效性，能够创造出品质高的预防药而对医药品产业做出较大贡献。
附图说明
图1A是皮下给药中的对A/Singapore/GP1908/2015株的IgG抗体效价。
图1B是皮下给药中的对B/Maryland/15/2015株的IgG抗体效价。
图2是皮下给药中的第35天时的对A/Singapore/GP1908/2015株的中和抗体效价。
图3A是经鼻给药中的对A/Singapore/GP1908/2015株的IgG抗体效价。
图3B是经鼻给药中的对B/Maryland/15/2015株的IgG抗体效价。
图4A是经鼻给药中的鼻腔清洗液中的对A/Singapore/GP1908/2015株的IgA抗体效价。
图4B是经鼻给药中的鼻腔清洗液中的对B/Maryland/15/2015株的IgA抗体效价。
图5是MIP03的NFκB的活化作用。
图6是MIP03的细胞因子(IL-1α)诱导作用。
图7是MIP03的细胞因子(IL-1β)诱导作用。
图8是MIP03的细胞因子(IL-13)诱导作用。
图9是MIP03的细胞因子(IFN-γ)诱导作用。
具体实施方式
以下详细地说明本发明的优选的实施方式。但是，本发明并不限定于以下的实施方式。
在本发明中，“结合有细胞膜穿透性肽的肽核酸”是指在细胞膜穿透性肽的N或C末端结合有肽核酸的分子，在本说明书中，也称为Mitsumata免疫刺激肽核酸(Mitsumata-Immunostimulatory Peptide nucleic acid，MIP)。
“细胞膜穿透性肽(CPP)”是指在与细胞共存的情况下通过细胞膜而转移至细胞内的肽。例如可举出表1所示的来自HIV的TAT的肽、来自果蝇的触角足的黑腹果蝇触足肽(Penetratin)、聚精氨酸、由神经肽甘丙肽衍生的转运肽(Transportan)、来自SV40的核定位信号(Nuclear Localization Signal，NLS)的Pep-1、抗菌肽LL37，但并不限定于它们。
优选可举出来自TAT的肽(由序列号1所示的氨基酸序列构成的肽)。
[表1]
“肽核酸(PNA)”是指具有将核酸的糖-磷酸骨架置换成N-(2-氨基乙基)甘氨酸的骨架，通过亚甲基羰基键键合碱基而成的核酸类似物。作为本发明中使用的肽核酸中的碱基(Base)，可以为腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶和尿嘧啶中的任一者，链长理想为通常的20mer以下，从佐剂活性的方面出发，优选为3～12mer，更优选为3～10mer。
下述式(1)示出3～12mer的肽核酸的结构。
[式中，碱基可以相同或不同，表示腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶或尿嘧啶，R表示-COOH或-CONH2，n表示1～10的整数]
在本发明中，作为优选的肽核酸，例如可举出具有至少1个鸟嘌呤或胞嘧啶作为碱基的3mer或10mer的肽核酸、具有3个鸟嘌呤作为碱基的3mer的肽核酸(G3PNA)、具有3个胞嘧啶作为碱基的3mer的肽核酸(C3PNA)、具有10个胞嘧啶作为碱基的10mer的肽核酸(C10PNA)等。
本发明的MIP可通过在细胞膜穿透性肽的N或C末端利用化学方法、例如介由两末端具有N-羟基丁二酰亚胺酯的交联剂使细胞膜穿透性肽与肽核酸的N末端间共价键合而制造。
应予说明，肽核酸例如可使用Fmoc型PNA单体单元等，利用该技术领域中公知的固相肽合成法进行合成。
作为本发明中使用的优选的MIP，可举出以下所示的来自HIV的TAT的由13个氨基酸构成的肽(序列号1)在其C末端与G3PNA共价键合而成的MIP01(式(2))、与C10PNA共价键合而成的MIP03(式(3))。
如后述实施例所示，在向小鼠注射和经粘膜给药将式(2)所示的MIP01与流感抗原(分裂抗原(split antigen))混合而制备的疫苗组合物的情况下，对该抗原的血中抗体效价高于MIP非添加组。另外，在经粘膜给药的情况下，粘膜的IgA效价也上升。另外，式(3)所示的MIP03中确认到NFκB的活化和IL-1β的较高的诱导能力，因此认为促进自然免疫系统的活化的可能性高，MIP为具有自然免疫的活化能力的佐剂。
即，MIP在注射或经粘膜给药抗原的情况下均具有提高血中或粘膜中的抗体诱导能力的佐剂活性，因此，MIP成为佐剂，另外，MIP包含药学上允许的载体的组合物可成为佐剂组合物。另外，MIP可用于制造佐剂或佐剂组合物。
在本发明中，“佐剂”是指在注射或经粘膜给药抗原的情况下，增加对抗原的免疫应答的物质。
这里，“粘膜”是指脊椎动物中的消化器官、呼吸道、泌尿生殖器官、眼等特别是外通性的内腔器官的内壁。因此，作为经粘膜给药，例如可举出口服给药、鼻腔给药(经鼻给药)、口腔给药、阴道内给药、上呼吸道给药、肺泡给药、滴眼给药等，但并不限定于这些。
本发明的佐剂或佐剂组合物可以与抗原组合给药，给药可以与抗原给药同时进行，也可以为抗原给药之前或之后。
本发明的佐剂或佐剂组合物的给药量可根据给药对象、给药方法、给药形态、抗原种类而适当地决定。
本发明的佐剂可以与抗原组合而制成疫苗组合物。本发明的疫苗组合物可通过将抗原和MIP混合而制备，也可以进一步适当地添加药学上允许的载体而制成适当的制剂。在本发明的疫苗组合物中，MIP可以处于与抗原或其它成分化学性结合的状态，也可以以游离的分子状态存在。
作为抗原，适合的是由病原体纯化而得的天然产物或者通过基因重组等方法人为制作的蛋白质或肽，具体而言，可举出作为完全病毒粒子的病毒体、不完全病毒粒子、病毒结构蛋白、病毒非结构蛋白、病原菌的全菌体、来自病原菌的蛋白质或糖蛋白、感染防御抗原、中和表位等，包含具有感染性的抗原和丧失感染性的抗原(灭活抗原)。作为灭活抗原，例如可举出通过物理(X射线照射、UV照射、热、超声波)、化学(福尔马林、β-丙内酯、双乙烯亚胺、汞、醇、氯)等操作而灭活的抗原，但并不限定于这些。从安全性的观点考虑，来自病原体的抗原理想为来自上述病毒或病原菌的灭活抗原。
作为病毒，例如可举出麻疹病毒、风疹病毒、腮腺炎病毒、脊髓灰白质炎病毒、轮状病毒、诺罗病毒、腺病毒、肠病毒、疱疹病毒、水痘病毒、严重急性呼吸道感染综合征(SARS)病毒、人类免疫缺陷病毒(HIV)、人类T细胞白血病病毒(HTLV-1)、人乳头状瘤病毒、日本脑炎病毒、西尼罗河病毒、黄热病毒、登革热病毒、齐卡病毒、甲型肝炎病毒、乙型肝炎病毒、丙型肝炎病毒、戊型肝炎病毒、RS病毒、流感病毒等，优选为流感病毒。
作为病原菌，可举出白喉杆菌、破伤风杆菌、百日咳杆菌、脑膜炎球菌、乙型流感菌、肺炎球菌、霍乱弧菌、结核菌、牙周病菌等。
作为疫苗组合物的剂型，例如可举出液剂、悬浮剂、冷冻干燥制剂等。作为液剂，可举出溶解于纯化水、缓冲液而成的液型等。作为悬浮剂，可举出与甲基纤维素、羟甲基纤维素、聚乙烯基吡咯烷酮、明胶、酪蛋白等一起悬浮于纯化水、缓冲液等而成的悬浮剂等。
可以根据需要向这些制剂添加通常使用的吸收促进剂、表面活性剂、保存剂、稳定剂、防湿剂、增溶剂等。
应予说明，本发明的疫苗组合物只要不损害该疫苗的免疫原性和安全性，则也可以含有MIP以外的佐剂。
本发明的疫苗组合物中所含的抗原的量只要为足以诱导特异性抗体应答的量就没有特别限定，也可考虑与所并用的MIP的比率而适当地设定。例如，在使用流感病毒的分裂抗原作为抗原的情况下，只要在1次的给药用量即1～60μgHA(HA换算)的范围内含有即可，更优选为9～15μgHA(HA换算)。上述浓度为通过利用一元放射免疫扩散试验法等按照WHO或国家的基准所确定的试验法测定HA蛋白质的浓度而得到的值。
本发明的疫苗组合物的给药路径没有特别限定，可以为利用注射的给药(皮下给药、肌肉内给药、皮内给药、静脉内给药)、口服给药、非口服给药(例如，鼻腔给药、滴眼给药、经阴道给药、舌下给药、经皮给药)中的任一者，例如通过对鼻腔内进行滴加、喷雾或喷射而进行给药。
本发明的佐剂组合物或疫苗组合物的给药对象可举出人和除人以外的哺乳动物，优选为人。作为除人以外的哺乳动物，例如可举出小鼠、大鼠、仓鼠、豚鼠、兔、猪、牛、山羊、绵羊、狗、猫、恒河猴、食蟹猴、猩猩、黑猩猩等。
实施例
以下，通过实施例具体地说明本发明，但本发明并不受它们任何限定。
＜CPP和MIP＞
委托肽研究所株式会社合成作为CPP的CPP01(来自TAT的肽(序列号1))、作为MIP的上述式(2)所示的MIP01和式(3)所示的MIP03。
实施例1MIP01的皮下给药时的佐剂活性评价
将流感HA疫苗“生研”的A/H1N1亚型(A/Singapore/GP1908/2015株)和B/维多利亚系统(Victoria lineage)(B/Maryland/15/2016株)的各原液作为分裂抗原(SV)，以每0.3mL中各株的血凝集素成为1μg的方式混合各分裂抗原，以成为1μg的方式向其中添加来自HIV的TAT的由13个氨基酸构成的细胞膜穿透性肽或者MIP01。另外，作为对照，也一并制备不添加佐剂的仅有抗原的给药液。
将如上制备的给药液(表2)以3周为间隔向BALB/c小鼠(雌，5周龄)的后背部皮下给药2次，每次0.3mL(各组5只)，从各给药起2周后进行全采血。将通过全采血而得到的血液进行离心分离，制备血清，测定血清的与A/Singapore/GP1908/2015株和B/Maryland/15/2016株特异性结合的IgG(总IgG)效价。另外，在2次给药后的血清中，也测定对A/Singapore/GP1908/2015株的中和抗体效价。
[表2]
各给药组的IgG效价如图1A和图1B所示。每次给药对分裂抗原添加1μg的MIP01，由此在1次和2次给药中，对各株的血中抗原特异性的IgG效价均高于佐剂非给药组。另外，与CPP01给药组相比，也确认到在MIP01给药组中，在1次和2次给药的两个时间点，对于任一株，血中抗原特异性的IgG效价均较高。在图2中，示出2次给药后的对A/Singapore/GP1908/2015株的中和抗体效价，但中和抗体效价的结果也为MIP01给药组最高，接着为佐剂非添加组，显示最低值的是CPP01给药组。因此确认到，通过给药MIP01，不仅血中抗原的特异性IgG效价，对病毒的排除较为重要的中和抗体效价也提高，认为使肽核酸与细胞膜穿透性肽结合而得的结构对于发挥MIP01的佐剂活性较为重要。
实施例2 MIP01的经鼻给药时的佐剂活性评价
与实施例1同样地将流感HA疫苗“生研”的A/H1N1亚型(A/Singapore/GP1908/2015株)和B/维多利亚系统(B/Maryland/15/2016株)的各原液作为分裂抗原，以每10μL中各株的血凝集素成为1μg的方式混合各分裂抗原，以成为10μg的方式向其中添加CPP01或者MIP01。另外，作为对照，也一并制备不添加佐剂的仅有抗原的给药液。
将如上制备的给药液(表3)以3周为间隔向BALB/c小鼠(雌，5周龄)的鼻腔给药2次，每10μL(一只鼻5μL)(各组5只)。从各给药起2周后进行全采血，通过离心分离制备血清。另外，在进行2次给药2周后的采血后，每个个体利用400μL的含蛋白酶抑制剂的D-PBS清洗鼻腔内，回收该清洗液作为鼻腔清洗液。将血清供于与A/Singapore/GP1908/2015株和B/Maryland/15/2016株特异性结合的IgG(总IgG)效价的测定，将鼻腔清洗液供于与A/Singapore/GP1908/2015株和B/Maryland/15/2016株特异性结合的IgA效价的测定。
[表3]
各株的IgG效价的结果如图3A和图3B所示，在经鼻给药时，也为在1次和2次给药的任一者中对各株的血中抗原特异性的IgG效价均高于佐剂非给药组。特别是对B/Maryland/15/2016株的IgG效价相对于佐剂非添加组而言确认到显著的抗体效价的上升(曼-惠特尼的U检定，p＜0.05)。另外，关于鼻腔清洗液的抗原特异性IgA效价，在任一给药时间点对各株的IgA效价均在MIP01给药组中成为最高值，接着为佐剂非添加组，最低值的是CPP01给药组(图4A、图4B)。因此，认为在肽核酸修饰细胞膜穿透性肽而成的MIP不仅在利用注射的给药中，在经粘膜给药中也具有佐剂活性，在经粘膜给药中，除促进血中的IgG以外，也促进粘膜中的IgA诱导。
实施例3 MIP03的自然免疫活化能力
在体外对MIP03的自然免疫活化能力进行评价。
以最终浓度成为10、20、40和80μM的方式对1×106cells/mL的Raw264.7报告细胞株(Reporter Cell Line)(Novus biologicals，NBP2-26261)添加MIP03，在5％CO2、37℃下培养2天后，回收培养液。将所回收的培养液以12000×g离心分离1分钟，测定其上清液的碱性磷酸酶和细胞因子。碱性磷酸酶的测定使用SEAP Reporter Gene Assay kitLuminescence(Cayman Chemical，600260)，细胞因子的测定使用Bio-Plex Pro小鼠细胞因子GI23-Plex(Bio-Rad，M60009RDPDB03)。另外，作为阴性对照(NC),添加培养基，在细胞因子测定中，作为阳性对照，添加作为Toll样受体7的激动剂的咪喹莫特(0.02μM)，与它们进行比较评价。
碱性磷酸酶的测定结果如图5所示，确认到依赖于MIP03的添加浓度的碱性磷酸酶的发光信号提高。因此可知MIP03促进NFκB的活化，表明使自然免疫活化。另外，图6～9示出细胞因子测定的结果，确认到依赖于MIP03的浓度，IL-1α和IL-1β诱导提高(图6和7)，特别是IL-1β与咪喹莫特相比，确认到非常高的诱导(图7)。另外，作为Th2和Th1相关细胞因子，将IL-13的测定结果示于图8，将IFN-γ的测定结果示于图9，任一细胞因子均确认到与作为阳性对照的咪喹莫特相同程度的诱导。
如上所述，由于具有NFκB的活化和IL-1β的高诱导能力，因此认为MIP03促进经由C型凝集素受体、Nod样受体的自然免疫系统的活化的可能性较高，其结果表现出与抗体应答相关的Th1/Th2细胞因子的诱导能力。因此，MIP为具有自然免疫的活化能力的佐剂。
序列表
<110> 电化株式会社
<120> 以肽核酸为基础的佐剂
<130> DK0008
<150> 2019-106444
<151> 2019-06-06
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<213> 人类免疫缺陷病毒
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Claims (12)
1.一种佐剂，由结合有细胞膜穿透性肽的肽核酸构成。
2.根据权利要求1所述的佐剂，其中，细胞膜穿透性肽选自由序列号1～7所示的氨基酸序列构成的肽。
3.根据权利要求1所述的佐剂，其中，细胞膜穿透性肽为由序列号1所示的氨基酸序列构成的肽。
4.根据权利要求1～3中任一项所述的佐剂，其中，肽核酸由下述式(1)表示，
式中，碱基可以相同或不同，表示腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶或尿嘧啶，R表示-COOH或-CONH2，n表示1～10的整数。
5.根据权利要求4所述的佐剂，其中，式(1)中，n为1～8的整数。
6.根据权利要求4或5所述的佐剂，其中，式(1)中，R为-CONH2。
7.根据权利要求4～6中任一项所述的佐剂，其中，式(1)中，碱基为鸟嘌呤或胞嘧啶。
8.一种佐剂组合物，含有结合有细胞膜穿透性肽的肽核酸和药学上允许的载体。
9.一种疫苗组合物，含有权利要求1～7中任一项所述的佐剂和抗原。
10.一种结合有细胞膜穿透性肽的肽核酸的用途，用于制造佐剂。
11.一种结合有细胞膜穿透性肽的肽核酸的作为佐剂的用途。
12.一种方法，使用结合有细胞膜穿透性肽的肽核酸作为佐剂。
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| US12016920B2 (en) | 2024-06-25 |
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| JPWO2020246584A1 (zh) | 2020-12-10 |
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