CN104338126B - Having a vaccine composition and treating or preventing viral hpv - Google Patents

Having a vaccine composition and treating or preventing viral hpv Download PDF

Info

Publication number
CN104338126B
CN104338126B CN201410382617.4A CN201410382617A CN104338126B CN 104338126 B CN104338126 B CN 104338126B CN 201410382617 A CN201410382617 A CN 201410382617A CN 104338126 B CN104338126 B CN 104338126B
Authority
CN
China
Prior art keywords
microspheres
antigen
hpv
emulsion
plga
Prior art date
Application number
CN201410382617.4A
Other languages
Chinese (zh)
Other versions
CN104338126A (en
Inventor
王连艳
刘永江
贾成成
马光辉
陈健平
潘勇昭
银飞
王雅君
Original Assignee
中国科学院过程工程研究所
北京康乐卫士生物技术股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN201310343768 priority Critical
Application filed by 中国科学院过程工程研究所, 北京康乐卫士生物技术股份有限公司 filed Critical 中国科学院过程工程研究所
Priority to CN201410382617.4A priority patent/CN104338126B/en
Publication of CN104338126A publication Critical patent/CN104338126A/en
Application granted granted Critical
Publication of CN104338126B publication Critical patent/CN104338126B/en

Links

Abstract

本发明涉及疫苗组合物及其在药物中的用途。 The present invention relates to vaccine compositions and their use in medicine. 尤其涉及由包被HPV L1蛋白的纳微球制成的疫苗组合物或疫苗制剂及其在治疗或预防HPV病毒药物中的应用。 In particular it relates to a vaccine composition made of HPV L1 protein coated microspheres or nanofiltration vaccine formulation and its application in treating or preventing HPV viruses. 具体而言,本发明针对HPV L1抗原蛋白,纳微球粒径大小及其组合、纳微球材料、纳微球对抗原装载方式对其免疫应答的影响,设计制备粒径均一、不同粒径的PLGA、PLA,PLGA/PC纳微球,以其包埋或吸附HPV L1抗原蛋白,制备成疫苗组合物或疫苗制剂,研究其对体内免疫应答强度和水平的影响,开发高效的HPV L1蛋白疫苗组合物。 Specifically, the present invention is directed to antigenic HPV L1 proteins, nano particle size microspheres, and combinations thereof, microspheres nano material, affect the loading nano microspheres for antigen on the immune response in a manner designed Preparation of Uniform, different particle sizes the PLGA, PLA, PLGA / PC nano microspheres, entrapping or its adsorbed HPV L1 protein antigen, or to prepare a vaccine formulation a vaccine composition, study their effects on the intensity and level of immune response, the development of efficient HPV L1 protein vaccine composition.

Description

一种具有治疗或预防HPV病毒的疫苗组合物及其应用 Having a vaccine composition and treating or preventing HPV virus

技术领域 FIELD

[0001]本发明涉及疫苗组合物及其在药物中的用途。 [0001] The present invention relates to vaccine compositions and their use in medicine. 尤其涉及由包被或吸附HPV Ll蛋白的纳微球制成的疫苗组合物或疫苗制剂及其在治疗或预防HPV病毒药物中的应用。 In particular it relates to a vaccine composition or vaccine formulation made from coated or adsorbed HPV Ll protein microspheres satisfied its application in treating or preventing HPV viruses. 技术背景 technical background

[0002] 人乳头瘤病毒(Human papillomaviruses,HPV)是无囊膜的双链DNA病毒,主要由病毒外壳和基因组DNA组成(BeraarcUBurk et al.2011) dHPV病毒外壳是由360个Ll蛋白(形成72个五聚体)和至多72个L2蛋白构成的二十面体结构,直径55-60nm (Howley and Lowy2007)。 [0002] Human Papillomavirus (Human papillomaviruses, HPV) are nonenveloped double-stranded DNA virus, the virus is mainly composed of a housing and consisting of genomic DNA (BeraarcUBurk et al.2011) dHPV viral coat protein is composed of 360 Ll (forming 72 ) and icosahedral up to 72 L2 proteins composed of pentamers, a diameter of 55-60nm (Howley and Lowy2007). 病毒外壳蛋白具有自组装特性,在体外LI蛋白单独或与L2蛋白共同自组装形成类病毒样颗粒(Virus-like Particle,VLP) (Chen,Garcea et al.2000,Finnen,Erickson et al.2003,Buck,Cheng et al.2008,Wang and Roden 2013)。 Viral coat proteins having self-assembling properties, in vitro LI protein alone or together with L2 proteins self-assemble into virus-like particles (Virus-like Particle, VLP) (Chen, Garcea et al.2000, Finnen, Erickson et al.2003, Buck, Cheng et al.2008, Wang and Roden 2013). 目前的HPV疫苗都是以VLP 作为革E抗原,已有两种基于HPV LlVLP预防性疫苗上市(Jansen and Shaw 2004,Howley and Lowy 2007?Buonaguro?Tomesello et al·2009,Harper 2009?Frazer?Leggatt et al.2011?Hariri ?Dunne et al·2011,Malagon,Drolet et al·2012,Lehtinen and Dillner 2013,Shaw 2013)。 The current HPV vaccines are based VLP antigen E as leather, it has been two HPV LlVLP preventive vaccine based on the market (Jansen and Shaw 2004, Howley and Lowy 2007? Buonaguro? Tomesello et al · 2009, Harper 2009? Frazer? Leggatt et al.2011? Hariri? Dunne et al · 2011, Malagon, Drolet et al · 2012, Lehtinen and Dillner 2013, Shaw 2013). 2006年6月8日,美国食品与药品管理局(FDA)正式批准美国Merck公司(即默沙东公司)生产的Gardasil HPV预防性疫苗上市;它是由酿酒酵母表达并纯化的HPV16/18/6/11L1VLP四价宫颈癌预防性疫苗,以无定形羟基磷酸铝铝硫酸盐(amorphous aluminum Hydroxyphosphatesulfate,AAHS)为佐剂,被批准用于预防6〜26岁女孩和妇女HPV16、18、6、11型感染所引起的宫颈癌、癌前病变和生殖器疣,这是FDA通过的世界上第一个肿瘤疫苗(Villa,Costa et al.2005,Villa,Ault et al.2006,Bryan 2007, 0188〇11,¥;!_113 6七31.2007,6〇1(18七〇116 311(1¥11〇(3〇1〇2012)。随后英国葛兰素史克(631〇公司生产的商品名为Cervarix的HPV预防性疫苗也成功上市,它是由来源于昆虫表达系统的HPV16/18L1VLP二价宫颈癌预防性疫苗,采用AS04佐剂(氢氧化铝复合MPL) (Paavonen, Jenkins et al.2007 ?Garcon ?MoreI et al·2011,Kreimer,Gonzalez et al.2011? Szare June 8, 2006, the US Food and Drug Administration (FDA) formally approved by the US Merck Company (ie, Merck) produced Gardasil HPV preventive vaccine on the market; it is expressed by S. cerevisiae and purified HPV16 / 18/6 / 11L1VLP tetravalent cancer preventive vaccines, amorphous aluminum phosphate aluminum hydroxyphosphate sulfate (amorphous aluminum Hydroxyphosphatesulfate, AAHS) adjuvant, was approved for the prevention of 6~26 years for girls and women HPV16,18,6,11 infection caused by cervical cancer, precancerous lesions and genital warts, which is the world's first cancer vaccine by the FDA (Villa, Costa et al.2005, Villa, Ault et al.2006, Bryan 2007, 0188〇11, ¥ ;!. _ 1136 seven 31.2007,6〇1 (18 seven 〇116 311 (1 ¥ 11〇 (3〇1〇2012) followed by British GlaxoSmithKline (631〇 company under the trade name of HPV preventive vaccine Cervarix also successful listing, which is HPV16 / 18L1VLP prophylactic cancer vaccine derived from a divalent insect expression system using AS04 adjuvant (aluminum hydroxide composite MPL) (Paavonen, Jenkins et al.2007? Garcon? MoreI et al · 2011, Kreimer, Gonzalez et al.2011? Szare wski 2012)。临床实验证明,上述两种预防性疫苗均具有良好的耐受性,且无严重不良反应(Paavonen,Jenkins et a 1.2007 ? Rei singer? Block et al. 2007 ? Perez ? Lazcano-Ponce et al.2008,Verstraeten,Descamps et al.2008,Paavonen,Naud et al.2009, Garcon,Morel et al.2011?Herrero?ffachoIder et al.2011?Kreimer?Gonzalez et al·2011,Seemann and Dodet 2011,Goldstone and Vuocolo 2012?Lehtinen?Paavonen et al.2012,Szarewski,Poppe et al.2012)c3但这两种预防性疫苗价格昂贵,且需要低温保存,极大限制了在发展中国家和落后地区的使用,因此开发一种低成本的高效价HPV疫苗就显得尤为重要(Jansen and Shaw 2004,Buonaguro,Tornesello et al.2009,Campo and Roden 2010 ?Frazer ? Leggatt et al.2011?Hariri ?Dunne et al·2011,Lehtinen and Dillner 2013,Shaw 2013)。 wski 2012). Clinical experiments show that both prophylactic vaccines have well tolerated, and no serious adverse reactions (Paavonen, Jenkins et a 1.2007? Rei singer? Block et al. 2007? Perez? Lazcano-Ponce et al.2008, Verstraeten, Descamps et al.2008, Paavonen, Naud et al.2009, Garcon, Morel et ffachoIder et al.2011 al.2011? Herrero?? Kreimer? Gonzalez et al · 2011, Seemann and Dodet 2011, Goldstone and Vuocolo 2012? Lehtinen? Paavonen et al.2012, Szarewski, Poppe et al.2012) c3 but both preventive vaccine is expensive, and requires cold storage, which greatly limits the use in developing countries and backward areas, Therefore, the development of high-titer HPV vaccine cost is particularly important (Jansen and Shaw 2004, Buonaguro, Tornesello et al.2009, Campo and Roden 2010? Frazer? Leggatt et al.2011? Hariri? Dunne et al · 2011, Lehtinen and Dillner 2013, Shaw 2013). 研究表明,以HPV LI五聚体蛋白免疫实验动物后可以诱导产生保护性的免疫应答(Rose,White et al.l998,Yuan,Estes et al.2001,0hlschlager,0sen et al.2003,Senger,Schadlich et al.2010,Wu,Gersch et al.2011),而基于原核生物E coli的表达技术可以大大降低HPV疫苗的生产成本。 Studies have shown that, in order to induce the protein-immunized animals HPV LI pentamer protective immune response (Rose, White et al.l998, Yuan, Estes et al.2001,0hlschlager, 0sen et al.2003, Senger, Schadlich et al.2010, Wu, Gersch et al.2011), and E coli-based expression technology prokaryotes can greatly reduce the production cost HPV vaccine. 在中国发明专利02129070.9《乳头瘤病毒衣壳蛋白的原核制备和应用》中提供HPV 16L1五聚体的空间三维结构,及利用大肠杆菌大量表达和制备人乳头瘤病毒衣壳蛋白Ll的技术方法。 HPV 16L1 provides five three-dimensional spatial structure mer in Chinese patent 02129070.9 "Preparation and application of prokaryotic papillomavirus capsid proteins" in a large number of E. coli and expression and preparation of human papillomavirus capsid protein Ll of the art methods.

[0003] 铝佐剂是目前应用最为广泛的一类佐剂,有着80多年的应用历史和数百亿人次的使用记录,也是长期以来一度是被美国食品和药品监督管理局(FDA)批准用于人类疫苗的唯一佐剂(Glenny,Pope et al · 1926,Gupta and Siber 1995,Gupta 1998,Baylor,Egan et al.2002,Clements and Griffiths 2002,Lindblad 2004,Lindblad 2004)。 [0003] Alum is currently the most widely used class of adjuvants, has 80 years of application history and a record ten billion people use, but also has long once by the US Food and Drug Administration (FDA) approved the use the only human vaccine adjuvant (Glenny, Pope et al · 1926, Gupta and Siber 1995, Gupta 1998, Baylor, Egan et al.2002, Clements and Griffiths 2002, Lindblad 2004, Lindblad 2004). 但其在HPV LI蛋白抗原的应用中存在一些不足。 But there are some deficiencies in the application of HPV LI protein antigen. 错盐佐剂仅在注射部位形成库效应(;Gupta,Chang et al.l996,Hem 2002,Verdier,Burnett et al.2005,Hem and Hogenesch 2007,Noe, Green et al.2010),通过炎症反应吸引树突状细胞(DC) (Morefield,Sokolovska et al.2005,Kool,Soullie et al.2008,Sharp,Ruane et al.2009,Flach,Ng et al.2011, Ghimire,Benson et al.2012)、巨细胞CM(J)) (Hamilton,Byrne et al.2000,Jordan, Mills et al.2004,Rimaniol,Gras et al.2004,Rimaniol,Gras et al.2007)等抗原呈递细胞(antigen-presenting cell,APC),这些细胞通过吞作用摄取抗原并经NLRP3炎性小体激活免疫应答(De Gregorio,Tritto et al.2008,Dostert,Petrilli et al.2008, Eisenbarth ? Colegio et al·2008,Franchi and Nunez 2008,Kool,Petri 11i et al.2008,Li,Willingham et al.2008,Cassel,Joly et al.2009,Demento,Eisenbarth et al.2009,Duewell,Kono et al.2010,Davis,Wen et al.2011)D同时,错佐剂通过刺激IL-10分泌等机制对细胞免疫 Complex salt is formed only adjuvant effect at the injection site library (; Gupta, Chang et al.l996, Hem 2002, Verdier, Burnett et al.2005, Hem and Hogenesch 2007, Noe, Green et al.2010), attracted by inflammation dendritic cells (DC) (Morefield, Sokolovska et al.2005, Kool, Soullie et al.2008, Sharp, Ruane et al.2009, Flach, Ng et al.2011, Ghimire, Benson et al.2012), giant cells CM (J)) (Hamilton, Byrne et al.2000, Jordan, Mills et al.2004, Rimaniol, Gras et al.2004, Rimaniol, Gras et al.2007) and other antigen presenting cells (antigen-presenting cell, APC ), these antigens uptake by endocytosis and by the NLRP3 inflammasome activation of the immune response (De Gregorio, Tritto et al.2008, Dostert, Petrilli et al.2008, Eisenbarth? Colegio et al · 2008, Franchi and Nunez 2008, kool, Petri 11i et al.2008, Li, Willingham et al.2008, Cassel, Joly et al.2009, Demento, Eisenbarth et al.2009, Duewell, Kono et al.2010, Davis, Wen et al.2011) D Meanwhile, error adjuvant by stimulating secretion of IL-10 on immune mechanisms 生抑制作用(Chen,Ni et al.2011),而对于病毒感染而言,细胞免疫效果将发挥更好的地免疫保护和免疫防治作用(O7Hagan and Valiante2003, Demento?Cui et al.2012?Levitz and Golenbock 2012?01ive 2012?Cain?Sanders et al.2013,HogenEsch 2013)。 Health inhibition (Chen, Ni et al.2011), while for viral infection, the immune cells will play a better effect of the immune protection and immune prevention and treatment (O7Hagan and Valiante2003, Demento? Cui et al.2012? Levitz and Golenbock 2012? 01ive 2012? Cain? Sanders et al.2013, HogenEsch 2013). 另外,铝佐剂可引起IgE介导的过敏反应(如注射部位肉芽肿) 以及神经系统不良反应等,从而引起人们对错佐剂安全性的担忧(Petrik,Wong et al.2007?Bystrianyk2009?Shaw and Petrik 2009?Munks?McKee et al.2010? Tomljenovic and Shaw 2011) 为此,需要针对HPV LI蛋白抗原,无论是对基于LI五聚体, 还是LlVLP的候选疫苗开发新型免疫佐剂(Campo and Roden 2010,Mariani and Venuti 2010,Chen,Ni et al.2011,Foged 2011,Gattoc,Nair et al.2013,Koff,Burton et al.2013,Shaw 2013,Tomljenovic,Spinosa et al.2013)〇 In addition, aluminum adjuvants can cause IgE-mediated allergic reactions (such as injection site granuloma) and adverse reactions of the nervous system, causing safety concerns wrong people adjuvant (Petrik, Wong et al.2007? Bystrianyk2009? Shaw and Petrik 2009? Munks? McKee et al.2010? Tomljenovic and Shaw 2011) for this reason, it is necessary for the HPV LI protein antigen, whether it is developing new immune adjuvant (Campo and Roden candidate vaccine based on LI pentamer, or the LlVLP 2010, Mariani and Venuti 2010, Chen, Ni et al.2011, Foged 2011, Gattoc, Nair et al.2013, Koff, Burton et al.2013, Shaw 2013, Tomljenovic, Spinosa et al.2013) billion

[0004] 近年来大量文献证实,体内可生物降解的聚合物纳微球通过包埋抗原,可将其转化为颗粒型抗原,有利于被抗原提呈细胞摄取,进而在胞内释放抗原,通过后续抗原加工、 提呈,增强免疫应答强度和水平(Langer,Clel and et al.1997,Johansen,Men et al·2000,Sahay,Alakhova et al.2010?De Temmerman,Rejman et al·2011,Danhier, Ansorena et al.2012) 例如,Torres MP等人制备了聚苷类微球,以卵清蛋白(OVA)为模型抗原,研究微球的佐剂效果,结果表明微球能够提高抗原提呈细胞表面MHC分子表达和相关细胞因子分泌,显示其具有一定的佐剂效应(Torres,Wilson_Welder et al.2011);又如Uto T.等人制备了生物可降解的聚谷氨酸纳微球(γ-PGA NPs)并从机制上研究了其佐剂性能,掲示此微球能通过TLR4 (Toll样受体)和MyD88信号通路诱发强有力的固有和获得性免疫应答反应(Uto,Akagi et al.2011h研究表 [0004] In recent years, large-documented, in vivo biodegradable polymeric microspheres by embedding nano antigens, can be converted to antigen particles conducive to uptake by antigen-presenting cells, and then releases the antigen intracellularly by follow antigen processing, presentation, and enhance the immune response intensity level (Langer, Clel and et al.1997, Johansen, Men et al · 2000, Sahay, Alakhova et al.2010? De Temmerman, Rejman et al · 2011, Danhier, Ansorena et al.2012) e.g., Torres MP, et al polyglycoside class of microspheres were prepared, ovalbumin (OVA) as a model antigen, microspheres adjuvant effect, results showed that the microspheres can be enhanced surface antigen presenting cells expression of MHC molecules and associated cytokine secretion, which display the effect of a certain adjuvant (Torres, Wilson_Welder et al.2011); the biodegradable microspheres Another example sodium polyglutamate Uto T. et al prepared ([gamma] PGA NPs) and studied the mechanism from its adjuvanticity, kei shown microspheres by this TLR4 (Toll-like receptors), and MyD88 signaling pathways induced strong innate and adaptive immune responses (Uto, Akagi et al.2011h Study table ,上述携载抗原的微粒型佐剂使可溶性抗原变成颗粒型抗原,而颗粒型抗原被APC摄取后,可以改变抗原的提呈途径,不仅可以激活CD4+T细胞,而且可以激活CD8+T细胞,大大提升细胞免疫功能,实现胞内感染的彻底清除,是有前景的病毒感染疫苗佐剂与递送系统(Wang and Singh 2011,Dierendonck,De Koker et al.2012)。此外,生物可降解聚合物微球可以提供大量抗原并保护其免受在生理条件下的快速降解,通过微球对所包埋抗原的持续或脉冲释放行为,有效模拟传统疫苗多次免疫程序,减少接种次数和免疫原的使用总量,因而改进了病人的适应性、降低了用药成本(Langer,Cleland et al.1997,Johansen,Estevez et al.2000,De Temmerman,Rejman et al.2011,Demento,Cui et al.2012)〇 , The above-described type adjuvant microparticles carrying the antigen, the soluble antigen into antigen particles, while the particles are ingested antigen APC, antigen presenting pathway may be changed, not only the activated CD4 + T cells, and can activate CD8 + T cells, greatly enhance the cellular immune function, to achieve complete removal of intracellular infection, viral infection is a promising vaccine adjuvants and delivery systems (Wang and Singh 2011, Dierendonck, De et al.2012 Koker). Further, the biodegradable polymeric can provide a lot of microspheres was antigen and protect it from rapid degradation under physiological conditions, the microspheres through a sustained or pulsatile release of the entrapped antigen acts effectively simulate traditional vaccines multiple immunization procedures, and reduce the number of immunogen inoculation the total amount used, thus improving the adaptability of the patient, reducing the cost of medication (Langer, Cleland et al.1997, Johansen, Estevez et al.2000, De Temmerman, Rejman et al.2011, Demento, Cui et al.2012 ) 〇

[0005] 作为抗原蛋白的佐剂与递送系统,聚合物颗粒的很多理化性质会影响其在体内与免疫细胞的相互作用,并最终影响免疫应答类型、强度和水平,包括粒径、形态、表面性质等(Gupta and Siber 1995,Hem and Hogenesch 2007,Mastelic,Ahmed et al.2010,Wang and Singh 2011,Korsholm,Andersen et al.2012)。 [0005] As the delivery system and an adjuvant antigen protein, many physical and chemical properties of the polymer particles will affect its interaction with immune cells in vivo, and ultimately affect the type of immune response, and the level of intensity, including particle size, morphology, surface nature of (Gupta and Siber 1995, Hem and Hogenesch 2007, Mastelic, Ahmed et al.2010, Wang and Singh 2011, Korsholm, Andersen et al.2012). 例如,Xiang SD等(Xiang,Scholzen et al. 2006)在综述文章里提到,表面带正电荷的颗粒与表面带负电荷和不带电荷的颗粒相比,更易于诱导高水平的抗体和CD8+T细胞应答;Foged C.等人(Foged,Brodin et al. 2005)研究了颗粒表面电荷对DC细胞摄取行为的影响,得到了类似结果,即表面带正电荷的颗粒更易于被DC摄取;而Yasuhiko T.等人(Tabata and Ikada 1988)的研究却得到了相反的结论,他们认为带负电荷的纤维素微球能够促进巨噬细胞的摄取。 E.g., Xiang SD, etc. (Xiang, Scholzen et al. 2006) is negatively charged in the review article mentioned, positively charged surface of the particle and the surface particles are not charged more easily performed than antibodies induced high levels and CD8 + T cell responses; Foged C. et al (. Foged, Brodin et al 2005) studied the effects of particle surface charge of DC uptake of cell behavior, to obtain similar results, i.e., positively charged surface of the particles are more readily taken DC; and Study Yasuhiko T. et al. (Tabata and Ikada 1988) has been the opposite conclusion, that they negatively charged cellulose microspheres can promote macrophage uptake. 同样,聚合物颗粒粒径对免疫学效应的影响也尚未形成统一定论。 Similarly, the effect of polymer particle size immunological effect has not yet formed a unified conclusion. Gutierro I.等人(Gutierro,Hernandez et al. 2002)发现,与200和500nm粒径相比,1相比,的PLGA微球可产生高水平抗体;Mann JSF等人(Mann,Shakir et al.2009)制备了载流感抗原的天然生物膜微囊(Biosome), 结果表明小粒径有利于诱导高效体液免疫应答,而大粒径则倾向诱导机体产生细胞免疫应答;WendorfJ等人(Wendorf,Chesko et al .2008)米用表面带负电的PLGA微球(IlOnm和IOOOnm)吸附HIV-I和诱发脑膜炎的MenB (Meningococcal B)蛋白,2个粒径产生了相似的免疫应答。 Gutierro I. et al (. Gutierro, Hernandez et al 2002) found that, compared with 200 and 500nm particle size, compared to 1, the PLGA microspheres can produce high levels of antibody; Mann JSF et al. (Mann, Shakir et al. 2009) natural biofilm microcapsules (Biosome) contained influenza antigen preparation results show that small particle size is conducive to efficient induction of humoral immune response, while the large particle size tends to induce a cellular immune response; WendorfJ et al (Wendorf, Chesko et al .2008) m with negatively charged surface of the PLGA microspheres (IlOnm and IOOOnm) adsorption of HIV-I induced meningitis and MenB (meningococcal B) protein, two particle size produced a similar immune response. 不同研究者针对颗粒同一性质得到不同的结果。 Different researchers have different results for the same nature of the particles. 本发明针对HPV Ll抗原蛋白,纳微球粒径大小对其免疫应答的影响,设计制备粒径均一、不同粒径的PLGA纳微球,以其包埋HPV Ll抗原蛋白,研究PLGA纳微球粒径对体内免疫应答强度和水平的影响,开发高效的HPV Ll蛋白疫苗组合物。 The present invention is directed to antigenic HPV Ll proteins, affect the particle size of microspheres is satisfied on the immune response, the preparation of uniform size design, different size nano PLGA microspheres, with its entrapped antigen HPV Ll proteins, nano research PLGA microspheres intensity and particle size of the response of the immune level, develop efficient HPV Ll protein vaccine composition.

发明内容 SUMMARY

[0006] 第一方面本发明提供一种疫苗组合物,包含与佐剂组合的抗原,抗原与佐剂的比例为10-80yg/mg,所述抗原包含HPV Ll蛋白,所述佐剂组合物包括聚乳酸-聚乙醇酸共聚物(PLGA)或聚乳酸(PLA)。 [0006] a first aspect the present invention provides a vaccine composition comprising an antigen and the proportion of antigen and adjuvant adjuvant combinations of 10-80yg / mg, the antigen comprises HPV Ll proteins, the adjuvant compositions It includes polylactic acid - glycolic acid (PLGA) or polylactic acid (PLA).

[0007] 在本发明的具体实施例中本发明的疫苗组合物,其中所述的HPV Ll蛋白包埋在PLGA中或吸附在PLGA表面,均以粒径分布在IOOnm〜ΙΟΟμπι之间的纳微球形式存在。 [0007] The vaccine compositions of the invention in the specific embodiment of the present invention, HPV Ll protein wherein said entrapped in or adsorbed on PLGA PLGA surface, the particle size distribution are satisfied between the micro IOOnm~ΙΟΟμπι there is a ball forms.

[0008] 在本发明的具体的实施例中疫苗组合物,其中所述的纳微球0-90 %分布在100_ 1 OOOnm、1-1 OymSlO-IOOynriEE—范围内粒径或其组合之间。 [0008] In a specific vaccine composition embodiment of the present invention, wherein the 0-90% satisfied microspheres distributed between 100_ 1 OOOnm, the particle size range 1-1 OymSlO-IOOynriEE- or combinations thereof.

[0009] 在本发明的具体的实施例中疫苗组合物,优选纳微球0-90 %分布在200-900nm、2-9μπι或20-90μπι任一粒径或其组合之间。 [0009] In a particular embodiment of the invention the vaccine composition, preferably 0-90% nano microspheres distributed between 200-900nm, 2-9μπι 20-90μπι or any one or a combination of grain size.

[0010] 在本发明的具体的实施例中疫苗组合物,优选纳微球0-90%分布在400-700nm、4-7μπι或40-70μπι任一粒径或其组合之间。 [0010] In a particular embodiment of the invention the vaccine composition, preferably 0-90% nano microspheres distributed between 400-700nm, 4-7μπι 40-70μπι or any one or a combination of grain size.

[0011] 在本发明的具体的实施例中疫苗组合物,所述的纳微球10-90 %分布在100_ 1 OOOnm、90-10 % 分布在1-1 ΟμίΉ。 [0011] In a particular embodiment of the invention the vaccine composition, the satisfied distribution of microspheres 10-90% 100_ 1 OOOnm, 90-10% distributed in 1-1 ΟμίΉ.

[0012] 在本发明的一个具体的实施例中的疫苗组合物,优选的纳微球10-90%分布在300-800nm、90-10% 分布在3-8μπι。 [0012] In a particular nano microspheres of the present invention, the embodiment of the vaccine composition, preferably 10 to 90% in the distribution of 300-800nm, 90-10% distribution 3-8μπι.

[0013] 在本发明的一个具体的实施例中的疫苗组合物,更优选所述的纳微球10-90%分布在500-600nm、90-10% 分布在5-6μπι。 [0013] In the present invention a particular embodiment of the vaccine composition, more preferably said nano microspheres distributed in 10-90% 500-600nm, 90-10% distribution 5-6μπι.

[0014] 在本发明的具体的实施例中的疫苗组合物,其中所述的纳微球10-90 %分布在1_ 1 Ομπι、90-10 % 分布在10-1 ΟΟμπι。 [0014] In a specific vaccine composition embodiment of the present invention, wherein the 10-90% satisfied microspheres distributed 1_ 1 Ομπι, 90-10% distributed 10-1 ΟΟμπι.

[0015] 在本发明的一个具体的实施例中的疫苗组合物,优选所述的纳微球10-90 %分布在3-8μπι、90-10% 分布在30-80μπι。 [0015] In the embodiment of the vaccine composition of the present invention a particular embodiment, the nanofiltration is preferably 10-90% Distribution of microspheres in 3-8μπι, 90-10% distribution 30-80μπι.

[0016] 在本发明的另一个具体的实施例中的疫苗组合物,优选其中所述的纳微球10-90%分布在5-6以111、90-10%分布在50-6(^111。 [0016] The vaccine composition In another specific embodiment of the present invention, preferably wherein said nano microspheres distributed in 10-90% to 5-6% 111,90-10 distributed 50-6 (^ 111.

[0017] 在本发明的具体的实施例中疫苗组合物,优选纳微球10-90%分布在100-1000nm、 90-10%分布在10-100μπι。 [0017] In a particular embodiment of the invention the vaccine composition, preferably 10-90% sodium microspheres distributed in 100-1000nm, 90-10% distribution 10-100μπι.

[0018] 在本发明的另一个具体的实施例中疫苗组合物,其中所述的纳微球10-90%分布在300-800nm、90-10% 分布在30-80μπι。 [0018] In another embodiment the vaccine compositions of specific embodiments of the present invention, wherein the 10-90% satisfied microspheres distributed 300-800nm, 90-10% distribution 30-80μπι.

[0019] 在本发明的另一个具体的实施例中的疫苗组合物,其中所述的纳微球10-90 %分布在500-600nm、90-10% 分布在50-60μπι。 [0019] The vaccine composition In another specific embodiment of the present invention, wherein the nano microspheres distributed in 10-90% 500-600nm, 90-10% distribution 50-60μπι.

[0020] 在本发明的具体的实施例中,其中所述的纳微球在每一个粒径范围内的粒径分散系数范围为0.005〜0.2。 [0020] In a particular embodiment of the present invention, the particle size dispersion coefficient range wherein said susceptance microspheres in each size range of 0.005~0.2.

[0021] 在本发明的一个具体的实施例中的疫苗组合物,其中所述佐剂中进一步包含如下免疫增强组分脂类物质:卵磷脂、硬脂胺、甘油磷脂中一种或几种的组合,其中脂类物质与PLGA 或PLA的比例为1 -99:99-1,优选1 -50:100。 [0021] In the embodiment of the vaccine composition of the present invention a particular embodiment, wherein the adjuvant further comprises the immune-enhancing lipid components: lecithin, stearylamine, one or several glycerophospholipid composition, wherein the ratio of PLGA or PLA and lipids is 1-99: 99-1, preferably 1-50: 100.

[0022] 更具体的,在本发明的具体的实施例中疫苗组合物,其中所述佐剂中进一步包含小分子免疫佐剂:CpG、MPLA、咪喹莫特、PolyI = C等中一种或几种的组合,其中小分子免疫佐剂与PLGA或PLA的比例为10-100yg/mg。 [0022] More specifically, in specific embodiments of the present invention, the vaccine composition, wherein said adjuvant further comprises a small molecule immunoadjuvant: of one CpG, MPLA, imiquimod, PolyI = C, etc. or more combinations thereof, wherein the ratio of small molecule immunomodulator adjuvant is PLGA or PLA 10-100yg / mg.

[0023] 在本发明的具体的实施例中的疫苗组合物,其中HPV LI蛋白是指HPV全长、截短或重组的Ll形成的VLP、五聚体或多聚体。 [0023] In a specific vaccine composition embodiment of the present invention, it refers to a protein wherein the HPV LI VLP HPV full length, truncated or recombinant Ll formed, pentamer or multimeric.

[0024] 在本发明的具体的实施例中疫苗组合物,其中HPV Ll蛋白是指6、11、16、18、26、 30、31、33、34、35、39、45,51、52、53、56、58、59、66、67、68、69、70、72、73或82、85、97型册乂中一种和/或几种的组合。 [0024] In a particular embodiment of the invention the vaccine composition, wherein the HPV Ll protein refers 6,11,16,18,26, 30,31,33,34,35,39,45,51,52, and 53,56,58,59,66,67,68,69,70,72,73 or 82,85,97 or combinations of one type of book qe / characteristics of.

[0025] 另一方面本发明公开一种疫苗制剂,该疫苗制剂由19所述的疫苗组合物与HPV Ll 蛋白及药用辅料制成,其中纳微球中HPV Ll与游离HPV Ll比例为10-90% :90-10%,进一步更优选10-50 % :90-50%。 [0025] another aspect of the present invention discloses a vaccine formulation, the vaccine formulation is made of a vaccine composition with a pharmaceutically acceptable adjuvant HPV Ll protein of claim 19, wherein the microspheres in sodium free HPV Ll and HPV Ll ratio of 10 -90%: 90 to 10%, even more preferably 10-50%: 90-50%.

[0026] 在本发明的具体的实施例中的疫苗制剂,所述的药用辅料为生理盐水或PBS缓冲液。 [0026] In a particular embodiment the vaccine formulation of the present invention, the pharmaceutical excipient is physiological saline or PBS buffer.

[0027] 另一方面在本发明的具体的实施例中疫苗组合物的制备方法主要包含包埋和吸附两种方法,其中该包埋方法包括如下步骤: [0027] In another aspect of this embodiment the step of preparing a vaccine composition comprising entrapped and adsorbed mainly two methods, wherein the method comprises embedding a particular embodiment of the present invention:

[0028] (1)含药乳滴的制备:将HPV LI蛋白和/或小分子免疫佐剂CpG等溶于缓冲盐体系制备成内水相(Wl); Preparation of emulsion droplets [0028] (1) containing drugs: the HPV LI proteins and / or small molecules and other immunological adjuvant CpG dissolved in buffered saline prepared aqueous phase system (Wl of the);

[0029] (2)将PLGA/PLA或PLGA/PLA与脂类物质溶于有机溶剂中制成油相(0),将所述水相(Wl)与油相(0)混合,制成油包水型(W1/0)预乳液,将此预乳液加入外水相(W2)中,制得Wl/ 0/W2型预复乳液,将所述W1/0/W2型预复乳液反复过膜得到尺寸均一的W1/0/W2型乳液; [0029] (2) The PLGA / PLA or PLGA / PLA with lipids dissolved in an organic solvent to prepare an oil phase (0), the aqueous phase (Wl of the) with oil (0) were mixed phase, an oil prepared water-in (W1 / 0) pre-emulsion, this pre-emulsion was added to the external aqueous phase (W2), the obtained Wl / 0 / W2 emulsion type pre complex, the W1 / 0 / W2-type double emulsion repeatedly through the pre- film obtained uniform size W1 / 0 / W2 emulsion;

[0030] (3)乳滴固化:将W1/0/W2型乳液常温固化,洗涤、干燥后制成疫苗组合物。 [0030] (3) curing the emulsion droplets: The W1 / 0 / W2 emulsion cured at room temperature, washed, and dried to prepare a vaccine composition.

[0031] 在本发明的具体的实施例中提供一种制备方法,步骤⑴所述的内水相pH值范围为4.0-12.0,步骤⑴所述的缓冲盐体系为枸橼酸-磷酸氢二钠、醋酸-醋酸钠缓冲液或磷酸盐缓冲液。 [0031] In particular there is provided a method for preparing an embodiment of the present invention, the step of ⑴ said inner aqueous phase pH in the range 4.0 to 12.0, the step of buffer salt system according ⑴ Citrate - hydrogen diphosphate sodium, acetic acid - sodium acetate buffer or a phosphate buffer.

[0032] 在本发明的一个具体的实施例中提供的制备方法,步骤⑴所述的内水相pH值范围为6.0-8.0。 [0032] In a particular embodiment of the present invention is provided a method for making the step ⑴ said inner aqueous phase pH in the range 6.0 to 8.0.

[0033] 在本发明的一个具体的实施例中步骤(2)所述油相为常温下呈液体与水不互溶的油性物质,优选为乙酸乙酯、丙酮、二氯甲烷、三氯甲烷或上述至少两种以上的混合物。 [0033] with as an oily substance with a water-immiscible liquid in step a particular embodiment of the present invention, the embodiment (2) the oil phase is room temperature, preferably ethyl acetate, acetone, methylene chloride, chloroform or said at least two or more thereof.

[0034] 在本发明的具体的实施例中步骤(2)所述的外水相(W2)为含有0.001-10%的PVA 水溶液,其醇解度为87〜90%,聚合链节数为1700-1750;或为含有0.001-10%的PVA-PEG混合水溶液,PVA与PEG的质量百分比为20-80:30-90。 [0034] In a specific embodiment of the present invention, the step (2) of the external aqueous phase (W2) containing 0.001% PVA aqueous solution, which alcoholysis degree 87~90%, the number of sections of polymeric chain 1700-1750; or a mixed aqueous solution comprising PVA-PEG 0.001 to 10%, the percentage of the mass of PVA and PEG 20-80: 30-90.

[0035] 在本发明的具体的实施例中步骤(2)中所述的油包水型(W1/0型)预乳液是通过采用均相乳化器或超声波乳化器制得的。 [0035] The particular step in an embodiment of the present invention, (2) water-in-oil type (W1 / 0 type) a homogeneous pre-emulsion is obtained by using an ultrasonic emulsifier or the emulsifier obtained.

[0036] 在本发明的具体的实施例中步骤(2)中所述的尺寸均一的W1/0/W2型乳液是通过将W1/0/W2型预乳液在较高压力作用下反复压过微孔膜得到的。 [0036] In a specific embodiment of the present invention, the step of uniform size (2) of the W1 / 0 / W2 emulsion is formed by W1 / 0 / W2 type pre-emulsion at a high pressure is repeatedly pressed through microporous film obtained.

[0037] 在本发明的具体的实施例中步骤(2)中所述的含抗原乳滴,水相(Wl)与油相(0)体积比为1:1〜1:50。 [0037] the step in a particular embodiment of the present invention (2) antigen-containing emulsion droplets and the aqueous phase (Wl of the) oil phase (0) volume ratio of 1: 1~1: 50.

[0038] 在本发明的具体的实施例中步骤⑶固化方式为溶剂蒸发法和溶剂萃取法。 [0038] In a particular embodiment of the present invention, the step of solvent evaporation ⑶ Cure and solvent extraction.

[0039] 在本发明的具体的实施例中,步骤⑶固化时间为lh_8h。 [0039] In a particular embodiment of the present invention, the step of curing time ⑶ lh_8h.

[0040] 另一方面在本发明的具体的实施例中公开另一种疫苗组合物的制备方法,即吸附方法包括如下步骤⑴将PLGA/PLA或PLGA/PLA与脂类物质溶于有机溶剂中制成油相(0),将此油相加入外水相(W2)中,制得0/W2型预复乳液; [0040] Another aspect discloses a method for preparing a vaccine composition in the particular embodiments of the present invention, i.e., the adsorption process comprising the steps ⑴ the PLGA / PLA or PLGA / PLA and lipid substances soluble in organic solvents formed oil phase (0), this oil phase was added external aqueous phase (W2), the obtained 0 / W2 emulsion type pre complex;

[0041] (2)将所述0/W2型预复乳液反复过膜得到尺寸均一的0/W2型乳液;洗涤、干燥后得PLGA/PLA 微球; [0041] (2) The 0 / W2-type double emulsion repeatedly through the pre-film obtained uniform size 0 / W2 emulsion; washed, and dried to obtain PLGA / PLA microspheres;

[0042] (3)将HPV Ll蛋白和/或小分子免疫佐剂CpG溶于缓冲盐体系中,得l-40yg/0.2-ImL的HPV抗原溶液,将PLGA/PLA微球溶于缓冲盐体系中,得1-lOmg/mL的溶液;将两溶液等体积混合,吸附过夜,得到吸附有HPV抗原的PLGA悬液,即为HPV疫苗组合物。 [0042] (3) The HPV Ll proteins and / or small molecule is dissolved in a buffered saline by CpG immune system to give l-40yg / 0.2-ImL an HPV antigen solution, the PLGA / PLA microspheres were dissolved in buffer salt system , yielding 1-lOmg / mL solution; the two solutions were mixed by volume, etc., adsorbed overnight, to give a suspension of PLGA adsorbed HPV antigens, namely HPV vaccine composition.

[0043] 上述提到吸附方法中的具体操作步骤可以参照包埋方法中的相应步骤。 [0043] The above-mentioned adsorption process of the specific steps may be embedded in the respective steps of the method reference.

[0044] 另一方面在本发明的具体的实施例中提供一种疫苗制剂的制备方法,在疫苗组合物的制备方法中的乳滴固化步骤后还包括如下步骤: [0044] In another aspect provides a vaccine formulation after the preparation of the specific embodiment of the present invention, the milk in the production of a vaccine composition in the curing step further comprises dropping the step of:

[0045] 疫苗制剂形成:取质量比10-90% :90-10%的疫苗组合物(纳微球)与游离的HPVLl 蛋白用生理盐水或I3BS缓冲液悬浮,配制成含有l-40yg/0.2-lmL的HPV抗原的微球悬液,制得HPV疫苗制剂。 [0045] The vaccine formulation formed: Take the mass ratio of 10-90%: 90-10% of the vaccine composition (nano microspheres) with the free HPVLl I3BS protein with normal saline or buffer suspension, formulated containing l-40yg / 0.2 the suspension of microspheres -lmL HPV antigen was prepared HPV vaccine formulation.

[0046] 在本发明的具体的实施例中提供的疫苗制剂的制备方法,PBS缓冲液PH值范围6-8,纳微球浓度为1-lOmg/mL。 Preparation Method [0046] The vaccine formulations provided in a particular embodiment of the present invention, PBS buffer PH value range 6-8, nano microsphere concentration of 1-lOmg / mL.

[0047] 另一方面在本发明的具体的实施例中公开疫苗组合物在制备治疗或预防HPV感染的药物中的应用。 Application of a medicament for treating or preventing HPV infections [0047] In another aspect of the disclosed vaccine compositions specific embodiments of the present invention.

[0048] 在本发明的具体的实施例中公开的疫苗制剂在制备治疗或预防HPV感染的药物中的应用。 [0048] The vaccine formulation disclosed In a specific embodiment of the present invention in the manufacture of a medicament for the treatment or prevention of HPV infections.

[0049] 在提供的任何方法的一个实施方案中,通过口服、皮下、肺部、鼻内、腹腔、淋巴、真皮内或肌肉给药施用该剂型。 [0049] In one embodiment of any of the methods provided by the oral, subcutaneous, pulmonary, intranasal, intraperitoneal, lymph node, intradermal, or intramuscular administration of the dosage form.

[0050] 在另一个实施方案中,提供一种人乳头瘤病毒的疫苗制剂,它是以包被了HPV LlVLP抗原的PLGA均一微球为主的一种疫苗制剂,同时可共包埋或复配其他分子佐剂,还可向疫苗中复配一定量的未包被的HPVL1VLP抗原,即由包被在纳微球的HPV Ll抗原与游离的HPV Ll抗原共同及临床或药学上可接受的药用辅料制成疫苗制剂。 [0050] In another embodiment, there is provided a vaccine formulation for human papillomavirus, which is coated with an antigen vaccine formulation HPV LlVLP PLGA microspheres uniformly oriented, while co-complex or embedded other molecules with adjuvant, the vaccine may also be multiplexed with the quantity of uncoated HPVL1VLP antigen, i.e. the packets are common and clinically or pharmaceutically acceptable in Na microspheres HPV Ll of HPV Ll antigen and free antigen pharmaceutical excipients prepared vaccine formulation.

[0051]在另一个实施方案中微球中抗原装载率为20ug/mg以上;包被抗原与游离抗原质量比为0-100%。 [0051] In another embodiment, the antigen loaded microspheres was 20ug / mg or more; coating antigen and free antigen ratio of 0-100% by mass.

[0052] 采用本发明所制备微球表面光滑平整,球形良好(图1,图2,图3),在每一个粒径范围内所制备的粒径均一可控(图4),粒径分散系数在范围为0.005〜0.2,优选0.005〜0.1, 更优选0.005〜0.05。 [0052] The microspheres prepared with the present invention, smooth surface, good spherical (FIG. 1, FIG. 2, FIG. 3), the particle size of each prepared in a uniform, controlled particle size range (FIG. 4), the dispersion particle diameter factor in the range of 0.005~0.2, preferably 0.005~0.1, more preferably 0.005~0.05. 载药量和包埋率高,动物实验表明本发明的新型HPV疫苗是安全的,对实验动物无可见的毒性,可有效的刺激动物产生较强的体液免疫和细胞免疫应答(图5,图6,图7,图8)可用于人乳头瘤病毒的相关疾病或感染的预防和治疗。 Embedding high drug loading, animal experiments show that the present invention is novel HPV vaccine is safe, no visible toxicity in experimental animals, can be effective to stimulate the animals to generate a strong humoral and cellular immune responses (FIG. 5, FIG. 6, FIG. 7, FIG. 8) may be used for human papillomavirus related disease or infection prevention and treatment.

[0053] 为了清楚理解本发明,以下将结合附图和实例进一步说明之,但以下的实施例并非限定本发明。 [0053] For a clear understanding of the present invention will be further described in conjunction with the accompanying drawings and examples, but not limited to the following embodiments of the present invention.

附图说明 BRIEF DESCRIPTION

[0054] 图IPLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(430nm) [0054] FIG IPLGA pentamer antigen entrapped HPVLl nano microspheres scanning electron micrographs (of 430 nm)

[0055] 图2PLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(1.38μπι) [0055] FIG 2PLGA pentamer antigen entrapped HPVLl nano microspheres scanning electron micrographs (1.38μπι)

[0056] 图3PLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(5.60μπι) [0056] FIG 3PLGA pentamer antigen entrapped HPVLl nano microspheres scanning electron micrographs (5.60μπι)

[0057] 图4PLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(400nm) [0057] FIG 4PLGA pentamer antigen entrapped HPVLl nano microspheres scanning electron micrographs (400 nm)

[0058] 图5PLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(800nm) [0058] FIG 5PLGA pentamer antigen entrapped HPVLl nano microspheres scanning electron micrographs (800 nm)

[0059] 图6PLGA包埋HPVLl五聚体抗原纳微球扫描电镜图(1.3抗原) [0059] FIG 6PLGA pentamer antigen entrapped HPVLl nano scanning electron micrographs of microspheres (1.3 antigen)

[0060] 图7PLGA/PC包埋HPVLl五聚体抗原纳微球扫描电镜图(400nm) [0060] FIG 7PLGA / PC antigen entrapped HPVLl pentamer nano microspheres scanning electron micrographs (400 nm)

[0061] 图8PLGA包裹HPV16L1五聚体纳微球扫描电镜图(1聚体) [0061] FIG 8PLGA wrapped HPV16L1 pentamer nano microspheres scanning electron micrographs (1 mer)

[0062] 图9PLGA包裹HPV16L1五聚体和CpG的纳微球(1纳微) [0062] FIG 9PLGA wrapped HPV16L1 pentamers and CpG satisfied microspheres (1 micro-nano)

[0063] 图10PLGA包埋HPVL1VLP抗原纳微球扫描电镜图(1原纳) [0063] FIG 10PLGA nano microspheres entrapped antigen HPVL1VLP scanning electron micrographs (1 original sodium)

[0064] 图11PLGA/HSPC包埋HPVL1VLP抗原纳微球扫描电镜图(1原纳) [0064] FIG 11PLGA / HSPC nano microspheres entrapped antigen HPVL1VLP scanning electron micrographs (1 original sodium)

[0065] 图12PLA包埋HPVL1VLP抗原纳微球扫描电镜图(1原纳) [0065] FIG 12PLA nano microspheres entrapped antigen HPVL1VLP scanning electron micrographs (1 original sodium)

[0066] 图13PLGA包埋HPVL1VLP抗原纳微球扫描电镜图(10纳微) [0066] FIG 13PLGA satisfied antigen entrapped HPVL1VLP scanning electron micrographs of microspheres (10 micro-nano)

[0067] 图14PLGA纳微球扫描电镜图(1微球) [0067] FIG 14PLGA nano microspheres scanning electron micrographs (1 microspheres)

[0068] 图15载HPVl 6L1五聚体抗原PLGA纳微球粒径分布图 [0068] FIG. 15 is contained HPVl 6L1 pentamer antigen PLGA microsphere size distribution nano

[0069] 图16不同粒径载抗原PLGA微球组免疫小鼠38天后血清中中和抗体滴度 [0069] FIG 16 different carrier particle group antigens PLGA microspheres after 38 days in the immunized mouse serum neutralizing antibody titers

[0070] 图17不同粒径载抗原PLGA微球组免疫小鼠38天后血清中特异性抗体IgG滴度 [0070] FIG 17 different carrier particle group antigens PLGA microspheres immunized mouse serum specific antibody IgG titers 38 days

[0071] 图18各组免疫小鼠后脾细胞上清中的细胞因子水平检测结果(Luminex技术) [0071] The cytokine levels of spleen cell supernatants after the detection result of each group of 18 mice immunized FIG (Luminex technology)

[0072] 图19各组免疫小鼠后脾细胞上清中IFN-小和IL-4水平检测结果(ELISA法) [0072] FIG. 19 mice in each group immunized IFN- and IL-4 levels small detection result (ELISA method) in spleen cell supernatants

[0073] 图20各组免疫小鼠后脾细胞IL-4和IFN-小酶联免疫斑点形成细胞数(SFC) (ELI SPOT 法) [0073] After each group of 20 mice immunized splenocytes FIG IFN- and IL-4 ELISpot small forming cells (SFC) (ELI SPOT method)

[0074] 图21HPV LI五聚体蛋白采用不同佐剂系统免疫小鼠后,血清结合抗体滴度 [0074] After the protein in mice immunized with different adjuvant system, serum antibody titers binding pentamers FIG 21HPV LI

[0075] 图22HPV Ll五聚体蛋白采用不同佐剂系统免疫小鼠后,中和抗体的滴度 [0075] After the protein in mice immunized with different adjuvant system, and the antibody titer in FIG 22HPV Ll pentamer

[0076] 图23HPV Ll五聚体蛋白采用不同佐剂系统免疫小鼠后,ELISP0T测定的细胞免疫应答结果 [0076] The cellular immune response to the protein results in mice immunized with different adjuvant system, ELISP0T measured FIG 23HPV Ll pentamer

[0077] 图24HPV Ll五聚体蛋白采用不同佐剂系统免疫小鼠,取其脾细胞在体外培养条件下经抗原刺激后,ELI SA测定IFN-A和IL-4细胞因子分泌情况 [0077] FIG 24HPV Ll protein pentamers different adjuvant system immunized mice, spleen cells after antigen stimulation, ELI SA assay IFN-A and IL-4 cytokine secretion in vitro culture

[0078] 图25HPV Ll五聚体蛋白采用不同佐剂系统免疫小鼠,取其脾细胞在体外培养条件下经抗原刺激后,Luminex方法测定细胞因子分泌情况。 [0078] FIG 25HPV Ll protein pentamers different adjuvant system immunized mice, spleen cells after antigen stimulation, the Luminex method for measuring cytokine secretion in vitro culture.

[0079] 图26PLGA复配CpG小分子佐剂包被HPV16L1五聚体的微球免疫小鼠后中和抗体滴度 After [0079] FIG 26PLGA complex of small molecule adjuvant CpG coated microsphere immunized mice HPV16L1 pentamer neutralizing antibody titers

[0080] 图27PLGA复配CpG小分子佐剂包被HPV16L1五聚体的微球免疫小鼠后结合抗体滴度 [0080] FIG 27PLGA complex of small molecule adjuvant CpG binding antibody titer after coated microsphere immunized mice HPV16L1 pentamer

[0081] 图28不同粒径及其组合的PLGA包被HPVl 6L1五聚体的微球免疫小鼠后结合抗体滴度 [0081] FIG 28 different sizes and combinations of PLGA-coated microspheres after binding antibody titers in mice immunized HPVl 6L1 pentamer

[0082] 图29不同粒径及其组合的PLGA包被HPVl 6L1VLP的微球免疫小鼠后中和抗体滴度 [0082] FIG 29 different sizes and combinations PLGA-coated microspheres after immunization of mice HPVl 6L1VLP neutralizing antibody titers

[0083] 图30PLGA、PLGA/HSPC、PLA包被HPV16L1VLP的微球免疫小鼠后中和抗体滴度 [0083] FIG 30PLGA, the PLGA / HSPC, PLA microspheres coated immunized mice and antibody titer in HPV16L1VLP

[0084] 图31PLGA、PLGA/HSPC、PLA包被HPV16L1VLP的微球免疫小鼠后结合抗体滴度 [0084] FIG 31PLGA, PLGA / HSPC, PLA binding antibody titer after coated microsphere immunized mice HPV16L1VLP

[0085] 图32PLGA微球吸附HPV16L1VLP后免疫小鼠后的结合抗体滴度 [0085] FIG 32PLGA microspheres adsorption HPV16L1VLP binding antibody titers in mice after immunization

[0086] 具体实例 [0086] Specific examples of

[0087] 本发明参照如下表格实施本发明的技术方案,本发明的技术方案包括但不限于如下实施例。 [0087] The embodiments of the present invention with reference to the table below aspect of the present invention, the aspect of the present invention include, but are not limited to the following examples.

[0088] 表1 [0088] TABLE 1

Figure CN104338126BD00111

Figure CN104338126BD00121

Figure CN104338126BD00131

Figure CN104338126BD00141

Figure CN104338126BD00151

Figure CN104338126BD00161

Figure CN104338126BD00171

[0101] 上述表格中涉及具体实施例如下: [0101] DETAILED embodiment relates to the above table are as follows:

[0102] 具体实施例一——IOOnmPLGA载HPV 6L1五聚体抗原微球的制备 [0102] DETAILED --IOOnmPLGA prepared a pentameric contained HPV 6L1 antigen microspheres Example

[0103] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0103] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为1.33mg/mL的HPV 6L1五聚体溶于枸橼酸-磷酸氢二钠缓冲盐溶液的抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为600r/min下搅拌2min。 Specific methods: EA 400mg of PLGA is dissolved in a solvent in the oil phase lO.OmL (0), 3mL was added at a concentration of 1.33mg / mL HPV 6L1 pentamer of citric acid was dissolved in - disodium hydrogen phosphate buffered saline the aqueous phase (Wl of the), disintegrator for colostrum protein of the antigen solution used as S-450D ultrasonic cell under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, the aqueous solution was then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) of NaCl (the external aqueous phase W2), a magnetic stirring min using pre-prepared complex emulsion (W1 / 0 / W2), emulsifying conditions 600r / stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以1.5M Pa压力的氮气将复乳液反复压过膜孔径为0.6μπι的SPG膜, 直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 1.5M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 0.6μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为400nm, PDI值为0.04,药物包埋率为90.8%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 400nm, PDI value was 0.04, and 90.8 percent of drug loading.

[0104] 具体实施例二一一包含游离抗原的HPV疫苗的制备 HPV vaccine preparation [0104] embodiment comprises two hundred and eleven free antigen

[0105] 称取实施例一所制备的平均粒径为IOOnm的载HPV 6L1五聚体抗原微球4.45mg复配120 · 3μΙ^度为1 · 333mg/mL的HPV 6L1五聚体抗原,加入pH=7 · 0的PBS缓冲液至lmL,配制得含有200yg/mL的且纳微球中包被抗原与游离抗原比为2:8的HPV抗原的微球悬液,制得HPV疫苗制剂。 [0105] Example weighed an average particle diameter of the prepared embodiment of IOOnm contained HPV 6L1 pentamer microspheres 4.45mg antigen complex degree of 120 · 3μΙ ^ 1 · 333mg / mL of pentamer HPV 6L1 antigen, was added pH = PBS 7 · 0 to lmL buffer, formulated to give microspheres and nano bag containing 200yg / mL of antigen and free antigen ratio of 2: suspension of microspheres 8 HPV antigen was prepared HPV vaccine formulation.

[0106] 具体实施例三——430nm PLGA载HPV 16L1五聚体抗原微球的制备 [0106] DETAILED prepared contained HPV 16L1 three --430nm PLGA microspheres five antigens embodiment mer

[0107] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0107] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为411^/1^的冊¥161^1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5% PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为600r/min下搅拌2min。 1 ^ ^ ^ fine 10.01111 4 was dissolved in a solvent as the oil phase (0), was added at a concentration of 31 ^ 411 ^ / 1 ^ 161 ^ ¥ album 1 pentameric protein antigen as an aqueous phase (Wl of the), with S- 450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / under ice-cooling aq v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 600r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以IM Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入7 8 0 m L的0.9 % N a C1溶液中,在室温下搅拌4 h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured uniformly 7 8 0 m 0.9% N a C1 L solution and stirred at room temperature for 4 h (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing deionized water washed by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为430nm,PDI值为0.03,药物包埋率为92.6 %。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 430nm, PDI 0.03, drug loading was 92.6%. 所制得微球扫描电镜图见图1。 The microspheres obtained SEM is shown in Figure 1.

[0108] 具体实施例五——1.38μΐΉ PLGA载HPV16L1五聚体抗原微球制备 [0108] DETAILED --1.38μΐΉ PLGA prepared according to a fifth embodiment HPV16L1 pentamer antigen Microspheres

[0109] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0109] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为6.67mg/mL的HPV 16L1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备Wl/Ο型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为400r/min下搅拌2min。 Specific methods: EA 400mg of PLGA is dissolved in a solvent in the oil phase lO.OmL (0), 3mL was added at a concentration of 6.67mg / mL in the five-mer HPV 16L1 protein antigen as an aqueous phase (Wl of the), with S -450D ultrasonic cell disrupter for colostrum of under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared Wl / Ο colostrum type, then the primary emulsion was poured into 65mL containing 1.5% PVA (w aqueous / v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为5.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 5.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为1.38μπι,PDI值为0.05,药物包埋率为93.1%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 1.38μπι, PDI value of 0.05, drug loading was 93.1%. 所制得载抗原纳微球扫描电镜图如图2所示。 The prepared antigen contained nano microspheres SEM shown in Figure 2.

[0110] 具体实施例六——Iym PLGA载HPV16L1五聚体抗原微球制备 [0110] Specific examples of six --Iym PLGA prepared HPV16L1 pentamer antigen Microspheres

[0111] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0111] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为6.67mg/mL的HPV 16L1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为400r/min下搅拌2min。 Specific methods: EA 400mg of PLGA is dissolved in a solvent in the oil phase lO.OmL (0), 3mL was added at a concentration of 6.67mg / mL in the five-mer HPV 16L1 protein antigen as an aqueous phase (Wl of the), with S -450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, and then poured into 65mL of the primary emulsion containing 1.5% PVA (w under ice-cooling aqueous / v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为5.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 5.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为Ιμπι,PDI值为0.05,药物包埋率为93.1 %。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of Ιμπι, PDI value of 0.05, drug loading was 93.1%. 所制得载抗原纳微球扫描电镜图如图8所示。 The prepared antigen contained nano microspheres SEM shown in Figure 8. FIG.

[0112] 具体实施例七——5.6μπι PLGA载HPV16L1五聚体抗原微球制备 [0112] DETAILED seventh embodiment --5.6μπι prepared HPV16L1 pentamer antigen contained PLGA microspheres

[0113] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0113] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为9.33mg/mL的HPV 16L1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为300r/min下搅拌2min。 Specific methods: EA 400mg of PLGA is dissolved in a solvent in the oil phase lO.OmL (0), 3mL was added at a concentration of 9.33mg / mL in the five-mer HPV 16L1 protein antigen as an aqueous phase (Wl of the), with S -450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, and then poured into 65mL of the primary emulsion containing 1.5% PVA (w under ice-cooling aqueous / v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 300r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以40kPa 压力的氮气将复乳液反复压过膜孔径为15μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, a pressure of 40kPa nitrogen emulsion was repeatedly pressed through the complex membrane pore size 15μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2 ). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为5.6ym,PDI值为0.08,药物包埋率为79.7%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 5.6ym, PDI value of 0.08, was 79.7% drug loading. 所制得载抗原纳微球扫描电镜图如图3所示。 The prepared antigen contained nano microspheres SEM shown in Figure 3.

[0114] 具体实施例十七——20.82μπι PLGA载HPV45L1五聚体抗原微球制备 [0114] Preparation of seventeen --20.82μπι PLGA contained HPV45L1 pentamer antigen specific embodiments microspheres

[0115] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0115] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为5.33311^/1^的册¥451^1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为250r/min下搅拌2min。 1 ^ ^ fine 10.01111 4 was dissolved in the solvent as an oil phase (0), was added at a concentration of 5.33311 ^ 31 ^ / 1 ^ 451 ^ ¥ album 1 pentameric protein antigen as an aqueous phase (Wl of the), with S-450D ultrasonic cell disrupter for colostrum of under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v ) aqueous solution of NaCl (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 250r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以5kPa压力的氮气将复乳液反复压过膜孔径为52μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, the pressure of nitrogen 5kPa emulsion was repeatedly pressed through the complex membrane pore size 52μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2 ). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况, 平均粒径为20.82μπι,PDI值为0.10,药物包埋率为89.2%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 20.82μπι, PDI is 0.10, drug loading was 89.2%.

[0116] 具体实施例十八——80.71μπι PLA载HPV58L1五聚体抗原微球制备 [0116] DETAILED Preparation Example eighteen --80.71μπι HPV58L1 pentamer antigen contained PLA microspheres

[0117] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0117] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为5.33311^/1^的册¥581^1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为250r/min下搅拌2min。 1 ^ ^ fine 10.01111 4 was dissolved in the solvent as an oil phase (0), was added at a concentration of 5.33311 ^ 31 ^ / 1 ^ 581 ^ ¥ album 1 pentameric protein antigen as an aqueous phase (Wl of the), with S-450D ultrasonic cell disrupter for colostrum of under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v ) aqueous solution of NaCl (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 250r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0. IkPa 压力的氮气将复乳液反复压过膜孔径为190.3μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, the pressure of nitrogen 0. IkPa emulsion was repeatedly pressed through the complex membrane pore size 190.3μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为80.71μπι,PDI值为0.18,药物包埋率为85.5%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 80.71μπι, PDI value of 0.18, drug loading was 85.5%.

[0118] 具体实施例十九——400nm PLGA/HSPC载HPV16L1五聚体抗原微球制备 [0118] Specific embodiments of nineteen --400nm PLGA / antigen prepared HPV16L1 pentamer microspheres contained HSPC

[0119] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0119] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将300mg的PLGA和IOOmg卵磷脂溶于lO.OmL的MC溶剂中作为油相(0),加入3mL浓度为1.333mg/ mL的HPV 6L1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(Wl/ 0/W2),乳化条件为500r/min下搅拌2min。 The specific method is: the MC and solvent PLGA 300mg lO.OmL IOOmg lecithin dissolved in an oil phase as (0), 3mL was added at a concentration of 1.333mg / mL HPV-6L1 pentameric the antigen protein as an aqueous phase (Wl of the ), carried out with 1.5 of colostrum (ultrasound power setting of 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, and then poured into 65mL of the primary emulsion under ice-cooling with S-450D ultrasonic cell disrupter % PVA (w / v) aqueous solution of NaCl (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (Wl / 0 / W2), with stirring under emulsifying conditions 500r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中, 以IM Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为400nm,PDI值为0.05,药物包埋率为92.8%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 400nm, PDI value of 0.05, was 92.8% drug loading. 所得微球扫描电镜图如7。 The microspheres as FIG. 7 SEM.

[0120] 具体实施例二十——800nm PLGA载HPV16L1五聚体抗原微球制备 [0120] DETAILED Preparation Example twenty --800nm ​​HPV16L1 pentamer antigen contained PLGA microspheres

[0121] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0121] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将30011^的? Specific methods are: the 30011 ^ of? 1^^溶于10.01^的如溶剂中作为油相(0),加入31^浓度为411^/1^的冊¥111^1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5% PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为500r/min下搅拌2min。 10.01 ^ 1 ^^ dissolved in a solvent such as an oil phase (0), was added at a concentration of 31 ^ 411 ^ / 1 ^ 111 ^ ¥ album 1 pentameric protein antigen as an aqueous phase (Wl of the), with S- 450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / under ice-cooling aq v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 500r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以IM Pa压力的氮气将复乳液反复压过膜孔径为9.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 9.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为800nm,PDI值为0.06,药物包埋率为88.2%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 800nm, PDI is 0.06, drug loading was 88.2%. 所得微球的扫描电镜图如图5。 Scanning electron micrographs of the resulting microspheres are shown in Figure 5.

[0122] 具体实施例二^^一——1300nm PLGA载HPV16L1五聚体抗原微球制备 [0122] DETAILED ^^ a second embodiment --1300nm PLGA prepared HPV16L1 pentamer antigen Microspheres

[0123] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0123] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将30011^的? Specific methods are: the 30011 ^ of? 1^^溶于10.01^的如溶剂中作为油相(0),加入31^浓度为411^/1^的冊¥111^1五聚体抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5% PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为500r/min下搅拌2min。 10.01 ^ 1 ^^ dissolved in a solvent such as an oil phase (0), was added at a concentration of 31 ^ 411 ^ / 1 ^ 111 ^ ¥ album 1 pentameric protein antigen as an aqueous phase (Wl of the), with S- 450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / under ice-cooling aq v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 500r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以IM Pa压力的氮气将复乳液反复压过膜孔径为9.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 9.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为1300nm,PDI值为0.06,药物包埋率为88.2%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 1300nm, PDI is 0.06, drug loading was 88.2%. 所得微球的扫描电镜图如图6。 Scanning electron micrograph of the resulting microspheres Figure 6.

[0124] 具体实施例二十六——IOOOnm PLGA载抗原纳微球共包埋CPG1826载HPV16L1五聚体抗原微球制备 [0124] Specific embodiments of an antigen contained twenty-six --IOOOnm PLGA microspheres were prepared nano antigen contained HPV16L1 CPG1826 microspheres embedded pentamer

[0125] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0125] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为411^/1^的冊¥161^1五聚体抗原蛋白和5yg CpG1826作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(Wl/ 0/W2),乳化条件为400r/min下搅拌2min。 1 ^ ^ ^ fine 10.01111 4 was dissolved in a solvent as the oil phase (0), was added at a concentration of 31 ^ 411 ^ / 1 ^ 1 ^ album ¥ 161 pentameric 5yg CpG1826 and the antigen protein as an aqueous phase (Wl of the), be of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then the primary emulsion was poured into 65mL of ice water bath under conditions containing S-450D with ultrasonic cell disrupter 1.5% PVA aqueous solution (w / v) of NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (Wl / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中, 以0.25M Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为l〇〇〇nm,PDI值为0.06,药物包埋率为87.8 %。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of l〇〇〇nm, PDI is 0.06, drug loading was 87.8%. 所得微球扫描电镜图如图9。 The microspheres SEM shown in Figure 9.

[0126] 具体实施例二十七——1.57μπι PLGA载抗原纳微球共包埋MPLA载HPV16L1五聚体抗原微球制备 [0126] DETAILED DESCRIPTION twenty-seven carrier --1.57μπι PLGA microspheres were entrapped antigen satisfied MPLA prepared HPV16L1 pentamer antigen Microspheres Example

[0127] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0127] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为6.67mg/mL的HPV 16L1五聚体抗原蛋白和5yg MPLA作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL 的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/ W2),乳化条件为400r/min下搅拌2min。 Specific methods are: 400mg PLGA is dissolved in EA lO.OmL solvent as the oil phase (0), 3mL was added to the concentration of 6.67mg / mL of pentamer HPV 16L1 protein antigen as an aqueous phase and 5yg MPLA (Wl of the) , disrupter of colostrum performed under ice-cooling with S-450D cells ultrasound (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then the primary emulsion was poured into 65mL containing 1.5% aqueous solution of NaCl PVA (w / v) (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为5.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 5.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为1.57μπι,PDI值为0.05,药物包埋率为82.2%〇 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 1.57μπι, PDI value of 0.05, was 82.2% drug loading billion

[0128] 具体实施例二十九——23.57μπι PLA载抗原纳微球共包埋?〇171:(:载冊¥451^1五聚体抗原微球制备 [0128] DETAILED DESCRIPTION twenty-nine antigen contained --23.57μπι PLA microspheres were embedded nano 〇171 :( Example: Preparation of 1 451 ^ pentamer antigen Microspheres volumes ¥?

[0129] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0129] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将400mg的PLGA溶于lO.OmL的EA溶剂中作为油相(0),加入3mL浓度为5.33mg/mL的HPV 16L1五聚体抗原蛋白和5yg PolyI = C作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备Wl/Ο型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(Wl/ 0/W2),乳化条件为400r/min下搅拌2min。 Specific methods: EA 400mg of PLGA is dissolved in a solvent in the oil phase lO.OmL (0), 3mL was added to the concentration of 5.33mg / mL of pentamer HPV 16L1 protein antigen and 5yg PolyI = C as an aqueous phase ( WL), S-450D with ultrasonic cell disrupter for colostrum of (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared Wl / Ο colostrum type under ice-cooling, and then poured into a primary emulsion containing 65mL of aqueous solution of NaCl 1.5% PVA (w / v) (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (Wl / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中, 以0.25M Pa压力的氮气将复乳液反复压过膜孔径为5.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 5.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为23.57μπι,PDI值为0.09,药物包埋率为86.3%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 23.57μπι, PDI value of 0.09, drug loading was 86.3%. 具体实施例三^^一——400nmPLGA载HPV 16L1五聚体抗原微球的制备 Microspheres prepared antigen-mer --400nmPLGA a carrier according to a third ^^ HPV 16L1 five DETAILED DESCRIPTION

[0130] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0130] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^仙4溶剂中作为油相(0),加入311^浓度为1.3311^/11^的耶¥ 16L1VLP抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为600r/min下搅拌2min。 1 ^ ^ ^ 4 10.01111 dissolved in the solvent as an oil phase Sin (0), was added at a concentration of 1.3311 ^ 311 ^ / 11 ^ of the antigenic protein Y. ¥ 16L1VLP as an aqueous phase (Wl of the), S-450D with ultrasonic cell disrupter be of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) of NaCl under ice-cooling aqueous solution (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 600r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以1.5M Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 1.5M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为400nm,PDI值为0.05,药物包埋率为90.5%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 400nm, PDI value of 0.05, was 90.5% drug loading. 所制备得到微球的扫描电镜图如图4所示 The microspheres prepared SEM shown in Figure 4

[0131] 具体实施例三十六——128nmPLGA载HPV 6L1VLP抗原微球的制备 [0131] Preparation Example thirty-six --128nmPLGA contained HPV 6L1VLP antigen specific embodiment Microspheres

[0132] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0132] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 11^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为1.3311^/1^的册¥61^1¥1^ 抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为600r/min下搅拌2min。 ^ 11 ^ fine 10.01111 4 was dissolved as an oil phase solvent (0) was added at a concentration of 31 ^ 1.3311 ^ / 1 ^ 1 album ¥ ¥ ^ 1 ^ 61 is the antigen protein as an aqueous phase (Wl of the), with S-450D ultrasonic cell disrupter for colostrum of under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v ) aqueous solution of NaCl (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 600r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以1.5M Pa压力的氮气将复乳液反复压过膜孔径为〇.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 1.5M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 〇.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入7 8 0 m L的0.9 % N a C1溶液中,在室温下搅拌4 h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured uniformly 7 8 0 m 0.9% N a C1 L solution and stirred at room temperature for 4 h (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing deionized water washed by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为128nm,PDI值为0.03,药物包埋率为91.7 %。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 128nm, PDI 0.03, drug loading was 91.7%.

[0133] 具体实施例三十七 [0133] Specific embodiments thirty-seven

[0134] 称取实施例三十六所制备的平均粒径为128nm的载HPV 6L1VLP抗原微球4.4mg复配12 · 03μΙ^度为13 · 33mg/mL的HPV 6L1五聚体抗原,加生理盐水或pH=7 · 0的PBS缓冲液至lmL,配制得含有200yg/mL的且纳微球中包被抗原与游离抗原比为2:8的HPV抗原的微球悬液,制得HPV疫苗制剂。 [0134] The average particle diameter of said prepared in Example thirty-six embodiment taken as an antigen contained HPV 6L1VLP microspheres 128nm 4.4mg of compound 12 · 03μΙ ^ degree of 13 · 33mg / mL of pentamer antigen HPV 6L1, plus physiological saline or buffer was added to pH = PBS lmL 7 · 0, formulation and have satisfied microspheres containing bag 200yg / mL of antigen and free antigen ratio of 2: 8 microsphere suspensions HPV antigen, HPV vaccine prepared preparation.

[0135] 具体实施例三十八——467nm PLGA载HPV IILlVLP抗原微球的制备 [0135] Preparation thirty-eight --467nm PLGA microspheres contained HPV IILlVLP antigen specific embodiment according

[0136] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0136] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 11^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为411^/1^的册¥111^1^抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/ v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为600r/min下搅拌2min。 ^ 11 ^ fine 10.01111 4 was dissolved as an oil phase solvent (0) was added at a concentration of 31 ^ 411 ^ / 1 ^ 111 ^ 1 album ¥ ^ with antigen proteins as an aqueous phase (Wl of the), with S-450D ultrasonic cell disrupter under ice-cooling in the primary emulsification (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) of aqueous solution of NaCl (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 600r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以IM Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm) 去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为467nm,PDI值为0.05,药物包埋率为89.2%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 467nm, PDI value of 0.05, drug loading was 89.2%.

[0137] 具体实施例四十——Ιμπι PLGA载HPV16L IVLP抗原微球制备 [0137] Preparation of PLGA Microspheres Example forty carrier HPV16L IVLP antigen --Ιμπι DETAILED DESCRIPTION

[0138] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0138] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^仙4溶剂中作为油相(0),加入311^浓度为6.6711^/11^的耶¥ 16L1VLP抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为400r/min下搅拌2min。 1 ^ ^ ^ 4 10.01111 dissolved in the solvent as an oil phase Sin (0), was added at a concentration of 6.6711 ^ 311 ^ / 11 ^ of the antigenic protein Y. ¥ 16L1VLP as an aqueous phase (Wl of the), S-450D with ultrasonic cell disrupter be of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) of NaCl under ice-cooling aqueous solution (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为5.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 5.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为Ιμπι,PDI值为0.05,药物包埋率为89.5 %。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of Ιμπι, PDI value of 0.05, drug loading was 89.5%. 所制得载抗原纳微球扫描电镜图如图10所示。 The prepared antigen contained nano microspheres scanning electron micrograph shown in Figure 10.

[0139] 具体实施例四十二——5.87μπι PLGA载HPV18L1VLP抗原微球制备 [0139] Preparation forty-two --5.87μπι PLGA microspheres contained HPV18L1VLP antigen specific embodiments

[0140] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0140] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^仙4溶剂中作为油相(0),加入311^浓度为9.3311^/11^的耶¥ 18L1VLP抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为300r/min下搅拌2min。 1 ^ ^ ^ 4 10.01111 dissolved in the solvent as an oil phase Sin (0), was added at a concentration of 9.3311 ^ 311 ^ / 11 ^ of the Y. ¥ 18 L1 VLP antigen proteins as an aqueous phase (Wl of the), S-450D with ultrasonic cell disrupter be of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) of NaCl under ice-cooling aqueous solution (external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 300r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以40kPa 压力的氮气将复乳液反复压过膜孔径为15μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, a pressure of 40kPa nitrogen emulsion was repeatedly pressed through the complex membrane pore size 15μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2 ). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为5.87μπι,PDI值为0.07,药物包埋率为89.2%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 5.87μπι, PDI 0.07, drug loading was 89.2%.

[0141] 具体实施例五十二——25.02μπι PLA载HPV45L1VLP抗原微球制备 [0141] Preparation fifty-two --25.02μπι PLA microspheres contained HPV45L1VLP antigen specific embodiments

[0142] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0142] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^溶于10.01111^细4溶剂中作为油相(0),加入311^浓度为5.3311^/1^的册¥451^¥1^ 抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为250r/min下搅拌2min。 1 ^ ^ fine 10.01111 4 was dissolved in the solvent as an oil phase (0), was added at a concentration of 5.3311 ^ 311 ^ / 1 ^ album ¥ 451 ¥ ^ 1 ^ with antigen proteins as an aqueous phase (Wl of the), with S-450D ultrasonic cell disruption system of colostrum performed under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) NaCl aqueous solution (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), emulsifying condition under 250r / min stirring 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以5kPa压力的氮气将复乳液反复压过膜孔径为52μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, the pressure of nitrogen 5kPa emulsion was repeatedly pressed through the complex membrane pore size 52μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2 ). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为25.02um,PDI值为0.06,药物包埋率为75.3%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 25.02um, PDI is 0.06, drug loading was 75.3%.

[0143] 具体实施例五十五——IOOOnm PLGA/HSPC载HPV16L1VLP抗原微球制备 [0143] Preparation of antigen contained HPV16L1VLP microspheres embodiment fifty-five --IOOOnm PLGA / HSPC DETAILED DESCRIPTION

[0144] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0144] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将300mg的PLGA和IOOmg卵磷脂溶于10·OmL的MC溶剂中作为油相(0),加入3mL浓度为4mg/mL的HPV 16L1VLP抗原蛋白作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2), 乳化条件为500r/min下搅拌2min。 The specific method is: the PLGA 300mg of the lecithin was dissolved in MC and IOOmg 10 · OmL the solvent as the oil phase (0), 3mL was added at a concentration of 4mg / mL antigen protein of HPV 16L1VLP as an aqueous phase (Wl of the), with S -450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, and then poured into 65mL of the primary emulsion containing 1.5% PVA (w under ice-cooling aqueous / v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 500r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以IM Pa压力的氮气将复乳液反复压过膜孔径为9.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/ W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, IM Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 9.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting complex emulsion droplets uniformly stirred at rt for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze-dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为l〇〇〇nm,PDI值为0.07,药物包埋率为80.6%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of l〇〇〇nm, PDI 0.07, drug loading was 80.6%. 所制得载抗原纳微球扫描电镜图如图11所示。 The prepared antigen contained nano microspheres SEM shown in Figure 11.

[0145] 具体实施例五十六——489nm PLGA载抗原纳微球共包埋CPG1826载HPVlILlVLP抗原微球制备 [0145] Specific embodiments of an antigen contained fifty-six --489nm PLGA microspheres prepared nano microspheres were entrapped antigen CPG1826 carrier HPVlILlVLP

[0146] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0146] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 11^溶于10.01111^细4溶剂中作为油相(0),加入31^浓度为411^/1^的册¥111^1^抗原蛋白和5yg CpG1826作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2), 乳化条件为400r/min下搅拌2min。 ^ 11 ^ fine 10.01111 4 was dissolved as an oil phase solvent (0) was added at a concentration of 31 ^ 411 ^ / 1 ^ 111 ^ 1 album ¥ ^ 5yg CpG1826 and the antigen protein as an aqueous phase (Wl of the), with S- 450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / under ice-cooling aq v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为489nm,PDI值为0.05,药物包埋率为90.8%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 489nm, PDI value of 0.05, was 90.8% drug loading.

[0147] 具体实施例六十六——IOOOnm PLA载抗原纳微球载HPV16L1VLP抗原微球制备 [0147] Specific embodiments of an antigen contained sixty-six --IOOOnm PLA microspheres prepared HPV16L1VLP satisfied antigen Microspheres

[0148] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0148] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^溶于10.01111^细4溶剂中作为油相(0),加入311^浓度为6.6711^/1^的册¥161^¥1^ 抗原蛋白和5yg CpG1826作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL 的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/ W2),乳化条件为400r/min下搅拌2min。 1 ^ ^ fine 10.01111 4 was dissolved in the solvent as an oil phase (0), was added at a concentration of 6.6711 ^ 311 ^ / 1 ^ album ¥ 161 ¥ ^ 1 ^ 5yg CpG1826 and the antigen protein as an aqueous phase (Wl of the), with S -450D for ultrasonic cell disrupter of colostrum (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, and then poured into 65mL of the primary emulsion containing 1.5% PVA (w under ice-cooling aqueous / v), NaCl (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为1.4μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 1.4μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为l〇〇〇nm,PDI值为0.04,药物包埋率为89.4%。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of l〇〇〇nm, PDI value was 0.04, drug loading was 89.4%. 所制得载抗原纳微球扫描电镜图如图12所示。 The prepared antigen contained nano microspheres FIG SEM shown in Figure 12.

[0149] 具体实施例六十八——IOym PLGA载抗原纳微球载HPV16L1VLP抗原微球制备 [0149] Specific embodiments of an antigen contained sixty-eight --IOym PLGA microspheres prepared HPV16L1VLP satisfied antigen Microspheres

[0150] 采用快速膜乳化技术结合复乳溶剂去除法制备载抗原纳微球。 [0150] The membrane emulsification technique rapid binding double emulsion solvent was removed Preparation of antigen contained nano microspheres. 具体方法为:将40011^的? Specific methods are: the 40011 ^ of? 1^^溶于10.01111^仙4溶剂中作为油相(0),加入311^浓度为6.6711^/11^的耶¥ 16L1VLP抗原蛋白和5yg CpG1826作为内水相(Wl),用S-450D超声波细胞破碎仪在冰水浴条件下进行初乳化(超声功率设定为30%,超声时间为60s)制备W1/0型初乳,然后将初乳液倒入65mL的含1.5%PVA (w/v)的NaCl的水溶液(外水相W2)中,采用磁力搅拌制备预复乳液(W1/0/W2),乳化条件为400r/min下搅拌2min。 1 ^ ^ ^ 4 10.01111 dissolved in the solvent as an oil phase Sin (0), was added at a concentration of 6.6711 ^ 311 ^ / 11 ^ ¥ 16L1VLP of Jesus and the antigen protein as an aqueous phase 5yg CpG1826 (Wl of the), with S-450D ultrasonic cell disruption system of colostrum performed under ice-cooling conditions (ultrasound power setting was 30%, ultrasonic time of 60s) was prepared W1 / 0 type colostrum, then poured into a primary emulsion containing 65mL of 1.5% PVA (w / v) NaCl aqueous solution (the external aqueous phase W2), the pre-complex prepared using a magnetic stirring emulsion (W1 / 0 / W2), with stirring under emulsifying conditions 400r / min 2min. 随后,将此预复乳液倒入快速膜乳化的储料罐中,以0.25M Pa压力的氮气将复乳液反复压过膜孔径为50.2μπι的SPG膜,直至得到均一的复乳液滴(W1/0/W2)。 Subsequently, this pre-emulsion was poured into a rapidly complex membrane emulsification in the storage tank, 0.25M Pa pressure with nitrogen the emulsion is repeatedly pressed through the complex membrane pore size 50.2μπι SPG membrane, the complex until a uniform emulsion droplets (W1 / 0 / W2). 将所制得的均一复乳液滴倒入780mL的0.9%NaCl溶液中,在室温下搅拌4h (磁力搅拌速率200rpm)去除有机溶剂,固化后的微球用去离子水离心洗涤3次,最后冷冻干燥制成成品。 The resulting double emulsion droplets was poured into 780mL uniform 0.9% NaCl solution is stirred at room temperature for 4H (magnetic stirring rate at 200 rpm) the organic solvent is removed, the microspheres after curing washed with deionized water by centrifugation 3 times, and finally freeze dried finished products. 用激光粒度仪测定粒径分布情况,平均粒径为1〇μπι,PDI值为0.07,药物包埋率为90.2%,所制得载抗原纳微球扫描电镜图如图13所不。 Particle size distribution measured using a laser particle size analyzer, the average particle diameter of 1〇μπι, PDI 0.07, drug loading was 90.2%, the resulting microspheres contained antigen satisfied SEM Figure 13 are not shown in FIG.

[0151] 具体实施例七十四 [0151] Specific embodiments seventy-four

[0152] 分别取实施例三^^一制备得到的430nm纳微球1.128mg和实施例五制备得到的1.38五制纳微球1.157mg配制成抗原质量比为50% : 50%的粒径组合,加入PH值为7的I3BS缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.31mg/mL混悬液。 [0152] Microspheres were collected 430nm satisfied 1.128mg prepared according to a third embodiment ^^ a five systems and 1.38 is satisfied five microspheres obtained in Preparation Example antigen formulated embodiment 1.157mg mass ratio of 50%: 50% particle diameter in combination , I3BS lmL added buffer PH value of 7, have prepared containing 100yg / nano and microspheres antigen mL concentration was 2.31mg / mL suspension.

[0153] 具体实施例七十九 [0153] Specific embodiments seventy-nine

[0154] 分别取实施例三^^一制备得到的430nm纳微球1.128mg和实施例三十三制备得到的20.65μπι纳微球1.147mg配制成抗原质量比为50% : 50 %的粒径组合,加入PH值为7的PBS 缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.27mg/mL混悬液。 [0154] The third embodiment are taken ^^ prepared in a nano 430nm 1.128mg microspheres prepared in Examples and thirty three nano microspheres obtained 20.65μπι 1.147mg antigen formulated mass ratio of 50%: 50% particle diameter composition, the addition of PBS buffer lmL PH value of 7, comprising preparing an antigen and have a concentration of sodium microsphere 100yg / mL was 2.27mg / mL suspension.

[0155] 具体实施例八十四 [0155] Specific embodiments eighty-four

[0156] 分别取实施例五制备得到的1.38μπι纳微球1.157mg和实施例三十三制备得到的20.65μπι纳微球1.147mg配制成抗原质量比为50% :50%的粒径组合,加入PH值为7的I3BS缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.31mg/mL混悬液。 [0156] Preparation Example pentahydrate were obtained 1.38μπι 1.157mg and nano microspheres prepared in Example thirty-three nano microspheres obtained 20.65μπι 1.147mg antigen formulated embodiments mass ratio of 50%: 50% particle diameter of the composition, I3BS buffer was added lmL PH value of 7, have prepared containing 100yg / mL antigen and nano microsphere concentration 2.31mg / mL suspension.

[0157] 具体实施例九十二 [0157] Specific embodiments ninety-two

[0158] 分别取实施例三^^一制备得到的430nm纳微球0.68mg、实施例五制备得到的1.38μ m纳微球0.69mg和实施例三十三制备得到的20.65μπι纳微球0.92mg配制成抗原质量比为30 % : 30 % : 40 %的粒径组合,加入PH值为7的PBS缓冲液ImL,制备得含有lOOyg/mL的抗原且纳微球浓度为2.29mg/mL混悬液。 [0158] 430nm were taken embodiment 0.68mg Example nano microspheres prepared in a three ^^, five 1.38μ m obtained in Preparation Example microspheres 0.69mg sodium and sodium 20.65μπι microspheres prepared in Example thirty-three embodiment obtained 0.92 mass ratio of mg antigen was formulated as a 30%: 30%: 40% particle diameter in combination, PBS buffer was added ImL PH value of 7, have prepared containing lOOyg / mL antigen and nano microsphere concentration 2.29mg / mL mix suspension.

[0159] 具体实施例一百零一 [0159] Specific embodiments of one hundred and one

[0160] 分别取实施例六十八制备得到的IOMi纳微球和实施例四十制备得到的Ιμπι纳微球配制成抗原质量比为20% :80%的粒径组合,加入PH值为7的PBS缓冲液lmL,制备得含有100 yg/mL的抗原且纳微球浓度为2.35mg/mL混悬液。 [0160] Microspheres were collected IOMi satisfied Preparation Example sixty-eight obtained in Preparation Example and forty nano microspheres obtained Ιμπι antigen formulated embodiment is 20% by mass ratio: 80% particle diameter of the composition, is added to PH 7 PBS buffer lmL, have prepared containing 100 yg / nano and microspheres antigen mL concentration was 2.35mg / mL suspension.

[0161] 具体实施例一百零二 [0161] Specific embodiments of one hundred and two

[0162] 分别取实施例六十八制备得到的ΙΟμπι纳微球和实施例四十制备得到的Ιμπι纳微球配制成抗原质量比为80% :20%的粒径组合,加入PH值为7的PBS缓冲液lmL,制备得含有100 yg/mL的抗原且纳微球浓度为2.35mg/mL混悬液。 [0162] Microspheres were collected ΙΟμπι satisfied Preparation Example sixty-eight obtained in Preparation Example and forty obtained Ιμπι nano microspheres formulated antigen mass ratio of 80% embodiments: 20% particle diameter in combination, is added to PH 7 PBS buffer lmL, have prepared containing 100 yg / nano and microspheres antigen mL concentration was 2.35mg / mL suspension.

[0163] 具体实施例一百零三 [0163] Specific embodiments of one hundred and three

[0164] 分别取实施例六十八制备得到的ΙΟμπι纳微球1.175mg和实施例四十制备得到的1μ m纳微球1.174mg配制成抗原质量比为50% :50%的粒径组合,加入PH值为7的PBS缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.35mg/mL混悬液。 [0164] Preparation Example were taken sixty-eight ΙΟμπι obtained nano and microspheres prepared in Example forty 1.175mg obtained 1μ m nano microspheres 1.174mg antigen formulated embodiments mass ratio of 50%: 50% particle diameter of the composition, PBS buffer was added lmL PH value of 7, have prepared containing 100yg / mL antigen and nano microsphere concentration 2.35mg / mL suspension.

[0165] 具体实施例一百零六 [0165] Specific embodiments of one hundred and six

[0166] 分别取实施例六十六制备得到的458nm纳微球1.175mg和实施例六十八制备得到的23.58μπι纳微球1.164mg配制成抗原质量比为50% :50%的粒径组合,加入PH值为7的PBS 缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.34mg/mL混悬液。 [0166] Preparation Example were taken sixty-six 458nm obtained nano and microspheres were prepared sixty-eight 1.175mg embodiment nano microspheres obtained 23.58μπι 1.164mg antigen formulated mass ratio of 50%: 50% particle diameter in combination , lmL PBS buffer was added to PH value of 7, have prepared containing 100yg / nano and microspheres antigen mL concentration was 2.34mg / mL suspension.

[0167] 具体实施例一百—I [0167] Specific embodiments of one hundred -I

[0168] 分别取实施例四十制备得到的1.69μπι纳微球1.174mg和实施例六十八制备得到的23.58μπι纳微球1.164mg配制成抗原质量比为50% :50%的粒径组合,加入PH值为7的PBS缓冲液lmL,制备得含有100yg/mL的抗原且纳微球浓度为2.34mg/mL混悬液。 [0168] Preparation Example embodiments were taken forty 1.69μπι obtained nano and microspheres 1.174mg Preparation Example sixty-eight nano microspheres obtained 23.58μπι 1.164mg antigen formulated mass ratio of 50%: 50% particle diameter in combination , lmL PBS buffer was added to PH value of 7, have prepared containing 100yg / nano and microspheres antigen mL concentration was 2.34mg / mL suspension.

[0169] 具体实施例一百二十 [0169] In particular embodiments of one hundred and twenty

[0170] 分别取实施例六十六制备得到的4 5 8 nm纳微球0.71 mg、实施例四十制备得到的1.69μηι纳微球1.174mg和实施例六十八制备得到的0.47μηι纳微球0.004mg配制成抗原质量比为30 % : 50 % : 20 %的粒径组合,加入PH值为7的I3BS缓冲液ImL,制备得含有I 原且纳微球浓度为2.35mg/mL混悬液。 [0170] Preparation Example were taken sixty-six obtained 4 5 8 nm nano microspheres embodiment 0.71 mg, 1.69μηι nano and microspheres 1.174mg sixty-eight Preparation Example Preparation Example forty obtained micro-nano obtained embodiment 0.47μηι 0.004mg antigen formulated ball mass ratio of 30%: 20% particle diameter of the composition, a buffer is added I3BS ImL PH value of 7, was prepared containing I have primary and nano microsphere concentration 2.35mg / mL suspension: 50% liquid.

[0171] 表格中其他实施例的具体实施步骤参照以上相关具体实施例施行。 [0171] Referring to the above table, specific examples related to the purposes of other embodiments of the procedure of Example particular embodiments. 实例一百二十五一一微球粒径及粒径分布表征 Examples of one hundred and twenty five hundred eleven microsphere size and size distribution characterization

[0172] 将其中一些实施例制备得到的微球悬浮液于蒸馏水中超声使其均匀分散,采用激光粒度仪CZeiasizer® 3000HS,UK)测定其粒径大小及分布,不同载抗原纳微球佐剂粒径分布图如图15所示。 [0172] The preparation of certain embodiments wherein the suspension of microspheres obtained in distilled water and ultrasonically dispersed uniformly, and the distribution of particle size measured using a laser particle size analyzer CZeiasizer® 3000HS, UK), sodium different carrier microsphere antigen adjuvant The particle size distribution shown in Figure 15.

[0173] 实施例一百二十六——HPV装载率测定 [0173] Example embodiments for determination of the loading one hundred twenty-six --HPV

[0174] 将制备好的所述冻干纳微球5mg悬浮于I.OmL含5% (w/v) SDS浓度为0. IM的NaOH溶液,室温下振荡24小时,使微球完全溶解(水解),溶液中蛋白质含量micro-BCA试剂盒进行测定。 [0174] The prepared nano the lyophilized microspheres was suspended in I.OmL 5mg containing 5% (w / v) SDS at a concentration of 0. IM NaOH solution, and shaken for 24 hours at room temperature to completely dissolve the microspheres ( hydrolysis), a solution of the protein content of micro-BCA assay kit.

[0175] 实施例一百二十七一一不同粒径PLGA载抗原微球免疫后抗体水平评价比较 [0175] Example one hundred twenty seven hundred and eleven Comparative Evaluation of antibody levels after different sizes PLGA microspheres contained antigen immunization

[0176] 取实施例五和实施例三制备好的所述冻干微球,悬浮于生理盐水中,得微球悬液, 使得微球悬液中抗原的含量为20邱/100此,即制得HPV疫苗制剂。 [0176] and the fifth embodiment taken good lyophilized microspheres prepared in Example three, suspended in physiological saline to give a suspension of the microspheres, such that the antigen in the microspheres is suspension Qiu 20/100 Herein, i.e. HPV vaccine formulations prepared.

[0177] 取实施例五和实施例三制得的两种纳微球疫苗制剂背部皮下免疫8-10周的雌性BALB/c小鼠,每只小鼠注射抗原剂量为20yg/100yL,同时设立以铝盐为佐剂的HPV五聚体抗原作为对照组。 Two kinds of formulations satisfied the back microsphere vaccine [0177] according to a fifth embodiment and taken according to a third embodiment prepared was subcutaneously immunized 8-10 weeks old female BALB / c mice, a dose of antigen was injected per mouse 20yg / 100yL, also set aluminum salt as adjuvant to pentamer HPV antigen as a control group.

[0178] 检测以上三组的体液免疫效果。 [0178] detecting humoral immune effect above three groups. 检测的指标包括不同粒径PLGA微球载抗原组免疫小鼠38天后的中和抗体水平和特异性抗体IgG水平,分别图16和图17所示。 Indicators include detection of different diameters PLGA Microspheres group of antigens of mice immunized after 38 days and the levels of neutralizing antibodies specific IgG levels, respectively, FIGS. 16 and 17 shown in FIG.

[0179] 不同粒径的PLGA载抗原纳微球免疫小鼠后产生的特异性抗体中和抗体水平不同。 [0179] Antibodies specific for different particle sizes produced after carrying PLGA microspheres satisfied antigen and antibody levels in immunized mice different. 由图16可知,小鼠免疫38天后1.38μπι载抗原PLGA微球产生的中和抗体水平和IgG水平均值都高于铝佐剂组,但无显著性差异;但1.38μπι载抗原PLGA组血清中中和抗体水平和IgG水平均显著高于430nm载抗原PLGA组(Ρ<0 · 05或Ρ<0 · 01) 〇 Seen from FIG. 16, the immunized mice 38 days after the neutralizing and IgG antibody levels average 1.38μπι water contained PLGA microspheres produced antigen are higher than the aluminum adjuvant group, but no significant difference; but 1.38μπι PLGA contained antigen in serum antibody levels and IgG levels were significantly higher than 430nm and an antigen contained in PLGA group (Ρ <0 · 05 or Ρ <0 · 01) square

[0180] 实施例一百二十八一一不同粒径PLGA载抗原微球免疫后细胞因子水平评价比较 One hundred and twenty eight hundred eleven cytokines evaluated after different sizes PLGA microspheres contained antigen immunization [0180] Comparative Example

[0181] 取实施例五和实施例三制得的两种纳微球疫苗制剂背部皮下免疫8-10周的雌性BALB/c小鼠,每只小鼠注射抗原剂量为20yg/100yL,同时设立以铝盐为佐剂的HPV五聚体抗原作为对照组。 Two kinds of formulations satisfied the back microsphere vaccine [0181] according to a fifth embodiment and taken according to a third embodiment prepared was subcutaneously immunized 8-10 weeks old female BALB / c mice, a dose of antigen was injected per mouse 20yg / 100yL, also set aluminum salt as adjuvant to pentamer HPV antigen as a control group.

[0182] 检测以上三组的细胞免疫效果。 [0182] Immune cells detected above three groups. 检测指标包括采用抗原体外刺激小鼠脾细胞72h 后,脾细胞上清中细胞因子水平(Luminex技术、ELISA法),结果如图18和图19所示;以及采用抗原体外刺激小鼠脾细胞36h后,IFN- γ和IL-4酶联免疫斑点形成细胞数(ELISP0T法), 结果如图20所不。 Detection index includes an antigen using in vitro stimulated spleen cells 72h, the spleen cells supernatant cytokine levels (Luminex technology, ELISA method), the results shown in FIGS. 18 and 19; and the use of in vitro antigen stimulated mouse splenocytes 36h after, IFN- γ and IL-4 ELISpot forming cells (ELISP0T method), results 20 are not shown in FIG.

[0183] IL-4和IL-6代表Th2型应答的细胞因子,IFN-γ和IL-2代表Thl型应答的细胞因子由图18、图19和图20均可以看出,430nm和1.38μπι两个粒径的载抗原PLGA微球组脾细胞上清中的IFN-γ水平均显著高于铝佐剂组(Ρ<0.05或Ρ<0.01),且430nm载抗原PLGA纳微球组脾细胞上清中的IFN- γ水平极显著高于1.38μπι载抗原PLGA纳微球组组(P<0.01);由图7可知,430nm载抗原PLGA纳微球组脾细胞上清中的IL-2水平也显著高于铝佐剂组(P<0.05), 且430nm载抗原PLGA纳微球组脾细胞上清中IL-2的分泌水平也高于1.38μπι载抗原PLGA组, 但二者无显著差异。 [0183] IL-4 and IL-6 on behalf of Th2-type cytokine responses, IFN-γ and IL-2 on behalf of Thl type cytokine responses by 18, 19 and 20 can be seen, of 430 nm and 1.38μπι carrying antigen levels were IFN-γ PLGA microspheres supernatant of splenocytes of two groups were significantly higher than the particle size of the aluminum adjuvant group (Ρ <0.05 or Ρ <0.01), and sodium 430nm antigen PLGA microspheres group contained splenocytes IFN- γ levels in the supernatants was significantly higher than that contained antigen 1.38μπι satisfied PLGA microspheres group group (P <0.01); understood from FIG. 7, 430nm PLGA microspheres satisfied spleen cell supernatants group contained in the IL-2 antigen levels were also significantly higher than Al adjuvant group (P <0.05), and the level of secreted antigen 430nm PLGA microspheres satisfied spleen cell supernatants of IL-2 group was also higher than the carrier contained 1.38μπι antigen PLGA group, but no significant both difference. 这表明,相比铝佐剂,载抗原PLGA纳微球组能够有效诱导Thl型免疫应答,增强细胞免疫应答,且粒径较小的PLGA载抗原纳微球(430nm)相比大粒径PLGA载抗原纳微球(1.38μπι)更能够有效诱导Thl型免疫应答,增强细胞免疫应答。 This shows that, compared to the aluminum adjuvant, an antigen contained in PLGA microspheres satisfied groups could induce Thl-type immune response, enhancing cellular immune response, PLGA small particle size carrier and the antigen is satisfied microspheres (of 430 nm) as compared to large particle size PLGA antigen contained nano microspheres (1.38μπι) can be more effective to induce Thl-type immune response, enhancing cellular immune response.

[0184] 在Th2型细胞因子分泌水平方面,不同粒径载抗原PLGA纳微球组能诱导与铝佐剂类似的IL-4分泌水平,无显著性差异,1.38μπι载抗原PLGA纳微球组脾细胞上清中的IL-4水平显著高于430nm载抗原PLGA纳微球组组(Ρ<0.05或Ρ<0.01) ; 1.38μπι载抗原PLGA纳微球组脾细胞上清中的IL-6水平显著高于铝佐剂组(Ρ<0.05),且1.38μπι载抗原PLGA纳微球组脾细胞上清中的IL-6水平极显著高于430nm载抗原PLGA纳米球组(Ρ<0.01)。 [0184] In the Th2 cytokine secretion levels, different particle carrying antigens PLGA microspheres group is satisfied with the aluminum adjuvant can induce similar levels of IL-4, no significant difference, 1.38μπι antigen contained sodium PLGA microspheres group IL-4 levels in the supernatants of spleen cells was significantly higher than 430nm antigen contained PLGA microspheres group satisfied group (Ρ <0.05 or Ρ <0.01); 1.38μπι antigen contained in PLGA microspheres satisfied spleen cell supernatants of IL-6 group significantly higher than aluminum adjuvant group (Ρ <0.05), and the antigen contained 1.38μπι PLGA microspheres satisfied spleen cell supernatants of IL-6 group significantly higher than the level of the antigen contained in the PLGA nanoparticles 430nm group (Ρ <0.01) .

[0185] 以上结果表明载抗原PLGA纳微球组可以诱导与铝佐剂相似的IL-4的分泌水平,可以有效诱导体液免疫,以及更高水平的IFN-γ水平,显著提升细胞免疫应答;而且大粒径的载抗原PLGA微球(1.38μπι)能更显著提高IL-6、IL-4的分泌水平,增强Th2型免疫应答,促进B 细胞活化产生抗体,有效地诱导体液免疫应答;而小粒径载抗原PLGA微球(430nm)显著提升IFN-γ的分泌水平,增强Thl型免疫应答,有助于细胞免疫应答的提升。 [0185] These results indicate that an antigen contained sodium PLGA microspheres can be set with an aluminum adjuvant induced similar levels of IL-4 secretion, can efficiently induce humoral immunity, as well as higher levels of IFN-γ levels, significantly improve the cellular immune response; large particle size carrier and the antigen PLGA microspheres (1.38μπι) are more significantly improved IL-6, and IL-4 secretion, enhanced Th2 immune response, activation of B cell generates antibodies efficiently induce humoral immune responses; and small particle carrying antigens PLGA microspheres (of 430 nm) significantly enhance the secretion of IFN-γ, enhanced Thl immune response, help to improve the immune response cells.

[0186] 实施例一百二十九——1.38ymPLGA载抗原纳微球复配游离抗原免疫小鼠后免疫效果分析 [0186] The mice immunized with the free antigen Analysis of immune effect one hundred twenty-nine --1.38ymPLGA microspheres contained antigen complex admittance Example

[0187] 取实施例六制得的微球制剂背部皮下免疫8-10周的雌性BALB/c小鼠,每只小鼠注射抗原剂量为20邱/100此,其中微球疫苗制剂中HPV抗原含量为IOyg/只,游离HPV抗原含量为IOyg/只,同时设立以铝盐为佐剂的HPV五聚体抗原和实施例5制备的1.38μπι PLGA载抗原纳微球作为对照组。 Back microsphere preparation [0187] Preparation according to a sixth embodiment was immunized subcutaneously 8-10 weeks old female BALB / c mice, a dose of antigen was injected per mouse Qiu 20/100 Here, wherein the microspheres HPV antigen in the vaccine formulation content IOyg / only, HPV antigen content of free IOyg / only, while aluminum salt as adjuvant in established HPV antigen and pentamer satisfied 1.38μπι PLGA microspheres prepared antigen contained in Example 5 as a control group. 检测以上几组的体液免疫和细胞免疫效果。 Humoral and cellular immune effects of the above test groups.

[0188] 结果显示1.38μπι PLGA载抗原纳微球复配游离抗原产生的中和抗体滴度和特异性抗体IgG滴度是1.38μπι PLGA载抗原纳微球组的5倍左右,这表明PLGA载抗原纳微球复配游离抗原可以有效地增强体液免疫效果。 [0188] The results are shown 1.38μπι PLGA microspheres contained antigen complex admittance free antigen generated antibody titers and specific IgG titers were approximately 5 times 1.38μπι PLGA microspheres contained sodium antigen group, indicating that PLGA with antigen complex nano microspheres free antigen can effectively enhance humoral immunity.

[0189] 实施例一百三十四一一五聚体及VLP纳微球的制备 Preparation Example one hundred thirty-four one hundred and fifteen polymer microspheres and nano VLP [0189] Embodiment

[0190] 参照以上具体实施方式,按照以下表格的配方制备不同比例、不同粒径及不同型别的五聚体及VLP的纳微球的制备。 DETAILED DESCRIPTION above [0190] with reference to the preparation of different sizes and different types other pentamers and VLP nano microspheres prepared according to the formulation in different proportions to the following table.

[0191] 表5载HPV16L1五聚体PLGA微球不同粒径的序号 [0191] Table 5 contains five different particle sizes HPV16L1-mer No. PLGA microspheres

[0192] [0192]

Figure CN104338126BD00271

[0193] 表6载HPV16L1五聚体抗原的PLGA微球不同粒径组合序号 [0193] Table 6 contains HPV16L1 pentamer antigen combinations of different particle sizes PLGA microspheres No.

[0194] [0194]

Figure CN104338126BD00272

[0195] [0195]

Figure CN104338126BD00281

[0196] 表7载HPVl 6L1五聚体抗原的PLGA微球不同HPV型别序号 [0196] Table 7 contains HPVl 6L1 pentamer antigen PLGA microspheres number of different HPV types

[0197] [0197]

Figure CN104338126BD00282

[0198] [0198]

Figure CN104338126BD00291

[0199] 表8载HPV16L1五聚体抗原的PLGA微球复配脂类物质序号 [0199] Table 8 pentamer contained HPV16L1 PLGA microspheres antigen complex lipids No.

[0200] [0200]

Figure CN104338126BD00292

[0203] [0203]

Figure CN104338126BD00301

[0204] 表10载HPV16LIVLP抗原的PLGA微球的不同粒径的序号 [0204] PLGA microspheres contained 10 HPV16LIVLP antigen table number of different size

[0205] [0205]

Figure CN104338126BD00302

[0206] [0206]

Figure CN104338126BD00311

[0207] 表11载HPV16L1VLP抗原的PLGA微球的不同粒径组合序号 [0207] Table 11 contains the PLGA microspheres HPV16L1VLP antigen combinations of different particle sizes No.

[0208] [0208]

Figure CN104338126BD00312

[0209] 表12载HPV16LIVLP抗原的PLGA微球的不同HPV型别序号 [0209] TABLE 12 PLGA microspheres contained HPV16LIVLP antigen different HPV types Reference

[0210] [0210]

Figure CN104338126BD00321

[0211] 表13载HPV16L1VLP抗原的PLGA微球的复配脂类物质序号 [0211] Table 13 contains the PLGA microspheres HPV16L1VLP antigen complex lipids No.

[0212] [0212]

Figure CN104338126BD00322

[0213] [0213]

Figure CN104338126BD00331

[0214] 表14载HPVl 6L1VLP抗原的PLGA微球的复配不同分子佐剂的序号 Complex of different molecular adjuvant [0214] PLGA microspheres contained in Table 14 Antigen number HPVl 6L1VLP

[0215] [0215]

Figure CN104338126BD00332

[0216] 实施例一百三十五一一不同粒径的纳微球HPV Ll五聚体免疫原性研究 [0216] Immunogenicity of one hundred thirty five hundred and eleven different size nano microspheres embodiments HPV Ll pentamer

[0217] 取实施例十九、二十、三十和二十一制备的样品,以400nm、800nm、1.3μηι三种不同粒径PLGA包埋的HPV Ll五聚体蛋白以及400nmPLGA/PC包被的HPV Ll五聚体蛋白免疫小鼠, 初步实验结果表明,该佐剂系统可以有效诱导包括中和抗体和细胞免疫在内的抗原特异性免疫应答。 [0217] Sample preparation twenty-one nineteen, twenty, thirty and embodiments taken to 400nm, 800nm, 1.3μηι three different particle PLGA embedded HPV Ll protein pentamers and 400nmPLGA / PC coated the mice immunized with HPV Ll protein pentamers, preliminary experimental results show that the adjuvant system may include efficiently induce antigen-specific immune neutralizing antibodies and cellular immune responses, including. 在图4中,分析血清中的抗体,结果表明,与传统铝盐佐剂组相比,400nm和800nm PLGA佐剂组的中和抗体滴度基本相同,但1.3μπι的中和抗体佐剂组以及400nmPLGA+卵磷脂(PLGA/PC)复合佐剂组的中和抗体和结合滴度较高;另800nm和1.3μ. 3nm的佐剂组的结合抗体滴度均显著高于400nm PLGA,但两者之间无显著性差异。 In Figure 4, analysis of serum antibodies, results showed that, compared with the conventional aluminum salt adjuvant group, 400 nm and 800nm ​​PLGA adjuvants and antibody titers of substantially the same group, but the neutralizing antibody 1.3μπι adjuvant group and 400nmPLGA + binding and neutralizing antibody titers high lecithin (PLGA / PC) compound adjuvant group;. 800nm ​​and another binding antibody titer 1.3μ 3nm adjuvant group were significantly higher than 400nm PLGA, but both no significant difference between the. 以ELISP0T方法检测HPV特异性CTL免疫应答,结果表明,传统铝佐剂组基本没有诱导出特异性CTL免疫应答,而PLGA佐剂组均有效地诱导出比较高水平的HPV特异性CTL免疫应答,其中尤以400nm和800nm PLGA佐剂组为最高(图2)。 ELISP0T to detect HPV-specific CTL immune response, results showed substantially no conventional aluminum adjuvant group induced specific CTL immune response, PLGA and adjuvant group were significantly induce high levels of HPV-specific CTL Comparative immune response, 400nm and 800nm ​​PLGA especially in the highest adjuvant group (FIG. 2). 取免疫后小鼠的脾细胞在体外培养条件下经抗原刺激后测定上清里的细胞因子分泌情况,结果表明,比较Thl型(刺激细胞免疫)细胞因子IFN-细和TNF-细等的分泌量,PLGA佐剂组均显著高于传统铝盐佐剂组;而在Th2型细胞因子(刺激体液/抗体免疫应答)IL-4方面,PLGA佐剂组基本与传统铝盐佐剂组相同,但使用PLGA+卵磷脂(PLGA/PC)的复合佐剂组则显著高于其它组。 Mouse splenocytes in the supernatant of cytokine secretion was measured after antigen stimulation in vitro culture taken after immunization, the results showed that, comparing Thl type (to stimulate cellular immunity) cytokine IFN- fine and thin like TNF- secretion , of PLGA adjuvant group were significantly higher than conventional aluminum salt adjuvant group; and IL-4 aspect, the adjuvant group of PLGA substantially same as a conventional aluminum salt adjuvant group Th2-type cytokines (stimulating humoral / antibody immune response), but use PLGA + lecithin (PLGA / PC) complex adjuvant group was significantly higher than other groups. 结果见图21、22、23、24、25。 The results shown in Figure 21, 22.

[0218] 实施例一百三十六——IOOOnm PLGA载抗原纳微球共包埋CPG1826联合免疫小鼠后免疫效果分析 After [0218] Example contained one hundred thirty-six --IOOOnm PLGA microspheres were entrapped antigen satisfied CPG1826 co-immunized mice Immune Analysis

[0219] 按照实施例二十六制备共包埋CpG1826的PLGA载抗原纳微球。 [0219] preparing dimethyl-sixth embodiment according to the carrier antigen were entrapped PLGA microspheres CpG1826 is satisfied. 将所制得的微球冻干后,悬浮于生理盐水中,得微球悬液,使得微球悬液中抗原的含量为20yg/100yL,即制得HPV疫苗制剂。 After the prepared microspheres lyophilized, suspended in physiological saline to give a suspension of the microspheres, such that the antigen in the microspheres suspension was 20yg / 100yL, i.e. to prepare HPV vaccine formulation. 将其背部皮下免疫8-10周的雌性BALB/c小鼠,每只小鼠注射抗原剂量为20μ g/100yL,同时设立以铝盐为佐剂的HPV五聚体抗原和IOOOnm PLGA载抗原纳微球作为对照组。 Which was immunized subcutaneously 8-10 weeks old female BALB / c mice, a dose of antigen was injected per mouse 20μ g / 100yL, also set to aluminum salt adjuvant and antigen HPV pentamer antigen IOOOnm PLGA contained sodium microspheres as controls. 检测以上几组的体液免疫和细胞免疫效果。 Humoral and cellular immune effects of the above test groups. PLGA复配CpG小分子佐剂包被HPV16L1五聚体的微球免疫小鼠后中和抗体和结合抗体滴度分别见图26和27,结果显示共包埋CpG的IOOOnm载抗原PLGA纳微球组可免疫小鼠后能诱导与铝盐佐剂水平相当的中和抗体水平。 PLGA small molecule adjuvant CpG compound coated microspheres after immunization of mice HPV16L1 pentameric antibody binding and neutralizing antibody titers are shown in Figures 26 and 27, the results demonstrate that the co-embedded CpG-carrier antigen IOOOnm satisfied PLGA microspheres after the set of mice immunized with aluminum adjuvant-induced comparable levels of neutralizing antibody. 而在结和抗体水平上,Iym PLGA包埋的HPV16L1五聚体蛋白在初次免疫时诱导了显著高于铝盐佐剂的结合抗体水平,在第二次免疫时能诱导与铝盐佐剂相当水平的结合抗体滴度,并且在添加CpG小分子佐剂后在第二次免疫时诱导的结合抗体水平显著高于不添加CpG组,同时在第一次和第二次免疫时诱导的结合抗体水平均显著高于传统铝盐佐剂。 And the antibody level in colorectal, Iym PLGA embedded HPV16L1 pentameric binding protein induced antibody levels were significantly higher than aluminum salt adjuvant in the primary immunization, can induce in the second immunization with an aluminum salt adjuvant considerable binding antibody titer levels, and after the addition of small molecule adjuvant CpG induced at the second immunization binding antibody levels were significantly higher than the group without addition of CpG, induced simultaneously in the first and second immunological binding antibodies were significantly higher than traditional aluminum salt adjuvant.

[0220] 实施例一百三十七——不同粒径组合的PLGA载抗原纳微球免疫小鼠后免疫效果分析 [0220] Example one hundred thirty-seven - combinations of the different sizes of PLGA contained nano microspheres antigen immunized mice Immune Analysis

[0221] 取实施例一百零一、一百零二、一百零三制得的不同粒径组合的微球制剂背部皮下免疫8-10周的雌性BALB/c小鼠,每只小鼠注射抗原剂量为20yg/100yL,同时设立以铝盐为佐剂的HPV五聚体抗原和实施例四十、六十八制备的不同粒径的PLGA载抗原纳微球作为对照组。 [0221] Example embodiments taken one hundred and one, one hundred and two, one hundred and three microspheres prepared in different particle sizes combined preparation subcutaneously immunized 8-10 weeks old female BALB / c mice, each mouse injection of antigen dose 20yg / 100yL, while aluminum salt as adjuvant in established HPV antigen pentamer and PLGA forty different sizes embodiment, the carrier prepared according sixty-eight nano microspheres antigen as a control group. 检测以上几组的体液免疫效果。 Humoral immune effect of the above test groups. 不同粒径及其组合的PLGA包被HPV16L1五聚体的微球免疫小鼠后结合抗体滴度和中和抗体滴度分别如28和29。 Different sizes and combinations PLGA-coated microsphere immunized mice after binding pentamers HPV16L1 antibody titer and neutralizing antibody titers as 28 and 29, respectively.

[0222] 结果显示Ιμπι或ΙΟμπι或两粒径按一定比例组合的PLGA包埋的HPV16L1VLP诱导的中和抗体滴度无显著差异,而Iym PLGA-HPV16L1VLP组在初次免疫后诱导的结合抗体滴度显著低于HPV16LlVLP-lym+10ym (0 · 5+0 · 5)组,在第二次免疫后显著低于HPV16LlVLP-lym+10y m (0 · 2+0 · 8)组和HPV16LlVLP-lym+10ym (0 · 8+0 · 2)组。 [0222] The results are shown Ιμπι two or ΙΟμπι particle diameter by a certain percentage or a combination of embedded HPV16L1VLP PLGA and induced no significant difference in antibody titers, whereas Iym PLGA-HPV16L1VLP group after the first immunization induced significant antibody titers binding below HPV16LlVLP-lym + 10ym (0 · 5 + 0 · 5) group, after the second immunization were significantly lower than HPV16LlVLP-lym + 10y m (0 · 2 + 0 · 8) group and HPV16LlVLP-lym + 10ym ( 8 + 0 · 0 · 2) groups.

[0223] 实施例一百三十八——不同佐剂的HPV 16L1VLP免疫效应的研究 Immune effect of HPV 16L1VLP different adjuvant - one hundred thirty-eight [0223] Example

[0224] 以PLGA,PLGA/HSPC,PLA包埋HPV 16L1VLP免疫小鼠后,诱导产生的中和抗体和结合抗体滴度均无显著差异,如图30和31。 [0224] In PLGA, PLGA / HSPC, after embedding PLA HPV 16L1VLP immunized mice, and the induction of antibody production and binding of antibody titers were not significantly different, 30 and 31.

[0225] 实施例一百三十九——PLGA的颗粒的制备 Preparation [0225] Example embodiments particles of one hundred thirty-nine --PLGA

[0226] 将IOOmg的PLGA溶于20mL的二氯甲烷中作为油相(0),配制1.0%的PVA溶液作为外水相(W2),将油相(0)加入至50mL的外水相(W2)中,制备得到水包油型的预乳液(W2/0),将此预复乳液采用快速膜乳化的方法反复过膜制备得到粒径均一的水包油的复乳液(0/W2); 固化3h,离心洗涤,冻干,得到干燥的PLGA微球,PLGA的SEM照片如图2所示。 [0226] The IOOmg of PLGA was dissolved in 20mL of dichloromethane as an oil phase (0), formulated as a 1.0% solution of PVA external aqueous phase (W2 of), the oil phase (0) 50mL was added to an external aqueous phase ( W2), a pre-prepared oil-in-water emulsion (W2 / 0), this pre-emulsion is multiplexing using flash membrane emulsification method for preparing a film obtained through repeated re oil in water emulsion of uniform particle size (0 / W2) ; curing 3h, washed by centrifugation and lyophilized to obtain dry PLGA microspheres, PLGA SEM photograph shown in Fig.

[0227] 实施例一百四十——PLGA的颗粒吸附HPV16L1VLP疫苗组合物的制备 Preparation of vaccine composition HPV16L1VLP particles [0227] Example Adsorption one hundred forty --PLGA

[0228] 将制备得到的PLGA微球溶于0.8mL的pH=5、10mM PBS缓冲溶液中,得20mg/mL的PLGA微球悬液;取一定量的HPV溶于0.8mL的pH = 5、IOmM PBS缓冲溶液中,得20ug/mL的HPV 抗原溶液,将两者等体积混合,吸附过夜,得到1.6mL吸附有HPV抗原的PLGA悬液,即为HPV疫苗组合物。 [0228] The preparation of PLGA microspheres obtained is dissolved in pH 0.8mL = 5,10mM PBS buffer solution to give 20mg / mL suspension of PLGA microspheres; HPV certain amount of the dissolved pH 0.8mL = 5, IOmM PBS buffer solution to give an HPV antigen solution 20ug / mL, the volume of the mixture of the two, etc., adsorbed overnight, to give a suspension of PLGA has 1.6mL adsorbed HPV antigens, namely HPV vaccine composition.

[0229] 实施例一百四十——PLGA微球对HPV 16L1VLP吸附免疫应答的研究 Immune responses [0229] Example one hundred forty --PLGA microspheres adsorption of HPV 16L1VLP

[0230] 取实施例一百三十九中1施例的PLGA吸附HPV 16L1VLP样品后免疫小鼠,PLGA-HPV 16L1VLP可以诱导与Merck公司的Gardasil疫苗相当水平的结合抗体滴度。 [0230] Example embodiments taken one hundred thirty-nine PLGA in Example 1 after the adsorption of HPV 16L1VLP sample immunized mice, PLGA-HPV 16L1VLP induce binding of the antibody titer of Merck vaccine Gardasil comparable levels. 结果如图32。 The results shown in FIG 32.

Figure CN104338126BD00351

Figure CN104338126BD00361

Figure CN104338126BD00371

Figure CN104338126BD00381

Figure CN104338126BD00391

Figure CN104338126BD00401

Figure CN104338126BD00411

Figure CN104338126BD00421

Figure CN104338126BD00431

Figure CN104338126BD00441

Claims (20)

1. 一种疫苗组合物,包含与佐剂组合的抗原,抗原与佐剂的比例为l〇-8〇yg/mg,所述抗原包含HPV Ll蛋白,所述佐剂组合物包括聚乳酸-聚乙醇酸共聚物PLGA或聚乳酸PLA,所述的HPV Ll蛋白包埋在PLGA或PLA中或吸附在PLGA或PLA表面,均以纳微球形式存在,所述的纳微球10-90%分布在1-10μπι、90-10%分布在10-100μπι,所述的纳微球在每一个粒径范围内的粒径分散系数范围为0.005〜0.2;所述佐剂中进一步包含卵磷脂,其中卵磷脂与PLGA 或PLA的比例为1-99:99-1;其中HPV Ll蛋白是指HPV全长、截短或重组的Ll形成的VLP或五聚体。 Ratio of 1. A vaccine composition comprising an antigen, an antigen and an adjuvant in combination with an adjuvant for l〇-8〇yg / mg, the antigen comprises HPV Ll proteins, the adjuvant composition comprising a polylactic acid - glycolic acid PLGA or PLA polylactic acid, the HPV Ll proteins embedded in PLGA or PLA, PLGA or PLA or adsorbed surface, are present in the form of microspheres is satisfied, said satisfied microspheres 10-90% distributed 1-10μπι, 90-10% distribution 10-100μπι, a particle size range of the scattering coefficient susceptance microspheres in each size range of 0.005~0.2; further comprising a lecithin in the adjuvant, wherein the ratio of lecithin and PLGA or PLA 1-99: 99-1; refers to a protein wherein the HPV Ll VLP or HPV pentamer full length, truncated or recombinant Ll formed.
2. 如权利要求1的疫苗组合物,其中所述的纳微球10-90%分布在3-8μπι、90-10%分布在30-80μπι。 2. The vaccine composition of claim 1, wherein said nano microspheres distributed in 10-90% 3-8μπι, 90-10% distribution 30-80μπι.
3. 如权利要求2的疫苗组合物,其中所述的纳微球10-90%分布在5-6μπι、90-10%分布在5〇-60ym。 3. A vaccine composition as claimed in claim 2, wherein said nano microspheres distributed in 10-90% 5-6μπι, 90-10% distributed 5〇-60ym.
4. 如权利要求3所述疫苗组合物,其中所述佐剂中进一步包含小分子免疫佐剂:CpG、 MPLA、咪喹莫特、PoIyI: C中的一种或几种的组合,其中小分子免疫佐剂与PLGA或PLA的比例为10-100yg/mg。 Wherein C is a combination of small or more of: 4. A vaccine composition as claimed in claim 3, wherein the adjuvant further comprises a small molecule immunoadjuvant: CpG, MPLA, imiquimod, PoIyI the proportion of molecules with PLGA or PLA immunological adjuvant is 10-100yg / mg.
5. 如权利要求1所述疫苗组合物,其中HPV LI蛋白是指6、11、16、18、26、30、31、33、34、 35、39、45,51、52、53、56、58、59、66、67、68、69、70、72、73或82、85、97型册¥中一种和/或几种的组合。 5. The vaccine composition as claimed in claim 1, wherein the HPV LI protein refers 6,11,16,18,26,30,31,33,34, 35,39,45,51,52,53,56, and 58,59,66,67,68,69,70,72,73 or 82,85,97 or combinations of one type of book ¥ / several of.
6. —种疫苗制剂,其特征在于该疫苗制剂由权利要求5所述的疫苗组合物与HPV Ll蛋白及药用辅料制成,其中纳微球中HPV Ll与游离HPV Ll比例为10-90% :90-10%。 6. - vaccines formulations, wherein the vaccine formulation of the vaccine composition prepared as claimed in claim 5, wherein the HPV Ll protein, and a pharmaceutically acceptable adjuvant, wherein the microspheres in sodium free HPV Ll and HPV Ll ratio of 10-90 %: 90 to 10%.
7. 如权利要求6所述的疫苗制剂,其特征在于所述的药用辅料为生理盐水或PBS缓冲液。 7. The vaccine formulation according to claim 6, wherein the pharmaceutical excipient is physiological saline or PBS buffer.
8. 如权利要求4所述疫苗组合物的制备方法,该方法包括如下步骤: (1) 含药乳滴的制备:将HPV LI蛋白和小分子免疫佐剂CpG溶于缓冲盐体系制备成pH值范围为4.0-12.0的内水相Wl; (2) 将PLGA或PLA与脂类物质溶于有机溶剂中制成油相0,将所述内水相Wl与油相0混合,制成油包水型W1/0预乳液,将此预乳液加入外水相中,制得W1/0/W2型预复乳液,将所述W1/0/W2型预复乳液反复过膜得到尺寸均一的W1/0/W2型乳液; ⑶乳滴固化:将W1/0/W2型乳液常温固化,洗涤、干燥后制成疫苗组合物。 8. The method of preparing the vaccine composition of claim 4 wherein said process comprising the steps of: (1) preparing a drug-containing emulsion droplets: the HPV LI proteins and small molecule immune system by CpG dissolved in a buffered saline pH prepared value within the range of 4.0 to 12.0 aqueous phase Wl; (2) the PLA or PLGA and lipid substances in an organic solvent to prepare an oil phase 0, the inner aqueous phase with the oil phase 0 Wl mixed to prepare an oil water-in-W1 / 0 pre-emulsion, this pre-emulsion was added to the outer aqueous phase to prepare a W1 / 0 / W2 emulsion type pre complex, the W1 / 0 / W2-type double emulsion repeatedly through the pre-film obtained uniform size W1 / 0 / W2 emulsion; ⑶ emulsion droplets curing: W1 / 0 / W2 emulsion cured at room temperature, washed, and dried to prepare a vaccine composition.
9. 如权利要求5所述疫苗组合物的制备方法,该方法包括如下步骤: (1)将PLGA或PLA与脂类物质溶于有机溶剂中制成油相0,将此油相加入外水相W2中,制得0/W2型预复乳液; ⑵将所述0/W2型预复乳液反复过膜得到尺寸均一的0/W2型乳液;洗涤、干燥后得PLGA 或PLA微球; (3) 将HPV Ll蛋白和小分子免疫佐剂CpG溶于缓冲盐体系中,得l-40yg/0.2-lmL的HPV 抗原溶液,将PLGA或PLA微球溶于缓冲盐体系中,得1-lOmg/mL的溶液;将两溶液等体积混合,吸附过夜,得到吸附有HPV抗原的PLGA悬液,即为HPV疫苗组合物。 9. The method of preparing the vaccine composition of claim, the method comprising the steps of: (1) the PLA or PLGA and lipid substances are dissolved in an organic solvent in the oil phase is made 0, this oil phase was added the outer aqueous phase W2, prepared 0 / W2 emulsion type pre complex; ⑵ the 0 / W2-type double emulsion repeatedly through the pre-film obtained 0 / W2 emulsion of uniform size; washed and dried to obtain PLA or PLGA microspheres; ( 3) the HPV Ll proteins and small molecule is dissolved in a buffered saline immunological adjuvant CpG system to give l-40yg / HPV antigen solution in 0.2-lmL, the PLA or PLGA microspheres soluble salt buffer system to give 1-lOmg / mL solution; the two solutions were mixed by volume, etc., adsorbed overnight, to give adsorbed HPV antigen PLGA suspension, namely HPV vaccine composition.
10. 如权利要求8或9所述疫苗组合物的制备方法,其特征在于所述缓冲盐体系为枸橼酸-磷酸氢二钠、醋酸一醋酸钠缓冲液或磷酸盐缓冲液。 10. A method of preparing 8 or the vaccine composition of claim 9, characterized in that the buffer system is a citric acid salt - disodium hydrogen phosphate, acetate, sodium acetate buffer or a phosphate buffer.
11. 如权利要求8所述疫苗组合物的制备方法,其特征在于,步骤(1)所述的内水相pH值范围为6.0-8.0。 11. The method of preparing the vaccine composition of claim 8, characterized in that, in step (1) of the aqueous phase within the pH range 6.0 to 8.0.
12. 如权利要求8或9所述疫苗组合物的制备方法,其特征在于,所述油相为常温下呈液体与水不互溶的油性物质。 12. The method as 8 or the vaccine composition of claim 9, characterized in that the oil was a liquid oily substance with a water-immiscible phase at ordinary temperature.
13. 如权利要求8或9所述疫苗组合物的制备方法,其特征在于,所述的外水相W2为含有0.001-10 %的PVA水溶液,其醇解度为87〜90%,聚合链节数为1700-1750;或为含有0.001-10%的PVA-PEG混合水溶液,PVA与PEG的质量百分比为20-80:30-90。 13. The method as 8 or the vaccine composition of claim 9, characterized in that the external aqueous phase W2 of an aqueous solution containing 0.001 to 10% of PVA which alcoholysis degree 87~90%, the polymeric chains section number is 1700-1750; or containing 0.001 to 10% of the mixed aqueous solution of PVA-PEG, PVA and PEG mass percentage of 20-80: 30-90.
14. 如权利要求8所述疫苗组合物的制备方法,其特征在于,步骤(2)中所述的油包水型Wl/Ο型预乳液是通过采用均相乳化器或超声波乳化器制得的。 14. The method of preparing the vaccine composition of claim 8, characterized in that, in step (2) in the water-in-oil Wl / Ο type a homogeneous pre-emulsion is obtained by using an ultrasonic emulsifier or emulsifier prepared of.
15. 如权利要求8所述疫苗组合物的制备方法,其特征在于,步骤(2)中所述的尺寸均一的W1/0/W2型乳液是通过将W1/0/W2型预乳液在压力作用下反复压过微孔膜得到的。 15. The method of preparing the vaccine composition of claim 8, characterized in that, in step (2) W1 / 0 / W2 emulsion in the size is uniform by W1 / 0 / W2 type pre-emulsion at a pressure repeatedly pressed through the action of the microporous film obtained.
16. 如权利要求8所述疫苗组合物的制备方法,其特征在于,固化方式为溶剂蒸发法或溶剂萃取法。 16. The method of preparing the vaccine composition of claim 8, characterized in that the method is curable solvent extraction or solvent evaporation method.
17. 如权利要求8所述疫苗组合物的制备方法,其特征在于,固化时间为lh-8h。 17. The method of preparing the vaccine composition of claim 8, characterized in that the curing time is lh-8h.
18. —种疫苗制剂的制备方法,其特征在于在如权利要求8所述疫苗组合物的制备方法中的乳滴固化步骤后还包括如下步骤: ⑷疫苗制剂形成:取质量比10-90% :90-10%的疫苗组合物与游离的HPVLl蛋白用生理盐水或PBS缓冲液悬浮,配制成含有l-40yg/0.2-lmL的HPV抗原的微球悬液,制得HPV疫苗制剂。 18. - Preparation of a vaccine formulation types, characterized in that the milk in the method of preparing 8 vaccine composition as claimed in claim dropwise post curing step further comprises the step of: forming ⑷ vaccine formulation: Take the mass ratio of 10-90% : 90 to 10% of the vaccine composition HPVLl protein free PBS buffer with saline or a suspension, the suspension is formulated into microspheres containing antigen HPV l-40yg / 0.2-lmL was prepared HPV vaccine formulation.
19. 如权利要求5所述的疫苗组合物在制备治疗或预防HPV感染的药物中的应用。 19. The vaccine composition of claim 5 preparation of a medicament for the treatment or prevention of HPV infection in the claims.
20. 如权利要求6或7所述的疫苗制剂在制备治疗或预防HPV感染的药物中的应用。 20. A vaccine formulation as claimed in claim 6 or 7 Application of a medicament for treating or preventing HPV infections requirements.
CN201410382617.4A 2013-08-08 2014-08-06 Having a vaccine composition and treating or preventing viral hpv CN104338126B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201310343768 2013-08-08
CN201410382617.4A CN104338126B (en) 2013-08-08 2014-08-06 Having a vaccine composition and treating or preventing viral hpv

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410382617.4A CN104338126B (en) 2013-08-08 2014-08-06 Having a vaccine composition and treating or preventing viral hpv

Publications (2)

Publication Number Publication Date
CN104338126A CN104338126A (en) 2015-02-11
CN104338126B true CN104338126B (en) 2018-05-04

Family

ID=52495117

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410382617.4A CN104338126B (en) 2013-08-08 2014-08-06 Having a vaccine composition and treating or preventing viral hpv

Country Status (1)

Country Link
CN (1) CN104338126B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106031794A (en) * 2015-03-20 2016-10-19 中国科学院过程工程研究所 Intracellular pH-response polylactic-acid nanometer microspheres and preparing method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103118700A (en) * 2010-05-26 2013-05-22 西莱克塔生物科技公司 Synthetic nanocarrier combination vaccines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103118700A (en) * 2010-05-26 2013-05-22 西莱克塔生物科技公司 Synthetic nanocarrier combination vaccines

Also Published As

Publication number Publication date
CN104338126A (en) 2015-02-11

Similar Documents

Publication Publication Date Title
Kim et al. Injectable, spontaneously assembling, inorganic scaffolds modulate immune cells in vivo and increase vaccine efficacy
Makidon et al. Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine
van der Lubben et al. Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice
Csaba et al. Nanoparticles for nasal vaccination
Hu et al. Nanoparticle-detained toxins for safe and effective vaccination
Slütter et al. Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen
AU2007217514B2 (en) Nanoparticles for immunotherapy
Perrie et al. Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens
De Koker et al. Designing polymeric particles for antigen delivery
US8323696B2 (en) Nanoparticles for immunotherapy
Chong et al. Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery
US20080311214A1 (en) Polymerized solid lipid nanoparticles for oral or mucosal delivery of therapeutic proteins and peptides
Mori et al. The vaccine adjuvant alum inhibits IL‐12 by promoting PI 3 kinase signaling while chitosan does not inhibit IL‐12 and enhances T h1 and T h17 responses
Gamvrellis et al. Vaccines that facilitate antigen entry into dendritic cells
Peek et al. Nanotechnology in vaccine delivery
Gupta et al. M-cell targeted biodegradable PLGA nanoparticles for oral immunization against hepatitis B
Jain et al. Mannosylated niosomes as adjuvant–carrier system for oral genetic immunization against Hepatitis B
Sarti et al. In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A
Petrovsky et al. Vaccine adjuvants: current state and future trends
O'Hagan et al. Poly (butyl-2-cyanoacrylate) particles as adjuvants for oral immunization
Lawson et al. Defending the mucosa: adjuvant and carrier formulations for mucosal immunity
HogenEsch Mechanism of immunopotentiation and safety of aluminum adjuvants
Kumar et al. Shape and size-dependent immune response to antigen-carrying nanoparticles
Kang et al. Pluronic® F127 enhances the effect as an adjuvant of chitosan microspheres in the intranasal delivery of Bordetella bronchiseptica antigens containing dermonecrotoxin
Bal et al. Efficient induction of immune responses through intradermal vaccination with N-trimethyl chitosan containing antigen formulations

Legal Events

Date Code Title Description
C06 Publication
C10 Entry into substantive examination
GR01