CN104338126A - Vaccine composition for treating or preventing HPV virus and application of vaccine composition - Google Patents

Abstract

The invention relates to a vaccine composition and application thereof in medicines, and in particular relates to a vaccine composition or a vaccine preparation which is prepared from nano microspheres coated with HPV L1 protein, and application of the vaccine composition or the vaccine preparation in medicines for treating or preventing HPV virus. Specifically, aiming at the influence of the particle size and combination of the nano microspheres, nano microsphere materials and a nano microsphere antigen loading mode on the immune response of HPV L1 antigen protein, PLGA, PLA, PLGA/PC nano microspheres of which the particle sizes are uniform or different are designed and prepared, the nano microspheres are coated with or adsorb HPV L1 antigen protein so as to prepare the vaccine composition or the vaccine preparation, the influence of the vaccine composition or the vaccine preparation on the in-vivo immune response intensity and the level is studied, and an efficient HPV L1 protein vaccine composition is developed.

Description

A kind of vaccine combination and application thereof with treatment or prevention HPV virus

Technical field

The present invention relates to vaccine combination and the purposes in medicine thereof.Particularly relate to by bag by or absorption HPV L1 albumen receive the vaccine combination or bacterin preparation and the application in treatment or prevention HPV virus drugs thereof that microsphere makes.

Technical background

Human papillomavirus (Human papillomaviruses, HPV) is the double-stranded DNA virus without cyst membrane, primarily of virus coat and genomic DNA composition (Bernard, Burk et al.2011).HPV virus coat is by 360 L1 albumen (forming 72 pentamers) and the icosahedral structure of virus that forms of 72 L2 albumen at the most, diameter 55-60nm (Howley and Lowy2007).Virus capsid protein has self assembly characteristic, L1 albumen separately or form viroid sample granule (Virus-like Particle with L2 albumen co-assemble in vitro, VLP) (Chen, Garcea et al.2000, Finnen, Erickson et al.2003, Buck, Cheng et al.2008, Wang and Roden 2013).Current HPV vaccine is all using VLP as target antigen, has two kinds based on HPV L1VLP preventative vaccine listing (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).On June 8th, 2006, the Gardasil HPV preventative vaccine listing that FDA Food and Drug Administration (FDA) official approval Merck company of the U.S. (i.e. MSD Corp.) is produced, it is expressed and the HPV16/18/6/11L1VLP tetravalence cancer, prophylaxis vaccine of purification by saccharomyces cerevisiae, with amorphous Adju-Phos aluminium sulfate (amorphous aluminum Hydroxyphosphatesulfate, AAHS) be adjuvant, be approved for prevention 6 ~ 26 years old girl and women HPV16, 18, 6, cervical cancer caused by 11 types infect, precancerous lesion and genital wart, this is first tumor vaccine (Villa in the world that FDA passes through, Costa et al.2005, Villa, Ault et al.2006, Bryan 2007, Olsson, Villa et al.2007, Goldstone and Vuocolo 2012).The HPV preventative vaccine that the commodity that Britain's GlaxoSmithKline PLC (GSK) company produces subsequently are called Cervarix also successfully goes on the market, it is the HPV16/18L1VLP bivalence cancer, prophylaxis vaccine that origin comes from insect expression system, adopt AS04 adjuvant (aluminium hydroxide compound MPL) (Paavonen, Jenkins et al.2007, Garcon, Morel et al.2011, Kreimer, Gonzalez et al.2011, Szarewski 2012).Clinical trial certificate, above-mentioned two kinds of preventative vaccines all have good toleration, and without serious adverse reaction (Paavonen, Jenkins et al.2007, Reisinger, 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, Wacholder 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).But these two kinds of preventative vaccines are expensive, and need cryopreservation, strongly limit the use in developing country and backward areas, therefore the high-titer HPV vaccine developing a kind of low cost just seems 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).Research shows; so that the immunne response (Rose, White et al.1998, the Yuan that produce protectiveness can be induced after HPV L1 pentamer protein immunization laboratory animal; Estes et al.2001; Ohlschlager, Osen et al.2003, Senger; Schadlich et al.2010; Wu, Gersch et al.2011), and the production cost of HPV vaccine greatly can be reduced based on the expression technology of prokaryote E coli.In Chinese invention patent 02129070.9 " the protokaryon Synthesis and applications of papillomatosis virus capsid protein ", provide the HPV 16L1 space three-dimensional structure of pentamer, and utilize the technical method of escherichia coli great expression and preparation human papillomavirus capsid protein L 1.

Aluminium adjuvant is the class adjuvant be most widely used at present, there are the applicating history of more than 80 year and the use record of tens billion of person-times, also be the unique adjuvant (Glenny being once used for human vaccination for a long time by U.S. food and Drug Administration (FDA) approval, Pope et al.1926, Gupta and Siber 1995, Gupta 1998, Baylor, Egan et al.2002, Clements and Griffiths 2002, Lindblad 2004, Lindblad 2004).But it comes with some shortcomings in the application of HPV L1 proteantigen.Alum adjuvant only forms Reservoir effect (Gupta in injection site, Chang et al.1996, Hem 2002, Verdier, Burnett et al.2005, Hem and Hogenesch 2007, Noe, Green et al.2010), attract dendritic cell (DC) (Morefield, Sokolovska et al.2005 by inflammatory reaction, Kool, Soullie et al.2008, Sharp, Ruane et al.2009, Flach, Ng et al.2011, Ghimire, Benson et al.2012), macrophage (Hamilton, Byrne et al.2000, Jordan, Mills et al.2004, Rimaniol, Gras et al.2004, Rimaniol, Gras et al.2007) etc. antigen-presenting cell (antigen-presenting cell, APC), these cells are by phagocytosis antigen uptaking and through NLRP3 inflammatory corpusculum activate immunity response (De Gregorio, Tritto et al.2008, Dostert, Petrilli et al.2008, Eisenbarth, Colegio et al.2008, Franchi and Nunez 2008, Kool, Petrilli 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).Simultaneously; aluminium adjuvant produces inhibitory action (Chen, Ni et al.2011) by stimulating the mechanism such as IL-10 secretion to cellular immunization, and for viral infection; Study On Cellular Immune will play better immunoprotection and immune protection effect (O ' Hagan and Valiante2003; Demento, Cui et al.2012, Levitz and Golenbock 2012; Olive 2012; Cain, Sanders et al.2013, HogenEsch 2013).In addition, the anaphylaxis (as injection site granuloma) that aluminium adjuvant can cause IgE to mediate and nervous system untoward reaction etc., thus cause people to the worry (Petrik of aluminium adjuvant safety, Wong et al.2007, Bystrianyk2009, Shaw and Petrik 2009, Munks, McKee et al.2010, Tomljenovic and Shaw 2011).No matter for this reason, need for HPV L1 proteantigen, be to based on L1 pentamer, or candidate vaccine development of new immunological adjuvant (Campo and Roden 2010, the Mariani and Venuti 2010, Chen of L1VLP, Ni et al.2011, Foged 2011, Gattoc, Nair et al.2013, Koff, Burton et al.2013, Shaw 2013, Tomljenovic, Spinosa et al.2013).

Lot of documents confirms in recent years, in body, biodegradable polymer nano microsphere is by embedding antigen, granular pattern antigen can be translated into, be conducive to being absorbed by antigen presenting cell, and then in born of the same parents released antigen, by follow-up antigen processing, offer, strengthen immune response strength and level (Langer, Cleland et al.1997, Johansen, Men et al.2000, Sahay, Alakhova et al.2010, De Temmerman, Rejman et al.2011, Danhier, Ansorena et al.2012).Such as, the people such as Torres M.P. have prepared poly-glycoside microsphere, with ovalbumin (OVA) for model antigen, the adjuvant effect of research microsphere, result shows that microsphere can improve antigen presenting cell surface MHC developed by molecule and relevant cell factor secretion, show it and there is certain adjuvant effect (Torres, Wilson-Welder et al.2011); And for example the people such as Uto T. has prepared biodegradable polyglutamic acid and has received microsphere (γ-PGA NPs) have studied its adjuvant properties from mechanism, disclose this microsphere and bring out strong intrinsic and Acquired immune response reaction (Uto, Akagi et al.2011) by TLR4 (Toll-like receptor) and MyD88 signal path.Research shows, the above-mentioned particle type adjuvant carrying antigen makes soluble antigen become granular pattern antigen, and after granular pattern antigen absorbed by APC, the approach of offering of antigen can be changed, not only can activate CD4+T cell, and CD8+T cell can be activated, greatly promoting cellular immune function, realize the thorough removing of intracellular infection, is promising viral infection vaccine adjuvant and delivery system (Wang and Singh 2011, Dierendonck, De Koker et al.2012).In addition; biodegradable polymer microsphere can provide a large amount of antigen and protect it from fast degradation in physiological conditions; by microsphere continuing or pulse release behavior embedded antigen; effective simulation traditional vaccine repeatedly immune programme for children; reduce inoculation times and immunogenic use total amount; thus improve the adaptability of patient, reduce drug cost (Langer; Cleland et al.1997; Johansen; Estevez et al.2000, De Temmerman, Rejman et al.2011; Demento, Cui et al.2012).

As adjuvant and the delivery system of antigen protein, a lot of physicochemical properties of polymer beads can affect its in vivo with the interaction of immunocyte, and finally affect type of immune response, intensity and level, comprise (Gupta and Siber 1995, the Hem and Hogenesch 2007, Mastelic such as particle diameter, form, surface nature, Ahmed et al.2010, Wang and Singh 2011, Korsholm, Andersen et al.2012).Such as, (the Xiang such as Xiang S.D., Scholzen et al.2006) to mention in survey article, granule and surface band negative charge and uncharged Particle Phase ratio of surface band positive charge, be easier to antibody and the CD8+T cell response of induced high levels; The people (Foged, Brodin et al.2005) such as Foged C. have studied the impact of Particle surface charge on the behavior of DC cellular uptake, and obtain similar results, namely the granule of surface band positive charge is easier to be absorbed by DC; And the research of the people (Tabata and Ikada 1988) such as Yasuhiko T. obtains contrary conclusion, they think that electronegative cellulose microsphere can promote the picked-up of macrophage.Equally, polymer beads particle diameter also not yet forms unified final conclusion to the impact of immunology effect.The people (Gutierro, Hernandez et al.2002) such as Gutierro I. finds, compare with 500nm particle diameter with 200,1 compares, PLGA microsphere can produce high-level antibody; People (the Mann such as Mann J.S.F., Shakir et al.2009) prepare the natural biological membrane vesicles (Biosome) of year influenza antigens, result shows that small particle diameter is conducive to inducement efficient humoral immunoresponse(HI), and Large stone is then inclined to induction body and produces cellullar immunologic response; The people such as WendorfJ (Wendorf, Chesko et al.2008) adopt surperficial electronegative PLGA microsphere (110nm and 1000nm) adsorb HIV-1 and bring out meningitic MenB (Meningococcal B) albumen, 2 particle diameters create similar immunne response.Different researcher obtains different results for particle identity matter.The present invention is directed to HPV L1 antigen protein, receive the impact of microspherulite diameter size on its immunne response, uniform particle diameter is prepared in design, the PLGA of different-grain diameter receives microsphere, with its embedding HPV L1 antigen protein, research PLGA receives the impact of microspherulite diameter on immune response strength in body and level, develops efficient HPV L1 protein vaccine composition.

Summary of the invention

First aspect the invention provides a kind of vaccine combination, comprise the antigen with adjuvant combination, the ratio of antigen and adjuvant is 10-80 μ g/mg, described antigen comprises HPV L1 albumen, and described adjunvant composition comprises PLGA (PLGA) or polylactic acid (PLA).

Vaccine combination of the present invention in a particular embodiment of the present invention, wherein said HPV L1 albumen is embedded in PLGA or is adsorbed on PLGA surface, all exists with the microspheres form of receiving of particle size distribution between 100nm ~ 100 μm.

Vaccine combination in specific embodiment of the present invention, the wherein said microsphere 0-90% that receives is distributed between 100-1000nm, 1-10 μm or 10-100 μm of arbitrary particle size range or its combination.

Vaccine combination in specific embodiment of the present invention, the microsphere 0-90% that preferably receives is distributed between 200-900nm, 2-9 μm or 20-90 μm of arbitrary particle diameter or its combination.

Vaccine combination in specific embodiment of the present invention, the microsphere 0-90% that preferably receives is distributed between 400-700nm, 4-7 μm or 40-70 μm of arbitrary particle diameter or its combination.

Vaccine combination in specific embodiment of the present invention, the described microsphere 10-90% that receives is distributed in 100-1000nm, 90-10% and is distributed in 1-10 μm.

Vaccine combination in a specific embodiment of the present invention, the microsphere 10-90% that preferably receives is distributed in 300-800nm, 90-10% and is distributed in 3-8 μm.

Vaccine combination in a specific embodiment of the present invention, more preferably the described microsphere 10-90% that receives is distributed in 500-600nm, 90-10% and is distributed in 5-6 μm.

Vaccine combination in specific embodiment of the present invention, wherein said receives that microsphere 10-90% is distributed in 1-10 μm, 90-10% is distributed in 10-100 μm.

Vaccine combination in a specific embodiment of the present invention, preferably describedly receives that microsphere 10-90% is distributed in 3-8 μm, 90-10% is distributed in 30-80 μm.

Vaccine combination in another specific embodiment of the present invention, preferably wherein saidly receives that microsphere 10-90% is distributed in 5-6 μm, 90-10% is distributed in 50-60 μm.

Vaccine combination in specific embodiment of the present invention, the microsphere 10-90% that preferably receives is distributed in 100-1000nm, 90-10% and is distributed in 10-100 μm.

Vaccine combination in another specific embodiment of the present invention, the wherein said microsphere 10-90% that receives is distributed in 300-800nm, 90-10% and is distributed in 30-80 μm.

Vaccine combination in another specific embodiment of the present invention, the wherein said microsphere 10-90% that receives is distributed in 500-600nm, 90-10% and is distributed in 50-60 μm.

In specific embodiment of the present invention, the wherein said particle size dispersion coefficient scope of microsphere in each particle size range of receiving is 0.005 ~ 0.2.

Vaccine combination in a specific embodiment of the present invention, following immunostimulant component lipid material is comprised further: one or more combination in lecithin, stearylamine, phosphoglyceride in wherein said adjuvant, wherein the ratio of lipid material and PLGA or PLA is 1-99: 99-1, preferred 1-50: 100.

More specifically, vaccine combination in specific embodiment of the present invention, small molecule immune adjuvant is comprised further: one or more combination in CpG, MPLA, imiquimod, PolyI:C etc., wherein the ratio of small molecule immune adjuvant and PLGA or PLA is 10-100 μ g/mg in wherein said adjuvant.

Vaccine combination in specific embodiment of the present invention, wherein HPV L1 albumen refers to HPV total length, the L1 of truncate or restructuring is formed VLP, pentamer or polymer.

Vaccine combination in specific embodiment of the present invention, wherein HPV L1 albumen refers to 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 or 82,85, combinations a kind of and/or several in 97 type HPV.

The present invention discloses a kind of bacterin preparation on the other hand, this bacterin preparation is made up of the vaccine combination described in 19 and HPV L1 albumen and pharmaceutic adjuvant, wherein receiving HPV L1 and free HPV L1 ratio in microsphere is 10-90%: 90-10%, further more preferably 10-50%: 90-50%.

Bacterin preparation in specific embodiment of the present invention, described pharmaceutic adjuvant is normal saline or PBS buffer.

In specific embodiment of the present invention, the preparation method of vaccine combination mainly comprises embedding and absorption two kinds of methods on the other hand, and wherein this embedding method comprises the steps:

(1) preparation of pastille emulsion droplet: HPV L1 albumen and/or small molecule immune adjuvant CpG etc. are dissolved in buffer salt system and are prepared into interior aqueous phase (W1);

(2) PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent make oil phase (O), described aqueous phase (W1) is mixed with oil phase (O), make water-in-oil type (W1/O) pre-emulsion, this pre-emulsion is added in outer aqueous phase (W2), the obtained pre-double emulsion of W1/O/W2 type, crosses the pre-double emulsion of described W1/O/W2 type the W1/O/W2 type emulsion that film obtains size uniformity repeatedly;

(3) emulsion droplet solidification: by the normal temperature cure of W1/O/W2 type emulsion, makes vaccine combination after washing, drying.

A kind of preparation method is provided in specific embodiment of the present invention, interior aqueous pH values scope described in step (1) is 4.0-12.0, and the buffer salt system described in step (1) is citric acid-sodium hydrogen phosphate, Acetic acid-sodium acetate buffer or phosphate buffer.

The preparation method provided in a specific embodiment of the present invention, the interior aqueous pH values scope described in step (1) is 6.0-8.0.

In a specific embodiment of the present invention, step (2) described oil phase is liquid and the immiscible oily matter of water under being room temperature, is preferably ethyl acetate, acetone, dichloromethane, chloroform or above-mentioned at least two or more mixture.

Outer aqueous phase (W2) in specific embodiment of the present invention described in step (2) is the PVA aqueous solution containing 0.001-10%, and its alcoholysis degree is 87 ~ 90%, and polymeric chain joint number is 1700-1750; Or be the PVA-PEG mixed aqueous solution containing 0.001-10%, the mass percent of PVA and PEG is 20-80: 30-90.

Water-in-oil type (W1/O type) pre-emulsion in specific embodiment of the present invention described in step (2) obtains by adopting homogeneous phase emulsator or ultrasonic emulsator.

In specific embodiment of the present invention, the W1/O/W2 type emulsion of the size uniformity described in step (2) obtains by W1/O/W2 type pre-emulsion is repeatedly pressed through microporous membrane under elevated pressures effect.

In specific embodiment of the present invention described in step (2) containing antigen emulsion droplet, aqueous phase (W1) and oil phase (O) volume ratio are 1: 1 ~ 1: 50.

In specific embodiment of the present invention, step (3) curing mode is solvent evaporated method and solvent extraction.

In specific embodiment of the present invention, step (3) hardening time is 1h-8h.

The preparation method of another kind of vaccine combination is disclosed on the other hand in specific embodiment of the present invention, namely adsorption method comprises the steps that PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent by (1) and makes oil phase (O), this oil phase is added in outer aqueous phase (W2), the obtained pre-double emulsion of O/W2 type;

(2) the pre-double emulsion of described O/W2 type is crossed the O/W2 type emulsion that film obtains size uniformity repeatedly; PLGA/PLA microsphere is obtained after washing, drying;

(3) HPV L1 albumen and/or small molecule immune adjuvant CpG are dissolved in buffer salt system, obtain the HPV antigenic solution of 1-40 μ g/0.2-1mL, PLGA/PLA microsphere is dissolved in buffer salt system, obtain the solution of 1-10mg/mL; By two solution equal-volume mixing, attach overnight, obtains the PLGA suspension being adsorbed with HPV antigen, is HPV vaccine combination.

The above-mentioned concrete operation step mentioned in adsorption method can with reference to the corresponding steps in embedding method.

A kind of preparation method of bacterin preparation is provided on the other hand in specific embodiment of the present invention, also comprises the steps: after the emulsion droplet curing schedule in the preparation method of vaccine combination

Bacterin preparation is formed: the vaccine combination (receive microsphere) getting mass ratio 10-90%: 90-10% suspends with free HPVL1 albumen normal saline or PBS buffer, be mixed with the microsphere suspensions of the HPV antigen containing 1-40 μ g/0.2-1mL, obtained HPV bacterin preparation.

The preparation method of the bacterin preparation provided in specific embodiment of the present invention, PBS ph value of buffer solution scope 6-8, microsphere concentration of receiving is 1-10mg/mL.

The application of open vaccine combination in the medicine that preparation is treated or prevention HPV infects in specific embodiment of the present invention on the other hand.

In the application of bacterin preparation disclosed in specific embodiment of the present invention in the medicine that preparation is treated or prevention HPV infects.

In an embodiment of any method provided, use this dosage form by oral, subcutaneous, pulmonary, intranasal, abdominal cavity, lymph, intradermal or intramuscular delivery.

In another embodiment, the bacterin preparation of a kind of human papillomavirus is provided, it is that wrapping by the homogeneous microsphere of PLGA of HPV L1VLP antigen is main a kind of bacterin preparation, can embed altogether or other molecule adjuvants composite simultaneously, also can in vaccine composite a certain amount of HPVL1VLP antigen not wrapping quilt, namely by be coated on receive the HPV L1 antigen of microsphere with free HPV L1 antigen common and clinical or pharmaceutically acceptable pharmaceutic adjuvant make bacterin preparation.

In microsphere, antigen charging ratio is more than 20ug/mg in another embodiment; Envelope antigen and free antigen mass ratio are 0-100%.

Adopt microsphere features smooth surface prepared by the present invention smooth, spherical good (Fig. 1, Fig. 2, Fig. 3), uniform particle diameter controlled (Fig. 4) prepared in each particle size range, particle size dispersion coefficient is 0.005 ~ 0.2 in scope, preferably 0.005 ~ 0.1, more preferably 0.005 ~ 0.05.Drug loading and embedding rate high, zoopery shows that novel HPV vaccine of the present invention is safe, to laboratory animal without visible toxicity, effectively can produce stronger humoral immunization and cellullar immunologic response (Fig. 5 by stimulating animal, Fig. 6, Fig. 7, Fig. 8) can be used for the relevant disease of human papillomavirus or the prevention and therapy of infection.

Understand the present invention in order to clear, further illustrate it below with reference to accompanying drawing and example, but following embodiment non-limiting the present invention.

Accompanying drawing explanation

Fig. 1 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (430nm)

Fig. 2 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (1.38 μm)

Fig. 3 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (5.60 μm)

Fig. 4 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (400nm)

Fig. 5 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (800nm)

Fig. 6 PLGA embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (1.3 antigen)

Fig. 7 PLGA/PC embeds HPVL1 pentamer antigen and receives microsphere scanning electron microscope (SEM) photograph (400nm)

Fig. 8 PLGA wraps up HPV16L1 pentamer and receives microsphere scanning electron microscope (SEM) photograph (1 aggressiveness)

What Fig. 9 PLGA wrapped up HPV16L1 pentamer and CpG receives microsphere (1 receive micro-)

Figure 10 PLGA embeds HPVL1VLP antigen and receives microsphere scanning electron microscope (SEM) photograph (1 former receive)

Figure 11 PLGA/HSPC embeds HPVL1VLP antigen and receives microsphere scanning electron microscope (SEM) photograph (1 former receive)

Figure 12 PLA embeds HPVL1VLP antigen and receives microsphere scanning electron microscope (SEM) photograph (1 former receive)

Figure 13 PLGA embeds HPVL1VLP antigen and receives microsphere scanning electron microscope (SEM) photograph (10 receive micro-)

Figure 14 PLGA receives microsphere scanning electron microscope (SEM) photograph (1 microsphere)

Figure 15 carries HPV16L1 pentamer antigen PLGA and receives microspherulite diameter scattergram

Figure 16 different-grain diameter carries antigen PLGA microsphere group immune mouse NAT in serum after 38 days

Figure 17 different-grain diameter carries antigen PLGA microsphere group immune mouse specific antibody IgG titre in serum after 38 days

Figure 18 respectively organizes the cytokine levels testing result (Luminex technology) after immune mouse in splenocyte supernatant

Figure 19 respectively to organize after immune mouse the little and IL-4 horizontal detection result (ELISA method) of IFN-in splenocyte supernatant

After Figure 20 respectively organizes immune mouse, the little ELISpot of splenocyte IL-4 and IFN-forms cell number (SFC) (ELISPOT method)

After Figure 21 HPV L1 pentamer albumen adopts different adjuvant system immune mouse, serum binding antibody titre

After Figure 22 HPV L1 pentamer albumen adopts different adjuvant system immune mouse, the titre of neutralizing antibody

After Figure 23 HPV L1 pentamer albumen adopts different adjuvant system immune mouse, the cellullar immunologic response result that ELISPOT measures

Figure 24 HPV L1 pentamer albumen adopts different adjuvant system immune mouse, get its splenocyte in vitro under condition of culture after antigenic stimulus, ELISA measures IFN-A and IL-4 cytokine secretion profile

Figure 25 HPV L1 pentamer albumen adopts different adjuvant system immune mouse, get its splenocyte in vitro under condition of culture after antigenic stimulus, Luminex method measures cytokine secretion profile.

Figure 26 PLGA composite CpG small molecule adjuvants bag is by NAT after the microsphere immune mouse of HPV16L1 pentamer

Figure 27 PLGA composite CpG small molecule adjuvants bag is by binding antibody titre after the microsphere immune mouse of HPV16L1 pentamer

The PLGA bag of Figure 28 different-grain diameter and combination thereof is by binding antibody titre after the microsphere immune mouse of HPV16L1 pentamer

The PLGA bag of Figure 29 different-grain diameter and combination thereof is by NAT after the microsphere immune mouse of HPV16L1VLP

Figure 30 PLGA, PLGA/HSPC, PLA bag is by NAT after the microsphere immune mouse of HPV16L1VLP

Figure 31 PLGA, PLGA/HSPC, PLA bag is by binding antibody titre after the microsphere immune mouse of HPV16L1VLP

Binding antibody titre after Figure 32 PLGA microsphere absorption HPV16L1VLP after immune mouse

Instantiation

The present invention implements technical scheme of the present invention with reference to following form, and technical scheme of the present invention includes but not limited to following embodiment.

Table 1

Table 2

Table 3

Table 4

Specific embodiment is related to as follows in above table:

Specific embodiment one---100nmPLGA carries the preparation of HPV 6L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 6L1 pentamer of 1.33mg/mL is dissolved in the antigen protein of citric acid-sodium hydrogen phosphate buffer salt solution as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 600r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1.5M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 0.6 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 400nm, PDI value is 0.04, and medicine embedding rate is 90.8%.

Specific embodiment two---comprise the preparation of the HPV vaccine of free antigen

The mean diameter taken prepared by embodiment one is the composite 120.3 μ L concentration of HPV 6L1 pentamer antigen microsphere 4.45mg of carrying of 100nm is the HPV 6L1 pentamer antigen of 1.333mg/mL, add the PBS buffer of pH=7.0 to 1mL, prepare containing 200 μ g/mL's and to receive envelope antigen in microsphere be the microsphere suspensions of the HPV antigen of 2: 8 with free antigen ratio, obtained HPV bacterin preparation.

Specific embodiment three---430nm PLGA carries the preparation of HPV 16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1 pentamer antigen protein of 4mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 600r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 430nm, PDI value is 0.03, and medicine embedding rate is 92.6%.Obtained microsphere scanning electron microscope (SEM) photograph is shown in Fig. 1.

Specific embodiment five---1.38 μm of PLGA carry the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1 pentamer antigen protein of 6.67mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 5.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1.38 μm, and PDI value is 0.05, and medicine embedding rate is 93.1%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in Figure 2.

Specific embodiment six---1 μm of PLGA carries the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1 pentamer antigen protein of 6.67mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 5.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1 μm, and PDI value is 0.05, and medicine embedding rate is 93.1%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in Figure 8.

Specific embodiment seven---5.6 μm of PLGA carry the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1 pentamer antigen protein of 9.33mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 300r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 40kPa pressure, double emulsion is pressed through the SPG film that membrane aperture is 15 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 5.6 μm, and PDI value is 0.08, and medicine embedding rate is 79.7%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in Figure 3.

Specific embodiment 17---20.82 μm of PLGA carry the preparation of HPV45L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 45L1 pentamer antigen protein of 5.333mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 250r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 5kPa pressure, double emulsion is pressed through the SPG film that membrane aperture is 52 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 20.82 μm, and PDI value is 0.10, and medicine embedding rate is 89.2%.

Specific embodiment 18---80.71 μm of PLA carry the preparation of HPV58L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 58L1 pentamer antigen protein of 5.333mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 250r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.1kPa pressure, double emulsion is pressed through the SPG film that membrane aperture is 190.3 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 80.71 μm, and PDI value is 0.18, and medicine embedding rate is 85.5%.

Specific embodiment 19---400nm PLGA/HSPC carries the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by PLGA and the 100mg lecithin of 300mg in the MC solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 6L1 pentamer antigen protein of 1.333mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 500r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 400nm, PDI value is 0.05, and medicine embedding rate is 92.8%.Thus obtained microsphere scanning electron microscope (SEM) photograph is as 7.

Specific embodiment 20---800nm PLGA carries the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 300mg in the MC solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 11L1 pentamer antigen protein of 4mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 500r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 9.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 800nm, PDI value is 0.06, and medicine embedding rate is 88.2%.The scanning electron microscope (SEM) photograph of thus obtained microsphere is as Fig. 5.

Specific embodiment 21---1300nm PLGA carries the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 300mg in the MC solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 11L1 pentamer antigen protein of 4mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 500r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 9.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1300nm, PDI value is 0.06, and medicine embedding rate is 88.2%.The scanning electron microscope (SEM) photograph of thus obtained microsphere is as Fig. 6.

Specific embodiment 26---1000nm PLGA carry antigen receive microsphere embed altogether CPG1826 carry HPV16L1 pentamer antigen microsphere preparation

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 16L1 pentamer antigen protein and 5 μ g CpG1826 that 3mL concentration is 4mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1000nm, PDI value is 0.06, and medicine embedding rate is 87.8%.Thus obtained microsphere scanning electron microscope (SEM) photograph is as Fig. 9.

Specific embodiment 27---1.57 μm of PLGA carry antigen and receive microsphere and embed MPLA altogether and carry the preparation of HPV16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 16L1 pentamer antigen protein and 5 μ g MPLA that 3mL concentration is 6.67mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 5.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1.57 μm, and PDI value is 0.05, and medicine embedding rate is 82.2%.

Specific embodiment 29---23.57 μm of PLA carry antigen and receive microsphere and embed PolyI:C altogether and carry the preparation of HPV45L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 16L1 pentamer antigen protein and 5 μ g PolyI:C that 3mL concentration is 5.33mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 5.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 23.57 μm, and PDI value is 0.09, and medicine embedding rate is 86.3%.Specific embodiment 31---400nmPLGA carries the preparation of HPV 16L1 pentamer antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1VLP antigen protein of 1.33mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 600r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1.5M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 400nm, PDI value is 0.05, and medicine embedding rate is 90.5%.Prepared by obtain the scanning electron microscope (SEM) photograph of microsphere as shown in Figure 4

Specific embodiment 36---128nmPLGA carries the preparation of HPV 6L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 6L1VLP antigen protein of 1.33mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 600r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1.5M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 0.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 128nm, PDI value is 0.03, and medicine embedding rate is 91.7%.

Specific embodiment 37

The mean diameter taken prepared by embodiment 36 is the composite 12.03 μ L concentration of HPV 6L1VLP antigen microsphere 4.4mg of carrying of 128nm is the HPV 6L1 pentamer antigen of 13.33mg/mL, add the PBS buffer of normal saline or pH=7.0 to 1mL, prepare containing 200 μ g/mL's and to receive envelope antigen in microsphere be the microsphere suspensions of the HPV antigen of 2: 8 with free antigen ratio, obtained HPV bacterin preparation.

Specific embodiment 38---467nm PLGA carries the preparation of HPV 11L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 11L1VLP antigen protein of 4mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 600r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 467nm, PDI value is 0.05, and medicine embedding rate is 89.2%.

Specific embodiment 40---1 μm of PLGA carries the preparation of HPV16L 1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1VLP antigen protein of 6.67mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 5.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1 μm, and PDI value is 0.05, and medicine embedding rate is 89.5%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in Figure 10.

Specific embodiment 42---5.87 μm of PLGA carry the preparation of HPV18L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 18L1VLP antigen protein of 9.33mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 300r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 40kPa pressure, double emulsion is pressed through the SPG film that membrane aperture is 15 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 5.87 μm, and PDI value is 0.07, and medicine embedding rate is 89.2%.

Specific embodiment 52---25.02 μm of PLA carry the preparation of HPV45L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLA of 400mg in the EA solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 45L1VLP antigen protein of 5.33mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 250r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 5kPa pressure, double emulsion is pressed through the SPG film that membrane aperture is 52 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 25.02um, PDI value is 0.06, and medicine embedding rate is 75.3%.

Specific embodiment 55---1000nm PLGA/HSPC carries the preparation of HPV16L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by PLGA and the 100mg lecithin of 300mg in the MC solvent of 10.0mL as oil phase (O), adding 3mL concentration is that the HPV 16L1VLP antigen protein of 4mg/mL is as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 500r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 1M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 9.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous double emulsion is dropped in stirred at ambient temperature 4h (magnetic agitation speed 200rpm) and remove organic solvent, the deionized water centrifuge washing 3 times of the microsphere after solidification, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1000nm, PDI value is 0.07, and medicine embedding rate is 80.6%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in figure 11.

Specific embodiment 56---489nm PLGA carry antigen receive microsphere embed altogether CPG1826 carry HPV11L1VLP antigen microsphere preparation

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 11L1VLP antigen protein and 5 μ g CpG1826 that 3mL concentration is 4mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 489nm, PDI value is 0.05, and medicine embedding rate is 90.8%.

Specific embodiment 66---1000nm PLA carry antigen receive microsphere carry HPV16L1VLP antigen microsphere preparation

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 16L1VLP antigen protein and 5 μ g CpG1826 that 3mL concentration is 6.67mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 1.4 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 1000nm, PDI value is 0.04, and medicine embedding rate is 89.4%.The obtained antigen that carries receives microsphere scanning electron microscope (SEM) photograph as shown in figure 12.

Specific embodiment 68---10 μm of PLGA carry antigen and receive microsphere and carry the preparation of HPV16L1VLP antigen microsphere

Adopt fast film emulsifying technology to carry antigen in conjunction with the preparation of double emulsion solvent removal method and receive microsphere.Concrete grammar is: be dissolved in by the PLGA of 400mg in the EA solvent of 10.0mL as oil phase (O), add HPV 16L1VLP antigen protein and 5 μ g CpG1826 that 3mL concentration is 6.67mg/mL as interior aqueous phase (W1), (ultrasonic power is set as 30% under ice-water bath condition, to carry out just emulsifying with S-450D ultrasonic cell disruption instrument, ultrasonic time is 60s) prepare W1/O type colostrum, then colostric fluid is poured in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, magnetic agitation is adopted to prepare pre-double emulsion (W1/O/W2), emulsification condition is stir 2min under 400r/min.Subsequently, this pre-double emulsion is poured in the storage tank of fast film emulsifying, with the nitrogen of 0.25M Pa pressure, double emulsion is pressed through the SPG film that membrane aperture is 50.2 μm repeatedly, until obtain homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured in the 0.9%NaCl solution of 780mL, at room temperature stir 4h (magnetic agitation speed 200rpm) and remove organic solvent, microsphere after solidification deionized water centrifuge washing 3 times, finished product is made in last lyophilization.Measure particle size distribution situation with laser particle analyzer, mean diameter is 10 μm, and PDI value is 0.07, and medicine embedding rate is 90.2%, obtained year antigen receive microsphere scanning electron microscope (SEM) photograph as Figure 13 no.

Specific embodiment 74

The 430nm that Example 31 prepares respectively receives 1.38 five systems that microsphere 1.128mg and embodiment five prepare and receives microsphere 1.157mg to be mixed with antigenic quality ratio be the mechanical fraction of 50%: 50%, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.31mg/mL suspension.

Specific embodiment 79

The 430nm that Example 31 prepares respectively 20.65 μm of receiving that microsphere 1.128mg and embodiment 33 prepare receive microsphere 1.147mg, and to be mixed with antigenic quality ratio be the mechanical fraction of 50%: 50%, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.27mg/mL suspension.

Specific embodiment 84

Example 5 1.38 μm of preparing 20.65 μm of receiving that microsphere 1.157mg and embodiment 33 prepare are received microsphere 1.147mg to be mixed with antigenic quality ratio are the mechanical fraction of 50%: 50% respectively, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.31mg/mL suspension.

Specific embodiment 92

The 430nm that Example 31 prepares respectively 1.38 μm of receiving that microsphere 0.68mg, embodiment five prepare 20.65 μm of receiving that microsphere 0.69mg and embodiment 33 prepare receive microsphere 0.92mg, and to be mixed with antigenic quality ratio be the mechanical fraction of 30%: 30%: 40%, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.29mg/mL suspension.

Specific embodiment 101

Example 68 10 μm of preparing 1 μm of receiving that microsphere and embodiment 40 prepare is received microsphere to be mixed with antigenic quality ratio is the mechanical fraction of 20%: 80% respectively, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.35mg/mL suspension.

Specific embodiment 102

Example 68 10 μm of preparing 1 μm of receiving that microsphere and embodiment 40 prepare is received microsphere to be mixed with antigenic quality ratio is the mechanical fraction of 80%: 20% respectively, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.35mg/mL suspension.

Specific embodiment 103

Example 68 10 μm of preparing 1 μm of receiving that microsphere 1.175mg and embodiment 40 prepare is received microsphere 1.174mg to be mixed with antigenic quality ratio is the mechanical fraction of 50%: 50% respectively, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.35mg/mL suspension.

Specific embodiment 106

The 458nm that Example 66 prepares respectively 23.58 μm of receiving that microsphere 1.175mg and embodiment 68 prepare receive microsphere 1.164mg, and to be mixed with antigenic quality ratio be the mechanical fraction of 50%: 50%, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.34mg/mL suspension.

Specific embodiment 111

Example 40 1.69 μm of preparing 23.58 μm of receiving that microsphere 1.174mg and embodiment 68 prepare are received microsphere 1.164mg to be mixed with antigenic quality ratio are the mechanical fraction of 50%: 50% respectively, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.34mg/mL suspension.

Specific embodiment 120

The 458nm that Example 66 prepares respectively 1.69 μm of receiving that microsphere 0.71mg, embodiment 40 prepare 0.47 μm of receiving that microsphere 1.174mg and embodiment 68 prepare receives microsphere 0.004mg, and to be mixed with antigenic quality ratio be the mechanical fraction of 30%: 50%: 20%, add the PBS buffer 1mL that pH value is 7, being prepared into the antigen containing 100 μ g/mL and receiving microsphere concentration is 2.35mg/mL suspension.

In form, the concrete implementation step of other embodiments is implemented with reference to above relevant specific embodiment.Example 125---microspherulite diameter and particle size distribution characterize

Microsphere suspension liquid some of them embodiment prepared is ultrasonic in distilled water makes it dispersed, employing laser particle analyzer ( 3000HS, UK) measure its size and distribution, the different antigen that carries receives microsphere adjuvant grain size distribution as shown in figure 15.

Embodiment 126---HPV charging ratio measures

It is the NaOH solution of 0.1M that the microsphere 5mg that the described lyophilizing prepared received is suspended in 1.0mL containing 5% (w/v) SDS concentration, vibrate 24 hours under room temperature, microsphere is dissolved (hydrolysis) completely, and protein content in solution micro-BCA test kit measures.

Embodiment 127---different-grain diameter PLGA carries antibody horizontal evaluation comparison after the immunity of antigen microsphere

The described lyophilizing microsphere that Example five and embodiment three prepare, is suspended in normal saline, obtains microsphere suspensions, makes the content of antigen in microsphere suspensions be 20 μ g/100 μ L, i.e. obtained HPV bacterin preparation.

Example five and obtained two kinds of embodiment three receive the female BAl BIc/c mice in microspheres vaccine preparation dorsal sc immunity 8-10 week, every injected in mice antigen dose is 20 μ g/100 μ L, sets up with the aluminum salt HPV pentamer antigen that is adjuvant as a control group simultaneously.

Detect the Humoral of above three groups.The index detected comprises different-grain diameter PLGA microsphere and carries the neutralizing antibody level of antigen group immune mouse after 38 days and specific antibody IgG level, respectively shown in Figure 16 and Figure 17.

The PLGA of different-grain diameter carries antigen, and to receive the specific antibody neutralizing antibody level that produces after microsphere immune mouse different.As shown in Figure 16, the neutralizing antibody level of mouse immune antigen PLGA microsphere generation in 1.38 μm years after 38 days and the horizontal average of IgG are all higher than aluminium adjuvant group, but there was no significant difference; But neutralizing antibody level and IgG level are all significantly higher than 430nm and carry antigen PLGA group (P < 0.05 or P < 0.01) in 1.38 μm years antigen PLGA group serum.

Embodiment 128---different-grain diameter PLGA carries cytokine levels evaluation comparison after the immunity of antigen microsphere

Example five and obtained two kinds of embodiment three receive the female BAl BIc/c mice in microspheres vaccine preparation dorsal sc immunity 8-10 week, every injected in mice antigen dose is 20 μ g/100 μ L, sets up with the aluminum salt HPV pentamer antigen that is adjuvant as a control group simultaneously.

Detect the Study On Cellular Immune of above three groups.After Testing index comprises employing antigen in vitro stimulation mouse boosting cell 72h, cytokine levels (Luminex technology, ELISA method) in splenocyte supernatant, result is as shown in Figure 18 and Figure 19; And after adopting antigen in vitro to stimulate mouse boosting cell 36h, IFN-γ and IL-4 ELISpot form cell number (ELISPOT method), and result as shown in figure 20.

IL-4 and IL-6 represents the cytokine of Th2 type response, IFN-γ and IL-2 represents the cytokine of Th1 type response by Figure 18, Figure 19 and Figure 20 all can find out, the IFN-γ level of carrying in antigen PLGA microsphere group splenocyte supernatant of 430nm and 1.38 μm of two particle diameters is all significantly higher than aluminium adjuvant group (P < 0.05 or P < 0.01), and 430nm carries the antigen PLGA IFN-γ level pole received in microsphere group splenocyte supernatant is significantly higher than 1.38 μm and carries antigen PLGA and receive microsphere group group (P < 0.01), as shown in Figure 7,430nm carries the antigen PLGA IL-2 level received in microsphere group splenocyte supernatant and is also significantly higher than aluminium adjuvant group (P < 0.05), and 430nm carries antigen PLGA receives the secretion level of IL-2 in microsphere group splenocyte supernatant and also carry antigen PLGA group higher than 1.38 μm, but the two is without significant difference.This shows, compare aluminium adjuvant, carry antigen PLGA to receive microsphere group and effectively can induce Th1 type immunne response, strengthen cellullar immunologic response, and the less PLGA of particle diameter carries antigen receives microsphere (430nm) and compare Large stone PLGA and carry antigen and receive microsphere (1.38 μm) and more effectively can induce Th1 type immunne response, strengthen cellullar immunologic response.

In Th2 cytokines secretion level, different-grain diameter carries antigen PLGA to be received microsphere group and can induce and the similar IL-4 secretion level of aluminium adjuvant, there was no significant difference, 1.38 μm carry the antigen PLGA IL-4 level received in microsphere group splenocyte supernatant and are significantly higher than 430nm and carry antigen PLGA and receive microsphere group group (P < 0.05 or P < 0.01); 1.38 μm carry the antigen PLGA IL-6 level received in microsphere group splenocyte supernatant and are significantly higher than aluminium adjuvant group (P < 0.05), and 1.38 μm carry the antigen PLGA IL-6 level pole received in microsphere group splenocyte supernatant and are significantly higher than 430nm and carry antigen PLGA nanometer set of balls (P < 0.01).

Above result shows to carry antigen PLGA and receives microsphere group and can induce the secretion level of the IL-4 similar to aluminium adjuvant, can effective elicit humoral immune, and higher levels of IFN-γ level, significantly promotes cellullar immunologic response; And year antigen PLGA microsphere (1.38 μm) of Large stone more can significantly improve the secretion level of IL-6, IL-4, strengthen Th2 type immunne response, promote that B cell activation produces antibody, elicit humoral immune is replied effectively; And small particle diameter carries the secretion level that antigen PLGA microsphere (430nm) significantly promotes IFN-γ, strengthen Th1 type immunne response, contribute to the lifting of cellullar immunologic response.

Embodiment 129---1.38 μm of PLGA carry antigen and receive Analysis of Immunity Effect after the composite free antigen immune mouse of microsphere

Female BAl BIc/c the mice in the microball preparation dorsal sc immunity 8-10 week that Example six is obtained, every injected in mice antigen dose is 20 μ g/100 μ L, wherein in microspheres vaccine preparation, HPV antigenic content is 10 μ g/, free HPV antigenic content be 10 μ g/ only, set up the 1.38 μm of PLGA prepared with the aluminum salt HPV pentamer antigen that is adjuvant and embodiment 5 to carry antigen simultaneously and receive microsphere as a control group.Detect humoral immunization and the Study On Cellular Immune of above several groups.

Result shows 1.38 μm of PLGA and carries antigen to receive NAT that the composite free antigen of microsphere produces and specific antibody IgG titre be that 1.38 μm of PLGA carry antigen and receive about 5 times of microsphere group, and this shows that PLGA carries antigen and receives the composite free antigen of microsphere and effectively can strengthen Humoral.

Embodiment 134---pentamer and VLP receive the preparation of microsphere

With reference to above detailed description of the invention, prepare the preparation receiving microsphere of other pentamer of different proportion, different-grain diameter and different shaped and VLP according to the formula of following form.

The sequence number of table 5 year HPV16L1 pentamer PLGA microsphere different-grain diameter

The PLGA microsphere different-grain diameter combination sequence number of table 6 year HPV16L1 pentamer antigen

The PLGA microsphere different HPV type sequence number of table 7 year HPV16L1 pentamer antigen

The composite lipid material sequence number of PLGA microsphere of table 8 year HPV16L1 pentamer antigen

The sequence number of the composite different molecular adjuvant of PLGA microsphere of table 9 year HPV16L1 pentamer antigen

The sequence number of the different-grain diameter of the PLGA microsphere of table 10 year HPV16L1VLP antigen

The different-grain diameter combination sequence number of the PLGA microsphere of table 11 year HPV16L1VLP antigen

The different HPV type sequence numbers of the PLGA microsphere of table 12 year HPV16L1VLP antigen

The composite lipid material sequence number of the PLGA microsphere of table 13 year HPV16L1VLP antigen

The sequence number of the composite different molecular adjuvant of the PLGA microsphere of table 14 year HPV16L1VLP antigen

Embodiment 135---different-grain diameter receive the research of microsphere HPV L1 pentamer immunogenicity

Sample prepared by Example 19,20,30 and 21, with 400nm, 800nm, the HPV L1 pentamer albumen of 1.3 μm of three kinds of different-grain diameter PLGA embeddings and the HPV L1 pentamer protein immunization mice of 400nmPLGA/PC bag quilt, preliminary experimental results shows, this adjuvant system can effectively induce the antigen-specific immune response comprising neutralizing antibody and cellular immunization.In the diagram, antibody in serum analysis, result shows, compared with tradition aluminium salt adjuvant group, the NAT of 400nm with 800nm PLGA adjuvant group is substantially identical, but the neutralizing antibody of the neutralizing antibody adjuvant group of 1.3 μm and 400nm PLGA+ lecithin (PLGA/PC) composite adjuvant group is with higher in conjunction with titre; The binding antibody titre of the adjuvant group of another 800nm and 1.3 μ .3nm is all significantly higher than 400nm PLGA, but there was no significant difference between the two.The response of HPV specific CTL immunity is detected with ELISPOT method, result shows, conventional aluminum adjuvant group does not induce specific CTL immunity response substantially, and PLGA adjuvant group induces the response of more high-caliber HPV specific CTL immunity all effectively, be wherein the highest (Fig. 2) with 400nm and 800nm PLGA adjuvant group especially.The splenocyte getting immunized mice measures the cytokine secretion profile in supernatant in vitro under condition of culture after antigenic stimulus, result shows, the relatively thin and TNF-of Th1 type (irritation cell immunity) cytokine IFN-secretory volume carefully etc., PLGA adjuvant group is all significantly higher than tradition aluminium salt adjuvant group; And in Th2 cytokines (stimulating body fluid/antibody mediated immunity response) IL-4, PLGA adjuvant group is basic identical with tradition aluminium salt adjuvant group, but the composite adjuvant group of PLGA+ lecithin (PLGA/PC) is used then to be significantly higher than other group.The results are shown in Figure 21,22,23,24,25.

Embodiment 136---1000nm PLGA carries antigen and receives microsphere and embed Analysis of Immunity Effect after CPG1826 combined immunization mice altogether

The PLGA embedding CpG1826 altogether according to embodiment 26 preparation carries antigen and receives microsphere.By obtained microballoon lyophilized after, be suspended in normal saline, obtain microsphere suspensions, make the content of antigen in microsphere suspensions be 20 μ g/100 μ L, be i.e. obtained HPV bacterin preparation.By the female BAl BIc/c mice in its dorsal sc immunity 8-10 week, every injected in mice antigen dose is 20 μ g/100 μ L, sets up to carry antigen with the aluminum salt HPV pentamer antigen that is adjuvant and 1000nm PLGA and receive microsphere as a control group simultaneously.Detect humoral immunization and the Study On Cellular Immune of above several groups.PLGA composite CpG small molecule adjuvants bag is shown in Figure 26 and 27 respectively by neutralizing antibody and binding antibody titre after the microsphere immune mouse of HPV16L1 pentamer, and the 1000nm that result display embeds CpG altogether carries antigen PLGA and receives the neutralizing antibody level that microsphere group can be induced after immune mouse and Alum adjuvant is on close level.And on knot and antibody horizontal, the HPV16L1 pentamer albumen of 1 μm of PLGA embedding when initial immunity induction of the binding antibody level being significantly higher than Alum adjuvant, can induce when second time immunity and the binding antibody titre of the suitable level of Alum adjuvant, and after adding CpG small molecule adjuvants, when second time immunity, the binding antibody level of induction is significantly higher than and does not add CpG group, and when first time and second time immunity, the binding antibody level of induction is all significantly higher than tradition aluminium salt adjuvant simultaneously.

The PLGA of embodiment 137---different-grain diameter combination carries antigen and receives Analysis of Immunity Effect after microsphere immune mouse

Female BAl BIc/c the mice in the microball preparation dorsal sc immunity 8-10 week of the different-grain diameter combination that Example 101,102,103 is obtained, every injected in mice antigen dose is 20 μ g/100 μ L, sets up the PLGA of the different-grain diameter prepared with the aluminum salt HPV pentamer antigen that is adjuvant and embodiment 40,68 to carry antigen simultaneously and receives microsphere as a control group.Detect the Humoral of above several groups.The PLGA of different-grain diameter and combination thereof wraps by binding antibody titre and NAT after the microsphere immune mouse of HPV16L1 pentamer respectively as 28 and 29.

Result shows the NAT of the HPV16L1VLP induction of the PLGA embedding that 1 μm or 10 μm or two particle diameters combine by a certain percentage without significant difference, and the binding antibody titre that 1 μm of PLGA-HPV16L1VLP group is induced after initial immunity is significantly lower than HPV16L1VLP-1 μm+10 μm (0.5+0.5) groups, remarkable in HPV16L1VLP-1 μm+10 μm (0.2+0.8) group and HPV16L1VLP-1 μm+10 μm (0.8+0.2) groups after second time immunity.

The research of the HPV 16L1VLP immunological effect of embodiment 138---different adjuvant

After embedding HPV 16L1VLP immune mouse with PLGA, PLGA/HSPC, PLA, induce the neutralizing antibody and binding antibody titre that produce all without significant difference, as Figure 30 and 31.

The preparation of the granule of embodiment 139---PLGA

The PLGA of 100mg is dissolved in the dichloromethane of 20mL as oil phase (O), the PVA solution of preparation 1.0% is as outer aqueous phase (W2), oil phase (O) is added in the outer aqueous phase (W2) of 50mL, prepare the pre-emulsion (W2/O) of oil-in-water type, adopt the method for fast film emulsifying repeatedly to cross the oil-in-water double emulsion (O/W2) that film preparation obtains uniform particle diameter this pre-double emulsion; Solidification 3h, centrifuge washing, lyophilizing, obtain dry PLGA microsphere, the SEM photo of PLGA as shown in Figure 2.

The preparation of the granular absorption HPV16L1VLP vaccine combination of embodiment 140---PLGA

The PLGA microsphere prepared is dissolved in pH=5,10mM PBS buffer solution of 0.8mL, obtains the PLGA microsphere suspensions of 20mg/mL; Get a certain amount of HPV to be dissolved in pH=5,10mM PBS buffer solution of 0.8mL, obtain the HPV antigenic solution of 20ug/mL, mixed by both equal-volumes, attach overnight, obtains the PLGA suspension that 1.6mL is adsorbed with HPV antigen, is HPV vaccine combination.

Embodiment 140---PLGA microsphere adsorbs the research of immunne response to HPV 16L1VLP

In Example 139,1 PLGA executing example adsorbs immune mouse after HPV 16L1VLP sample, and PLGA-HPV 16L1VLP can induce the binding antibody titre with the suitable level of the Gardasil vaccine of Merck company.Result is as Figure 32.

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Claims (34)

1. a vaccine combination, comprise the antigen with adjuvant combination, the ratio of antigen and adjuvant is 10-80 μ g/mg, and described antigen comprises HPV L1 albumen, and described adjunvant composition comprises PLGA (PLGA) or polylactic acid (PLA).

2. vaccine combination as claimed in claim 1, wherein said HPV L1 albumen is embedded in PLGA or is adsorbed on PLGA surface, all exists at the microspheres form of receiving between 100nm ~ 100 μm with particle size distribution.

3. vaccine combination as claimed in claim 2, the wherein said particle size dispersion coefficient scope of microsphere in each particle size range of receiving is 0.005 ~ 0.2.

4. vaccine combination as claimed in claim 3, the wherein said microsphere 0-90% that receives is distributed between 100-1000nm, 1-10 μm or 10-100 μm of arbitrary particle size range or its combination.

5. vaccine combination as claimed in claim 4, the wherein said microsphere 0-90% that receives is distributed between 200-900nm, 2-9 μm or 20-90 μm of arbitrary particle diameter or its combination.

6. vaccine combination as claimed in claim 5, the wherein said microsphere 0-90% that receives is distributed between 400-700nm, 4-7 μm or 40-70 μm of arbitrary particle diameter or its combination.

7. vaccine combination as claimed in claim 4, the wherein said microsphere 10-90% that receives is distributed in 100-1000nm, 90-10% and is distributed in 1-10 μm.

8. vaccine combination as claimed in claim 7, the wherein said microsphere 10-90% that receives is distributed in 300-800nm, 90-10% and is distributed in 3-8 μm.

9. vaccine combination as claimed in claim 8, the wherein said microsphere 10-90% that receives is distributed in 500-600nm, 90-10% and is distributed in 5-6 μm.

10. vaccine combination as claimed in claim 4, wherein said receives that microsphere 10-90% is distributed in 1-10 μm, 90-10% is distributed in 10-100 μm.

11. as the vaccine combination of claim 10, wherein said receives that microsphere 10-90% is distributed in 3-8 μm, 90-10% is distributed in 30-80 μm.

12. as the vaccine combination of claim 11, wherein said receives that microsphere 10-90% is distributed in 5-6 μm, 90-10% is distributed in 50-60 μm.

13. vaccine combinations as claimed in claim 4, the wherein said microsphere 10-90% that receives is distributed in 100-1000nm, 90-10% and is distributed in 10-100 μm.

14. as the vaccine combination of claim 13, and the wherein said microsphere 10-90% that receives is distributed in 300-800nm, 90-10% and is distributed in 30-80 μm.

15. as the vaccine combination of claim 14, and the wherein said microsphere 10-90% that receives is distributed in 500-600nm, 90-10% and is distributed in 50-60 μm.As described in as arbitrary in claim 4 to 15 vaccine combination, following immunostimulant component lipid material is comprised further: one or more combination in lecithin, stearylamine, phosphoglyceride, wherein the ratio of lipid material and PLGA or PLA is 1-99: 99-1 in wherein said adjuvant.

16. vaccine combinations as claimed in claim 15, small molecule immune adjuvant is comprised further: one or more combination in CpG, MPLA, imiquimod, PolyI:C etc., wherein the ratio of small molecule immune adjuvant and PLGA or PLA is 10-100 μ g/mg in wherein said adjuvant.

17. as the vaccine combination of claims 14 or 15, wherein HPV L1 albumen refers to HPV total length, the L1 of truncate or restructuring is formed VLP, pentamer or polymer.

18. vaccine combinations as claimed in claim 17, wherein HPV L1 albumen refers to 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 or 82,85, combinations a kind of and/or several in 97 type HPV.

19. 1 kinds of bacterin preparations, is characterized in that this bacterin preparation is made up of the vaccine combination described in 18 and HPV L1 albumen and pharmaceutic adjuvant, and wherein receiving HPV L1 and free HPV L1 ratio in microsphere is 10-90%: 90-10%.

20. bacterin preparations as claimed in claim 19, is characterized in that described pharmaceutic adjuvant is normal saline or PBS buffer.

The preparation method of 21. vaccine combinations as claimed in claim 18, the method comprises the steps:

(1) preparation of pastille emulsion droplet: HPV L1 albumen and/or small molecule immune adjuvant CpG etc. being dissolved in buffer salt system, to be prepared into pH value range be aqueous phase (W1) in 4.0-12.0;

(2) PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent make oil phase (O), described aqueous phase (W1) is mixed with oil phase (O), make water-in-oil type (W1/O) pre-emulsion, this pre-emulsion is added in outer aqueous phase (W2), the obtained pre-double emulsion of W1/O/W2 type, crosses the pre-double emulsion of described W1/O/W2 type the W1/O/W2 type emulsion that film obtains size uniformity repeatedly;

(3) emulsion droplet solidification: by the normal temperature cure of W1/O/W2 type emulsion, makes vaccine combination after washing, drying.

The preparation method of 22. vaccine combinations as claimed in claim 18, the method comprises the steps:

(1) PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent make oil phase (O), this oil phase is added in outer aqueous phase (W2), the obtained pre-double emulsion of O/W2 type;

(2) the pre-double emulsion of described O/W2 type is crossed the O/W2 type emulsion that film obtains size uniformity repeatedly; PLGA/PLA microsphere is obtained after washing, drying;

(3) HPV L1 albumen and/or small molecule immune adjuvant CpG are dissolved in buffer salt system, obtain the HPV antigenic solution of 1-40 μ g/0.2-1mL, PLGA/PLA microsphere is dissolved in buffer salt system, obtain the solution of 1-10mg/mL; By two solution equal-volume mixing, attach overnight, obtains the PLGA suspension being adsorbed with HPV antigen, is HPV vaccine combination.

23. preparation methoies as described in claim 21 or 22, is characterized in that described buffer salt system is citric acid-sodium hydrogen phosphate, Acetic acid-sodium acetate buffer or phosphate buffer.

24. preparation methoies as claimed in claim 21, is characterized in that, the interior aqueous pH values scope described in step (1) is 6.0-8.0.

25. as described in claim 21 or 22, it is characterized in that, described oil phase is in liquid and the immiscible oily matter of water under room temperature, is preferably ethyl acetate, acetone, dichloromethane, chloroform or above-mentioned at least two or more mixture.

26. as described in claim 21 or 22, it is characterized in that, described outer aqueous phase (W2) is the PVA aqueous solution containing 0.001-10%, and its alcoholysis degree is 87 ~ 90%, and polymeric chain joint number is 1700-1750; Or be the PVA-PEG mixed aqueous solution containing 0.001-10%, the mass percent of PVA and PEG is 20-80: 30-90.

27. as claimed in claim 21, it is characterized in that, water-in-oil type (W1/O type) pre-emulsion described in step (2) obtains by adopting homogeneous phase emulsator or ultrasonic emulsator.

28. as claimed in claim 21, it is characterized in that, the W1/O/W2 type emulsion of the size uniformity described in step (2) obtains by W1/O/W2 type pre-emulsion is pressed through microporous membrane under pressure repeatedly.

29. as claim 21 or as described in, it is characterized in that, described in step (2) containing antigen emulsion droplet, aqueous phase (W1 or W2) and oil phase (O) volume ratio are 1: 1 ~ 1: 50.

30. as described in claim 21 or 22, it is characterized in that, curing mode is solvent evaporated method and solvent extraction.

31. as described in claim 21 or 22, it is characterized in that, hardening time is 1h-8h.

The preparation method of 32. 1 kinds of bacterin preparations, also comprises the steps: after it is characterized in that the emulsion droplet curing schedule in the preparation method of vaccine combination as described in claim 21 or 22

(4) bacterin preparation is formed: the vaccine combination (receive microsphere) getting mass ratio 10-90%: 90-10% suspends with free HPVL1 albumen normal saline or PBS buffer, be mixed with the microsphere suspensions of the HPV antigen containing 1-40 μ g/0.2-1mL, obtained HPV bacterin preparation.

The application of 33. vaccine combinations as claimed in claim 18 in the medicine that preparation is treated or prevention HPV infects.

Application in the medicine that 34. bacterin preparations as described in claim 19 or 20 infect in preparation treatment or prevention HPV.