CN104338126B - It is a kind of that there is the vaccine combination for treating or preventing HPV viruse and its application - Google Patents

Abstract

Purposes the present invention relates to vaccine combination and its in medicine.More particularly to vaccine combination or bacterin preparation and its application in HPV viruse medicine is treated or prevented made of microballoon received by coating HPV L1 albumen.Specifically, the present invention is directed to HPV L1 antigen proteins, receive microspherulite diameter size and combinations thereof, the influence of micro-sphere material, the former load mode of microballoon of receiving confrontation to its immune response of receiving, design prepares uniform particle diameter, PLGA, PLA of different-grain diameter, PLGA/PC receives microballoon, with its embedding or absorption HPV L1 antigen proteins, is prepared into vaccine combination or bacterin preparation, it is studied to vivo immunization response intensity and horizontal influence, develops efficient HPV L1 protein vaccine compositions.

Description

It is a kind of that there is the vaccine combination for treating or preventing HPV viruse and its application

Technical field

Purposes the present invention relates to vaccine combination and its in medicine.More particularly to by being coated with or adsorbing HPV L1 albumen Receive vaccine combination made of microballoon or bacterin preparation and its application in HPV viruse medicine is treated or prevented.

Technical background

Human papilloma virus (Human papillomaviruses, HPV) is the double-stranded DNA virus of no cyst membrane, mainly by Virus coat and genomic DNA composition (Bernard, Burk et al.2011).HPV viruse shell is by 360 L1 albumen (forming 72 pentamers) and the icosahedral structure of virus that at most 72 L2 albumen is formed, diameter 55-60nm (Howley and Lowy2007).Virus capsid protein has self assembly characteristic, and L1 albumen is independent in vitro or is formed with L2 albumen co-assembles Viroid sample particle (Virus-like Particle, VLP) (Chen, Garcea et al.2000, Finnen, Erickson Et al.2003, Buck, Cheng et al.2008, Wang and Roden 2013).Current HPV vaccines are all with VLP As target antigen, existing two kinds based on HPV L1VLP preventative vaccines 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, (FDA) the official approval U.S. of U.S. Food and Drug Administration The Gardasil HPV preventative vaccines listing of Merck companies (i.e. MSD Corp.) production；It is expressed simultaneously by saccharomyces cerevisiae The HPV16/18/6/11L1VLP tetravalence cancer, prophylaxis vaccines of purifying, with amorphous Adju-Phos aluminium sulfate (amorphous aluminum Hydroxyphosphatesulfate, AAHS) is adjuvant, is approved for prevention 6~26 years old Girl and caused cervical carcinoma, precancerous lesion and the genital wart of the type of women HPV16,18,6,11 infection, this be FDA by In the world first tumor vaccine (Villa, Costa et al.2005, Villa, Ault et al.2006, Bryan 2007, Olsson, Villa et al.2007, Goldstone and Vuocolo 2012).Subsequent Britain's GlaxoSmithKline PLC (GSK) is public The HPV preventative vaccines for taking charge of the trade name Cervarix of production also successfully list, it is that origin comes from insect expression system HPV16/18L1VLP divalence cancer, prophylaxis vaccines, using AS04 adjuvants (the compound MPL of aluminium hydroxide) (Paavonen, Jenkins et al.2007, Garcon, Morel et al.2011, Kreimer, Gonzalez et al.2011, Szarewski 2012).Clinical experiments have proved that above two preventative vaccine is respectively provided with good tolerance, and without seriously not Good 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 both preventative vaccines are expensive, and need low temperature Preserve, strongly limit the use in developing country and backward areas, therefore develop a kind of inexpensive high-titer HPV vaccines Just it is particularly important (Jansen and Shaw 2004, Buonaguro, Tornesello et al.2009, Campo and Roden 2010, Frazer, Leggatt et al.2011, Hariri, Dunne et al.2011, Lehtinen and Dillner 2013, Shaw 2013).Research shows, can induce generation after HPV L1 pentamer albumen immunization experiment animals Immune response (Rose, White et al.1998, Yuan, Estes the et al.2001, Ohlschlager, Osen of protectiveness Et al.2003, Senger, Schadlich et al.2010, Wu, Gersch et al.2011), and it is based on prokaryotes E The expression technology of coli can substantially reduce the production cost of HPV vaccines.In Chinese invention patent 02129070.9《Papillomatosis The protokaryon of virus capsid protein prepares and application》The space three-dimensional structure of middle offer HPV 16L1 pentamers, and utilize Escherichia coli Great expression and the technical method for preparing human papilloma virus capsid protein L 1.

Aluminium adjuvant is a kind of adjuvant being most widely used at present, have more than 80 years applicating history and tens billion of person-times Usage record, and be once that human vaccination is used for by U.S.'s food and Drug Administration (FDA) approval for a long time Unique adjuvant (Glenny, Pope et al.1926, Gupta and Siber 1995, Gupta 1998, Baylor, Egan Et al.2002, Clements and Griffiths 2002, Lindblad 2004, Lindblad 2004).But it is in HPV Come with some shortcomings in the application of L1 proteantigens.Alum adjuvant only forms Reservoir effect (Gupta, Chang in injection site Et al.1996, Hem 2002, Verdier, Burnett et al.2005, Hem and Hogenesch 2007, Noe, Green et al.2010), Dendritic Cells (DC) (Morefield, Sokolovska et is attracted by inflammatory reaction Al.2005, 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 presentation Cell (antigen-presenting cell, APC), these cells absorb antigen and small through NLRP3 inflammatories by phagocytosis Body activation immune 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).Meanwhile aluminium adjuvant is by stimulating IL- The mechanism such as 10 secretions produce cellular immunity inhibitory action (Chen, Ni et al.2011), and for virus infects, carefully Born of the same parents' immune effect will play more preferable ground 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, aluminium adjuvant can cause the allergic reaction (such as injection site granuloma) that IgE is mediated And nervous system adverse reaction etc., so as to cause worry (Petrik, Wong et of the people to aluminium adjuvant security Al.2007, Bystrianyk2009, Shaw and Petrik 2009, Munks, McKee et al.2010, Tomljenovic and Shaw 2011).For this reason, it may be necessary to be directed to HPV L1 proteantigens, either to based on L1 pentamers, Or candidate vaccine exploitation immunologic adjuvant (Campo and Roden 2010, Mariani the and Venuti of L1VLP 2010, Chen, Ni et al.2011, Foged 2011, Gattoc, Nair et al.2013, Koff, Burton et Al.2013, Shaw 2013, Tomljenovic, Spinosa et al.2013).

Lot of documents confirms in recent years, and internal biodegradable polymer receives microballoon by embedding antigen, can be by its turn Granular pattern antigen is turned to, is conducive to be absorbed by antigen presenting cell, and then in intracellular released antigen, processed by follow-up antigen, Offer, enhancing 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).For example, Torres M.P. et al. are prepared for poly- glycoside microballoon, with ovalbumin (OVA) for mould Type antigen, studies the adjuvant effect of microballoon, the results showed that microballoon can improve the MHC molecule expression of antigen presenting cell surface and phase Cytokine secretion is closed, shows it with certain adjuvant effect (Torres, Wilson-Welder et al.2011)；And for example Uto T. et al. are prepared for biodegradable polyglutamic acid and receive microballoon (γ-PGA NPs) and to have studied its adjuvant from mechanism Performance, disclosing this microballoon can be strong intrinsic and acquired by TLR4 (Toll-like receptor) and the induction of MyD88 signal paths Immune response (Uto, Akagi et al.2011).Research shows that the above-mentioned particle type adjuvant for carrying antigen makes solubility Antigen becomes granular pattern antigen, and after granular pattern antigen is absorbed by APC, thus it is possible to vary antigen offers approach, can not only swash CD4+T cells living, and CD8+T cells can be activated, cellular immune function is greatly promoted, realizes the thorough clear of intracellular infection Remove, be promising virus infection vaccine adjuvant and delivery system (Wang and Singh 2011, Dierendonck, De Koker et al.2012).In addition, biodegradable polymer microballoon can provide a large amount of antigens and protected from physiology Under the conditions of fast degradation, by microballoon to embedded antigen continue or pulse release behavior, effectively simulate traditional vaccine it is more Secondary immune programme, reduces the use total amount of inoculation times and immunogene, thus improve the adaptability of patient, reduce medication into This (Langer, Cleland et al.1997, Johansen, Estevez et al.2000, De Temmerman, Rejman Et al.2011, Demento, Cui et al.2012).

As the adjuvant and delivery system of antigen protein, many physicochemical properties of polymer beads can influence its in vivo with The interaction of immunocyte, and type of immune response, intensity and level are finally influenced, including particle diameter, form, surface nature etc. (Gupta and Siber 1995, Hem and Hogenesch 2007, Mastelic, Ahmed et al.2010, Wang And Singh 2011, Korsholm, Andersen et al.2012).For example, (Xiang, the Scholzen such as Xiang S.D. Et al.2006) mentioned in survey article, the positively charged particle in surface and the negatively charged and uncharged particle in surface Compare, it is easier to the antibody and CD8+T cell responses of induced high levels；Foged C. et al. (Foged, Brodin et Al.2005 influence of the Particle surface charge to DC cellular uptake behaviors) is have studied, has obtained similar results, i.e. surface positively charged The particle of lotus is easier to be absorbed by DC；And the research of Yasuhiko T. et al. (Tabata and Ikada 1988) obtains Opposite conclusion, they think that negatively charged cellulose microsphere can promote the intake of macrophage.Equally, polymer beads Influence of the particle diameter to immunology effect also not yet forms unified final conclusion.Gutierro I. et al. (Gutierro, Hernandez Et al.2002) find, compared with 200 and 500nm particle diameters, 1 compares, PLGA microballoons can produce high-level antibody；Mann Et al. J.S.F. (Mann, Shakir et al.2009) is prepared for carrying the natural biological membrane vesicles (Biosome) of influenza antigens, The result shows that small particle is conducive to inducement efficient humoral immune response, and big particle diameter is then inclined to induction body generation cellular immunity and answers Answer；WendorfJ et al. (Wendorf, Chesko et al.2008) using the electronegative PLGA microballoons in surface (110nm and 1000nm) adsorb HIV-1 and induce MenB (Meningococcal B) albumen of meningitis, 2 particle diameters generate similar exempt from Epidemic disease response.Different researchers obtain different results for particle identity matter.The present invention is directed to HPV L1 antigen proteins, receives micro- Influence of the ball particle size to its immune response, design are prepared uniform particle diameter, the PLGA of different-grain diameter and receive microballoon, embedded with it HPV L1 antigen proteins, research PLGA receive microspherulite diameter to vivo immunization response intensity and horizontal influence, develop efficient HPV L1 protein vaccine compositions.

The content of the invention

First aspect is the present invention provide a kind of vaccine combination, comprising the antigen with adjuvant combination, the ratio of antigen and adjuvant Example is 10-80 μ g/mg, and the antigen includes HPV L1 albumen, and the adjunvant composition includes polylactide-polyglycolic acid copolymer (PLGA) or polylactic acid (PLA).

Vaccine combination of the invention in a specific embodiment of the present invention, wherein the HPV L1 albumen is embedded in In PLGA or adsorb on PLGA surfaces, exist with receive microspheres form of the particle diameter distribution between 100nm~100 μm.

The vaccine combination in the specific embodiment of the present invention, wherein the microballoon 0-90% that receives is distributed in 100- Between 1000nm, 1-10 μm or 10-100 μm of any particle size range or its combination.

The vaccine combination in the specific embodiment of the present invention, the microballoon 0-90% that preferably receives are distributed in 200-900nm, 2- Between 9 μm or 20-90 μm of any particle diameter or its combination.

The vaccine combination in the specific embodiment of the present invention, the microballoon 0-90% that preferably receives are distributed in 400-700nm, 4- Between 7 μm or 40-70 μm of any particle diameter or its combination.

The vaccine combination in the specific embodiment of the present invention, the microballoon 10-90% that receives are distributed in 100- 1000nm, 90-10% are distributed in 1-10 μm.

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

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

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

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

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

The present invention specific embodiment in vaccine combination, the microballoon 10-90% that preferably receives be distributed in 100-1000nm, 90-10% is distributed in 10-100 μm.

The vaccine combination in another specific embodiment of the present invention, wherein the microballoon 10-90% that receives is distributed 30-80 μm is distributed in 300-800nm, 90-10%.

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

In the specific embodiment of the present invention, wherein described particle size dispersion of the microballoon in each particle size range of receiving Coefficient range is 0.005~0.2.

Vaccine combination in the specific embodiment of the present invention, wherein being further included in the adjuvant as follows Immune-enhancing effect component lipid material：One or more of combination in lecithin, stearylamine, glycerophosphatide, wherein lipid material with The ratio of PLGA or PLA is 1-99: 99-1, preferably 1-50: 100.

More specifically, the vaccine combination in the specific embodiment of the present invention, wherein being further included in the adjuvant Small molecule immune adjuvant：CpG, MPLA, imiquimod, PolyI：One or more of combination in C etc., wherein small molecule immune are helped The ratio of agent and PLGA or PLA are 10-100 μ g/mg.

The present invention specific embodiment in vaccine combination, wherein HPV L1 albumen refer to HPV total lengths, truncate or VLP, pentamer or the polymer that the L1 of restructuring is formed.

The vaccine combination in the specific embodiment of the present invention, wherein HPV L1 albumen refer to 6,11,16,18,26, 30th, in 31,33,34,35,39,45,51,52,53,56,58,59,66,67,68,69,70,72,73 or 82,85,97 type HPV A kind of and/or several combination.

On the other hand the present invention discloses a kind of bacterin preparation, and the bacterin preparation is as the vaccine combination described in 19 and HPV L1 Albumen and pharmaceutic adjuvant are made, wherein it is 10-90%: 90-10% to receive HPV L1 and free HPV L1 ratios in microballoon, further More preferably 10-50%: 90-50%.

Bacterin preparation in the specific embodiment of the present invention, the pharmaceutic adjuvant buffer for physiological saline or PBS Liquid.

On the other hand the preparation method of vaccine combination mainly comprising embedding and is inhaled in the specific embodiment of the present invention Attached two methods, the wherein embedding method include the following steps：

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

(2) PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent and oil phase (O) is made, by the water phase (W1) mixed with oil phase (O), water-in-oil type (W1/O) pre-emulsion is made, this pre-emulsion is added in outer aqueous phase (W2), W1/ is made The pre- double emulsion of W1/O/W2 types, is crossed film and obtains the W1/O/W2 type lotions of size uniformity by the pre- double emulsion of O/W2 types repeatedly；

(3) emulsion droplet cures：By W1/O/W2 type lotion normal temperature cures, vaccine combination is made after washing, drying.

A kind of preparation method, the inner aqueous phase pH value range described in step (1) are provided in the specific embodiment of the present invention For 4.0-12.0, the buffer salt system described in step (1) is citric acid-disodium hydrogen phosphate, Acetic acid-sodium acetate buffer solution or phosphoric acid Salt buffer.

The preparation method provided in the specific embodiment of the present invention, the inner aqueous phase pH value model described in step (1) Enclose for 6.0-8.0.

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

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

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

The W1/O/W2 type lotions of size uniformity in the specific embodiment of the present invention described in step (2) are to pass through W1/O/W2 types pre-emulsion is pressed through what microporous barrier obtained repeatedly under elevated pressures effect.

Emulsion droplet containing antigen in the specific embodiment of the present invention described in step (2), water phase (W1) and oil phase (O) body Product is than being 1: 1~1: 50.

Step (3) curing mode is solvent evaporated method and solvent extraction in the specific embodiment of the present invention.

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

On the other hand the preparation method of another vaccine combination disclosed in the specific embodiment of the present invention, that is, adsorb Method includes the following steps that PLGA/PLA or PLGA/PLA and lipid material are dissolved in organic solvent by (1) and oil phase (O) is made, and incites somebody to action This oil phase is added in outer aqueous phase (W2), and the pre- double emulsion of O/W2 types is made；

(2) the pre- double emulsion of O/W2 types is crossed to film repeatedly and obtains the O/W2 type lotions of size uniformity；After washing, drying PLGA/PLA microballoons；

(3) HPV L1 albumen and/or small molecule immune adjuvant CpG are dissolved in buffer salt system, obtain 1-40 μ g/0.2- The HPV antigenic solutions of 1mL, PLGA/PLA microballoons are dissolved in buffer salt system, obtain the solution of 1-10mg/mL；By two solution etc. Volume mixture, absorption overnight, obtain being adsorbed with the PLGA suspensions of HPV antigens, are HPV vaccine combinations.

Concrete operation step in adsorption method mentioned above is referred to the corresponding steps in embedding method.

On the other hand a kind of preparation method of bacterin preparation is provided in the specific embodiment of the present invention, in vaccine combination Following steps are further included after emulsion droplet curing schedule in the preparation method of thing：

Bacterin preparation is formed：Take the vaccine combination (receive microballoon) of mass ratio 10-90%: 90-10% and free HPVL1 Albumen physiological saline or PBS buffer suspend, and are configured to the microsphere suspensions of the HPV antigens containing 1-40 μ g/0.2-1mL, system Obtain HPV bacterin preparations.

The preparation method of the bacterin preparation provided in the specific embodiment of the present invention, PBS buffer pH range 6- 8, microballoon concentration of receiving is 1-10mg/mL.

On the other hand the vaccine combination disclosed in the specific embodiment of the present invention is preparing treatment or prevention HPV infection Medicine in application.

Bacterin preparation disclosed in the specific embodiment of the present invention is in the medicine for treating or preventing HPV infection is prepared Application.

In an embodiment of any method of offer, by oral, subcutaneous, lung, intranasal, abdominal cavity, lymph, true Intracutaneous or intramuscular delivery applies the formulation.

In another embodiment, there is provided a kind of bacterin preparation of human papilloma virus, it is to be coated with HPV A kind of bacterin preparation based on the homogeneous microballoons of PLGA of L1VLP antigens, while other molecule adjuvants can be embedded or compound altogether, may be used also Compound a certain amount of not coated HPVL1VLP antigens into vaccine, i.e., by be coated on receive microballoon HPV L1 antigens with it is free HPV L1 antigens are common and clinical or bacterin preparation is made in pharmaceutically acceptable pharmaceutic adjuvant.

Microballoon moderate resistance original charging ratio is more than 20ug/mg in another embodiment；Envelope antigen and free antigen matter It is 0-100% to measure ratio.

It is smooth using microsphere features smooth surface prepared by the present invention, spherical good (Fig. 1, Fig. 2, Fig. 3), in each particle diameter model It is controllable (Fig. 4) to enclose interior prepared uniform particle diameter, particle size dispersion coefficient is 0.005~0.2, preferably 0.005~0.1 in scope, More preferably 0.005~0.05.Drugloading rate and embedding rate are high, zoopery show the new HPV vaccines of the present invention be it is safe, it is right Experimental animal effectively can produce stronger humoral immunity and cellullar immunologic response (Fig. 5, figure by stimulating animal without visible toxicity 6, Fig. 7, Fig. 8) it can be used for the relevant disease of human papilloma virus or the prevention and treatment of infection.

In order to be clearly understood that the present invention, it is further illustrated below with reference to attached drawing and example, but following embodiment is simultaneously The non-limiting present invention.

Brief description of the drawings

Fig. 1 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (430nm)

Fig. 2 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (1.38 μm)

Fig. 3 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (5.60 μm)

Fig. 4 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (400nm)

Fig. 5 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (800nm)

Fig. 6 PLGA embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (1.3 antigen)

Fig. 7 PLGA/PC embedding HPVL1 pentamer antigens receive microballoon scanning electron microscope (SEM) photograph (400nm)

Fig. 8 PLGA parcel HPV16L1 pentamers receive microballoon scanning electron microscope (SEM) photograph (1 aggressiveness)

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

Figure 10 PLGA embedding HPVL1VLP antigens receive microballoon scanning electron microscope (SEM) photograph (1 original is received)

Figure 11 PLGA/HSPC embedding HPVL1VLP antigens receive microballoon scanning electron microscope (SEM) photograph (1 original is received)

Figure 12 PLA embedding HPVL1VLP antigens receive microballoon scanning electron microscope (SEM) photograph (1 original is received)

Figure 13 PLGA embedding HPVL1VLP antigens receive microballoon scanning electron microscope (SEM) photograph (10 receive micro-)

Figure 14 PLGA receive microballoon scanning electron microscope (SEM) photograph (1 microballoon)

Figure 15 carries HPV16L1 pentamer antigens PLGA and receives microspherulite diameter distribution map

Figure 16 different-grain diameters carry antigen PLGA microballoon groups and mouse neutralizing antibody titers in serum after 38 days are immunized

Figure 17 different-grain diameters carry antigen PLGA microballoon groups and mouse specific antibody IgG titres in serum after 38 days are immunized

The cytokine levels testing result (Luminex technologies) in splenocyte supernatant after mouse is immunized in Figure 18 each groups

Figure 19 each groups are immunized after mouse that IFN- in splenocyte supernatant is small and the horizontal testing results of IL-4 (ELISA method)

The small elisa of splenocyte IL-4 and IFN- forms cell number (SFC) after mouse is immunized in Figure 20 each groups (ELISPOT methods)

After mouse is immunized using different adjuvant systems in Figure 21 HPV L1 pentamer albumens, serum binding antibody titre

After mouse is immunized using different adjuvant systems in Figure 22 HPV L1 pentamer albumens, the titre of neutralizing antibody

After mouse is immunized using different adjuvant systems in Figure 23 HPV L1 pentamer albumens, the cellular immunity of ELISPOT measure Response result

Mouse is immunized using different adjuvant systems in Figure 24 HPV L1 pentamer albumens, takes its splenocyte condition of culture in vitro It is lower after antigenic stimulus, ELISA measure IFN-A and IL-4 cytokine secretion profiles

Mouse is immunized using different adjuvant systems in Figure 25 HPV L1 pentamer albumens, takes its splenocyte condition of culture in vitro It is lower after antigenic stimulus, Luminex methods measure cytokine secretion profile.

Neutralizing antibody drips after mouse is immunized in the microballoon of Figure 26 PLGA compounding CpG small molecule adjuvants coating HPV16L1 pentamers Degree

Binding antibody drips after mouse is immunized in the microballoon of Figure 27 PLGA compounding CpG small molecule adjuvants coating HPV16L1 pentamers Degree

Binding antibody drips after mouse is immunized in the microballoon of the PLGA coating HPV16L1 pentamers of Figure 28 different-grain diameters and combinations thereof Degree

Neutralizing antibody titers after mouse are immunized in the microballoon of the PLGA coatings HPV16L1VLP of Figure 29 different-grain diameters and combinations thereof

Neutralizing antibody titers after mouse are immunized in Figure 30 PLGA, the microballoon of PLGA/HSPC, PLA coating HPV16L1VLP

Binding antibody titre after mouse is immunized in Figure 31 PLGA, the microballoon of PLGA/HSPC, PLA coating HPV16L1VLP

The binding antibody titre after mouse is immunized after Figure 32 PLGA microballoons absorption HPV16L1VLP

Instantiation

The present invention implements technical scheme with reference to following form, and technical scheme includes but not limited to such as Lower embodiment.

Table 1

Table 2

Table 3

Table 4

Specific embodiment is as follows involved in above table：

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

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 6L1 five that 3mL concentration is 1.33mg/mL Aggressiveness is dissolved in the antigen protein of citric acid-disodium hydrogen phosphate buffer salt solution as inner aqueous phase (W1), thin with S-450D ultrasonic waves Born of the same parents crush instrument and just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) preparation W1/O types are carried out under the conditions of ice-water bath Colostrum, then pours into colostric fluid in the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, using magnetic force Stirring prepares pre- double emulsion (W1/O/W2), and emulsification condition is to stir 2min under 600r/min.Then, this pre- double emulsion is poured into In the storage tank of fast film emulsification, double emulsion is pressed through as 0.6 μm of SPG films by membrane aperture using the nitrogen of 1.5M Pa pressure repeatedly, Until obtaining homogeneous emulsion drop (W1/O/W2).The 0.9%NaCl that obtained homogeneous emulsion drop is poured into 780mL is molten In liquid, 4h (magnetic agitation speed 200rpm) is stirred at room temperature and removes organic solvent, microballoon deionized water after curing from The heart washs 3 times, and finished product is made in finally freeze-drying.With laser particle analyzer measure particle diameter distribution situation, average grain diameter 400nm, PDI values are 0.04, and medicine embedding rate is 90.8%.

Specific embodiment two --- the preparation of the HPV vaccines comprising free antigen

The load HPV 6L1 pentamer antigen microballoons 4.45mg that the average grain diameter prepared by embodiment one is 100nm is weighed to answer With the HPV 6L1 pentamer antigens that 120.3 μ L concentration are 1.333mg/mL, the PBS buffer of pH=7.0 is added to 1mL, is prepared G/mL's and the microsphere suspensions of envelope antigen in microballoon and HPV antigens that free antigen ratio is 2: 8 must be received containing 200 μ, be made HPV bacterin preparations.

Specific embodiment three --- 430nm PLGA carry the preparation of HPV 16L1 pentamer antigen microballoons

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), and it is poly- to add the HPV 1,6L1 five that 3mL concentration is 4mg/mL Body antigen protein carries out just emulsification under the conditions of ice-water bath with S-450D ultrasonic cell disruption instruments and (surpasses as inner aqueous phase (W1) Acoustical power is set as 30%, ultrasonic time 60s) prepare W1/O type colostrums, then by colostric fluid pour into 65mL contain 1.5% In the aqueous solution (outer aqueous phase W2) of the NaCl of PVA (w/v), pre- double emulsion (W1/O/W2), emulsification condition are prepared using magnetic agitation To stir 2min under 600r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1M Pa pressure Double emulsion is pressed through membrane aperture and is 1.4 μm of SPG films by nitrogen repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).By institute Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, and 4h (magnetic agitation speeds are stirred at room temperature 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.With Laser particle analyzer measures particle diameter distribution situation, and average grain diameter 430nm, PDI value are 0.03, and medicine embedding rate is 92.6%.Institute Microballoon scanning electron microscope (SEM) photograph is made and sees Fig. 1.

Specific embodiment five --- 1.38 μm of PLGA carry HPV16L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 1,6L1 five that 3mL concentration is 6.67mg/mL Aggressiveness antigen protein carries out just emulsification with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 0.25M Double emulsion is pressed through membrane aperture and is 5.2 μm of SPG films by the nitrogen of Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic agitations are stirred at room temperature Speed 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finally freeze-drying is made into Product.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 1.38 μm, and PDI values are 0.05, and medicine embedding rate is 93.1%.It is as shown in Figure 2 that obtained load antigen receives microballoon scanning electron microscope (SEM) photograph.

Specific embodiment six --- 1 μm of PLGA carries HPV16L1 pentamer antigens microballoon and prepares

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 1,6L1 five that 3mL concentration is 6.67mg/mL Aggressiveness antigen protein carries out just emulsification with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 0.25M Double emulsion is pressed through membrane aperture and is 5.2 μm of SPG films by the nitrogen of Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic agitations are stirred at room temperature Speed 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finally freeze-drying is made into Product.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 1 μm, and PDI values are 0.05, and medicine embedding rate is 93.1%. It is as shown in Figure 8 that obtained load antigen receives microballoon scanning electron microscope (SEM) photograph.

Specific embodiment seven --- 5.6 μm of PLGA carry HPV16L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 1,6L1 five that 3mL concentration is 9.33mg/mL Aggressiveness antigen protein carries out just emulsification with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 300r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 40kPa Double emulsion is pressed through membrane aperture and is 15 μm of SPG films by the nitrogen of pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).4h (magnetic agitation speed 200rpm) is stirred at room temperature in obtained homogeneous double emulsion drop and removes organic solvent, is cured Finished product is made in the deionized water centrifuge washing 3 times of microballoon afterwards, finally freeze-drying.Particle diameter distribution feelings are measured with laser particle analyzer Condition, average grain diameter are 5.6 μm, and PDI values are 0.08, and medicine embedding rate is 79.7%.Obtained load antigen receives microballoon scanning electron microscope Figure is as shown in Figure 3.

Specific embodiment 17 --- 20.82 μm of PLGA carry HPV45L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 4,5L1 five that 3mL concentration is 5.333mg/mL Aggressiveness antigen protein carries out just emulsification with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 250r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, is pressed with 5kPa Double emulsion is pressed through membrane aperture and is 52 μm of SPG films by the nitrogen of power repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2). 4h (magnetic agitation speed 200rpm) is stirred at room temperature in obtained homogeneous double emulsion drop and removes organic solvent, after curing Finished product is made in microballoon deionized water centrifuge washing 3 times, finally freeze-drying.Particle diameter distribution situation is measured with laser particle analyzer, Average grain diameter is 20.82 μm, and PDI values are 0.10, and medicine embedding rate is 89.2%.

Specific embodiment 18 --- 80.71 μm of PLA carry HPV58L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 5,8L1 five that 3mL concentration is 5.333mg/mL Aggressiveness antigen protein carries out just emulsification with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 250r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 0.1kPa Double emulsion is pressed through membrane aperture and is 190.3 μm of SPG films by the nitrogen of pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).4h (magnetic agitation speed 200rpm) is stirred at room temperature in obtained homogeneous double emulsion drop and removes organic solvent, is cured Finished product is made in the deionized water centrifuge washing 3 times of microballoon afterwards, finally freeze-drying.Particle diameter distribution feelings are measured with laser particle analyzer Condition, average grain diameter are 80.71 μm, and PDI values are 0.18, and medicine embedding rate is 85.5%.

Specific embodiment 19 --- 400nm PLGA/HSPC carry HPV16L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will PLGA the and 100mg lecithin of 300mg, which is dissolved in the MC solvents of 10.0mL, is used as oil phase (O), and it is 1.333mg/ to add 3mL concentration The HPV 6L1 pentamers antigen proteins of mL are as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments in ice-water bath condition Lower progress just emulsifies (ultrasonic power is set as 30%, ultrasonic time 60s) and prepares W1/O type colostrums, then pours into colostric fluid In the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, pre- double emulsion (W1/ is prepared using magnetic agitation O/W2), emulsification condition is to stir 2min under 500r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, Double emulsion is pressed through as 1.4 μm of SPG films by membrane aperture using the nitrogen of 1M Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).It is organic molten that 4h (magnetic agitation speed 200rpm) removals are stirred at room temperature in obtained homogeneous double emulsion drop Agent, microballoon after curing deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.Grain is measured with laser particle analyzer Footpath distribution situation, average grain diameter 400nm, PDI value are 0.05, and medicine embedding rate is 92.8%.Thus obtained microsphere scanning electron microscope (SEM) photograph Such as 7.

Specific embodiment 20 --- 800nm PLGA carry HPV16L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 300mg, which is dissolved in the MC solvents of 10.0mL, is used as oil phase (O), and it is poly- to add the HPV 1,1L1 five that 3mL concentration is 4mg/mL Body antigen protein carries out just emulsification under the conditions of ice-water bath with S-450D ultrasonic cell disruption instruments and (surpasses as inner aqueous phase (W1) Acoustical power is set as 30%, ultrasonic time 60s) prepare W1/O type colostrums, then by colostric fluid pour into 65mL contain 1.5% In the aqueous solution (outer aqueous phase W2) of the NaCl of PVA (w/v), pre- double emulsion (W1/O/W2), emulsification condition are prepared using magnetic agitation To stir 2min under 500r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1M Pa pressure Double emulsion is pressed through membrane aperture and is 9.2 μm of SPG films by nitrogen repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).By institute Obtained homogeneous double emulsion drop is stirred at room temperature 4h (magnetic agitation speed 200rpm) and removes organic solvent, the microballoon after curing With deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.Particle diameter distribution situation is measured with laser particle analyzer, it is average Particle diameter is 800nm, and PDI values are 0.06, and medicine embedding rate is 88.2%.The scanning electron microscope (SEM) photograph of thus obtained microsphere such as Fig. 5.

Specific embodiment 21 --- 1300nm PLGA carry HPV16L1 pentamer antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 300mg, which is dissolved in the MC solvents of 10.0mL, is used as oil phase (O), and it is poly- to add the HPV 1,1L1 five that 3mL concentration is 4mg/mL Body antigen protein carries out just emulsification under the conditions of ice-water bath with S-450D ultrasonic cell disruption instruments and (surpasses as inner aqueous phase (W1) Acoustical power is set as 30%, ultrasonic time 60s) prepare W1/O type colostrums, then by colostric fluid pour into 65mL contain 1.5% In the aqueous solution (outer aqueous phase W2) of the NaCl of PVA (w/v), pre- double emulsion (W1/O/W2), emulsification condition are prepared using magnetic agitation To stir 2min under 500r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1M Pa pressure Double emulsion is pressed through membrane aperture and is 9.2 μm of SPG films by nitrogen repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).By institute Obtained homogeneous double emulsion drop is stirred at room temperature 4h (magnetic agitation speed 200rpm) and removes organic solvent, the microballoon after curing With deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.Particle diameter distribution situation is measured with laser particle analyzer, it is average Particle diameter is 1300nm, and PDI values are 0.06, and medicine embedding rate is 88.2%.The scanning electron microscope (SEM) photograph of thus obtained microsphere such as Fig. 6.

Specific embodiment 26 --- 1000nm PLGA carry antigen and receive microballoon to be embedded CPG1826 to carry HPV16L1 five poly- altogether It is prepared by body antigen microballoon

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), and it is poly- to add the HPV 1,6L1 five that 3mL concentration is 4mg/mL Body antigen protein and 5 μ g CpG1826 are as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments under the conditions of ice-water bath Carry out just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) and prepare W1/O type colostrums, then pour into colostric fluid In the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, pre- double emulsion (W1/ is prepared using magnetic agitation O/W2), emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, Double emulsion is pressed through as 1.4 μm of SPG films by membrane aperture using the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous double emulsion Drip (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h is stirred at room temperature (magnetic agitation speed 200rpm) removes organic solvent, and microballoon after curing deionized water centrifuge washing 3 times, finally freezing is dry It is dry that finished product is made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter 1000nm, PDI value are 0.06, medicine embedding Rate is 87.8%.Thus obtained microsphere scanning electron microscope (SEM) photograph such as Fig. 9.

Specific embodiment 27 --- 1.57 μm of PLGA carry antigens and receive microballoon to be embedded MPLA and carries HPV16L1 pentamers altogether It is prepared by antigen microballoon

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 1,6L1 five that 3mL concentration is 6.67mg/mL Aggressiveness antigen protein and 5 μ g MPLA as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments under the conditions of ice-water bath into Just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums to row, and colostric fluid then is poured into 65mL The NaCl containing 1.5%PVA (w/v) aqueous solution (outer aqueous phase W2) in, pre- double emulsion (W1/O/ is prepared using magnetic agitation W2), emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with Double emulsion is pressed through membrane aperture and is 5.2 μm of SPG films by the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic is stirred at room temperature Power stir speed (S.S.) 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, is finally freeze-dried Finished product is made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 1.57 μm, and PDI values are 0.05, medicine embedding rate For 82.2%.

Specific embodiment 29 --- 23.57 μm of PLA carry antigens and receive microballoon embeds PolyI altogether：It is poly- that C carries HPV45L1 five It is prepared by body antigen microballoon

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 1,6L1 five that 3mL concentration is 5.33mg/mL Aggressiveness antigen protein and 5 μ g PolyI：C is as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments in ice-water bath condition Lower progress just emulsifies (ultrasonic power is set as 30%, ultrasonic time 60s) and prepares W1/O type colostrums, then pours into colostric fluid In the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) of 65mL, pre- double emulsion (W1/ is prepared using magnetic agitation O/W2), emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, Double emulsion is pressed through as 5.2 μm of SPG films by membrane aperture using the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous double emulsion Drip (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h is stirred at room temperature (magnetic agitation speed 200rpm) removes organic solvent, and microballoon after curing deionized water centrifuge washing 3 times, finally freezing is dry It is dry that finished product is made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 23.57 μm, and PDI values are 0.09, medicine bag Rate is buried as 86.3%.Specific embodiment 31 --- 400nmPLGA carries the preparation of HPV 16L1 pentamer antigen microballoons

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV that 3mL concentration is 1.33mg/mL 16L1VLP antigen proteins carry out colostrum with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Change (ultrasonic power is set as 30%, ultrasonic time 60s) and prepare W1/O type colostrums, colostric fluid is then poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 600r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1.5M Double emulsion is pressed through membrane aperture and is 1.4 μm of SPG films by the nitrogen of Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic agitations are stirred at room temperature Speed 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finally freeze-drying is made into Product.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter 400nm, PDI value are 0.05, and medicine embedding rate is 90.5%.It is prepared obtain microballoon scanning electron microscope (SEM) photograph it is as shown in Figure 4

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

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 6L1VLP that 3mL concentration is 1.33mg/mL Antigen protein carries out just emulsification (ultrasound with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Power setting is 30%, ultrasonic time 60s) W1/O type colostrums are prepared, then contain 1.5%PVA by what colostric fluid poured into 65mL (w/v) in the aqueous solution (outer aqueous phase W2) of NaCl, pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, emulsification condition is 2min is stirred under 600r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1.5M Pa pressure Double emulsion is pressed through membrane aperture and is 0.4 μm of SPG films by nitrogen repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).By institute Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, and 4h (magnetic agitation speeds are stirred at room temperature 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.With Laser particle analyzer measures particle diameter distribution situation, and average grain diameter 128nm, PDI value are 0.03, and medicine embedding rate is 91.7%.

Specific embodiment 37

The load HPV 6L1VLP antigen microballoons 4.4mg that the average grain diameter prepared by embodiment 36 is 128nm is weighed to answer With the HPV 6L1 pentamer antigens that 12.03 μ L concentration are 13.33mg/mL, add the PBS buffer of physiological saline or pH=7.0 extremely 1mL, g/mL's and receives the microballoon of envelope antigen and the HPV antigens that free antigen ratio is 2: 8 in microballoon and hangs with being made containing 200 μ Liquid, is made HPV bacterin preparations.

Specific embodiment 38 --- 467nm PLGA carry the preparation of HPV 11L1VLP antigen microballoons

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 11L1VLP that 3mL concentration is 4mg/mL and resists Former albumen carries out just emulsification (ultrasonic work(with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Rate is set as 30%, ultrasonic time 60s) W1/O type colostrums are prepared, then colostric fluid is poured into (the w/ containing 1.5%PVA of 65mL V) in the aqueous solution (outer aqueous phase W2) of NaCl, pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, emulsification condition is 2min is stirred under 600r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with the nitrogen of 1M Pa pressure Double emulsion is pressed through membrane aperture and is 1.4 μm of SPG films by gas repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).Will be made The homogeneous emulsion drop obtained is poured into the 0.9%NaCl solution of 780mL, and 4h (magnetic agitation speed 200rpm) is stirred at room temperature Remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finished product is made in finally freeze-drying.With laser grain Instrument measure particle diameter distribution situation is spent, average grain diameter 467nm, PDI value are 0.05, and medicine embedding rate is 89.2%.

Specific embodiment 40 --- 1 μm of PLGA carries HPV16L 1VLP antigens microballoon and prepares

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV that 3mL concentration is 6.67mg/mL 16L1VLP antigen proteins carry out colostrum with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Change (ultrasonic power is set as 30%, ultrasonic time 60s) and prepare W1/O type colostrums, colostric fluid is then poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 0.25M Double emulsion is pressed through membrane aperture and is 5.2 μm of SPG films by the nitrogen of Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic agitations are stirred at room temperature Speed 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, finally freeze-drying is made into Product.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 1 μm, and PDI values are 0.05, and medicine embedding rate is 89.5%. It is as shown in Figure 10 that obtained load antigen receives microballoon scanning electron microscope (SEM) photograph.

Specific embodiment 42 --- 5.87 μm of PLGA carry HPV18L1VLP antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV that 3mL concentration is 9.33mg/mL 18L1VLP antigen proteins carry out colostrum with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Change (ultrasonic power is set as 30%, ultrasonic time 60s) and prepare W1/O type colostrums, colostric fluid is then poured into containing for 65mL In the aqueous solution (outer aqueous phase W2) of the NaCl of 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, breast Change condition is to stir 2min under 300r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 40kPa Double emulsion is pressed through membrane aperture and is 15 μm of SPG films by the nitrogen of pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).4h (magnetic agitation speed 200rpm) is stirred at room temperature in obtained homogeneous double emulsion drop and removes organic solvent, is cured Finished product is made in the deionized water centrifuge washing 3 times of microballoon afterwards, finally freeze-drying.Particle diameter distribution feelings are measured with laser particle analyzer Condition, average grain diameter are 5.87 μm, and PDI values are 0.07, and medicine embedding rate is 89.2%.

Specific embodiment 52 --- 25.02 μm of PLA carry HPV45L1VLP antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 45L1VLP that 3mL concentration is 5.33mg/mL Antigen protein carries out just emulsification (ultrasound with S-450D ultrasonic cell disruption instruments as inner aqueous phase (W1) under the conditions of ice-water bath Power setting is 30%, ultrasonic time 60s) W1/O type colostrums are prepared, then contain 1.5%PVA by what colostric fluid poured into 65mL (w/v) in the aqueous solution (outer aqueous phase W2) of NaCl, pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, emulsification condition is 2min is stirred under 250r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with the nitrogen of 5kPa pressure Double emulsion is pressed through to membrane aperture repeatedly and is 52 μm of SPG films, until obtaining homogeneous emulsion drop (W1/O/W2).By obtained by Homogeneous double emulsion drop 4h (magnetic agitation speed 200rpm) be stirred at room temperature remove organic solvent, the microballoon after curing spends Finished product is made in ionized water centrifuge washing 3 times, finally freeze-drying.Particle diameter distribution situation, average grain diameter are measured with laser particle analyzer For 25.02um, PDI values are 0.06, and medicine embedding rate is 75.3%.

Specific embodiment 55 --- 1000nm PLGA/HSPC carry HPV16L1VLP antigens microballoon and prepare

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will PLGA the and 100mg lecithin of 300mg, which is dissolved in the MC solvents of 10.0mL, is used as oil phase (O), and it is 4mg/mL's to add 3mL concentration HPV 16L1VLP antigen proteins are carried out as inner aqueous phase (W1) with S-450D ultrasonic cell disruption instruments under the conditions of ice-water bath Just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into 65mL's In the aqueous solution (outer aqueous phase W2) of NaCl containing 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, Emulsification condition is to stir 2min under 500r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with 1M Double emulsion is pressed through membrane aperture and is 9.2 μm of SPG films by the nitrogen of Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/ W2).4h (magnetic agitation speed 200rpm) is stirred at room temperature in obtained homogeneous double emulsion drop and removes organic solvent, is cured Finished product is made in the deionized water centrifuge washing 3 times of microballoon afterwards, finally freeze-drying.Particle diameter distribution feelings are measured with laser particle analyzer Condition, average grain diameter 1000nm, PDI value are 0.07, and medicine embedding rate is 80.6%.Obtained load antigen receives microballoon scanning electron microscope Figure is as shown in figure 11.

Specific embodiment 56 --- 489nm PLGA carry antigen receive microballoon embed altogether CPG1826 carry HPV11L1VLP resist It is prepared by former microballoon

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 11L1VLP that 3mL concentration is 4mg/mL and resists Former albumen and 5 μ g CpG1826 are carried out as inner aqueous phase (W1) with S-450D ultrasonic cell disruption instruments under the conditions of ice-water bath Just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums, and colostric fluid then is poured into 65mL's In the aqueous solution (outer aqueous phase W2) of NaCl containing 1.5%PVA (w/v), pre- double emulsion (W1/O/W2) is prepared using magnetic agitation, Emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with Double emulsion is pressed through membrane aperture and is 1.4 μm of SPG films by the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic is stirred at room temperature Power stir speed (S.S.) 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, is finally freeze-dried Finished product is made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter 489nm, PDI value are 0.05, medicine embedding rate For 90.8%.

Specific embodiment 66 --- 1000nm PLA, which carry antigen and receive microballoon, to be carried HPV16L1VLP antigens microballoons and prepares

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV 16L1VLP that 3mL concentration is 6.67mg/mL Antigen protein and 5 μ g CpG1826 as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments under the conditions of ice-water bath into Just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) prepares W1/O type colostrums to row, and colostric fluid then is poured into 65mL The NaCl containing 1.5%PVA (w/v) aqueous solution (outer aqueous phase W2) in, pre- double emulsion (W1/O/ is prepared using magnetic agitation W2), emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storage tank of fast film emulsification, with Double emulsion is pressed through membrane aperture and is 1.4 μm of SPG films by the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, 4h (magnetic is stirred at room temperature Power stir speed (S.S.) 200rpm) remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times, is finally freeze-dried Finished product is made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter 1000nm, PDI value are 0.04, medicine embedding rate For 89.4%.It is as shown in figure 12 that obtained load antigen receives microballoon scanning electron microscope (SEM) photograph.

Specific embodiment 68 --- 10 μm of PLGA, which carry antigens and receive microballoon, to be carried HPV16L1VLP antigens microballoon and prepares

Prepared using fast film emulsifying technology combination double emulsion solvent removal method and carry antigen and receive microballoon.Specific method is：Will The PLGA of 400mg, which is dissolved in the EA solvents of 10.0mL, is used as oil phase (O), adds the HPV that 3mL concentration is 6.67mg/mL 16L1VLP antigen proteins and 5 μ g CpG1826 are as inner aqueous phase (W1), with S-450D ultrasonic cell disruption instruments in ice-water bath bar Just emulsification (ultrasonic power is set as 30%, ultrasonic time 60s) is carried out under part and prepares W1/O type colostrums, then falls colostric fluid In the aqueous solution (outer aqueous phase W2) of the NaCl containing 1.5%PVA (w/v) for entering 65mL, pre- double emulsion is prepared using magnetic agitation (W1/O/W2), emulsification condition is to stir 2min under 400r/min.Then, this pre- double emulsion is poured into the storing of fast film emulsification In tank, double emulsion is pressed through as 50.2 μm of SPG films by membrane aperture using the nitrogen of 0.25M Pa pressure repeatedly, until obtaining homogeneous Emulsion drop (W1/O/W2).Obtained homogeneous emulsion drop is poured into the 0.9%NaCl solution of 780mL, is stirred at room temperature Mix 4h (magnetic agitation speed 200rpm) and remove organic solvent, microballoon after curing deionized water centrifuge washing 3 times is last cold It is lyophilized dry finished product to be made.Particle diameter distribution situation is measured with laser particle analyzer, average grain diameter is 10 μm, and PDI values are 0.07, medicine bag It is 90.2% to bury rate, and obtained load antigen receives microballoon scanning electron microscope (SEM) photograph such as Figure 13 institutes not.

Specific embodiment 74

The 430nm that Example 31 is prepared respectively receives microballoon 1.128mg and embodiment five is prepared 1.38 5 system receive microballoon 1.157mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS delay Fliud flushing 1mL, it is 2.31mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 79

The 430nm that Example 31 is prepared respectively receives microballoon 1.128mg and embodiment 33 is prepared 20.65 μm receive microballoon 1.147mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS Buffer solution 1mL, it is 2.27mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 84

Be prepared 1.38 μm of Example five receive microballoon 1.157mg respectively and embodiment 33 is prepared 20.65 μm receive microballoon 1.147mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS delay Fliud flushing 1mL, it is 2.31mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 92

The 430nm that Example 31 is prepared respectively receives 1.38 μ that microballoon 0.68mg, embodiment five are prepared M receives microballoon 0.69mg and embodiment 33 is prepared 20.65 μm receive microballoon 0.92mg and be configured to antigenic quality ratio and be 30%: 30%: 40% mechanical fraction, add pH value be 7 PBS buffer 1mL, be prepared into the antigen containing 100 μ g/mL and Microballoon concentration of receiving is 2.29mg/mL suspensions.

Specific embodiment 101

Respectively be prepared 10 μm of Example 68 receive microballoon and example IV ten is prepared 1 μm receive microballoon The mechanical fraction that antigenic quality ratio is 20%: 80% is configured to, the PBS buffer 1mL that pH value is 7 is added, is prepared into containing 100 The antigen of μ g/mL and microballoon concentration of receiving are 2.35mg/mL suspensions.

Specific embodiment 102

Respectively be prepared 10 μm of Example 68 receive microballoon and example IV ten is prepared 1 μm receive microballoon The mechanical fraction that antigenic quality ratio is 80%: 20% is configured to, the PBS buffer 1mL that pH value is 7 is added, is prepared into containing 100 The antigen of μ g/mL and microballoon concentration of receiving are 2.35mg/mL suspensions.

Specific embodiment 103

1 μ that be prepared 10 μm of Example 68 receive microballoon 1.175mg respectively and example IV ten is prepared M receive microballoon 1.174mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS buffer 1mL, it is 2.35mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 106

The 458nm that Example 66 is prepared respectively receives microballoon 1.175mg and embodiment 68 is prepared 23.58 μm receive microballoon 1.164mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS Buffer solution 1mL, it is 2.34mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 111

Be prepared 1.69 μm of Example 40 receive microballoon 1.174mg respectively and embodiment 68 is prepared 23.58 μm receive microballoon 1.164mg be configured to antigenic quality ratio be 50%: 50% mechanical fraction, add pH value be 7 PBS delay Fliud flushing 1mL, it is 2.34mg/mL suspensions to be prepared into the antigen containing 100 μ g/mL and receive microballoon concentration.

Specific embodiment 120

The 458nm that Example 66 is prepared respectively receives microballoon 0.71mg, example IV ten and is prepared 1.69 μm receive microballoon 1.174mg and embodiment 68 is prepared 0.47 μm receive microballoon 0.004mg and be configured to antigenic quality Than the mechanical fraction for 30%: 50%: 20%, the PBS buffer 1mL that pH value is 7 is added, is prepared into anti-containing 100 μ g/mL It is former and to receive microballoon concentration be 2.35mg/mL suspensions.

The specific implementation step of other embodiment related specific embodiment with reference to more than is implemented in form.Example 120 Five --- microspherulite diameter and particle diameter distribution characterization

The microsphere suspension liquid that some of embodiments are prepared ultrasound in distilled water makes its dispersed, using sharp Light particle size analyzer (3000HS, UK) measure its particle size and distribution, difference carries antigens and receives microballoon adjuvant particle diameter distribution Figure is as shown in figure 15.

Embodiment 126 --- HPV charging ratios measure

Described lyophilized receive microballoon 5mg by what is prepared to be suspended in 1.0mL molten containing the NaOH that 5% (w/v) SDS concentration is 0.1M Liquid, when vibration 24 is small at room temperature, makes microballoon be completely dissolved (hydrolysis), and protein content in solution micro-BCA kits carry out Measure.

Embodiment 127 --- different-grain diameter PLGA carries antigen microballoon and rear antibody level evaluation comparison is immunized

The lyophilized microballoon that Example five and embodiment three prepare, is suspended in physiological saline, obtains microsphere suspensions, So that the content of antigen is 20 μ g/100 μ L in microsphere suspensions, that is, HPV bacterin preparations are made.

Receive microspheres vaccine preparation dorsal sc made from Example five and embodiment three female of 8-10 weeks is immunized for two kinds BALB/c mouse, every mouse injections of antigens dosage is 20 μ g/100 μ L, while the HPV pentamers set up using aluminium salt as adjuvant resist Original is as a control group.

More than detection three groups of Humoral.The index of detection includes different-grain diameter PLGA microballoons load antigen group and is immunized Neutralizing antibody of the mouse after 38 days is horizontal and specific antibody IgG is horizontal, respectively shown in Figure 16 and Figure 17.

The PLGA of different-grain diameter carries antigen and receives microballoon that the specific antibody neutralizing antibody produced after mouse is immunized is horizontal different. As shown in Figure 16, mouse immune carries neutralizing antibody level and the horizontal averages of IgG that antigen PLGA microballoons produce for 1.38 μm after 38 days All it is higher than aluminium adjuvant group, but there was no significant difference；But 1.38 μm carry neutralizing antibody level and IgG levels in antigen PLGA group serum It is all remarkably higher than 430nm and carries antigen PLGA groups (P ＜ 0.05 or P ＜ 0.01).

Embodiment 128 --- different-grain diameter PLGA carries antigen microballoon and rear cytokine levels evaluation comparison is immunized

Receive microspheres vaccine preparation dorsal sc made from Example five and embodiment three female of 8-10 weeks is immunized for two kinds BALB/c mouse, every mouse injections of antigens dosage is 20 μ g/100 μ L, while the HPV pentamers set up using aluminium salt as adjuvant resist Original is as a control group.

More than detection three groups of Study On Cellular Immune.Testing index includes stimulating mouse boosting cell 72h using antigen in vitro Afterwards, cytokine levels (Luminex technologies, ELISA method) in splenocyte supernatant, as a result as shown in Figure 18 and Figure 19；And adopt After stimulating mouse boosting cell 36h with antigen in vitro, IFN-γ and IL-4 elisa form cell number (ELISPOT methods), As a result it is as shown in figure 20.

IL-4 and IL-6 represents the cell factor of Th2 type responses, and IFN-γ and IL-2 represent the cell factor of Th1 type responses By Figure 18, Figure 19 and Figure 20 it can be seen from 430nm and 1.38 μm of two particle diameter load antigen PLGA microballoon group splenocyte supernatants In IFN-γ level be all remarkably higher than aluminium adjuvant group (P ＜ 0.05 or P ＜ 0.01), and 430nm carries antigen PLGA and receives microballoon group The horizontal pole of IFN-γ in splenocyte supernatant, which is significantly higher than 1.38 μm and carries antigen PLGA, receives microballoon group group (P ＜ 0.01)；Can by Fig. 7 Knowing, 430nm carries that IL-2 that antigen PLGA receives in microballoon group splenocyte supernatant is horizontal to be also significantly greater than aluminium adjuvant group (P ＜ 0.05), And 430nm carries antigen PLGA and receives also above 1.38 μm load antigen PLGA groups of secretion level of IL-2 in microballoon group splenocyte supernatant, But the two is without significant difference.This shows, compared to aluminium adjuvant, carry antigen PLGA receive microballoon group can effectively induce Th1 types to be immunized should Answer, strengthen cellullar immunologic response, and the less PLGA of particle diameter carries antigen and receives microballoon (430nm) and carries antigen compared to big particle diameter PLGA and receive Microballoon (1.38 μm) more can effectively induce Th1 type immune responses, strengthen cellullar immunologic response.

In terms of Th2 cytokines secretion levels, different-grain diameter, which carries antigen PLGA, to be received microballoon group and can induce and aluminium adjuvant Similar IL-4 secretion levels, there was no significant difference, and 1.38 μm carry the IL-4 water that antigen PLGA receive in microballoon group splenocyte supernatant It is flat be significantly higher than 430nm and carry antigen PLGA receive microballoon group group (P ＜ 0.05 or P ＜ 0.01)；1.38 μm carry antigen PLGA and receive microballoon IL-6 levels in group splenocyte supernatant are significantly higher than aluminium adjuvant group (P ＜ 0.05), and 1.38 μm carry antigen PLGA and receive microballoon group The horizontal poles of IL-6 in splenocyte supernatant are significantly higher than 430nm and carry antigen PLGA nanospheres group (P ＜ 0.01).

Result above, which shows to carry antigen PLGA, which receives microballoon group, can induce the secretion level of the IL-4 similar to aluminium adjuvant, can It is immunized with effective induction body fluid, and higher levels of IFN-γ is horizontal, is obviously improved cellullar immunologic response；It is and large-sized The secretion level of IL-6, IL-4 can more be significantly improved by carrying antigen PLGA microballoons (1.38 μm), strengthen Th2 type immune responses, promote B Cell activation produces antibody, effectively induction body fluid immune response；And small particle carries antigen PLGA microballoons (430nm) and is obviously improved The secretion level of IFN-γ, strengthens Th1 type immune responses, contributes to the lifting of cellullar immunologic response.

Embodiment 129 --- 1.38 μm of PLGA load antigens are received after mouse is immunized in microballoon compounding free antigen and are immunized Effect analysis

Female BAl BIc/c the mouse of 8-10 weeks, every mouse note is immunized in microball preparation dorsal sc made from Example six It is 20 μ g/100 μ L to penetrate antigen dose, and HPV antigenic contents are 10 μ g/ wherein in microspheres vaccine preparation, and dissociate HPV antigenic contents For 10 μ g/ only, while 1.38 μm of PLGA prepared by HPV pentamers antigen and embodiment 5 using aluminium salt as adjuvant are set up carry antigen Receive microballoon as a control group.More than detection several groups of humoral immunity and Study On Cellular Immune.

1.38 μm of PLGA of the results show carry antigens and receive the neutralizing antibody titers and specificity that microballoon compounding free antigen produces IgG antibody titre is that 1.38 μm of PLGA carry antigens and receive 5 times or so of microballoon group, this shows that PLGA carries antigen and receives microballoon compounding trip It can effectively strengthen Humoral from antigen.

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

With reference to above embodiment, different proportion, different-grain diameter and different shaped are prepared according to the formula of below table The preparation for receiving microballoon of other pentamer and VLP.

Table 5 carries the sequence number of HPV16L1 pentamer PLGA microballoon different-grain diameters

Table 6 carries the PLGA microballoons different-grain diameter combination sequence number of HPV16L1 pentamer antigens

Table 7 carries the PLGA microballoon difference HPV type sequence numbers of HPV16L1 pentamer antigens

Table 8 carries the PLGA microballoons compounding lipid material sequence number of HPV16L1 pentamer antigens

Table 9 carries the sequence number of the PLGA microballoons compounding different molecular adjuvant of HPV16L1 pentamer antigens

Table 10 carries the sequence number of the different-grain diameter of the PLGA microballoons of HPV16L1VLP antigens

Table 11 carries the different-grain diameter combination sequence number of the PLGA microballoons of HPV16L1VLP antigens

Table 12 carries the different HPV types sequence numbers of the PLGA microballoons of HPV16L1VLP antigens

Table 13 carries the compounding lipid material sequence number of the PLGA microballoons of HPV16L1VLP antigens

Table 14 carries the sequence number of the compounding different molecular adjuvant of the PLGA microballoons of HPV16L1VLP antigens

Embodiment 135 --- different-grain diameter receives microballoon HPV L1 pentamer immunogenicity researchs

Sample prepared by Example 19,20,30 and 21, with 400nm, 800nm, 1.3 μm of three kinds of differences Mouse is immunized in the HPV L1 pentamer albumens and the coated HPV L1 pentamer albumens of 400nmPLGA/PC of particle diameter PLGA embeddings, Preliminary experimental results show that the adjuvant system can effectively induce the antigentic specificity including neutralizing antibody and cellular immunity Immune response.In Fig. 4, the antibody in serum is analyzed, the results showed that, compared with tradition aluminium salt adjuvant group, 400nm and 800nm The neutralizing antibody titers of PLGA adjuvant groups are essentially identical, but 1.3 μm of neutralizing antibody adjuvant group and 400nmPLGA+ lecithin (PLGA/PC) neutralizing antibody of composite adjuvant group and combination titre are higher；The combination of the adjuvant group of another 800nm and 1.3 μ .3nm resists Body titre is all remarkably higher than 400nm PLGA, but there was no significant difference between the two.With ELISPOT methods detection HPV specificity CTL immune responses, the results showed that, traditional aluminium adjuvant group does not induce specific CTL immunity response substantially, and PLGA adjuvant groups More high-caliber HPV specific CTL immunities response is effectively induced, wherein especially with 400nm and 800nm PLGA adjuvants Group is highest (Fig. 2).The splenocyte of immunized mice is taken to be measured in vitro after antigenic stimulus under condition of culture thin in supernatant Intracellular cytokine secretes situation, the results showed that, compare that Th1 types (stimulation cellular immunity) cell factor IFN- is thin and TNF- secretions carefully etc. Amount, PLGA adjuvant groups are all remarkably higher than tradition aluminium salt adjuvant group；And (stimulate body fluid/antibody mediated immunity should in Th2 cytokines Answer) IL-4 aspects, PLGA adjuvants group is substantially identical with tradition aluminium salt adjuvant group, but answering using PLGA+ lecithin (PLGA/PC) Combination adjuvant group is then significantly higher than other groups.The result is shown in Figure 21,22,23,24,25.

Embodiment 136 --- 1000nm PLGA carry antigen and receive microballoon embeds CPG1826 combined immunization mouse altogether Analysis of Immunity Effect afterwards

Prepared according to embodiment 26 and embed the PLGA of CpG1826 altogether and carry antigen and receive microballoon.Obtained microballoon is frozen After dry, it be suspended in physiological saline, obtain microsphere suspensions so that the content of antigen is 20 μ g/100 μ L in microsphere suspensions, that is, is made HPV bacterin preparations.Its dorsal sc is immunized to the female BAl BIc/c mouse of 8-10 weeks, every mouse injections of antigens dosage is 20 μ G/100 μ L, at the same set up using aluminium salt as adjuvant HPV pentamers antigen and 1000nm PLGA carry antigen receive microballoon as compare Group.More than detection several groups of humoral immunity and Study On Cellular Immune.PLGA compounding CpG small molecule adjuvants coatings HPV16L1 five is poly- The microballoon of body is immunized neutralizing antibody and binding antibody titre after mouse and sees Figure 26 and 27 respectively, and the results show embeds CpG's altogether 1000nm loads antigen PLGA receives after mouse can be immunized in microballoon group and can induce the neutralizing antibody level being on close level with Alum adjuvant.And On knot and antibody level, the HPV16L1 pentamer albumens of 1 μm of PLGA embedding are in initial immunity induction of being significantly higher than aluminium The binding antibody of salt adjuvant is horizontal, can induce when second immune with the comparable levels of binding antibody titre of Alum adjuvant, and And be significantly higher than in the binding antibody level induced after adding CpG small molecule adjuvants when second immune and do not add CpG groups, together When for the first time and the binding antibody level that induces of when second immune is all remarkably higher than tradition aluminium salt adjuvant.

Embodiment 137 --- the PLGA of different-grain diameter combination, which carries antigen and receives microballoon, is immunized immune effect after mouse Analysis

The microball preparation back skin that different-grain diameter made from Example 101,102,103 combines Female BAl BIc/c the mouse of 8-10 weeks are immunized down, every mouse injections of antigens dosage is 20 μ g/100 μ L, while is set up with aluminium salt The PLGA of the different-grain diameter prepared for the HPV pentamers antigen and example IV ten, 68 of adjuvant carries antigen and receives microballoon conduct Control group.More than detection several groups of Humoral.The PLGA of different-grain diameter and combinations thereof is coated with the micro- of HPV16L1 pentamers Binding antibody titre and neutralizing antibody titers are immunized after mouse respectively such as 28 and 29 in ball.

In the HPV16L1VLP inductions for the PLGA embeddings that 1 μm or 10 μm of the results show or two particle diameters combine by a certain percentage With antibody titer without significant difference, and the binding antibody titre that 1 μm of PLGA-HPV16L1VLP group induces after initial immunity is shown Work is less than HPV16L1VLP-1 μm+10 μm (0.5+0.5) groups, and HPV16L1VLP-1 μm+10 μ is substantially less than after immune at second M (0.2+0.8) is organized and HPV16L1VLP-1 μm+10 μm (0.8+0.2) groups.

The research of the HPV 16L1VLP immunological effects of embodiment 138 --- different adjuvants

With PLGA, after mouse is immunized in PLGA/HSPC, PLA embedding HPV 16L1VLP, the neutralizing antibody and knot of generation are induced Antibody titer is closed without significant difference, such as Figure 30 and 31.

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

The PLGA of 100mg is dissolved in the dichloromethane of 20mL to the PVA solution for as oil phase (O), preparing 1.0% as outer Water phase (W2), oil phase (O) is added into the outer aqueous phase (W2) of 50mL, the pre-emulsion (W2/O) of oil-in-water type is prepared, will This pre- double emulsion crosses film preparation using the method for fast film emulsification and obtains the oil-in-water double emulsion (O/W2) of uniform particle diameter repeatedly； Cure 3h, centrifuge washing, freezes, obtain dry PLGA microballoons, the SEM photograph of PLGA is as shown in Figure 2.

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

The PLGA microballoons being prepared are dissolved in the pH=5 of 0.8mL, 10mM PBS buffer solutions, obtain 20mg/mL's PLGA microsphere suspensions；Take a certain amount of HPV to be dissolved in the pH=5 of 0.8mL, 10mM PBS buffer solutions, obtain the HPV of 20ug/mL Antigenic solution, both are mixed in equal volume, and absorption overnight, obtains the PLGA suspensions that 1.6mL is adsorbed with HPV antigens, are HPV epidemic diseases Seedling composition.

Research of embodiment 140 --- the PLGA microballoons to HPV 16L1VLP absorption immune responses

Mouse, PLGA-HPV is immunized after adsorbing HPV 16L1VLP samples in 1 PLGA for applying example in Example 139 16L1VLP can be induced and the comparable levels of binding antibody titre of the Gardasil vaccines of Merck companies.As a result such as Figure 32.

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Claims (20)

1. a kind of vaccine combination, comprising the antigen with adjuvant combination, the ratio of antigen and adjuvant is 10-80 μ g/mg, described anti- Original includes HPV L1 albumen, and the adjunvant composition includes polylactide-polyglycolic acid copolymer PLGA or polylactic acid PLA, described HPV L1 albumen be embedded in PLGA or PLA or adsorb on PLGA or PLA surfaces, exist to receive microspheres form, it is described The microballoon 10-90% that receives be distributed in 1-10 μm, 90-10% be distributed in 10-100 μm, described receives microballoon in each particle size range Interior particle size dispersion coefficient scope is 0.005~0.2；Lecithin, wherein lecithin and PLGA are further included in the adjuvant Or the ratio of PLA is 1-99：99-1；Wherein HPV L1 albumen refers to the VLP or five that HPV total lengths, truncation or the L1 of restructuring are formed Aggressiveness.

2. vaccine combination as claimed in claim 1, wherein the microballoon 10-90% that receives is distributed in 3-8 μm, 90-10% distributions At 30-80 μm.

3. vaccine combination as claimed in claim 2, wherein the microballoon 10-90% that receives is distributed in 5-6 μm, 90-10% distributions At 50-60 μm.

4. vaccine combination as claimed in claim 3, wherein further including small molecule immune adjuvant in the adjuvant：CpG、 MPLA, imiquimod, PolyI:One or more of combinations in C, the wherein ratio of small molecule immune adjuvant and PLGA or PLA For 10-100 μ g/mg.

5. vaccine combination as claimed in claim 1, wherein HPV L1 albumen refer to 6,11,16,18,26,30,31,33,34, 35th, it is a kind of and/or several in 39,45,5l, 52,53,56,58,59,66,67,68,69,70,72,73 or 82,85,97 type HPV Combination.

6. a kind of bacterin preparation, it is characterised in that the bacterin preparation is as the vaccine combination described in claim 5 and HPV L1 eggs Bletilla pharmaceutic adjuvant is made, wherein it is 10-90% to receive HPV L1 and free HPV L1 ratios in microballoon：90-10%.

7. bacterin preparation as claimed in claim 6, it is characterised in that the pharmaceutic adjuvant buffers for physiological saline or PBS Liquid.

8. the preparation method of vaccine combination as claimed in claim 4, this method comprises the following steps：

(1) preparation of drug containing emulsion droplet：HPV L1 albumen and small molecule immune adjuvant CpG are dissolved in buffer salt system and are prepared into pH value Scope is the inner aqueous phase W1 of 4.0-12.0；

(2) PLGA or PLA and lipid material are dissolved in organic solvent and oil phase O is made, the inner aqueous phase W1 and oil phase O is mixed Close, water-in-oil type W1/O pre-emulsions are made, this pre-emulsion is added in outer aqueous phase, the pre- double emulsion of W1/O/W2 types is made, by described in The pre- double emulsion of W1/O/W2 types crosses film and obtains the W1/O/W2 type lotions of size uniformity repeatedly；

(3) emulsion droplet cures：By W1/O/W2 type lotion normal temperature cures, vaccine combination is made after washing, drying.

9. the preparation method of vaccine combination as claimed in claim 5, this method comprises the following steps：

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

(2) the pre- double emulsion of O/W2 types is crossed to film repeatedly and obtains the O/W2 type lotions of size uniformity；PLGA is obtained after washing, drying Or PLA microballoons；

(3) HPV L1 albumen and small molecule immune adjuvant CpG are dissolved in buffer salt system, obtain the HPV of 1-40 μ g/0.2-1mL Antigenic solution, PLGA or PLA microballoons are dissolved in buffer salt system, obtain the solution of 1-10mg/mL；Two solution are mixed in equal volume Close, absorption overnight, obtains being adsorbed with the PLGA suspensions of HPV antigens, is HPV vaccine combinations.

10. the preparation method of vaccine combination as described in claim 8 or 9, it is characterised in that the buffer salt system is citron Acid-disodium hydrogen phosphate, Acetic acid-sodium acetate buffer solution or phosphate buffer.

11. the preparation method of vaccine combination as claimed in claim 8, it is characterised in that the inner aqueous phase pH value described in step (1) Scope is 6.0-8.0.

12. the preparation method of vaccine combination as described in claim 8 or 9, it is characterised in that the oil phase be room temperature under be in liquid Body and the immiscible oily matter of water.

13. the preparation method of vaccine combination as described in claim 8 or 9, it is characterised in that the outer aqueous phase W2 be containing The PVA aqueous solutions of 0.001-10%, its alcoholysis degree are 87~90%, and polymerization chain number is 1700-1750；Or to contain 0.001- The mass percent of 10% PVA-PEG mixed aqueous solutions, PVA and PEG are 20-80：30-90.

14. the preparation method of vaccine combination as claimed in claim 8, it is characterised in that the water-in-oil type described in step (2) W1/O type pre-emulsions are by using made from homogeneous emulsifier or ultrasonic emulsator.

15. the preparation method of vaccine combination as claimed in claim 8, it is characterised in that the size uniformity described in step (2) W1/O/W2 types lotion obtained by the way that W1/O/W2 types pre-emulsion is pressed through microporous barrier repeatedly under pressure.

16. the preparation method of vaccine combination as claimed in claim 8, it is characterised in that curing mode for solvent evaporated method or Solvent extraction.

17. the preparation method of vaccine combination as claimed in claim 8, it is characterised in that hardening time 1h-8h.

18. a kind of preparation method of bacterin preparation, it is characterised in that in the preparation method of vaccine combination as claimed in claim 8 In emulsion droplet curing schedule after further include following steps：

(4) bacterin preparation is formed：Take mass ratio 10-90%：The vaccine combination of 90-10% is given birth to free HPVL1 albumen Manage brine or PBS buffer suspends, be configured to the microsphere suspensions of the HPV antigens containing 1-40 μ g/0.2-1mL, HPV vaccines are made Preparation.

19. application of the vaccine combination as claimed in claim 5 in the medicine for treating or preventing HPV infection is prepared.

20. application of the bacterin preparation as claimed in claims 6 or 7 in the medicine for treating or preventing HPV infection is prepared.