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

Abstract

The present invention relates to vaccine compositions and their use in medicine. In particular, the invention relates to a vaccine composition or vaccine preparation made of nano-microspheres coated with HPV L1 protein and its application in medicine for treating or preventing HPV virus. Specifically, the present invention aims at HPV L1 antigen protein, nano-microsphere particle size and its combination, nano-microsphere material, and the impact of nano-microsphere on antigen loading mode and its immune response, and designs and prepares PLGA, PLA, and PLGA/PC nano-microspheres are used to embed or adsorb HPV L1 antigenic protein to prepare vaccine compositions or vaccine preparations, to study their effects on the intensity and level of immune responses in vivo, and to develop highly efficient HPV L1 proteins Vaccine composition.

Description

A vaccine composition for treating or preventing HPV virus and its application

technical field

The present invention relates to vaccine compositions and their use in medicine. In particular, the invention relates to a vaccine composition or vaccine preparation made of nano-microspheres coated or adsorbed with HPV L1 protein and its application in medicines for treating or preventing HPV virus.

technical background

Human papillomaviruses (HPV) are non-enveloped double-stranded DNA viruses, mainly composed of viral shell and genomic DNA (Bernard, Burk et al. 2011). The HPV viral coat is an icosahedral structure composed of 360 L1 proteins (forming 72 pentamers) and up to 72 L2 proteins, with a diameter of 55-60 nm (Howley and Lowy 2007). Viral coat protein has self-assembly properties, and in vitro L1 protein alone or together with L2 protein self-assembles to form virus-like particles (Virus-like Particle, VLP) (Chen, Garcea et al.2000, Finnen, Erickson et al.2003, Buck , Cheng et al. 2008, Wang and Roden 2013). The current HPV vaccines all use VLP as the target antigen, and two HPV L1VLP-based preventive vaccines have been marketed (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 8, 2006, the U.S. Food and Drug Administration (FDA) officially approved the marketing of the Gardasil HPV preventive vaccine produced by Merck (ie Merck); it is expressed and purified by Saccharomyces cerevisiae. 11L1VLP quadrivalent cervical cancer preventive vaccine, with amorphous aluminum hydroxyphosphate aluminum sulfate (AAHS) as an adjuvant, is approved to prevent HPV16, 18, 6, 11 infection in girls and women aged 6 to 26 Caused by cervical cancer, precancerous lesions and genital warts, this is the world's first tumor vaccine approved by the FDA (Villa, Costa et al.2005, Villa, Ault et al.2006, Bryan 2007, Olsson, Villa et al .2007, Goldstone and Vuocolo 2012). Subsequently, the HPV preventive vaccine produced by British GlaxoSmithKline (GSK) company under the trade name of Cervarix has also been successfully listed. It is derived from the HPV16/18L1VLP bivalent cervical cancer preventive vaccine derived from insect expression system, using AS04 adjuvant ( Aluminum hydroxide composite MPL) (Paavonen, Jenkins et al. 2007, Garcon, Morel et al. 2011, Kreimer, Gonzalez et al. 2011, Szarewski 2012). Clinical trials have shown that the above two preventive vaccines are well tolerated and have no serious adverse reactions (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 , Paavone et al. 2012, Szarewski, Poppe et al. 2012). However, these two preventive vaccines are expensive and require low temperature storage, which greatly limits their use in developing countries and backward areas. Therefore, it is particularly important to develop a low-cost, high-titer HPV vaccine (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). Studies have shown that immunization of experimental animals with HPV L1 pentamer protein can induce protective immune responses (Rose, White et al.1998, Yuan, Estes et al.2001, Ohlschlager, Ose et al.2003, Senger, Schadlich et al. al.2010, Wu, Gersch et al.2011), and the expression technology based on prokaryotic Ecoli can greatly reduce the production cost of HPV vaccine. In the Chinese invention patent 02129070.9 "Prokaryotic preparation and application of papillomavirus capsid protein", the three-dimensional structure of HPV 16L1 pentamer is provided, and the technical method of mass expression and preparation of human papillomavirus capsid protein L1 is provided by Escherichia coli.

Aluminum adjuvant is the most widely used adjuvant at present. It has a history of more than 80 years and tens of billions of use records. It has also been approved by the US Food and Drug Administration (FDA) for human vaccines for a long time. (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 has some shortcomings in the application of HPVL1 protein antigen. Aluminum salt adjuvants form a depot effect only at the injection site (Gupta, Changet al. 1996, Hem 2002, Verdier, Burnett et al. 2005, Hem and Hogenesch 2007, Noe, Green et al. 2010), attracting tree Dendritic cells (DC) (Morefield, Sokolovska et al.2005, Kool, Soullie et al.2008, Sharp, Ruane et al.2009, Flach, Ng et al.2011, Ghimire, Benson et al.2012), macrophages (Hamilton, Byrne et al.2000, Jordan, Mills et al.2004, Rimaniol, Gras et al.2004, Rimaniol, Gras et al.2007) and other antigen-presenting cells (antigen-presenting cells, APCs), these cells through phagocytosis Acts to uptake antigen and activate immune response via NLRP3 inflammasome (De Gregorio, Tritto et al. , 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). At the same time, aluminum adjuvant can inhibit cellular immunity by stimulating IL-10 secretion and other mechanisms (Chen, Ni et al.2011), and for virus infection, the effect of cellular immunity will exert better immune protection and immune prevention role (O'Hagan and Valiante 2003, Demento, Cui et al. 2012, Levitz and Golenbock 2012, Olive 2012, Cain, Sanders et al. 2013, HogenEsch 2013). In addition, aluminum adjuvants can cause IgE-mediated allergic reactions (such as injection site granuloma) and adverse reactions in the nervous system, which cause people to worry about the safety of aluminum adjuvants (Petrik, Wong et al.2007, Bystrianyk2009, Shaw and Petrik 2009, Munks, McKee et al. 2010, Tomljenovic and Shaw 2011). To this end, novel immune adjuvants need to be developed against HPV L1 protein antigens, whether based on L1 pentamer or L1 VLP candidate vaccines (Campo and Roden 2010, Mariani and Venuti 2010, Chen, Ni et al. 2011, Foged 2011, Gattoc, Nair et al. 2013, Koff, Burton et al. 2013, Shaw 2013, Tomljenovic, Spinosa et al. 2013).

In recent years, a large number of literatures have confirmed that biodegradable polymer nanospheres in the body can convert antigens into particulate antigens by embedding antigens, which is beneficial for uptake by antigen-presenting cells, and then release antigens in cells, and through subsequent antigen processing , presentation, and enhanced 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. prepared polyglycoside microspheres, and used ovalbumin (OVA) as a model antigen to study the adjuvant effect of the microspheres. The results showed that the microspheres can improve the expression of MHC molecules on the surface of antigen-presenting cells and the expression of related cells. Factor secretion, showing that it has a certain adjuvant effect (Torres, Wilson-Welder et al.2011); and Uto T. et al. prepared biodegradable polyglutamic acid nanospheres (γ-PGA NPs) and Mechanistically studied its adjuvant properties, revealing that the microspheres can induce strong innate and adaptive immune responses through TLR4 (Toll-like receptor) and MyD88 signaling pathways (Uto, Akagi et al.2011). Studies have shown that the above-mentioned antigen-carrying particulate adjuvant makes the soluble antigen into a granular antigen, and the granular antigen can change the way of antigen presentation after being ingested by APC, which can not only activate CD4+ T cells, but also activate CD8 +T cells can greatly enhance the cellular immune function and achieve the complete elimination of intracellular infection. They are promising adjuvants and delivery systems for virus infection vaccines (Wang and Singh 2011, Dierendonck, DeKoker et al. 2012). In addition, biodegradable polymer microspheres can provide a large number of antigens and protect them from rapid degradation under physiological conditions. Through the continuous or pulse release behavior of the microspheres to the embedded antigens, it can effectively simulate the multiple immunization procedures of traditional vaccines. Reduce the number of vaccinations and the total amount of immunogen used, thereby improving patient adaptability and reducing drug costs (Langer, Cleland et al.1997, Johansen, Estevez et al.2000, De Temmerman, Rejman et al.2011, Demento, Cui et al. 2012).

As an adjuvant and delivery system for antigenic proteins, many physical and chemical properties of polymer particles will affect their interaction with immune cells in vivo, and ultimately affect the type, intensity and level of immune responses, including particle size, shape, surface properties, 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 S.D. et al. (Xiang, Scholzen et al. 2006) mentioned in a review article that particles with positively charged surfaces are more likely to induce high levels of antibody and CD8+ T cell response; Foged C. et al. (Foged, Brodin et al.2005) studied the effect of particle surface charge on the uptake behavior of DC cells, and obtained similar results, that is, particles with positive charges on the surface are more likely to be taken up by DC; while Yasuhiko The study by T. et al. (Tabata and Ikada 1988) got the opposite conclusion. They believed that negatively charged cellulose microspheres could promote the uptake by macrophages. Similarly, the impact of polymer particle size on immunological effects has not yet reached a unified conclusion. Gutierro I. et al. (Gutierro, Hernandez et al.2002) found that compared with 200 and 500nm particle size, PLGA microspheres of 1 can produce high levels of antibodies; MannJ.S.F. et al. (Mann, Shakir et al. 2009) prepared natural biofilm microcapsules (Biosome) carrying influenza antigens, the results showed that small particle size is conducive to the induction of high-efficiency humoral immune response, while large particle size tends to induce the body to produce cellular immune response; WendorfJ et al (Wendorf, Chesko et al.2008) used negatively charged PLGA microspheres (110nm and 1000nm) to adsorb HIV-1 and the Meningococcal B (Meningococcal B) protein that induces meningitis, and the two particle sizes produced similar immune responses. Different researchers have obtained different results for the same particle properties. The present invention aims at HPV L1 antigenic protein, the impact of nano-microsphere particle size on its immune response, designs and prepares PLGA nano-microspheres with uniform particle size and different particle sizes, uses it to embed HPV L1 antigenic protein, and studies PLGA nano-microspheres The effect of particle size on the intensity and level of immune response in vivo, and the development of highly effective HPVL1 protein vaccine composition.

Contents of the invention

In the first aspect, the present invention provides a vaccine composition comprising an antigen combined with an adjuvant, the ratio of the antigen to the adjuvant being 10-80 μg/mg, the antigen comprising HPV L1 protein, and the adjuvant composition comprising polylactic acid - Polyglycolic acid copolymer (PLGA) or polylactic acid (PLA).

In a specific embodiment of the present invention, the vaccine composition of the present invention, wherein the HPV L1 protein is embedded in PLGA or adsorbed on the surface of PLGA, exists in the form of nanospheres with a particle size distribution between 100nm and 100μm .

In a specific embodiment of the present invention, in the vaccine composition, wherein 0-90% of the nanospheres are distributed in any range of particle size of 100-1000 nm, 1-10 μm or 10-100 μm or a combination thereof.

In a specific embodiment of the present invention, in the vaccine composition, preferably 0-90% of the nano-microspheres are distributed in any particle size of 200-900 nm, 2-9 μm or 20-90 μm or a combination thereof.

In a specific embodiment of the present invention, in the vaccine composition, preferably 0-90% of the nano-microspheres are distributed in any particle size of 400-700 nm, 4-7 μm or 40-70 μm or a combination thereof.

In the specific embodiment of the vaccine composition of the present invention, 10-90% of the nanospheres are distributed at 100-1000 nm, and 90-10% are distributed at 1-10 μm.

In the vaccine composition in a specific embodiment of the present invention, preferably 10-90% of the nanospheres are distributed at 300-800 nm, and 90-10% are distributed at 3-8 μm.

In the vaccine composition in a specific embodiment of the present invention, more preferably, 10-90% of the nanospheres are distributed at 500-600 nm, and 90-10% are distributed at 5-6 μm.

In the vaccine composition of the specific embodiment of the present invention, 10-90% of the nanospheres are distributed in 1-10 μm, and 90-10% are distributed in 10-100 μm.

In the vaccine composition in a specific embodiment of the present invention, preferably, 10-90% of the nanospheres are distributed at 3-8 μm, and 90-10% are distributed at 30-80 μm.

In another specific embodiment of the vaccine composition of the present invention, preferably, 10-90% of the nanospheres are distributed at 5-6 μm, and 90-10% are distributed at 50-60 μm.

In a specific embodiment of the present invention, in the vaccine composition, preferably 10-90% of the nanospheres are distributed at 100-1000 nm, and 90-10% are distributed at 10-100 μm.

In another specific embodiment of the present invention is the vaccine composition, wherein 10-90% of the nanospheres are distributed at 300-800 nm, and 90-10% are distributed at 30-80 μm.

In another specific embodiment of the vaccine composition of the present invention, 10-90% of the nanospheres are distributed at 500-600 nm, and 90-10% are distributed at 50-60 μm.

In a specific embodiment of the present invention, the particle size dispersion coefficient of the nano-microspheres in each particle size range is in the range of 0.005-0.2.

The vaccine composition in a specific embodiment of the present invention, wherein the adjuvant further includes the following immune-enhancing component lipids: one or a combination of lecithin, stearylamine, and glycerophospholipids, Wherein the ratio of lipids to PLGA or PLA is 1-99:99-1, preferably 1-50:100.

More specifically, in a specific embodiment of the present invention, the vaccine composition, wherein the adjuvant further includes small molecule immune adjuvants: one or more of CpG, MPLA, imiquimod, PolyI:C, etc. The combination of small molecule immune adjuvant and PLGA or PLA ratio of 10-100μg/mg.

In the vaccine composition of the specific embodiment of the present invention, wherein the HPV L1 protein refers to the VLP, pentamer or multimer formed by HPV full-length, truncated or recombinant L1.

In the specific embodiment of the present invention, the vaccine composition, wherein the HPV L1 protein refers to 6, 11, 16, 18, 26, 30, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56 , 58, 59, 66, 67, 68, 69, 70, 72, 73 or 82, 85, 97 HPV types and/or a combination of several.

On the other hand, the present invention discloses a vaccine preparation, which is made from the vaccine composition described in 19, HPV L1 protein and pharmaceutical excipients, wherein the ratio of HPV L1 to free HPV L1 in nanospheres is 10-90% : 90-10%, more preferably 10-50%: 90-50%.

In the vaccine formulation of the specific embodiment of the present invention, the pharmaceutical adjuvant is physiological saline or PBS buffer.

On the other hand, in a specific embodiment of the present invention, the preparation method of the vaccine composition mainly includes two methods of embedding and adsorption, wherein the embedding method includes the following steps:

(1) Preparation of drug-containing emulsion droplets: Dissolving HPV L1 protein and/or small molecule immune adjuvant CpG in a buffer salt system to prepare an inner water phase (W1);

(2) PLGA/PLA or PLGA/PLA and lipids are dissolved in an organic solvent to make an oil phase (O), and the water phase (W1) is mixed with the oil phase (O) to make a water-in-oil type (W1/O) pre-emulsion, this pre-emulsion is added to the external water phase (W2) to obtain a W1/O/W2 type pre-emulsion, and the W1/O/W2 type pre-emulsion is repeatedly passed through the membrane to obtain the size Uniform W1/O/W2 emulsion;

(3) Emulsion solidification: the W1/O/W2 type emulsion is solidified at room temperature, washed and dried to make a vaccine composition.

A preparation method is provided in a specific embodiment of the present invention, the pH value range of the internal aqueous phase described in step (1) is 4.0-12.0, and the buffer salt system described in step (1) is citric acid-hydrogen phosphate Disodium, acetic acid-sodium acetate buffer, or phosphate buffer.

In the preparation method provided in a specific embodiment of the present invention, the pH value of the internal aqueous phase in step (1) is in the range of 6.0-8.0.

In a specific embodiment of the present invention, the oil phase in step (2) is an oily substance that is liquid and water-immiscible at normal temperature, preferably ethyl acetate, acetone, dichloromethane, chloroform or at least two of the above mixture of the above.

In a specific embodiment of the present invention, the external water phase (W2) described in step (2) is a PVA aqueous solution containing 0.001-10%, its degree of alcoholysis is 87-90%, and the number of polymer chains is 1700-1750 ; or a mixed aqueous solution containing 0.001-10% PVA-PEG, the mass percentage of PVA and PEG is 20-80:30-90.

In a specific embodiment of the present invention, the water-in-oil type (W1/O type) pre-emulsion described in step (2) is prepared by using a homogeneous emulsifier or an ultrasonic emulsifier.

In a specific embodiment of the present invention, the W1/O/W2 type emulsion with uniform size described in step (2) is by repeatedly pressing the W1/O/W2 type pre-emulsion through the microporous membrane under the action of higher pressure owned.

In a specific embodiment of the present invention, in the antigen-containing emulsion droplets described in step (2), the volume ratio of the water phase (W1) to the oil phase (O) is 1:1-1:50.

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

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

On the other hand, a preparation method of another vaccine composition is disclosed in a specific embodiment of the present invention, that is, the adsorption method includes the following steps (1) dissolving PLGA/PLA or PLGA/PLA and lipid substances in an organic solvent to prepare Form an oil phase (O), add this oil phase in the external water phase (W2), and make O/W2 type pre-emulsion;

(2) the O/W2 type pre-complex emulsion is repeatedly passed through the membrane to obtain a uniformly sized O/W2 type emulsion; after washing and drying, PLGA/PLA microspheres are obtained;

(3) Dissolving HPV L1 protein and/or small molecule immune adjuvant CpG in buffer salt system to obtain HPV antigen solution of 1-40 μg/0.2-1mL, dissolving PLGA/PLA microspheres in buffer salt system to obtain 1-10 mg/mL solution; mix the two solutions in equal volumes, and absorb overnight to obtain a PLGA suspension adsorbed with HPV antigen, which is the HPV vaccine composition.

The specific operation steps in the adsorption method mentioned above can refer to the corresponding steps in the embedding method.

On the other hand, a method for preparing a vaccine preparation is provided in a specific embodiment of the present invention, which also includes the following steps after the emulsion solidification step in the method for preparing the vaccine composition:

Vaccine preparation formation: take the vaccine composition (nanomicrospheres) and free HPVL1 protein with a mass ratio of 10-90%: 90-10% and suspend them with physiological saline or PBS buffer solution, and prepare the vaccine containing 1-40 μg/0.2-1mL The microsphere suspension of HPV antigen is prepared HPV vaccine preparation.

In the preparation method of the vaccine preparation provided in the specific embodiment of the present invention, the pH value of the PBS buffer solution is in the range of 6-8, and the concentration of the nanospheres is 1-10 mg/mL.

On the other hand, the application of the vaccine composition in the preparation of medicines for treating or preventing HPV infection is disclosed in specific embodiments of the present invention.

The application of the vaccine preparation disclosed in the specific embodiments of the present invention in the preparation of medicines for treating or preventing HPV infection.

In one embodiment of any of the methods provided, the dosage form is administered by oral, subcutaneous, pulmonary, intranasal, intraperitoneal, lymphatic, intradermal or intramuscular administration.

In another embodiment, a vaccine formulation of human papillomavirus is provided, which is a vaccine formulation mainly based on PLGA homogeneous microspheres coated with HPVL1VLP antigen, and can be co-embedded or compounded with other molecular adjuvants at the same time. A certain amount of uncoated HPV L1 VLP antigen can also be compounded into the vaccine, that is, the HPV L1 antigen coated in the nanosphere and the free HPV L1 antigen are combined and clinically or pharmaceutically acceptable. into vaccine preparations.

In another embodiment, the antigen loading rate in the microsphere is above 20ug/mg; the mass ratio of coated antigen to free antigen is 0-100%.

The surface of the microspheres prepared by the present invention is smooth and smooth, and the spherical shape is good (Fig. 1, Fig. 2, Fig. 3), and the prepared particle diameters in each particle diameter range are uniform and controllable (Fig. 4), and the particle diameter dispersion coefficient is in the range of It is 0.005 to 0.2, preferably 0.005 to 0.1, more preferably 0.005 to 0.05. Drug loading and embedding rate are high, and animal experiments show that the novel HPV vaccine of the present invention is safe, has no visible toxicity to experimental animals, and can effectively stimulate animals to produce stronger humoral immunity and cellular immune response (Fig. 5, Fig. 6, Figure 7, Figure 8) can be used for the prevention and treatment of human papillomavirus-related diseases or infections.

In order to clearly understand the present invention, it will be further described below in conjunction with the accompanying drawings and examples, but the following embodiments do not limit the present invention.

Description of drawings

Figure 1 PLGA-embedded HPVL1 pentamer antigen nano-microsphere scanning electron micrograph (430nm)

Figure 2 SEM image of PLGA-embedded HPVL1 pentamer antigen nanospheres (1.38 μm)

Figure 3 SEM images of PLGA-embedded HPVL1 pentamer antigen nanospheres (5.60 μm)

Figure 4 PLGA-embedded HPVL1 pentamer antigen nanosphere scanning electron micrograph (400nm)

Fig. 5 Scanning electron micrograph (800nm) of PLGA-embedded HPVL1 pentamer antigen nanospheres

Figure 6 SEM image of PLGA-embedded HPVL1 pentamer antigen nanospheres (1.3 Antigen)

Figure 7 PLGA/PC embedded HPVL1 pentamer antigen nano-microsphere scanning electron micrograph (400nm)

Figure 8 SEM image of PLGA-wrapped HPV16L1 pentamer nanospheres (1-mer)

Figure 9 PLGA-wrapped HPV16L1 pentamer and CpG nanospheres (1 nanometer)

Figure 10 Scanning electron micrograph of PLGA-embedded HPVL1VLP antigen nanospheres (1 original nanometer)

Figure 11 Scanning electron micrograph of PLGA/HSPC-embedded HPVL1VLP antigen nanospheres (1 original nanometer)

Fig. 12 Scanning electron micrograph of PLA-embedded HPVL1VLP antigen nanospheres (1 original nanometer)

Figure 13 Scanning electron micrograph of PLGA-embedded HPVL1VLP antigen nanospheres (10 nanometers)

Figure 14 SEM image of PLGA nanospheres (1 microsphere)

Figure 15 Particle size distribution of PLGA nanospheres loaded with HPV16L1 pentamer antigen

Figure 16 Neutralizing antibody titers in serum after 38 days of immunization of mice with antigen-loaded PLGA microspheres with different particle sizes

Figure 17 Specific antibody IgG titers in serum after 38 days of immunization of mice with antigen-loaded PLGA microspheres with different particle sizes

Fig. 18 Detection results of cytokine levels in spleen cell supernatant after immunizing mice in each group (Luminex technology)

Figure 19 Detection results of IFN-small and IL-4 levels in splenocyte supernatant after immunizing mice in each group (ELISA method)

Figure 20 Number of spleen cells IL-4 and IFN-small ELISPOT forming cells (SFC) after immunizing mice in each group (ELISPOT method)

Figure 21 After HPV L1 pentameric protein was used to immunize mice with different adjuvant systems, the serum binding antibody titer

Figure 22 The titers of neutralizing antibodies after HPV L1 pentameric protein was immunized with different adjuvant systems

Figure 23 After the HPV L1 pentameric protein is used to immunize mice with different adjuvant systems, the results of the cellular immune response determined by ELISPOT

Figure 24 HPV L1 pentameric protein was used to immunize mice with different adjuvant systems, and the splenocytes were cultured in vitro and stimulated by antigens, and ELISA was used to measure the secretion of IFN-A and IL-4 cytokines

Figure 25 HPV L1 pentameric protein was used to immunize mice with different adjuvant systems, and the splenocytes were cultured in vitro and stimulated by antigens, and the secretion of cytokines was measured by Luminex method.

Figure 26 PLGA complex CpG small molecule adjuvant coated HPV16L1 pentamer microspheres immunized mice with neutralizing antibody titers

Figure 27 PLGA complex CpG small molecule adjuvant coated with HPV16L1 pentamer microspheres after immunization of mice binding antibody titer

Figure 28 The binding antibody titer after immunizing mice with PLGA-coated HPV16L1 pentamer microspheres of different particle sizes and their combinations

Figure 29 Neutralizing antibody titers after immunizing mice with PLGA-coated HPV16L1VLP microspheres of different particle sizes and their combinations

Figure 30 PLGA, PLGA/HSPC, and PLA-coated HPV16L1VLP microspheres immunized mice with neutralizing antibody titers

Figure 31 PLGA, PLGA/HSPC, PLA-coated HPV16L1VLP microspheres after immunization of mice binding antibody titers

Figure 32 The binding antibody titer after immunizing mice after PLGA microspheres adsorbed HPV16L1VLP

specific example

The present invention implements the technical solutions of the present invention with reference to the following tables, and the technical solutions of the present invention include but are not limited to the following examples.

Table 1

Table 2

table 3

Table 4

The specific examples involved in the above table are as follows:

Specific Example 1——Preparation of 100nmPLGA-loaded HPV 6L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400mg of PLGA in 10.0mL of EA solvent as the oil phase (O), add 3mL of HPV 6L1 pentamer with a concentration of 1.33mg/mL and dissolve it in citric acid-disodium hydrogen phosphate buffered saline solution The antigenic protein was used as the inner water phase (W1), and the S-450D ultrasonic cell disruptor was used to carry out colostrum under ice-water bath conditions (the ultrasonic power was set to 30%, and the ultrasonic time was 60s) to prepare W1/O type colostrum, and then Pour the primary emulsion into 65mL of NaCl aqueous solution (outer water phase W2) containing 1.5% PVA (w/v), and prepare the pre-multiplex emulsion (W1/O/W2) by magnetic stirring, and the emulsification condition is 600r/min Stir for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 0.6 μm by the nitrogen gas of 1.5M Pa pressure until uniform double emulsion drops (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 400nm, the PDI value was 0.04, and the drug embedding rate was 90.8%.

Specific embodiment two - the preparation of the HPV vaccine that comprises free antigen

Weigh 4.45 mg of HPV 6L1 pentamer antigen-loaded microspheres with an average particle size of 100 nm prepared in Example 1 and compound 120.3 μL of HPV 6L1 pentamer antigen with a concentration of 1.333 mg/mL, and add PBS buffer with pH=7.0 solution to 1 mL, and prepared a microsphere suspension containing 200 μg/mL of HPV antigen with a ratio of coated antigen to free antigen in the nanomicrospheres of 2:8 to prepare an HPV vaccine preparation.

Specific Example Three——Preparation of 430nm PLGA-loaded HPV 16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 16L1 pentamer antigen protein with a concentration of 4 mg/mL as the internal water phase (W1), and use S-450D Ultrasonic cell breaker was used to perform colostrum in ice-water bath condition (ultrasonic power was set to 30%, ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65mL containing 1.5%PVA (w/v ) in an aqueous solution of NaCl (outer water phase W2), using magnetic stirring to prepare a pre-multiplex emulsion (W1/O/W2), the emulsification condition is 600r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 1.4 μm by nitrogen gas at a pressure of 1 MPa until uniform double emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 430nm, the PDI value was 0.03, and the drug embedding rate was 92.6%. The SEM images of the prepared microspheres are shown in Figure 1.

Specific Example Five——Preparation of 1.38 μm PLGA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 16L1 pentamer antigen protein with a concentration of 6.67 mg/mL as the internal water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 400 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 5.2 μm with nitrogen gas at a pressure of 0.25 MPa until uniform complex emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured by a laser particle size analyzer, the average particle size was 1.38 μm, the PDI value was 0.05, and the drug embedding rate was 93.1%. The scanning electron micrograph of the prepared antigen-loaded nanospheres is shown in FIG. 2 .

Specific Example 6—Preparation of 1 μm PLGA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 16L1 pentamer antigen protein with a concentration of 6.67 mg/mL as the internal water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 400 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 5.2 μm with nitrogen gas at a pressure of 0.25 MPa until uniform complex emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1 μm, the PDI value was 0.05, and the drug embedding rate was 93.1%. The scanning electron micrograph of the prepared antigen-loaded nanomicrospheres is shown in FIG. 8 .

Specific Example 7——Preparation of 5.6 μm PLGA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 16L1 pentamer antigen protein with a concentration of 9.33 mg/mL as the internal water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 300 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 15 μm by the nitrogen of 40kPa pressure until uniform double emulsion drops (W1/O/W2 ). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured by a laser particle size analyzer, the average particle size was 5.6 μm, the PDI value was 0.08, and the drug embedding rate was 79.7%. The scanning electron micrograph of the prepared antigen-loaded nanospheres is shown in FIG. 3 .

Specific Example 17——Preparation of 20.82 μm PLGA-loaded HPV45L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 45L1 pentamer antigen protein with a concentration of 5.333 mg/mL as the internal water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 250 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 52 μm with nitrogen at a pressure of 5 kPa until uniform double emulsion droplets (W1/O/W2 ). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 20.82 μm, the PDI value was 0.10, and the drug embedding rate was 89.2%.

Specific Example Eighteen——Preparation of 80.71 μm PLA-loaded HPV58L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 58L1 pentamer antigen protein with a concentration of 5.333 mg/mL as the internal water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 250 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 190.3 μm with nitrogen at a pressure of 0.1 kPa until uniform complex emulsion droplets (W1/O /W2). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured by a laser particle size analyzer, the average particle size was 80.71 μm, the PDI value was 0.18, and the drug embedding rate was 85.5%.

Specific Example Nineteen——Preparation of 400nm PLGA/HSPC Carrying HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: 300 mg of PLGA and 100 mg of lecithin are dissolved in 10.0 mL of MC solvent as the oil phase (O), and 3 mL of HPV 6L1 pentamer antigen protein with a concentration of 1.333 mg/mL is added as the internal water phase (W1) , use the S-450D ultrasonic cell breaker to perform colostrum under ice-water bath conditions (the ultrasonic power is set to 30%, and the ultrasonic time is 60s) to prepare W1/O type colostrum, and then pour the colostrum into 65mL of 1.5% In the NaCl aqueous solution of PVA (w/v) (external water phase W2), the pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 500r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 1.4 μm by nitrogen gas at a pressure of 1 MPa until uniform double emulsion droplets (W1/O /W2). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 400nm, the PDI value was 0.05, and the drug embedding rate was 92.8%. The SEM image of the obtained microspheres is shown in Figure 7.

Specific Example Twenty——Preparation of 800nm PLGA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 300 mg of PLGA in 10.0 mL of MC solvent as the oil phase (O), add 3 mL of HPV 11L1 pentamer antigen protein with a concentration of 4 mg/mL as the internal water phase (W1), and use S-450D Ultrasonic cell breaker was used to perform colostrum in ice-water bath condition (ultrasonic power was set to 30%, ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65mL containing 1.5%PVA (w/v ) in an aqueous solution of NaCl (outer water phase W2), using magnetic stirring to prepare a pre-multiplex emulsion (W1/O/W2), the emulsification condition is 500r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 9.2 μm by nitrogen gas at a pressure of 1 MPa until uniform double emulsion droplets (W1/O /W2). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 800nm, the PDI value was 0.06, and the drug embedding rate was 88.2%. The scanning electron microscope image of the obtained microspheres is shown in Fig. 5 .

Specific Example 21——Preparation of 1300nm PLGA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 300 mg of PLGA in 10.0 mL of MC solvent as the oil phase (O), add 3 mL of HPV 11L1 pentamer antigen protein with a concentration of 4 mg/mL as the internal water phase (W1), and use S-450D Ultrasonic cell breaker was used to perform colostrum in ice-water bath condition (ultrasonic power was set to 30%, ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65mL containing 1.5%PVA (w/v ) in an aqueous solution of NaCl (outer water phase W2), using magnetic stirring to prepare a pre-multiplex emulsion (W1/O/W2), the emulsification condition is 500r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 9.2 μm by nitrogen gas at a pressure of 1 MPa until uniform double emulsion droplets (W1/O /W2). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1300nm, the PDI value was 0.06, and the drug embedding rate was 88.2%. The scanning electron micrograph of the obtained microspheres is shown in Fig. 6 .

Specific Example 26—Preparation of 1000nm PLGA Antigen-Carrying Nanomicrospheres Co-embedded with CPG1826 Carrying HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: 400 mg of PLGA is dissolved in 10.0 mL of EA solvent as the oil phase (O), and 3 mL of HPV 16L1 pentamer antigen protein and 5 μg of CpG1826 are used as the inner water phase (W1) with a concentration of 4 mg/mL. The S-450D ultrasonic cell disruptor performs colostrum under the condition of ice-water bath (the ultrasonic power is set to 30%, and the ultrasonic time is 60s) to prepare W1/O type colostrum, and then pour the colostrum into 65mL containing 1.5%PVA ( w/v) NaCl aqueous solution (external water phase W2), using magnetic stirring to prepare the pre-multiplex emulsion (W1/O/W2), the emulsification condition is 400r/min stirring 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 1.4 μm by the nitrogen gas of 0.25M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1000nm, the PDI value was 0.06, and the drug embedding rate was 87.8%. The scanning electron micrograph of the obtained microspheres is shown in Figure 9 .

Specific Example 27—Preparation of 1.57 μm PLGA-loaded Antigen Nanomicrospheres Co-embedded MPLA-loaded HPV16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: 400 mg of PLGA is dissolved in 10.0 mL of EA solvent as the oil phase (O), and 3 mL of HPV 16L1 pentamer antigen protein and 5 μg MPLA are added as the inner water phase (W1) at a concentration of 6.67 mg/mL. Use the S-450D ultrasonic cell breaker to perform colostrum under ice-water bath conditions (the ultrasonic power is set to 30%, and the ultrasonic time is 60s) to prepare W1/O type colostrum, and then pour the colostrum into 65 mL of 1.5% PVA-containing (w/v) NaCl aqueous solution (external water phase W2), using magnetic stirring to prepare the pre-multiplex emulsion (W1/O/W2), the emulsification condition is 400r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 5.2 μm by the nitrogen gas of 0.25M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1.57 μm, the PDI value was 0.05, and the drug embedding rate was 82.2%.

Specific Example 29—Preparation of 23.57 μm PLA antigen-carrying nanomicrospheres co-embedded with PolyI:C carrying HPV45L1 pentamer antigen microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: 400 mg of PLGA is dissolved in 10.0 mL of EA solvent as the oil phase (O), and 3 mL of HPV 16L1 pentamer antigen protein and 5 μg of PolyI:C are added as the inner water phase (W1 ), use the S-450D ultrasonic cell breaker to perform primary emulsification under ice-water bath condition (ultrasonic power is set to 30%, ultrasonic time is 60s) to prepare W1/O type colostrum, and then pour the colostrum into 65mL containing 1.5 %PVA (w/v) NaCl aqueous solution (external water phase W2), using magnetic stirring to prepare pre-multiplex emulsion (W1/O/W2), the emulsification condition is 400r/min stirring 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 5.2 μm by the nitrogen gas of 0.25M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 23.57 μm, the PDI value was 0.09, and the drug embedding rate was 86.3%. Specific Example 31——Preparation of 400nm PLGA-loaded HPV 16L1 Pentamer Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV16L1VLP antigen protein with a concentration of 1.33 mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell disruption The instrument carries out colostrum under the condition of ice-water bath (ultrasonic power is set to 30%, ultrasonic time is 60s) to prepare W1/O type colostrum, then pour the colostrum into 65mL of NaCl containing 1.5%PVA (w/v) In the aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 600 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 1.4 μm with nitrogen gas at a pressure of 1.5 MPa until uniform complex emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 400nm, the PDI value was 0.05, and the drug embedding rate was 90.5%. The scanning electron microscope image of the prepared microspheres is shown in Figure 4

Specific Example Thirty-Six——Preparation of 128nmPLGA-loaded HPV 6L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 6L1VLP antigen protein with a concentration of 1.33 mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell The breaker was used to perform colostrum under ice-water bath conditions (ultrasonic power was set to 30%, and the ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 600 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 0.4 μm by the nitrogen gas of 1.5M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 128nm, the PDI value was 0.03, and the drug embedding rate was 91.7%.

Specific embodiment thirty-seven

Weigh 4.4 mg of HPV 6L1 VLP antigen-loaded microspheres with an average particle size of 128 nm prepared in Example 36 and compound 12.03 μL of HPV 6L1 pentamer antigen with a concentration of 13.33 mg/mL, add physiological saline or pH=7.0 PBS buffer solution to 1 mL, prepared a microsphere suspension containing 200 μg/mL of HPV antigen with a ratio of coated antigen to free antigen in the nanomicrospheres of 2:8, and prepared HPV vaccine preparation.

Specific Example Thirty-Eight——Preparation of 467nm PLGA-loaded HPV 11L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400mg of PLGA in 10.0mL of EA solvent as the oil phase (O), add 3mL of HPV 11L1VLP antigen protein with a concentration of 4mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell disruption The instrument carries out colostrum under the condition of ice-water bath (ultrasonic power is set to 30%, ultrasonic time is 60s) to prepare W1/O type colostrum, then pour the colostrum into 65mL of NaCl containing 1.5%PVA (w/v) In the aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 600 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 1.4 μm by nitrogen gas at a pressure of 1 MPa until uniform double emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 467nm, the PDI value was 0.05, and the drug embedding rate was 89.2%.

Specific Example 40—Preparation of 1 μm PLGA-loaded HPV16L 1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV16L1VLP antigen protein with a concentration of 6.67 mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell disruption The instrument carries out colostrum under the condition of ice-water bath (ultrasonic power is set to 30%, ultrasonic time is 60s) to prepare W1/O type colostrum, then pour the colostrum into 65mL of NaCl containing 1.5%PVA (w/v) In the aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 400 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 5.2 μm with nitrogen gas at a pressure of 0.25 MPa until uniform complex emulsion droplets (W1/O /W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1 μm, the PDI value was 0.05, and the drug embedding rate was 89.5%. The scanning electron micrograph of the prepared antigen-loaded nanomicrospheres is shown in FIG. 10 .

Specific Example 42——Preparation of 5.87 μm PLGA-loaded HPV18L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV18L1VLP antigen protein with a concentration of 9.33 mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell disruption The instrument carries out colostrum under the condition of ice-water bath (ultrasonic power is set to 30%, ultrasonic time is 60s) to prepare W1/O type colostrum, then pour the colostrum into 65mL of NaCl containing 1.5%PVA (w/v) In the aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 300 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 15 μm by the nitrogen of 40kPa pressure until uniform double emulsion drops (W1/O/W2 ). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 5.87 μm, the PDI value was 0.07, and the drug embedding rate was 89.2%.

Specific Example 52——Preparation of 25.02 μm PLA-loaded HPV45L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400mg of PLA in 10.0mL of EA solvent as the oil phase (O), add 3mL of HPV 45L1VLP antigen protein with a concentration of 5.33mg/mL as the internal water phase (W1), and use S-450D ultrasonic cell The breaker was used to perform colostrum under ice-water bath conditions (ultrasonic power was set to 30%, and the ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 250 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 52 μm with nitrogen at a pressure of 5 kPa until uniform double emulsion droplets (W1/O/W2 ). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 25.02um, the PDI value was 0.06, and the drug embedding rate was 75.3%.

Specific Example 55——Preparation of 1000nm PLGA/HSPC-loaded HPV16L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is as follows: 300 mg of PLGA and 100 mg of lecithin are dissolved in 10.0 mL of MC solvent as the oil phase (O), and 3 mL of HPV 16L1VLP antigenic protein with a concentration of 4 mg/mL is added as the internal water phase (W1). A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (outer water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 500 r/min and stirring for 2 min. Subsequently, this pre-multiplex emulsion is poured into the storage tank for rapid membrane emulsification, and the complex emulsion is repeatedly pressed through the SPG membrane with a membrane pore size of 9.2 μm with nitrogen gas at a pressure of 1 MPa until uniform complex emulsion droplets (W1/O/ W2). The obtained homogeneous complex emulsion was stirred at room temperature for 4 hours (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally freeze-dried to obtain a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1000nm, the PDI value was 0.07, and the drug embedding rate was 80.6%. The scanning electron micrograph of the prepared antigen-loaded nanospheres is shown in FIG. 11 .

Specific Example 56—Preparation of 489nm PLGA Antigen-Carrying Nanomicrospheres Co-embedded with CPG1826 Carrying HPV11L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 11L1 VLP antigen protein with a concentration of 4 mg/mL and 5 μg of CpG1826 as the internal water phase (W1), and use S-450D Ultrasonic cell breaker was used to perform colostrum in ice-water bath condition (ultrasonic power was set to 30%, ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65mL containing 1.5%PVA (w/v ) in an aqueous solution of NaCl (outer water phase W2), using magnetic stirring to prepare a pre-multiplex emulsion (W1/O/W2), the emulsification condition is 400r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 1.4 μm by the nitrogen gas of 0.25M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 489nm, the PDI value was 0.05, and the drug embedding rate was 90.8%.

Specific example sixty-six——Preparation of 1000nm PLA antigen-loaded nanomicrospheres loaded with HPV16L1VLP antigen microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV 16L1 VLP antigen protein with a concentration of 6.67 mg/mL and 5 μg of CpG1826 as the inner water phase (W1), and use S- A 450D ultrasonic cell disruptor was used to perform colostrum in an ice-water bath (the ultrasonic power was set at 30%, and the ultrasonic time was 60 s) to prepare W1/O type colostrum, and then the colostrum was poured into 65 mL of 1.5% PVA (w/ v) In the NaCl aqueous solution (external water phase W2), a pre-multiplex emulsion (W1/O/W2) was prepared by magnetic stirring, and the emulsification condition was 400 r/min and stirring for 2 min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 1.4 μm by the nitrogen gas of 0.25M Pa pressure until a uniform double emulsion droplet (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer, the average particle size was 1000nm, the PDI value was 0.04, and the drug embedding rate was 89.4%. The scanning electron micrograph of the prepared antigen-loaded nanomicrospheres is shown in FIG. 12 .

Specific Example 68——Preparation of 10 μm PLGA Antigen-Carrying Nanomicrospheres Carrying HPV16L1VLP Antigen Microspheres

Antigen-loaded nanospheres were prepared by rapid membrane emulsification technology combined with double emulsion solvent removal method. The specific method is: dissolve 400 mg of PLGA in 10.0 mL of EA solvent as the oil phase (O), add 3 mL of HPV16L1VLP antigen protein with a concentration of 6.67 mg/mL and 5 μg of CpG1826 as the internal water phase (W1), and use S-450D Ultrasonic cell breaker was used to perform colostrum in ice-water bath condition (ultrasonic power was set to 30%, ultrasonic time was 60s) to prepare W1/O type colostrum, and then the colostrum was poured into 65mL containing 1.5%PVA (w/v ) in an aqueous solution of NaCl (outer water phase W2), using magnetic stirring to prepare a pre-multiplex emulsion (W1/O/W2), the emulsification condition is 400r/min and stirring for 2min. Subsequently, this pre-multiple emulsion is poured into the storage tank of rapid membrane emulsification, and the double emulsion is repeatedly pressed through the SPG membrane with a membrane aperture of 50.2 μm by the nitrogen gas of 0.25M Pa pressure until uniform double emulsion droplets (W1/ O/W2). The obtained homogeneous double emulsion was poured dropwise into 780 mL of 0.9% NaCl solution, stirred at room temperature for 4 h (magnetic stirring rate 200 rpm) to remove the organic solvent, and the solidified microspheres were centrifuged and washed 3 times with deionized water, and finally frozen Dried to make a finished product. The particle size distribution was measured with a laser particle size analyzer. The average particle size was 10 μm, the PDI value was 0.07, and the drug embedding rate was 90.2%.

Specific embodiment seventy-four

Take 1.128 mg of the 430nm nanospheres prepared in Example 31 and 1.157 mg of the 1.385 nanospheres prepared in Example 5 to form a particle size combination with an antigen mass ratio of 50%:50%, and add the pH value Using 1 mL of PBS buffer solution of 7, prepare a suspension containing 100 μg/mL of antigen and nanosphere concentration of 2.31 mg/mL.

Specific embodiment seventy-nine

Take 1.128 mg of the 430nm nanospheres prepared in Example 31 and 1.147 mg of the 20.65 μm nanospheres prepared in Example 33 to form a particle size combination with an antigen mass ratio of 50%:50%, and add PH 1 mL of PBS buffer solution with a value of 7 was prepared to prepare a suspension containing 100 μg/mL of antigen and a nanosphere concentration of 2.27 mg/mL.

Specific embodiment eighty-four

Take 1.157 mg of the 1.38 μm nanospheres prepared in Example 5 and 1.147 mg of the 20.65 μm nanospheres prepared in Example 33 to form a particle size combination with an antigen mass ratio of 50%:50%, and add a pH value Using 1 mL of PBS buffer solution of 7, prepare a suspension containing 100 μg/mL of antigen and nanosphere concentration of 2.31 mg/mL.

Specific embodiment ninety-two

Take 0.68 mg of 430 nm nanospheres prepared in Example 31, 0.69 mg of 1.38 μm nanospheres prepared in Example 5, and 0.92 mg of 20.65 μm nanospheres prepared in Example 33 to prepare antigenic mass The particle size combination with a ratio of 30%:30%:40% was added to 1 mL of PBS buffer with a pH value of 7 to prepare a suspension containing 100 μg/mL of antigen and a nanosphere concentration of 2.29 mg/mL.

Specific embodiment one hundred and one

The 10 μm nanospheres prepared in Example 68 and the 1 μm nanospheres prepared in Example 40 were respectively prepared into a particle size combination with an antigen mass ratio of 20%:80%, and PBS buffer with a pH value of 7 was added. 1 mL of the solution, and a suspension containing 100 μg/mL of antigen and a concentration of nanomicrospheres of 2.35 mg/mL was prepared.

Specific embodiment one hundred and two

Take the 10 μm nanospheres prepared in Example 68 and the 1 μm nanospheres prepared in Example 40 to form a particle size combination with an antigen mass ratio of 80%:20%, and add PBS buffer with a pH value of 7 1 mL of the solution, and a suspension containing 100 μg/mL of antigen and a concentration of nanomicrospheres of 2.35 mg/mL was prepared.

Specific embodiment one hundred and three

Take 1.175 mg of the 10 μm nanospheres prepared in Example 68 and 1.174 mg of the 1 μm nanospheres prepared in Example 40 to form a particle size combination with an antigen mass ratio of 50%:50%, and add a pH value of 7 of PBS buffer solution 1mL to prepare a suspension containing 100 μg/mL antigen and nanosphere concentration of 2.35 mg/mL.

Specific embodiment one hundred and six

Take 1.175 mg of the 458nm nanospheres prepared in Example 66 and 1.164 mg of the 23.58 μm nanospheres prepared in Example 68 to form a particle size combination with an antigen mass ratio of 50%:50%, and add PH 1 mL of PBS buffer with a value of 7 was prepared to prepare a suspension containing 100 μg/mL of antigen and a nanosphere concentration of 2.34 mg/mL.

Specific embodiments one hundred and eleven

Take 1.174 mg of the 1.69 μm nanospheres prepared in Example 40 and 1.164 mg of the 23.58 μm nanospheres prepared in Example 68 to form a particle size combination with an antigen mass ratio of 50%:50%, and add PH 1 mL of PBS buffer with a value of 7 was prepared to prepare a suspension containing 100 μg/mL of antigen and a nanosphere concentration of 2.34 mg/mL.

Specific embodiment one hundred and twenty

Take 0.71 mg of 458nm nanospheres prepared in Example 66, 1.174 mg of 1.69 μm nanospheres prepared in Example 40, and 0.004 mg of 0.47 μm nanospheres prepared in Example 68 to prepare antigens The particle size combination with a mass ratio of 30%:50%:20% was added to 1 mL of PBS buffer with a pH value of 7 to prepare a suspension containing 100 μg/mL of antigen and a nanosphere concentration of 2.35 mg/mL.

The specific implementation steps of other embodiments in the table are implemented with reference to the above related specific embodiments. Example 125—Characterization of Microsphere Particle Size and Particle Size Distribution

The microsphere suspension prepared in some of the examples was ultrasonically dispersed in distilled water to make it uniformly dispersed, and a laser particle size analyzer ( 3000HS, UK) to measure the particle size and distribution, and the particle size distribution diagrams of different antigen-loaded nanosphere adjuvants are shown in Figure 15.

Embodiment 126——Determination of HPV loading rate

Suspend 5 mg of the prepared freeze-dried nano-microspheres in 1.0 mL of NaOH solution containing 5% (w/v) SDS at a concentration of 0.1 M, and shake at room temperature for 24 hours to completely dissolve (hydrolyze) the microspheres. Protein content was determined with micro-BCA kit.

Example 127—Comparison of antibody level evaluation after immunization with PLGA antigen-loaded microspheres with different particle sizes

Take the freeze-dried microspheres prepared in Example 5 and Example 3 and suspend them in physiological saline to obtain a microsphere suspension, so that the content of the antigen in the microsphere suspension is 20 μg/100 μL, and the HPV vaccine preparation is obtained .

Take the two nanosphere vaccine preparations prepared in Example 5 and Example 3 and subcutaneously immunize female BALB/c mice for 8-10 weeks on the back. Each mouse is injected with an antigen dose of 20 μg/100 μL. HPV pentamer antigen as adjuvant was used as control group.

The humoral immunity effects of the above three groups were detected. The detected indicators include the level of neutralizing antibody and specific antibody IgG level after 38 days of immunization of mice with antigens loaded on PLGA microspheres with different particle sizes, as shown in Figure 16 and Figure 17, respectively.

The levels of neutralizing antibodies of specific antibodies produced by PLGA-loaded antigen nanomicrospheres with different particle sizes after immunization of mice were different. It can be seen from Figure 16 that the neutralizing antibody level and IgG level produced by the 1.38 μm antigen-loaded PLGA microspheres were higher than those of the aluminum adjuvant group after 38 days of mice immunization, but there was no significant difference; but the 1.38 μm antigen-loaded PLGA group serum The levels of neutralizing antibody and IgG were significantly higher than those of 430nm antigen-loaded PLGA group (P<0.05 or P<0.01).

Example 128—Comparison of evaluation of cytokine levels after immunization with PLGA antigen-loaded microspheres with different particle sizes

Take the two nanosphere vaccine preparations prepared in Example 5 and Example 3 and subcutaneously immunize female BALB/c mice for 8-10 weeks on the back. Each mouse is injected with an antigen dose of 20 μg/100 μL. HPV pentamer antigen as adjuvant was used as control group.

The cellular immunity effects of the above three groups were detected. The detection indicators include the level of cytokines in the spleen cell supernatant (Luminex technology, ELISA method) after stimulating mouse splenocytes with antigen in vitro for 72 hours, the results are shown in Figure 18 and Figure 19; After that, IFN-γ and IL-4 enzyme-linked immunospot forming cell numbers (ELISPOT method), the results are shown in Figure 20.

IL-4 and IL-6 represent cytokines responding to Th2 type, and IFN-γ and IL-2 represent cytokines responding to Th1 type. The IFN-γ levels in the splenocyte supernatant of the antigen-loaded PLGA microsphere group were significantly higher than those in the aluminum adjuvant group (P<0.05 or P<0.01), and the 430nm antigen-loaded PLGA nanosphere group splenocyte supernatant The level of IFN-γ was significantly higher than that of the 1.38 μm antigen-loaded PLGA nanosphere group (P<0.01); as can be seen from Figure 7, the IL-2 level in the splenocyte supernatant of the 430nm antigen-loaded PLGA nanosphere group was also significantly It was higher than aluminum adjuvant group (P<0.05), and the secretion level of IL-2 in splenocyte supernatant of 430nm antigen-loaded PLGA nanosphere group was also higher than that of 1.38μm antigen-loaded PLGA group, but there was no significant difference between the two. This shows that, compared with aluminum adjuvant, the antigen-loaded PLGA nanosphere group can effectively induce Th1 type immune response and enhance cellular immune response, and the PLGA antigen-loaded nanospheres with smaller particle size (430nm) are more effective than the large particle size PLGA Antigen-loaded nanospheres (1.38 μm) are more effective in inducing Th1-type immune responses and enhancing cellular immune responses.

In terms of the secretion level of Th2 cytokines, the antigen-loaded PLGA nanosphere group with different particle sizes can induce IL-4 secretion level similar to that of aluminum adjuvant, and there is no significant difference. The level of IL-4 in serum was significantly higher than that of the 430nm antigen-loaded PLGA nanosphere group (P<0.05 or P<0.01); the IL-6 level in the supernatant of splenocytes in the 1.38μm antigen-loaded PLGA nanosphere group was significantly higher In the aluminum adjuvant group (P<0.05), the level of IL-6 in the splenocyte supernatant of the 1.38μm antigen-loaded PLGA nanosphere group was significantly higher than that of the 430nm antigen-loaded PLGA nanosphere group (P<0.01).

The above results show that the antigen-loaded PLGA nanosphere group can induce the secretion level of IL-4 similar to that of aluminum adjuvant, can effectively induce humoral immunity, and a higher level of IFN-γ level, which can significantly improve the cellular immune response; and large particles Antigen-loaded PLGA microspheres (1.38 μm) can significantly increase the secretion level of IL-6 and IL-4, enhance Th2 immune response, promote the activation of B cells to produce antibodies, and effectively induce humoral immune response; while small particle size Antigen-loaded PLGA microspheres (430nm) significantly increase the secretion level of IFN-γ, enhance Th1 immune response, and contribute to the improvement of cellular immune response.

Example 129——Analysis of immune effect after immunizing mice with free antigen compounded with 1.38 μm PLGA antigen-loaded nanomicrospheres

Take the microsphere preparation prepared in Example 6 and subcutaneously immunize female BALB/c mice for 8-10 weeks on the back. The dose of antigen injected into each mouse is 20 μg/100 μL, and the HPV antigen content in the microsphere vaccine preparation is 10 μg/mouse , the content of free HPV antigen was 10 μg/body, and the HPV pentamer antigen with aluminum salt as an adjuvant and the 1.38 μm PLGA antigen-carrying nanomicrospheres prepared in Example 5 were set up as control groups. The effects of humoral immunity and cellular immunity of the above groups were detected.

The results show that the neutralizing antibody titer and specific antibody IgG titer produced by 1.38 μm PLGA antigen-loaded nanospheres compounded with free antigen are about 5 times that of the 1.38 μm PLGA-loaded antigen nanospheres group, which shows that PLGA-loaded antigen nanoparticles The compounded free antigen can effectively enhance the effect of humoral immunity.

Example 134——Preparation of Pentamer and VLP Nanomicrospheres

Referring to the above specific implementation methods, the preparation of nano-microspheres of pentamers and VLPs with different proportions, different particle sizes and different types was prepared according to the formula in the following table.

Table 5 contains the serial numbers of different particle sizes of HPV16L1 pentameric PLGA microspheres

Table 6 Combination numbers of different particle sizes of PLGA microspheres loaded with HPV16L1 pentamer antigen

Table 7 The serial numbers of different HPV types of PLGA microspheres loaded with HPV16L1 pentamer antigen

Table 8 Sequence number of PLGA microsphere compound lipid substance containing HPV16L1 pentamer antigen

Table 9 The serial numbers of different molecular adjuvants compounded with PLGA microspheres loaded with HPV16L1 pentamer antigen

Table 10 carries the serial numbers of the different particle sizes of the PLGA microspheres of HPV16L1VLP antigen

Table 11 Combination numbers of different particle sizes of PLGA microspheres loaded with HPV16L1VLP antigen

Table 12 The different HPV type numbers of the PLGA microspheres loaded with HPV16L1VLP antigen

Table 13 Sequence number of compound lipids of PLGA microspheres loaded with HPV16L1VLP antigen

Table 14 The sequence numbers of different molecular adjuvants in the compounding of PLGA microspheres loaded with HPV16L1VLP antigen

Example 135—Study on Immunogenicity of HPV L1 Pentamer of Nanospheres and Microspheres with Different Particle Sizes

Take the samples prepared in Examples 19, 20, 30, and 21, HPV L1 pentamer protein embedded in PLGA with three different particle sizes of 400nm, 800nm, and 1.3 μm, and HPV L1 coated with 400nmPLGA/PC The mice were immunized with pentameric protein, and preliminary experimental results showed that the adjuvant system can effectively induce antigen-specific immune responses including neutralizing antibodies and cellular immunity. In Figure 4, the antibodies in the serum were analyzed, and the results showed that, compared with the traditional aluminum salt adjuvant group, the neutralizing antibody titers of the 400nm and 800nm PLGA adjuvant groups were basically the same, but the 1.3μM neutralizing antibody adjuvant group and The neutralizing antibody and binding titer of 400nmPLGA+lecithin (PLGA/PC) composite adjuvant group were higher; the binding antibody titers of the other 800nm and 1.3μ.3nm adjuvant groups were significantly higher than 400nm PLGA, but the difference between the two No significant difference between. The HPV-specific CTL immune response was detected by ELISPOT. The results showed that the traditional aluminum adjuvant group basically did not induce a specific CTL immune response, while the PLGA adjuvant group effectively induced a relatively high level of HPV-specific CTL immune response. Among them, the 400nm and 800nm PLGA adjuvant groups were the highest (Figure 2). The splenocytes of the immunized mice were cultured in vitro and stimulated by antigens to measure the secretion of cytokines in the supernatant. , the PLGA adjuvant group was significantly higher than the traditional aluminum salt adjuvant group; and in terms of Th2 cytokines (stimulating humoral/antibody immune response) IL-4, the PLGA adjuvant group was basically the same as the traditional aluminum salt adjuvant group, but The compound adjuvant group using PLGA+lecithin (PLGA/PC) was significantly higher than the other groups. The results are shown in Figures 21, 22, 23, 24, and 25.

Example 136——Analysis of Immune Effect of 1000nm PLGA Antigen-loaded Nanospheres Co-embedded with CPG1826 and Combined Immunization of Mice

According to Example 26, PLGA antigen-loaded nanospheres co-embedded with CpG1826 were prepared. After the prepared microspheres were freeze-dried, they were suspended in physiological saline to obtain a microsphere suspension, so that the antigen content in the microsphere suspension was 20 μg/100 μL, and the HPV vaccine preparation was obtained. Subcutaneously immunize female BALB/c mice for 8-10 weeks on the back, and inject the antigen dose of 20μg/100μL into each mouse. At the same time, HPV pentamer antigen with aluminum salt as adjuvant and 1000nm PLGA-carried antigen nanometer were established. balls as a control group. The effects of humoral immunity and cellular immunity of the above groups were detected. The titers of neutralizing antibody and binding antibody after immunizing mice with HPV16L1 pentamer-coated microspheres coated with PLGA compound CpG small molecule adjuvant are shown in Figures 26 and 27, respectively. After immunizing mice, the group can induce neutralizing antibody levels comparable to the level of aluminum salt adjuvant. In terms of binding antibody level, 1 μm PLGA-embedded HPV16L1 pentameric protein induced significantly higher binding antibody levels than aluminum salt adjuvant in the first immunization, and could induce a level comparable to that of aluminum salt adjuvant in the second immunization. Level of binding antibody titer, and the level of binding antibody induced in the second immunization after adding CpG small molecule adjuvant was significantly higher than that in the group without CpG, while the binding antibody induced in the first and second immunization The levels were significantly higher than traditional aluminum salt adjuvants.

Example 137—Analysis of immune effect after immunization of mice with PLGA antigen-loaded nanospheres with different particle size combinations

Get the female BALB/c mice of 8-10 weeks subcutaneously immunized on the back with the microsphere preparations of different particle size combinations prepared in Examples 101, 102, and 103, and inject the antigen dose into each mouse 20 μg/100 μL, HPV pentamer antigen with aluminum salt as adjuvant and PLGA antigen-loaded nanospheres with different particle sizes prepared in Examples 40 and 68 were set up as control groups. The humoral immunity effects of the above groups were detected. The binding antibody titers and neutralizing antibody titers after immunizing mice with PLGA-coated HPV16L1 pentamer microspheres of different particle sizes and their combinations were 28 and 29, respectively.

The results showed that PLGA-embedded HPV16L1VLPs with 1 μm or 10 μm or a combination of the two particle sizes in a certain proportion induced no significant difference in neutralizing antibody titers, while the 1 μm PLGA-HPV16L1VLP group induced significantly lower binding antibody titers than HPV16L1VLP after primary immunization The -1μm+10μm(0.5+0.5) group was significantly lower than the HPV16L1VLP-1μm+10μm(0.2+0.8) group and the HPV16L1VLP-1μm+10μm(0.8+0.2) group after the second immunization.

Example 138—Study on the immune effect of HPV 16L1 VLP with different adjuvants

After immunizing mice with PLGA, PLGA/HSPC, and PLA-embedded HPV 16L1 VLP, there was no significant difference in the induced neutralizing antibody and binding antibody titers, as shown in Figures 30 and 31.

Embodiment 139——Preparation of particles of PLGA

Dissolve 100mg of PLGA in 20mL of dichloromethane as the oil phase (O), prepare a 1.0% PVA solution as the external water phase (W2), add the oil phase (O) to 50mL of the external water phase (W2) , prepare the pre-emulsion (W2/O) of oil-in-water type, this pre-multiplex emulsion adopts the method for fast film emulsification to repeatedly pass through the membrane to prepare the multi-emulsion (O/W2) of oil-in-water with uniform particle size; solidify for 3h , washed by centrifugation, and freeze-dried to obtain dry PLGA microspheres. The SEM photo of PLGA is shown in Figure 2.

Example 140——Preparation of PLGA particle-adsorbed HPV16L1VLP vaccine composition

Dissolve the prepared PLGA microspheres in 0.8mL pH=5, 10mM PBS buffer solution to obtain a 20mg/mL PLGA microsphere suspension; take a certain amount of HPV and dissolve it in 0.8mL pH=5, 10mM PBS buffer solution In the solution, 20ug/mL HPV antigen solution was obtained, the two were mixed in equal volumes, and adsorbed overnight to obtain 1.6mL PLGA suspension with HPV antigen adsorbed, which was the HPV vaccine composition.

Example 140——Study of PLGA Microspheres Adsorbing Immune Response to HPV 16L1 VLP

The PLGA-HPV16L1VLP sample in Example 1 of Example 139 was taken to immunize mice after absorbing the HPV 16L1VLP sample, and the PLGA-HPV16L1VLP could induce a binding antibody titer comparable to Merck's Gardasil vaccine. The result is shown in Figure 32.

Baylor, NW, W. Egan and P. Richman (2002). "Aluminum salts in vaccines--US perspective." Vaccine 20 Suppl 3: S18-23.

Bernard, H.-U., RD Burk, E.-M. de Villiers and H. zur Hausen (2011). Family Papillomaviridae. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses . AM King, MJ Adams, EBCarstens and E. Lefkowitz , Elsevier: 235-248.

Bryan, JT(2007). "Developing an HPV vaccine to prevent cervical cancer and genital warts." Vaccine 25(16):3001-3006.

Buck, CB, N. Cheng, CD Thompson, DRLowy, ACSteven, JTSchiller and B.L. Trus (2008). "Arrangement of L2 within the papillomavirus capsid." J Virol 82(11):5190-5197.

Buonaguro, FM, MLTornesello and L. Buonaguro(2009). "Virus-likeparticle vaccines and adjuvants: the HPV paradigm." Expert Rev Vaccines 8(10):1379-1398.

Bystrianyk, R. (2009, 21 September 2009). "Study clearly demonstrates that aluminum found in vaccines can cause neurologic damage." Retrieved September 3, 2012, 2012, from http://www.healthsentinel.com/joomla/index.php? option=com_content &view=article&id=2598:stu dy-clearly-demonstrates-that-aluminum- found-in-vaccines-can-cause-neurologic-damage&catid=5:original&Itemid=24 .

Cain, DW, SESanders, MMCunningham and G. Kelsoe(2013). "Disparateadjuvant properties among three formulations of"alum"." Vaccine 31(4):653-660.

Campo, MS and RB Roden(2010). "Papillomavirus prophylactic vaccines:established successes, new approaches." J Virol 84(3):1214-1220.

Cassel, SL, S. Joly and FSSutterwala(2009). "The NLRP3inflammasome: asensor of immune danger signals." Semin Immunol 21(4):194-198.

Chen, J., G. Ni and XS Liu(2011). "Papillomavirus virus like particle-based therapeutic vaccine against human papillomavirus infection related diseases: immunological problems and future directions." Cell Immunol 269(1):5-9.

Chen, XS, RL Garcea, I. Goldberg, G. Casini and SC Harrison (2000). "Structure of small virus-like particles assembled from the L1protein of human papillomavirus 16." Mol Cell 5(3):557-567.

Clements, CJ and E. Griffiths(2002). "The global impact of vaccines containing aluminum adjuvants." Vaccine 20 Suppl 3:S24-33.

Danhier, F., E. Ansorena, JMSilva, R. Coco, A. Le Breton and V. Preat (2012). "PLGA-based nanoparticles: An overview of biomedical applications." J Control Release 161(2):505- 522.

Davis, BK, H. Wen and JPTing (2011). "The inflammasome NLRs inimmunity, inflammation, and associated diseases." Annu Rev Immunol 29:707-735.

De Gregorio, E., E. Tritto and R. Rappuoli(2008). "Alum adjuvanticity: unraveling a century old mystery." Eur J Immunol 38(8):2068-2071.

De Temmerman, ML, J. Rejman, J. Demeester, DJ Irvine, B. Gander and SC De Smedt (2011). "Particulate vaccines: on the quest for optimal delivery and immune response." Drug Discov Today 16(13-14):569- 582.

Demento,SL,W.Cui,JMCriscione,E.Stern,J.Tulipan,SMKaech andT.M.Fahmy(2012)."Role of sustained antigen release from nanoparticles vaccinesin shaping the T cell memory phenotype." Biomaterials 33(19) :4957-4964.

Demento, SL, SCEisenbarth, HGFoellmer, C.Platt, MJCaplan, W.MarkSaltzman, I.Mellman, M.Ledizet, E.Fikrig, RAFlavell and TMFahmy (2009). "Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy." Vaccine 27(23):3013-3021.

Dierendonck, M., S. De Koker, C. Vervaet, JPRemon and BGDe Geest(2012). "Interaction between polymeric multilayer capsules and immune cells." J Control Release 161(2):592-599.

Dostert, C., V. Petrilli, R. Van Bruggen, C. Steele, BTMossman and J. Tschopp (2008). "Innate immune activation through Nalp3inflammasome sensing of asbestos and silica." Science 320(5876):674-677.

Duewell, P., H. Kono, KJ Rayner, CMSirois, G. Vladimer, FG Bauernfeind, GSAbela, L. Franchi, G. Nunez, M. Schnurr, T. Espevik, E. Lien, KAFitzgerald, KLRock, KJ Moore, SDWright, V .Hornung and E.Latz(2010). "NLRP3inflammasomes are required for atherogenesis and activated by cholesterol crystals." Nature 464(7293):1357-1361.

Eisenbarth, SC, OR Colegio, W. O'Connor, FS Sutterwala and RAFlavell (2008). "Crucial role for the Nalp3inflammasome in the immunostimulatory properties of aluminum adjuvants." Nature 453(7198):1122-1126.

Finnen, RL, KD Rickson, XS Chen and RL Garcea (2003). "Interactions between papillomavirus L1 and L2 capsid proteins." J Virol 77(8):4818-4826.

Flach, TL, G. Ng, A. Hari, MD Desrosiers, P. Zhang, SMWard, MESeamone, A. Vilaysane, ADMucsi, Y. Fong, E. Prenner, CCLing, J. Tschopp, DAMuruve, MWAmrein and Y. Shi ( 2011). "Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity." Nat Med 17(4):479-487.

Foged, C.(2011). "Subunit vaccines of the future: the need for safe, customized and optimized particulate delivery systems." The Deliv 2(8):1057-1077.

Foged, C., B. Brodin, S. Frokjaer and A. Sundblad(2005). "Particle size and surface charge affect particle uptake by human dendritic cells in an in vitromodel." Int J Pharm 298(2):315-322.

Franchi, L. and G. Nunez(2008). "The Nlrp3inflammasome is critical for aluminum hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity." Eur J Immunol 38(8):2085-2089.

Frazer, IH, GRLeggatt and SRMattarollo (2011). "Prevention and treatment of papillomavirus-related cancers through immunization." Annu Rev Immunol 29:111-138.

Garcon, N., S. Morel, A. Didierlaurent, D. Descamps, M. Wettendorff and M. Van Mechelen (2011). "Development of an AS04-adjuvanted HPV vaccine with the adjuvant system approach." BioDrugs 25(4):217 -226.

Gattoc, L., N. Nair and K. Ault (2013). "Human Papillomavirus Vaccination: Current Indications and Future Directions." Obstetrics and Gynecology Clinics of North America (0).

Ghimire, TR, RABenson, P. Garside and JM Brewer(2012). "Aluminum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro." Immunol Lett 147(1-2):55-62.

Glenny, AT, CG Pope, H. Waddington and U. Wallace (1926). "Immunological notes. XVII–XXIV." The Journal of Pathology and Bacteriology 29(1):31-40.

Goldstone, SE and S. Vuocolo(2012). "A prophylactic quadrivalent vaccine for the prevention of infection and disease related to HPV-6,-11,-16 and-18." Expert Rev Vaccines 11(4):395-406.

Gupta, RK(1998). "Aluminum compounds as vaccine adjuvants." Advanced Drug Delivery Reviews 32(3):155-172.

Gupta, RK, ACChang, P.Griffin, R.Rivera and GRSiber(1996). "In vivodistribution of radioactivity in mice after injection of biodegradablepolymer microspheres containing 14C-labeled tetanus toxoid." Vaccine 14(15):1412-1416.

Gupta, RK and GR Siber(1995). "Adjuvants for human vaccines--current status, problems and future prospects." Vaccine 13(14):1263-1276.

Gutierro, I., RM Hernandez, M. Igartua, AR Gascon and JLPedraz(2002). "Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres." Vaccine 21(1-2):67-77.

Hamilton, JA, R.Byrne and G.Whitty(2000). "Particulate adjuvants caninduce macrophage survival, DNA synthesis, and a synergistic proliferative response to GM-CSF and CSF-1." J Leukoc Biol 67(2):226-232 .

Hariri, S., E. Dunne, MSaraiya, E. Unger and L. Markowitz (2011). Chapter 5: Human Papillomavirus. Manual for the Surveillance of Vaccine-Preventable Diseases, 5th Edition (Centers for Disease Control and Prevention, Atlanta, GA, 2008.) . SWRoush, L. McIntyre and LMBaldy.

Harper, DM(2009). "Currently approved prophylactic HPV vaccines." Expert Rev Vaccines 8(12):1663-1679.

Hem, SL(2002). "Elimination of aluminum adjuvants." Vaccine 20 Suppl 3:S40-43.

Hem, SLand H. Hogenesch(2007). "Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation." Expert Rev Vaccines 6(5):685-698.

Herrero, R., S. Wacholder, AC Rodriguez, D. Solomon, P. Gonzalez, ARKreimer, C. Porras, J. Schussler, S. Jimenez, MESherman, W. Quint, JTSchiller, DRLowy, M. Schiffman, A. Hildesheim and G. Costa Rica Vaccine Trial(2011). "Prevention of persistent human papillomavirus infection by an HPV16/18 vaccine: a community-based randomized clinical trial in Guanacaste, Costa Rica." Cancer Discov 1(5):408-419.

Hogen Esch, H.(2013). "Mechanism of immunopotentiation and safety of aluminum adjuvants." Frontiers in Immunology 3.

Howley, PM and DRLowy(2007).Chapter 62:Papillomaviruses. Fields Virology,5th Edition.D.Knipe and P.Howley.Philadelphia,PA,Lippencott,Williams&Wilkins.

Jansen, KU and ARShaw (2004). "Human papillomavirus vaccines and prevention of cervical cancer." Annu Rev Med 55:319-331.

Johansen, P., F. Estevez, R. Zurbriggen, HP Merkle, R. Gluck, G. Corradin and B. Gander (2000). "Towards clinical testing of a single-administration tetanus vaccine based on PLA/PLGA microspheres." Vaccine 19( 9-10): 1047-1054.

Johansen, P., Y. Men, HP, Merkle and B. Gander(2000). "Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination." Eur J Pharm Biopharm 50(1):129-146.

Jordan, MB, DM Mills, J. Kappler, P. Marrack and JCCambier(2004). "Promotion of B cell immune responses via an alum-induced myeloid cell population." Science 304(5678):1808-1810.

Koff, WC, DR Burton, PR Johnson, BD Walker, CRKing, GJ Nabel, R. Ahmed, MKBhan and SAPlotkin (2013). "Accelerating next-generation vaccine development for global disease prevention." Science 340(6136):1232910.

Kool, M., V. Petrilli, T. De Smedt, A. Rolaz, H. Hammad, M. van Nimwegen, IM Bergen, R. Castillo, BN Lambrecht and J. Tschopp (2008). "Cutting edge: alumadjuvant stimulates inflammatory dendritic cells through activation of the NALP3inflammasome." J Immunol 181(6):3755-3759.

Kool, M., T. Soullie, M. van Nimwegen, MA Willart, F. Muskens, S. Jung, H CH Hoogsteden, H. Hammad and BN Lambrecht (2008). "Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells." J Exp Med 205(4):869-882.

Korsholm, KS, PL Andersen and D. Christensen(2012). "Cationicliposomal vaccine adjuvants in animal challenge models: overview and current clinical status." Expert Rev Vaccines 11(5):561-577.

Kreimer, AR, P. Gonzalez, HA Katki, C. Porras, M. Schiffman, AC Rodriguez, D. Solomon, S. Jimenez, JTSchiller, DRLowy, LJ van Doorn, L. Struijk, W. Quint, S. Chen, S. Wacholder , A. Hildesheim, R. Herrero and CVTV Group (2011). "Efficacy of a bivalent HPV 16/18 vaccine against anal HPV 16/18 infection among young women: a nested analysis within the Costa Rica Vaccine Trial." Lancet Oncol 12(9):862 -870.

Langer, R., JL Cleland and J. Hanes (1997). "New advances in microsphere-based single-dose vaccines." Adv Drug Deliv Rev 28(1):97-119.

Lehtinen, M. and J. Dillner(2013). "Clinical trials of human papillomavirus vaccines and beyond." Nat Rev Clin Oncol .

Lehtinen, M., J. Paavonen, CM Wheeler, U. Jaisamrarn, SM Garland, X. Castellsague, SRSkinner, D. Apter, P. Naud, J. Salmeron, SNChow, H. Kitchener, JCTeixeira, J. Hedrick, G. Limson , A. Szarewski, B. Romanowski, FYAoki, TFSchwarz, WAPoppe, NSDe Carvalho, MJ Germar, K. Peters, A. Mindel, P. DeSutter, FXBosch, MPDavid, D. Descamps, F. Struyf and G. Dubin (2012) ."Overallefficacy of HPV-16/18AS04-adjuvanted vaccine against grade 3or greatercervical intraepithelial neoplasia: 4-year end-of-study analysis of theranomised, double-blind PATRICIA trial." Lancet Oncol 13(1):89-99.

Levitz, SM and DT Golenbock(2012). "Beyond empiricism:informing vaccine development through innate immunity research." Cell 148(6):1284-1292.

Li, H., SB Willingham, JPTing and F. Re(2008). "Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3." J Immunol 181(1):17-21.

Lindblad, EB(2004). "Aluminium adjuvants--in retrospect and prospect." Vaccine 22(27-28):3658-3668.

Lindblad, EB(2004). "Aluminium compounds for use in vaccines." Immunol Cell Biol 82(5):497-505.

Malagon, T., M. Drolet, MC Boily, ELFranco, M. Jit, J. Brisson and M. Brisson (2012). "Cross-protective efficacy of two human papillomavirus vaccines: a systematic review and meta-analysis." Lancet Infect Dis .

Mann, JF, E. Shakir, KCCarter, ABMullen, J.Alexander and VAFerro(2009). "Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2bias in the immune response and protection against tinfection." Vaccine 27(27) :3643-3649.

Mariani, L. and A. Venuti(2010). "HPV vaccine: an overview of immune response, clinical protection, and new approaches for the future." J Transl Med 8:105.

Mastelic, B., S. Ahmed, WMEgan, G. Del Giudice, H. Golding, I. Gust, P. Neels, SG Reed, RL Sheets, CA Siegrist and PHLambert (2010). "Mode of action of adjuvants: implications for vaccine safety and design." Biologicals 38(5):594-601.

Morefield, GL, A. Sokolovska, D. Jiang, H. Hogen Esch, JP Robinson and S. L. Hem (2005). "Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro." Vaccine 23(13):1588- 1595.

Munks , MW, ASMcKee, MK Macleod, RL Powell, JL Degen, NA Reisdorph, JW Kappler and P. Marrack (2010).

Noe, SM, MAGreen, H. HogenEsch and SLHem(2010). "Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by therelationship between antigen retention at the inoculation site and the immune response." Vaccine 28(20):3588-3594.

O'Hagan, DT and NM Valiante (2003). "Recent advances in the discovery and delivery of vaccine adjuvants." Nat Rev Drug Discov 2(9):727-735.

Ohlschlager, P., W. Osen, K. Dell, S. Faath, RL Garcea, I. Jochmus, M. Muller, M. Pawlita, K. Schafer, P. Sehr, C. Staib, G. Sutter and L. Gissmann (2003). "Humanpapillomavirus type 16L1capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6mice." J Virol 77(8):4635-4645.

Olive, C.(2012). "Pattern recognition receptors: sentinels in innateimmunity and targets of new vaccine adjuvants." Expert Rev Vaccines 11(2):237-256.

Olsson, SE, LL Villa, RL Costa, CA Petta, RP Andrade, C. Malm, OE Iversen, J. Hoye, M. Steinwall, G. Riis-Johannessen, A. Andersson-Ellstrom, K. Elfgren, G. von Krogh, M. Lehtinen , J.Paavonen, GMTamms, K.Giacoletti, L.Lupinacci, MTEsser, SCVuocolo, AJSaah and E.Barr(2007). -like particle (VLP) vaccine." Vaccine 25(26):4931-4939.

Paavonen, J., D. Jenkins, FXBosch, P. Naud, J. Salmeron, CM Wheeler, SNChow, DLApter, HCKitchener, X. Castellsague, NS de Carvalho, SRSkinner, DM Harper, JA Hedrick, U. Jaisamrarn, GALimson, M. Dionne, W. Quint, B. Spiessens, P. Peeters, F. Struyf, SL Wieting, MOLehtinen, G. Dubin and HPsgroup (2007). "Efficacy of a prophylactic adjuvanted bivalent L1virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial." Lancet 369(9580):2161-2170.

Paavonen, J., P. Naud, J. Salmeron, CM Wheeler, SNChow, D. Apter, H. Kitchener, X. Castellsague, JCTeixeira, SRSkinner, J. Hedrick, U. Jaisamrarn, G. Limson, S. Garland, A. .Szarewski, B.Romanowski, FYAoki, TFSchwarz, WAPoppe, FXBosch, D.Jenkins, K.Hardt, T.Zahaf, D.Descamps, F.Struyf, M.Lehtinen, G.Dubin and HPSGroup (2009)."Efficacy of human papillomavirus(HPV)-16/18AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types(PATRICIA): final analysis of a double-blind, randomized study in young women." Lancet 374(9686):301-314.

Perez, G., E. Lazcano-Ponce, M. Hernandez-Avila, PJ Garcia, N. Munoz, LL Villa, J. Bryan, FJ Taddeo, S. Lu, MTEsser, S. Vuocolo, C. Sattler and E. Barr (2008 )."Safety,immunogenicity,and efficacy of quadrivalent humanpapillomavirus(types 6,11,16,18)L1virus-like-particle vaccine in Latin American women." Int J Cancer 122(6):1311-1318.

Petrik, MS, MC Wong, RCTabata, RFGarry and CAShaw(2007). "Aluminum adjuvant linked to Gulf War illness induces motor neuron death inmice." Neuromolecular Med 9(1):83-100. Reisinger, KS, SLBlock, E. Lazcano -Ponce, R. Samakoses, MTEsser, J. Erick, D. Puchalski, KEGiacoletti, HLSings, S. Lukac, FBA Alvarez and E. Barr (2007). "Safety and persistent immunogenicity of aquadrivalent human papillomavirus types 6,11,16, 18L1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial." Pediatr Infect Dis J 26(3):201-209.

Rimaniol, AC, G. Gras and P. Clayette(2007). "In vitro interactions between macrophages and aluminum-containing adjuvants." Vaccine 25(37-38):6784-6792.

Rimaniol, AC, G. Gras, F. Verdier, F. Capel, VB Grigoriev, F. Porcheray, E. Sauzeat, JG Fournier, P. Clayette, CA Siegrist and D. Dormont (2004). "Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen -presenting cell type." Vaccine 22(23-24):3127-3135.

Rose, RC, WI White, M. Li, JASuzich, C. Lane and RL Garcea (1998). "Human papillomavirus type 11 recombinant L1 capsomeres induce virus-neutralizing antibodies." J Virol 72(7):6151-6154.

Sahay, G., DYAlakhova and AV Kabanov(2010). "Endocytosis of nanomedicines." Journal of Controlled Release 145(3):182-195.

Seemann, S. and B. Dodet(2011). "IABS international scientific workshop--Mode of Action of Adjuvants: Implications for vaccine safety and design, April29-30, 2010, Bethesda, MD, USA." Vaccine 29(17) :3340.

Senger, T., L. Schadlich, S. Textor, C. Klein, KM Michael, CBBuck and L. Gissmann (2010). "Virus-like particles and capsomeres are potent vaccines against cutaneous alpha HPVs." Vaccine 28(6):1583- 1593.

Sharp, FA, D. Ruane, B. Claass, E. Creagh, J. Harris, P. Malyala, M. Singh, DTO' Hagan, V. Petrilli, J. Tschopp, LAO'Neill and EC Lavelle (2009)." Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3inflammasome." Proc Natl Acad Sci USA 106(3):870-875.

Shaw, AR(2013). "Human papillomavirus vaccines six years after approval." Annu Rev Med 64:91-100.

Shaw, CA and MS Petrik (2009). "Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration." J Inorg Biochem 103(11):1555-1562.

Szarewski, A.(2012). "Cervarix(R): a bivalent vaccine against HPV types16and 18, with cross-protection against other high-risk HPV types." Expert Rev Vaccines 11(6):645-657.

Szarewski, A., WAPoppe, SRSkinner, CM Wheeler, J. Paavonen, P. Naud, J. Salmeron, SNChow, D. Apter, H. Kitchener, X. Castellsague, JCTeixeira, J. Hedrick, U. Jaisamrarn, G. Limson , S. Garland, B. Romanowski, FYAoki, TFSchwarz, FXBosch, DM Harper, K. Hardt, T. Zahaf, D. Descamps, F. Struyf, M. Lehtinen, G. Dubin and HPS Group (2012). "Efficacy of the human papillomavirus(HPV)-16/18AS04-adjuvanted vaccine in women aged 15-25years with and without serological evidence of previous exposure to HPV-16/18." Int J Cancer 131(1):106-116.

Tabata, Y. and Y. Ikada(1988). "Effect of the size and surface charge of polymer microspheres on their phagocytosis by macrophage." Biomaterials 9(4):356-362.

Tomljenovic, L. and CAShaw(2011). "Do aluminum vaccine adjuvants contribute to the rising prevalence of autism?" J Inorg Biochem 105(11):1489-1499.

Tomljenovic, L., JPSpinosa and CAShaw (2013). "Human papillomavirus (HPV) vaccines as an option for preventing cervical malignancies: (how) effective and safe?" Curr Pharm Des 19(8):1466-1487.

Torres, MP, JH Wilson-Welder, SK Lopac, Y. Phanse, B. Carrillo-Conde, AERamer-Tait, BH Bellaire, MJ Wannemuehler and B. Narasimhan (2011). "Polyanhydride microparticles enhance dendritic cell antigen presentation and activation." Acta Biomater 7( 7): 2857-2864.

Uto, T., T. Akagi, K. Yoshinaga, M. Toyama, M. Akashi and M. Baba (2011). "The induction of innate and adaptive immunity by biodegradable poly(gamma-glutamic acid) nanoparticles via a TLR4 and MyD88 signaling pathway .” Biomaterials 32(22):5206-5212.

Verdier, F., R. Burnett, C. Michelet-Habchi, P. Moretto, F. Fievet-Groyne and E. Sauzeat (2005). "Aluminium assay and evaluation of the local reaction at several time points after intramuscular administration of aluminum containing vaccines in the Cynomolgus monkey." Vaccine 23(11):1359-1367.

Verstraeten, T., D. Descamps, MP David, T. Zahaf, K. Hardt, P. Izurieta, G. Dubin and T. Breuer (2008). "Analysis of adverse events of potential autoimmune aetiology in a large integrated safety database of AS04 adjuvanted vaccines .” Vaccine 26(51):6630-6638.

Villa, LL, KAAult, ARGiuliano, RLCosta, CA Petta, RP Andrade, DR Brown, A. Ferenczy, DM Harper, LA Koutsky, RJ Kurman, M. Lehtinen, C. Malm, SEOlsson, BMRonnett, FESkjeldestad, M. Steinwall, MHStoler, CM Wheeler, FJ Taddeo, J. Yu, L. Lupinacci, R. Railkar, R. Marchese, MTEsser, J. Bryan, KU Jansen, HLSings, GMTamms, AJ Saah and E. Barr (2006). "Immunologic responses following administration of a vaccine targeting humanpapillomavirus Types 6,11 , 16, and 18." Vaccine 24(27-28):5571-5583.

Villa, LL, RLCosta, CA Petta, RPAndrade, KAAult, ARGiuliano, CM Wheeler, LA Koutsky, C. Malm, M. Lehtinen, FESkjeldestad, SEOlsson, M. Steinwall, DR Brown, RJ Kurman, BMRonnett, MHStoler, A. Ferenczy, DM Harper, GMTamms, J. Yu, L. Lupinacci, R. Railkar, FJ Taddeo, KU Jansen, MTEsser, HLSings, AJ Saah and E. Barr (2005). "Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1virus-like particle vaccine inyoung women: a randomised double-blind placebo-controlled multicentre phase II efficacy trial." Lancet Oncol 6(5):271-278.

Wang, JW and RB Roden(2013). "Virus-like particles for the prevention of human papillomavirus-associated malignancies." Expert Rev Vaccines 12(2):129-141.

Wang, W. and M. Singh(2011). "Selection of adjuvants for enhanced vaccine potency." World Journal of Vaccines 1(2):33-78.

Wendorf, J., J. Chesko, J. Kazzaz, M. Ugozzoli, M. Vajdy, D. O'Hagan and M. Singh (2008). "A comparison of anionic nanoparticles and microparticles as vaccine delivery systems." Hum Vaccin 4 (1):44-49.

Wu, WH, E. Gersch, K. Kwak, S. Jagu, B. Karanam, WK Huh, RL Garcea and R.B. Roden (2011). "Capsomer vaccines protect mice from vaginal challenge with human papillomavirus." PLoS One 6(11) :e27141.

Xiang, SD, A. Scholzen, G. Minigo, C. David, V. Apostolopoulos, PLMottramand M. Plebanski (2006). "Pathogen recognition and development of particulate vaccines: does size matter?" Methods 40(1):1-9 .

Yuan, H., PA Estes, Y. Chen, J. Newsome, VA Olcese, RL Garcea and R. Schlegel (2001). "Immunization with a pentameric L1fusion protein protects against papillomavirus infection." J Virol 75(17):7848-7853.

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.