Antigenic nanoparticles, methods of making and vaccine preparation applications thereof

Abstract

The application provides an antigen nanoparticle and a preparation method and vaccine preparation application thereof, and relates to the technical field of biotechnology.The antigen nanoparticle provided by the application comprises an antigen and poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol, the antigen nanoparticle has good stability and high immunocompetence, and can be used for the preparation of a vaccine.The preparation method of the antigen nanoparticle provided by the application is convenient to operate, parameters are controllable, reproducibility is good, and the reaction is carried out in a water environment, the condition is mild, and the influence on the structure of the antigen is small.The vaccine provided by the application comprises the antigen nanoparticle and an adjuvant, and the inventor has found that the antigen nanoparticle and the adjuvant have a synergistic effect, and the combination of the two can further improve the immunization effect of the vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an antigen nanoparticle, a preparation method thereof and a vaccine preparation application. BACKGROUND

[0002] AIDS is a major infectious disease caused by infection with human immunodeficiency virus, which attacks the human immune system, mainly killing CD4 + helper T lymphocytes that play an important role in the human immune system, leading to immunodeficiency, thereby weakening the body's defense against many infections and some cancers, and ultimately leading to death. Since the first case of HIV infection was discovered in 1985, the number of new infections has increased year by year, and the mortality rate is high, highlighting the great pressure and difficulties faced by AIDS prevention and treatment today.

[0003] Currently, there is no effective vaccine for AIDS, although highly active antiretroviral therapy can effectively inhibit HIV-1 replication and has been successfully applied to clinical treatment, but the latent property of HIV-1 infection leads to the inability of antiviral drugs to completely eliminate viruses in HIV-1 infected individuals, and the side effects are large after long-term medication and drug resistance is easy to produce. The development of an AIDS vaccine faces many challenges, including the inability to eliminate latent infections caused by the virus integrating its genome into the host cell's chromosome through reverse transcription, killing important CD4 + helper T lymphocytes of the immune system, high genetic variability, inability to induce broad-spectrum neutralizing antibody immune response, etc. Although scientists have tried various methods of vaccine research, no successful AIDS vaccine has been developed. Recently, several similar natural state trimeric bodies such as SOSIP.664, NFL trimer, UFO trimer, etc. have been obtained internationally, which can produce self 2-level strain neutralization reactions in guinea pigs, rabbits and monkeys, respectively. This shows that the natural Env structure plays an important role in inducing broad-spectrum neutralization reactions.

[0004] However, the current HIV envelope protein trimer vaccine design has the following disadvantages:

[0005] 1. Long time and multiple immunizations are required to induce a certain level of neutralizing antibody titers;

[0006] 2. Dependence on immune adjuvants to enhance immune response;

[0007] 3. Unable to induce high-titer broad-spectrum neutralizing antibodies.

[0008] Vaccine adjuvant materials play a crucial role in developing effective vaccines, as they can significantly enhance the immunogenicity of viral antigens as immune enhancers, thereby improving the immunoprotective function of vaccines. Since the 1920s, only six adjuvants (aluminum adjuvant, MF59, AS01, AS03, AS04, and CpG 1018) have been approved for marketing, so exploring innovative potential adjuvant molecules to increase the immunoreactivity of viral antigens and prolong the duration of the immune response, i.e., immunological memory, will help the development of broad-spectrum COVID-19 vaccines and AIDS vaccines.

[0009] Therefore, the present application is proposed. SUMMARY

[0010] The first object of the present application is to provide an antigen nanoparticle with high immunological activity to solve at least one of the above problems.

[0011] The second object of the present application is to provide a method for preparing the above antigen nanoparticle.

[0012] The third object of the present application is to provide the use of the above antigen nanoparticle in the preparation of a vaccine.

[0013] The fourth object of the present application is to provide a vaccine to solve at least one of the above problems.

[0014] In a first aspect, the present application provides an antigen nanoparticle comprising an antigen and polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene), the antigen being combined with the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) by reaction.

[0015] As a further technical solution, the antigen comprises at least one of a viral protein, a tumor vaccine protein, or an Alzheimer's vaccine protein;

[0016] Preferably, the viral protein comprises an HIV vaccine protein, a hepatitis B virus protein, a COVID-19 virus protein, or an influenza virus protein;

[0017] Preferably, the HIV vaccine protein comprises an HIV envelope protein trimer;

[0018] Preferably, the HIV envelope protein trimer comprises at least one of SOSIP.664, NFL trimer, or UFO trimer.

[0019] As a further technical solution, the method for preparing the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) comprises: reacting mPEG-NH2 and PMHC 18 to prepare the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene).

[0020] As a further technical solution, the molecular weight of the mPEG-NH2 is 2000-5000 Da;

[0021] Preferably, the PMHC 18 has a molecular weight of 30000-50000 Da;

[0022] Preferably, the molar ratio of the mPEG-NH2 and PMHC 18 is 1:8-1:12, preferably 1:10;

[0023] Preferably, the reaction of the mPEG-NH2 and PMHC 18 is carried out in an organic solvent, which includes dichloromethane;

[0024] Preferably, the reaction conditions of the mPEG-NH2 and PMHC 18 include stirring at a speed of 200-300 rpm for 20-28 h;

[0025] Preferably, the catalyst of the reaction of the mPEG-NH2 and PMHC 18 includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine;

[0026] Preferably, the reaction of the mPEG-NH2 and PMHC 18 further includes a purification step after the reaction;

[0027] Preferably, the purification step includes dialysis with a dialysis bag with a molecular weight cut-off of 14 kDa.

[0028] In a second aspect, the present application provides a method for preparing an antigen nanoparticle, comprising the following steps: a. dissolving polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) in water to prepare a suspension;

[0029] b. adding the suspension of step a. dropwise to an antigen solution to prepare an antigen nanoparticle after mixing.

[0030] As a further technical solution, in step a., the suspension further includes a cosolvent;

[0031] Preferably, the cosolvent includes dimethyl sulfoxide.

[0032] As a further technical solution, the mass ratio of the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) and the antigen is 4:1-6:1, preferably 5:1;

[0033] Preferably, the concentration of the antigen solution is 0.8-1.2 mg / mL.

[0034] As a further technical solution, in the step b, the mixing comprises stirring at a rotation speed of 200-300 rpm for 1.6-2.4 h;

[0035] Preferably, in the step b, the mixing further comprises a purification step after the mixing.

[0036] Preferably, when the antigen is an HIV envelope protein trimer, the purification step comprises dialysis using a dialysis bag with a molecular weight cut-off of 100 kDa.

[0037] In a third aspect, the present application provides a use of the antigen nanoparticle as described above in the preparation of a vaccine.

[0038] In a fourth aspect, the present application provides a vaccine comprising the antigen nanoparticle as described above and an adjuvant.

[0039] Preferably, the adjuvant is an AS03 adjuvant.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] The antigen nanoparticle provided by the present application comprises an antigen and polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene), wherein the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) and the antigen are combined through hydrophobic interaction, which has little effect on the spatial structure of the antigen, can improve the stability of the antigen, and realizes the controllable release of the antigen. The antigen nanoparticle of the present application has good stability and high immunological activity, and can be used for the preparation of a vaccine.

[0042] The preparation method of the antigen nanoparticle provided by the present application is simple to operate, controllable in parameters, good in reproducibility, and the reaction is carried out in a water environment, which is mild in conditions and has little effect on the structure of the antigen.

[0043] The vaccine provided by the present application comprises the antigen nanoparticle as described above and an adjuvant, and the inventors have found that the antigen nanoparticle and the adjuvant have a synergistic effect, and the combination of the two can further improve the immunological effect of the vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0045] Figure 1 NMR spectrum of the polymer PEG-PHC 18 ;

[0046] Figure 2 Strategy for immunizing New Zealand rabbits with HIV vaccine proteins;

[0047] Figure 3 Evaluation of binding antibody titers in sera from immunized rabbits;

[0048] Figure 4 Evaluation of neutralizing antibody titers in sera from immunized rabbits that neutralize HIV pseudovirus MW965.26_C;

[0049] Figure 5 Evaluation of neutralizing antibody titers in sera from immunized rabbits that neutralize HIV Tier 1 pseudovirus;

[0050] Figure 6 Evaluation of neutralizing antibody titers in sera from immunized rabbits that neutralize HIV Tier 2 pseudovirus;

[0051] Figure 7 Evaluation of broad neutralizing antibody titers in sera from four immunized rabbits that neutralize HIV Tier 2 pseudovirus. DETAILED DESCRIPTION

[0052] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and embodiments, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained by commercial purchase.

[0053] In a first aspect, the present application provides an antigen nanoparticle, comprising an antigen and polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene), the antigen being combined with the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) by reaction.

[0054] The polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) is an amphiphilic polymer that can bind to the antigen by hydrophobic interaction, can achieve controlled release of the antigen, and can stimulate the immune system more persistently.

[0055] The antigen nanoparticle of the present application has good stability and high immunological activity, and can be used for the preparation of vaccines.

[0056] In some preferred embodiments, the antigen includes, but is not limited to, at least one of viral proteins (HIV vaccine protein, hepatitis B virus protein, SARS-CoV-2 protein, influenza virus protein, etc.), tumor vaccine protein, or Alzheimer's vaccine protein.

[0057] This invention does not have specific requirements for vaccine proteins. Based on the fact that antigen surfaces generally have hydrophilic and hydrophobic sites, polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene) can bind to most antigens through hydrophobic interactions, thereby producing nanoprotein vaccines.

[0058] Preferably, the HIV vaccine protein includes an HIV envelope protein trimer;

[0059] Preferably, the HIV envelope protein trimer includes at least one of SOSIP.664, NFL trimer, UFO trimer, or other trimers.

[0060] In some preferred embodiments, the method for preparing the polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene) includes: mixing mPEG-NH2 and PMHC 18 The reaction yielded polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene).

[0061] In this invention, the polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene) is mainly composed of the hydrophilic polymer mPEG-NH2 and the hydrophobic polymer PMHC. 18 The material was prepared by reaction and is amphiphilic.

[0062] In some preferred embodiments, the molecular weight of the mPEG-NH2 is 2000-5000 Da, preferably 5000 Da;

[0063] Preferably, the PMHC 18 The molecular weight is 30,000-50,000 Da;

[0064] Preferably, the mPEG-NH2 and PMHC 18 The molar ratio can be, for example, but not limited to, 1:8, 1:9, 1:10, 1:11 or 1:12, preferably 1:10.

[0065] Preferably, the mPEG-NH2 and PMHC 18 The reaction is carried out in an organic solvent, including dichloromethane, in which the reaction favors the reaction of mPEG-NH2 and PMHC. 18 The reaction.

[0066] Preferably, the mPEG-NH2 and PMHC18 The reaction conditions include stirring at a rotation speed of 200-300 rpm for 20-28 h using a stirring device such as, but not limited to, a magnetic stirrer.

[0067] Preferably, the mPEG-NH2 and PMHC 18 The catalyst for the reaction includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine.

[0068] Preferably, the mPEG-NH2 and PMHC 18 After the reaction, a purification step is further included to remove unreacted raw materials, catalysts and other impurities.

[0069] Preferably, the purification step includes dialysis using a dialysis bag with a molecular weight cut-off of 100 kDa.

[0070] By further optimizing and adjusting the preparation conditions of the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene), the yield of mPEG-NH2 and PMHC 18 The reaction yield is improved, and the preparation cost is reduced.

[0071] In a second aspect, the present application provides a preparation method of an antigen nanoparticle, comprising the following steps: a. dissolving the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) in water to prepare a suspension;

[0072] b. adding the suspension of step a. into an antigen solution, and mixing to prepare an antigen nanoparticle.

[0073] The preparation method of the antigen nanoparticle provided by the present application is simple to operate, controllable in parameters, good in reproducibility, and the reaction is carried out in a water environment, which is mild in conditions and has little influence on the structure of the antigen.

[0074] In some preferred embodiments, in step a., the suspension further includes a cosolvent, which is mixed with the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) and water to promote the dissolution of the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) and improve the stability of the suspension.

[0075] Preferably, the cosolvent is dimethyl sulfoxide.

[0076] In some preferred embodiments, the mass ratio of the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) to the antigen can be, but is not limited to, 4:1, 5:1 or 6:1, and is preferably 5:1.

[0077] Preferably, the concentration of the antigen solution can be, but is not limited to, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL or 1.2 mg / mL.

[0078] Through further optimization and adjustment of the antigen concentration and the ratio of the antigen to the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene), the prepared vaccine has better immunocompetence and higher stability.

[0079] In some preferred embodiments, in the step b, the mixing manner is not particularly limited, for example, a magnetic stirrer can be used for mixing, and the stirring speed is 200-300 rpm, and the stirring time is 1.6-2.4 h.

[0080] In some preferred embodiments, in the step b, the mixing is followed by a purification step, and the purification step is used to remove unreacted antigens, polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) and other impurities.

[0081] Preferably, when the antigen is an HIV envelope protein trimer, the purification step comprises dialysis using a dialysis bag with a molecular weight cut-off of 100 kDa.

[0082] In a third aspect, the present application provides an application of the above-mentioned antigen nanoparticles in the preparation of a vaccine.

[0083] The antigen nanoparticles of the present application have good stability and high immunocompetence, and can be used for the preparation of a vaccine.

[0084] In a fourth aspect, the present application provides a vaccine comprising the above-mentioned antigen nanoparticles and an adjuvant.

[0085] The vaccine provided by the present application comprises the above-mentioned antigen nanoparticles and an adjuvant, and the inventors have found that the antigen nanoparticles and the adjuvant have a synergistic effect, and the combination of the two can further improve the immunization effect of the vaccine.

[0086] Preferably, the adjuvant is an AS03 adjuvant.

[0087] The present application will be further described below through specific examples and comparative examples, but it should be understood that these examples are only used for more detailed description, and should not be understood as limiting the present application in any form.

[0088] In the following examples or test examples, HIV envelope protein trimer UFO trimer is taken as an example for research.

[0089] Example 1

[0090] An HIV envelope protein nanoparticle is prepared by the following method: An HIV envelope protein nanoparticle is prepared by the following method:

[0091] (1) Select mPEG-NH2 with a molecular weight of 5000 Da and PMHC with a molecular weight of 30000-50000 Da. 18 Among them, mPEG-NH2 and PMHC 18 The molar ratio is 1:10, PMHC 18 Dissolved in dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred for 30 min for catalysis. Then, mPEG-NH2 and triethylamine were added, and the mixture was stirred with a magnetic stirrer at 250 rpm for 24 h. The resulting reaction product was dried under nitrogen to remove the organic solvent. It was then reconstituted with water, and the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed against pure water, with the water changed every 2-3 h for one day. The purified reaction product was collected from the dialysis bag, frozen at -80°C overnight, and then freeze-dried for 24 h to obtain a white flocculent substance, which was the amphiphilic polymer. NMR confirmed PMHC. 18 The ratio of mPEG-NH2 and the results are as follows Figure 1 As shown, the polymer was dissolved in deuterated chloroform for NMR detection. The results were compared between 3.8–3.5 ppm (for -CH2 groups in PEG) and 1.1–1.3 ppm (for C... 18 The peak area of ​​the -CH2 group in the chain was determined and anchored to PMHC. 18 The average number of PEG groups on the chain resulted in a PEG incorporation rate of 7.05%. In the figure, "p" represents the -CH2 group in the PEG, and "q" represents the -CH2 group in the C18 chain.

[0092] (2) Dissolve the polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene) prepared in step (1) in water to obtain a suspension.

[0093] (3) Add the suspension droplets prepared in step (2) to an HIV envelope protein trimer solution with a concentration of 1 mg / mL (the mass ratio of poly(maleic anhydride-ALT-1-octadecene) in the suspension to HIV envelope protein trimer solution is 5:1), and stir on a magnetic stirrer for 2 hours at a speed of 250 rpm.

[0094] (4) After the reaction is completed, the reaction mixture obtained in step (3) is put into a dialysis bag with a molecular weight cutoff of 100kDa and dialyzed in PBS. Fresh pure water is replaced every 2-3 hours. After dialysis for 12 hours, HIV nanoparticles are obtained.

[0095] Example 2

[0096] An HIV envelope protein nanoparticle, the preparation method of which is as follows:

[0097] (1) Select mPEG-NH2 with a molecular weight of 3000 Da and PMHC with a molecular weight of 30000-50000 Da. 18 Among them, mPEG-NH2 and PMHC 18 With a molar ratio of 1:8, PMHC 18 The product was dissolved in dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred for 30 min for catalysis. Then, mPEG-NH2 and triethylamine were added, and the mixture was stirred with a magnetic stirrer at 200 rpm for 20 h. The resulting product was dried under nitrogen to remove the organic solvent. It was then reconstituted with water, and the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed against pure water, with the water changed every 2-3 h for one day. The purified product was collected from the dialysis bag, frozen at -80°C overnight, and then freeze-dried for 20 h to obtain a white flocculent substance, which was the amphiphilic polymer.

[0098] (2) The polyethylene glycol-grafted poly(maleic anhydride-ALT-1-octadecene) prepared in step (1) was added to dimethyl sulfoxide and dissolved in water to obtain a suspension.

[0099] (3) Add the suspension droplets prepared in step (2) to a HIV envelope protein trimer solution with a concentration of 0.8 mg / mL (wherein the mass ratio of poly(maleic anhydride-ALT-1-octadecene) in the suspension to HIV envelope protein trimer solution is 4:1), and stir on a magnetic stirrer for 1.6 h at a stirring speed of 200 rpm.

[0100] (4) After the reaction is completed, the reaction mixture obtained in step (3) is put into a dialysis bag with a molecular weight cutoff of 100kDa and dialyzed in PBS. Fresh pure water is replaced every 2-3 hours. After dialysis for 12 hours, HIV nanoparticles are obtained.

[0101] Example 3

[0102] An HIV envelope protein nanoparticle, the preparation method of which is as follows:

[0103] (1) Select mPEG-NH2 with a molecular weight of 4000 Da and PMHC with a molecular weight of 30000-50000 Da. 18 Among them, mPEG-NH2 and PMHC 18 With a molar ratio of 1:12, PMHC 18The reaction mixture was stirred for 30 min with 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride as catalyst, then mPEG-NH2 and triethylamine were added, and the reaction mixture was stirred with a magnetic stirrer at 300 rpm for 28 h. The reaction product was dried by blowing nitrogen gas, and the organic solvent was removed. Then the reaction product was dissolved in water, and the solution was put into a dialysis bag with a molecular weight cut-off of 14 kDa and dialyzed in pure water. The dialysis water was changed every 2-3 h, and the dialysis was performed for one day. The purified reaction product was collected in the dialysis bag, frozen overnight at -80°C, and then freeze-dried in a freeze dryer for 28 h to obtain a white flocculent substance, i.e. the amphiphilic polymer.

[0104] (2) The polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) prepared in step (1) was dissolved in water to obtain a suspension.

[0105] (3) The suspension prepared in step (2) was added dropwise to a solution of HIV envelope protein trimer with a concentration of 1.2 mg / mL (wherein the mass ratio of poly(maleic anhydride-ALT-1-octadecene) in the suspension to HIV envelope protein trimer in the solution of HIV envelope protein trimer was 6:1), and stirred on a magnetic stirrer at 300 rpm for 2.4 h.

[0106] (4) After the reaction was completed, the reaction mixture obtained in step (3) was put into a dialysis bag with a molecular weight cut-off of 100 kDa and dialyzed in PBS. The dialysis water was changed every 2-3 h, and the dialysis was performed for 12 h to obtain HIV nanoparticles.

[0107] Example 4

[0108] An HIV nano-vaccine comprising the HIV envelope protein nanoparticles of Example 1 and AS03 adjuvant.

[0109] Example 5

[0110] An HIV nano-vaccine comprising the HIV envelope protein nanoparticles of Example 2 and AS01 adjuvant.

[0111] Example 6

[0112] An HIV nano-vaccine comprising the HIV envelope protein nanoparticles of Example 3 and AS04 adjuvant.

[0113] Test Example 1

[0114] Positive control: HIV envelope protein trimer + AS03 adjuvant.

[0115] Immunogen: HIV envelope protein trimer.

[0116] Polymer: PMHC 18 (molecular weight 30-50 kDa), mPEG-NH2 (molecular weight 5 kDa).

[0117] Animals: Female New Zealand rabbits (over 12 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were performed in accordance with the guidelines for protecting animal life and were approved by the Animal Ethics Committee of Jinan University. They were housed under 12 hours light / 12 hours dark conditions with free access to food and water purified, and were acclimated for 7-10 days. Experimental methods:

[0118] HIV nano vaccine immune response research.

[0119] Immunization strategy: Blood was taken once a week before immunization as a blank control. One week later, immunization began, with immunization once every four weeks, for a total of four immunizations. Blood was taken from the marginal ear vein twice a week after each immunization, and the specific experimental times are shown in Figure 2

[0120] Grouping: New Zealand rabbits were randomly divided into three groups, with 3 rabbits in each group.

[0121] Experimental design: 1) Grouping: ① Group 1: HIV envelope protein trimer + AS03 adjuvant; ② Group 2: HIV envelope protein trimer nanoparticle (Example 1); ③ Group 3: HIV envelope protein trimer nanoparticle (Example 1) + AS03 adjuvant. There were 3 rabbits in each group, and the experiment was performed by subcutaneous injection immunization, in which Group 1 and Group 3 were injected with 100 μL of antigen plus 100 μL of adjuvant for each rabbit, for a total of 200 μL of pharmaceutical agent, containing 55 μg of antigen. Group 2 was injected with 100 μL of pharmaceutical agent for each rabbit, containing 55 μg of antigen. Blood was taken from the marginal ear vein two weeks after each immunization, and serum samples were obtained by separation.

[0122] 2) Evaluation of binding antibody titers: The obtained serum samples were analyzed by ELISA to explore the HIV-specific binding antibody titers in the rabbit serum after immunization.

[0123] 3) Evaluation of neutralizing antibody response: The obtained serum samples were subjected to pseudovirus neutralization experiments to determine the neutralizing antibody titers in the rabbit serum after immunization that could neutralize HIV pseudovirus. Experimental results:

[0124] The results are shown in Figure 3 (ELISA detection of HIV-specific binding antibodies in rabbit serum after immunization. The initial dilution of each serum sample was 1:100, followed by serial dilution at 1:10. The data are expressed as mean ± standard deviation. Group 1: HIV envelope protein trimer + AS03 adjuvant; Group 2: HIV envelope protein trimer nanoparticle; Group 3: HIV envelope protein trimer nanoparticle + AS03 adjuvant.) from Figure 3 ​It can be seen that the binding antibody titers produced after the fourth immunization in Group 1, Group 2 and Group 3 were 1:2,722, 1:2,441 and 1:19,409, respectively, indicating that the HIV envelope protein trimer prepared into a nano vaccine combined with an adjuvant can significantly improve the antibody titer. At the same time, the binding antibody titer of Group 3 after the third immunization reached 1:10,245, which was much higher than the binding antibody titers of the rabbits in Group 1 and Group 2 after four immunizations. This further indicates that the nano vaccine combined with an adjuvant method can shorten the time to reach a high-titer immune response, so that the same effect can be achieved with fewer immunization times.

[0125] The results of the evaluation of the neutralizing antibody titers of the rabbit serum after immunization against HIV pseudovirus MW965.26_C are shown in Table 2. Figure 4 (The neutralizing antibody titers were detected using HIV pseudovirus MW965.26_C on TZM-bl cells. The initial dilution of each serum was 1:20, followed by serial dilution at a ratio of 1:5. The black horizontal line is the average within the group. Group 1: HIV envelope protein trimer + AS03 adjuvant; Group 2: HIV envelope protein trimer nanoparticle; Group 3: HIV envelope protein trimer nanoparticle + AS03 adjuvant.) Figure 4 It can be seen that after the fourth immunization, the antibody titer of Group 3 against MW965.26_C (1:428.9) was higher than that of Group 1 (1:168.6) and Group 2 (1:185.8), indicating that the HIV envelope protein trimer vaccine prepared into a nano vaccine combined with an adjuvant can improve the titer of the neutralizing antibodies produced by immunization. At the same time, the combination of nano materials and adjuvants significantly shortens the time to induce neutralizing antibodies: after the second immunization, Group 2 and Group 3 have already produced low-titer neutralizing antibodies, with titers of 1:25.9 and 1:29.2, respectively, while the control Group 1 did not detect neutralizing antibodies against MW965.26_C. After the third immunization, the antibody titer of Group 3 against MW965.26_C (1:274.9) was much higher than that of Group 1 (1:39.4) and Group 2 (1:78). After the fourth immunization, the antibody titer of Group 3 against MW965.26_C remained higher than that of Group 1 and Group 2.

[0126] The results of the evaluation of the broad spectrum of neutralizing antibodies against HIV pseudovirus produced by the rabbit serum after immunization are shown in Table 3. Figures 5-6and Table 1 (neutralizing antibody titers were determined on TZM-bl cells with HIV pseudoviruses, including Tier 1 pseudoviruses (MW965.26_C, SF162.LS_B, 92RW020.2_A) that are easily neutralized, Tier 2 pseudoviruses (398F1_A, X2278_B, TRO11_B, CE0217_C, CE1176_C, 25710_C, CNE8_CRF01, CNE55_CRF01, CH119_CRF07) that are difficult to neutralize, and murine leukemia pseudovirus (SVA-MLV) as a control. Each serum was initially diluted 1 :20, followed by serial dilutions at 1 :5. Group 1 : HIV Env protein trimers + AS03 adjuvant; Group 2: HIV Env protein trimers nanoparticle; Group 3: HIV Env protein trimers nanoparticle + AS03 adjuvant.

[0127] Table 1 Summary of neutralization of HIV Tier 2 pseudoviruses by rabbit sera post-immunization

[0128]

[0129]

[0130] From the results of the breadth evaluation, it can be seen that after the second immunization, the sera from Group 2 and Group 3 containing the nanoparticles can specifically neutralize the HIV Tier 1 pseudovirus MW965.26_C, while Group 1 has not shown neutralization activity against the tested viruses after the second immunization; after the third immunization, the sera from Group 2 and Group 3 containing the nanoparticles can specifically neutralize 1 Tier 1 pseudovirus (MW965.26_C) and 3 Tier 1 pseudoviruses (MW965.26_C, SF162.LS_B, 92RW020.2_A), respectively, while the sera from Group 1 can specifically neutralize 2 Tier 1 pseudoviruses (MW965.26_C, 92RW020.2_A) and 1 Tier 2 pseudovirus (CH119_CRF07); after the fourth immunization, the sera from Group 2 can specifically neutralize 2 Tier 1 pseudoviruses (MW965.26_C, SF162.LS_B) and 4 Tier 2 pseudoviruses (X2278_B, TRO11_B, CE0217_C, CNE55_CRF01), the sera from Group 3 can specifically neutralize 2 Tier 1 pseudoviruses (MW965.26_C, SF162.LS_B) and 2 Tier 2 pseudoviruses (X2278_B, TRO11_B), while the sera from Group 1 can only specifically neutralize 1 Tier 1 pseudovirus (MW965.26_C) and 1 Tier 2 pseudovirus (X2278_B). The sera from Group 1 can non-specifically neutralize SVA-MLV (1:48), and thus the lower neutralization titers against the five other Tier 2 pseudoviruses (398F1_A, TRO11_B, CE0217_C, CNE1176_C, CH119_CRF07) are also non-specific neutralization reactions. These results show that the nanoparticle method can relatively increase the number of neutralization pseudoviruses induced by the HIV envelope protein trimer to produce neutralizing antibodies, which suggests that the use of the nanoparticle can induce the production of broadly neutralizing antibodies. In addition, according to the data, we further evaluated the degree of the production of the broadly neutralizing antibodies of the sera from the rabbits after the fourth immunization Figure 6 to neutralize HIV Tier 2 pseudoviruses. Figure 7 When the sera have non-specific neutralization activity against SVA-MLV, the titers to neutralize HIV pseudoviruses are 2.5 times and above the titers to neutralize SVA-MLV to determine that the sera have specific neutralization activity against HIV pseudoviruses. When comparing the neutralization titers between groups, when the neutralization titer is <1:20, the neutralization titer of 1:10 is taken for calculation, and the black line in the group is the average value. Group 1: HIV envelope protein trimer + AS03 adjuvant; Group 2: HIV envelope protein trimer nanoparticles; Group 3: HIV envelope protein trimer nanoparticles + AS03 adjuvant. As Figure 7As shown, the average of neutralizing antibody titers against X2278_B in sera of rabbits in group 2 and group 3 after the fourth immunization were 1:56.5 and 1:83.5, respectively, which were higher than that of group 1 (1:31). Meanwhile, sera of rabbits in group 2 could neutralize other three HIV Tier 2 pseudoviruses (TRO11_B, CE0217_C, CNE55_CRF01), and sera of rabbits in group 3 could also neutralize another HIV Tier 2 pseudovirus (TRO11_B), while sera of rabbits in group 1 failed to neutralize these HIV Tier 2 pseudoviruses, which further demonstrated that the nano-vaccine method could induce relatively broad-spectrum neutralizing antibodies, and was expected to achieve better immune response against HIV.

[0131] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An antigenic nanoparticle, characterized in that, poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol, wherein the antigen is reacted with the poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol; the antigen is an HIV envelope protein trimer; The preparation method of the polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) comprises: mixing mPEG-NH2 and PMHC 18 to prepare polyethylene glycol grafted poly(maleic anhydride-ALT-1-octadecene) by reaction; the molecular weight of the mPEG-NH2 is 2000-5000 Da; the molecular weight of the PMHC 18 is 30000-50000 Da; the molar ratio of the mPEG-NH2 and the PMHC 18 is 1:8-1:12; the molar ratio of the mPEG-NH2 and the PMHC 18 After the reaction is completed, a purification step is further included; the purification step comprises dialysis using a dialysis bag with a molecular weight cut-off of 14 kDa; the method for preparing the antigen nanoparticle comprises the following steps: a. dissolving the poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol in water to prepare a suspension; b. adding the suspension of step a. into an antigen solution, and mixing to prepare the antigen nanoparticle; the mass ratio of the poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol to the antigen is 4:1-6:1; the concentration of the antigen solution is 0.8-1.2 mg / mL; the HIV envelope protein trimer is one of SOSIP.664, NFL trimer or UFO trimer.

2. The antigenic nanoparticle of claim 1, wherein, The molar ratio of the mPEG-NH2 and PMHC 18 was 1:

10.

3. The antigenic nanoparticle of claim 1, wherein, The mPEG-NH2 and PMHC 18 The reaction is carried out in an organic solvent, including dichloromethane.

4. The antigenic nanoparticle of claim 3, wherein, The mPEG-NH2 and PMHC 18 The conditions of the reaction include stirring at a rotation speed of 200-300 rpm for 20-28 h.

5. The antigenic nanoparticle of claim 1, wherein, The mPEG-NH2 and PMHC 18 The catalysts for the reaction include 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and triethylamine.

6. The antigenic nanoparticle of claim 1, wherein, in step a., the suspension further comprises a cosolvent; the cosolvent comprises dimethyl sulfoxide.

7. The antigenic nanoparticle of claim 1, wherein, the mass ratio of the poly( maleic anhydride-ALT-1-octadecene) grafted with polyethylene glycol to the antigen is 5:

1.

8. The antigenic nanoparticle of claim 1, wherein, in step b., the mixing comprises stirring at a rotation speed of 200-300 rpm for 1.6-2.4 h.

9. The antigenic nanoparticle of claim 1, wherein, in step b., the mixing further comprises a purification step after the mixing. the purification step comprises dialysis using a dialysis bag with a molecular weight cut-off of 100 kDa.

10. Use of the antigen nanoparticle of any one of claims 1-9 in the preparation of a vaccine.

11. A vaccine comprising a polynucleotide of claim 1. the vaccine comprises the antigen nanoparticle of any one of claims 1-9 and an adjuvant.

12. The vaccine of claim 11, characterized in that, the adjuvant is an AS03 adjuvant.

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