Preparation method and application of HPV (human papillomavirus) recombinant protein therapeutic vaccine

Abstract

The invention relates to a preparation method and application of an HPV (human papillomavirus) recombinant protein therapeutic vaccine, and belongs to the technical field of biological medicine, and the preparation method of the HPV recombinant protein therapeutic vaccine comprises the following steps: simultaneously wrapping and delivering an HPV16HPV E6E7 recombinant protein antigen and a nucleic acid TLR (toll-like receptor) agonist adjuvant molecule by using composite lipid nanoparticles prepared from various lipids. The recombinant protein therapeutic vaccine prepared by the invention is beneficial to inducing strong T cell immune response aiming at the HPV16E6E7 antigen and killing cervical cancer cells infected by HPV. Besides, different from lipid nanoparticles (LNP) for delivering mRNA vaccines, cations or ionizable lipids do not exist in the formula, the safety is obviously higher than that of the mRNA vaccines, and the lipid nanoparticles have greater advantages than mRNA therapeutic vaccines, and are low in toxic and side effects and stable in vaccine effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a preparation method and application of an HPV recombinant protein therapeutic vaccine. Background Art

[0002] Cervical cancer is a common gynecological malignancy, ranking second in incidence among gynecological malignancies in China, second only to breast cancer. It is now clear that persistent infection with high-risk HPV is a necessary factor for the development of cervical cancer and precancerous lesions, and plays a crucial role in the progression of cervical cancer. HPV is a double-stranded circular DNA virus. There are over 200 HPV subtypes, and infection with 17 HPV types is causally linked to the development of cervical cancer. HPV types 16 and 18 are associated with over 70% of cervical cancers, followed by subtypes 45, 33, 58, 31, and 52. The remaining 10 HPV genotypes—HPV types 35, 59, 39, 56, 51, 68, 73, 26, 69, and 82—can also cause cervical cancer. The infection rate of high-risk HPV in the female population is as high as 10-20%. Persistent HPV infection is a necessary condition for causing cervical cancer. Therefore, early detection and early treatment are particularly necessary for the prevention and control of cervical cancer.

[0003] Currently approved cervical cancer vaccines are all preventive and ineffective for treating precancerous lesions and cervical cancer following infection. For example, GSK's bivalent HPV16 / 18 VLP vaccine can prevent over 70% of cervical precancerous lesions and cancer caused by HPV16 / 18; Merck's quadrivalent VLP vaccine can prevent cervical cancer caused by HPV16 / 18 and genital warts caused by HPV6 / 11; and Merck's 9-valent VLP vaccine can prevent over 90% of cervical cancer caused by HPV infection. All three vaccines are based on virus-like particles (VLPs) formed by the HPV L1 protein. The progression from HPV infection to cervical cancer is a gradual process that can take years to decades. This progression is generally believed to proceed through several stages: mild, moderate, and severe intraepithelial neoplasia, carcinoma in situ, and invasive cancer. Currently, there are no specific treatments for cervical precancerous lesions. Treatments include recombinant human interferon and anti-HPV biopharmaceutical proteins to alleviate symptoms. Surgical treatments include loop electrosurgical excision surgery (LES), cold knife conization, and hysterectomy, depending on the patient's progression. However, these treatments are unsatisfactory for advanced or recurrent cervical cancer that has metastasized, with a 5-year survival rate of only 16.8%. Therefore, developing new treatment modalities is crucial to halt the progression of cervical cancer and improve the prognosis for patients with advanced and recurrent cervical cancer.

[0004] HPV therapeutic vaccines achieve the goal of treating HPV precancerous lesions and cervical cancer by activating the body's own immune system. It is currently considered the safest, most effective, and most economical treatment. HPV therapeutic vaccines differ from the currently widely used preventive vaccines in the following aspects: (1) Therapeutic vaccines are mainly used for patients with precancerous lesions and cervical cancer after infection, while preventive vaccines can only be used for normal people who are not infected. Once infected with the virus, the preventive vaccine is ineffective; (2) The two have different mechanisms of action. Preventive vaccines mainly work through humoral immunity, while therapeutic vaccines mainly induce cellular immune responses to eliminate virus-infected cells or tumor cells; (3) The vaccine targets are different. Preventive vaccines are all based on virus-like particles formed by HPV L1 protein, while therapeutic vaccines mainly target E6E7 proteins that are continuously expressed during latent infection. The HPV genome is composed of three regions: the early (E) region that encodes viral proteins that regulate the viral life cycle and cellular functions of infected epithelial cells, the late (L) region that encodes structural proteins of the viral icosahedral capsid that mediates cell entry, and the LCR (long control region), which contains cis-acting sequences that mediate viral replication and transcription. Preventive vaccines such as and Stimulating the formation of neutralizing antibodies against the capsid protein L1 prevents viral entry and spread of infection. Early oncogenic proteins E6 and E7 are the most targeted antigens for HPV therapeutic vaccines currently in development because: 1) E6 and E7 are abundantly and exclusively expressed in precancerous and cancerous lesions, so the risk of targeting healthy tissues is not significant; 2) E6 and E7 are required for transformation and maintenance of infected cells, so the risk of immune escape mediated by antigen loss is not significant; 3) central tolerance mechanisms to E6 and E7 have not been documented; and 4) immune responses to E6 and E7 have been characterized preclinically and clinically.

[0005] Currently, no HPV therapeutic vaccine is available on the market, and most therapeutic vaccines are still in Phase II clinical trials. Product formats include recombinant protein vaccines, peptide vaccines, chimeric vaccines, and nucleic acid vaccines. Similar products under development domestically and internationally include MGIPhmrma Biologics' plasmid DNA vaccines E-7101 and XYC-101, currently in Phase III clinical trials; Gentice's protein vaccines GTL-001, currently in Phase I clinical trials; Xenova's TA-CIN, currently in Phase III clinical trials; Inovio's DNA vaccine HPV-16 E7 inhibitor INO-3112, currently in Phase II clinical trials; and Advaxis' HPV-16 E7 protein inhibitor ADXS-HPV, currently in Phase III clinical trials. No similar products have been approved for marketing domestically or internationally. Inovio's DNA vaccine VGX-3100 is the most advanced, with a higher response rate in the treatment group than in the placebo group in Phase III clinical trials. Among them, 27.6% (37 / 134) of the participants in the treatment group achieved the primary endpoint (histopathological outcome and viral clearance), while the rate in the placebo group was 8.7% (6 / 69), achieving a statistically significant difference (p=0.001). In particular, in terms of viral clearance, the viral clearance rate in the treatment group was 37.3% (50 / 134), while that in the placebo group was 8.7% (6 / 69). It is currently believed that the use of mRNA technology to prepare HPV therapeutic vaccines has good prospects. The gene encoding the HPV antigen E6E7 is prepared into mRNA and given to the patient for expression in the body, stimulating the body to produce a cellular immune response with antigenic characteristics, thereby achieving the purpose of treating the disease. Compared with DNA vaccines, it is safer (no risk of gene integration) and simpler to deliver (no need to enter the cell nucleus). In addition, the production cycle is short and the cost is low. However, mRNA vaccines also have some difficult-to-overcome drawbacks, such as high instability. To be effective, mRNA vaccines must enter cells. However, due to the presence of numerous RNases in the blood and tissues, mRNA can be degraded during delivery before entering cells, rendering it ineffective in exerting its immune-preventive effects. mRNA vaccines also exhibit some toxicity, primarily stemming from synthetic raw materials and the materials used to encapsulate the mRNA, such as cationic lipids. These materials can enter cells along with the mRNA, potentially causing toxicity or impairing the cellular immune response, posing a potential risk.

[0006] Recombinant protein vaccines offer advantages such as high stability and safety, offering distinct advantages over nucleic acid vaccines. Two key challenges must be addressed when using recombinant proteins as therapeutic vaccines: first, how to induce an immune response primarily based on T cells, with cell-mediated immunity predominantly responsible for tumor cell destruction in vivo; and second, how to employ appropriate adjuvants to enhance the immunogenicity of the recombinant protein. Addressing these two issues effectively will provide recombinant protein therapeutic vaccines with significant advantages over mRNA vaccines. To address the immunogenicity of recombinant protein vaccines, nanoparticles are often employed. Nanoparticles use nanomaterials as carriers to deliver specific antigens and adjuvants for therapeutic or preventive purposes. Nanoparticles typically range in size from 1 to 1000 nm (10 to 200 nm). This size advantage allows them to more readily accumulate in lymphoid organs such as the lymph nodes and spleen. Their size, similar to that of the pathogen, makes them readily taken up by antigen-presenting cells (APCs), activating specific T or B cells. Artificial nanoparticles come in various forms, including self-assembling protein nanoparticles, polymeric or lipid nanoparticles, inorganic nanoparticles, and biomimetic nanoparticles. Lipid-based nanoparticles are the most widely used due to their ease of preparation, safety, and high biocompatibility. TLRs are innate immune receptors that directly or indirectly detect pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways. Naturally occurring and synthetic TLR agonists can exploit these endogenous immune signaling pathways to enhance and modulate vaccine responses, making them excellent vaccine adjuvants. Each TLR has its own specific tissue localization and downstream gene signaling pathways. TLR agonists can be combined with other TLRs or alternative adjuvants to produce combination adjuvants with synergistic or modulatory effects. The TLR receptor family comprises six transmembrane TLRs (TLR-1, 2, 4, 5, 6, and 10) and four TLRs localized to endosomal membranes (TLR-3, 7, 8, and 9). Each PAMP is recognized by a different TLR, namely lipopolysaccharide (TLR4), lipopeptides (TLR2 and TLR6 or TLR1), flagellin (TLR5), single-stranded RNA (TLR7 / 8), double-stranded RNA (TLR3) and DNA containing CpG motifs (TLR9). Among them, nucleic acid-based TLR receptor agonists have been shown to have a strong adjuvant effect. TLR3 agonists such as Poly (I: C) can activate immune responses against viral infections and also show certain effects against tumors. Studies have shown that it can promote the maturation of DC cells, thereby enhancing anti-tumor T cell responses. TLR9 agonists such as CpG oligonucleotides have been shown to stimulate anti-tumor specific T cell responses and have achieved initial success in combination with cancer vaccines. Therefore, providing a preparation method and application of an HPV recombinant protein therapeutic vaccine to solve the low immunogenicity and safety issues of existing recombinant protein vaccines is of great practical significance. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method and application of an HPV recombinant protein therapeutic vaccine to solve the problems of low immunogenicity and safety of existing recombinant protein vaccines.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] The first aspect of the present application provides an HPV recombinant protein therapeutic vaccine, comprising composite lipid nanoparticles, HPV16 E6E7 recombinant protein and a nucleic acid TLR agonist adjuvant.

[0010] As a preferred technical solution of the present invention, the preparation method of the composite lipid nanoparticles comprises: dissolving DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol.

[0011] Furthermore, the molar ratio of DSPC, cholesterol and DMG-PEG2000 is 57-61:37.5-41.5:1.3-1.7; and the amount of anhydrous ethanol added is such that the total concentration of the composite lipid nanoparticles is 5-40 mM.

[0012] Preferably, the molar ratio of DSPC, cholesterol and DMG-PEG2000 is 59:39.5:1.5; and the amount of anhydrous ethanol added is such that the total concentration of the composite lipid nanoparticles is 15 mM.

[0013] As a preferred technical solution of the present invention, the HPV16 E6E7 recombinant protein includes fusion expression or single expression; it can be produced in different expression systems, including Escherichia coli (prokaryotic system), yeast (eukaryotic system) or mammalian cells (higher eukaryotic system).

[0014] Furthermore, the HPV16 E6E7 recombinant protein was mutated at the binding sites of the tumor suppressor proteins p53 and pRb to avoid potential tumorigenicity.

[0015] As a preferred technical solution of the present invention, the nucleic acid TLR agonist adjuvant includes any one of double-stranded RNA (dsRNA), single-stranded RNA (ssRNA) or double-stranded DNA (dsDNA), which activates TLR3, TLR7 / 8 and TLR9 receptors respectively.

[0016] Furthermore, the nucleic acid TLR agonist adjuvant includes a TLR3 agonist.

[0017] Furthermore, the TLR3 agonist comprises polyI:C.

[0018] The second aspect of the present application provides a method for preparing an HPV recombinant protein therapeutic vaccine, comprising the following steps:

[0019] Composite lipid nanoparticles prepared from multiple lipids are used to simultaneously encapsulate and deliver HPV16 HPV E6E7 recombinant protein antigens and nucleic acid TLR agonist adjuvant molecules.

[0020] As a preferred technical solution of the present invention, the preparation method specifically comprises the following steps:

[0021] (1) dissolving a nucleic acid TLR agonist adjuvant in PBS buffer containing HPV16 E6E7 recombinant protein to prepare an aqueous phase;

[0022] (2) The composite lipid nanoparticles were mixed as the lipid phase and the aqueous phase, and the HPV recombinant protein therapeutic vaccine was prepared using a microfluidic method.

[0023] In some embodiments, the method for preparing the PBS buffer containing the HPV16 E6E7 recombinant protein in step (1) comprises: dissolving the HPV16 E6E7 recombinant protein in PBS buffer to prepare the PBS buffer containing the HPV16 E6E7 recombinant protein.

[0024] Furthermore, the concentration of the PBS buffer containing the HPV16 E6E7 recombinant protein is 0.1-1 mg / mL.

[0025] Preferably, the concentration of the PBS buffer containing the HPV16 E6E7 recombinant protein is 0.2 mg / mL.

[0026] Furthermore, in the aqueous phase of step (1), the concentration of the nucleic acid TLR agonist adjuvant is 0.1-5 mg / mL.

[0027] Preferably, the concentration of the nucleic acid TLR agonist adjuvant is 2 mg / mL.

[0028] Furthermore, in the microfluidic method of step (2), the flow rate ratio of the lipid phase to the aqueous phase is 1:1-5, and the total flow rate is 6-20 mL / min.

[0029] Preferably, the flow rate ratio of the lipid phase to the aqueous phase is 1:3; the total flow rate is 12 mL / min.

[0030] The third aspect of the present application provides a recombinant HPV protein therapeutic vaccine that is applicable to all subtypes related to cervical cancer, including HPV16, 18, 45, 33, 58, 31, 52, 35, 59, 39, 56, 51, 68, 73, 26, 69, and 82.

[0031] Furthermore, HPV recombinant protein therapeutic vaccines are used to treat latent infections, precancerous lesions and invasive cervical cancer of HPV-related subtypes.

[0032] Furthermore, the HPV recombinant protein therapeutic vaccine prepared in this way can be delivered together with the nucleic acid TLR adjuvant to any tumor antigen protein, tumor antigen synthetic peptide or tumor cell lysate containing tumor antigen.

[0033] Beneficial effects of the present invention:

[0034] This application uses composite lipid nanoparticles to encapsulate and deliver HPV16 E6E7 recombinant protein and nucleic acid TLR agonists, which is beneficial to the uptake and activation of vaccine antigens by antigen-presenting cells. The recombinant protein is degraded and presented in the cell, which is more conducive to activating cellular immune responses, especially CD8 T cell immune responses. The receptors of nucleic acid TLR agonists are on the lysosomal membrane and enter the lysosome through the intervention of composite lipid nanoparticles. They have a more powerful adjuvant effect than soluble nucleic acid TLR receptor agonists. At the same time, the composite lipid nanoparticles themselves have a very strong adjuvant effect. This nano vaccine preparation method is beneficial for inducing a strong T cell immune response against HPV16E6E7 antigens, killing cervical cancer cells infected with HPV. In addition, unlike lipid nanoparticles (LNPs) that deliver mRNA vaccines, there are no cationic or ionizable lipids in the formula, and the safety is significantly higher than that of mRNA vaccines. It has greater advantages than mRNA therapeutic vaccines, low toxicity and side effects, and stable vaccine effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 This is a structural diagram of the recombinant protein nanovaccine of the present invention;

[0037] Figure 2 Schematic diagram of the expression and purification of HPV16 E6E7 fusion protein;

[0038] Figure 3 For HPV recombinant protein nano vaccine testing;

[0039] Figure 4 This is the ELISPOT test result of HPV16 recombinant protein nanovaccine;

[0040] Figure 5 The protective effect of HPV16 recombinant protein nanovaccine on animals. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0042] Example 1: Preparation of HPV16 E6E7 recombinant protein immunogen

[0043] First, prepare the HPV16 E6E7 fusion protein as a recombinant protein therapeutic vaccine. The sequence of the HPV16 E6E7 fusion protein is as follows:

[0044] MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDI ILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRF

[0045] HNIRGRWTGRCMSCCRSSRTRRETQL

[0046] GPGPGHGDTPTLHEYmLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP

[0047] The codon-optimized HPV16 E6E7 fusion protein gene was inserted into the E. coli expression vector pET-30a. After E. coli transformation, resistance screening, and IPTG-induced expression verification, a strain that correctly expressed the target protein was obtained. The target protein was expressed in the form of inclusion bodies. The inclusion bodies were first cleaned and then denatured, renatured, and refolded to obtain the target protein for vaccine preparation. The target protein (such as 100mg / L) was finally obtained by gel filtration and reverse phase chromatography purification technology. Figure 2 ).

[0048] Example 2: Preparation of HPV recombinant protein nanovaccine

[0049] (1) HPV recombinant protein nanovaccine was prepared by microfluidics. Composite lipid nanoparticles were used as the lipid phase. The lipid phase consisted of three lipids: DSPC, cholesterol, and DMG-PEG2000. DSPC, cholesterol, and DMG-PEG2000 were dissolved in anhydrous ethanol at a molar ratio of 59:39.5:1.5. The total concentration of the three lipids was 15 mM. 2 mg of HPV16 E6E7 recombinant protein was dissolved in 10 mL of PBS buffer, and poly I:C was then added to 1 mg / mL to prepare the aqueous phase.

[0050] (2) The lipid phase was mixed with the aqueous phase at a flow rate ratio of 1:3 and a total flow rate of 12 mL / min, and the recombinant protein nanovaccine (e.g. Figure 1 ), the initial product contained 25% ethanol, and the ethanol concentration was immediately diluted with PBS to below 2.5% after preparation. The particle size and uniformity were measured using a dynamic light scattering (DLS) instrument.

[0051] The average particle size of the recombinant protein nanoparticle vaccine was 82 nm ( Figure 3 ), PDI 0.1, which meets the design requirements of recombinant protein nanovaccine.

[0052] The recombinant protein nanovaccine product is transferred directly into a dialysis cassette (Thermo Slide-A-Lyzer™ dialysis cassette, 20K MWCO, is recommended) or a 20 kDa pre-treated dialysis bag using a syringe. The dialysis cassette or bag containing the nanovaccine product is placed in at least 50 volumes of PBS buffer and dialyzed at room temperature for at least 6 hours, with one change of buffer. Ultrafiltration is then performed using a 30 kDa ultrafiltration tube, followed by centrifugation, sterilization through a 0.22 μM aqueous filter membrane, and aliquoting. Finally, the ultrafiltered sample is placed in PBS buffer containing 10% sucrose. The prepared recombinant protein nanovaccine product can be stored at 4°C.

[0053] Example 3: Analysis of Immunogenicity of HPV Recombinant Protein Nanovaccine

[0054] ELISPOT assay was used to detect the immunogenicity of HPV recombinant protein nanovaccine

[0055] Six-week-old female mice were immunized with 100 μL of the HPV recombinant protein nanovaccine prepared in Example 2 above as the primary immunization. Two weeks later, a booster injection of the same dose was given. One week after the termination of immunization, the spleens of the mice were removed, and lymphocytes were separated by a cell strainer. Red blood cell lysis buffer was added and allowed to stand for 5 minutes. The cells were centrifuged at 500 g for 15 minutes, washed once with PBS, and resuspended in RPMI-1640 medium containing 10% FBS to a volume of 1×10 6 cells / mL.

[0056] The specific T cell immune response induced by the vaccine was detected using Dakota's pre-coated ELISPOT kit. First, 200 μL / well 1640 culture medium was used to activate the pre-coated plate. 100 μL of 1x10 5 Cells were plated at 400 μL / well, and samples and HPV16 E6E7 synthetic peptide stimulation were added. The cells were cultured in a 37°C, 5% CO2 incubator for 16-24 hours. Cells and culture medium were discarded from the wells, and 100 μL / well of deionized water was added. The cells were incubated at 4°C for 10 minutes. 260 μL / well of Washing Buffer was added, and the solution was discarded. This was repeated six times. Biotinylated Antibody working solution was added to the laboratory wells at 100 μL / well and incubated at 37°C for 1 hour. After washing, Streptavidin-HRP working solution was added to each experimental well at 100 μL / well and incubated at 37°C for 1 hour. 260 μL / well of Washing Buffer was added and repeated six times. Add freshly prepared AEC color development solution to the experimental wells, 100 μL / well; incubate at room temperature in the dark for 5-30 minutes. Stop color development when spots appear, wash each experimental well with deionized water, and count the spots on the ELISPOT plate after drying for statistical analysis.

[0057] Animal experimental results show that HPV16 recombinant protein nanovaccine has very strong immunogenicity ( Figure 4 ).

[0058] Example 4: HPV16 recombinant protein nanovaccine animal protection experiment

[0059] The protective effect of HPV recombinant protein nanovaccine was tested using the TC-1 mouse tumor model.

[0060] TC-1 cells are a mouse lung epithelial tumor cell line that expresses HPV16 E6 and E7 proteins. Six-week-old female mice were selected and 5x10 6 A tumor model was established using TC-1 cells; the initial vaccine was injected intramuscularly on the 3rd day, followed by booster immunizations 10 and 21 days later. The tumor volume of the vaccine-immunized mice and the control group mice was continuously tracked and measured; the experiment was terminated when all experimental animals in the negative control group died or the predetermined experimental purpose was achieved; statistical analysis was performed after the data were obtained.

[0061] The experimental results are as follows Figure 5 As shown in the results, tumors completely disappeared after immunization of mice with the HPV16 recombinant protein nanovaccine, fully demonstrating the immune protection of the recombinant protein nanovaccine. Furthermore, the recombinant protein nanovaccine was safe in animal experiments, with no significant local or systemic adverse reactions observed.

[0062] Throughout the specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0063] The above content is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A HPV recombinant protein therapeutic vaccine, characterized in that: It includes composite lipid nanoparticles, HPV16E6E7 recombinant protein and nucleic acid TLR agonist adjuvant.

2. The HPV recombinant protein therapeutic vaccine according to claim 1, characterized in that The preparation method of the composite lipid nanoparticles comprises the following steps: dissolving DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol.

3. The HPV recombinant protein therapeutic vaccine according to claim 2, characterized in that The molar ratio of DSPC, cholesterol and DMG-PEG2000 is 57-61:37.5-41.5:1.3-1.7; the amount of anhydrous ethanol added is such that the total concentration of the composite lipid nanoparticles is 5-40 mM; Preferably, the molar ratio of DSPC, cholesterol and DMG-PEG2000 is 59:39.5:1.5; and the amount of anhydrous ethanol added is such that the total concentration of the composite lipid nanoparticles is 15 mM.

4. The HPV recombinant protein therapeutic vaccine according to claim 1, characterized in that The HPV16E6E7 recombinant protein includes fusion expression or single expression.

5. The HPV recombinant protein therapeutic vaccine according to claim 1, characterized in that The nucleic acid TLR agonist adjuvant includes any one of double-stranded RNA (dsRNA), single-stranded RNA (ssRNA) or double-stranded DNA (dsDNA), which activates TLR3, TLR7 / 8 and TLR9 receptors respectively.

6. The HPV recombinant protein therapeutic vaccine according to claim 5, characterized in that The nucleic acid TLR agonist adjuvant includes a TLR3 agonist, and the TLR3 agonist includes polyI:C.

7. A method for preparing the HPV recombinant protein therapeutic vaccine according to any one of claims 1 to 6, characterized in that: The steps include: Composite lipid nanoparticles prepared from multiple lipids are used to simultaneously encapsulate and deliver HPV16 HPV E6E7 recombinant protein antigens and nucleic acid TLR agonist adjuvant molecules.

8. The method for preparing the HPV recombinant protein therapeutic vaccine according to claim 7, characterized in that: The preparation method specifically comprises the following steps: (1) dissolving a nucleic acid TLR agonist adjuvant in PBS buffer containing HPV16 E6E7 recombinant protein to prepare an aqueous phase; (2) The composite lipid nanoparticles were mixed as the lipid phase and the aqueous phase, and the HPV recombinant protein therapeutic vaccine was prepared using a microfluidic method.

9. The method for preparing the HPV recombinant protein therapeutic vaccine according to claim 8, characterized in that: The concentration of the PBS buffer containing the HPV16 E6E7 recombinant protein in step (1) is 0.1-1 mg / mL; preferably, the concentration of the PBS buffer containing the HPV16 E6E7 recombinant protein is 0.2 mg / mL; in the aqueous phase in step (1), the concentration of the nucleic acid TLR agonist adjuvant is 0.1-5 mg / mL; preferably, the concentration of the nucleic acid TLR agonist adjuvant is 2 mg / mL.

10. A use of the HPV recombinant protein therapeutic vaccine according to any one of claims 1 to 6, characterized in that: Applicable to all subtypes associated with cervical cancer, including HPV16, 18, 45, 33, 58, 31, 52, 35, 59, 39, 56, 51, 68, 73, 26, 69, and 82.

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