Preparation method and application of HPV recombinant protein therapeutic vaccine

Abstract

The present application relates to a kind of preparation method and application of HPV recombinant protein therapeutic vaccine, belong to biological medicine technical field, the preparation method of the HPV recombinant protein therapeutic vaccine described includes: a plurality of complex lipid nanoparticles prepared by lipid are simultaneously wrapped and delivered HPV16HPV E6E7 recombinant protein antigen and nucleic acid TLR agonist adjuvant molecule.This application is beneficial to the recombinant protein therapeutic vaccine prepared to induce strong T cell immune response to HPV16E6E7 antigen, kill the cervical cancer cell infected by HPV.In addition, unlike the lipid nanoparticle (LNP) of mRNA vaccine delivery, there is no cation or ionizable lipid in the formula, significantly higher than mRNA vaccine in safety, with greater advantage than mRNA therapeutic vaccine, and low toxicity and side effects, vaccine effect is stable.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying a recombinant HPV protein therapeutic vaccine. Background Technology

[0002] Cervical cancer is one of the most common gynecological malignancies, ranking second in incidence among gynecological malignancies in China, after breast cancer. Currently, it is clear that persistent infection with high-risk HPV is a necessary factor for the development of cervical cancer and precancerous lesions. Persistent HPV infection is a crucial link in the process of cervical cancer development. HPV is a double-stranded circular DNA virus with over 200 subtypes. Seventeen HPV types are causally related to cervical cancer, with HPV 16 and 18 associated with over 70% of cervical cancers. Other subtypes, in descending order, are 45, 33, 58, 31, and 52. The remaining ten HPV genotypes, including HPV 35, 59, 56, 51, 68, 73, 26, 69, and 82, can also lead to cervical cancer. High-risk HPV infection rates are as high as 10-20% in women. Persistent HPV infection is a necessary condition for cervical cancer, so early detection and treatment are particularly important for the prevention and control of cervical cancer.

[0003] Currently approved cervical cancer vaccines are all preventative vaccines and are ineffective against precancerous lesions and cervical cancer after infection. For example, GSK's bivalent HPV16 / 18 VLPs vaccine can prevent more than 70% of precancerous lesions and cervical cancer caused by HPV16 / 18; Merk's quadrivalent VLPs vaccine can prevent cervical cancer caused by HPV16 / 18 and genital warts caused by HPV6 / 11; and Merk's nonavalent VLPs can prevent more than 90% of cervical cancer caused by HPV infection. All three vaccines are based on virus-like particles formed from the HPV L1 protein. The progression of HPV infection to cervical cancer is a gradual process that can take years to decades. This process is generally considered to involve several stages: mild, moderate, and severe intraepithelial neoplasia, carcinoma in situ, and invasive carcinoma. Currently, there are no specific drugs for precancerous cervical lesions; drug treatment uses recombinant human interferon and anti-HPV biological proteins to alleviate the condition. Surgical treatments, depending on the patient's progression, include procedures such as loop electrosurgical excision procedure (LEEP), cold knife conization, and total hysterectomy. However, for advanced or recurrent cervical cancer with metastasis, these treatments are not very effective, with a 5-year survival rate of only 16.8%. Therefore, researching new treatment modalities is crucial for halting the progression of cervical cancer and improving the prognosis of patients with advanced and recurrent cervical cancer.

[0004] HPV therapeutic vaccines work by activating the body's own immune system to treat HPV precancerous lesions and cervical cancer. They are currently considered the safest, most effective, and most economical treatment. HPV therapeutic vaccines differ from the preventive vaccines widely used in clinical practice 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 uninfected normal people. Once infected with the virus, the preventive vaccine is ineffective; (2) Their mechanisms of action are different. Preventive vaccines mainly work through humoral immunity, while therapeutic vaccines mainly induce cellular immune responses to clear virus-infected cells or tumor cells; (3) Their vaccine targets are different. Preventive vaccines are all based on virus-like particles formed by HPV L1 protein, while therapeutic vaccines mainly target the E6E7 protein that is continuously expressed in latent infection. The HPV genome consists of three regions: the early (E) region, which encodes viral proteins that regulate the viral life cycle and the cellular function of infecting epithelial cells; the late (L) region, which encodes structural proteins that mediate the viral icosahedral capsid; and the LCR (long control region), which contains cis-acting sequences that mediate viral replication and transcription. Preventive vaccines such as and Stimulates the formation of neutralizing antibodies against capsid protein L1, preventing viral entry and infection spread. Early-stage carcinogenic proteins E6 and E7 are the most targeted antigens for currently developed HPV therapeutic vaccines because: 1) E6 and E7 are expressed extensively and specifically in precancerous and cancerous lesions, thus the risk of targeting healthy tissue is not significant; 2) E6 and E7 are essential for transformation and maintaining the transformation of infected cells, thus the risk of antigen loss-mediated immune escape is not significant; 3) Central tolerance mechanisms to E6 and E7 have not been documented; 4) Immune responses to E6 and E7 have been characterized preclinically and clinically.

[0005] Currently, there are no marketed HPV treatment vaccines; most therapeutic vaccines are still in Phase II clinical trials. Product forms include recombinant protein vaccines, peptide vaccines, chimeric vaccines, and nucleic acid vaccines. Domestic and international companies developing similar products include MGIPhmrma Biologics' plasmid DNA vaccines E-7101 and XYC-101 (Phase III clinical trials); Gentice's protein vaccines GTL-001 (Phase I clinical trials) and Xenova TA-CIN (Phase III clinical trials); Inovio's HPV-16E7 inhibitor, INO-3112 (Phase II clinical trials); and Advaxis's HPV-16E7 protein inhibitor, ADXS-HPV (Phase III clinical trials). No similar products have been approved for marketing domestically or internationally. Inovio's DNA vaccine VGX-3100 has made the most progress, with a higher response rate in the treatment group than the placebo group in its Phase III clinical trial. In the treatment group, 27.6% (37 / 134) of participants achieved the primary endpoint (histopathological outcome and viral clearance), compared to 8.7% (6 / 69) in the placebo group, a statistically significant difference (p = 0.001). Particularly in viral clearance, the clearance rate in the treatment group was 37.3% (50 / 134), while it was 8.7% (6 / 69) in the placebo group. Currently, the use of mRNA technology to prepare HPV therapeutic vaccines is considered promising. This involves preparing the gene encoding the HPV antigen E6E7 into mRNA and administering it to patients, allowing it to be expressed in vivo and stimulating the body to produce an antigen-specific cellular immune response, thereby achieving the goal of treating the disease. Compared to DNA vaccines, it is safer (no risk of gene integration) and simpler to deliver (no need to enter the cell nucleus). Furthermore, it has a shorter production cycle and lower cost. However, mRNA vaccines also have some insurmountable drawbacks, such as high instability. mRNA vaccines need to enter the body's cells to be effective, but because there are large amounts of RNases in the body's blood and tissues, the mRNA may be degraded during delivery before entering cells, thus failing to exert its immunoprotective effect. mRNA vaccines also have some toxicity, mainly from synthetic raw materials and mRNA encapsulation materials, such as cationic lipids. These materials can enter cells along with the mRNA, potentially causing toxicity or affecting the cellular immune response, posing a potential risk.

[0006] Recombinant protein vaccines offer advantages in stability and safety compared to nucleic acid vaccines. Two key challenges need to be addressed in developing recombinant protein vaccines for therapeutic purposes: first, how to induce a T-cell-based immune response to kill and eliminate tumor cells primarily through cellular immunity; and second, how to utilize appropriate adjuvants to enhance the immunogenicity of recombinant proteins. Solving these two problems will give recombinant protein therapeutic vaccines a significant advantage over mRNA vaccines. To address the immunogenicity issue of recombinant protein vaccines, nanoparticle vaccines are commonly used. These vaccines utilize nanomaterials as carriers to deliver specific antigens and adjuvants, achieving therapeutic or preventative effects. Nanoparticles typically range from 1-1000 nm (10-200 nm) in size, making them easier to concentrate in lymphatic organs such as lymph nodes and the spleen. Their size, similar to that of pathogens, allows for easy uptake by antigen-presenting cells (APCs), activating specific T or B cells. Artificial nanoparticles exist in various forms, including self-assembled protein nanoparticles, polymer or lipid nanoparticles, inorganic nanoparticles, and biomimetic nanoparticles. Lipid-based nanoparticles are the most widely used due to their simple preparation, high safety, and 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 utilize these endogenous immune signaling pathways to enhance and regulate 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 regulatory effects. The TLR receptor family includes six transmembrane TLRs (TLR-1, 2, 4, 5, 6, and 10) and four endosome membrane-localized TLRs (TLR-3, 7, 8, and 9). Each PAMP is recognized by a different TLR, namely lipopolysaccharide (TLR4), lipopeptide (TLR2 and TLR6 or TLR1), flagellin (TLR5), single-stranded RNA (TLR7 / 8), double-stranded RNA (TLR3), and DNA containing a CpG motif (TLR9). Nucleic acid TLR receptor agonists have shown potent adjuvant effects. TLR3 agonists, such as Poly(I:C), can activate immune responses against viral infections and have also shown some effectiveness against tumors. Studies have shown that they can promote the maturation of dendritic cells (DCs), 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 preliminary success in combination with cancer vaccines. Therefore, providing a method for preparing and applying a recombinant HPV protein therapeutic vaccine to address the low immunogenicity and safety issues of existing recombinant protein vaccines is of significant practical importance. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a recombinant HPV protein therapeutic vaccine, thereby addressing the issues of low immunogenicity and safety of existing recombinant protein vaccines.

[0008] The objective of this invention can be achieved through the following technical solutions:

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

[0010] As a preferred embodiment of the present invention, the method for preparing the composite lipid nanoparticles includes: dissolving DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol.

[0011] Further, 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.

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

[0013] As a preferred embodiment 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, HPV16 E6E7 recombinant protein mutations are located at sites where tumor suppressor proteins p53 and pRb bind to avoid potential tumorigenicity.

[0015] As a preferred embodiment 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 respectively activate TLR3, TLR7 / 8, and TLR9 receptors.

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

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

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

[0019] Composite lipid nanoparticles prepared from multiple lipids simultaneously encapsulate and deliver HPV16 / E6 / E7 recombinant protein antigens and nucleic acid TLR agonist adjuvant molecules.

[0020] As a preferred embodiment of the present invention, the preparation method specifically includes the following steps:

[0021] (1) The nucleic acid TLR agonist adjuvant was dissolved 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 by microfluidic method.

[0023] In some embodiments, the method for preparing the PBS buffer containing HPV16 E6E7 recombinant protein in step (1) includes: dissolving the HPV16 E6E7 recombinant protein in PBS buffer to obtain 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 described in 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 described in 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 this application provides an HPV recombinant protein therapeutic vaccine applicable to all cervical cancer-related subtypes, including HPV16, 18, 45, 33, 58, 31, 52, 35, 59, 39, 56, 51, 68, 73, 26, 69, and 82.

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

[0032] Furthermore, the HPV recombinant protein therapeutic vaccine prepared thereby can deliver, together with a nucleic acid TLR adjuvant, any tumor antigen protein, tumor antigen synthetic peptide, or tumor cell lysate containing tumor antigens.

[0033] The beneficial effects of this invention are:

[0034] This application utilizes composite lipid nanoparticles to encapsulate and deliver recombinant HPV16 E6E7 protein and a nucleic acid-based TLR agonist, which facilitates the uptake and activation of vaccine antigens by antigen-presenting cells. The intracellular degradation and presentation of the recombinant protein further enhances the activation of cellular immune responses, particularly CD8 T cell immune responses. The receptors for nucleic acid-based TLR agonists are located on the lysosomal membrane; the composite lipid nanoparticles, through their intervention, enter the lysosome, exhibiting a stronger adjuvant effect than soluble nucleic acid TLR receptor agonists. Simultaneously, the composite lipid nanoparticles themselves possess a very strong adjuvant effect. This nanovaccine preparation method is beneficial for inducing a robust T-cell immune response against the HPV16 E6E7 antigen, killing HPV-infected cervical cancer cells. Furthermore, unlike lipid nanoparticles (LNPs) used to deliver mRNA vaccines, this formulation does not contain cationic or ionizable lipids, resulting in significantly higher safety than mRNA vaccines. It offers greater advantages than mRNA therapeutic vaccines, with lower toxicity and more stable vaccine efficacy. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0037] Figure 2 This is a schematic diagram illustrating the expression and purification of the HPV16 E6E7 fusion protein.

[0038] Figure 3 For the detection of HPV recombinant protein nanovaccine;

[0039] Figure 4 The results of ELISPOT testing for the HPV16 recombinant protein nanovaccine;

[0040] Figure 5 To assess the protective effect of HPV16 recombinant protein nanovaccine on animals. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

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

[0043] First, the HPV16 E6E7 fusion protein was prepared for use as a recombinant protein therapeutic vaccine for immunization. The HPV16 E6E7 fusion protein sequence is shown below:

[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 selection, and IPTG-induced expression verification, strains correctly expressing the target protein were obtained. The target protein was expressed in inclusion body form. First, the inclusion bodies were washed, and then underwent denaturation, renaturation, and refolding processes to finally obtain the target protein for vaccine preparation. High-purity target protein (e.g., [missing information]) was finally obtained through gel filtration and reverse-phase chromatography purification techniques. Figure 2 ).

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

[0049] (1) HPV recombinant protein nanovaccine was prepared using a microfluidic method. The composite lipid nanoparticles were used as the lipid phase, which 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, and 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 then poly I:C was added to a concentration of 1 mg / mL to obtain the aqueous phase.

[0050] (2) The lipid phase and aqueous phase were mixed at a flow rate ratio of 1:3 and a total flow rate of 12 mL / min. Recombinant protein nanoparticles (such as...) were then prepared using a microfluidic method. Figure 1 The initial product contained 25% ethanol. Immediately after preparation, the ethanol concentration was reduced to below 2.5% by diluting with PBS. The particle size and uniformity were measured using a dynamic light scattering (DLS) instrument.

[0051] The recombinant protein nanovaccine was tested and found to have an average particle size of 82 nm (e.g., ...). Figure 3 The PDI is 0.1, which meets the design requirements for recombinant protein nanovaccines.

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

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

[0054] Immunogenicity of HPV recombinant protein nanovaccine was detected using enzyme-linked immunospot assay (ELISPOT).

[0055] Six-week-old female mice were immunized with an initial injection of 100 μL of the HPV recombinant protein nanovaccine prepared in Example 2 above, followed by a booster injection of the same dose two weeks later. One week after immunization was terminated, the spleens of the mice were harvested, and lymphocytes were isolated using a cell filter. Red blood cell lysis buffer was added, and the cells were incubated for 5 minutes, centrifuged at 500g for 15 minutes, washed once with PBS, and resuspended in RPMI-1640 medium containing 10% FBS to a concentration of 1 x 10⁻⁶ μL. 6 Cells / mL.

[0056] The vaccine-induced specific T-cell immune response was detected using the Dakoway pre-coated ELISPOT kit. First, the pre-coated plates were activated with 200 μL / well 1640 medium. Then, 100 μL of 1x10⁻⁶ medium was added to both the experimental and control groups. 5 Add sample and HPV16 E6E7 synthetic peptide stimulant to each well, and incubate at 37°C in a 5% CO2 incubator for 16-24 hours. Discard the cells and culture medium from the wells, add 100 μL of deionized water per well, and incubate at 4°C for 10 minutes to lyse the cells. Add 260 μL of Washing Buffer per well, discard the liquid, and repeat six times. Add 100 μL of Biotinylated Antibody working solution to the laboratory and incubate at 37°C for 1 hour. After washing the plate, incubate with ELISA. Add 100 μL of Streptavidin-HRP working solution to each well and incubate at 37°C for 1 hour. Add 260 μL of Washing Buffer working solution to each well, and repeat six times. Add freshly prepared AEC chromogenic solution to each well (100 μL / well); let stand at room temperature in the dark for 5-30 minutes. Once spots appear, stop the chromogenic process. Wash each well with deionized water, air dry, count the spots on the ELISPOT plate, and perform statistical analysis.

[0057] Animal experiments have shown that the HPV16 recombinant protein nanovaccine has very strong immunogenicity. Figure 4 ).

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

[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 cell tumor cell line expressing HPV16 E6 and E7 proteins; 5 x 10 cells were subcutaneously injected into the back of each 6-week-old female mouse. 6 A tumor model was established using TC-1 cells; the initial intramuscular injection of the vaccine was administered on day 3, followed by booster immunizations on days 10 and 21. The tumor volume of the vaccine-immunized mice and the control group mice was continuously monitored and measured; the experiment was terminated when all experimental animals in the negative control group died, or when the predetermined experimental objective was achieved; statistical analysis was performed on the obtained data.

[0061] Experimental results are as follows Figure 5 As shown, the tumors completely disappeared in mice immunized with the HPV16 recombinant protein nanovaccine, fully demonstrating the immunoprotective effect of the recombinant protein nanovaccine. Furthermore, the recombinant protein nanovaccine was safe in animal experiments, with no obvious local or systemic adverse reactions observed.

[0062] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above 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 one or more embodiments or examples.

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

Claims

1. An HPV recombinant protein therapeutic vaccine, characterized in that, It is composed of composite lipid nanoparticles, HPV16 E6E7 recombinant protein, and nucleic acid-based TLR agonist adjuvant; The composite lipid nanoparticles are prepared by dissolving DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol at a molar ratio of 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. The HPV16 E6E7 recombinant protein sequence is shown below: MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQ RFHNIRGRWTGRCMSCRSRTRRETQLGPGPGHGDTPTLHEYmLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP; The adjuvant for the nucleic acid TLR agonist is polyI:C.

2. The HPV recombinant protein therapeutic vaccine according to claim 1, characterized in that... The molar ratio of DSPC, cholesterol, and DMG-PEG2000 is 59:39.5:1.5; The amount of anhydrous ethanol added was such that the total concentration of the composite lipid nanoparticles was 15 mM.

3. A method for preparing an HPV recombinant protein therapeutic vaccine as described in any one of claims 1-2, characterized in that, Includes the following steps: Composite lipid nanoparticles prepared from multiple lipids simultaneously encapsulate and deliver HPV16 E6E7 recombinant protein antigen and nucleic acid TLR agonist adjuvant molecules.

4. The method for preparing the HPV recombinant protein therapeutic vaccine according to claim 3, characterized in that, The preparation method specifically includes the following steps: (1) The nucleic acid TLR agonist adjuvant was dissolved 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 by microfluidic method.

5. The method for preparing the HPV recombinant protein therapeutic vaccine according to claim 4, characterized in that, The concentration of the PBS buffer containing HPV16 E6E7 recombinant protein in step (1) is 0.1-1 mg / mL; the concentration of the nucleic acid TLR agonist adjuvant in the aqueous phase in step (1) is 0.1-5 mg / mL.

6. The method for preparing the HPV recombinant protein therapeutic vaccine according to claim 5, characterized in that, The concentration of the PBS buffer containing the HPV16E6E7 recombinant protein is 0.2 mg / mL; the concentration of the nucleic acid TLR agonist adjuvant in the aqueous phase is 2 mg / mL.

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