Mutant of HPV53 E6 protein and application thereof

Abstract

The invention discloses a mutant of HPV53 E6 protein and application of the mutant. Compared with wild type E6, the mutant has the following three mutation sites: K79W, S133P and N8K, the K79W mutation design enhances the stability of a hydrophobic space and forms new hydrophobic interaction with Y108 / H130, S133P enables the flexibility of a loop region to be more stable, N8K mutation forms a salt bridge with E10 / E11, and the relation between the loop region and helix is enhanced. The mutation designs can maintain the stability of the whole structure, significantly improve the protein stability and immunogenicity, break through the technical bottlenecks of low in-vivo expression quantity and weak immunogenicity of the natural E6 protein, and provide a new way for immunotherapy of HPV53 related lesions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a mutant of HPV53 E6 protein and application thereof. BACKGROUND

[0002] Human Papillomavirus (HPV) is a virus with double-stranded DNA structure, which is closely related to many malignant tumors such as cervical cancer, vaginal cancer, anal cancer, head and neck cancer, and there is no therapeutic vaccine on the market. So far, more than 200 types of HPV have been identified, which are divided into two categories of high-risk and medium-risk types according to their carcinogenic potential. Persistent HPV infection can lead to precancerous lesions in the cervix, anus, vulva, vagina, and head and neck, and may cause cancer due to persistent infection.

[0003] Notably, epidemiological studies and clinical data in recent years have shown that the infection rate of HPV53 type ranks first among non-preventive vaccine-covered subtypes, and is frequently detected in cervical precancerous lesion and cancer tissue samples. In 2021, two nationwide studies in China revealed that the infection rate of HPV53 type was second only to HPV16, 52 and other subtypes included in the preventive vaccine, becoming the type with the highest infection rate among non-vaccine-covered high-risk and medium-risk HPV subtypes. In addition, a survey on the infection of non-vaccine HPV subtypes in Shanghai also pointed out that in the patient population without lesions and CIN1 (cervical intraepithelial neoplasia 1), the infection rate of HPV53 type ranked first, and in the CIN2 (cervical intraepithelial neoplasia 2) patients, its infection rate also ranked third. These data all suggest that HPV53 type infection has become a potential risk factor for cervical precancerous lesions. However, as of now, there has been no related report on the development of preventive or therapeutic vaccines against HPV53 type, therefore, it is particularly important to strengthen the monitoring and attention on HPV53 type infection and to develop HPV53 type therapeutic vaccine.

[0004] E6 protein is one of the early proteins expressed after HPV infection, and plays an important role as an antigen in the immunotherapy of HPV infection. However, the natural E6 protein has low expression level and limited immunogenicity, which limits its effect as an immunogen.

[0005] Therefore, there is an urgent need for an improved E6 protein mutant that can not only improve the structural stability and expression level, but also significantly enhance the immune response effect, and combine with mRNA vaccine platform and delivery system to provide a new treatment approach for HPV53 and related lesions, and also has reference significance for the design of therapeutic vaccines for other HPV types. SUMMARY

[0006] To solve the technical problem, the application provides a mutant of HPV53 E6 protein and application thereof, the structure stability and expression level of which are significantly improved by modifying the amino acid sequence of wild-type E6 protein, thereby enhancing the immune response effect.

[0007] To achieve the above object, the application adopts the following technical solutions: In the first aspect of the application, a mutant of HPV53 E6 protein is provided, which comprises at least one of the following mutations relative to wild-type HPV53 E6 protein: N8K, K79W, S133P; Preferably, the mutant comprises at least two mutations, and more preferably, simultaneously comprises three mutations.

[0008] Further, the mutant of HPV53 E6 protein further comprises a homologous variant of one of N8K, K79W, S133P with conservative amino acid substitution.

[0009] The homologous variant refers to a sequence comprising conservative amino acid substitution, deletion or insertion without affecting the function of E6 mutant. It is known to those skilled in the art that a small amount of conservative substitution will not change the expression and antigenicity of E6, and therefore the above sequences all belong to the protection scope of the application.

[0010] In the second aspect of the application, the application provides a DNA molecule encoding the mutant of HPV53 E6 protein.

[0011] The DNA can be a codon-optimized sequence, or a variant having at least 80% homology with any one of SEQ ID NO: 5-SEQ ID NO: 8 and capable of encoding the mutant.

[0012] In the third aspect of the application, an mRNA encoding the mutant of HPV53 E6 protein is provided.

[0013] Further, the mRNA of the mutant comprises a 5' cap structure (Cap1), a regulatory sequence facilitating translation initiation (e.g. Kozak sequence), a 5' untranslated region (5'UTR), an open reading frame (ORF), a 3' untranslated region (3'UTR), and a PolyA tail. Further, the mRNA can comprise modified nucleosides (e.g. Ψ, m1Ψ, 5mC) or a PolyA tail of optimized length to improve stability and translation efficiency. The mRNA of the present application preferably adopts a cap1 capping structure to enhance translation efficiency. However, the present application is not limited to cap1, and other common 5' end capping methods, including cap0, cap2, anti-reverse cap analog (ARCA), etc., as well as any artificial or enzymatic capping method that can achieve efficient translation initiation and translation efficiency, can be applied to the present application and fall within the scope of the present application.

[0014] Further, the sequence of the ORF is selected from one of SEQ ID NO: 5-SEQ ID NO: 8.

[0015] In a fourth aspect of the present application, a therapeutic vaccine composition is provided, comprising the mRNA.

[0016] Further, the composition further comprises at least one of: a delivery system of lipid nanoparticle (LNP), polymer nanoparticle, virus-like particle (VLP), lipid-polymer hybrid particle, cationic emulsion, or other carriers capable of achieving in vivo delivery.

[0017] Further, the composition can also be used in combination with E7 protein, L1 protein or E2 protein of HPV53, or antigens of other high-risk types of HPV, in the form of a fusion protein or multiple components. It is known in the art that the combination of E6 and E7 antigens can enhance the immune effect, and the mutant E6 of the present application can also adopt a similar strategy to further improve the level of immune protection.

[0018] In a fifth aspect of the present application, the E6 mutant, the mRNA encoding the same, or the vaccine composition can be used for treating pathologies associated with HPV53 infection, including but not limited to persistent HPV53 infection, cervical intraepithelial lesions (CIN I-III) associated with HPV53 infection, and intraepithelial lesions of the vulva, vagina, anus or throat region, and in some embodiments, tumors that can be caused by HPV53 infection.

[0019] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: The HPV53 E6 protein mutant provided by this invention shows that the modified E6 mutant mRNA expresses a more stable E6 protein in cells than the native E6 protein sequence, and can induce higher levels of humoral and cellular immunity, suggesting enhanced immune response indicators related to viral clearance and cancer cell clearance. Specifically: (1) Significantly enhanced protein expression and stability: The K79W mutation enhances hydrophobic interactions (with Y108 / H130), S133P stabilizes the loop region conformation, and N8K forms a salt bridge (E10 / E11). These three factors work together to maintain the overall folding stability of the E6 protein. In HEK293 cells, the mean fluorescence intensity (MFI) of the triple mutant (E6-04) reached 24,523±758.1, which was 206% higher than that of the wild type (8,000.9±941.3).

[0020] (2) The mutant induces high antibody titers and maintains them for a long time: In the mouse model, the antibody titer (GMT) was >10 28 days after immunization with the triple mutant mRNA-LNP. 5 The antibody titer was more than 10 times higher than the wild type; after 90 days, the titer of the triple mutant antibody remained at 10. 5 above.

[0021] (3) The mutant efficiently activates T-cell immunity: IFN-γ secretion level: ELISApot assay showed that the number of IFN-γ spots on spleen cells reached 156.0±48.9 / 2x10 after immunization with the triple mutant. 5 Cells, compared to wild-type (25.8±4.8), showed a 504% increase ( p The number of spots in the triple mutant (E6-04) was 84% ​​higher than that of the single mutant (E6-02 S133P) with the best cellular immunogenicity (156.0±48.9 vs 84.7±12.9), confirming the synergistic effect of the mutation sites. Therefore, the mutants of this invention can significantly improve protein stability and immunogenicity. More importantly, the triple mutant showed a much greater improvement in immune response than the single mutant or wild type, an effect that exceeded the reasonable expectations of those skilled in the art, demonstrating the inventiveness of this invention. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1Serum antibody titer after immunizing mice with mutant mRNA-LNP Figure 2 ELISpot was used to detect IFN-γ production in mice immunized with mutant mRNA-LNP. Detailed Implementation

[0024] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0027] The overall concept of this invention is as follows: According to one embodiment of the present invention, a mutant of the E6 protein of wild-type HPV53 is provided, comprising one or more of the following amino acid mutation sites: The K79W mutation design enhances the stability of the hydrophobic space and forms new hydrophobic interactions with Y108 / H130, while S133P makes the loop region more stable. The N8K mutation forms a salt bridge with E10 / E11, strengthening the connection between the loop region and the helix.

[0028] These mutations can maintain the stability of the overall structure, increase intracellular expression, and thus promote antibody production and presentation of immune epitopes. The mutant can be a triple mutant containing all the above mutations, and its amino acid sequence is shown in SEQ ID NO:4, and the corresponding DNA coding sequence is SEQ ID NO:8.

[0029] According to another typical embodiment of the present invention, an mRNA encoding the above-mentioned E6 protein mutant is provided, which comprises the following structure: 5' Cap Structure (Cap1); 5' Untranslated Region (5'UTR); Regulatory sequences that promote translation initiation, such as the Kozak sequence; Encoding the open reading frame (ORF) of the mutant; 3' Untranslated Region (3'UTR); PolyA tail sequence.

[0030] These structural units can be any structure known in the art.

[0031] We synthesized, reverse transcribed, purified, and encapsulated sequences containing different mutations into lipid nanoparticles (LNPs) to obtain the corresponding mRNA-LNP complexes (wild-type E6 protein without mutations served as a negative control). Next, we evaluated the expression of different sequences in mammalian cells and the levels of humoral and cellular immunity after immunizing mice.

[0032] Evaluation results showed that the HPV53 E6 mutant protein of this invention exhibited significantly higher protein stability, antibody titer, and cellular immunity levels than the wild-type E6. This indicates that the E6 protein expressed by the mRNA of the modified E6 mutant is more stable in cells than the native E6 protein, and can induce higher levels of humoral and cellular immunity, thereby enhancing the ability to clear viruses and pathogenic cells.

[0033] According to a typical embodiment of the present invention, a therapeutic vaccine composition is also provided, comprising the above-described mRNA and lipid nanoparticle (LNP) delivery system, wherein the LNP contains any combination of cationic lipids, PEGylated lipids, cholesterol, etc.

[0034] According to a typical embodiment of the present invention, the use of the above-described vaccine composition in the preparation of a drug for treating HPV53-related lesions (including but not limited to persistent viral infection, cervical squamous intraepithelial lesions, etc.) is also provided.

[0035] Those skilled in the art will understand that, without altering the basic concept of the present invention, the mRNA can also be modified by nucleoside, optimized by the length of the PolyA tail, adjusted by the structure of the untranslated region, or used in combination with other HPV antigens to achieve better immune effects.

[0036] Furthermore, since the modification strategy of the present invention mainly acts on key structural sites of the E6 protein, even if there are individual amino acid substitutions or nucleotide changes at other positions (i.e., ≥80% homologous variants), it can maintain similar effects in terms of stability and immunogenicity improvement, and those skilled in the art can reasonably expect its functional equivalence.

[0037] Finally, existing research in this field has shown that combined immunization with E6 and antigens such as E7, L1, and E2 can significantly improve the therapeutic effect of vaccines. The E6 mutant of this invention, when combined with the aforementioned antigens, can further enhance the body's immune reactivity, thereby achieving better therapeutic or preventative effects.

[0038] The following will provide a detailed description of a mutant of the HPV53 E6 protein according to the present application, in conjunction with examples and experimental data.

[0039] Example 1: Preparation of mRNA-LNP encoding the E6 protein mutant (1) mRNA synthesis We first synthesized the DNA sequences corresponding to the amino acid sequences of five E6 protein mutants (E6-WT, E6-01, E6-02, E6-03, and E6-04). WT represents the wild-type E6 protein, whose sequence is derived from E6 protein [Humanpapillomavirus 53], with the protein sequence number P36815 on the UniProt protein database website and Gene ID: 1489464 on the NCBI database, as shown in SEQ ID NO: 10. E6-01's amino acid sequence is that of the E6 protein with the K79W mutation, as shown in SEQ ID NO: 1. E6-02's sequence is that of the E6 protein with the S133P mutation, as shown in SEQ ID NO: 2. E6-03's sequence is that of the E6 protein with the N8K mutation, as shown in SEQ ID NO: 3. E6-04 is the amino acid sequence of the E6 protein with three mutations: K79W, S133P, and N8K, as shown in SEQ ID NO.4.

[0040] The amino acid sequences of SEQ ID NO. 1-4 were codon-optimized, taking into account both the codon fitness index (CAI) and minimum free energy (MFE) of the mRNA. The optimized sequences resulted in structurally more stable mRNAs, extending the mRNA half-life and protein expression time, thereby increasing the final yield of the antigen-encoding protein in mammalian cells. The resulting optimized DNA sequences are SEQ ID NO. 5-8. The amino acid sequence of the wild-type E6 protein was codon-optimized to obtain SEQ ID NO. 9.

[0041] Table 1

[0042] Five synthesized DNA sequences (SEQ ID NO. 5-8 and the wild-type sequence SEQ ID NO. 9) were inserted between the Bgl I and Mlu I restriction sites of the pUC57-Kan plasmid vector, respectively, and then transformed into... E. coli Positive clones with high expression were cultured and selected.

[0043] After shake-flask expansion culture, bacterial cells were collected. High-quality templates were obtained after alkaline lysis, neutralization, clarification, concentration, medium replacement, purification, linearization digestion, and template purification for in vitro transcription. Furthermore, all templates consisted of a DNA template containing an open reading frame surrounded by 5' and 3' untranslated regions (UTRs), ending with a sequence encoding a poly(A) tail. Templates E6-WT, E6-01, E6-02, E6-03, and E6-04 were transcribed in vitro using preferred T7 RNA polymerase and transcription conditions, with the introduction of modified nucleotides (such as N1-methylpseudouridine) to improve mRNA translation efficiency, yielding the corresponding mRNA. The resulting mRNA had a Cap1 5' end and a poly(A) tail of approximately 120 bases at the 3' end. Cap1 capping was preferred, but other capping methods known in the art (such as cap0, cap2, ARCA, etc.) could also be used.

[0044] (2) Purification of mRNA The synthesized mRNA was purified by oligodT affinity chromatography, followed by aseptic filtration through transverse flow filtration to replace the buffer with sodium acetate. RNA integrity was determined using a microfluidic parallel capillary electrophoresis system (Fragment Analyzer system 5200, Agilent), and the concentration, pH, residual DNA, protein, and double-stranded RNA impurities were also measured. The 5' capping efficiency and 3'-polyadenosine (poly A) tail of the mRNA were analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0045] (3) mRNA encapsulation mRNAs were encapsulated using lipid nanoparticles (LNPs). Five mRNAs were encapsulated into LNPs (Maier) using an ethanol-soluble mixture of lipids (ionic cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol lipids). et al , MolTher, 2013 21(8):1570-8). The products were analyzed and characterized, including particle size and polydispersity, encapsulation efficiency, pH value, endotoxin and bioburden determination, and are suitable for in vivo and in vitro scientific studies in the preclinical stage. They were named E6-WT-LNP, E6-01-LNP, E6-02-LNP, E6-03-LNP and E6-04-LNP, respectively.

[0046] Example 2: Expression of HPV53 E6 mutant in HEK293 cells The mRNAs of E6-WT, E6-01, E6-02, E6-03, and E6-04 were transfected into HEK293 cells. Six groups were set up: a PBS control group, and groups of E6-WT, E6-01, E6-02, E6-03, and E6-04. The transfection dose of mRNA per well was 5 μg. After 24 hours of transfection, the expression of HPV53 E6 protein was detected by flow cytometry. The specific experimental methods are as follows: (1) HEK293 cells were seeded in 24-well culture dishes. When the cells reached 80%, 5 μg of mRNA of E6-WT, E6-01, E6-02, E6-03 and E6-04 and 10 μl of Lipofectamine MessengerMax™ TransfectionReagent (LMRNA015, Invitrogen) were added respectively. After 24 h of transfection, the cells were collected.

[0047] (2) At room temperature, cells were fixed with 4% paraformaldehyde for 15 minutes, and then treated with 0.2% Saponin for 10 minutes to break the cell membrane. Then, the E6 protein in the cells was labeled with anti-E6 monoclonal antibody and incubated at room temperature for 1 h.

[0048] (3) The primary antibody was washed away with PBS, and the secondary fluorescent antibody was added. The cells were incubated at room temperature for 1 h. After washing the cells with PBS, the proportion of positive cells and the fluorescence intensity were detected by flow cytometry. The results are shown in Table 2.

[0049] Table 2. Expression of mRNA encoding HPV53 E6 mutant in HEK293.

[0050] As shown in Table 2: More than 85% of cells transfected with five mRNAs—E6-WT, E6-01, E6-02, E6-03, and E6-04—expressed the E6 protein. The mean fractional protein index (MFI) of cells transfected with E6-01, E6-02, E6-03, and E6-04 was more than 50% higher than that of E6-WT. Among them, E6-04 had the highest MFI, exceeding E6-WT by more than 200%, suggesting that the mRNA encoding E6-04 (i.e., the mutant containing a 3-amino acid mutation) expresses the most HPV53 E6 protein in cells.

[0051] Example 3: In vivo immunogenicity study of HPV53 E6 mutant mRNA vaccine Six-week-old BALB / c mice were randomly divided into groups of five: control group (empty LNP group (eLNP)), E6-WT-LNP, E6-01-LNP, E6-02-LNP, E6-03-LNP, and E6-04-LNP. The mRNA-LNP vaccine was administered via intramuscular injection into the right hind leg at a dose of 5 μg / mouse, with an injection volume of 50 μL / mouse. A booster immunization was performed on day 14 using the same method and dosage. Blood samples were collected from the orbital sinus on days 28 and 90. The collected blood was allowed to stand at 2-8°C for at least 12 hours, then centrifuged at 3000 rpm for 10 minutes. The supernatant serum was aspirated and transferred to 1.5 ml sterile centrifuge tubes, inactivated by incubation at 56°C for 30 minutes before use. Specific experimental methods are as follows: (1) The HPV53 E6 protein was coated onto an ELISA plate, diluted with PBS to 5 μg / mL, 100 μL per well, and incubated overnight at 4°C.

[0052] (2) The next day, wash once with PBS, discard the washing solution, add PBS blocking solution containing 5% fetal bovine serum, and place at 37°C for 1 hour.

[0053] (3) Discard the blocking solution and wash three times with PBST washing solution. Add mouse serum diluted 1:1000, and make a 3-fold serial dilution. Set up a total of 7 gradients, 100 μL per well, and incubate at 37°C for 1 hour.

[0054] (4) Discard the supernatant, wash 3 times with PBST washing buffer, add an appropriate concentration of HRP-labeled goat anti-mouse secondary antibody, 100 μL per well, and incubate at 37°C for 1 hour.

[0055] (5) Add ELISA colorimetric solution and incubate at room temperature in the dark for 15 minutes. Add 2 mol / L H2SO4 to stop the colorimetric reaction, and measure the absorbance at 450 nm using a microplate reader.

[0056] (6) Result determination: The average OD value of the blank control and negative serum (eLNP group) should not be higher than 0.1. The cut-off value is calculated based on the average OD value of the negative serum. If the average OD value of the negative serum is not higher than 0.05, it is calculated as 0.05. Cut-off value = average OD value of negative serum × 2.1. If the average OD value of the test serum is higher than the cut-off value, it is judged as positive. The highest dilution of the test serum judged as positive is the antibody titer of the serum.

[0057] The results are as follows Figure 1The results showed that after mice were immunized with five groups of vaccines, namely E6-WT-LNP, E6-01-LNP, E6-02-LNP, E6-03-LNP and E6-04-LNP, all mice produced antibodies against E6 protein on day 28 after the initial immunization. Among them, E6-04 produced the highest antibody titer and the antibody level remained at a high level 90 days after the initial immunization.

[0058] Example 4: Detection of cellular immune levels in HPV53 E6 mutant mRNA vaccine The immunization procedure was the same as in Example 3. On day 28 post-immunization, the spleens of mice in each group were collected to detect antigen-specific cellular immune responses. The specific experimental methods are as follows: (1) Grind the extracted mouse spleen through a 200-mesh filter, transfer the cell septum filter to a 15 mL centrifuge tube, and centrifuge at 1800 rpm and 4℃ for 5 minutes.

[0059] (2) Discard the supernatant, resuspend the cells in red blood cell lysis buffer, lyse red blood cells on ice for 15 min, add 10 mL PBS solution to stop the reaction, and centrifuge at 1800 rpm and 4℃ for 5 min.

[0060] (3) Discard the supernatant, add 1 mL of PBS solution to resuspend the cells, remove the flocculent material with a 200 μL pipette tip, and count the spleen cells in each group.

[0061] (4) Prepare RPMI-1640 basal medium containing peptide pool, i.e., dilute peptide pool with basal 1640 medium and the final concentration of peptide pool is 1 mg / mL.

[0062] (5) Take out the ELISpot plate, add 200 μL of 1640 basal medium without peptide pool, and let it stand at room temperature for 10 min to activate.

[0063] (6) Discard the culture medium and add 150 μL of 1640 medium containing the peptide pool to each well. The positive control group is added with 140 μL of 1640 medium and 10 μL of positive stimulant, while the negative control group is added with only 150 μL of 1640 basal medium. Take 5 × 10⁵ spleen single-cell suspensions from each group and add them to ELISpot wells. Gently tap to disperse the cells and incubate in a cell culture incubator for 18-24 h.

[0064] (7) Remove the incubated plate, invert and discard the culture medium, add 200 μL of pre-cooled deionized water, and incubate at 4°C for 10 min to lyse the cells. Invert and discard the deionized water, add 200 μL of 1×Washing Buffer (the 50×Washing Buffer in the kit is diluted 50 times with deionized water) to wash 3 times, each time with 1×Washing Buffer for 1 min.

[0065] (8) Add 100 μL of diluted primary antibody solution (the biotin-labeled antibody in the kit is diluted 100 times with Dilutionbuffer R) and incubate at 37°C for 1 h. Remove the liquid from the plate, add 200 μL of 1×Washing Buffer and wash 5 times, each time with 1×Washing Buffer for 1 min.

[0066] (9) Add 100 μL of diluted enzyme-labeled avidin (diluted 100 times with Dilution buffer R in the kit) and incubate at 37°C for 1 h. Remove the liquid from the plate and wash 5 times with 200 μL of 1×Washing Buffer, each time for 1 min.

[0067] (10) Add 100 μL of the prepared ACE solution (mix 20×AEC solution Ⅰ, 20×AEC solution Ⅱ, 200×AEC solution Ⅲ and AEC dilution in the kit at a ratio of 10:10:1:180) and incubate at 37℃ for 15-30 min.

[0068] (11) Observe with the naked eye until the color develops to a suitable level (observe every 5 minutes), then invert the plate to discard the liquid, rinse the inside of the plate and the base with deionized water, and place it in a dark place to air dry naturally.

[0069] (12) Use an automated ELISPOT reader (Mabtech IRIS FluoroSpot / ELISpot reader) to count the spots inside the wells.

[0070] If the number of spots in the negative control well is 0-5, the number of spots in the experimental group well minus the number of spots in the negative control well must be ≥6; if the number of spots in the negative control well is ≥6, the number of spots in the experimental group well must be ≥2 times the number of spots in the negative control well, and this shall be used as the positive standard.

[0071] The results are as follows Figure 2 The results showed that, compared with E6-WT-LNP (25.8 ± 4.8), the number of IFN-γ secreted spots in spleen cells of mice immunized with E6-01-LNP (47.0 ± 12.5) and E6-03-LNP (49.2 ± 14.8) was increased, but the differences were not statistically significant (ns p>0.05). However, the E6-02-LNP (84.7 ± 12.9) group significantly increased the number of IFN-γ secreted spots. E6-04-LNP (**) group (**) showed a significantly increased number of IFN-γ secreted spots. p<0.01). E6-04-LNP (156.0 ± 48.9) resulted in the highest number of IFN-γ secreted by spleen cells in mice after immunization, more than 6 times that of E6-WT-LNP (**** , p The result (<0.0001) indicates that the RNA vaccine prepared based on the E6 variant with three amino acid mutations exhibits the best cellular immunity in mice. This improvement significantly surpasses the effects of common single-point mutation modifications, demonstrating an unexpected technical achievement.

[0072] In the embodiments of this invention, the expression levels and immunogenicity of single-mutation and triple-mutation sites were verified respectively. Experimental results showed that single-point mutation could improve the stability or immune response of the E6 protein to some extent, while the triple mutant exhibited the most significant enhancement effect. Those skilled in the art will understand that when two mutation sites are introduced simultaneously, their effect is reasonably expected to be between that of single-point mutation and triple mutation, and can produce a synergistic optimization effect. Therefore, even if experimental data for the double mutant are not separately listed in this application, its functionality is supported by common knowledge in the art and falls within the scope of protection of this invention.

[0073] In summary, the mutant of wild-type HPV53 E6 protein and its application provided by this invention, through modification of the amino acid sequence of the expressed HPV53E6 protein, results in the mutant mRNA expressing a higher level of the modified protein in vivo than the natural protein, inducing more and more effective humoral and cellular immunity, thereby enhancing viral clearance and the clearance of cancerous cells.

[0074] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0075] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A mutant of the HPV53 E6 protein, characterized in that, The mutant of the HPV53 E6 protein, relative to the wild-type HPV53 E6 protein, includes at least one of the following mutations: N8K, K79W, S133P; The amino acid sequence of the mutant HPV53 E6 protein is selected from one of SEQ ID NO.1-SEQ ID NO.

4.

2. A DNA nucleic acid molecule encoding a mutant of the HPV53 E6 protein of claim 1.

3. An mRNA encoding the HPV53 E6 protein mutant of claim 1.

4. The mRNA according to claim 3, characterized in that, The mRNA contains a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a PolyA tail.

5. The mRNA according to claim 4, characterized in that, The sequence of the ORF is selected from one of SEQ ID NO:5-SEQ ID NO:8, or a sequence variant that has at least 80% homology with it and retains E6 functional activity.

6. A therapeutic vaccine composition, characterized in that, The composition comprises the mRNA according to any one of claims 3-5.

7. The therapeutic vaccine composition according to claim 6, characterized in that, The composition further includes at least one of the following: Delivery systems for lipid nanoparticles, polymer nanoparticles, virus-like particles, lipid-polymer hybrid particles, cationic emulsions, or other carriers capable of in vivo delivery.

8. The therapeutic vaccine composition according to claim 6, characterized in that, The composition further includes at least one of the following: HPV53's E7, L1, or E2 proteins.

9. Use of the mutant of HPV53 E6 protein of claim 1, or the mRNA of any one of claims 3-5, or the vaccine composition of any one of claims 6-8 in the preparation of a medicament for treating HPV53 infection-related lesions, the preparation of a multivalent HPV vaccine, or an antigen diagnostic kit.

10. The use according to claim 9, characterized in that, The lesions were selected from: (i) Persistent HPV53 infection; (ii) Cervical intraepithelial lesions; (iii) Other intraepithelial lesions, including intraepithelial lesions in the vulva, vagina, anus or pharynx.

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