Recombinant proteins and their use in hpv prevention

Abstract

The application discloses a recombinant protein and application thereof in HPV prevention, and belongs to the field of biological medicine. The recombinant protein comprises one or more of L2 protein fragments of five serotypes of HPV, wherein the L2 protein of the five serotypes of HPV is selected from 6(13-75), 16(11-75), 18(12-75), 31(11-75) and 39(12-75). The recombinant protein and a composition combined with different adjuvants can simultaneously produce good preventive and / or therapeutic effects on 17 subtypes of HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 73, and high-efficiency prevention and / or treatment of HPV are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a recombinant protein and its application in HPV prevention. Background Technology

[0002] Human papillomavirus (HPV) belongs to the Papillomavacuolar virus family. It is a spherical DNA virus that can cause proliferation of squamous epithelium in human skin and mucous membranes and is currently the only confirmed carcinogen. Current research has confirmed that high-risk HPV types can be detected in the cervical tissue of almost all cervical cancer patients. Internationally, there are currently bivalent, quadrivalent, and nonavalent vaccines that can prevent infection with 2, 4, and 9 HPV types, respectively. However, there are over 100 types of HPV, and different types cause different clinical manifestations. The prevalent HPV types also differ in different countries and regions. Simply increasing the number of vaccine target antigens to completely prevent infection with different HPV types in different regions is unrealistic. Furthermore, for patients who are positive for HPV in the later stages of infection, the HPV viral DNA integrates into the host cell DNA, resulting in the loss of expression of capsid proteins L1 and L2; this renders vaccines and treatments targeting L1 and L2 ineffective. At the same time, the overexpression of oncogene proteins E6 / E7 enhances the carcinogenic effect, causing unlimited cell proliferation and transforming cervical tissue from a pathological stage into cancer.

[0003] Persistent infection with high-risk HPV leads to the integration of its DNA with the host's basement membrane cell DNA, resulting in the deletion of the HPV E2 fragment (E2 protein is a major viral transcription factor involved in transcriptional regulation). This deletion accelerates the progression of cervical lesions and increases the likelihood of malignant transformation. The deletion of the E2 fragment causes the expression of HPV E6 / E7 proteins, which can inactivate tumor suppressor proteins such as P53, RB, and P21, promoting and maintaining the integration of HPV DNA with cervical basal cell DNA, causing abnormal proliferation of cervical basal cells, thereby transforming normal host cells into malignant ones. In addition, E6 / E7 proteins can allow HPV to escape the host's immune surveillance and interfere with the body's immune response.

[0004] To date, approximately 200 HPV subtypes have been identified, with high-risk HPV types including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, and 58. HPV16 and HPV18 are the most prevalent circulating strains. Patients often have co-infection with multiple HPV types, thus necessitating the development of therapeutic drugs targeting various HPV types.

[0005] Therefore, there is an urgent need to research a recombinant protein that can prevent and treat more types of HPV infection, so that it can be used efficiently and easily for the prevention or treatment of HPV. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant protein and its application in HPV prevention and / or treatment, which has preventive and / or therapeutic effects on more types of HPV infection, achieving highly efficient prevention and / or treatment of HPV.

[0007] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0008] On one hand, the present invention provides a recombinant protein comprising one or more of the L2 protein fragments of the five HPV serotypes, wherein the L2 protein fragments of the five HPV serotypes are selected from 6(13-75), 16(11-75), 18(12-75), 31(11-75), and 39(12-75).

[0009] Preferably, the recombinant protein comprises any two, three, four, or five of the L2 protein fragments of the five HPV serotypes mentioned above.

[0010] Preferably, the recombinant protein comprises (1): 6 (13-75) and (2): 16 (11-75), 18 (12-75), 31 (11-75) and 39 (12-75) connected in any order from nitrogen terminus to carbon terminus.

[0011] Preferably, the recombinant protein comprises (1): 16 (11-75) and (2): 6 (13-75), 18 (12-75), 31 (11-75), and 39 (12-75) connected in any order from nitrogen terminus to carbon terminus.

[0012] Preferably, the recombinant protein comprises (1): 18 (12-75) and (2): 6 (13-75), 16 (11-75), 31 (11-75), and 39 (12-75) connected in any order from nitrogen terminus to carbon terminus.

[0013] Preferably, the recombinant protein comprises (1): 31 (11-75) and (2): 6 (13-75), 16 (11-75), 18 (12-75), and 39 (12-75) connected in any order from nitrogen terminus to carbon terminus.

[0014] Preferably, the recombinant protein comprises (1): 39 (12-75) and (2): 6 (13-75), 16 (11-75), 18 (12-75), and 31 (11-75) connected in any order from nitrogen terminus to carbon terminus.

[0015] Preferably, the L2 protein fragments are directly linked together.

[0016] Preferably, the L2 protein fragments are indirectly linked by amino acids or polypeptides.

[0017] Preferably, the recombinant protein sequence is SEQ ID NO.1.

[0018] SEQ ID NO.1:

[0019] ASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLRASVTDLYKTCKQSGTCPPDVVPKVEGTTLADK ILQWSSLGIFLGGLGIGTGSGTGGRTGYIPLKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLRASATDLYRTCKQSGTCPPDVVDKVEGTTLADKILQWTSLGIFLGGLGIGTGTGTGGTGRTGYIPL.

[0020] Terminology Explanation: In this invention,

[0021] Unless otherwise specified, “linkage” includes direct linking or linking via endpoints. Protein fragments in the same protein sequence may have both direct links and endpoint links, or all may be directly linked or all may be linked via endpoints.

[0022] "6(13-75)" represents amino acids 13-75 of the HPV 6 L2 protein; the specific sequence is ASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPL.

[0023] "16(11-75)" represents amino acids 11-75 of the HPV 16 L2 protein; the specific sequence is KRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPL.

[0024] "18(12-75)" represents amino acids 12-75 of the HPV 18 L2 protein; the specific sequence is RASVTDLYKTCKQSGTCPPDVVPKVEGTTLADKILQWSSLGIFLGGLGIGTGSGTGGRTGYIPL.

[0025] “31(11-75)” represents amino acids 11-75 of the HPV 31 L2 protein; the specific sequence is KRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPL.

[0026] “39(12-75)” represents amino acids 12-75 of the HPV 39 L2 protein; the specific sequence is RASATDLYRTCKQSGTCPPDVVDKVEGTTLADKILQWTSLGIFLGGLGIGTGTGTGGRTGYIPL.

[0027] "6(13-75) connected with 16(11-75), 18(12-75), 31(11-75), 39(12-75) in any order" means that 6(13-75) is the first digit, followed by 16(11-75), 18(12-75), 31(11-75), 39(12-75) in any order. Examples, but not limited to: 6(13-75) / 16(11-75) / 18(12-75) / 31(11-75) / 39(12-75); 6(13-75) / 16(11-75) / 31(11-75) / 18(12-75) / 39(12-75); 6(13-75) / 18(12-75) / 16(11-75) / 39(12-75) / 31(11-75); 6(13-75) / 18(12-75) / 39(12-75) / 16(11-75) / 31(11-75), etc. Here, " / " indicates "connection," and the specific meaning of "connection" is as described above.

[0028] The following statements have the same meaning as those mentioned above: “16(11-75) connected with 6(13-75), 18(12-75), 31(11-75), 39(12-75) in any order”; “18(12-75) connected with 6(13-75), 16(11-75), 31(11-75), 39(12-75) in any order”; “31(11-75) connected with 6(13-75), 16(11-75), 18(12-75), 39(12-75) in any order”; “39(12-75) connected with 6(13-75), 16(11-75), 18(12-75), 31(11-75) in any order”.

[0029] In another aspect, the present invention provides an immune composition comprising the above-mentioned recombinant protein and MF59 adjuvant.

[0030] Preferably, the volume ratio of the recombinant protein to the MF59 adjuvant is 1:0.1-2, more preferably 1:0.5-2, and even more preferably 1:1.

[0031] In another aspect, the present invention provides an immune composition comprising the above-mentioned recombinant protein and aluminum adjuvant.

[0032] Preferably, the aluminum adjuvant is selected from at least one of aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum, and potassium alum.

[0033] Preferably, the mass ratio of the recombinant protein to the aluminum adjuvant is 1:0.5-5, more preferably 1:1-2.5, and most preferably 1:2.

[0034] In another aspect, the present invention provides the use of the above-mentioned recombinant protein or composition in the preparation of drugs and vaccines for the prevention and / or treatment of HPV infection.

[0035] In another aspect, the present invention provides a medicament for preventing and / or treating HPV infection, comprising the above-mentioned recombinant protein or composition.

[0036] The types of drugs mentioned include, but are not limited to, injectable drugs or mucosal drugs.

[0037] The administration methods of the drug include, but are not limited to: inhalation, transmucosal or transdermal administration, and parenteral (intradermal, intramuscular, subcutaneous, intravenous or intraperitoneal) administration.

[0038] The drug can be administered alone, or it can be administered simultaneously or sequentially with one or more further complexes.

[0039] Finally, the present invention provides a vaccine for the prevention and / or treatment of HPV infection, comprising the above-described recombinant protein or composition.

[0040] The vaccine comprises the aforementioned T-cell epitope peptides and / or tandem polypeptides and / or chimeric or fusion polypeptides and / or biological vectors and / or Ii-key hybrid constructs.

[0041] The vaccine also includes pharmaceutically acceptable carriers, excipients, diluents, adjuvants, lyophilization stabilizers, wetting or emulsifying agents, pH buffers, gelling or thickening additives, and preservatives.

[0042] Examples of pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited to, demineralized or distilled water, saline solutions, vegetable oils, silicone oils, volatile organosilicones, mineral oils, lower polyalkylene glycols or lower alkyl glycols, fatty acid esters, polyvinylpyrrolidone, agar, carrageenan, tragacanth gum, gum arabic, and petrolatum.

[0043] Furthermore, the vegetable oils mentioned include, but are not limited to: peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, or coconut oil.

[0044] Furthermore, the silicone oil includes, but is not limited to, polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane, and methylphenyl polysiloxane.

[0045] Furthermore, the mineral oil includes, but is not limited to, p-hydroxysiloxane.

[0046] The lower polyalkyl glycols or lower alkyl glycols mentioned include, but are not limited to: polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol, or glycerol.

[0047] The fatty acid esters mentioned include, but are not limited to, isopropyl palmitate, isopropyl myristate, or ethyl oleate.

[0048] In some embodiments, the vaccine complex may be a liquid formulation, such as an aqueous solution, a water-in-oil emulsion, or an oil-in-water emulsion, or a formulation for subcutaneous, intradermal, intramuscular, or intravenous administration, such as a sterile suspension or emulsion. Liquid formulations may also include suspensions and emulsions containing suspending agents or emulsifiers. Routes of administration may include transdermal, mucosal, intradermal, intramuscular, subcutaneous, intravenous, or intraperitoneal administration.

[0049] In some embodiments, the vaccine is in the form of a vaccination kit. The present invention also provides a vaccination kit comprising an optional sterile container including an immunogenic dose of the vaccine, a device for administering the vaccine, and optional instructions including management information on the immunogenic dose of the complex for treating and / or preventing HPV infection-related diseases.

[0050] The beneficial effects of this invention are as follows:

[0051] This invention provides a recombinant protein and its application in HPV prevention and / or treatment. Compared with the existing nine-valent vaccine, this recombinant protein and its composition with different adjuvants can simultaneously produce good preventive and / or therapeutic effects against 17 HPV subtypes, namely HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73, achieving highly efficient prevention and / or treatment of HPV. Attached Figure Description

[0052] Figure 1 The chromatogram is for purification by chromatography 1. Detailed Implementation

[0053] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and such changes should also fall within the scope of protection claimed in this application.

[0054] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0055] In the following examples, the aluminum phosphate used was purchased from CRODA, catalog number 21645-51-2; and the MF59 used was purchased from Invitrogen, catalog number Vac-adx-10.

[0056] Example 1: Preparation of Recombinant Protein

[0057] (1) Preparation of recombinant HPV target antigen

[0058] Preparation of pET-L2 recombinant plasmid: The gene sequence SEQ ID NO.1 was synthesized and constructed into the Pet expression vector, transformed into the E. coli cloning vector, and after culturing, the recombinant plasmid was extracted using a plasmid extraction kit.

[0059] The pET L2 recombinant plasmid was transformed into an E. coli expression strain. Recombinants were picked and sequenced. Single colonies of the verified recombinants were inoculated into LB liquid medium and cultured overnight at 37°C with constant temperature shaking at 220 rpm. 200 μL of the medium was transferred to a medium containing antibiotics and cultured under the same conditions. When the absorbance A value of the culture medium reached 0.6, IPTG was added to induce the expression of the fusion protein. The bacterial cells were then collected and stored at -80°C for later use.

[0060] (2) Purification of recombinant HPV target antigen

[0061] After bacterial resuspension, the cells were homogenized using an autoclave, and the supernatant was removed by centrifugation. Inclusion body precipitates were washed with washing buffer and dissolved in guanidine hydrochloride solution. The cells were then equilibrated with a hydrophobic equilibrium buffer and eluted with buffer solutions of different concentrations. The collected flow-through, P1, and P2 collection solutions were analyzed by 10% SDS-PAGE to assess the purification efficiency of the target protein. The first step, hydrophobic chromatography, used packing materials including, but not limited to, Capto Phenyl, Capto Phenyl Impress, Phenyl-Sepharose 6 Fast Flow, and Fractogel EMD Phenyl, among other phenyl-related hydrophobic packing materials. The second step, cation chromatography, used packing materials including, but not limited to, Capto S, Capto SP Impress, and Fractogel. EMD SO3-(M), Fractogel EMDSE Hicap(M), Fractogel EMD COO-(M), etc.; third step molecular sieve chromatography, S200, Superdex 200 pg, Superdex 75 pg, etc. See chromatography chromatogram 1. Figure 1 .

[0062] The sequence of the prepared recombinant protein is shown in SEQ ID NO.1.

[0063] SEQ ID NO.1:

[0064] ASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLRASVTDLYKTCKQSGTCPPDVVPKVEGTTLADK ILQWSSLGIFLGGLGIGTGSGTGGRTGYIPLKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLRASATDLYRTCKQSGTCPPDVVDKVEGTTLADKILQWTSLGIFLGGLGIGTGTGTGGTGRTGYIPL.

[0065] The prepared recombinant protein was subjected to relevant tests, including content, purity, sterility, endotoxin, host DNA residue, and host protein residue. The test results are shown in the table below.

[0066]

[0067] Example 2: Preparation of the Immunocomposition

[0068] Adjuvant-free group: The recombinant protein obtained in Example 1 was diluted with physiological saline to 0.2 mg / ml.

[0069] MF59 adjuvant group: The recombinant protein solution obtained in Example 1 was mixed with MF59 adjuvant at a volume ratio of 1:2.

[0070] AL adjuvant group: The recombinant protein obtained in Example 1 was mixed with AL adjuvant at a mass ratio of 1:2.

[0071] Example 3: Immune Process

[0072] The immunogenicity of the prepared immunogenic composition was compared with that of HPV 9 (Gardasil 9 group) (rabbit).

[0073] Rabbits were administered medication via intramuscular injection (lateral thigh muscle, including but not limited to the gluteus maximus and quadriceps femoris muscles; in this experiment, the gluteus maximus was used). Each injection consisted of 0.5 ml per rabbit (containing 0.1 mg of recombinant protein), administered three times daily on days 0, 21, and 42, with each injection being 0.5 ml. Blood samples were collected from the marginal ear vein on days 2, 40, and 61, ensuring a minimum blood volume of 1 ml. Serum was stored at -80°C for subsequent experimental analysis. The titer of anti-L2 antibodies in rabbit serum was detected using ELISA. The neutralizing titer in serum was determined using a pseudovirus neutralization assay. Analysis of variance was performed on the data from each group; P < 0.05 was considered statistically significant.

[0074] The detection process is based on patent CN112439059B, and the immunogenicity evaluation method is a standard technical means in this field. In this application, the more specific operation method of the pseudovirus neutralization test is as follows:

[0075] 2×10 6 MCF 10A cells per ml were seeded into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. After treatment with cell lysis buffer, the cell matrix membrane was prepared.

[0076] The prepared HPV pseudovirus was inoculated into a cell matrix membrane and then cultured in a 37°C, 5% CO2 incubator for 72 h. CHO-furin cells expressing furinase were added to the upper phase, and titrated diluted serum was added. The cells were incubated at 37°C for 12 h. Then, pgsA-745 cells were inoculated into the matrix membrane and cultured at 37°C, 5% CO2 for 72 h. The cell culture plate was removed, and the reaction solution was taken out according to the luciferase detection kit. The luminescence intensity value was read in a 96-well chemiluminescence detection plate.

[0077] Neutralization inhibition rate:

[0078] Inhibition rate = [1 - (mean luminescence intensity of test sample group - mean CC of cell control) / (mean luminescence intensity of virus control VC - mean CC of cell control)] × 100%.

[0079] Based on the neutralization inhibition rate results, IC was calculated using the Reed-Muench method. 50 .

[0080] Formula 1: Distance ratio = (Percentage of inhibition rate higher than 50% - 50%) / (Percentage of inhibition rate higher than 50% - Percentage of inhibition rate lower than 50%);

[0081] Formula 2: lg(neutralizing antibody titer) = logarithm of the inhibition rate of dilutions above 50% + distance ratio × logarithm of dilution factor.

[0082] Neutralizing antibody titers were calculated, and comparisons were performed against 17 HPV types: 6 / 11 / 16 / 18 / 31 / 33 / 45 / 39 / 45 / 51 / 52 / 56 / 58 / 59 / 66 / 68 / 73. The results are as follows:

[0083]

[0084]

[0085] The results show that the recombinant protein of the present invention can simultaneously produce good preventive and / or therapeutic effects against 17 HPV subtypes, including HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 73. Furthermore, the effect can be further enhanced when used in combination with adjuvants. Moreover, compared to Gardasil group 9 (HPV 9-valent), the recombinant protein of the present invention has been shown to be more effective in preventing and / or treating HPV by testing for an additional 8 high-risk HPV subtypes.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that, The recombinant protein sequence is SEQ ID NO.

1.

2. An immune composition, characterized in that, It includes the recombinant protein of claim 1 and MF59 adjuvant.

3. The immune composition according to claim 2, characterized in that, The volume ratio of the recombinant protein to MF59 adjuvant is 1:0.1-2.

4. An immune composition, characterized in that, Includes the recombinant protein of claim 1 and the aluminum adjuvant.

5. The immune composition according to claim 4, characterized in that, The aluminum adjuvant is selected from at least one of aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum, and potassium alum.

6. The immune composition according to claim 4, characterized in that, The mass ratio of the recombinant protein to the aluminum adjuvant is 1:0.5-5.

7. The use of the recombinant protein of claim 1 or the composition of any one of claims 2-6 in the preparation of a medicament for the prevention and / or treatment of HPV infection.

8. The application according to claim 7, characterized in that, The drugs mentioned include vaccines.

9. A drug for preventing and / or treating HPV infection, characterized in that, Includes the recombinant protein of claim 1 or the composition of any one of claims 2-6.

10. A vaccine for the prevention and / or treatment of HPV infection, characterized in that, Includes the recombinant protein of claim 1 or the composition of any one of claims 2-6.

Images