WO2016026401A1 - 增强hpv抗原表位肽免疫原性的方法及类病毒颗粒、颗粒制备方法与应用 - Google Patents

增强hpv抗原表位肽免疫原性的方法及类病毒颗粒、颗粒制备方法与应用 Download PDF

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WO2016026401A1
WO2016026401A1 PCT/CN2015/086636 CN2015086636W WO2016026401A1 WO 2016026401 A1 WO2016026401 A1 WO 2016026401A1 CN 2015086636 W CN2015086636 W CN 2015086636W WO 2016026401 A1 WO2016026401 A1 WO 2016026401A1
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hpv
hbc
antigen
vlp
enhancing
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French (fr)
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邵忠琦
李军强
蒋蓉
杨鸣鸣
朱涛
毛慧华
宇学峰
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天津康希诺生物技术有限公司
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Priority to US15/438,790 priority Critical patent/US10125175B2/en

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]

Definitions

  • the invention relates to the technical field of vaccine preparation, in particular to a method for enhancing the immunogenicity of an HPV antigen epitope peptide, a virus-like particle, a particle preparation method and an application thereof.
  • Human papillomavirus belongs to the genus Papillomavirus of the papillomavirus, which is a non-enveloped double-stranded circular epithelial DNA virus with a small molecular weight, which invades the reproductive tract scale. Epithelial cells and epithelial cells of other tissues and organs cause differentiation of cells, causing local epithelial hyperplasia, thickening or papillary appearance.
  • the HPV genome is a double-stranded circular DNA, which exists in the form of a covalently closed supercoiled structure, an open circular structure, and a linear molecule.
  • the HPV genome encodes nine open reading frames and is divided into three functional regions, the early transcribed region. Late transcribed regions and non-transcribed regions.
  • the early transcribed region also known as the E region, encodes eight early proteins such as E1, E2, E3, E4, E5, E6, E7, and E8, and is involved in viral DNA replication, transcription, translational regulation, and cell transformation.
  • the late transcribed region encodes the major capsid protein L1 and the minor capsid protein L2.
  • HPV L1 can be expressed in yeast or insect cells and self-assembled into VLPs of the same size as real virus particles. It can also be co-assembled into VLPs with L2 proteins. Fluorescence analysis of VLP from morphology and antigen is almost identical to natural viruses. It is identical and exhibits good immunogenicity and tolerance. After immunizing the body, it can induce high titer neutralizing antibodies. This method can produce HPV L1 VLP vaccine.
  • HPV vaccine The most mature HPV vaccine is designed for the L1 antigen, and the representative product is Gardasil, which is produced by Merck, which is marketed, including the four-valent vaccine of HPV 6, 11, 16, 18, and the Cervarix---HPV16 of GSK.
  • 18-valent vaccine in addition to the 9-valent HPV vaccine developed by Merck, has completed Phase III clinical studies, and the results show that the protection of cervical, vaginal and vulvar precancerous lesions caused by HPV-31, 33, 45, 52, 58 About 97%, and the immune response to HPV-6, 11, 16, 18 is no less than Gardasil, it is expected that the 9-valent vaccine can also be approved soon.
  • HPV's L2 is immunogenic and can stimulate the body to produce neutralizing antibodies
  • the research on prophylactic vaccine against HPV L2 has made little progress, partly due to the neutralization of HPV L2 neutralizing epitopes. It is not related to the immune cause of the body.
  • HPV16 L2 aa17-36 polypeptide-immunized mouse serum was able to neutralize HPV5, HPV6, HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, HPV58 pseudoviruses. It has been confirmed that L2 has multiple cross-neutralizing epitopes, and the peptide sequences with cross-neutralizing activity are linked together, which can improve the rendering efficiency of L2 cross-neutralizing epitopes. It is a promising broad-spectrum HPV vaccine development. One of the ways, but the only regret is that the pure peptide coupling molecular weight is too small to effectively stimulate the body's immune response.
  • the technical problem to be solved by the present invention is to provide a method for enhancing the immunogenicity of an HPV epitope peptide.
  • Another technical problem to be solved by the present invention is to obtain a viroid-like particle which can display a human papillomavirus antigen peptide by the method of enhancing the immunogenicity of an HPV epitope peptide.
  • Another technical problem to be solved by the present invention is to provide a method for preparing the virus-like particles.
  • Another technical problem to be solved by the present invention is to provide an application of the virus-like particles.
  • the technical solution of the present invention is:
  • HPV antigen gene is assembled into a gene of HBc (a protein carried by hepatitis B virus, which is also a core antigen of hepatitis B virus), and the fusion protein is expressed in vitro, and the integrated gene is passed.
  • HBc a protein carried by hepatitis B virus, which is also a core antigen of hepatitis B virus
  • the fusion protein is expressed in vitro, and the integrated gene is passed.
  • Exogenous expression causes HBc to form VLPs (viral-like particles), and the VLP surface displays multiple copies of HPV antigen, resulting in HBc-HPV VLPs.
  • the method for enhancing the immunogenicity of an HPV epitope peptide wherein the HPV antigen gene encodes a major capsid protein L1 or a late transcribed region of HPV encoding a minor capsid protein L2.
  • the above method for enhancing the immunogenicity of an HPV epitope peptide wherein the HPV antigen gene encodes a secondary capsid protein L2.
  • the above method for enhancing the immunogenicity of an HPV epitope peptide wherein the HPV antigen expressed on the surface of the VLP is an antigen peptide of L1 or L2, or a plurality of antigen peptides of L1 or L2, or a plurality of L1 or L2 Coupling of antigenic peptides.
  • the HPV antigen expressed on the surface of the VLP is a coupling of two antigen peptides of L2, and the sequences of the two antigen peptides are
  • the above method for enhancing the immunogenicity of an HPV epitope peptide the HPV antigen peptide is completely violent It is exposed on the surface of the viroid-like particles formed by HBc, and the surface of the VLP exhibits more than one copy of the HPV antigen peptide.
  • the above method for enhancing the immunogenicity of the HPV epitope peptide can insert more foreign genes into the HBc gene without affecting the formation of HBc virus particles.
  • the above method for enhancing the immunogenicity of the HPV epitope peptide obtains a viroid-like particle capable of expressing a human papillomavirus antigen peptide, which is an HBc-L2 fusion protein having a sequence represented by ⁇ 400>8 in the sequence table, that is, in the embodiment The whole genome sequence in Table 1.
  • the above method for enhancing the immunogenicity of the HPV epitope peptide obtains a viroid-like particle which can display a human papillomavirus antigen peptide which can self-assemble the VLP after being solublely expressed in an E. coli expression system.
  • the cloning vector was transformed into DH5a, and the monoclonal was picked, inoculated into 5 ml of LB medium, and cultured at 37 ° C and 200 rpm overnight;
  • the plasmid was extracted according to the plasmid extraction kit instructions, and the plasmid extraction effect was identified by 1% agarose gel electrophoresis;
  • the ligation product was transformed into BL21 (DE3), and the monoclonal was picked for PCR identification. Then the positive clone was inoculated into LB medium and the plasmid was extracted for enzyme digestion.
  • the cloning vector of the step (1) is constructed by life technology company, and the outsourcing company synthesizes the target gene (including the BamHI and NdeI restriction sites at both ends) and ligates the T vector to constitute a cloning vector.
  • the specific steps of expression and purification of the HBc-HPV VLP are:
  • IPTG was added to induce expression of the target protein, wherein the induction condition was 1 mM IPTG, Low temperature induction at 25 ° C;
  • Protein precipitation protein supernatant was slowly added (NH 4 ) 2 SO 4 powder under magnetic stirring, the final concentration was 10% w / v, after all the powder was dissolved, the beaker was placed at 4 ° C for 2 h;
  • Membrane package washing 100KDa membrane package is repeatedly washed and filtered, the purpose of which is to remove small molecular weight protein, while concentrating the volume of the resuspension, and after concentrating, the volume is concentrated to about 10 ml, and the filter membrane is passed through 0.22 ⁇ m;
  • Protein purity and VLP particle size were identified by molecular sieve high performance liquid chromatography (SEC-HPLC) and HPLC-multi-angle laser light scattering (MALS).
  • the above-mentioned virus-like particles are used in the preparation of a HPV liquid injection vaccine which does not contain any adjuvant and protective agent, and the specific steps are as follows:
  • Buffer replacement The buffer solution of the vaccine stock solution was replaced with 20 mg/ml NaCl, or 4 mM PB or 8 mg/ml NaCl + 2 mM PB (2), and the protein concentration was determined. The concentration measurement method was determined by reference protein concentration. Kit instructions;
  • the above-mentioned virus-like particles are used in the preparation of an HPV vaccine, wherein the vaccine final product comprises an aluminum hydroxide adjuvant, and the specific steps are as follows:
  • Buffer replacement The buffer solution of the vaccine stock solution was replaced with 20 mg/ml NaCl, or 4 mM PB or 8 mg/ml NaCl + 2 mM PB (2), and the protein concentration was determined. The concentration measurement method was determined by reference protein concentration. Kit instructions;
  • the above method for enhancing the immunogenicity of the HPV epitope peptide overcomes the disadvantages of poor immunogenicity of the prior art antigen peptide, overcomes the influence of the insertion sequence on the assembly of HBc VLP, and rationally designs the insertion site of the L2 antigen peptide in HBc.
  • the surface of the HBc-VLP is covered with not only one copy number of the L2 antigen peptide, that is, the system is on the outer surface of the pseudovirus, and can present not only one copy number of the L2 antigen peptide, but also the HPV L2 neutralizing epitope completely by the HBc pseudovirus.
  • the particles are displayed on the surface of the virion, thereby effectively enhancing the immunogenicity of the L2 neutralizing epitope polypeptide; the obtained HBc-L2 fusion protein can be automatically assembled into a VLP after being soluble, thereby eliminating the cumbersome formation of VLP by in vitro renaturation of the protein in vitro.
  • the process greatly improves the recovery of antigenic VLPs; the resulting HBc-L2 fusion protein has important applications in the prevention of vaccine production for HPV infection.
  • Figure 1 Enzyme map of HBc-L2-pET 9a expression vector, showing that the DNA fragment of about 670 bp is obtained by enzyme digestion, which is consistent with the theoretical size of the fusion gene;
  • FIG. 2 HBc-L2 induced expression identification, the figure shows that after IPTG induced expression, the cells were collected by centrifugation, and the supernatant and supernatant electrophoresis after sonication showed that the target proteins were mainly in the supernatant, indicating that the constructed fusion gene was mainly Expressing expression of the target protein by soluble expression;
  • FIG. 3 HPLC identification of HBc-L2 VLPs, which shows that the experimentally designed HPV L2 fusion polypeptide has only 52 amino acids, theoretically a molecular weight of approximately 22 KD, but by SEC-HPLC analysis (using TSK G5000), the molecular weight of the purified product obtained ( Retention time 14min) is much larger than BSA protein (molecular weight 66KD, retention time is about 23min), suggesting that the fusion protein self-assembly forms VLP particles;
  • FIG. 4 MALS identifies HBc-L2 VLPs, which show that combined SEC-HPLC and MALS results can be It can be seen that there are macromolecular particles with uniform molecular weight in the expression product;
  • FIG. 5 Electron micrograph of HBc-L2 VLP.
  • the electron micrograph shows that the fusion protein successfully self-assembled into VLP, which is consistent with the results of SEC-HPLC and MALS.
  • the fusion protein self-assembly
  • the efficiency of VLP particles is high, and the uniformity of VLP particles is good;
  • FIG. 6 CL-4B separation of 100KD membrane package ultrafiltrate purification map, the figure shows 100KDa ultrafiltration concentrate, CLLP can effectively separate VLP, the arrow peak in the figure is the fusion protein obtained by CL-4B VLP particles;
  • Figure 7 HBc-L2 VLP particle purity analysis (HPLC), the results show the purity analysis of VLP purified antigen SEC-HPLC, the purity of the target peak indicated by the arrow in the figure reaches 85%;
  • FIG. 8 Hepatitis B virus HBc protein VLP assembly diagram, showing that the HBc protein of hepatitis B virus (HBV) can be automatically assembled into VLP particles after expression in E. coli;
  • Figure 9 Simulation of the HBc-L2 VLP vaccine design. The figure shows that the anterior segment of the HBc protein can be inserted into the anterior and posterior segments without affecting the formation of VLPs. More importantly, the inserted antigens are assembled by HBc protein VLPs. Show all outside.
  • Outsourcing Life technology synthesizes HBc-L2 fusion gene (designed with restriction enzyme sites at both ends of the fusion gene), fusion gene-linked T vector, and fusion gene information as shown in Table 1:
  • the electrophoresis picture provided in this embodiment is only an electrophoresis picture for firstly exploring whether the expression vector is soluble or not, and does not represent the true expression amount in the embodiment. After optimization of the medium and adjustment of the fermentation process, the expression amount can be obtained.
  • the electrophoresis picture shows a process several times higher.
  • the cells were collected by fermentation, and after sonication, the supernatant collected by centrifugation was simply and purely purified, and macromolecular particles were present in the supernatant by SEC-HPLC and MALS.
  • the crude sample was identified by electron microscopy to confirm the presence of VLP particles.
  • the cells were collected by centrifugation, and the cells were resuspended in 400 mL of TGE buffer (50 mM Tris, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol), and ultrasonically disrupted in an ice bath;
  • TGE buffer 50 mM Tris, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol
  • VLP particle identification the concentrate was subjected to SEC-HPLC and MALS detection. The results are shown in Figure 3 and Figure 4. The macromolecular particles were analyzed by SEC-HPLC and MALS. After identification by electron microscopy, the macromolecular particles were identified. The VLP structure, the results are shown in Figure 5.
  • Example 2 On the basis of Example 2, the purification method of HBc-L2 VLP antigen was further improved.
  • the 100KD membrane was coated with ultrafiltration concentrate and passed through molecular sieve; after purification by CL-4B or sepharose-4FF, the purity of target antigen VLP was at least 80.
  • the purification process has the characteristics of simple process, high recovery rate and easy amplification; specifically, the specific steps of the VLP purification process of the present invention are as follows:
  • Protein precipitation protein supernatant was slowly added (NH 4 ) 2 SO 4 powder under magnetic stirring, the final concentration was 10% w / v, after all the powder was dissolved, the beaker was placed at 4 ° C for 2 h;
  • Membrane package washing 100KDa membrane pack is repeatedly washed and filtered, the purpose of which is to remove small molecular weight protein, while concentrating the volume of the resuspension, and after concentrating, the concentration is concentrated to about 10 ml;
  • the designed vaccine volume is 0.5ml, and the protein content of each dose containing VLP is 25 ⁇ g or 50ug.
  • the adjuvant may or may not be added to the preparation; if the adjuvant is added, it is aluminum hydroxide adjuvant, preparation buffer For NaCl or NaCl and PB, see Tables 3 and 4.
  • an antigen of at least 80% purity of the HBc-L2 VLP antigen was prepared and immunized against the mouse.
  • the L2 antigen peptide with GST tag was constructed in the experiment, and GST-L2 was used as the control in the VLP immunogenicity study. table 5.
  • the serum of the second and third exempted samples were collected, and the serum titer of the mice was detected by ELISA.
  • the ELISA used was a commonly used experimental method in the laboratory.
  • the experiment selected HPV-L2 antigen polypeptide as the coating antigen, and tested the anti-L2 IgG antibody titer in the serum of the mouse, which was coated at a concentration of 5 ug/ml, 100 ul/well, and the coating condition was 4 ° C overnight; with 2% BSA
  • the ELISA plate was blocked in a 200 ul/well manner.
  • the reaction time of serum and secondary antibody was 1 h, and after washing at the end of each reaction, it was washed 5 times with 0.1% PBST. After the end of the secondary antibody reaction, TMB was added for color development, and the results of OD450 were measured, as shown in Table 6.
  • the HBc-L2 VLP antigenicity study showed that the immune effect of HBc-L2 VLP was much better than that of the GST-L2 antigen alone.
  • the serum triad free effect was stronger than that of the second exemption, suggesting that the HBc-L2 VLP has an immunopotentiating effect.
  • aluminum adjuvant can significantly improve the immune response of the antigen.
  • the results of animal experiments show that the L2 antigen polypeptide is constructed by HBc-L2 recombination, and the VLP particles can stimulate the body to produce an obvious immune response, and the antibody level can provide sufficient protection.
  • HPV-specific neutralizing antibodies in the serum of three-free mice were determined by HPV pseudovirus neutralization assay, in order to reflect the protective effect of the vaccine.
  • the host cell for neutralization assay is 293FT.
  • the specific experimental steps are as follows:
  • the experimentally designed L2 antigen peptide is derived from HPV16, it can be seen that the antigenic epitope peptide has cross-neutralizing activity, and can simultaneously neutralize HPV16 and HPV18 type pseudovirus efficiently.
  • HBV protein of HBV After expression in E. coli, it can be assembled into VLP particles automatically.
  • the exogenous antigen can be inserted into the anterior, posterior and intermediate portions of the HBc protein without affecting the formation of VLP. More importantly, as shown in Fig. 9, the inserted antigen is all exposed to the outside of the HBc VLP.
  • HBc-L2 VLP can effectively improve the immunogenicity of L2 antigen peptide, and the insertion of L2 gene does not affect the formation of HBc VLP, the inserted epitope peptide It can be the same, or it can be different, and it can be a fusion gene of two epitopes.
  • the fusion protein comprises a linker LE between the vaccine vector and an HPV L2 neutralizing epitope, and the addition of the flexible linker prevents the antigenic protein and HBc from forming a VLP.
  • the mutual influence helps to form the correct conformation; in addition, the foreign gene is not only one at the insertion site of HBc, but these sites can be inserted into the same polypeptide, and different polypeptides can be inserted; the foreign gene can be inserted in front of HBc. , intermediate and posterior, and does not affect the formation of HBc VLP; the foreign gene inserted into HBc can be a neutralizing epitope, or two neutralizing epitopes, which can be inserted into one site or inserted into different sites. More preferably, two neutralizing epitopes are expressed in the same locus.
  • the design of the HPV L2 neutralizing epitope peptide enables the HBc-L2 VLP antigen to have a broad spectrum of antiviral ability, expanding the serotype restriction for the L1 designed vaccine; and preparing the HBc-L2VLP particle size.
  • the present invention provides a broader spectrum of prophylactic HPV vaccines
  • HPV vaccines currently marketed are designed for the L1 antigen and have significant serum specificity, while the HPV16/18 bivalent vaccine can only inhibit about 70% of HPV infections. In order to make the vaccine spectrum more comprehensive, it needs to be designed to include more Multi-serotype HPV multivalent vaccines, such as the tetravalent vaccine developed by Merck and the currently completed 9-valent vaccine. Studies have found that L2 has multiple cross-neutralizing epitopes, either alone or in combination with cross-neutralizing epitopes, to prevent more serotypes of HPV.
  • the present invention selects an antigen peptide having a broader cross-neutralizing epitope as a target gene, and obtaining an HPV virus in which HBc-L2 VLP can neutralize a plurality of serotypes is a HPV vaccine having a broader spectrum of prevention.
  • the present invention provides an experimental method for enhancing immunity of an antigen polypeptide
  • the molecular weight of the neutralizing epitope antigen polypeptide of HPV L2 is very small, and it is difficult to stimulate the body to produce a sufficiently strong immune response by single immunization, although a large number of reports have reported that HPV L2 can produce neutralizing antibodies, and there is a cross-neutralizing epitope on L2. But the reason for the slow progress of the HPV L2 vaccine.
  • HBc-VLP antigen The delivery vector, the designed HBc-L2 VLP antigen firstly increases the molecular weight and spatial structure of the antigen, can effectively stimulate the immune response of the organism, and more importantly based on the insertion site setting in the embodiment of the present invention, HBc VLP The surface is covered by the HPV L2 antigen peptide.
  • the L2 cross-neutralizing epitope antigen peptide has a large number of copies on the surface of the HBc VLP, and the structure of the HBc VLP ensures that the L2 cross-neutralizing epitope antigenic peptide active site is completely exposed, based on the implementation of the present invention.
  • the designed HBc-L2 VLP immunized mice can stimulate the body to produce a good immune response.
  • HPV L1 expression can self-assemble VLPs in vitro, the self-assembly of L1 antigens has a low recovery rate of pseudovirions.
  • the HPV virus envelope is an icosahedral structure formed by the formation of 72 L1 protein pentamers.
  • the L1 protein expressed in vitro constitutes a pseudoviral particle similar to the HPV virus, which requires five L1 monomers to form pentamers and 72 pentamers.
  • the VLP formed by self-assembly is only partially icosahedral.
  • the L2 antigen polypeptide is presented based on the HBc VLP, which greatly increases the VLP yield, and is expected to greatly reduce the cost of the HPV vaccine.
  • the present invention does not affect the assembly efficiency of HBc VLPs while inserting L2 and epitope gene sequences.
  • HBc-VLP antigen-presenting vector
  • the fusion protein can be self-assembled into a VLP after being soluble in Escherichia coli, and the assembly efficiency of the VLP is not lower than the yield of the HBc VLP alone; the uniformity of the VLP is also very good by electron microscopic analysis. it is good.
  • the neutralizing epitope of the L2 antigen has multiple ways at the insertion site of HBc.
  • the insertion site of L2 in HBc may be the front end, the middle portion or the rear end of the HBc protein, and the L2 epitope peptide may be inserted into one site, or two or three bits may be inserted at the same time.
  • one antigen may be inserted into the epitope, and two epitopes may be inserted; similarly, the insertion sites of the two fusion epitope peptides in this embodiment may be the same site or different sites.
  • the similar products developed have also achieved great success.
  • the method for carrying out the enhanced antigen polypeptide provided by the present embodiment can be applied to an immunogenic polypeptide-enhancing example such as L2 neutralizing epitope and the like.
  • HBc-VLP hepatitis B core antigen VLP
  • HBc-VLP hepatitis B core antigen VLP
  • HBc-VLP is a potent antigenic vector, and antigen presented on HBc-VLP can eliminate the need for adjuvants and produce Th1 immune responses.
  • HBc-VLP is non-infectious. Unlike other VLPs, they are not involved in viral receptor interactions and are not neutralizing antibody targets, so vector inhibition is not a major challenge.
  • the use of HBc-VLP as a vaccine delivery system can effectively overcome the shortcomings of small molecular weight polypeptide sequences that cannot effectively stimulate immunity.
  • Inserting the neutralizing epitope of HPV L2 into HBc and expressing the fusion protein in vitro can present the target antigen on the surface of the pseudovirus particle while forming the HBc VLP, forming VLP particles covering the HPV L2 neutralizing epitope and enhancing immunity. reaction.
  • the method for enhancing the immune response of the HPV epitope peptide and the method for preparing the virus-like particles are described in an illustrative manner, and are not limited thereto, and may be enumerated according to the limited range.
  • the method of the present invention can be applied to the development of an immunogenicity of a peptide of the same type of antigen, and thus variations and modifications without departing from the general inventive concept are within the scope of the present invention.

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Abstract

本发明公开了一种增强HPV抗原表位肽免疫原性的方法，把HPV抗原基因组装到HBc的基因上，将整合之后的基因通过外源表达系统表达融合蛋白，融合蛋白将自动组装形成类病毒颗粒VLP，且所述VLP表面展示多于一个拷贝的HPV抗原，得到HBc-HPV VLP。

Description

增强HPV抗原表位肽免疫原性的方法及类病毒颗粒、颗粒制备方法与应用 技术领域
本发明涉及疫苗制备技术领域，尤其是增强HPV抗原表位肽免疫原性的方法及类病毒颗粒、颗粒制备方法与应用。
背景技术
人乳头瘤病毒(human papillomavirus，HPV)属于乳头多瘤空泡病毒科乳头瘤病毒属，它是一类分子量较小的无包膜的双链环状嗜上皮性DNA病毒，通过入侵生殖道鳞状上皮细胞以及其他组织器官的上皮细胞，使细胞发生分化失调，引起局部上皮增生、增厚或呈乳头状。
HPV基因组是双链环状DNA，以共价闭合的超螺旋结构、开放的环状结构、线性分子3种形式存在，HPV基因组编码9个开放阅读框，分为3个功能区即早期转录区、晚期转录区和非转录区。早期转录区又称为E区，分别编码E1、E2、E3、E4、E5、E6、E7、E8等8个早期蛋白，参与病毒DNA的复制、转录、翻译调控和细胞转化等功能。晚期转录区编码主要衣壳蛋白L1和次要衣壳蛋白L2。HPV衣壳是由72个L1蛋白五聚体构成的T＝7的二十面体结构，因此一个病毒粒子上含有360个L1蛋白单体，L2蛋白位于L1构成的VLP(类病毒颗粒)上，每个病毒粒子上含有12个或者更多拷贝的L2蛋白。
HPV的L1可以在酵母菌或者昆虫细胞中表达，并自我组装成与真正病毒颗粒大小相同的VLP，也可以与L2蛋白共组装成VLP，VLP从形态学和抗原的荧光分析跟天然的病毒几乎完全相同，并表现出了很好的免疫原性及耐受性，免疫机体后可诱导产生高滴度的中和抗体，利用此方法可以生产HPV L1 VLP疫苗。
最为成熟的HPV疫苗是针对L1抗原设计的，代表产品为已获批上市的由Merck公司生产的Gardasil---包括HPV6、11、16、18的四价疫苗，和GSK的Cervarix---HPV16、18二价疫苗，此外Merck开发的9价HPV疫苗已经完成了III期临床研究，结果表明，对HPV-31、33、45、52、58引起的宫颈、阴道和外阴癌前病变的保护性大约有97％，而对HPV-6、11、16、18的免疫应答也不亚于Gardasil，预计9价疫苗也可以很快获批上市。
虽然有多篇文章报道HPV的L2具有免疫原性，可以激发机体产生中和抗体，但是针对HPV L2的预防性疫苗的研究进展不大，部分原因与单独蛋白表达HPV L2的中和表位，不能有效激发机体的免疫原因有关。
1999年，Kawana等首先报道在HPV16 L2 aa108—120存在HPV6的交叉中和表位，该表位的合成肽免疫的鼠血清能够同时抑制HPV16假病毒和HPV6假病毒的感染。Embers等从体内水平上验证了该交叉中和表位的合成肽能够诱导中和性抗体，并抑制乳头瘤病毒的感染。2007年，Gambhira等发现HPV16 L2 aa17—36多肽免疫的鼠血清能够中和HPV5、HPV6、HPV11、HPV16、HPV18、HPV31、HPV45、HPV52、HPV58假病毒。现已证实L2存在多个交叉中和表位，将具有交叉中和活性的肽段序列链接在一起，可提高L2交叉中和表位的呈递效率，是极具潜力的广谱性HPV疫苗研发途径之一，但唯一的遗憾是,纯粹的肽段偶联分子量太小，不能有效激发机体的免疫反应。
发明内容
本发明所要解决的技术问题在于提供增强HPV抗原表位肽免疫原性的方法。
本发明所要解决的另一技术问题在于利用所述增强HPV抗原表位肽免疫原性的方法获得可以展示人乳头瘤病毒抗原肽的类病毒颗粒。
本发明所要解决的另一技术问题在于提供所述类病毒颗粒的制备方法。
本发明所要解决的另一技术问题在于提供所述类病毒颗粒的应用。
为解决上述技术问题，本发明的技术方案是：
一种增强HPV抗原表位肽免疫原性的方法，把HPV抗原基因组装到HBc(乙肝病毒携带的蛋白，也是乙肝病毒的核心抗原)的基因上，体外表达融合蛋白，则整合之后的基因通过外源表达使HBc形成VLP(类病毒颗粒),且所述VLP表面展示多个拷贝的HPV抗原，得到HBc-HPV VLP。
优选的，上述增强HPV抗原表位肽免疫原性的方法，所述HPV抗原基因为HPV晚期转录区编码主要衣壳蛋白L1或HPV晚期转录区编码次要衣壳蛋白L2。
优选的，上述增强HPV抗原表位肽免疫原性的方法，所述HPV抗原基因为HPV晚期转录区编码次要衣壳蛋白L2。
优选的，上述增强HPV抗原表位肽免疫原性的方法，所述VLP表面表达的HPV抗原是L1或L2的一个抗原肽，或L1或L2的多个抗原肽，或L1或L2的多个抗原肽的偶联。
优选的，上述增强HPV抗原表位肽免疫原性的方法，所述VLP表面表达的HPV抗原是L2的两个抗原肽的偶联，所述两个抗原肽的序列为
ASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQ和GTGGRTGYIPLGTRPPT。
优选的，上述增强HPV抗原表位肽免疫原性的方法，所述HPV抗原肽完全暴 露在HBc形成的类病毒颗粒的表面，且VLP表面展示的HPV抗原肽多于一个拷贝。
优选的，上述增强HPV抗原表位肽免疫原性的方法，可以在HBc基因上插入更多的外源基因，且不影响HBc类病毒颗粒的形成。
上述增强HPV抗原表位肽免疫原性的方法获得可以表达人乳头瘤病毒抗原肽的类病毒颗粒，为HBc-L2融合蛋白，具有序列表中<400>8所示的序列，即实施例中表1中的全基因组序列。
优选的，上述增强HPV抗原表位肽免疫原性的方法获得可以展示人乳头瘤病毒抗原肽的类病毒颗粒，所述HBc-L2融合蛋白在大肠杆菌表达系统中可溶性表达后可自组装VLP。
上述类病毒颗粒的表达载体构建方法，具体步骤为：
(1)克隆载体转化DH5a，挑取单克隆，接种于5ml LB培养基中，37℃200rpm过夜培养；
(2)按照质粒提取试剂盒说明书提取质粒，1％琼脂糖凝胶电泳鉴定质粒提取效果；
(3)BamHI和NdeI双酶切克隆载体上的目标基因；
(4)按照胶回收试剂盒说明书回收目标基因；
(5)将回收的目标基因和经BamHI和NdeI双酶切后的表达载体质粒pET 9a通过连接酶进行连接，16℃过夜；
(6)连接产物转化BL21(DE3)，挑取单克隆进行PCR鉴定，继而将阳性克隆接种LB培养基后提取质粒进行酶切鉴定；
(7)阳性菌种扩增保存。
所述步骤(1)所述克隆载体由life technology公司构建，外包公司合成目标基因(-包括两端的BamHI和NdeI酶切位点)并连接T载体构成克隆载体。
优选的，上述类病毒颗粒的制备方法，所述HBc-HPV VLP的表达和纯化的具体步骤为：
Ⅰ、表达实验步骤为：
(1)菌种复苏：将10ul冻存菌液接种到50ml LB培养基中，37℃，250rpm摇菌过夜；
(2)菌种扩增：以5ml复苏菌液接种100mlLB培养基的比例扩大培养，2L三角瓶中加入1L的LB培养基，37℃250rpm摇菌至OD600＝0.6---0.7；
(3)诱导表达：加入IPTG诱导目标蛋白表达，其中诱导条件为1mM IPTG， 25℃低温诱导；
(4)菌体收获：诱导4h后，诱导结束，8000rpm离心10min，收集菌体沉淀；
(5)超声破碎：菌体用400mL TGE buffer(50mM Tris，0.5mM EDTA，50mM Nacl，5％甘油)重悬，冰浴超声破碎，镜检确保菌体完全破碎；
Ⅱ、纯化的实验步骤为：
(1)蛋白收集：超声破碎后，8000rpm离心10min，收集上清；
(2)蛋白沉淀：蛋白上清在磁力搅拌状态下缓慢加入(NH4)2SO4粉末，终浓度为10％w/v，待所有粉末溶解后将烧杯置于4℃静置2h；
(3)离心收集沉淀，计算沉淀重量，以每1g沉淀用60mL buffer的比例以TGE buffer重悬溶解；
(4)重悬液再次离心，收集上清，过0.22μm滤膜；
(5)膜包洗涤：100KDa的膜包反复洗滤，其目的是除去小分子量蛋白质，同时浓缩重悬液体积，洗滤结束后浓缩体积至10ml左右，过0.22μm滤膜；
(6)CL-4B凝胶分离纯化浓缩液，收集第一峰；
(7)采用分子筛高效液相色谱(SEC-HPLC)以及HPLC-多角度激光光散射(MALS)联用技术鉴定蛋白纯度和VLP颗粒大小。
上述类病毒颗粒在制备HPV疫苗方面的应用。
优选的，上述类病毒颗粒在制备不含有任何佐剂和保护剂的HPV液体针剂疫苗方面的应用，具体步骤如下：
(1)缓冲液置换：将疫苗原液的缓冲液置换为20mg/ml的NaCl，或者4mM的PB或者8mg/ml的NaCl+2mM的PB(2)，测定蛋白浓度，浓度测定方法参考蛋白浓度测定试剂盒说明书；
(3)补加相应的缓冲液至抗原浓度为100ug/ml，或者50ug/ml；
(4)无菌过滤，得到疫苗半成品；
(5)自动分装机分装疫苗半成品，0.5ml/剂；
(6)封口，如果疫苗产品分装在安剖瓶中的话，需要封口，如果分装在西林瓶中的话，需要压盖；
(7)贴签，4℃冷冻保存。
优选的，上述类病毒颗粒在制备HPV疫苗方面的应用，其中疫苗终产品包括氢氧化铝佐剂，具体的步骤如下：
(1)缓冲液置换：将疫苗原液的缓冲液置换为20mg/ml的NaCl，或者4mM的PB或者8mg/ml的NaCl+2mM的PB(2)，测定蛋白浓度，浓度测定方法参考蛋白浓度测定试剂盒说明书；
(3)补加相应的缓冲液至抗原浓度为900ug/ml，或者450ug/ml；
(4)无菌过滤，得到疫苗半成品；
(5)加入1％的氢氧化铝佐剂，补加缓冲液使得佐剂的终浓度＜0.1％，蛋白抗原的终浓度为100ug/ml，50ug/ml；
(5)自动分装机分装疫苗半成品，0.5ml/剂；
(6)封口，如果疫苗产品分装在安剖瓶中的话，需要封口，如果分装在西林瓶中的话，需要压盖；
(7)贴签，4℃冷冻保存。
本发明的有益效果是：
上述增强HPV抗原表位肽免疫原性的方法，克服了现有技术抗原肽免疫原性差的缺点，克服了插入序列对HBc VLP组装的影响，合理设计了L2抗原肽在HBc中的插入位点，使HBc-VLP表面覆盖着不仅一个拷贝数的L2抗原肽，即本系统在假病毒的外表面，可以呈递不仅一个拷贝数的L2抗原肽，使HPV L2中和表位完全被HBc假病毒颗粒展现在病毒粒子表面，从而有效的增强L2中和表位多肽的免疫原性；所得HBc-L2融合蛋白可溶表达后可自动组装为VLP，免除了体外蛋白以变性复性形成VLP的繁琐过程，极大的提高了抗原VLP的回收率；所得HBc-L2融合蛋白在预防HPV感染的疫苗生产方面具有重要应用。
附图说明
图1：HBc-L2-pET 9a表达载体酶切图，图中显示酶切得到670bp左右的DNA片段，和融合基因的理论大小相一致；
图2：HBc-L2诱导表达鉴定，图中显示IPTG诱导表达结束后，离心收集菌体，超声破碎后上清和沉淀电泳，结果表明，目标蛋白主要分别在上清中，说明构建的融合基因主要以可溶表达的形成表达目标蛋白；
图3：HPLC鉴定HBc-L2 VLP，图中显示实验设计的HPV L2融合多肽只有52个氨基酸，理论上分子量约22KD，但是经SEC-HPLC分析(使用TSK G5000)，获得的纯化产物的分子量(保留时间14min)远远大于BSA的蛋白(分子量66KD,保留时间大约在23min)，提示融合蛋白自组装形成VLP颗粒；
图4：MALS鉴定HBc-L2 VLP，图中显示结合SEC-HPLC以及MALS结果可以 看出，表达产物中存在分子量均一性好的大分子颗粒；
图5：HBc-L2 VLP电镜照片，图中显示电镜图片直观显示融合蛋白成功自组装为VLP，和SEC-HPLC以及MALS的结果相一致；此外，从电镜图片中可以看出，融合蛋白自组装VLP颗粒的效率高，VLP颗粒均一性好；
图6：CL-4B分离100KD膜包超滤液纯化图，图中显示100KDa超滤浓缩液，经CL-4B可以有效的分离得到VLP，图中箭头峰则为CL-4B分离得到的融合蛋白VLP颗粒；
图7：HBc-L2 VLP颗粒纯度分析(HPLC)，图中显示VLP纯化抗原SEC-HPLC纯度分析结果，图中箭头所指目标峰的纯度达到85％；
图8：乙肝病毒HBc蛋白VLP组装图，图中显示乙肝病毒(HBV)的HBc蛋白在大肠杆菌表达后，可以自动组装成VLP颗粒；
图9：HBc-L2 VLP疫苗设计模拟图，图中显示HBc蛋白的前段，后端以及中间可以插入外源抗原，且不影响VLP的形成，更重要的是插入抗原经HBc蛋白VLP组装后，全部展示于外面。
具体实施方式
实施例1 HBc-L2 VLP重组融合蛋白的构建
(1)HBc-L2基因序列的合成
外包Life technology合成HBc-L2融合基因(融合基因两端设计有酶切位点)，融合基因链接T载体，融合基因信息见表1：
表1 HBc-L2融合蛋白基因信息
Figure PCTCN2015086636-appb-000001
Figure PCTCN2015086636-appb-000002
(2)HBc-VLP诱导表达
BamHI,NdeI双酶切T载体上的目标序列，酶切片段连接pET 9a表达载体，转化BL21(DE3)，如图1所示，经酶切以及PCR验证转化成功。
(3)IPTG诱导表达
10ul冻存菌液接种50ml LB培养基，过夜复苏菌种，第二天早上以5ml复苏菌液接种100ml LB培养基的比例，转接扩大培养，待OD600＝0.6时，加入1mM的IPTG诱导表达，诱导温度为25℃；诱导4h后，离心收集沉淀，超声破碎，分别取破碎后的上清以及沉淀进行SDS-PAGE电泳分析，结果如图2所示，构建的表达载体主要以可溶表达的方式表达目标蛋白。
需要说明的是，在本实施例中基因合成，表达载体构建以及诱导表达、超声破碎和SDS-PAGE分析表达结果均为本领域常规的实验方法，任何熟悉该领域的人都掌握此实验技能，而整个诱导表达过程中使用的培养基则为常规的LB培养基。
并且，本实施例提供的电泳图片仅为初次探索表达载体是否可溶表达的电泳图片，并不代表本实施例中真正的表达量，经过培养基优化以及发酵工艺调整后，可以获得表达量为电泳图片所示数倍高的工艺。
实施例2 HBc-L2重组抗原融合蛋白VLP鉴定
发酵收集菌体，超声破碎后，离心收集的上清经简单粗纯，以SEC-HPLC以及MALS分析上清中存在大分子颗粒，将此粗纯样品经电镜分析鉴定，确认存在VLP颗粒。
HBc-L2重组蛋白VLP鉴定粗纯工艺：
(1)发酵结束后，离心收集菌体，菌体用400mL TGE buffer(50mM Tris，0.5mM EDTA，50mM NaCl，5％甘油)重悬，冰浴超声破碎；
(2)离心收集上清，磁力搅拌状态下缓慢加入10％w/v(NH4)2SO4粉末，待 所有粉末溶解后将烧杯置于4℃静置2h；
(3)离心收集沉淀，计算沉淀重量，后每1g用60mL buffer重悬溶解；
(4)离心收集上清，上清用0.22μm滤膜过滤，之后用100KD的膜包超滤；
(5)VLP颗粒鉴定，浓缩液进行SEC-HPLC与MALS检测，结果如图3和图4所示，经SEC-HPLC以及MALS分析存在大分子颗粒；后经电镜鉴定，明确此大分子颗粒具有VLP结构，结果如图5所示。
实施例3 HBc-L2重组抗原融合蛋白VLP纯化
在实施例2的基础上，进一步完善了HBc-L2 VLP抗原纯化方法，将100KD膜包超滤浓缩液，过分子筛；经CL-4B或者sepharose-4FF纯化后，目标抗原VLP的纯度至少达80％；此纯化工艺具有流程简单，回收率高，易于放大的特点；具体来讲，本发明所述VLP纯化工艺具体步骤如下：
(1)蛋白收集：超声破碎后，8000rpm离心10min，收集上清；
(2)蛋白沉淀：蛋白上清在磁力搅拌状态下缓慢加入(NH4)2SO4粉末，终浓度为10％w/v，待所有粉末溶解后将烧杯置于4℃静置2h；
(3)离心收集沉淀，计算沉淀重量，以每1g沉淀用60mL buffer的比例以TGE buffer重悬溶解；
(4)重悬液再次离心，收集上清，过0.22um的滤膜；
(5)膜包洗涤：100KDa的膜包反复洗滤，其目的是除去小分子量蛋白质，同时浓缩重悬液体积，洗滤结束后浓缩体积至10ml左右；
(6)CL-4B或者sepharose 4FF凝胶分离纯化浓缩液，收集第一峰；
(7)SEC-HPLC以及MALS鉴定蛋白纯度，100KD膜包超滤液过分子筛，分管收集，以SEC-HPLC分析蛋白分子量以及纯度，结果表明大分子颗粒分布于第一个峰中，且纯度在80％以上，结果如图6和图7所示，分析条件见下表2。
表2
Figure PCTCN2015086636-appb-000003
Figure PCTCN2015086636-appb-000004
实施例4 HBc-L2 VLP疫苗制剂
设计疫苗装量为0.5ml，每剂含有VLP的蛋白含量为25μg或者50ug，制剂中可以加入佐剂，也可以不含有佐剂；若加入佐剂，则为氢氧化铝佐剂，制剂缓冲液为NaCl或者NaCl和PB，见表3和表4。
表3 HBc-L2 VLP疫苗制剂(低剂量)
Figure PCTCN2015086636-appb-000005
表4 HBc-L2 VLP疫苗制剂(高剂量)
Figure PCTCN2015086636-appb-000006
实施例5 HBc-L2 VLP抗原小鼠免疫
制备HBc-L2 VLP抗原纯度至少80％的抗原，进行小鼠免疫。为了易于比较多肽经HBc有效包装VLP后抗原性增强的效果，实验单纯构建了带有GST标签的L2抗原肽，VLP免疫原性研究中以GST-L2作为对照；实验设计的小鼠免疫方案见表5。
表5 VLP抗原免疫原性研究实验方案
Figure PCTCN2015086636-appb-000007
采集二免以及三免后的血清，以ELISA检测小鼠血清效价，所采用的ELISA为实验室常用的实验方法。实验选择以HPV-L2抗原多肽为包被抗原，检测小鼠血清中抗L2 IgG抗体效价，其中包被浓度为5ug/ml，100ul/well，包被条件为4℃过夜；以2％BSA，200ul/well的方式封闭ELISA板。血清检测时，血清以及二抗的反应时间为1h，每次反应结束后以0.1％PBST洗涤5次，二抗反应结束后加入TMB显色，OD450测定实验结果，见表6。
表6 VLP抗原免疫原性实验结果
Figure PCTCN2015086636-appb-000008
HBc-L2 VLP抗原性研究结果表明，HBc-L2 VLP的免疫效果远远好于单独GST-L2抗原多肽。将HBc-L2 VLP免疫小鼠，小鼠血清三免效果强于二免，提示HBc-L2 VLP存在免疫加强效果。此外，铝佐剂可以明显提高抗原的免疫反应。动物实验结果表明，L2抗原多肽经HBc-L2重组构建，形成VLP颗粒后可激发机体产生明显的免疫反应，其抗体水平可提供足够强的保护力。
实施例6 保护性实验
采用HPV假病毒中和实验法测定三免鼠血清中HPV特异性中和抗体，借以反应疫苗的保护性。
其中中和实验检测用的宿主细胞为293FT，具体的实验步骤如下：
(1)胰酶消化细胞瓶中的293FT细胞，调整细胞浓度到1.5×105，将此细胞加入96孔细胞板，每孔100ul，37℃、5％CO2培养箱培养4h；
(2)待测血清系列梯度稀释，假病毒按照预先确定的稀释倍数稀释；
(3)血清和假病毒颗粒1:1混合，各50ul，总体积为100ul，4℃中和2h；
(4)中和液加入96孔细胞培养板，置于37℃、5％CO2培养箱培养72h；
(5)荧光显微镜观察结果，感染抑制率大于50％的孔为阳性孔，记录实验结果见表7。
表7：HPV疫苗免疫小鼠中和抗体测定结果
Figure PCTCN2015086636-appb-000009
结果表明，GST-L2抗原多肽免疫血清中难以检测到中和抗体，估计与GST-L2抗原多肽难以激发有效的免疫反应有关。
尽管实验设计的L2抗原肽来自于HPV16，但是可以看到给抗原表位肽具有交叉中和活性，可同时有效的中和HPV16以及HPV18型假病毒。
本发明的设计原理说明及有益效果分析：
本发明所述HBc-L2 VLP通用疫苗的设计原理如图8所示，HBV的HBc蛋白 在大肠杆菌表达后，可以自动组装成VLP颗粒。在实施过程中，HBc蛋白的前段，后端以及中间均可以插入外源抗原，且不影响VLP的形成，更重要的是，如图9所示，插入抗原全部暴露于HBc VLP的外面。因此基于HBc表达异源抗原形成VLP的特性，设计表达HBc-L2 VLP，可有效提高L2抗原肽的免疫原性，且L2基因的插入并不影响HBc VLP的形成，所插入的抗原表位肽可以是同一个，也可以是不同的，更可以是两个表位的融合基因。
在本发明的实施例1中，所述融合蛋白，包括位于所述疫苗载体与HPV L2中和表位之间的连接体LE，此柔性连接体的加入可以避免抗原蛋白质与HBc在形成VLP时的相互影响，有助于形成正确的构象；此外，外源基因在HBc的插入位点不仅一个，这些位点可以插入同一个多肽，也可以插入不同的多肽；外源基因可以插入HBc的前面，中间以及后面，且不影响HBc VLP的形成；插入HBc的外源基因可以是一个中和表位，也可以是两个中和表位，可以插入一个位点，也可以插入不同的位点；更可以是两个中和表位融合表达在同一个位点。
由本发明的实施例可以看出，HPV L2中和表位肽的设计使得HBc-L2 VLP抗原具有广谱的抗病毒能力，扩大了针对L1设计疫苗的血清型限制；制备的HBc-L2VLP颗粒大小均一，批次一致性好；所提供的VLP抗原纯化方法，工艺设计简单，可以获得VLP纯度在80％以上的抗原；所制备的HBc-L2 VLP抗原免疫小鼠后，获得滴度远高于L2的抗原反应。
为了更好的理解本发明所述技术方案，对下述几个问题进行阐述：
(1)本发明提供了一种潜在的预防谱更广的HPV疫苗
目前上市的HPV疫苗均是针对L1抗原设计的，具有明显的血清特异性，而HPV16/18双价疫苗只能抑制约70％的HPV感染，为了让疫苗的预防谱更广，需要设计包括更多血清型的HPV多价疫苗，比如Merck开发的四价疫苗以及目前刚完成临床的9价疫苗。研究发现L2存在多个交叉中和表位，单独或者联合交叉中和表位，可以预防更多血清型的HPV病毒。本发明选择具有更广交叉中和表位的抗原肽作为目标基因，获得HBc-L2 VLP可以中和多个血清型的HPV病毒，是一种预防谱更广的HPV疫苗。
(2)本发明提供了抗原多肽增强免疫力的实验方法
HPV L2的中和表位抗原多肽分子量很小，单独免疫很难激发机体产生足够强的免疫反应，这也是虽然大量的文献报道HPV L2可以产生中和抗体，且L2上存在交叉中和表位，但是针对HPV L2疫苗进展缓慢的原因。通过HBc-VLP抗原 递呈载体，设计的HBc-L2 VLP抗原首先增大了抗原的分子量和空间结构，可有效的激发机体的免疫反应，更为重要的基于本发明实施例中的插入位点设置，HBc VLP的表面被HPV L2抗原肽覆盖。
在本发明的实施例中，L2交叉中和表位抗原肽在HBc VLP表面存在大量拷贝数，且HBc VLP的结构保证L2交叉中和表位抗原肽活性位点完全暴露在外，基于本发明实施例设计的HBc-L2 VLP免疫小鼠后可以激发机体产生很好的免疫反应。
(3)本发明实施例极大提高了HBc–L2 VLP的得率
虽然HPV L1体外表达可以自组装VLP，但是基于L1抗原的自组装为假病毒颗粒的回收率很低。HPV病毒外壳是由72个L1蛋白五聚体形成的二十面体结构，体外表达的L1蛋白组成类似HPV病毒的假病毒颗粒需要经过5个L1单体形成五聚体以及72个五聚体形成二十面体的过程，往往自组装形成的VLP只有部分为二十面体结构。而本实施例基于HBc VLP呈递L2抗原多肽，极大提高VLP得率，有望极大的降低HPV疫苗的成本。
(4)本发明在插入L2中和表位基因序列的同时并不影响HBc VLP的组装效率
以HBc-VLP作为抗原呈递载体的研究多有报道，但是限于插入序列可能会影响HBc VLP的形成，基于该载体设计的疫苗产品并不多，本研究实施例基于前期大量分子生物学、结构生物学的研究，优化HBc插入位点，确保L2碱基的插入并不影响VLP的形成；
在本发明的实施例中融合蛋白经大肠杆菌可溶表达后，可自组装为VLP，其VLP的组装效率并不低于单独HBc VLP的得率；经电镜分析鉴定，VLP大小均一性也非常好。
(5)本发明实施例L2抗原中和表位在HBc的插入位点有多种方式
本发明的实施例中L2在HBc中的插入位点可以是HBc蛋白的前端、中间、也可以是后端，L2抗原表位肽可以插入一个位点，也可以同时插入两个或者三个位点；可以插入一个抗原中和表位，也可以插入两个抗原表位；同样，本实施例中两个融合抗原表位肽的插入位点可以是同一位点，也可以是不同位点，基于本实施例的研发思路，开发的同类产品也取得巨大的成功。此外，基于本实施例提供的增强抗原多肽的实施方法，可以应用到如L2中和表位等类似的抗原多肽免疫原性增强例中。
综上所述，纳米大小的乙肝核心抗原VLP(HBc-VLP)颗粒易于被抗原递呈细胞吞噬。HBc-VLP是一个有效的抗原载体，递呈在HBc-VLP上的抗原可以消除对佐剂的需要，且能产生Th1免疫反应。HBc-VLP是非感染性的，不同于其他的VLP，他们并不参与病毒受体间相互作用，也不是中和抗体靶位，因此载体抑制并不是主要的挑战。应用HBc-VLP作为疫苗投递系统可以有效的克服小分子量多肽序列不能有效激发机体免疫的缺点。将HPV L2的中和表位插入HBc中，体外表达融合蛋白，可以在形成HBc VLP的同时，在假病毒颗粒的表面呈递目标抗原，形成外表覆盖HPV L2中和表位的VLP颗粒，增强免疫反应。
上述参照具体实施方式对该增强HPV抗原表位肽免疫反应的方法及类病毒颗粒、颗粒制备方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，本发明所述方法可以应用到提高同类抗原表位肽免疫原性的研发中，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。

Claims (10)

  1. 一种增强HPV抗原表位肽免疫原性的方法，其特征在于：把HPV抗原基因组装到HBc的基因上，将整合之后的基因通过外源表达获得融合蛋白，融合蛋白自动组装形成类病毒颗粒VLP,且所述VLP表面展示HPV抗原，得到HBc-HPVVLP。
  2. 根据权利要求1所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：所述HPV抗原基因为HPV晚期转录区编码主要衣壳蛋白L1或HPV晚期转录区编码次要衣壳蛋白L2。
  3. 根据权利要求1所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：所述HPV抗原基因为HPV晚期转录区编码次要衣壳蛋白L2。
  4. 根据权利要求1所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：所述VLP表面展示的HPV抗原是L1或L2的一个抗原肽，或L1或L2的多个抗原肽，或L1或L2的多个抗原肽的偶联。
  5. 根据权利要求4所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：所述VLP表面展示的HPV抗原是L2的两个抗原肽的偶联，所述两个抗原肽的序列为ASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQ和GTGGRTGYIPLGTRPPT。
  6. 根据权利要求1所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：所述HPV抗原肽完全暴露在HBc形成的类病毒颗粒的表面，且展示在HBc VLP表面的抗原肽为多个拷贝。
  7. 根据权利要求1所述的增强HPV抗原表位肽免疫原性的方法，其特征在于：在HBc基因上插入更多的外源基因，且不影响HBc类病毒颗粒的形成。
  8. 权利要求1所述的增强HPV抗原表位肽免疫原性的方法获得的可以表达人乳头瘤病毒抗原肽的类病毒颗粒，其特征在于：为HBc-L2融合蛋白，具有序列表中<400>8所示的序列。
  9. 根据权利要求8所述的可以展示人乳头瘤病毒抗原肽的类病毒颗粒，其特征在于：所述HBc-L2融合蛋白在大肠杆菌表达系统中可溶性表达后可自组装VLP。
  10. 权利要求8所述的类病毒颗粒在制备HPV疫苗方面的应用。
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