CN102349996B - Human papilloma virus pharmaceutical composition and application thereof - Google Patents
Human papilloma virus pharmaceutical composition and application thereof Download PDFInfo
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- CN102349996B CN102349996B CN201110313514.9A CN201110313514A CN102349996B CN 102349996 B CN102349996 B CN 102349996B CN 201110313514 A CN201110313514 A CN 201110313514A CN 102349996 B CN102349996 B CN 102349996B
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Abstract
The invention relates to a human papilloma virus pharmaceutical composition for preventing and treating related diseases caused by human papilloma virus. The pharmaceutical composition is characterized by comprising the following components: human papilloma virus virus-like particles (HPV VLPs) used as an active ingredient, an adjuvant for promoting immune response, a cell factor immune accelerant for activating cell immunity to durably and efficiently generate immune response, a salt solution for maintaining the integral spatial configuration and certain dissolubility of VLPs, a physiologically miscible buffer for maintaining a certain pH value, a non-ionic surfactant for protecting VLPs from violent shaking which can lead to activity decrease and the balance of water. The pharmaceutical composition has the characteristics that: HPV VLPs are prepared into a liquid pharmaceutical composition with integral spatial configuration, efficient immunity and stable storage property; the composition is used for preventing and treating various diseases caused by human papilloma virus; and the composition has the characteristics of safe use, convenience, economy, no side effect and the like.
Description
Technical field
The present invention relates to a kind of gene engineering method preparing technical field, particularly from pharmaceutical composition of the virus-like particle (VLPs) of human papilloma (HPV) and preparation method thereof.
Background technology
Human papillomavirus (HPV) is a class double-chain small molecule DNA viruses, has strict species specificity, and main infection people's skin and mucous membrane tissue cause the proliferative lesion of corresponding site epithelial tissue.Clinically, vary in size and HPV can be divided into low risk and the large class of high-risk-type two according to HPV hypotype pathogenicity size or carcinogenic risk.Low risk HPV mainly causes exophytic wart class pathological changes and the low cervix uteri intraepithelial neoplasia of skin of anus and male external genital organs, the large nympha of women, urethral orifice, vagina hypomere, and its virus subtype mainly contains HPV6,11,30,39,42,43 types and HPV44 type.High-risk HPV causes external genitalia cancer, cervical cancer and height cervix uteri intraepithelial neoplasia except causing external genitalia wart, the more important thing is, its virus subtype mainly contains HPV16,18,31,33,35,45,51,52,56,58 types and HPV61 type.
Current research shows HPV except directly causing cervical cancer, also has significant relationship with lung bronchogenic carcinoma, rectal cancer, oral cancer and skin carcinoma.Show according to the interrelated data of the WHO of WHO, except causing cervical cancer, HPV infects the skin carcinoma that can also cause 60%, 60% the esophageal carcinoma, mankind's major diseases such as 50% pulmonary carcinoma, 50% breast carcinoma, 25% oral cancer.
HPV coding comprise eight early stage (E1～E7) and two late periods (L1～L2) gene, L1 albumen is main capsid protein, and there is the molecular weight of 55～60KDa, L1 albumen or L1 and L2 protein combination are at yeast, and the expression in insect cell, mammalian cell or antibacterial can cause the self assembly of virus-like particle (VLPs) (referring to Papillomaviruses Reviews:Current Research on Papillomaviruses; Lacey, ed.Leeds, UK:Leeds Medical Information, pp 101-12 (1996)), VLPs is in form similar to real virion, once and give animals or humans, can cause the neutralizing antibody of high titre, because VLPs does not comprise potential carcinogenic viral genome, therefore there is very high safety as HPV vaccine., be well known in the art as potential vaccine prevention HPV with HPV virus-like particle (VLPs).
Now verified, it is effective that inoculation VLPs bivalence HPV16 and HPV18 and tetravalence HPV6,11,16 and 18 vaccine produce immunne response in patient body, but successfully vaccine is not only required in generation immunne response in body, but also excitating organism produces immunological memory effectively, in the time again running into pathogen, produce rapidly and effectively immunne response.Immunological memory maintain the cellular immunization and the humoral immunization that depend on antigenic specificity.T lymphocyte is the effector lymphocyte of antigenic specificity cell immune system, mainly comprise helper T lymphocyte (CD4+) and cytotoxic T cell (CTL) (CD8+), the two plays an important role in the process of removing or infection control.CD4+T cell is mainly brought into play auxiliary bone-marrow-derived lymphocyte (TH2) the generation antibody of MHC-II quasi-molecule restriction and is produced and maintain the effect of antigenic specificity CD8+T memory cell (TH1). and it regulates activity mainly to comprise: (1) assosting effect to B cell, promote the antibody subtype conversion of B cell, the B cell differentiation of homologous chromosome sudden change and germinal center is plasma cell and Memorability cell; (2) CD4+T cell pair
the assosting effect of CD8+T cell, promotes it to responsiveness cell and Memorability cell differentiation, and maintains functional status; (3) regulating action of CD4+T cell to macrophage and APCs.And the CD8+T cell response with the relatively short effective stage is directly brought into play cytotoxic T lymphocyte (CTL) effect of MHC-I quasi-molecule restriction to target cell or stop growth and the survival of pathogen by the release cells factor, its effector function is competence exertion under antigen exists only, and the existence of visible antigen is the switch as the effector T cell with instant opening ways survival.Along with the clean-up effect function of antigen stops and forms Memorability CD8+T cell, be positioned the mucosa position of pathogen invasion, in the time again running into antigen, both developed rapidly and become effector lymphocyte.The Multiple components of humoral immunoresponse(HI) is being brought into play different effects in the defence process of pathogenic infection.By the plasma cell secretion multi-specificity antibody (natural antibody) of B1 group B development and cell differentiation, form pathogenic infection defence the first line of defence, these antibody have multiple effect: as the diffusion of the starting stage restriction pathogen infecting, form immune complex and activate Acquired immune response, be enrolled into germinal center and activating complement, play the function served as bridge that connects the natural immunity and acquired immunity simultaneously; Be second defence line by the Memorability antibody of long-life plasma cell secretion, be mainly distributed in peripheral circulation and mucosa position, there is high-affinity and high specific, can more effective defence pathogenic infection; The three lines of defence of Memorability B cellularity immune defence, performance has two kinds of effects, in the time again running into antigen, produce on the one hand rapidly and effectively responsing reaction, on the other hand as plasmacytic back-up system of long-life, keep the balance of long-life plasma cell quantity.Therefore, the target of present stage exploitation is under the prerequisite that keeps complete VLPs configuration, extend the pot-life of VLPs, not only want the immunne response of enhancing body to VLPs simultaneously, but also will be by adding Related Component, excitating organism produces immunological memory to VLPs, and in patient body, produces long-acting immunity.
Adjuvant is the material that a class and antigen share and can strengthen and regulate antigen immune response.In vaccine, add adjuvant can make vaccine better play a role effect and improve protection efficiency, therefore adjuvant is an important link in vaccine research.So far the new adjuvant that document is delivered, approximately has hundreds of, different adjuvants to have the mechanism of different Promote immunity reactions.
Adjuvant can change immune function, affects generation and the regulatory function of cytokine.Some adjuvants can be strengthened whole immune function; Most adjuvants can be strengthened secretion or the inhibition of some cytokine.The immune t-cell of existing known person and Mus has Th1 and Th2.Th1 mainly promotes complement fixation antibody and slow type anaphylaxis, and relevant cytokine has IFN-γ, IL-2, IL-12 etc.; Th2 promotes circulating antibody, and analytical type antibody comprises the generation of IgE, and relevant cell factor has IL-4, IL-5 and IL-10 etc.Some adjuvants can be strengthened Th1, and some adjuvants are strengthened Th2.
Although adjuvant is of a great variety, not all adjuvant is all applicable to human body and uses.The adjuvant that present stage goes through to go on the market is also only with several.And certain adjuvant is also only approved for certain specific vaccine, and do not mean that and can be used for other vaccines.Traditional adjuvant, for new generation vaccine, is also differed and plays surely good immune effect, and all novel vaccines and the compatibility of adjuvant must, through strict zoopery and human clinical's experiment, could determine whether it has practical value.
Cytokine is the heterogeneous peptide class regulatory factor that the interior immunocyte of body or the generation of nonimmune cell one group has extensive biologic activity, can activate and regulate in vivo immunity cell, and generation and adjusting to immunne response have important function.The regulating action of every kind of cytokine all has multiformity.At present, although still unintelligible to the definite mechanism of action of various cytokines, show by large quantity research, cytokine is irritation cell immunity effectively, combines to use body is produced to good immunologic enhancement with vaccine.And there has been cytokine profiles approval listing present stage, uses for many years clinically, distincter to its action effect and untoward reaction, set it as the component of vaccine, be considered to safe and reliable.
But cytokine in vivo the half-life short and involve great expense, as used separately with targeting antigen, its action time and immune response asynchronism(-nization) step, therefore cannot reach good immune effect.As combined use with traditional adjuvant, can effectively avoid the drawback of self, extend its half-life in vivo, and can reduce consumption.Also can effectively supplement the defect of the cellular immunization deficiency of traditional adjuvant self to body simultaneously.
Therefore present stage need to find a kind of can keep for a long time HPV VLPs stability and strengthen its immunogenic new pharmaceutical composition, specify compatibility relationship, and production technology is simple, production cost is lower, is reliably vital to human-body safety.
Summary of the invention
The object of this invention is to provide pharmaceutical composition and application thereof that a kind of stable storing also can play human papilloma (HPV) virus-like particle (VLPs) of long-acting immunity.Draw and in VLPs solution, use suitable salinity, buffer and pH value can effectively prevent the absorption of VLPs according to our many experiments, reduce the gathering of VLPs and increase its stability, do not affect its immunogenicity, wherein add the immunogenicity of this medicine of increase that relevant cytokine immunopotentiating agent can significance, this medicine making can be efficiently lasting stimulation human body autoimmune system also, produce the neutrality antibody of high titre, and produce immunological memory, thereby reach prevention to relevant disease that HPV causes and the object for the treatment of.The plurality of advantages such as the pharmaceutical composition that therefore the present invention uses, has production cost low, and technique is simple, safe and reliable.
The object of the present invention is achieved like this: the invention provides a kind ofly for preventing and treating by HPV and infect and cause the human papilloma virus pharmaceutical composition of relevant disease, it comprises following composition and amount ranges composition:
(1) HPV virus-like particle (VLPs) is adsorbed in adjuvant, and its VLPs concentration range is 10～60 μ g/ml;
(2) cytokine immunopotentiating agent, its field of activity is 1～5000 million international units;
(3) NaCl saline solution, its concentration range is 0.1M～0.4M;
(4) buffer for injection, its concentration range 5mM～20mM, keeps the pH of vaccine 5.5～7.5;
(5) non-ionic surface active agent, its concentration range 0.0005%～0.05%;
(6) dissolve with water for injection.
The present invention as required, also adds 0.1 of pharmaceutical composition total amount～10 ‰ pH adjusting agent.
The selected immunogenic substance of the present invention comprises: the one of HPV6a, HPV6b, HPV11, HPV16, HPV18, HPV31 or HPV32, two or more combination in any, and the VLPs that forms of other HPV hypotypes, owing to forming, mechanism is identical, is equally also applicable to this pharmaceutical composition.The optimum content that shows according to the study wherein various HPV VLPs is 10～60 μ g/ml, selects content as lower than 10 μ g/ml, cannot produce the neutrality antibody of high titre, thereby cannot reach the object of long-acting immunity in body.As reached identical immune effect, need frequent drug administration in a short time, so not only increase patient suffering, immunity will reduce the immunity of body repeatedly, thereby destroys patient's autoimmune system.Selecting content as higher than 60 μ g/ml, will there is slight untoward reaction in body, and as higher than 100 μ g/ml, Body adverse reaction is more serious.
The selected adjuvant of the present invention comprises: aluminium adjuvant, Ca3 (PO
4)
2, MF59, SAF, QS-21,3-O-one, two or more combination in any of removing acidylate monophosphoryl lipid (3D-MPL), immunostimulating complex (ISCOM), poly-the third Acetic acid, hydroxy-, bimol. cyclic ester (PLG), AGP, lipid A derivant and CpG.Wherein preferred adjuvant is that aluminium adjuvant, ISCOM adjuvant, 3-O-remove acidylate monophosphoryl lipid (3D-MPL) adjuvant, Ca
3(PO
4)
2adjuvant or MF59 adjuvant.
Aluminium adjuvant to comprise scope more extensive, comprise aluminium hydroxide (Al (OH)
3) adjuvant, aluminum phosphate (AlPO
4) adjuvant, Adju-Phos adjuvant, amorphous AAHS (AAHS) adjuvant, Alumen (KAl (SO
4)-12H
2o) adjuvant or nano-class aluminum adjuvant.
For a long time, aluminium adjuvant is most widely used adjuvant on vaccine always, and in the time that the dosage with suitable uses, it is the most safe and reliable that aluminium adjuvant is considered to.Now most countries is ratified in the world, uses with adjuvant aluminium adjuvant as people.Although present stage aluminium adjuvant Study on mechanism be not also very clear, it has been generally acknowledged that antigen is adsorbed on aluminium adjuvant, aluminium adjuvant can effectively stimulate humoral immunization, produces IgG, the IgE of higher titre, activates Th2 cell.
Cytokine immunopotentiating agent of the present invention is selected from interferon (IFN), interleukin (IL), one, two or more combination in any of granulocyte-macrophage colony stimutaing factor (GM-CSF) or chemotactic factor (TCA).
Cytokine immunopotentiating agent of the present invention is selected from interleukin (IL), and its interleukin (IL) is preferably interleukin (II).
The application of pharmaceutical composition of the present invention, its route of administration is intradermal injection, subcutaneous injection, hypodermoclysis, intramuscular injection, intravenous injection or venous transfusion.
The selected cytokine immunopotentiating agent of the present invention is that a class is by the immunocyte (lymphocyte activating, mononuclear phagocyte etc.) and relevant cell (fibrocyte, endotheliocyte etc.) produce there is the high activity of cell function of adjusting, multifunctional protein peptide molecule or glycoprotein, they are by interacting with the specific receptor of high-affinity, can activate and regulate immunologically competent cell in vivo and make it hypertrophy, thereby strengthen humoral immunization, cellular immunization and non-specific immunity, make body HPV neutrality antibody and the immunological memory of the high titre of generation rapidly and efficiently at short notice.Combine use to relevant adjuvant, can effectively avoid cytokine in vivo action time short, stimulate the impermanent end of keeping away.Extend its retention time in local organization, play the effect of slow release.
Wherein IL-2 can act on various kinds of cell, comprises T, bone-marrow-derived lymphocyte, macrophage and NK cell etc., and immunne response is had to rise effect widely.Show according to the study by targeting antigen and IL-2 intramuscular injection Mice Body the antibody titer obtaining and CD4
+t cell proliferation all improves more than 100 times, the amount of the various cytokines of vitro detection splenocyte secretion, find that IL-2 has strengthened the secretion of IFN-γ, IL-2 significantly, and IL-4 is only had to slight reinforcement, illustrate that IL-2 is mainly the Th1 type immunne response of having strengthened body, and contribute to break immunologic tolerance, it is generally acknowledged the prevention of most of infectious disease and the treatment of tumor, the vaccine that can excitating organism produces Th1 type immunne response is only effectively, and therefore the preferential selection of this patent is combined use by aluminium adjuvant with IL-2.Aluminium adjuvant can effectively be made up like this and cannot activate the defect of body Th1 cell.
IL-12 mainly participates in cellullar immunologic response in vivo, and targeting antigen and IL-12 muscle are injected in Mice Body, and T cell obviously increases, and CTL activity significantly improves.But humoral immunoresponse(HI) is played to inhibitory action, show as antibody titer and decline and B Leukopenia.
GM-CSF mainly strengthens immune response strength by the quantity and the function that regulate antigen presenting cell in immune response.Inject together with targeting antigen in Mice Body, the positive rate of rotation of antibody obviously improves, after testing its specific C D4
+the propagation of T cell and CTL activity all strengthen.
IFN-γ mainly breaks up to regulate immunne response by participating in Th cell to Th1 type, but humoral immunoresponse(HI) is inhibitory action or is had no effect.
TCA3 is a member in β chemoattracting cytoking family, can raise and activated mononuclear cell, macrophage and neutrophilic granulocyte.Inject together with targeting antigen in Mice Body and can specific CTL effect significantly improve, make IgG2a slightly increase, illustrate that TCA3 can strengthen the former Th1 type immunne response inducing.
The selected saline solution of the present invention is NaCl, being chosen as of this saline solution is well known in the art, consider from the adaptability of body, the suitableeest salinity is 0.1～0.2M, but find according to the study, the ionic strength that increases saline solution can improve stability and the dissolubility of HPV VLPs, and then effectively prevents the mutual gathering raising between the VLPs causing due to temperature.Because high salt concentration is restricted in injection, therefore from the viewpoint of stability, preferred salt concentration is 0.25～0.35M.Can address the above problem although increase salinity, the injection of high salt concentration can cause the osmolality disturbance of blood plasma, cannot carry out intravenous route administration, when subcutaneous equally, muscle, abdominal channels administration, will increase the pain of human body.Therefore the salinity of this patent preferred pharmaceutical compositions is: HPV VLPs concentration is selected during lower than 100 μ g/ml etc. and to be oozed NaCl solution, and concentration is 0.15M.It is 0.32M that HPV VLPs concentration is selected NaCl solution concentration during higher than 200 μ g/ml.
In the present invention, selected solvent pH value is in 5.5～7.5 scope, and its HPV VLPs could keep stablizing and forming complete space conformation.Show according to the study, low pH value can make to bring identical charges between high concentration HPV VLPs, reduces and assembles, and avoids coagulation and the albuminous degeneration that causes, thereby makes vaccine lose immunogenicity.Therefore the pH value of this patent preferred pharmaceutical compositions is: it is 6.5～7.5 that HPV VLPs concentration is selected pH value during lower than 120 μ g/ml.It is 5.5～6.5 that HPV VLPs concentration is selected pH value during higher than 180 μ g/ml.
In the present invention, selected buffer solution is phosphate buffer or histidine buffering liquid, and the concentration of buffer is 5mM～20mM.Phosphate buffer is the buffer system that this area is commonly used, and its buffering range is pH value 6～8.The buffering range of histidine buffering liquid is pH value 5.5～6.5.Show according to the study, phosphate buffer, because the ion of its generation can interact with aluminium adjuvant, causes the stability decreases of vaccine injecta, and therefore itself and aluminium adjuvant can not use simultaneously.As need be used simultaneously, need to increase the concentration of aluminium adjuvant in pharmaceutical composition.Phosphate buffer, in the time of cryopreservation, can cause phosphatic sucking-off simultaneously, causes the drift of injection pH value.High concentration HPV VLPs can be due to the unstable of pH value coagulation.Thereby cause immunogenicity to decline.The buffering range of histidine buffering liquid is applicable to the preservation of high concentration HPV VLPs, low temperature can not cause the unstable of buffer system, and therefore the buffer system of this patent preferred pharmaceutical compositions is: HPV VLPs concentration is selected phosphate buffer during lower than 130 μ g/ml.HPV VLPs concentration is selected histidine buffering liquid during higher than 200 μ g/ml.
In the present invention, selected surfactant is non-ionic surface active agent, comprising polyoxyethylene sorbitan monoleate (as: TWEEN
), polysorbate 20 (as: TWEEN
).Wherein preferred activating agent is polyoxyethylene sorbitan monoleate.Show according to the study, polyoxyethylene sorbitan monoleate not only can play increases the dissolubility of HPV VLPs in pharmaceutical composition, and can also protect HPV VLPs aluminium adjuvant in the process of transportation, avoids the reduction of the protein active causing due to violent concussion.But because the polyoxyethylene sorbitan monoleate of excessive concentrations can play slight blood pressure lowering and the effect of haemolysis in body, therefore the concentration range of the preferred polyoxyethylene sorbitan monoleate of this patent in pharmaceutical composition is 0.0005%～0.5% (weight/volume).
Feature of the present invention is application method provided by the invention, can produce the HPV polyvalent vaccine that a kind of immunogenicity is good, the compatibility is good, the storage time is long and stable.The present invention also has the features such as preparation method is simple, production cost is low, effect is good.
Accompanying drawing explanation
Fig. 1 is that HPV16 VLPs is adsorbed on different aluminum adjuvant, is kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
Fig. 2 is that HPV18 VLPs mixes with MPL, is adsorbed on different aluminum adjuvant, is kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
Fig. 3 be HPV6 VLPs with
mix, be adsorbed on different aluminum adjuvant, be kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
Fig. 4 is that HPV11 VLPs and QS-21 and MPL are adsorbed on respectively on Alumen, is kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
Fig. 5 is that HPV16 VLPs is adsorbed on Ca
3(PO
4)
2on adjuvant, be kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
Fig. 6 is that HPV18 VLPs is adsorbed on MF59 adjuvant, is kept under the condition of 2～8 ℃ different pharmaceutical composition stable comparable situation.
The specific embodiment
Below will by embodiment, the present invention will be further described, but following example is only the present invention's example wherein, does not represent the interest field that the present invention limits.
HPV6 VLPs, HPV11 VLPs, HPV16 VLPs, HPV18 VLPs (purity is more than 95%) are kept at respectively to 0.5M NaCl, 0.003%Tween 80, in the solution of pH6.2, aseptic filtration, being diluted to concentration is 1mg/ml, under the condition of-70 ℃, stores.
Embodiment 1: aluminium adjuvant compounding pharmaceutical compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Pharmaceutical composition study on the stability:
Preparation is with Al (OH)
3hPV16 VLPs as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This sterile pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV16 VLPs pharmaceutical composition.
The HPV16 VLPs of preparation using AAHS as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV16 VLPs pharmaceutical composition.
Will be with Al (OH)
3be placed in the refrigerator of 2～8 ℃ as the HPV16 VLPs pharmaceutical composition of adjuvant and each 100 of the HPV16VLPs pharmaceutical composition using AAHS as adjuvant, the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV16 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV6 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 1.
Embodiment 2: select novel MPL adjuvant compounding pharmaceutical compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Pharmaceutical composition study on the stability:
Preparation is with MPL/Al (OH)
3hPV18 VLPs as adjuvant:
Surplus is water for injection.
Prepare by aforementioned pharmaceutical compositions formula, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV18 VLPs pharmaceutical composition.
Preparation is with MPL/AlPO
4hPV18 VLPs as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV18 VLPs pharmaceutical composition.
Will be with MPL/Al (OH)
3as the HPV18 VLPs pharmaceutical composition of adjuvant and with MPL/AlPO
4each 100 of the HPV18 VLPs pharmaceutical composition as adjuvant is placed in the refrigerator of 2～8 ℃, and the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV18 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV18 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 2.
Embodiment 3: select novel
adjuvant compounding pharmaceutical compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Pharmaceutical composition study on the stability:
Preparation with
/ AAHS is as the HPV6 VLPs of adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV6 VLPs pharmaceutical composition.
Preparation with
/ AlPO
4hPV6 VLPs as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV6 VLPs pharmaceutical composition.
Will be with
/ AAHS as the HPV6 VLPs pharmaceutical composition of adjuvant with
/ AlPO
4each 100 of the HPV6 VLPs pharmaceutical composition as adjuvant is placed in the refrigerator of 2～8 ℃, and the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV6 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV6 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 3.
Embodiment 4: select novel QS-21 adjuvant compounding pharmaceutical compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Pharmaceutical composition study on the stability:
The HPV11 VLPs of preparation using QS-21/MPL/ Alumen as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds this pharmaceutical composition of 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV11 VLPs pharmaceutical composition.
The HPV11 VLPs of preparation using QS-21/ Alumen as adjuvant:
Surplus is water for injection.
Prepare by the formula of aforementioned pharmaceutical compositions, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV11 VLPs pharmaceutical composition.
HPV11 VLPs pharmaceutical composition using QS-21/MPL/ Alumen as adjuvant and each 100 of the HPV11 VLPs pharmaceutical composition using QS-21/ Alumen as adjuvant are placed in to the refrigerator of 2～8 ℃, and the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV11 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV11 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 4.
Embodiment 5: select Ca
3(PO
4)
2adjuvant preparation pharmaceutical vaccine compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Preparation related solution:
Preparation is with Ca
3(PO
4)
2hPV31 VLPs as adjuvant:
Surplus is water for injection.
Prepare by aforementioned pharmaceutical compositions formula, regulate pH value to 7.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV31 VLPs pharmaceutical composition.
Will be with Ca
3(PO
4)
2each 100 of the HPV31 VLPs pharmaceutical composition as adjuvant is placed in the refrigerator of 2～8 ℃, and the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV31 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV31 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 5.
Embodiment 6: select novel MF59 adjuvant preparation pharmaceutical vaccine compositions, long-term stability and the related application thereof of preserving of monitoring HPV VLPs.
Pharmaceutical composition study on the stability:
The HPV33 VLPs of preparation using MF59 as adjuvant:
Surplus is water for injection.
Prepare by aforementioned pharmaceutical compositions formula, regulate pH value to 6.2 ± 0.1, be settled to 55ml.
This pharmaceutical composition is divided and is filled in aseptic cillin bottle, and every bottle adds 0.5ml, 100 of subpackages, and the nitrogen by aseptic filtration in whole process for preparation, to get rid of air in bottle, finally rolls lid and makes HPV33 VLPs pharmaceutical composition.
Each 100 of HPV33 VLPs pharmaceutical composition using MF59 as adjuvant is placed in to the refrigerator of 2～8 ℃, and the percentage composition of BIAcore analysis (utilizing the neutrality antibody of a kind of HPV33 VLPs to analyze) destination protein is carried out in regularly sampling.When each detection using the HPV33 VLPs pharmaceutical composition of-70 ℃ of preservations as positive control.Testing result is shown in Fig. 6.
The immunogenicity experiments of embodiment 7:VLPs:
Test for relevant immune detection and animal immune according to the pharmaceutical composition formulated HPV16 VLPs pharmaceutical composition described in above-described embodiment 1～4.
Mouse immune experiment:
Choose 8～12 weeks BALB/c mouse, random packet, 12 every group; By following proposal (in table one) muscle place injecting immune before right side of mice footpath.
The calibrating of immune serum antibody:
Immune latter 14 days for the second time, mice is extractd to eyeball and get blood, collect blood with the disposable centrifuge tube of 15ml, under 4 ℃ of conditions, the centrifugal 30min of 10000g, collects serum, and-20 ℃ save backup.Make envelope antigen with HPV16 VLPs, ELISA method detects the HPV16 antibody titer in immune serum.Wherein, A group immune serum does not all detect antibody activity, the antibody titer of B group immune serum approximately 1: 1000, the antibody titer of C group immune serum approximately 1: 3000, the antibody titer of D, E, F group immune serum approximately 1: 5000, the antibody titer of G, H group immune serum approximately 1: 7000.
Mouse red blood cell blood clotting suppresses experiment:
Because HPV virus has the ability of coagulation mouse red blood cell, but this characteristic can be suppressed by corresponding specific antibody, so we utilize its characteristic, contrived experiment detects in the serum of immune mouse, whether to have produced specific antibody and its content for VLPs.
The HPV16 VLPs of 1% mouse red blood cell suspension and doubling dilution interacts after testing, and result is in the time diluting for 1024 times, and VLPs makes Mice red cell be the highly diluted multiple of " ++ " coagulation, is defined as a viral unit.It is that VLPs dilutes by 1: 256 (618.54ng/ hole) that 4 viral units are chosen in blood clotting inhibition experiment.
Carry out red cell agglutination with 618.54ng/ hole VLPs from the mouse immune serum of different doubling dilutions and suppress experiment, experimental result (in table two), it is 1: 8 that the blood clotting of B group immune serum suppresses to tire, it is 1: 16 that the blood clotting of C group immune serum suppresses to tire, it is 1: 32 that the blood clotting of D, E, F group immune serum suppresses to tire, and it is 1: 128 that the blood clotting of G, H group immune serum suppresses to tire.
The antibody capable that immune mouse produces is correctly combined and can be stoped VLPs and erythrocyte surface receptors bind with VLPs.The result of this experiment has confirmed the antibody capable identification VLPs comformational epitope of mouse immune serum, and it is active to have blood clotting inhibition.
Embodiment 8: the impact that in vaccine, different proportionings change mice Th1/Th2 cytokines
According to the drug regimen composition formula described in above-described embodiment 1, adopt Al (OH)
3adjuvant adds related immune promoter preparation HPV16 VLPs pharmaceutical composition for relevant immune detection and animal immune experiment.
Mouse immune experiment:
Choose 8～12 weeks BALB/c mouse, divide at random 6 groups, 25 every group.Wherein set up matched group (Al (OH)
3adjuvant), IL-2 group, IL-12 group, GM-CSF group, IFN-γ group, totally 6 groups of TCA3 groups, only, immunization protocol is 0,30 day to the each 150 μ l/ of mice leg muscle vaccinate, 2 pin immunity, respectively at 1,2 after initial immunity, 3, after 4 weeks and booster immunization the 2nd week, all mices all, through eye socket venous blood sampling, were stored in-20 ℃ after separation of serum.
Detect Cytokine of Serum level:
Use BDTM Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine Kit and flow cytometer (FACS Calibur), different blood serum samples is carried out to 5 cytokine (TNF simultaneously, IFN-γ, IL-5, IL-4, IL-2) quantitative assay.
After experimental result confirms that immunopotentiating agent adds, can stimulate mice to produce stronger cellullar immunologic response, the Th1/Th2 cytokines that induction produces all changes over time.Five kinds of cytokines all reached maximum at the 4th week.Wherein IL-2 and IFN-γ group can show that at the 2nd, 3,4 weeks inducing mouse produces higher cytokine levels, apparently higher than matched group.Illustrate that this kind of immunopotentiating agent initial immunity can strengthen the nonspecific immune response of vaccine, the immunogenicity that strengthens vaccine is had to good effect.
The clinical experiment in vitro of embodiment 9:HPV infected patient:
The serum antibody ELISA of Patients with Cervical Cancer detects:
The coating buffer 100 μ l/ holes of appropriate HPV16 VLPs PBS dilution are added in 96 hole ELISA Plate, be placed in 4 ℃ of coated spending the night.The liquid in hole that inclines, with PBS washing liquid washing 3 times.Add subsequently 1%BSA-PBS 100 μ l/ holes, PBS washing liquid washing 3 times after room temperature sealing 1hr.Add with 0.1%BSA-PBS1: the patients serum of 3000 dilutions, PBS washing liquid washing 3 times after incubated at room 1hr.Add afterwards with 0.1%BSA-PBS1: the sheep anti-mouse igg 100 μ l/ holes of the HRP labelling of 3000 dilutions.After room temperature leaves standstill 1hr, PBS washing liquid is washed 4 times.Every hole adds 100 μ l OPD substrate buffer solutions, lucifuge reaction 10min.Last every hole adds 100 μ l stop buffer cessation reactions.Detect every hole optical density value with microplate reader and 490nm wavelength, patients serum's testing result is positive, and patients serum's antibody titer is more than 1: 3000.
Detected and shown that the serum antibody of Patients with Cervical Cancer and HPV16 VLPs produce immunoreation by ELISA, result shows that patients serum's antibody titer is higher, and confirms by experiment in vitro, and HPV16 VLPs can produce corresponding antibody in patient body.Thereby corresponding eligible patients is reached to the object of prevention and treatment.
Wherein, infected and the various diseases that causes by HPV, comprising by relevant diseases such as the caused cervicitis of HPV, cervical polyp, cervix uteri hypertrophy, condyloma acuminatum, breast carcinoma, lung bronchogenic carcinoma, rectal cancer, esophageal carcinoma, pharyngeal cancer, oral cancer, skin carcinomas.Can utilize said method, carry out clinical experiment in vitro but be not limited to said method.
Claims (4)
1. a human papilloma virus pharmaceutical composition, for preventing and treating by the caused relevant disease of human papillomavirus, is characterized in that: it is made up of following composition and amount ranges:
(1) HPV16 virus-like particle is adsorbed in adjuvant, and HPV16 virus-like particle concentration is 160 μ g/ml;
(2) cytokine immunopotentiating agent, its field of activity is 1～5000 million international units;
(3) NaCl saline solution, its concentration range is 0.1M～0.4M;
(4) buffer for injection, its concentration range 5mM～20mM, keeps the pH of pharmaceutical composition 5.5～7.5;
(5) non-ionic surface active agent, its concentration range 0.0005%～0.05%;
(6) dissolve with water for injection;
Cytokine immunopotentiating agent is interleukin II,
Adjuvant is selected from aluminium adjuvant, Ca
3(PO
4)
2, MF59, SAF, QS21,3D-MPL, ISCOM, PLG, AGP, lipid A derivant or CpG one, two or more combination in any.
2. pharmaceutical composition according to claim 1, is characterized in that: aluminium adjuvant is selected from Al (OH)
3adjuvant, AlPO
4adjuvant, Adju-Phos adjuvant, amorphous AAHS (AAHS) adjuvant, KAl (SO
4)
212H
2o adjuvant or nano-class aluminum adjuvant.
3. pharmaceutical composition according to claim 1, is characterized in that: buffer is selected from phosphate buffer or histidine buffering liquid.
4. pharmaceutical composition according to claim 1, is characterized in that: non-ionic surface active agent is selected from polyoxyethylene sorbitan monoleate or polysorbate 20.
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| CN112439059B (en) * | 2019-09-02 | 2022-02-08 | 怡道生物科技(苏州)有限公司 | Recombinant human papilloma virus vaccine composition and application thereof |
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| WO2004091652A1 (en) * | 2003-04-18 | 2004-10-28 | Unihart Corporation | Pharmaceutical composition containing interferon for the treatment of hpv infections |
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| WO2004091652A1 (en) * | 2003-04-18 | 2004-10-28 | Unihart Corporation | Pharmaceutical composition containing interferon for the treatment of hpv infections |
| CN101181636A (en) * | 2006-11-14 | 2008-05-21 | 北京金迪克生物技术研究所 | Compound vaccine composition for preventing and controlling human viral infection, compound vaccine vaginal mist and uses thereof |
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