Immunogenic composition and vaccine for preventing diphtheria, pertussis and tetanus as well as preparation method and application of immunogenic composition and vaccine

Abstract

The invention provides a pertussis (five components), diphtheria and tetanus combined vaccine, and particularly relates to a pertussis and tetanus combined vaccine with improved immune response. The immunogenic composition and the vaccine provided by the invention contain specially selected double aluminum-based adjuvants, so that the prepared composition vaccine can induce an organism to generate a good immune response, and meanwhile, the safety of the vaccine is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and particularly relates to an immunogenic composition containing a double aluminum-based adjuvant, and a vaccine prepared thereby. Background Art

[0002] An adjuvant (derived from the Latin word "adjuvare", meaning to help) is a substance that enhances the immune response by physically or chemically binding to an antigen, and is particularly helpful in enhancing the specific immune response to the antigen contained in the vaccine. Adjuvants can not only assist antigens in inducing long-term and effective specific immune responses in the body, thereby achieving higher vaccine efficacy and prolonging the protection time of the immune response, but also reduce the amount of antigen used, production costs and the number of immunizations. Hundreds of materials have been used as adjuvants, including bacterial metabolites, mineral oils / surfactants and immunostimulants, microparticles, nucleic acids, liposome precipitations and polysaccharides. However, only aluminum-based adjuvants are used continuously and widely around the world.

[0003] Aluminum-based adjuvants have been used in the field of vaccines for nearly a hundred years and are the first adjuvants approved by the U.S. Food and Drug Administration (FDA) for use in human vaccines. Aluminum hydroxide and aluminum phosphate are the main forms of aluminum used as vaccine adjuvants. The method of adding adjuvants to vaccines to enhance immune responses has a long history. Early vaccines contained many other impurities in addition to antigen components, which affected the immune effect. With the advent of recombinant DNA technology and synthetic chemistry, highly purified antigens can be produced to induce more specific immune responses. The use of highly purified antigens improves the safety and tolerability of vaccines and enables simpler characterization of vaccines, but it usually also leads to reduced immunogenicity. Therefore, this antigen preparation requires additional adjuvants to achieve protective immunity. In 1926, Glenny et al. found that diphtheria toxoid (DT) adsorbed with aluminum had better immunogenicity than toxoid alone. This pioneering research promoted the use of aluminum as an adjuvant in vaccines. At present, aluminum-based adjuvants have been widely used in a variety of vaccines, including diphtheria-tetanus-pertussis vaccine, polio vaccine, 13-valent pneumococcal conjugate vaccine, HPV vaccine, etc.

[0004] Although aluminum-based adjuvants have been widely used in the field of vaccines, there are still many challenges in designing and preparing vaccines using aluminum-based adjuvants because the mechanism by which aluminum-based adjuvants exert their beneficial effects is still not fully understood. At present, common explanations for the ability of aluminum-based adjuvants to effectively enhance the immunogenicity of antigens include the belief that the addition of aluminum-based adjuvants increases the surface area of ​​the antigen, prolongs the stimulation time of the antigen, and enhances the uptake of the antigen at the injection site, thereby exerting its function of enhancing the immune response of the antigen. The physical and chemical properties of aluminum hydroxide-based adjuvants and aluminum phosphate-based adjuvants are significantly different, and these differences lead to differences in the immune responses induced by these two chemical substances. In addition, the in vivo experimental results of aluminum hydroxide-based adjuvants and aluminum phosphate-based adjuvants are also different. For example, researchers found that aluminum phosphate adjuvants are more easily dissolved after being injected into experimental animals. This difference in results will ultimately affect the nature of the immune response induced by the two adjuvants. Therefore, different aluminum-based adjuvants will cause different reactions. The types of aluminum-based adjuvants selected for different antigen properties are also different. According to different antigen characteristics, content and combination methods, different aluminum-based adjuvants should be selected to achieve the best immune response requirements.

[0005] There are related studies and vaccine products that use AP and AH dual aluminum-based adjuvants to enhance antigen immunogenicity. The choice of AH and AP adjuvants depends largely on the nature of the antigen and the requirements for adsorption to achieve the best immune response. Therefore, different types of vaccines and different antigen compositions in the vaccine will result in different specific types and contents of adjuvants required to achieve the best immune response. At present, the aluminum-based adjuvants widely used in diphtheria, pertussis and tetanus vaccines are mostly single aluminum phosphate-based adjuvants or aluminum hydroxide-based adjuvants. For example, the aluminum-based adjuvants used in Boostrix® and Daptacel® are both single aluminum hydroxide adjuvants. In addition, there are patents that disclose that diphtheria, pertussis and tetanus vaccines can be prepared using dual aluminum-based adjuvants including aluminum hydroxide and aluminum phosphate, such as patent application WO2014135651A1, which discloses that aluminum hydroxide and aluminum phosphate can be used as adjuvants for diphtheria, pertussis and tetanus vaccines at the same time, but does not disclose the specific ratio of AP and AH; patent application CN1295481A discloses a DTaP-Hib vaccine, in which the ratio of adjuvant aluminum phosphate and aluminum hydroxide is between 1:1-20:1.

[0006] The antigen components and contents of DTP vaccines vary according to the age of the applicable population. If a new vaccine containing different antigen compositions and contents is developed, it is necessary to re-study and establish the type and specific composition of adjuvants suitable for the vaccine. Therefore, it is still necessary to further develop a DTP combined vaccine containing aluminum-based adjuvants. Summary of the invention

[0007] The present invention provides an immune composition using a dialuminum-based adjuvant and a vaccine prepared therefrom, in particular a diphtheria-tetanus-pertussis combined vaccine comprising a dialuminum-based adjuvant. The dialuminum-based adjuvant provided by the present invention is a mixture comprising aluminum hydroxide and aluminum phosphate, wherein the content of aluminum hydroxide in the dialuminum-based adjuvant is higher than the content of aluminum phosphate. The immune composition and vaccine provided by the present invention comprise the above-mentioned aluminum-based adjuvant. The present invention also provides the use of the above-mentioned aluminum-based adjuvant and immune composition in the preparation of a medicament for treating a disease and the use in the treatment or prevention of a disease.

[0008] It should be understood that the present invention is not limited to the specific methods and experimental conditions described, because these methods and conditions may vary. It should also be understood that the terminology used in this specification is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0009] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications mentioned in this specification are incorporated by reference in their entirety.

[0010] The terms used in this specification have meanings that are recognized and known to those skilled in the art. However, for the convenience and completeness of the description, specific terms and their meanings are given below and throughout the specification.

[0011] As used in this specification and the appended claims, terms may be construed in both the narrower and broader senses unless the context clearly dictates otherwise. Thus, for example, the term "method" includes one or more methods and / or steps, such as those described herein and / or which will become apparent to one of ordinary skill in the art upon reading this specification, etc.

[0012] The term “comprising” is intended to include “including” and “consisting of”. For example, a composition “comprising” X may consist of X alone or may include other substances, such as X+Y.

[0013] The term "about" or "approximately" means "within a statistically significant range of values". Such ranges can be within an order of magnitude of the specified value or range, typically within 20%, more specifically within 10%, and even more specifically within 5%. The permissible variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily determined by one skilled in the art. If a range is specified in the body of the present application, each integer within the range is also provided as an embodiment of the present invention.

[0014] The term "antigen" generally refers to a biological molecule, usually a protein, peptide, polysaccharide, lipid or conjugate, which contains at least one epitope to which an antibody that recognizes it can selectively bind; or, in some cases, an immunogenic substance that is capable of stimulating antibody production or a T cell response or both in an animal, including compositions that are injected into or ingested by an animal. An immune response can be induced on the entire molecule or on one or more different parts of the molecule (e.g., epitopes or haptens). The term can be applied to a single molecule, or to a homogeneous or heterogeneous population of antigenic molecules. Antigens are recognized by antibodies, T cell receptors or other elements of specific humoral and / or cellular immunity. The term "antigen" includes all related antigenic epitopes. Epitopes of a particular antigen can be identified using various methods well known in the art. For example, conformational epitopes can be identified by determining the spatial conformation of amino acids, for example, by methods such as X-ray crystallography and 2D nuclear magnetic resonance. In addition, for the purposes of the present invention, the term "antigen" can also be applied to proteins with modifications such as deletions, additions and substitutions (usually conservative in nature, but they may not be conservative) in the native sequence, provided that the protein retains the ability to elicit an immune response. Such modifications may be intentional, for example as a result of site-directed mutagenesis, or due to the use of specific synthetic methods or genetic engineering methods, or may be accidental, for example due to mutations in the host producing the antigen. In addition, the antigen may be produced, obtained or isolated from a microorganism such as a bacterium, or may be an intact microorganism. Likewise, oligonucleotides or polynucleotides expressing the antigen, for example in nucleic acid immunization protocols, are also included in the definition. Also included are synthetic antigens, for example, polytopes, flanking epitopes and other recombinant or synthetic antigens.

[0015] The term "immunogenic composition" refers to a composition containing at least one antigen that induces an immune response in an animal.

[0016] The term "adjuvant" refers to a compound or mixture that elicits an enhanced immune response to an antigen, as further described herein.

[0017] As used herein, the term "treatment" (including variants thereof, such as "treating" or "treated") refers to any one or more of the following: (i) prevention of infection or reinfection, such as in the case of conventional vaccines, (ii) reduction of severity or elimination of symptoms; and (iii) substantial or complete elimination of a particular pathogen or disease. Thus, treatment may be performed prophylactically (before infection) or therapeutically (after infection). According to the present invention, prophylactic or therapeutic treatment may be used. According to a specific embodiment of the present invention, compositions and methods are provided for treating host animals against infections caused by microorganisms (e.g., Clostridium tetani, etc.), including by prophylactic and / or therapeutic immunization. The methods of the present invention can be used to induce immunity in patients prophylactically and / or therapeutically. For the purpose of biomedical research, the methods of the present invention may also be applied to patients.

[0018] The term "patient" refers to a mammal, bird, fish, reptile or any other animal. The term "patient" also includes humans. The term "patient" also includes pets. Non-limiting examples of pets include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles and frogs. The term "patient" also includes pets. Non-limiting examples of domestic animals include: alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese and turkeys.

[0019] Aluminum-based adjuvants In a first aspect of the present invention, an aluminum-based adjuvant is provided, wherein the aluminum-based adjuvant comprises an aluminum salt, such as aluminum phosphate, aluminum hydroxide, or a combination of aluminum phosphate and aluminum hydroxide.

[0020] The aluminum-based adjuvant of the present invention can use adjuvants called aluminum hydroxide and aluminum phosphate. Preferably, the aluminum-based adjuvant of the present invention is a composition of aluminum hydroxide and aluminum phosphate. The aluminum phosphate and aluminum hydroxide of the present invention can be obtained by purchasing commercial products or prepared by existing methods. The preparation method of the aluminum adjuvant can refer to the method disclosed in patent US2009 / 0016946A1.

[0021] The aluminum-based adjuvant of the present invention may contain a buffer (e.g., phosphate or histidine or Tris buffer), or may not contain a buffer. The aluminum-based adjuvant of the present invention is preferably sterile and pyrogen-free. The aluminum-based adjuvant of the present invention may contain free aqueous phosphate ions, such as at a concentration of 1.0-20 mM, preferably 5-15 mM, and more preferably about 10 mM. The aluminum-based adjuvant may also contain sodium chloride.

[0022] Preferably, Al in the aluminum hydroxide in the aluminum-based adjuvant of the present invention is 3+ The content is higher than Al in aluminum phosphate 3+ content.

[0023] Furthermore, the Al in the aluminum hydroxide in the aluminum-based adjuvant of the present invention 3+ and Al in aluminum phosphate 3+ The weight percentage of is at least 2:1, for example ≥5:1, ≥6:1, ≥7:1, ≥8:1, ≥9:1, ≥10:1, ≥11:1, ≥12:1, ≥13:1, ≥14:1, ≥15:1, ≥16:1, ≥17:1, ≥18:1, ≥19:1, ≥20:1, ≥21:1, ≥22:1, ≥23:1, ≥24:1, ≥25:1, ≥26:1, ≥ 1, ≥27:1, ≥28:1, ≥29:1, ≥30:1, ≥31:1, ≥32:1, ≥33:1, ≥34:1, ≥35:1, ≥36:1, ≥37:1, ≥38:1, ≥39:1, ≥40:1, ≥41:1, ≥42:1, ≥43:1, ≥44:1, ≥45:1, ≥46:1, ≥47:1, ≥48:1, ≥49:1, ≥50:1, etc.

[0024] Preferably, Al in aluminum phosphate in the aluminum-based adjuvant of the present invention 3+ and Al in aluminum hydroxide 3+ The weight percentage of aluminum phosphate in the aluminum-based adjuvant of the present invention is about 1:20 to 1:50. 3+ and Al in aluminum hydroxide 3+ When the content ratio is within this range, the effect is better.

[0025] Specifically, Al in aluminum phosphate in the aluminum-based adjuvant of the present invention 3+ and Al in aluminum hydroxide 3+ The content ratio is about 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49 or 1:50.

[0026] More preferably, Al in aluminum phosphate in the aluminum-based adjuvant of the present invention 3+ and Al in aluminum hydroxide 3+ The weight percentage is about 1:25 to 1:45. The aluminum phosphate in the aluminum-based adjuvant of the present invention is Al 3+ and Al in aluminum hydroxide 3+ When the weight ratio is within this range, the vaccine prepared has better immune effect.

[0027] Specifically, Al in aluminum phosphate in the aluminum-based adjuvant of the present invention 3+and Al in aluminum hydroxide 3+ The content ratio is about 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44 or 1:45.

[0028] The aluminum-based adjuvant of the present invention can be used for the preparation of immunogenic compositions and vaccine preparations. The aluminum-based adjuvant of the present invention adsorbs with antigens to achieve optimal immune response of the antigens.

[0029] The aluminum-based adjuvant of the present invention can be particularly used for the preparation of diphtheria, pertussis and tetanus vaccine preparations.

[0030] Immunogenic compositions and vaccines The second aspect of the present invention provides an immunogenic composition and a vaccine prepared therefrom.

[0031] The immunogenic composition of the present invention comprises the aluminum-based adjuvant described above. The aluminum-based adjuvant in the immunogenic composition can enhance the immune response induced in patients receiving the composition.

[0032] Aluminum bases include hydroxides, phosphates, sulfates, etc., and salts in any suitable form (e.g., gelatinous, crystalline, amorphous, etc.). These salts are preferably used for adsorption. Adjuvants called aluminum hydroxide and / or aluminum phosphate may be used. Combinations of aluminum hydroxide and aluminum phosphate may be used in the present invention.

[0033] The vaccine of the present invention is preferably a diphtheria, pertussis and tetanus vaccine, which comprises an acellular pertussis antigen.

[0034] The DTP vaccine of the present invention provides protection against diseases caused by the following causes: Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis. It is usually composed of diphtheria toxoid (DT), tetanus toxoid (TT) and acellular pertussis (Pa).

[0035] The diphtheria antigen is typically a diphtheria toxoid. The preparation of diphtheria toxoid (DT) is well documented. Any suitable diphtheria toxoid may be used. For example, DT may be produced by purification of the toxin from culture of Corynebacterium diphtheriae followed by chemical detoxification, but may alternatively be prepared by purification of a recombinant or genetically detoxified analog of the toxin.

[0036] Bordetella pertussis is a gram-negative, non-spore-forming aerobic bacterium that can cause whooping cough. Vaccines against Bordetella pertussis have been available for many years and are classified into two categories: cellular and acellular. Cellular vaccines contain whole Bordetella pertussis cells that have been killed and inactivated (e.g., by treatment with formalin and / or heat), while acellular vaccines contain specifically purified Bordetella pertussis, either purified from natural bacilli or purified after expression in a recombinant host. The pertussis antigen in the immunogenic composition or vaccine of the present invention is an acellular pertussis antigen.

[0037] The DTP vaccine of the present invention contains five components of acellular pertussis antigens, wherein the Bordetella pertussis antigens include: (1) detoxified pertussis toxin (pertussis toxoid or "PT"); (2) filamentous hemagglutinin ("FHA"); (3) pertactin (PRN); and (4) fimbriae proteins 2 and 3 (FIM2 / 3, collectively referred to as fimbriae proteins in the present invention, and sometimes referred to as FIM in the present invention. FIM and FIM2 / 3 have the same meaning and are used interchangeably).

[0038] Prior to use in the present invention, FHA and pertussis adhesin may be treated with formaldehyde. Preferably, PT is detoxified by treatment with formaldehyde and / or glutaraldehyde. As an alternative to this chemical detoxification method, PT may be a mutant PT with reduced enzyme activity by mutagenesis, but detoxification by chemical treatment is preferred.

[0039] The tetanus antigen of the present invention is typically tetanus toxoid. Methods for preparing tetanus toxoid (TT) are well known in the art. TT can be produced by purifying the toxin from a culture. Clostridium tetani is subsequently chemically detoxified, or it can be prepared by purifying a recombinant or genetically detoxified toxin analog. Any suitable tetanus toxoid may be used in the immunogenic composition or vaccine of the present invention. "Tetanus toxoid" may encompass immunogenic fragments of the full-length protein.

[0040] Specifically, the immunogenic composition or vaccine of the present invention comprises the following components: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN) and pertussis fimbriae proteins, wherein the pertussis fimbriae proteins are fimbriae protein 2 and fimbriae protein 3; (ii) an aluminum-based adjuvant, wherein the aluminum-based adjuvant is a composition comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immune composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content).

[0041] The present invention determines the appropriate ratio range of aluminum phosphate to aluminum hydroxide in aluminum-based adjuvants through a large number of research experiments to obtain an immunogenic composition and vaccine preparation that achieves the best immune response.

[0042] In the present invention, the content of aluminum hydroxide added to the immunogenic composition is higher than that of aluminum phosphate, and the ratio of aluminum phosphate to aluminum hydroxide is about 1:20 to 1:50. The content ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of the present invention has a better effect when it is within the above ratio range.

[0043] Specifically, the ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of the present invention is about 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49 or 1:50.

[0044] Preferably, the weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:25-1:45.

[0045] Specifically, the ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of the present invention is about 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44 or 1:45.

[0046] The inventors of the present application speculate that the aluminum-based adjuvant of the present invention may affect the immunogenicity of the antigen by affecting the specific structure of the vaccine antigen. Antigen structure analysis can be performed by methods such as X-ray crystallography or nuclear magnetic resonance (NMR), and the thermal stability of the antigen before and after adsorption can be determined and evaluated by methods such as differential scanning calorimetry (DSC) or differential scanning fluorescence (DSF). In the present invention, the adsorption capacity of the aluminum-based adjuvant for each specific antigen in the diphtheria, pertussis and tetanus vaccine is characterized by measuring the adsorption rate of the aluminum-adsorbed antigen, and the antibody response in the animal is measured by immunization of an animal model to characterize the effect of a specific aluminum-based adjuvant on the immune response.

[0047] In order to find the optimal content of aluminum salt adjuvant for making immunogenic compositions, the adsorption rate of each antigen component at each adjuvant concentration was analyzed. As a result, when the final total aluminum ion concentration at the time of final binding was lower than 0.6 mg / ml, the adsorption rate of each antigen was relatively reduced.

[0048] When the aluminum-based adjuvant of the present invention is used in the immunogenic composition, Al 3+ The preferred range of addition amount in the composition for administration to a patient is from 0.6 mg / ml to 1.4 mg / ml. 3+ The total content is 0.3-0.7 mg / dose.

[0049] In some embodiments, the immunogenic composition comprises a concentration of about 0.1 mg / mL to about 1.4 mg / mL, about 0.1 mg / mL to about 1.3 mg / mL, about 0.1 mg / mL to about 1.2 mg / mL, about 0.1 mg / mL to about 1.1 mg / mL, about 0.1 mg / mL to about 1.0 mg / mL, about 0.1 mg / mL to about 0.9 mg / mL, about 0.1 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.7 mg / mL, about 0.1 mg / mL to about 0.6 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 0.4 mg / mL, from about 0.1 mg / mL to about 0.3 mg / mL, from about 0.1 mg / mL to about 0.2 mg / mL, about 0.25 mg / mL to about 0.95 mg / mL, about 0.25 mg / mL to about 0.85 0.5 mg / ml, about 0.25 mg / ml to about 0.75 mg / ml, about 0.25 mg / ml to about 0.65 mg / ml, about 0.25 mg / ml to about 0.55 mg / ml, about 0.25 mg / ml to about 0.45 mg / ml, about 0.25 mg / ml to about 0.35 mg / ml, about 0.5 mg / ml to about 1.0 mg / ml, about 0.5 mg / ml to about 0.9 mg / ml, about 0.5 mg / ml to about 0.8 mg / ml, about 0.5 mg / ml to about 0.75 mg / ml, about 0.5 mg / ml to about 0.7 mg / ml, about 0.5 mg / ml to about 0.65 mg / ml, about 0.5 mg / ml to about 0.6 mg / ml, about 0.6 mg / ml to about 1.0 mg / ml, about 0.6 mg / ml to about 1.2 mg / ml, about 0.6 mg / ml to about 1.3 mg / ml, about 0.6 1.4 mg / ml, about 0.75 mg / ml to about 1.0 mg / ml of Al3+ In a preferred embodiment, the immunogenic composition contains Al at a concentration of about 0.6 mg / ml to about 1.4 mg / ml. 3+ Total concentration, most preferably about 1.0 mg / ml Al 3+ Total concentration.

[0050] In a preferred embodiment, Al is added to the immunogenic composition. 3+ The total concentration is approximately 1 mg / mL (or 0.5 mg / dose).

[0051] The content of diphtheria toxoid (DT) in the immunogenic composition of the present invention is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 3-6 μg / dose.

[0052] In some specific embodiments, the content of diphtheria toxoid (DT) in the immunogenic composition of the present invention is 2.0, 2.5 or 3.0 Lf / dose; the content of tetanus toxoid (TT) in the immunogenic composition of the present invention is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 Lf / dose. Lf / dose; the content of pertussis toxin (PT) in the immunogenic composition is 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 μg / dose; the content of filamentous hemagglutinin (FHA) in the immunogenic composition of the present invention is 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 μg / dose; the content of pertussis borrelidin (PRN) in the immunogenic composition of the present invention is 2.0, 2.5, 3.0, 3.5 or 4.0 μg / dose; the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition of the present invention is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 Lf / dose.

[0053] Preferably, the content of diphtheria toxoid (DT) in the immunogenic composition of the present invention is 2.5 Lf / dose; the content of tetanus toxoid (TT) in the immunogenic composition is 5 Lf / dose; the content of pertussis toxin (PT) in the immunogenic composition is 8 μg / dose; the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 8 μg / dose; the content of pertussis pilin (PRN) in the immunogenic composition is 3 μg / dose; the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 5 μg / dose. When the content of each antigen in the immunogenic composition of the present invention is the specific content in the above preferred embodiment, the Al content of the adjuvant contained in the immunogenic composition is 1.5 μg / dose. 3+ The total content is preferably 1 mg / ml (or 0.5 mg / dose).

[0054] The aluminum salt adjuvant of the present invention is adsorbed onto part or all of the antigens in the composition.

[0055] The adsorption can be understood as respectively adsorbing different antigens onto the aluminum phosphate and aluminum hydroxide in the adjuvant and then mixing them. The mixing may further include a further adsorption step.

[0056] The step of adsorbing the aluminum-based adjuvant and the antigen of the present invention can be performed according to the following steps: firstly, the aluminum-based adjuvant and different antigens are adsorbed separately, and then the substances obtained after the adsorption are mixed.

[0057] The adsorption of the present invention may optionally include the step of adding an adjuvant again after mixing to adsorb the antigen.

[0058] Unless otherwise specified, steps involving mixing two or more components do not require a particular order of mixing. Thus, the components can be mixed in any order. If there are three components, two components can be mixed first, followed by the third component, and so on.

[0059] In order to ensure that the prepared vaccine achieves the best immune response, the present invention preferably allows the aluminum phosphate in the aluminum-based adjuvant to adsorb to the antigen FHA, and the aluminum hydroxide to adsorb to the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT. The present invention adopts this adsorption method in a specific embodiment.

[0060] In some specific embodiments, the step of adsorbing the aluminum salt adjuvant and the antigen of the present invention is carried out as follows: aluminum phosphate is adsorbed with FHA, aluminum hydroxide is adsorbed with PT, PRN, DT, TT, and FIM2 / 3, and the adsorbed antigens obtained above are mixed.

[0061] The meaning of "adsorbed antigens" in the present invention is considered to mean that not less than 90% is adsorbed. The adsorption rate of FHA, PT, PRN, FIM, DT, and TT in the composition or vaccine preparation after the adsorption antigens are mixed in the present invention can reach more than 90%, preferably more than 95%, and more preferably more than 99%.

[0062] The pH of the immunogenic compositions described herein is maintained at about 5.5 to about 9.5, e.g., about 5.5 to about 9.0, about 5.5 to about 8.5, about 5.5 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 5.5 to about 6.5, about 6.0 to about 9.5, about 6.0 to about 9.0, about 6.0 to about 8.5, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, about 6.0 to about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, and about 6.5 to about 8.0.

[0063] Preferably, the pH of the immunogenic composition of the present invention is maintained at 5.5-8.0.

[0064] Specifically, the pH of the immunogenic composition of the present invention can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0065] The vaccine may be buffered at this pH. A stable pH may be maintained by using a buffering agent.

[0066] The diphtheria, tetanus and pertussis combined vaccine with aluminum-based adjuvant of the present invention can be prepared by the following method steps: adsorbing aluminum phosphate with FHA, adsorbing aluminum hydroxide with PT, PRN, DT, TT and FIM2 / 3, and mixing the adsorbed substances obtained above. 3+ The final concentration is 0.6 mg / ml-1.4 mg / ml, and the AP / AH ratio ranges from 1:20-1:50, preferably 1:25-1:45.

[0067] Optionally, the present invention relates to a method for preparing an immune composition or vaccine, the method comprising the step of adding the remaining adsorbent within the range of 0.6 to 1.4 mg / ml of the aluminum ion concentration after the antigen is adsorbed onto the aluminum ion. In a preferred embodiment of the present invention, the inherent adsorption of the component antigens in the immune composition or vaccine can be maintained and the immune response can be enhanced.

[0068] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:20-1:50, Al 3+ The total content is 0.6-1.4 mg / ml.

[0069] The content of diphtheria toxoid (DT) in the immunogenic composition is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 3-6 μg / dose.

[0070] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml.

[0071] The content of diphtheria toxoid (DT) in the immunogenic composition is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 3-6 μg / dose.

[0072] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml.

[0073] The content of diphtheria toxoid (DT) in the immunogenic composition is 2.5 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 5 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 8 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 8 μg / dose; and / or, the content of pertussis pilus protein (FIM2 / 3) in the immunogenic composition is 5 μg / dose.

[0074] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:20-1:50, Al 3+ The total content is 0.6-1.4 mg / ml, and in the immunogenic composition, the antigen FHA is adsorbed with aluminum phosphate, and the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed with aluminum hydroxide.

[0075] The content of diphtheria toxoid (DT) in the immunogenic composition is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 3-6 μg / dose.

[0076] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The weight percentage is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml, and in the immunogenic composition, the antigen FHA is adsorbed with aluminum phosphate, and the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed with aluminum hydroxide.

[0077] The content of diphtheria toxoid (DT) in the immunogenic composition is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM2 / 3) in the immunogenic composition is 3-6 μg / dose.

[0078] Preferably, the present invention provides an immunogenic composition or vaccine, the immunogenic composition or vaccine comprising: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3); (ii) an aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture of aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition (measured as Al 3+ The content of aluminum phosphate (calculated as Al 3+ Calculation of content); The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+The percentage (calculated by weight) is 1:25-1:45, the total content of Al3+ is 0.6-1.4 mg / ml, and the antigen FHA in the immunogenic composition is adsorbed with aluminum phosphate, and the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed with aluminum hydroxide.

[0079] The content of diphtheria toxoid (DT) in the immunogenic composition is 2.5 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 5 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 8 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 8 μg / dose; and / or, the content of pertussis pilus protein (FIM2 / 3) in the immunogenic composition is 5 μg / dose.

[0080] In addition to the above-mentioned antigen and adjuvant components, the immunogenic composition or vaccine of the present invention generally also contains one or more "pharmaceutically acceptable carriers or excipients", which include any excipient that does not itself induce the production of antibodies that are harmful to the individual receiving the vaccine. Suitable excipients are generally large, slowly metabolized macromolecules, such as proteins, carbohydrates, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. The vaccine may also contain a diluent, such as water, saline, glycerol, etc. In addition, auxiliary substances may be present, such as wetting agents or emulsifiers, pH buffer substances, etc. Sterile pyrogen-free, phosphate-buffered saline is a typical carrier. A comprehensive discussion of pharmaceutically acceptable excipients can be found in the reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0081] The immunogenic compositions of the invention are in aqueous form, i.e. solutions or suspensions. This type of liquid formulation allows the composition to be administered directly from its packaged form without the need for reconstitution in an aqueous medium and is therefore very suitable for injection. The compositions may be present in vials, or they may be present in ready-to-fill syringes. The syringes may be needle-free or needle-free. A syringe will contain a single dose of the composition, whereas a vial may contain a single dose or multiple doses (e.g. 2 doses).

[0082] The immunogenic composition of the present invention may also contain a small amount of wetting agent, filler, emulsifier or pH regulator according to actual needs. Unless any conventional media or agents are incompatible with the active ingredients, they will be used in the immunogenic composition of the present invention.

[0083] Other "immunomodulators" that may be included in the immunogenic composition or vaccine include, for example, one or more interleukins 1-alpha, 1-beta, 2, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17 and 18 (and mutant forms thereof); interferons-alpha, beta and gamma; granulocyte-macrophage colony stimulating factor (GM-CSF); granulocyte colony stimulating factor (G-CSF); or tumor necrosis factor alpha and beta. Other adjuvants that can be used in the immunogenic compositions described herein include chemokines, including but not limited to MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules, such as selectins, such as L-selectin, P-selectin, and E-selectin; mucin-like molecules, such as CD34, GlyCAM-1, and MadCAM-1; integrin family members, such as LFA-1, VLA-1, Mac-1, and p150.95; members of the immunoglobulin superfamily, such as PECAM, ICAM, such as ICAM-1, ICAM-2, and ICAM-3, C-reactive protein, and immunoglobulin superfamily members. D2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40, CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6; and caspase (ICE).

[0084] The vaccine of the present invention can be packaged in unit dose form or in multiple dose form (e.g., 2 doses). For multiple dose forms, vials are preferred over prefilled syringes. The effective dose volume can be routinely determined, but a typical human dose of a composition for injection has a volume of 0.5 ml. The volume of "each dose" described in the present invention is 0.5 ml.

[0085] The immunogenic composition or vaccine of the invention may be isotonic to humans.

[0086] Treatment and administration methods The immunogenic composition or vaccine of the present invention is suitable for use in human patients. The present invention provides a method of inducing an immune response in a patient, which comprises administering the composition or vaccine of the present invention to the patient.

[0087] Immunogenic compositions containing aluminum-based adjuvants tend to phase separate over time; aluminum particles separate from the liquid and settle during storage. To ensure successful administration, the immunogenic composition must be visually homogeneous and resuspended to a homogeneous state prior to administration.

[0088] The present invention also provides the use of the above immunogenic composition in the production of a medicament for inducing an immune response in a patient; the medicament comprises a vaccine. These methods and uses are generally used to induce an antibody response, preferably a protective antibody response. Methods for analyzing antibody responses, neutralizing capacity and protective effects after vaccine immunization are well known in the art.

[0089] The compositions of the present invention can be administered in a variety of ways. The most preferred immunization route is intramuscular injection (e.g., injection into the arm or thigh), but other available routes also include subcutaneous injection, intranasal administration, oral administration, intradermal injection, transdermal injection, transdermal injection, inhalation administration, etc.

[0090] The vaccine prepared according to the method of the present invention can be used to prevent the infection of pertussis, tetanus and diphtheria bacilli in adolescents and adults. Therefore, the patient can be 6 years old and above.

[0091] Treatment can be carried out in a single dose regimen or in a multiple dose regimen. Multiple dose regimens can be used for primary immunization and / or booster immunization. In multiple dose regimens, different doses can be administered by the same or different routes.

[0092] Immunogenicity or an effective amount of an immunogenic composition can be determined in a dose response study in which patients are immunized with increasing amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dose. The initial dose in the study can be determined based on immune data in animal models. The dose may vary depending on the individual circumstances. The amount can be determined in routine experiments by methods known to those skilled in the art.

[0093] An immunologically effective amount of the immunogenic composition is administered to a patient in an appropriate dosage to initiate an immune response. The amount of the dose may vary depending on individual circumstances, such as age and weight. The amount can be determined in routine experiments by methods known to those skilled in the art.

[0094] Beneficial effects of the present invention: 1. The dual aluminum-based adjuvant of the present invention has a strong auxiliary immune enhancement effect on the antigen components of the vaccine. Its immune effect is much better than that of Al(OH)3 or AlPO4 alone as a vaccine adjuvant, and is also much better than the compatibility of Al(OH)3 and AlPO4 with other vaccine active ingredients. This shows that not only does the combined use of Al(OH)3 and AlPO4 in a specific ratio have a synergistic effect, but also there is a synergistic effect between the composite adjuvant of Al(OH)3 and AlPO4 and the antigen components in the vaccine of the present invention.

[0095] 2. The Al(OH)3 and AlPO4 used in the dual aluminum-based adjuvant of the present invention are both types of vaccine adjuvants that have been widely used in humans and are highly safe.

[0096] 3. The preparation process of the double aluminum-based adjuvant of the present invention is mature and low in price. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 The IgG antibody titer for antigen FHA under different AP / AH conditions; Figure 2 It is the IgG antibody titer for antigen PT detection under different AP / AH conditions; Figure 3 is the IgG antibody titer for antigen PRN under different AP / AH conditions; Figure 4 IgG antibody titer for antigen FIM detection under different AP / AH conditions; Figure 5 The IgG antibody titer for antigen DT under different AP / AH conditions; Figure 6 The IgG antibody titer for antigen TT under different AP / AH conditions; Figure 7 The IgG antibody titer against antigen FHA was tested under different aluminum content conditions when AP / AH=1 / 30; Figure 8 The IgG antibody titer against antigen PT was tested under different aluminum content conditions when AP / AH=1 / 30; Fig. 9 The IgG antibody titer against the antigen PRN was detected under different aluminum content conditions when AP / AH=1 / 30; Fig.10 The IgG antibody titer against antigen FIM was tested under different aluminum content conditions when AP / AH=1 / 30; Fig.11 The IgG antibody titer against antigen DT was tested under different aluminum content conditions when AP / AH=1 / 30; Fig.12The IgG antibody titer against antigen TT was tested under different aluminum content conditions when AP / AH=1 / 30; Fig.13 is the antigen adsorption rate for different antigens (FHA, PT, PRN, FIM, DT, TT) under different AP / AH conditions; Fig.14 Antigen adsorption rate test results; AP / AH=1 / 30, aluminum-based adjuvant content 1mg / ml, antigen content per ml contains DT: 4Lf; TT: 6Lf; PT: 12μg; FHA: 12μg; PRN: 4μg; FIM2 / 3: 6μg; Fig.15 Antigen adsorption rate test results; AP / AH=1 / 30, aluminum-based adjuvant content 1mg / ml, antigen content per milliliter contains DT: 6Lf; TT: 12Lf; PT: 20μg; FHA: 20μg; PRN: 8μg; FIM2 / 3: 12μg. DETAILED DESCRIPTION

[0098] The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to be decisive for the terms and scope of the present invention. It should be understood that where typical reaction conditions (e.g., temperature, reaction time, etc.) are given, conditions outside or below the specified ranges may also be used, although less convenient. Unless otherwise indicated, all parts and percentages given in this specification are by weight and all temperatures are in degrees Celsius.

[0099] In addition, the following examples use standard methods that are well known and routine to those skilled in the art, unless otherwise specified. As mentioned above, the following examples are provided for illustrative purposes and should not be construed as limiting the scope of the present invention in any way.

[0100] Example 1: Preparation of adsorbed antigen and diphtheria, pertussis and tetanus vaccine The PRN, PT, DT, TT, and FIM antigens are sterilized and filtered into a reaction container, respectively, and reacted with the filtered aluminum hydroxide adjuvant for 1-10 hours to obtain adsorbed PRN, adsorbed PT, adsorbed DT, adsorbed TT, and adsorbed FIM stock solutions. The FHA antigen is sterilized and filtered into a reaction container, and reacted with the filtered aluminum phosphate adjuvant in the reaction container for 70-90 hours to obtain an adsorbed FHA stock solution. The above-mentioned adsorbed antigens are mixed, stirred evenly, and packaged to prepare a diphtheria and pertussis vaccine preparation. Each dose of the vaccine preparation contains 8μg, 8μg, 3μg, 5μg, 2.5Lf, and 5Lf of antigens PT, FHA, PRN, FIM, DT, and TT, respectively.

[0101] Example 2: Immune effects of different ratios of aluminum phosphate / aluminum hydroxide combined vaccines DPT samples containing aluminum-based adjuvants with different ratios of aluminum phosphate / aluminum hydroxide were prepared according to the method of Example 1. The ratios of aluminum phosphate (AP) to aluminum hydroxide (AH) were 2:3, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, and 1:55, respectively. 3+ The content was 0.5 mg per dose, and the adjuvants of total aluminum phosphate and total aluminum hydroxide were set as the experimental control group. NIH mice in good health were selected and randomly divided into groups of 10 mice each. Each mouse was immunized with 1 / 3 HD test sample, one injection, and the eyeballs were removed 14 days later to collect serum. The serum antibody titer was determined by ELISA method, and statistical analysis was performed by GraphPad Prism 8.0 software.

[0102] For specific results, see Figure 1-6 . The results show that as the AP / AH ratio decreases, the FHA antibody titer decreases accordingly. When AP:AH is in the range of 1:15~1:50, the FHA antibody titer is at a higher level; as the AP / AH ratio decreases, the PT antibody titer increases accordingly. When AP:AH is in the range of 1:20~1:55, the PT antibody titer level is high; as the AP / AH ratio decreases, the PRN antibody titer increases accordingly. When AP:AH is in the range of 1:20~1:55, the PRN antibody titer level is high. According to the above results, when AP:AH is in the range of 1:20~1:50, the FHA, PT, PRN, FIM, DT, and TT antibodies are all at a high level and the antibody titers are not inferior to those of the full AP control and the full AH control.

[0103] Example 3: Same aluminum adjuvant, different Al 3+ The immune effect of diphtheria, pertussis and tetanus combined vaccine Samples containing an aluminum-based adjuvant with a ratio of aluminum phosphate to aluminum hydroxide of 1:30 and different aluminum contents (0.1, 0.3, 0.5, and 0.7 mg aluminum / dose, and a volume of 0.5 ml per dose) were prepared according to the method of Example 1. NIH mice in good health were selected and randomly divided into groups, with 10 mice in each group. Each mouse was immunized with 1 / 3 HD test sample for one injection. After 14 days, the eyeballs were removed to collect serum. The serum antibody titer was determined by ELISA method, and statistical analysis was performed using GraphPad Prism 8.0 software.

[0104] For specific results, see Figure 7-12 The results show that as the aluminum content increases, the IgG antibody titers produced by each antigen in mice increase accordingly. There are no significant differences in FHA, FIM, DT, and TT. The PT and PRN antibody titers at high doses of aluminum are significantly different from those at 0.1 mg aluminum.

[0105] Example 4: Adsorption rate of each antigen at different AP / AH DPT samples containing aluminum-based adjuvants with different ratios of aluminum phosphate / aluminum hydroxide were prepared according to the method of Example 1. The ratios of aluminum phosphate (AP) to aluminum hydroxide (AH) were 2:3, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, and 1:55, respectively. 3+ The total amount was 1.0 mg / ml. The antigen adsorption rate under each condition was tested. The results are as follows Fig.13 shown.

[0106] Example 5: Antigen adsorption rates of different antigen contents under the condition of AP / AH=1 / 30 The diphtheria and tetanus pertussis sample containing an aluminum-based adjuvant with an aluminum phosphate / aluminum hydroxide ratio of 1 / 30 was prepared according to the method of Example 1. 3+ The total amount was 1.0 mg / ml, the specific content of each antigen was adjusted, and the antigen adsorption rate under each condition was tested. Figure 14-15 shown.

[0107] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0108] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An immunogenic composition, characterized in that The immunogenic composition comprises: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM 2 / 3); (ii) a dialuminum-based adjuvant, which is a mixture of aluminum hydroxide and aluminum phosphate; The weight ratio of aluminum phosphate to aluminum hydroxide in the immunogenic composition is 1:20-1:

50. 3+ The total content of aluminum hydroxide is 0.6-1.4 mg / ml, and the aluminum hydroxide is Al 3+ The aluminum phosphate is calculated based on the content of Al 3+ Calculation of content.

2. The immunogenic composition according to claim 1, characterized in that The weight ratio of aluminum phosphate to aluminum hydroxide in the immunogenic composition is 1:25-1:

45.

3. The immunogenic composition according to any one of claims 1 or 2, characterized in that The content of diphtheria toxoid (DT) in the immunogenic composition is 2-3 Lf / dose; and / or, the content of tetanus toxoid (TT) in the immunogenic composition is 3-6 Lf / dose; and / or, the content of pertussis toxin (PT) in the immunogenic composition is 6-10 μg / dose; and / or, the content of filamentous hemagglutinin (FHA) in the immunogenic composition is 6-10 μg / dose; and / or, the content of pertussis borrelidin (PRN) in the immunogenic composition is 2-4 μg / dose; and / or, the content of pertussis fimbriae protein (FIM 2 / 3) in the immunogenic composition is 3-6 μg / dose.

4. A pertussis, diphtheria and tetanus combined vaccine, characterized in that: The vaccine comprises the immunogenic composition according to any one of claims 1 to 3 above.

5. The vaccine according to claim 4, characterized in that The vaccine further comprises a pharmaceutically acceptable excipient.

6. Use of the immunogenic composition according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating a disease.

7. The use according to claim 6, characterized in that The medicine is a vaccine.

8. The use according to claim 7, characterized in that The disease is diphtheria, tetanus or pertussis.

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