Immunogenic composition and vaccine for preventing diphtheria, pertussis, and tetanus, preparation method therefor, and use thereof

Abstract

The present invention provides a combination vaccine against pertussis (five-component), diphtheria, and tetanus. The vaccine of the present invention comprises a specifically selected dual-aluminum-based adjuvant.

Description

An immunogenic composition, vaccine, preparation method and application for the prevention of diphtheria, pertussis and tetanus. Technical Field

[0001] This invention relates to the field of vaccine technology, and to an immunogenic composition containing a bis-aluminum adjuvant, and a vaccine prepared therefrom. Background Technology

[0002] An adjuvant (derived from the Latin word "adjuvare," meaning assistant) is a substance that enhances the immune response by physically or chemically binding to an antigen, particularly helping to strengthen a specific immune response against antigens contained in vaccines. Adjuvants not only assist antigens in inducing a long-lasting and effective specific immune response in the body, thereby achieving higher vaccine efficacy and extending the protective time of the immune response, but also reduce the amount of antigen used, production costs, and the number of immunizations required. Hundreds of materials have been used as adjuvants, including bacterial metabolites, mineral oils / surfactants and immunostimulants, microparticles, nucleic acids, liposomes, and polysaccharides. However, only aluminum-based adjuvants have been consistently and widely used globally.

[0003] Aluminum-based adjuvants have been used in the vaccine field for nearly a century and were the first adjuvant 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 the immune response has a long history. Early vaccines contained many impurities in addition to the antigenic components, affecting the immunization effect. With the advent of recombinant DNA technology and synthetic chemistry, highly purified antigens could be produced to induce more specific immune responses. Using highly purified antigens improved vaccine safety and tolerability, and allowed for simpler vaccine characterization, but this often led to reduced immunogenicity. Therefore, such antigen preparations required the addition of adjuvants to achieve protective immunity. In 1926, Glaney et al. discovered 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. Currently, aluminum-based adjuvants are widely used in various vaccines, including DPT (diphtheria-tetanus-pertussis) vaccine, polio vaccine, 13-valent pneumococcal conjugate vaccine, and HPV vaccine.

[0004] Although aluminum adjuvants are widely used in the vaccine field, the mechanisms by which they exert their beneficial effects are not yet fully understood, thus posing significant challenges in designing and preparing vaccines using aluminum adjuvants. Common explanations for the effectiveness of aluminum adjuvants in enhancing antigen immunogenicity include the belief that their addition increases antigen surface area, prolongs antigen stimulation time, and enhances antigen uptake at the injection site, thereby enhancing the antigen-immune response. Aluminum hydroxide and aluminum phosphate adjuvants have significantly different physical and chemical properties, leading to differences in the immune responses they elicit. Furthermore, in vivo experimental results for aluminum hydroxide and aluminum phosphate adjuvants differ; for example, researchers have found that aluminum phosphate adjuvants dissolve more readily after injection into experimental animals. These differences ultimately affect the nature of the immune responses induced by the two adjuvants. Therefore, different aluminum adjuvants can elicit different responses. Different types of aluminum adjuvants are selected based on the properties of the antigen; different aluminum adjuvants should be chosen according to the characteristics, content, and combination of different antigens to achieve the optimal immune response.

[0005] Existing research and vaccine products utilize dual aluminum adjuvants (AP and AH) to enhance antigen immunogenicity. The choice between AH and AP adjuvants largely depends on the properties of the antigen and adsorption requirements to achieve the optimal immune response. Therefore, different vaccine types and varying antigenic compositions within vaccines lead to differences in the specific type and amount of adjuvant required to achieve the best immune response. Currently, the aluminum adjuvants widely used in DPT vaccines are mostly single aluminum phosphate or aluminum hydroxide adjuvants. For example, and All aluminum-based adjuvants used are single aluminum hydroxide adjuvants. In addition, some patents disclose the use of dual aluminum-based adjuvants, including aluminum hydroxide and aluminum phosphate, to prepare DTaP vaccines. For example, patent application WO2014135651A1 discloses the simultaneous use of aluminum hydroxide and aluminum phosphate as adjuvants for DTaP vaccines, but does not disclose the specific ratio of AP to AH; patent application CN1295481A discloses a DTaP-Hib vaccine with an adjuvant aluminum phosphate to aluminum hydroxide ratio between 1:1 and 20:1.

[0006] The antigenic components and amounts included in DPT vaccines vary depending on the age and other factors of the target population. Developing new vaccines with different antigenic compositions and amounts requires re-evaluating and establishing the appropriate adjuvant type and specific composition. Therefore, further development of DPT combined vaccines containing aluminum-based adjuvants is still needed. Summary of the Invention

[0007] This invention provides an immune composition using a bis-aluminum adjuvant and a vaccine prepared therefrom, particularly a DPT combined vaccine containing a bis-aluminum adjuvant. The bis-aluminum adjuvant provided by this invention is a mixture comprising aluminum hydroxide and aluminum phosphate, wherein the aluminum hydroxide content in the bis-aluminum adjuvant is higher than the aluminum phosphate content. The immune composition and vaccine provided by this invention contain the aforementioned aluminum adjuvant. This invention also provides the use of the aforementioned aluminum adjuvant and immune composition in the preparation of medicaments for treating diseases and in the treatment or prevention of diseases.

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

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

[0010] The terms used in this specification have meanings that are generally accepted and known to those skilled in the art; however, for the sake of convenience and completeness, specific terms and their meanings are given below and throughout the specification.

[0011] When used in this specification and the appended claims, terms may be interpreted in both narrow and broad senses unless the context clearly specifies otherwise. Thus, for example, the term "method" includes one or more methods and / or steps, such as those described herein and / or methods and / or steps that will become apparent to a person skilled in the art upon reading this specification, etc.

[0012] The term “comprising” means both “including” and “consisting of”. For example, a composition that “comprising” X can consist of only X or may contain other substances, such as X+Y.

[0013] The term "approximately" or "about" means "within a range of values ​​that are statistically significant." Such a range can be within an order of magnitude of the specified value or range, typically within 20%, more specifically within 10%, or even more specifically within 5%. The permissible variation included in the term "approximately" or "about" depends on the specific system being studied and can be readily determined by those skilled in the art. If a range is specified in the text of this application, each integer within that range is also provided as an embodiment of the invention.

[0014] The term "antigen" generally refers to a biomolecule, typically a protein, peptide, polysaccharide, lipid, or conjugate, that contains at least one epitope to which an antibody recognizing it may selectively bind; or, in some cases, an immunogenic substance capable of stimulating antibody production or a T-cell response, or both, in an animal, including compositions injected into or ingested by an animal. An immune response can be induced on the whole molecule or on one or more different parts of the molecule (e.g., epitopes or haptens). The term can be used for a single molecule, or a group of homogeneous or heterogeneous antigen molecules. Antigens are recognized by antibodies, T-cell receptors, or other elements of specific humoral and / or cellular immunity. The term "antigen" includes all relevant antigenic epitopes. Epitopes of a particular antigen can be identified using a variety of 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. Furthermore, for the purposes of this invention, the term "antigen" can also be used for proteins having modifications such as deletions, additions, and substitutions (generally conserved in nature, but they may not be conserved), in their native sequence, provided that the protein retains the ability to elicit an immune response. Such modifications may be intentional, such as as a result of site-directed mutagenesis, or due to the use of specific synthetic or genetic engineering methods, or they may be accidental, such as due to mutations in the host that produces the antigen. Furthermore, antigens can be produced, obtained, or isolated from microorganisms such as bacteria, or they can be from the entire microorganism. Similarly, oligonucleotides or polynucleotides expressing antigens, such as in nucleic acid immunization protocols, are also included in this definition. Synthetic antigens are also included, such as multiepitopes, 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 animals.

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

[0017] As used herein, the term "treatment" (including its variations, such as "treatment" or "being treated") means any one or more of the following: (i) prevention of infection or reinfection, as in the case of routine vaccination; (ii) reduction of severity or elimination of symptoms; and (iii) substantial or complete elimination of a specific pathogen or disease. Therefore, treatment can be preventative (pre-infection) or therapeutic (post-infection). According to the invention, both preventative and therapeutic treatments can be used. According to one specific embodiment of the invention, compositions and methods for treating host animals against infections caused by microorganisms (e.g., Clostridium tetani) are provided, including through preventative and / or therapeutic immunization. The methods of the invention can be used to preventatively and / or therapeutically induce immunity in patients. The methods of the invention can also be applied to patients for biomedical research purposes.

[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 domesticated 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

[0020] A first aspect of the present invention provides an aluminum-based adjuvant. The aluminum-based adjuvant comprises an aluminum salt, such as aluminum phosphate, aluminum hydroxide, or a combination of aluminum phosphate and aluminum hydroxide.

[0021] The aluminum-based adjuvant of this invention can be an adjuvant known as aluminum hydroxide and aluminum phosphate. Preferably, the aluminum-based adjuvant of this invention is a combination of aluminum hydroxide and aluminum phosphate. The aluminum phosphate and aluminum hydroxide of this invention can be obtained by purchasing commercially available products or prepared by existing methods. The preparation method of the aluminum adjuvant can refer to the method disclosed in patent US2009 / 0016946A1.

[0022] The aluminum-based adjuvant of the present invention may contain a buffer (e.g., phosphate, 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 at a concentration of, for example, 1.0-20 mM, preferably 5-15 mM, and more preferably about 10 mM. The aluminum-based adjuvant may also contain sodium chloride.

[0023] Preferably, the aluminum hydroxide in the aluminum-based adjuvant of the present invention contains Al. 3+ The content is higher than that of aluminum phosphate. 3+ content.

[0024] Furthermore, the aluminum hydroxide in the aluminum-based adjuvant of the present invention contains Al. 3+ Al in aluminum phosphate 3+The weight percentage must be 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, ≥ 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.

[0025] Preferably, the aluminum-based adjuvant of the present invention contains aluminum phosphate in the form of Al. 3+ Al in aluminum hydroxide 3+ The weight percentage of aluminum phosphate in the aluminum-based adjuvant of this invention is approximately 1:20 to 1:50. 3+ Al in aluminum hydroxide 3+ The content ratio of [specific ingredient] within this range has the best effect.

[0026] Specifically, the aluminum-based adjuvant of the present invention contains aluminum phosphate in the form of Al. 3+ Al in aluminum hydroxide 3+ The content ratio is approximately 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.

[0027] More preferably, the aluminum phosphate in the aluminum-based adjuvant of the present invention contains Al 3+ Al in aluminum hydroxide 3+ The weight percentage of aluminum in the aluminum-based adjuvant of this invention is approximately 1:25 to 1:45. 3+ Al in aluminum hydroxide 3+ Vaccines prepared with a weight ratio within this range have better immunizing effects.

[0028] Specifically, the aluminum-based adjuvant of the present invention contains aluminum phosphate in the form of Al. 3+ Al in aluminum hydroxide 3+The content ratio is approximately 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.

[0029] The aluminum-based adjuvant described in this invention can be used in the preparation of immunogenic compositions and vaccine formulations. The aluminum-based adjuvant of this invention achieves an optimal immune response by adsorbing onto the antigen.

[0030] The aluminum-based adjuvant described in this invention can be used specifically in the preparation of DPT vaccine formulations.

[0031] Immunogenic compositions and vaccines

[0032] A second aspect of the present invention provides an immunogenic composition and a vaccine prepared therefrom.

[0033] The immunogenic composition of the present invention includes the aluminum-based adjuvant described above, which can enhance the immune response induced in patients receiving the composition.

[0034] Aluminum-based salts include hydroxides, phosphates, sulfates, and salts in any suitable form (e.g., gel-like, crystalline, amorphous, etc.). Adsorption with these salts is preferred. Adjuvants called aluminum hydroxide and / or aluminum phosphate can be used. Combinations of aluminum hydroxide and aluminum phosphate can be used in this invention.

[0035] The vaccine described in this invention is preferably a DPT vaccine, which contains acellular pertussis antigen.

[0036] The DPT vaccine described in this invention provides protection against diseases caused by Corynebacterium diphtheriae, Clostridium tetani, and Bordetella pertussis. It typically consists of diphtheria toxoid (DT), tetanus toxoid (TT), and acellular pertussis (Pa).

[0037] Diphtheria antigen is usually a diphtheria toxoid. The preparation of diphtheria toxoids (DTs) is well documented. Any suitable diphtheria toxoid can be used. For example, DTs can be produced by purifying the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, but optionally by recombinant toxin or purification of a genetically modified detoxification analog.

[0038] Bordetella pertussis is a Gram-negative, nonspore-forming aerobic bacterium that causes wheezing and coughing. Vaccines against Bordetella pertussis have existed for many years and are classified into cellular and acellular types. Cellular vaccines contain intact 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, purified from natural bacilli or purified after expression in a recombinant host. The pertussis antigen described in the immunogenic compositions or vaccines of this invention is an acellular pertussis antigen.

[0039] The DPT vaccine of the present invention contains a 5-component acellular pertussis antigen, wherein the Bordetella pertussis antigen includes: (1) detoxifying pertussis toxin (pertussis toxoid or "PT"); (2) filamentous hemagglutinin ("FHA"); (3) pertussis adhesin (pertactin, PRN); and (4) fimbriae proteins 2 and 3 (FIM2 / 3, which are collectively referred to as fimbriae proteins in the present invention, and are sometimes referred to as FIM in the present invention. FIM and FIM2 / 3 have the same meaning and are used interchangeably).

[0040] Prior to use in this invention, FHA and Bordetella pertussis adhesin can be treated with formaldehyde. PT is preferably detoxified by treatment with formaldehyde and / or glutaraldehyde. As an alternative to this chemical detoxification method, PT can be a mutant PT with reduced enzyme activity through mutagenesis, but chemical detoxification is preferred.

[0041] The tetanus antigen of this invention is typically tetanus toxoid. Methods for preparing tetanus toxoid (TT) are well known in the art. TT can be produced by purifying the toxoid from a culture. Clostridium tetani is then chemically detoxified, or it can be prepared by purifying recombinant or genetically detoxified toxoid analogs. Any suitable tetanus toxoid can be used in the immunogenic compositions or vaccines described in this invention. "Tetanus toxoid" can encompass the immunogenic fragment of the full-length protein.

[0042] Specifically, the immunogenic composition or vaccine of the present invention comprises the following components:

[0043] (i) Diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; wherein the acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN) and pertussis fimbriae, wherein the pertussis fimbriae are fimbriae 2 and fimbriae 3.

[0044] (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 is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al).3+ (Content calculation).

[0045] This invention determines the appropriate ratio range of aluminum phosphate to aluminum hydroxide in aluminum-based adjuvants through extensive research and experimentation, in order to obtain immunogenic compositions and vaccine formulations that achieve optimal immune responses.

[0046] In this 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 ranges from approximately 1:20 to 1:50. The ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of this invention within the above-mentioned range has optimal effects.

[0047] Specifically, the ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of the present invention is approximately 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.

[0048] Preferably, the weight ratio of aluminum phosphate to aluminum hydroxide in the immunogenic composition (in Al) 3+ The weight calculation percentage is 1:25-1:45.

[0049] Specifically, the ratio of aluminum phosphate to aluminum hydroxide added to the immunogenic composition of the present invention is approximately 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.

[0050] The inventors of this application hypothesize that the aluminum-based adjuvant of this invention may affect the immunogenicity of the vaccine antigen by influencing its specific structure. The antigen structure can be analyzed using 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 using methods such as differential scanning calorimetry (DSC) or differential scanning fluorescence (DSF). In this invention, the adsorption capacity of the aluminum-based adjuvant for each specific antigen in the DPT vaccine is characterized by measuring the adsorption rate of the aluminum-adjuvanted antigen, and the effect of the specific aluminum-based adjuvant on the immune response is characterized by measuring the antibody response in animal models after immunization.

[0051] To determine the optimal content of aluminum salt adjuvant for manufacturing immunogenic compositions, the adsorption rate of each antigen component at each adjuvant concentration was analyzed. The results showed that when the final total aluminum ion concentration at the point of final binding was below 0.6 mg / ml, the adsorption rate of each antigen decreased relatively.

[0052] When the aluminum-based adjuvant of the present invention is used in the immunogenic composition, Al 3+ The preferred range for the amount added to the composition for administration to patients is from 0.6 mg / ml to 1.4 mg / ml. The immunogenic composition contains Al 3+ The total content is 0.3-0.7 mg / dose.

[0053] In some specific embodiments, the immunogenic composition comprises concentrations 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, and about 0.1 mg / mL. From about 0.1 mg / mL to about 0.6 mg / mL, from about 0.1 mg / mL to about 0.5 mg / mL, from 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, from about 0.25 mg / mL to about 0.95 mg / mL, from about 0.25 mg / mL to about 0.85 mg / mL, from 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 mg / ml to about 1.4 mg / ml, about 0.75 mg / ml to about 1.0 mg / ml of Al 3+Total concentration. 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+ The optimal total concentration of Al is approximately 1.0 mg / ml. 3+ Total concentration.

[0054] 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).

[0055] The immunogenic composition of the present invention contains diphtheria toxoid (DT) at a content of 2-3 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a content of 3-6 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a content of 6-10 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a content of 6-10 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a content of 2-4 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a content of 3-6 μg / dose.

[0056] 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; 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 diphtheria toxoid (DT) in the immunogenic composition of the present invention ...2.0, 2.5, or 3.0 Lf / dose. The immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 μg / dose; the immunogenic composition of the present invention contains Bordetella pertussis adhesin (PRN) at a concentration of 2.0, 2.5, 3.0, 3.5, or 4.0 μg / dose; and the immunogenic composition of the present invention contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 Lf / dose.

[0057] Preferably, the immunogenic composition of the present invention contains 2.5 Lf / dose of diphtheria toxoid (DT); 5 Lf / dose of tetanus toxoid (TT); 8 μg / dose of pertussis toxin (PT); 8 μg / dose of filamentous hemagglutinin (FHA); 3 μg / dose of Bordetella pertussis adhesin (PRN); and 5 μg / dose of pertussis fimbriae 2 / 3. When the content of each antigen in the immunogenic composition of the present invention is the specific content in the above preferred embodiments, the adjuvant A1 contained in the immunogenic composition... 3+ The total content is preferably 1 mg / ml (or 0.5 mg / dose).

[0058] The aluminum salt adjuvant of the present invention adsorbs some or all of the antigen in the composition.

[0059] The adsorption can be understood as adsorbing different antigens separately with aluminum phosphate and aluminum hydroxide in the adjuvant and then mixing them. The mixture may also include a further adsorption step.

[0060] The adsorption step of the aluminum-based adjuvant and antigen described in this invention can be carried out according to the following steps: first, the aluminum-based adjuvant and different antigens are adsorbed separately, and then the substances obtained after adsorption are mixed.

[0061] The adsorption process described in this invention may optionally include a step of adding an adjuvant again after mixing to perform antigen adsorption.

[0062] Unless otherwise specified, steps involving mixing two or more components do not require a specific mixing order. Therefore, the components can be mixed in any order. If there are three components, then two components can be mixed first, and then the third component can be mixed, and so on.

[0063] To ensure that the prepared vaccine achieves the best immune response, the present invention preferably adsorbs aluminum phosphate in the aluminum-based adjuvant with antigen FHA, and aluminum hydroxide with antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT. This adsorption method is adopted in the specific embodiments of the present invention.

[0064] In some specific embodiments, the adsorption step of the aluminum salt adjuvant and antigen according to the present invention is carried out according to the following steps: 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.

[0065] In this invention, "adsorbed antigen" is considered to mean that at least 90% is adsorbed. In the composition or vaccine formulation of the present invention after mixing the various adsorbed antigens, the adsorption rates of FHA, PT, PRN, FIM, DT, and TT can all reach 90% or more, preferably 95% or more, and more preferably 99% or more.

[0066] The pH of the immunogenic composition of the present invention is maintained at about 5.5 to about 9.5, for example, 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 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, and about 7.0 to about 8.0.

[0067] Preferably, the pH of the immunogenic composition of the present invention is maintained at 5.5-8.0.

[0068] 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.

[0069] The vaccine can be buffered at this pH level. A stable pH can be maintained by using buffers.

[0070] The DPT combined vaccine with aluminum-based adjuvant described in this invention can be prepared by the following steps: adsorbing aluminum phosphate with FHA, adsorbing aluminum hydroxide with PT, PRN, DT, TT, and FIM2 / 3, and mixing the adsorbed substances to obtain the final product. 3+ The final concentration is 0.6 mg / ml-1.4 mg / ml, and the AP / AH ratio ranges from 1:20 to 1:50, preferably from 1:25 to 1:45.

[0071] Optionally, the present invention relates to a method for preparing an immune composition or vaccine, the method comprising the step of adding residual adsorbent within a range not exceeding 0.6–1.4 mg / ml of aluminum ion concentration after the antigen has been adsorbed onto aluminum ions. In a preferred embodiment of the present invention, the inherent adsorption of the antigen components in the immune composition or vaccine can be maintained and the immune response enhanced.

[0072] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0073] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0074] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0075] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The percentage (calculated by weight) is 1:20-1:50, Al 3+ The total content is 0.6-1.4 mg / ml.

[0076] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2-3 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 3-6 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 2-4 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 3-6 μg / dose.

[0077] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0078] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0079] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0080] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The percentage (calculated by weight) is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml.

[0081] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2-3 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 3-6 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 2-4 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 3-6 μg / dose.

[0082] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0083] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0084] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0085] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The percentage (calculated by weight) is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml.

[0086] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2.5 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 5 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 8 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 8 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 3 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 5 μg / dose.

[0087] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0088] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0089] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0090] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The percentage (calculated by weight) is 1:20-1:50, Al 3+ The total content is 0.6-1.4 mg / ml, and the antigen FHA in the immunogenic composition is adsorbed by aluminum phosphate, while the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed by aluminum hydroxide.

[0091] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2-3 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 3-6 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 2-4 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 3-6 μg / dose.

[0092] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0093] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0094] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0095] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The percentage (calculated by weight) is 1:25-1:45, Al 3+ The total content is 0.6-1.4 mg / ml, and the antigen FHA in the immunogenic composition is adsorbed by aluminum phosphate, while the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed by aluminum hydroxide.

[0096] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2-3 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 3-6 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 6-10 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 2-4 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 3-6 μg / dose.

[0097] Preferably, the present invention provides an immunogenic composition or vaccine, said immunogenic composition or vaccine comprising:

[0098] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0099] (ii) An aluminum-based adjuvant, wherein the aluminum salt adjuvant is a mixture comprising aluminum hydroxide and aluminum phosphate, and the content of aluminum hydroxide in the immunogenic composition is (in Al...) 3+ The content of (calculated as Al) is higher than that of aluminum phosphate (in terms of Al). 3+ (Content calculation);

[0100] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (as Al) 3+ The weight percentage of the components 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, while the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT are adsorbed with aluminum hydroxide.

[0101] The immunogenic composition contains diphtheria toxoid (DT) at a concentration of 2.5 Lf / dose; and / or, the immunogenic composition contains tetanus toxoid (TT) at a concentration of 5 Lf / dose; and / or, the immunogenic composition contains pertussis toxin (PT) at a concentration of 8 μg / dose; and / or, the immunogenic composition contains filamentous hemagglutinin (FHA) at a concentration of 8 μg / dose; and / or, the immunogenic composition contains Bordetella pertussis adhesin (PRN) at a concentration of 3 μg / dose; and / or, the immunogenic composition contains pertussis fimbriae protein (FIM2 / 3) at a concentration of 5 μg / dose.

[0102] In addition to the antigen and adjuvant components described above, the immunogenic compositions or vaccines of the present invention typically contain one or more "pharmaceutically acceptable carriers or excipients," including any excipient that does not itself induce antibodies harmful to the recipient. Suitable excipients are typically large, slowly metabolizing macromolecules, such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those skilled in the art. Vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, excipients may be present, such as wetting agents or emulsifiers, pH buffers, etc. Sterile, pyrogen-free, phosphate-buffered saline is a typical carrier. A comprehensive discussion of pharmaceutically acceptable excipients can be found in reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0103] The immunogenic compositions of the present 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, making it ideal for injection. The compositions may be present in vials or in prepared filled syringes. The syringes may be needle-equipped or needle-free. The syringes will contain a single dose of the composition, while the vials may contain a single dose or multiple doses (e.g., two doses).

[0104] The immunogenic compositions of the present invention may also contain small amounts of wetting agents, fillers, emulsifiers, or pH adjusters as needed. Unless any conventional media or reagents are incompatible with the active ingredient, they will be used in the immunogenic compositions of the present invention.

[0105] Other “immunomodulators” that may be included in an immunogenic composition or vaccine include, for example, one or more interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12, 13, 14, 15, 16, 17 and 18 (and their mutant forms); interferon-α, β and γ; granulocyte-macrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF); or tumor necrosis factor α and β. Other adjuvants that may 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; and members of the immunoglobulin superfamily, such as PECAM and ICAM, such as ICAM-1, ICAM-2, and ICAM-3, C D2 and LFA-3; co-stimulatory molecules such as B7-1, B7-2, CD40, and CD40L; growth factors including angiogenic 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).

[0106] The vaccine of the present invention can be packaged in single-dose or multi-dose form (e.g., 2 doses). For multi-dose forms, vials are preferred over pre-filled syringes. The effective dose volume can be conventionally determined, but a typical human dose of the injectable composition has a volume of 0.5 ml. The volume of "each dose" as described in this invention is 0.5 ml.

[0107] The immunogenic compositions or vaccines of the present invention may be isotonic for humans.

[0108] Treatment methods and administration methods

[0109] The immunogenic compositions or vaccines of the present invention are suitable for human patients. The present invention provides a method for inducing an immune response in a patient, the method comprising administering the compositions or vaccines of the present invention to the patient.

[0110] Immunogenic compositions containing aluminum-based adjuvants are prone to phase separation over time; aluminum particles separate from the liquid and precipitate during storage. To ensure successful administration, the immunogenic composition must be visually homogeneous and resuspended to a homogeneous state before administration.

[0111] The present invention also provides the use of the above-described immunogenic compositions in the production of medicaments for inducing an immune response in a patient; said medicaments comprising a vaccine. These methods and uses are generally used to induce antibody responses, preferably protective antibody responses. Methods for analyzing antibody responses, neutralizing capacity, and protective effects following vaccine immunization are well known in the art.

[0112] The compositions of the present invention can be administered via a variety of routes. The most preferred route of administration is intramuscular injection (e.g., into the arm or thigh), but other available routes include subcutaneous injection, intranasal administration, oral administration, intradermal injection, percutaneous injection, transdermal injection, inhalation administration, etc.

[0113] The vaccine prepared according to the method of the present invention can be used to prevent infection with pertussis, tetanus, and diphtheria bacilli in adolescents and adults. Therefore, patients can be individuals aged 6 years and older.

[0114] Treatment can be administered using a single-dose or 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 via the same or different routes.

[0115] Immunogenicity, or an effective amount, of the immunogenic composition can be determined in a dose-response study, in which patients are immunized with incremental increments 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 immunization data from animal models. The dose may vary depending on individual circumstances. This amount can be determined in routine trials using methods known to those skilled in the art.

[0116] An immunologically effective amount of the immunogenic composition is administered to the patient at an appropriate dose to initiate an immune response. The dosage may vary depending on individual circumstances, such as age and weight. This amount can be determined in routine testing using methods known to those skilled in the art.

[0117] The beneficial effects of this invention are:

[0118] 1. The dual aluminum adjuvant of the present invention has a strong auxiliary immune enhancement effect on vaccine antigen components. Its immune effect is far better than Al(OH)3 or AlPO4 alone as vaccine adjuvants, and also far better than the compatibility effect of Al(OH)3 and AlPO4 with other active vaccine components. This shows that not only do Al(OH)3 and AlPO4 have a synergistic effect when used in combination in a specific ratio, but the Al(OH)3 and AlPO4 compound adjuvant also has a synergistic effect with the antigen components in the vaccine of the present invention.

[0119] 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 have high safety.

[0120] 3. The preparation process of the bisaluminum-based adjuvant described in this invention is mature and inexpensive. Attached image description:

[0121] Figure 1 shows the IgG antibody titers for the detection of antigen FHA under different AP / AH conditions;

[0122] Figure 2 shows the IgG antibody titer for antigen PT detection under different AP / AH conditions;

[0123] Figure 3 shows the IgG antibody titers for the detection of antigen PRN under different AP / AH conditions;

[0124] Figure 4 shows the IgG antibody titer for antigen FIM detection under different AP / AH conditions;

[0125] Figure 5 shows the IgG antibody titer for antigen DT under different AP / AH conditions;

[0126] Figure 6 shows the IgG antibody titer for antigen TT under different AP / AH conditions;

[0127] Figure 7 shows the IgG antibody titer against antigen FHA under different aluminum contents when AP / AH = 1 / 30.

[0128] Figure 8 shows the IgG antibody titer against antigen PT under different aluminum contents when AP / AH = 1 / 30.

[0129] Figure 9 shows the IgG antibody titer against antigen PRN under different aluminum contents when AP / AH = 1 / 30.

[0130] Figure 10 shows the IgG antibody titer against antigen FIM under different aluminum contents when AP / AH = 1 / 30.

[0131] Figure 11 shows the IgG antibody titer against antigen DT under different aluminum contents when AP / AH = 1 / 30.

[0132] Figure 12 shows the IgG antibody titer against antigen TT under different aluminum contents with AP / AH = 1 / 30.

[0133] Figure 13 shows the antigen adsorption rates for different antigens (FHA, PT, PRN, FIM, DT, TT) under different AP / AH conditions;

[0134] Figure 14. Antigen adsorption rate detection results; AP / AH = 1 / 30, aluminum adjuvant content 1 mg / ml, antigen content per milliliter: DT: 4 Lf; TT: 6 Lf; PT: 12 μg; FHA: 12 μg; PRN: 4 μg; FIM2 / 3: 6 μg;

[0135] Figure 15. Results of antigen adsorption rate detection; AP / AH = 1 / 30, aluminum adjuvant content 1 mg / ml, antigen content per milliliter: DT: 6 Lf; TT: 12 Lf; PT: 20 μg; FHA: 20 μg; PRN: 8 μg; FIM2 / 3: 12 μg. Detailed Implementation

[0136] 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 definitive in defining the terminology and scope of the invention. It should be understood that conditions outside or below the specified range may also be used, although less convenient, given typical reaction conditions (e.g., temperature, reaction time, etc.). Unless otherwise stated, all parts and percentages given in this specification are by weight, and all temperatures are in degrees Celsius.

[0137] Furthermore, the following embodiments utilize standard methods well-known and conventional to those skilled in the art, unless otherwise stated. As described above, the following embodiments are provided for illustrative purposes and should not be construed as limiting the scope of the invention in any way.

[0138] Example 1: Preparation of Adsorbed Antigen and DPT Vaccine

[0139] PRN, PT, DT, TT, and FIM antigens were sterilized and filtered into reaction vessels, and reacted with filtered aluminum hydroxide adjuvant for 1-10 hours to obtain adsorbed PRN, PT, DT, TT, and FIM stock solutions. FHA antigen was sterilized and filtered into reaction vessels, and reacted with filtered aluminum phosphate adjuvant for 70-90 hours to obtain adsorbed FHA stock solution. The above adsorbed antigens were mixed, stirred thoroughly, and dispensed to prepare the DPT vaccine formulation. Each dose of vaccine formulation contained 8 μg, 8 μg, 3 μg, 5 μg, 2.5 Lf, and 5 Lf of antigens PT, FHA, PRN, FIM, DT, and TT, respectively.

[0140] Example 2: Immunogenic effects of DPT combined vaccines with different ratios of aluminum phosphate / aluminum hydroxide

[0141] DPT samples containing aluminum-based adjuvants with different aluminum phosphate / aluminum hydroxide ratios were prepared according to the method in 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 dosage was 0.5 mg per dose, and total aluminum phosphate and total aluminum hydroxide adjuvants were used as experimental control groups. Healthy NIH mice were selected and randomly divided into groups of 10 mice each. Each mouse was immunized with 1 / 3 of the test product. After one injection, serum was collected by enucleation 14 days later. Serum antibody titers were determined by ELISA and statistical analysis was performed using GraphPad Prism 8.0 software.

[0142] The specific results are shown in Figures 1-6. The results show that as the AP / AH ratio decreases, the FHA antibody titer decreases accordingly. When the AP:AH ratio is in the range of 1:15–1:50, the FHA antibody titer is at a relatively high level. Similarly, the PT antibody titer increases as the AP / AH ratio decreases, and when the AP:AH ratio is in the range of 1:20–1:55, the PT antibody titer is relatively high. Likewise, the PRN antibody titer increases as the AP / AH ratio decreases, and when the AP:AH ratio is in the range of 1:20–1:55, the PRN antibody titer is relatively high. Based on these results, when the AP:AH ratio is in the range of 1:20–1:50, the FHA, PT, PRN, FIM, DT, and TT antibodies are all at relatively high levels, and their antibody titers are not inferior to the all-AP control and the all-AH control.

[0143] Example 3: Same aluminum adjuvant, different Al 3+ The immunization effect of DPT combined vaccine with high concentration

[0144] Samples containing aluminum-based adjuvants with a ratio of aluminum phosphate to aluminum hydroxide of 1:30 and different aluminum contents (0.1, 0.3, 0.5, 0.7 mg aluminum / dose, 0.5 ml per dose) were prepared according to the method in Example 1. Healthy NIH mice were selected and randomly divided into groups of 10 mice each. Each mouse was immunized with 1 / 3 of the HD test sample. After one injection, serum was collected by enucleation 14 days later. The serum antibody titer was determined by ELISA and statistical analysis was performed using GraphPad Prism 8.0 software.

[0145] The specific results are shown in Figure 7-12. The results show that as the aluminum content increases, the titers of IgG antibodies produced by each antigen in mice also increase. There were no significant differences in FHA, FIM, DT, and TT antibody titers, but the titers of PT and PRN antibodies were significantly different at high-dose aluminum compared to 0.1 mg aluminum.

[0146] Example 4: Adsorption rates of different AP / AH antigens

[0147] DPT samples containing aluminum-based adjuvants with different aluminum phosphate / aluminum hydroxide ratios were prepared according to the method in 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 concentration was 1.0 mg / ml. The antigen adsorption rate was measured under various conditions. The results are shown in Figure 13.

[0148] Example 5: Adsorption rates of various antigens with different antigen contents under the condition of AP / AH = 1 / 30

[0149] A DPT sample containing an aluminum-based adjuvant with an aluminum phosphate / aluminum hydroxide ratio of 1:30 was prepared according to the method in Example 1. 3+ The total concentration was 1.0 mg / ml. The specific concentrations of each antigen were adjusted, and the antigen adsorption rate under various conditions was measured. The results are shown in Figures 14-15.

[0150] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0151] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An immunogenic composition, characterized in that, The immunogenic composition includes: (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigen; said acellular pertussis antigen comprises pertussis toxin (PT), filamentous hemagglutinin (FHA), Bordetella pertussis adhesin (PRN), and pertussis fimbriae protein (FIM 2 / 3); (ii) A bisaluminum adjuvant, wherein the bisaluminum adjuvant is a mixture comprising 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 is 0.6-1.4 mg / ml, and the aluminum hydroxide is in the form of Al. 3+ The content calculation of the aluminum phosphate is based on 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 to 1:

45.

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

4. A combined vaccine for pertussis, diphtheria, and tetanus, characterized in that, The vaccine comprises the immunogenic composition as described in any one of claims 1-3 above.

5. The vaccine according to claim 4, characterized in that, The vaccine also contains pharmaceutically acceptable excipients.

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

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

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

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