An immunogenic composition, vaccine for preventing diphtheria, pertussis and tetanus, and preparation method and application thereof

Abstract

The present invention provides a five-component pertussis, diphtheria, and tetanus combined vaccine, particularly a diphtheria, pertussis, and tetanus vaccine with enhanced immune response. The immunogenic composition and vaccine of the present invention contain a specially selected bi-aluminum-based adjuvant. The resulting vaccine composition is capable of inducing a robust immune response while ensuring vaccine safety.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, in particular 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. It is particularly helpful in enhancing the specific immune response to the antigen contained in the vaccine. Adjuvants not only help antigens induce long-term and effective specific immune responses in the body, thereby achieving higher vaccine efficacy and prolonging the protective effect 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 precipitates, and polysaccharides. However, only aluminum-based adjuvants are used consistently and widely around the world.

[0003] Aluminum-based adjuvants have been used in vaccines for nearly a century and were the first adjuvants approved by the US 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 use of adjuvants in vaccines to enhance immune responses is a long-standing practice. Early vaccines contained many impurities in addition to the antigen, which compromised their effectiveness. The advent of recombinant DNA technology and synthetic chemistry has enabled the production of highly purified antigens to induce more specific immune responses. While the use of highly purified antigens improves vaccine safety and tolerability and simplifies vaccine characterization, it often results in reduced immunogenicity. Therefore, such antigen preparations require the addition of an adjuvant to achieve protective immunity. In 1926, Glenny et al. discovered that diphtheria toxoid (DT) adsorbed with aluminum exhibited greater immunogenicity than the toxoid alone. This groundbreaking 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 vaccines, the mechanisms by which they exert their beneficial effects remain largely unknown, leading to numerous challenges in designing and formulating vaccines using them. Common explanations for the effectiveness of aluminum-based adjuvants in enhancing antigen immunogenicity include the belief that their addition increases antigen surface area, prolongs antigen stimulation duration, and enhances antigen uptake at the injection site, thereby enhancing the immune response. Aluminum hydroxide-based adjuvants and aluminum phosphate-based adjuvants exhibit distinct physical and chemical properties, which contribute to the differences in immune responses elicited by these two chemical compounds. Furthermore, in vivo experimental results between aluminum hydroxide-based and aluminum phosphate-based adjuvants differ; for example, researchers have found that aluminum phosphate adjuvants dissolve more readily after injection into experimental animals. These differences ultimately influence the nature of the immune responses elicited by the two adjuvants. Therefore, different aluminum-based adjuvants can elicit different responses. Different types of aluminum-based adjuvants are selected based on the specific antigens, their content, and their combination to achieve the optimal immune response.

[0005] Related research and vaccine products have used dual aluminum-based adjuvants, AP and AH, to enhance antigen immunogenicity. The choice of AH and AP adjuvants depends largely on the properties of the antigen and the adsorption requirements to achieve an optimal immune response. Therefore, different vaccine types and antigen compositions within the vaccine will result in different adjuvant types and amounts required to achieve an optimal immune response. Currently, the aluminum-based adjuvants widely used in DTP vaccines are mostly single aluminum phosphate or aluminum hydroxide adjuvants. For example, Boostrix® and Daptacel® both use a single aluminum hydroxide adjuvant. Furthermore, patents disclose the use of dual aluminum-based adjuvants, including aluminum hydroxide and aluminum phosphate, in the preparation of DTP vaccines. For example, patent application WO2014135651A1 discloses the simultaneous use of aluminum hydroxide and aluminum phosphate as adjuvants in DTP vaccines, but does not disclose the specific ratio of AP to AH. Patent application CN1295481A discloses a DTaP-Hib vaccine with an adjuvant ratio of aluminum phosphate to aluminum hydroxide ranging from 1:1 to 20:1.

[0006] The antigen components and content of DTP vaccines vary depending on the age of the intended population. Developing new vaccines with different antigen compositions and content requires re-examination and confirmation of the appropriate adjuvant type and specific composition. Therefore, further development of DTP combination vaccines containing aluminum-based adjuvants is necessary. Summary of the Invention

[0007] The present invention provides an immune composition using a dialuminum-based adjuvant and a vaccine prepared therefrom, particularly a diphtheria-tetanus-pertussis combined vaccine comprising the dialuminum-based adjuvant. The dialuminum-based adjuvant provided herein is a mixture comprising aluminum hydroxide and aluminum phosphate, wherein the aluminum hydroxide content in the dialuminum-based adjuvant is higher than the aluminum phosphate content. The immune composition and vaccine provided herein comprise the aforementioned aluminum-based adjuvant. The present invention also provides the use of the aforementioned aluminum-based adjuvant and immune composition in the preparation of a medicament for treating a disease, as well as 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, as these methods and conditions may vary. It should also be understood that the terminology used in this specification is for the purpose of describing specific embodiments only 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 are to 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 skilled in the art upon reading this specification, and so forth.

[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 a range 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 variation encompassed by the term "about" or "approximately" depends on the specific system under investigation and can be readily determined by one 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 present invention.

[0014] The term "antigen" generally refers to a biological molecule, typically a protein, peptide, polysaccharide, lipid, or conjugate, that contains at least one epitope to which an antibody that recognizes it can selectively bind; or, in certain cases, an immunogenic substance capable of stimulating antibody production, a T-cell response, or both in an animal, including compositions injected into or ingested by an animal. An immune response can be induced by the entire molecule or by one or more distinct portions 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" encompasses 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. Furthermore, for the purposes of this invention, the term "antigen" also applies to proteins with modifications, such as deletions, additions, and substitutions (usually conservative in nature, but which may not be conservative), to 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 or genetic engineering methods, or they may be accidental, for example, 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 intact microorganisms. Similarly, oligonucleotides or polynucleotides that express antigens, for example, in nucleic acid immunization protocols, are also included in this 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 "treat" (including variants thereof, such as "treating" or "treated") refers to any one or more of the following: (i) preventing infection or reinfection, as in the case of conventional vaccines, (ii) reducing the severity or eliminating symptoms; and (iii) substantially or completely eliminating a particular pathogen or disease. Thus, treatment can be performed prophylactically (before infection) or therapeutically (after infection). According to the present invention, prophylactic or therapeutic treatments can be used. According to a specific embodiment of the present invention, compositions and methods are provided for treating a host animal against infection caused by a microorganism (e.g., Clostridium tetani, etc.), including through prophylactic and / or therapeutic immunization. The methods of the present invention can be used to induce immunity in a patient prophylactically and / or therapeutically. For the purposes of biomedical research, the methods of the present invention can 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 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] In a first aspect, the present invention provides an aluminum-based adjuvant comprising an aluminum salt, such as aluminum phosphate, aluminum hydroxide, or a combination of aluminum phosphate and aluminum hydroxide.

[0021] 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 combination 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.

[0022] 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, for example, 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.

[0023] Preferably, the 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.

[0024] 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 the present invention 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, ≥ 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, 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.

[0026] Specifically, the Al in the aluminum phosphate in the aluminum-based adjuvant of the present invention is 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.

[0027] More preferably, the Al in the aluminum phosphate in the aluminum-based adjuvant of the present invention is 3+ and Al in aluminum hydroxide 3+ The weight percentage of aluminum phosphate in the aluminum-based adjuvant of the present invention is about 1:25 to 1:45. 3+ and Al in aluminum hydroxide 3+ When the weight ratio of the vaccine is within this range, the vaccine prepared has better immune effect.

[0028] Specifically, the Al in the aluminum phosphate in the aluminum-based adjuvant of the present invention is3+ 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.

[0029] 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 can achieve the best immune response to the antigen by adsorption with the antigen.

[0030] The aluminum-based adjuvant of the present invention can be particularly used for the preparation of diphtheria, tetanus and pertussis vaccine preparations.

[0031] Immunogenic compositions and vaccines

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

[0033] 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.

[0034] Aluminum bases include hydroxides, phosphates, sulfates, and the like, as well as any suitable salt form (e.g., gelatinous, crystalline, amorphous, etc.). These salts are preferably used for adsorption. Adjuvants such as aluminum hydroxide and / or aluminum phosphate may be used. Combinations of aluminum hydroxide and aluminum phosphate may be used in the present invention.

[0035] The vaccine of the present invention is preferably a diphtheria, pertussis and tetanus vaccine, which contains acellular pertussis antigens.

[0036] The DTP vaccine of the present invention provides protection against diseases caused by the following: Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis. It is generally composed of diphtheria toxoid (DT), tetanus toxoid (TT) and acellular pertussis (Pa).

[0037] The diphtheria antigen is typically 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 purifying the toxin from culture of Corynebacterium diphtheriae followed by chemical detoxification, but may alternatively be produced by recombinant production of the toxin or purification of a genetically detoxified analog.

[0038] Bordetella pertussis is a Gram-negative, non-spore-forming aerobic bacterium that causes wheezing. Vaccines against Bordetella pertussis have been available for many years and are classified into two types: cellular and acellular. Cellular vaccines contain whole B. pertussis cells that have been killed and inactivated (e.g., by treatment with formalin and / or heat), while acellular vaccines contain specifically purified B. pertussis, either from native bacteria or after expression in a recombinant host. The pertussis antigens in the immunogenic compositions or vaccines of the present invention are acellular pertussis antigens.

[0039] The diphtheria, pertussis and tetanus (DTP) vaccine of the present invention contains five components of acellular pertussis antigens, wherein the Bordetella pertussis antigens include: (1) detoxified pertussis toxin (pertussis toxinoid 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).

[0040] Prior to use in the present invention, FHA and pertactin 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 in which the enzymatic activity has been reduced by mutagenesis, but detoxification by chemical treatment is preferred.

[0041] 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 then chemically detoxified, or it can be produced by purifying a recombinant or genetically detoxified toxin analog. Any suitable tetanus toxoid can be used in the immunogenic composition or vaccine of the present invention. "Tetanus toxoid" can encompass immunogenic fragments 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 antigens; the acellular pertussis antigens comprise 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;

[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 (expressed as Al 3+ The content of aluminum phosphate (calculated as Al3+ Calculation of content).

[0045] 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 immunogenic compositions and vaccine preparations that achieve optimal immune responses.

[0046] 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 ranges from approximately 1:20 to 1:50. The immunogenic composition of the present invention exhibits optimal effects when the ratio of aluminum phosphate to aluminum hydroxide is within this range.

[0047] 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.

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

[0049] 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.

[0050] The inventors of this application hypothesize that the aluminum-based adjuvants of the present invention may influence the immunogenicity of vaccine antigens by affecting their specific structure. Antigen structure analysis can be performed using methods such as X-ray crystallography or nuclear magnetic resonance (NMR), and the thermal stability of antigens before and after adsorption can be assessed using methods such as differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF). In this study, the adsorption capacity of the aluminum-based adjuvants for specific antigens in the diphtheria, pertussis, and tetanus vaccine was characterized by measuring the adsorption rate of aluminum-adsorbed antigens. Furthermore, the effects of specific aluminum-based adjuvants on the immune response were characterized by immunizing animal models and measuring the antibody responses in the animals.

[0051] To find the optimal aluminum salt adjuvant content for the preparation of 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 time of final binding was lower than 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 of addition 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.

[0053] In some embodiments, the immunogenic composition comprises an immunogenic agent at 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 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 mg / ml to about 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.

[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 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 adhesin (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.

[0056] In some 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 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 borrelial adhesin (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.

[0057] 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 adhesin (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 0.05. 3+ The total content is preferably 1 mg / ml (or 0.5 mg / dose).

[0058] The aluminum salt adjuvant of the present invention is adsorbed on part or all of the antigens in the composition.

[0059] 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 an adsorption step.

[0060] The step of adsorbing the aluminum-based adjuvant and the antigen of the present invention can be performed as follows: first, the aluminum-based adjuvant and different antigens are adsorbed separately, and then the substances obtained after adsorption are mixed.

[0061] The adsorption of the present invention may optionally include the step of adding an adjuvant again after mixing to adsorb the antigen.

[0062] Unless otherwise indicated, 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.

[0063] 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 with the antigen FHA, and the aluminum hydroxide to adsorb with the antigen PRN, antigen FIM2 / 3, antigen PT, antigen DT, and antigen TT. This adsorption method is adopted in the specific embodiment of the present invention.

[0064] 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.

[0065] The term "adsorbed antigen" as used herein means at least 90% adsorbed. The adsorption rate of FHA, PT, PRN, FIM, DT, and TT in the composition or vaccine formulation of the present invention, after mixing the adsorbed antigens, can be greater than 90%, preferably greater than 95%, and more preferably greater than 99%.

[0066] 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 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. A stable pH can be maintained by using a buffering agent.

[0070] The diphtheria, tetanus and pertussis combined vaccine with aluminum-based adjuvant of the present 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 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.

[0071] Alternatively, the present invention relates to a method for preparing an immune composition or vaccine, comprising the step of adding a residual adsorbent after the antigen is adsorbed onto aluminum ions, within a range of aluminum ion concentrations not exceeding 0.6 to 1.4 mg / ml. In preferred embodiments 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.

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

[0073] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0074] (ii) 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);

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

[0076] 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 pilin (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.

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

[0078] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0079] (ii) 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);

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

[0081] 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 pilin (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.

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

[0083] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0084] (ii) 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);

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

[0086] 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 pilin (PRN) in the immunogenic composition is 3 μg / dose; and / or, the content of pertussis fimbrial protein (FIM2 / 3) in the immunogenic composition is 5 μg / dose.

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

[0088] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0089] (ii) 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);

[0090] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The 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.

[0091] 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 pilin (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.

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

[0093] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0094] (ii) 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);

[0095] The weight of aluminum phosphate and aluminum hydroxide in the immunogenic composition (in terms of Al 3+ The 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.

[0096] 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 pilin (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.

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

[0098] (i) diphtheria toxoid, tetanus toxoid and acellular pertussis antigens; the acellular pertussis antigens include pertussis toxin (PT), filamentous hemagglutinin (FHA), pertussis adhesin (PRN), and pertussis fimbriae protein (FIM2 / 3);

[0099] (ii) 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 Al3+ Calculation of content);

[0100] 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.

[0101] 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 pilin (PRN) in the immunogenic composition is 3 μg / dose; and / or, the content of pertussis fimbrial protein (FIM2 / 3) in the immunogenic composition is 5 μg / dose.

[0102] In addition to the antigen and adjuvant components described above, the immunogenic compositions or vaccines of the present invention typically also contain one or more "pharmaceutically acceptable carriers or excipients," which include any excipient that does not itself induce the production of antibodies harmful to the individual receiving the vaccine. Suitable excipients are typically large, slowly metabolized macromolecules, such as proteins, carbohydrates, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. Vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may be present. Sterile, pyrogen-free, phosphate-buffered saline is a typical carrier. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th ed., 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 packaging without requiring reconstitution in an aqueous medium, making it well-suited for injection. The compositions can be presented in vials, or they can be presented in ready-to-fill syringes. Syringes can be needle-less or with needles. A syringe will contain a single dose of the composition, while a vial can contain a single dose or multiple doses (e.g., two doses).

[0104] The immunogenic composition of the present invention may further contain a small amount of a wetting agent, a filler, an emulsifier or a pH adjuster according to actual needs. Unless any conventional media or agents are incompatible with the active ingredient, they will be used in the immunogenic composition of the present invention.

[0105] 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-selectin, and C-reactive protein. D2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40, 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 vaccines of the present invention can be packaged in unit dose form or in multiple dose form (e.g., two doses). For multiple doses, vials are preferred over prefilled syringes. While effective dose volume can be routinely determined, a typical human dose of an injectable composition has a volume of 0.5 ml. As used herein, the volume of "each dose" is 0.5 ml.

[0107] The immunogenic composition or vaccine of the invention may be isotonic to humans.

[0108] Treatment and administration methods

[0109] The immunogenic composition or vaccine of the present invention is suitable for use in human patients. The present invention provides a method for inducing an immune response in a patient, which comprises administering the composition or vaccine of the present invention to the patient.

[0110] 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.

[0111] The present invention also provides the use of the above-described 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 efficacy following vaccine immunization are well known in the art.

[0112] 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 include subcutaneous injection, intranasal administration, oral administration, intradermal injection, transdermal injection, transdermal injection, inhalation administration, etc.

[0113] 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.

[0114] Treatment can be performed using a single dose regimen or a multiple dose regimen. Multiple dose regimens can be used for primary immunization and / or booster immunization. In a multiple dose regimen, different doses can be administered by the same or different routes.

[0115] Immunogenicity or the effective amount of an immunogenic composition can be determined in dose-response studies, 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 immunological data from animal models. The dose may vary depending on the individual's circumstances. This amount can be determined in routine experiments using methods known to those skilled in the art.

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

[0117] Beneficial effects of the present invention:

[0118] 1. The dual aluminum-based adjuvant of the present invention has a strong auxiliary immune enhancement effect on the vaccine antigen components. 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 effect 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 there is also a synergistic effect between the composite adjuvant of Al(OH)3 and AlPO4 and 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 are highly safe.

[0120] 3. The preparation process of the double aluminum-based adjuvant of the present invention is mature and the price is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1 is the IgG antibody titer for antigen FHA under different AP / AH conditions;

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

[0123] Figure 3 IgG antibody titer for antigen PRN under different AP / AH conditions;

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

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

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

[0127] Figure 7 The IgG antibody titer against antigen FHA was tested under different aluminum content conditions when AP / AH=1 / 30;

[0128] Figure 8 The IgG antibody titer against antigen PT was tested under different aluminum content conditions with AP / AH=1 / 30;

[0129] Figure 9 The IgG antibody titer against the antigen PRN was tested under different aluminum content conditions when AP / AH=1 / 30;

[0130] Figure 10The IgG antibody titer against antigen FIM was tested under different aluminum content conditions when AP / AH=1 / 30;

[0131] Figure 11 The IgG antibody titer against antigen DT was tested under different aluminum content conditions with AP / AH=1 / 30;

[0132] Figure 12 The IgG antibody titer against antigen TT was tested under different aluminum content conditions when AP / AH=1 / 30;

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

[0134] Figure 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;

[0135] Figure 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

[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 of the terminology and scope of the present invention. It should be understood that where typical reaction conditions (e.g., temperature, reaction time, etc.) are given, conditions above 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.

[0137] 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.

[0138] Example 1: Preparation of adsorbed antigens and diphtheria, pertussis and tetanus vaccines

[0139] Sterile-filter the PRN, PT, DT, TT, and FIM antigens separately into a reaction vessel and react with the filtered aluminum hydroxide adjuvant for 1-10 hours to produce adsorbed PRN, PT, DT, TT, and FIM stock solutions. Sterile-filter the FHA antigen into a reaction vessel and react with the filtered aluminum phosphate adjuvant for 70-90 hours to produce an adsorbed FHA stock solution. Combine the adsorbed antigens, stir evenly, and aliquot to prepare a diphtheria, pertussis, and tetanus vaccine formulation. Each dose of the vaccine formulation contains 8μg of PT, 8μg of FHA, 3μg of PRN, 5μg of DT, and 2.5Lf of TT, respectively.

[0140] Example 2: Immune effects of diphtheria, tetanus and pertussis combined vaccines with different ratios of aluminum phosphate / aluminum hydroxide

[0141] According to the method of Example 1, diphtheria and pertussis samples containing aluminum-based adjuvants with different ratios of aluminum phosphate / aluminum hydroxide were prepared. 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 a control group using full aluminum phosphate and full aluminum hydroxide adjuvants was used. NIH mice in good health were randomly divided into 10 groups. Each mouse was immunized with a single injection of 1 / 3 HD test article. Serum was collected 14 days after eye removal. Serum antibody titers were determined by ELISA, and statistical analysis was performed using GraphPad Prism 8.0 software.

[0142] Specific results can be found in Figure 1-6 The results show that FHA antibody titers decreased with decreasing AP / AH ratios, reaching higher levels when the AP:AH ratio was between 1:15 and 1:50. PT antibody titers increased with decreasing AP / AH ratios, reaching higher levels when the AP:AH ratio was between 1:20 and 1:55. PRN antibody titers also increased with decreasing AP / AH ratios, reaching higher levels when the AP:AH ratio was between 1:20 and 1:55. Based on these results, FHA, PT, PRN, FIM, DT, and TT antibody titers were all at higher levels when the AP:AH ratio was between 1:20 and 1:50, and were not inferior to those of the all-AP and all-AH controls.

[0143] Example 3: Same aluminum adjuvant, different Al 3+ Immune effect of combined diphtheria, tetanus and pertussis vaccine

[0144] 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 per dose, with a volume of 0.5 ml per dose) were prepared according to the method in Example 1. NIH mice in good health were selected and randomly divided into groups of 10. Each mouse was immunized with a single injection of 1 / 3 HD test sample. 14 days later, the eyeballs were removed and serum was collected. Serum antibody titers were determined by ELISA, and statistical analysis was performed using GraphPad Prism 8.0 software.

[0145] Specific results can be found in Figure 7-12 The results showed that as the aluminum content increased, the IgG antibody titers produced by each antigen in mice increased accordingly. There was no significant difference in FHA, FIM, DT, and TT. However, the PT and PRN antibody titers showed significant differences when compared with 0.1 mg aluminum at high doses.

[0146] Example 4: Antigen adsorption rates of different AP / AH

[0147] According to the method of Example 1, diphtheria and pertussis samples containing aluminum-based adjuvants with different ratios of aluminum phosphate / aluminum hydroxide were prepared. 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 were as follows Figure 13 shown.

[0148] Example 5: Antigen adsorption rates at different antigen contents under the condition of AP / AH=1 / 30

[0149] According to the method of Example 1, a diphtheria and pertussis sample containing an aluminum-based adjuvant with a ratio of aluminum phosphate to aluminum hydroxide of 1 / 30 was prepared. 3+ The total amount was 1.0 mg / ml, and the specific content of each antigen was adjusted to test the antigen adsorption rate under each condition. Figure 14-15 shown.

[0150] 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, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection 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 appropriate 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, filamentous hemagglutinin, pertussis adhesin and pertussis pilin; (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 1:25-1:45, and Al 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; The content of diphtheria toxoid in the immunogenic composition is 2-3 Lf / dose; the content of tetanus toxoid in the immunogenic composition is 3-6 Lf / dose; the content of pertussis toxin in the immunogenic composition is 6-10 μg / dose; the content of filamentous hemagglutinin in the immunogenic composition is 6-10 μg / dose; the content of pertussis pilus protein in the immunogenic composition is 2-4 μg / dose; the content of pertussis pilus protein in the immunogenic composition is 3-6 μg / dose; the adsorption steps of the adjuvant and the antigen are: adsorbing aluminum phosphate with filamentous hemagglutinin, adsorbing aluminum hydroxide with pertussis toxin, pertussis pilus protein, diphtheria toxoid, tetanus toxoid, and pertussis pilus protein, and mixing the adsorbed antigens obtained above.

2. A pertussis, diphtheria, and tetanus combined vaccine, characterized in that: The vaccine comprises the immunogenic composition according to claim 1 above.

3. The vaccine according to claim 2, characterized in that The vaccine further comprises a pharmaceutically acceptable excipient.

4. Use of the immunogenic composition according to claim 1 in preparing a medicament for preventing or treating a disease, characterized in that: The disease is diphtheria, tetanus or pertussis.

5. The use according to claim 4, characterized in that The medicine is a vaccine.

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