Immunogenic composition of haemophilus influenzae conjugated to protein d

Abstract

This invention relates to a multivalent vaccine composition comprising antigens against diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP), acellular pertussis (aP), poliomyelitis / inactivated poliomyelitis (IPV), hepatitis (Hep), and Haemophilus influenzae (Hib). Haemophilus influenzae is coupled to protein D or a fragment of protein D as a carrier protein. The invention also relates to a method for its preparation and an immunogenic composition comprising these components, wherein protein D induces a protective response against invasive infections caused by Haemophilus influenzae.

Description

Technical Field

[0001] This invention relates to a multivalent vaccine composition comprising antigens against diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B, and Haemophilus influenzae; wherein the antigens are derived from Haemophilus influenzae type b (Hib). Haemophilus influenzae The capsular polysaccharide antigen of Hib is coupled to protein D or a fragment of protein D as a carrier protein. The present invention also relates to methods for preparing these and immunogenic compositions comprising them, wherein protein D induces a protective response against invasive infections caused by Haemophilus influenzae. Background Technology

[0002] Haemophilus influenzae is a major cause of high morbidity and mortality rates in children under five years old worldwide. In 1933, Fothergill and Wright demonstrated that the vast majority of Hib meningitis cases occurred in children under five years old.

[0003] The first-generation Hib vaccine contained pure polysaccharides from the Hib capsule. These polysaccharide vaccines were introduced in the early 1980s, but exhibited poor immunogenicity in children under 18 months of age, the age group with the highest Hib disease burden. A Hib-drug conjugate vaccine was introduced in 1985.

[0004] Polysaccharides can directly stimulate B cells to produce antibodies without T cells. The disease burden caused by bacteria encapsulated in polysaccharides is most severe in the first year of life, but these common polysaccharides are generally not immunogenic, limiting their use as vaccines. Covalently coupling carbohydrate antigens to proteins that provide T-cell epitopes overcomes this limitation.

[0005] Haemophilus influenzae is an important human-specific pathogen found in the mucous membranes of the upper respiratory tract. Haemophilus influenzae isolates can be divided into two main categories: encapsulated strains and non-encapsulated / untyped (NT) strains. Encapsulated Haemophilus influenzae is more likely to cause invasive diseases, while non-encapsulated / untyped Haemophilus influenzae (NT Haemophilus influenzae) is more likely to cause most mucosal Haemophilus influenzae infections. The encapsulated form can penetrate the nasopharyngeal epithelium and invade capillaries; its polysaccharide capsule protects it from immune phagocytosis and complement-mediated lysis in non-immune hosts. Six serotypes (af) of Haemophilus influenzae have been identified; Haemophilus influenzae type b (Hib) is the main cause of invasive infections in children.

[0006] In regions of the world where no conjugate vaccine is available, sepsis, meningitis, and pneumonia caused by Hib remain major disease burdens.

[0007] Unspecimenized strains of Haemophilus influenzae are rarely associated with invasive diseases in healthy children and adults, but are associated with their respiratory infections. These strains are frequently (up to 40% probability) a cause of acute otitis media (AOM) in children. Furthermore, *H. influenzae* NT is commonly found in the purulent secretions of patients with cystic fibrosis and chronic obstructive pulmonary disease. Therefore, the disease burden caused by *H. influenzae* NT can be prevented through vaccination. The potential of many *H. influenzae* NT proteins as vaccine antigens has been evaluated in preclinical studies. Selecting an ideal *H. influenzae* NT vaccine candidate is not easy because *H. influenzae* NT produces extended sequences and variant antigens, such as outer membrane proteins, adhesins, lipopolysaccharides, and secreted virulence factors, when interacting with the immune system.

[0008] Haemophilus influenzae (NT) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and sometimes invasive infections. The Hib-conjugate vaccine does not prevent infections caused by NT Haemophilus influenzae. 。 Because NT Haemophilus influenzae is unencapsulated. 。 Approximately one-third of otitis media cases are caused by Haemophilus influenzae (NT), and this bacterium is the most common cause of recurrent otitis media.

[0009] Developing vaccines against polysaccharide-encapsulated pathogens such as Haemophilus influenzae type b is challenging because polysaccharides do not elicit strong and durable immune responses (e.g., non-T cell-dependent immune responses). This can be overcome by conjugating polysaccharides to protein carriers (e.g., tetanus toxoid, cross-reactive material 197 [CRM]), which greatly improves the immune response and induces memory of the polysaccharide (T cell-dependent immune response).

[0010] Here is a list of some US vaccines that contain Hib antigens conjugated to a carrier protein:

[0011] The following is a review of the multi-component combination vaccine compositions containing Hib antigens developed to date, with a focus on the binding properties of Haemophilus type b polysaccharide antigens.

[0012]

[0013] Based on existing technologies, only four carrier proteins have been used to develop conjugated Hib vaccines: diphtheria toxoid (PRP-D), tetanus toxoid (PRP-T), mutant diphtheria toxoid (HbOC / CRM), and meningococcal outer membrane protein (PRP-OMP). PRP-T conjugation was only used to achieve protective antibody levels after the second dose.

[0014] Commercially available vaccine compositions containing Hib antigen do not contain Hib that is coupled to protein D, which serves as a carrier protein.

[0015] WO200056360 also cites the drawbacks of using these commonly used protein vectors. Although these vectors are commonly used and successful in inducing anti-polysaccharide antibody responses, they also have some disadvantages.

[0016] It is known that if antibodies against protein carriers are present, antigen-specific immune responses will be suppressed (epitope suppression). For example, in most populations, because routine vaccines contain DT and TT, most people have immunity to DT and TT.

[0017] Furthermore, for vaccines requiring regular booster immunizations, the use of highly immunogenic vectors such as TT and DT may suppress polysaccharide antibody responses after several injections. With repeated vaccinations, negative reactions may also occur, such as delayed hyperresponsiveness.

[0018] Therefore, polysaccharide vaccines need to balance the necessity of the vector, the induction of high levels of anti-polysaccharide antibody responses, and the selection of the vector protein.

[0019] Protein D is an antigenically conserved, surface-localized outer membrane protein of all Haemophilus influenzae species, including untyped (NT) Haemophilus influenzae.

[0020] According to existing technology reports, the UK began increasing the use of acellular pertussis combined vaccines in late 1999, which led to a large number of Hib vaccine failures.

[0021] Therefore, one object of the present invention is to develop a multi-component combined vaccine composition wherein Hib is conjugated to the carrier protein protein D. Summary of the Invention

[0022] A key aspect of this invention is the development of a multi-component combined vaccine composition in which Hib is conjugated to a carrier protein, protein D, or a fragment of protein D, wherein protein D induces a protective response against invasive infection caused by Haemophilus influenzae.

[0023] The present invention also includes a method for coupling Hib with protein D or protein D fragments as carrier proteins.

[0024] The present invention also relates to a combination vaccine composition containing structurally unrelated antigens, comprising a Hib antigen conjugated to protein D, wherein in addition to responses to other antigens, a protective response against protein D is induced for invasive infections caused by untyped Haemophilus influenzae.

[0025] The present invention also relates to a combined vaccine composition comprising a Hib antigen conjugated to protein D, wherein the vaccine composition does not have problems such as vector-induced epitope inhibition, antigen competition, immune interference, and epitope loading. Attached Figure Description

[0026] Figure 1: Anti-PRP response of anti-Hib polysaccharide.

[0027] Figure 2: Antiprotein D response to the carrier protein PrD.

[0028] Figure 3: Bactericidal activity of vaccines containing Hib and PrD protein conjugates. . Detailed Implementation

[0029] The purpose of this invention is to study the dual role of protein D or protein D fragments as a conjugation carrier for polysaccharide antigens and as a protective antigen.

[0030] This invention relates to a combined vaccine containing an antigen mixture for the prevention of disease.

[0031] In particular, the present invention relates to a combined vaccine composition comprising a capsular polysaccharide antigen from Haemophilus influenzae type b (Hib) conjugated to protein D.

[0032] The present invention also relates to a combined vaccine composition comprising a Haemophilus influenzae type b (Hib) capsular polysaccharide antigen conjugated to protein D, wherein protein D induces a protective response against invasive infection caused by Haemophilus influenzae.

[0033] The present invention includes quadrivalent and / or pentavalent and / or hexavalent and / or multivalent vaccine compositions containing antigens against diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B, and Haemophilus influenzae; wherein a capsular polysaccharide antigen from Haemophilus influenzae type b (Hib) is coupled to protein D or a fragment of protein D as a carrier protein.

[0034] The present invention also includes a multi-component vaccine composition comprising antigens diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP) / acellular pertussis (aP), hepatitis (Hep), and Haemophilus influenzae (Hib) conjugated to protein D or protein D fragments as carrier proteins.

[0035] The present invention also includes a multi-component vaccine composition comprising antigenic diphtheria toxoid (DT), tetanus toxoid (TT), whole-celled pertussis (wP) / acellular pertussis (aP) and Haemophilus influenzae (Hib) conjugated to protein D or a fragment of protein D as a carrier protein.

[0036] The present invention also relates to an immunogenic multicomponent vaccine composition comprising antigen diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP) / acellular pertussis (aP), poliomyelitis, and Haemophilus influenzae (Hib), which are coupled to protein D or protein D fragments as carrier proteins.

[0037] The present invention also includes a method for coupling Hib with protein D as a carrier protein.

[0038] The present invention also provides a kit for a multi-component combined vaccine containing all antigen components in a single vial / pre-filled syringe, or a kit containing two different containers, vials, pre-filled syringes or dual-chamber syringes.

[0039] A first aspect of the present invention is to provide a multivalent vaccine composition comprising a Hib antigen coupled to protein D as a carrier protein.

[0040] The present invention also provides a combined vaccine comprising antigens for protecting subjects against at least diphtheria ('D'), tetanus ('T'), pertussis ('P'), and Haemophilus influenzae type b ('Hib'), wherein the Hib antigen is coupled to protein D or a fragment of protein D as a carrier protein.

[0041] In one embodiment, the present invention provides a combined vaccine comprising a vaccine for protecting a subject against at least diphtheria ('D'), tetanus ('T'), pertussis ('P'), hepatitis B surface antigen, and Haemophilus influenzae type b (Hib) antigen; wherein the Hib antigen is coupled to protein D or a fragment of protein D as a carrier protein.

[0042] In one embodiment, the present invention provides a combination vaccine comprising an antigen for protecting a subject against at least diphtheria ('D'), tetanus ('T'), hepatitis B surface antigen, and Haemophilus influenzae type b (Hib) antigen; wherein the Hib antigen is coupled to protein D or a fragment of protein D as a carrier protein.

[0043] In one embodiment, the present invention provides a combination vaccine comprising an antigen for protecting a subject against at least diphtheria ('D'), tetanus ('T'), pertussis ('P'), hepatitis B surface antigen (HepB), inactivated polio antigen (IPV), and Haemophilus influenzae type b (Hib); wherein the Hib antigen is coupled to protein D or a fragment of protein D as a carrier protein.

[0044] According to another aspect of the invention, the inactivated polio antigen is one or more Salk strains selected from Mahoney 1, MEF2 and Saukett 3, or one or more Sabin strains selected from Sabin 1, 2 and 3 and polio virus-like particle molecules that mimic viruses but are not infectious.

[0045] This invention provides quadrivalent, pentavalent, and hexavalent combination vaccine compositions, wherein the Hib antigen is coupled to protein D as a carrier protein. The vaccine composition may further comprise one or more antigens derived from hepatitis (A, C, D, E, F, and G strains), meningitis A, B, or C, influenza, pneumococcus, streptococcus, anthrax, dengue fever, malaria, measles, mumps, rubella, BCG, respiratory syncytial virus (RSV), Japanese encephalitis, rotavirus, smallpox, yellow fever, typhoid fever, varicella-zoster virus, etc.

[0046] The present invention also provides a pure liquid combination vaccine composition, or a vaccine composition for which the Hib antigen needs to be formulated immediately before use.

[0047] The pertussis antigen used in this invention can be cellular (e.g., whole-cell) or non-cellular.

[0048] Another aspect of the present invention relates to a method for coupling Hib and protein D.

[0049] According to one embodiment of the present invention, the Hib antigen is extracted from capsular polysaccharide, and the Hib antigen preparation used in the vaccine of the present invention contains the Hib antigen, preferably coupled to protein D as a carrier protein.

[0050] According to another embodiment of the present invention, protein D is derived from *Escherichia coli* (…). E. coli ) strain or Haemophilus influenzae (Hib) or other recombinant methods.

[0051] The polysaccharide conjugates of Hib and protein D can be prepared using any known conjugation technique. For example, the polysaccharides can be conjugated via thioether bonds. This conjugation method relies on activating the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Therefore, the activated polysaccharide can be conjugated directly or via a spacer group to an amino group on the carrier protein. The conjugates can also be prepared by direct reductive amination. Another method involves activating the polysaccharide with cyanogen bromide (CNBr), followed by derivatization with adipic dihydrazide (ADH), and then conjugating it to a protein carrier via a carbodiimide condensation reaction. Any other known method can be used to prepare the polysaccharide conjugates used in the vaccines of this invention.

[0052] Other antigens of the vaccine composition of the present invention Diphtheria is caused by Corynebacterium diphtheriae, a Gram-positive, non-spore-forming aerobic bacterium. This organism expresses an ADP-ribosylated exotoxin (“diphtheria toxin”) encoded by a prophage, which can be treated (e.g., using formaldehyde) to produce a toxoid. This toxoid is no longer toxic but retains its antigenicity and can stimulate the production of specific antitoxin antibodies after injection. The diphtheria antigen formulation used in the vaccine of the present invention preferably comprises diphtheria toxoid.

[0053] Tetanus is caused by Clostridium tetani, a Gram-positive, sporulating bacillus. This organism expresses an endopeptidase (“tetanus toxin”), which can be processed to produce a toxoid that is no longer toxic. However, it retains its antigenicity and can stimulate the production of specific antitoxin antibodies after injection. The tetanus antigen formulation used in the vaccine of this invention preferably comprises tetanus toxoid.

[0054] Whooping cough is caused by Bordetella pertussis (Botrytis cinerea). Bordetella pertussis Caused by… Whole-cell pertussis (wP) antigen can be prepared from Bordetella pertussis, which may cause infection. The wP antigen of the present invention can be inactivated by various methods such as using chemicals. However, inactivated Bordetella pertussis (wP) can be used in the vaccine composition of the present invention, preferably by heat inactivation at 56±1°C for 30 minutes. The wP antigen formulation used in the vaccine of the present invention is preferably made from Bordetella pertussis 134, 509, and 10536. Single-batch harvests of strains 10536, 509, and 134 are preferably mixed in a 1:1:1 ratio based on their turbidity.

[0055] Acellular pertussis (aP) antigen can be generated from any known Bordetella pertussis (Bacillus pertussis). Bordetella pertussis The aP antigen is obtained from *Bordetella pertussis* strain Tohama for the purposes of this invention. Any suitable culture medium can be used for the isolation, culturing, proliferation, and fermentation of the culture. For the purposes of this invention, a modified Stainer-Scholte is preferred. The acellular pertussis (aP) antigen used in the vaccine of this invention comprises at least one or more antigens selected from pertussis toxoid (PT), filamentous hemagglutinin (FHA), *Bordetella pertussis* adhesin (P69 or PRN), and FIM (fimbriae antigen-1, 2, or 3). However, according to a preferred embodiment of the invention, the aP antigen formulation used in the vaccine of this invention comprises PT, FHA, and PRN (P69) antigens.

[0056] Hepatitis is caused by various hepatitis strains such as A, B, C, D, E, F, or G, with hepatitis B virus (HBV) being one of the main causes of viral hepatitis. The HBV virion consists of a core surrounded by an outer protein coat or capsid. The main component of the capsid is a protein called HBV surface antigen, or more commonly, "HBsAg." When this antigen is administered to the recipient, it stimulates the production of anti-HBsAg antibodies, which protect against HBV infection. According to a preferred aspect of the invention, the hepatitis (Hep) antigen preparation used in the vaccine of the present invention comprises a Hep antigen derived from the surface antigen (HBsAg) of a hepatitis B virus strain.

[0057] For vaccine production, HBsAg can be prepared by purifying particulate antigen from the plasma of chronic hepatitis B carriers, since large amounts of HBsAg are synthesized in the liver and released into the bloodstream during HBV infection, or by expressing the protein using recombinant DNA methods. HBsAg used in the vaccine of this invention can be prepared using either of the above methods.

[0058] Poliomyelitis is caused by the poliovirus. The vaccine of the present invention may contain the Sabin (Sabin1 and / or Sabin2, and / or Sabin2) or Salk strain of the poliovirus. According to a preferred embodiment of the invention, the vaccine of the present invention contains the Salk strain. There are three Salk strains that can cause polio. These three types are similar and cause the same symptoms, but they are very different antigenically, and infection with one type does not prevent infection with the other types. The Salk poliovirus includes three types: poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-I strain), and poliovirus type 3 (e.g., Saukett strain). According to a preferred embodiment of the invention, the vaccine of the present invention may contain one or more of the said Salk strains.

[0059] Poliovirus can be grown in cell cultures. The Vero cell line, a continuous cell line derived from monkey kidney cells, can be used to grow poliovirus. After growth, virus particles can be purified using known techniques. Virus inactivation can be performed. The amount of poliovirus is typically expressed in 'continuous units' ("D antigen units"). Preferably, an IPV antigen formulation for producing the vaccine of the present invention is prepared to contain one or more strains used for vaccine production. This batch formulation is then used to formulate the vaccine of the present invention.

[0060] Other antigens envisioned in this invention include VLP, multi-epitope designer antigens, multimeric vaccine candidates, subunits, cleavage or purified proteins of poliovirus, HPV, COVID, hepatitis, varicella, shingles, etc.

[0061] Non-antigen components: In addition to antigenic components, vaccines may contain many non-antigenic components, which are pharmaceutically acceptable excipients. These include, but are not limited to, pH adjusters, buffers, adjuvants, preservatives, carriers, and tension modifiers.

[0062] Aluminum-based adjuvants are the most commonly used. These adjuvants have also been approved by the FDA for use in vaccines. Studies have shown that many aluminum-containing vaccines elicit higher and longer antibody responses than their counterparts without aluminum adjuvants. The benefits of adjuvants are typically observed during the initial immunization series rather than during booster doses. Aluminum phosphate is the preferred adjuvant for use in the vaccine compositions of this invention.

[0063] The vaccine may also contain novel adjuvants selected from emulsified adjuvants TLR3, TLR4, TLR5, TLR7, TLR8, TLR9 and TLR10 agonists.

[0064] Vaccines are susceptible to bacterial contamination. Therefore, to avoid life-threatening contamination from harmful microorganisms (which could introduce contamination into the vaccine during accidental contamination events), preservatives may be added to the vaccine composition during formulation. Preservatives already in use include phenomenon chloride, thimerosal, phenol, and 2-phenoxyethanol (2-POE). 2-Phenoxyethanol is the preferred preservative used in the vaccine compositions of this invention.

[0065] The compositions of the present invention can be administered via any conventional route used in the field of vaccines, particularly via systemic administration, i.e., non-enteric administration, such as subcutaneous, intramuscular, intradermal or intravenous routes.

[0066] Embodiments of the present invention Example 1: A quadrivalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0067] Composition: The composition of diphtheria toxoid, tetanus toxoid, hepatitis B (rDNA) and Haemophilus influenzae vaccine is shown in Table 1 below.

[0068] Table 1

[0069] NLT: Not less than, NMT: Not more than Preparation of vaccine stock solution: The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0070] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and whole-cell pertussis. Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0071] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (25% of the required volume) and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0072] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the Hib component to the above mixture, and incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0073] After incubation, the final vaccine stock solution is stored at 2-8°C, awaiting filling. The final vaccine stock solution is then filled into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and finally sealed with aluminum-plastic composite caps.

[0074] Storage temperature: Store at 2-8℃.

[0075] In the above embodiments, protein D was used as a carrier. However, it can also induce an immune response against untyped Haemophilus influenzae. Mouse experiments revealed that PD inoculation induced high serum IgG and IgA levels, as well as significant bactericidal activity against both homologous and heterologous strains.

[0076] Example 2: Determination of antibody (IgG) titers against Hib-PRP and protein D.

[0077] Animal immunization: Swiss mice were immunized with Haemophilus influenzae PRP conjugated to tetanus toxoid or protein D, alone or in combination with a five-component combination. One group of ten Swiss mice was administered the test vaccine subcutaneously on days 0, 14, and 28. Another group of ten Swiss mice remained unvaccinated. Blood was collected 10 days after the third administration. Serum was separated by centrifugation at 4000 rpm for 15 minutes. Serum samples were processed using an ELISA method to determine IgG antibodies against polynucleotide phosphate (PRP) and Haemophilus influenzae protein D.

[0078] Antibody assay: The IgG antibodies against PRP and Haemophilus influenzae type b protein D in mouse serum samples were measured using an internal ELISA method.

[0079] Test serum samples diluted 100-fold were added to wells coated with PRP or protein D antigen. A specific horseradish peroxidase (HRP)-conjugated secondary antibody was added, followed by the substrate, which was catalyzed by the enzyme to cause a color change, indicating the presence of the antibody. Results are shown in Table 2, Figure 1, and Figure 2.

[0080] Table 2: Geometric mean of antibody (IgG) titers against Hib-PRP and protein D:

[0081] This indicates that the anti-PRP response (i.e., anti-Hib polysaccharide) of Hib-TT and Hib-PrD is comparable in nature. As shown in Figure 1, the five-drug combination based on Hib-TT and Hib-PrD is comparable in nature.

[0082] As shown in Figure 2, the anti-protein D response (i.e. against the carrier protein PrD) of the five-drug combination based on Hib-PrD is comparable in nature.

[0083] Therefore, in addition to the anti-PRP response, Hib-PrD and its combination vaccines can also induce the anti-protein D response.

[0084] Example 3: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0085] Composition: The composition of diphtheria toxoid, tetanus toxoid, inactivated whole-cell pertussis, IPV (Sabin strain), hepatitis B (rDNA) and Haemophilus influenzae vaccine is shown in Table 3 below.

[0086] Table 3

[0087] vaccine stock solution The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0088] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and whole-cell pertussis. Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0089] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (25% of the required volume) and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0090] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the Hib and IPV components to the above mixture, and incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0091] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0092] The final vaccine stock solution was filled into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0093] Storage temperature: Store at 2-8℃.

[0094] Example 4: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0095] Composition: The composition of diphtheria toxoid, tetanus toxoid, inactivated whole-cell pertussis virus, hepatitis B virus (rDNA), Haemophilus influenzae and inactivated poliovirus (Salk) is shown in Table 4 below.

[0096] Table 4

[0097] NLT: Not less than, NMT: Not more than Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0098] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and whole-cell pertussis. Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0099] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (25% of the required volume) and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0100] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the Hib and IPV components to the above mixture, and incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0101] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0102] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0103] Storage temperature: Store at 2-8℃.

[0104] Example 5: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0105] Composition: The composition of diphtheria toxoid, tetanus toxoid, inactivated whole-cell pertussis virus, hepatitis B virus (rDNA), Haemophilus influenzae and inactivated poliovirus (Salk) is shown in Table 5 below.

[0106] Table 5

[0107] NLT: Not less than & NMT: Not more than Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0108] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and whole-cell pertussis. Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate at 25 ± 2 °C with stirring at 150 rpm for at least 18 hours.

[0109] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of adjuvant (25% of the required volume) and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Bring the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate at 25 ± 2 °C with stirring at 150 rpm for at least 18 hours.

[0110] Mixing of different components After incubation, mix components I and II, and incubate the resulting final block again at 25℃±2℃ for at least 18 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the Hib and VLP components of the IPV to the above mixture, and incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0111] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0112] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0113] Storage temperature: Store at 2-8℃.

[0114] Example 6: A pentavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0115] Composition: The composition of diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B (rDNA) and Haemophilus influenzae vaccine is shown in Table 6 below.

[0116] Table 6

[0117] NLT: Not less than & NMT: Not more than Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0118] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the calculated volumes of adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required volumes of tetanus toxoid, diphtheria toxoid, and acellular pertussis components (pertussis toxin (PT), filamentous hemagglutinin (FHA), and Bordetella pertussis adhesin (PRN)). Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the overall pH to 6.0 ± 0.5 and incubate at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0119] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the calculated volumes of adjuvant (25% of the required volume) and phenoxyethanol (50% of the required volume). Then, add the required volume of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0120] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the Hib component to the above mixture, and make up the volume of the resulting mixture to the required batch size with buffered saline. Incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours while stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0121] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0122] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0123] Storage temperature: Store at 2-8℃.

[0124] Example 7: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0125] Composition: Compositions of diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B (rDNA), Haemophilus influenzae and inactivated poliovirus (Sabin) are listed in Table 7 below.

[0126] Table 7

[0127] NLT: Not less than & NMT: Not more than Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0128] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel and add the required amounts of aluminum phosphate adjuvant and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and acellular pertussis components (pertussis toxin (PT), filamentous hemagglutinin (FHA), and Bordetella pertussis adhesin (PRN)). Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the overall pH to 6.0 ± 0.5 and incubate at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0129] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of aluminum hydroxide adjuvant and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0130] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the IPV and Hib components to the above mixture, and make up the volume of the resulting mixture to the required batch size with buffered saline. Incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0131] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0132] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0133] Storage temperature: Store at 2-8℃.

[0134] Example 8: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0135] Composition: The composition of diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B (rDNA), Haemophilus influenzae and inactivated poliovirus (Salk) is shown in Table 8 below.

[0136] Table 8

[0137] NLT: Not less than & NMT: Not more than Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients are calculated and determined based on the batch size and labeled quantity.

[0138] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel, and add the calculated volumes of aluminum adjuvant (75% of the required volume) and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required volumes of tetanus toxoid, diphtheria toxoid, and acellular pertussis components (pertussis toxin (PT), filamentous hemagglutinin (FHA), and Bordetella pertussis adhesin (PRN)). Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the overall pH to 6.0 ± 0.5 and incubate at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0139] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of aluminum adjuvant (25% of the required amount) and phenoxyethanol (50% of the required amount). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Make up the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate it at 25 ± 2 °C for at least 18 hours while stirring at 150 rpm.

[0140] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the IPV and Hib components to the above mixture, and make up the volume of the resulting mixture to the required batch size with buffered saline. Incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0141] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0142] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0143] Storage temperature: Store at 2-8℃.

[0144] Example 9: A hexavalent vaccine composition conjugated with Hib antigen and protein D as a carrier protein and its preparation method.

[0145] Composition: The composition of virus-like particles of diphtheria toxoid, tetanus toxoid, acellular pertussis, hepatitis B (rDNA), Haemophilus influenzae and poliovirus is shown in Table 9 below.

[0146] Table 9

[0147] NLT: Not less than & NMT: Not more than The dosage of type 1, 2, and 3 poliovirus-like particles (VLPs) was measured by protein content, and the content of their specific antigens was determined by the D-AgELISA method.

[0148] Vaccine bulk preparation The required amounts of each active pharmaceutical ingredient and other excipients shall be calculated and determined based on the batch size and labeled quantity.

[0149] Component 1 Transfer the calculated volume of buffer solution to a preparation vessel and add the required amounts of aluminum phosphate adjuvant and phenoxyethanol (50% of the required volume). Then, under constant stirring, add the required amounts of tetanus toxoid, diphtheria toxoid, and acellular pertussis components (pertussis toxin (PT), filamentous hemagglutinin (FHA), and Bordetella pertussis adhesin (PRN)). Make up the volume of the resulting mixture to 60% of the required batch size with buffered saline. Adjust the overall pH to 6.0 ± 0.5 and incubate at 25 ± 2 °C for at least 18 hours with stirring at 150 rpm.

[0150] Component 2 Transfer the calculated volume of buffer solution to a preparation vessel, and add the required amounts of aluminum hydroxide adjuvant and phenoxyethanol (50% of the required volume). Then, add the required amount of hepatitis B antigen while maintaining constant stirring. Bring the volume of the resulting mixture to 15% of the required batch size using buffered saline. Adjust the pH of the stock solution to 6.0 ± 0.5 and incubate at 25 ± 2 °C for 8 hours while stirring at 150 rpm.

[0151] Mixing of different components After incubation, mix components I and II, and incubate the resulting final stock solution again at 25℃±2℃ for at least 18 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5. After incubation, add the IPV and Hib components to the above mixture, and make up the volume of the resulting mixture to the required batch size with buffered saline. Incubate the resulting final stock solution again at 25℃±2℃ for at least 4 hours with stirring at 150 rpm. If necessary, adjust the pH of the stock solution to 6.5±0.5.

[0152] After incubation, the final vaccine stock solution is stored at 2-8℃, awaiting filling.

[0153] The final vaccine stock solution was poured into USPI-type tubular glass vials, sealed with bromobutyl rubber stoppers, and then sealed with aluminum-plastic combination caps.

[0154] Storage temperature: Store at 2-8℃.

[0155] Example 10: Determination of functional antibodies against NTHi The bactericidal activity level of immune serum is determined by a bactericidal assay. The serum bactericidal assay (SBA) is used to determine the ability of vaccine-induced antibodies to couple with complement and kill bacteria. The results of this assay demonstrate whether a specific serum sample has a sufficient level (titer) of bactericidal antibodies to reach a protective threshold.

[0156] To conduct the experiment, serum was incubated at 56°C for 30 minutes to remove complement activity. NTHi strain 3655 was grown to the logarithmic growth phase in brain heart infusion broth and at a growth rate of 5 × 10⁻⁶. 4 CFU / ml was suspended in sterile PCM buffer (PBS containing 0.15 mM CaCl2 and 1 mM MgCl2) containing 1% BSA.

[0157] Subsequently, approximately 20 μl of bacteria, 20 μl of serially diluted antiserum, 20 μl of rabbit complement, and the final 40 μl of PCM buffer containing 1% BSA were mixed. The initial bacterial concentration was determined on chocolate agar plates. The sample was then incubated at 37°C with gentle shaking for 1 hour, and 10 μl of sample was plated in duplicate. Serum and complement controls were also included to ensure the validity of the assay.

[0158] The samples from each group were incubated overnight at 37°C on agar plates. The bactericidal activity of serially diluted antiserum was assessed by measuring the CFU count on the second day. The dilution factor at which 50% bactericidal activity was achieved was recorded for comparison of the activity of different sera. The average value of the serum dilutions producing 50% bactericidal activity was recorded for comparison. The experimental results are shown in Table 10 and Figure 3.

[0159] Table 10

[0160] The bactericidal titer is defined as the reciprocal of the highest serum dilution that produces 50% bacterial death compared to the control group.

[0161] Based on the bactericidal activity of serum collected from vaccine samples, the samples of Hib conjugate with protein D showed significantly higher activity than those of vaccine samples containing Hib conjugate with tetanus toxoid.

Claims

1. An immunogenic multi-component vaccine composition comprising a polysaccharide antigen against diphtheria and / or tetanus and / or pertussis and / or poliomyelitis and / or hepatitis and / or Haemophilus influenzae type b, said polysaccharide antigen being conjugated to protein D or a fragment thereof.

2. The immunogenic multi-component vaccine composition according to claim 1, comprising Haemophilus influenzae type b protein D polysaccharide conjugate antigen.

3. The immunogenic multicomponent vaccine composition according to claim 1, comprising a capsular polysaccharide conjugated against Haemophilus influenzae type b, wherein at least one polysaccharide carrier protein is protein D.

4. The immunogenic multi-component vaccine composition according to claim 3, wherein, The antigen of Haemophilus influenzae type b is coupled to protein D, which is a carrier protein.

5. The immunogenic multi-component vaccine composition according to claim 1, wherein, The polysaccharide-protein D conjugate antigen is adsorbed onto an aluminum-based adjuvant.

6. The immunogenic multi-component vaccine composition according to claim 1, comprising a quadrivalent and / or pentavalent and / or hexavalent and / or multivalent vaccine composition.

7. The immunogenic multicomponent vaccine composition according to claim 6, comprising diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP) / acellular pertussis (aP), hepatitis B (Hep), and Haemophilus influenzae (Hib) antigens coupled to protein D as a carrier protein.

8. The immunogenic multicomponent vaccine composition according to claim 6, comprising diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP) / acellular pertussis (aP) and Haemophilus influenzae (Hib) antigens coupled to protein D as a carrier protein.

9. The immunogenic multi-component vaccine composition according to claim 6, comprising diphtheria toxoid (DT), tetanus toxoid (TT), whole-cell pertussis (wP) / acellular pertussis (aP), inactivated polio antigen (IPV), and Haemophilus influenzae (Hib) conjugated to protein D as a carrier protein.

10. The immunogenic multi-component vaccine composition according to claim 1, provided in the following form: All-liquid composition; Alternatively, a kit in which all components are contained in a single vial / pre-filled syringe; Alternatively, a kit containing two different containers: vials, pre-filled syringes, or dual-chamber syringes.