WO2025224732A1 - Vaccin combiné hexavalent méningococcique - Google Patents

Vaccin combiné hexavalent méningococcique

Info

Publication number
WO2025224732A1
WO2025224732A1 PCT/IN2024/052447 IN2024052447W WO2025224732A1 WO 2025224732 A1 WO2025224732 A1 WO 2025224732A1 IN 2024052447 W IN2024052447 W IN 2024052447W WO 2025224732 A1 WO2025224732 A1 WO 2025224732A1
Authority
WO
WIPO (PCT)
Prior art keywords
serogroup
polysaccharide
combination vaccine
meningitidis
hexavalent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IN2024/052447
Other languages
English (en)
Inventor
Syed S Ahmed
Safia SYED
Varnik Kumar
Arnabjyoti Bhagawati
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Techinvention Lifecare Private Ltd
Original Assignee
Techinvention Lifecare Private Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Techinvention Lifecare Private Ltd filed Critical Techinvention Lifecare Private Ltd
Publication of WO2025224732A1 publication Critical patent/WO2025224732A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Definitions

  • the present invention relates to biopharmaceuticals.
  • the present invention relates to a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.
  • the present invention relates to a hexavalent combination vaccine comprising serogroups A, B, C, Y, W- 135, X of Neisseria meningitidis.
  • the present invention relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W-135, X and recombinant proteins of serogroup B of Neisseria meningitidis.
  • the present invention specifically relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W-135, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.
  • the present invention further relates to the composition, use and process of preparation of hexavalent combination vaccine.
  • Neisseria meningitidis is a Gram-negative bacterium that can cause severe infections, including sepsis and meningitis. This bacterium causes 10-15% of community acquired meningitis cases in adults, of which most cases occur in younger adults. The case fatality rate has been reported to be 3-4% (van Soest TM et.,al, Lancet Reg Health Eur. 2023 Apr 28;30: 100640).
  • Meningococcal meningitis is characterized by sudden onset of headache, fever, and neck stiffness, sometimes accompanied by nausea, vomiting, photophobia, or altered mental status. Meningococcal disease progresses rapidly and has a case-fatality rate of 10%— 15% , even with antimicrobial drug treatment. Without rapid treatment, fatality rates can be much higher. Approximately 30% of people with meningococcal disease present with meningococcal sepsis, known as meningococcemia. Symptoms of meningococcemia can include abrupt onset of fever, chills, vomiting, diarrhea, and a petechial or purpuric rash, which can progress to purpura fulminans.
  • Meningococcemia often involves hypotension, acute adrenal hemorrhage, and multiorgan failure (https://wwwnc.cdc.gov/travel/yellowbook/2024/infections-diseases/meningococcal- disease#epi accessed on March 25, 2024)
  • meningococcal vaccine protects against infections caused by the bacteria Neisseria meningitidis (meningococci). Meningococcal infections can lead to meningitis (an infection of tissue covering the brain), dangerously low blood pressure (shock), and death. These bacteria are the leading cause of bacterial meningitis in children and the second leading cause of bacterial meningitis in adults. (https://www.msdmanuals.com/enin/home/infections/immunization/meningococcal- vaccine)
  • IMD Invasive meningococcal disease
  • IMD is characterised by a wide range of clinical manifestations, including meningitis, meningococcaemia, or both, in addition to other clinical presentations, with a case fatality rate of roughly 6-10%.
  • IMD can have long-term effects in survivors, including deafness, brain damage, and amputations of limbs or fingers. It is difficult to make an early diagnosis of IMD since at the time of the disease's onset, symptoms like sore throat may resemble those of common viral respiratory infections while rash and meningeal indications may not be evident. (Piccini, G et.al., Expert review of vaccines, 15(11), 1393-1407)
  • Serogroups A, B, C, X, Y, and W135 are mostly responsible for meningococcal disease. Regional differences affect each serogroup's relative importance. Humans are the only natural host of meningococci, and about 5-10% of adults are asymptomatic meningococcal carriers (Frasch, C et.al., Human vaccines & immunotherapeutics, 8(6), 715-724).
  • Vaccines containing recombinant proteins only (Trumenba, Pfizer), or polysaccharide-protein conjugates only (MenFive, Serum Institute of India), or recombinant proteins combined with OMVs (Bexsero, GSK Vaccines), or containing both meningococcal polysaccharide of serogroups C and OMPs of serogroup B (VA- Mengoc-BC, Finley Institute) have been developed and licensed.
  • Meningococcal conjugate vaccines in which the capsular polysaccharide is covalently linked to a carrier protein, were developed as a result of the shortcomings of the polysaccharide vaccines, which included poor immunogenicity in children under the age of two, a lack of immunological memory, and, in some instances, hypo responsiveness. (Berti, F et.al., Glycoconjugate Journal, 38(4), 401-409).
  • the current conjugate vaccines include different carrier proteins as well as other elements including formulation technology and conjugation method. MenACWY-D and MenACWY-CRM197 were approved earlier than MenACWY-TT, which is the most recently licenced of the three MenACWY conjugate vaccines that are now on the market. (Tontini, M et.al., Vaccine, 31(42), 4827- 4833).
  • Neisseria meningitidis (N. meningitidis), viz., A, B, C, W, X, and Y, are responsible for most of the infections. There are no vaccines that target all six of them. In the present invention, a hexavalent meningococcal vaccine combination targeting all six serogroups is disclosed.
  • Objects of the present invention are to provide a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.
  • An object of the present invention is to provide a hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.
  • An object of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis.
  • Another object of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein conjugate of serogroup A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.
  • Another object of the present invention is to provide a pharmaceutical composition of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.
  • Another object of the present invention is to provide process of preparation of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.
  • Yet another object of the present invention provides use of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis for use in protecting a human against infection by N. meningitidis.
  • aspects of the present invention relates to a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.
  • the present invention relates to a hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis.
  • the present invention relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and recombinant proteins of serogroup B of Neisseria meningitidis.
  • the present invention specifically relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.
  • the present invention further relates to the composition, use and process of preparation of hexavalent combination vaccine.
  • Further aspect of the present invention is to provide a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein the polysaccharides of serogroup A, C, Y, W and X are individually conjugated to carrier protein
  • the carrier protein is selected from diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesion protein (PsaA).
  • OMPC outer membrane complex c
  • PspA pneumococcal surface protein A
  • PsaA pneumococcal adhesion protein
  • the mutated fHbp is selected from the group consisting of (i) A02 comprising the amino acid sequence of SEQ ID NO: 1, or comprising at least 90% sequence identity to SEQ ID NO: 1; (ii) A42 comprising the amino acid sequence of SEQ ID NO: 2, or comprising at least 90% sequence identity to SEQ ID NO: 2; (iii) A46 comprising the amino acid sequence of SEQ ID NO: 3, or comprising at least 90% sequence identity to SEQ ID NO: 3; (iv) B44 comprising the amino acid sequence of SEQ ID NO: 4, or comprising at least 90% sequence identity to SEQ ID NO: 4; (v) B107 comprising the amino acid sequence of SEQ ID NO: 5, or comprising at least 90% sequence identity to SEQ ID NO: 5; and (iv) combinations thereof.
  • the present invention relates to a process of preparation of mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis serogroup B fHbp comprising steps of: a. Cloning of the individual fHbp gene with the signal sequence into expression vector, b. Plasmid extraction, c. Transformation d. Induction of the respective fHbp’ s e. Purification of respective fHbp’s
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the hexavalent combination vaccine, and a pharmaceutically acceptable excipient such as but not limited to a carrier, a diluent or an adjuvant.
  • a pharmaceutical composition comprising, wherein based on the unit dose of administration each dose of the composition comprises of: a. polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis in an amount of 2pg-10pg per dose; b. polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis in an amount of 2pg-10pg per dose; c.
  • an adjuvant in an amount of 150 pg- 500 pg per dose; h. sucrose in an amount of 10-30 mg per dose ; i. an emulsifier in an amount of 0.01 mg- 0.04 mg per dose - ; j. a -preservative in an amount of Ipl - 4 pl per dose ; k. a buffer to make the pH 6.8 ⁇ 0.4; and
  • Another aspect of the present invention is to provide process of preparation of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.
  • the present invention relates to a method of using the hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis for use in protecting a subject against infection by N. meningitidis comprising administering to the subject a therapeutically effective amount of the hexavalent combination vaccine or a composition comprising the same according to the invention, wherein the method is preferably immunization.
  • Figure 12 Western Blot Results of recombinant fHbp protein sequences for Serotype B
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • protein refers to large biomolecules and macromolecules that comprise one or more long chains of amino acid residues.
  • the chain amino acid residue may be of any length and may be linear or branched. This term encompasses all the naturally occurring or modified/ recombinant proteins.
  • Neisseria meningitidis or “TV. meningitidis ' according to the present invention means, but not limited to, refers to diplococcal, Gram-negative, and human commensal bacterium of the upper respiratory tract.
  • the pathogen can invade the mucosa and gain access to the bloodstream, resulting meningitis, severe sepsis, or localized infections in joints and heart.
  • adjuvant/adjuvant component means, but not limited to, refers to an adjuvant or an adjuvant component in the broadest sense is typically a (e.g., pharmacological or immunological) agent or composition that may modify, e.g., enhance, the efficacy of other agents, such as a drug or vaccine.
  • a (e.g., pharmacological or immunological) agent or composition that may modify, e.g., enhance, the efficacy of other agents, such as a drug or vaccine.
  • the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds.
  • adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention.
  • adjuvant or “adjuvant component” has the same meaning and can be used mutually.
  • Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof.
  • adjuvant is typically understood not to comprise agents which confer immunity by themselves.
  • An adjuvant assists the immune system unspecifically to enhance the antigen- specific immune response by e.g., promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response.
  • an adjuvant may preferably e.g., modulate the antigen- specific immune response by e.g., shifting the dominating Th2-based antigen specific response to a more Thl-based antigen specific response or vice versa and/or by inducing of mucosal immune responses and/or increased IgA titers. Accordingly, an adjuvant may favourably modulate cytokine ex-pression/secretion, antigen presentation, type of immune response etc.
  • Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccines. These may be co -administered by any route, e.g., intramuscularly, subcutaneous, IV or intradermal injections.
  • therapeutically effective amount means, but not limited to, refers to an amount of the active ingredient (i.e., therapeutic protein or antibody) sufficient to produce the desired therapeutic effect in a human or animal, e.g., the amount necessary to treat, cure, prevent, or inhibit development and progression of disease or the symptoms thereof and/or the amount necessary to ameliorate symptoms or cause regression of disease.
  • an amount of the active ingredient i.e., therapeutic protein or antibody
  • Such a therapeutically effective amount may vary depending on the structure and potency of the active ingredient and the contemplated mode of administration.
  • treatment means, but not limited to, refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those individuals, such as humans and animals, already with the disorder or condition to be treated as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • treatment also includes reduction of the likelihood of obtaining the disorder, reduction of the severity of the disorder in those already afflicted, and the induction of regression of the disorder or symptoms thereof.
  • pharmaceutically-acceptable carrier means, but not limited to, refers to a liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of pharmaceutically acceptable carriers well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions including phosphate buffered saline, emulsifiers, isotonic saline, and pyrogen free water.
  • pharmaceutically acceptable carriers may contain different components such as a buffer, sterile water for injection, normal saline or phosphate-buffered saline, sucrose, histidine, salts and polysorbate.
  • a buffer sterile water for injection
  • normal saline or phosphate-buffered saline sucrose
  • histidine phosphate-buffered saline
  • salts and polysorbate.
  • In an embodiment of the present invention is to provide a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.
  • hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis.
  • the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.
  • a hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis
  • the ratio of glycoconjugate of serogroups A, C, Y, W, and X of Neisseria meningitidis is 1 : 1 : 1 : 1 : 1.
  • the ratio of glycoconjugate of serogroups A, C, Y, W, and X and the proteins of serogroup B of Neisseria meningitidis is 1: 4.5 to 5.0.
  • the present invention provides a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein the polysaccharides of serogroup A, C, Y, W and X are individually conjugated to carrier protein selected from diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E.
  • coli ST and exotoxin A from Pseudomonas aeruginosa
  • Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA) and like thereof.
  • OMPC outer membrane complex c
  • PspA pneumococcal surface protein A
  • PsaA pneumococcal adhesin protein
  • the present invention provides a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein the polysaccharides of serogroups A, C, Y, W and X are individually conjugated to carrier protein selected from diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, and like thereof.
  • the hexavalent combination vaccine wherein the ratio of polysaccharide and carrier protein in each of the glycoconjugate is 1:1 to 3.0.
  • the present invention provides a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis wherein the process for the preparation of polysaccharide- protein conjugate of serogroups A, C, Y, W, X comprises the steps of: a. Fermentation of polysaccharides b. Activation of Polysaccharides and carrier protein c. Polysaccharide Protein Conjugation d. Purification
  • a method of preparing the polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis as claimed in anyone of claims 1-5 comprises the steps of: a) cultivating meningitis serogroup strains A, C, Y, W and X in presence of l-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) for about 12-20 hours to produce meningococcal A, C, Y, W, and X polysaccharides; b) purifying the meningococcal A, C, Y, W, and X polysaccharides by processes selected from ultrafiltration, cetyl-trimethylammonium bromide (CTAB) precipitation, centrifugation, depth filtration, ethanol, and salt precipitation; c) activating each of the purified meningococcal A, C, Y, W, and
  • a modified variant of proteins of serogroup B of Neisseria meningitidis is provided.
  • the modified variant of proteins of serogroup B of Neisseria meningitidis is mutated factor H binding proteins (fHbp) of N. meningitidis serogroup B.
  • fHbp mutated factor H binding proteins
  • the said variant polypeptide sequence is as set forth in at least one of the SEQ ID NOs. 1, 2, 3, 4 and 5.
  • the said variant is codon optimized for expression in a prokaryotic expression system.
  • the modified variant of proteins of serogroup B of Neisseria meningitidis is selected from modified variant of factor H binding proteins A02, A42, A46, B44 and B107 of A. meningitidis fHbp family.
  • a DNA construct comprising a gene encoding a polypeptide having at least one of the amino acid SEQ ID NOs. 1, 2, 3, 4 and 5.
  • an expression vector comprising a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of the SEQ ID NOs. 1, 2, 3, 4 and 5.
  • a recombinant host cell comprising an expression vector, wherein said expression vector comprises a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of SEQ ID NOs. 1, 2, 3, 4 and 5.
  • Hexavalent Composition comprising: (a) obtaining a recombinant host cell comprising an expression vector, wherein said expression vector comprises a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of SEQ ID NOs. 1, 2, 3, 4 and 5;
  • a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of sero group B is mutated factor H binding proteins (fHbp) of N. meningitidis serogroup B.
  • the present invention provides a hexavalent combination vaccine comprising (a) a combination of polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis', and (b) one or more proteins of serogroup B of N. meningitidis wherein the ratio of (a) and (b) is 1:4.5 to 5.0.
  • a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of serogroup B is selected from mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis fHbp family having at least 90% sequence identity to SEQ ID NOs. 1, 2, 3, 4 and 5 respectively.
  • fHbp mutated factor H binding proteins
  • a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroup A, C, Y, W, X and proteins of serogroups B of Neisseria meningitidis wherein proteins of serogroup B involves a process of preparation of mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis serogroup B fHbp comprising steps of: a. Cloning of the individual fHbp gene with the signal sequence into expression vector, b. Plasmid extraction, c. Transformation d. Induction of the respective fHbp’ s e. Purification of respective fHbp’s
  • a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of serogroup B comprise of at least two of the following: a) mutated protein of fHbp family A02 of N. meningitidis (SEQ ID NO: 1); b) mutated protein of fHbp family A42 of N. meningitidis (SEQ ID NO: 2); c) mutated protein of fHbp family A46 of N.
  • Another embodiment of the present invention is to provide a composition of hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis.
  • Another embodiment of the present invention is to provide a composition of hexavalent combination vaccine comprising: a. serogroup A-protein conjugate of Neisseria meningitides', b. serogroup C-protein conjugate of Neisseria meningitidis', c. serogroup Y-protein conjugate of Neisseria meningitidis', d. serogroup W-protein conjugate of Neisseria meningitidis', e. serogroup X-protein conjugate of Neisseria meningitidis', f. mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis', and g. one or more pharmaceutically acceptable excipients.
  • fHbp mutated Factor H binding proteins
  • the mutated Factor H binding proteins (fHbp) of serogroup B is selected from mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis fHbp family having at least 90% sequence identity to sequence ID nos. 1, 2, 3, 4 and 5 respectively.
  • the one or more pharmaceutically acceptable excipients include, but not limited to, adjuvant(s), emulsifier(s), preservative(s), chelating agent(s), pH modifying agent(s), antioxidant(s), buffer(s), stabilizing agent(s) or stabilizer, isotonic agent(s), chelating agent(s), and any mixtures thereof.
  • the composition can include one or more adjuvant(s).
  • Suitable adjuvant(s) include but not limited to aluminum phosphate, Aluminium hydroxide, Potassium aluminium Sulfate, Squalene, AS03 and a combination thereof.
  • Adjuvant(s) may be present at concentrations of from 0.1 to 1 mg of Aluminum content per dose.
  • the composition can include an emulsifier.
  • Suitable emulsifier include but not limited to PS -80, and PS20 and a combination thereof.
  • Emulsifier may be present at concentrations of from 0.010 to 0.1 mg per dose
  • the composition can include a stabilizer. Suitable stabilizer include but not limited to Sucrose, Lactorse, , mannitol, Glycine or Gutamic acid sodium salt and a combination thereof. Stabilizer may be present at concentrations of from 3 mg to 50 mg per dose.
  • Exemplary stabilizing agent or stabilizer suitable for use in the composition of the present disclosure includes, but not limited to, mannitol, sucrose, lactose, mannitol, histidine, glutamic acid and glycine.
  • the pH value of the composition may be adjusted if necessary to pH 6 to 7.
  • the pH can be adjusted by adding a pH modifying agent.
  • pH modifying agents include sodium hydroxide, sodium bicarbonate, hydrochloric acid and a combination thereof.
  • the composition can include one or more preservatives.
  • Suitable preservatives include but not limited to 2 phenoxy ethanol benzyl alcohol, methylparaben, propylparaben, phenol, cresol and a combination thereof.
  • Preservative(s) may be present at concentrations of from 0.5 mg -2.5 mg per dose.
  • the composition can include a buffer and the buffer may also be used to adjust the pH of the composition.
  • Suitable buffers range may be adjusted to pH 6-pH 7.2 using suitable buffers include but not limited to sodium and potassium phosphates, sodium and potassium citrates, mono-, di- and triethanolamines, bicarbonate buffer, carbonate buffer, histidine buffer, tartrate buffer, Tris buffer and mixtures thereof.
  • Buffer(s) may be present at concentrations of from 0.05 to 2% w/v.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup Y of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup W of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup X of N. meningitidis', one or more proteins of serogroup B of N. meningitides; an adjuvant; sucrose; an emulsifier; a stabilizer; a buffer to make the pH 6-7.2;
  • the pharmaceutical composition may be presented in unit-dose or multi-dose forms.
  • the unit dose of the pharmaceutical composition formulated as a sterile solution suitable for administration is 0.5 ml.
  • Each unit dose can contain a. polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis in an amount of 2pg-10pg per dose; b. polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis in an amount of 2pg-10pg per dose; c. polysaccharide-carrier protein glycoconjugates of serogroup Y of N.
  • an emulsifier in an amount of 0.01 mg- 0.04 mg per dose - ; j. a -preservative in an amount of Ipl - 4 pl per dose ; k. a buffer to make the pH 6.8 ⁇ 0.4; and
  • the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis is in fully liquid form or lyophilized form or a combination of liquid and lyophilized form thereof.
  • the unit dose can be administered to a human subject once or twice.
  • One of skill in that art will realize that the particular administration schedule will depend on the human subject and the dosage being used.
  • the administration schedule can also be different for individual subjects or change during the course of the therapy depending on the subject's reaction.
  • the unit dose is administered twice.
  • the pharmaceutical composition of this invention is presented in liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
  • the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis which is administered directly to a patient via pharmaceutical injection (e.g. Subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule.
  • Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule.
  • a primary dose schedule may be followed by a booster dose schedule.
  • Suitable timing between priming doses e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.
  • the pharmaceutical composition of this invention is presented in unit dose form in ampoules, vials or pre-filled syringes.
  • the pharmaceutical composition of this invention has a long shelf-life.
  • the shelf-life may be about 12 to about 36 months.
  • the aqueous injectable composition has a shelf-life of 18 months.
  • the present invention provides a process of preparing the pharmaceutical composition comprising the steps of: (a) Adding Adjuvant (b) providing a combination of polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis; (c) adding one or more proteins of serogroup B of A. meningitidis ; (d) adding excipients, emulsifier, stabilizer, and buffer one by one.
  • the present invention provides a method of treating a subject, comprising administering to the patient a therapeutically effective amount of a composition according to the invention.
  • the method is preferably immunization.
  • Example 1- Determination of Sialic acid content in meningococcal polysaccharide of serotypes C, W135 and Y.
  • the standard NANA (N- Acetylneuraminic acid) solution was diluted was demonstrated in the table above. Dilutions ranging from 20 - 100 pg/ml of the standard NANA solution was prepared using purified water. In case of dry polysaccharide, a solution containing about 1 mg/ml of polysaccharide in purified water was prepared; the test sample was diluted 1: 10-fold to get a concentration of 100 pg to be used as test sample. From the above standard and sample dilutions preparation, 100 pl of each standard and sample was taken in duplicates.
  • reagent E comprising (1 ml each of 3 M sulphuric acid, 2.5% ammonium molybdate, 10 % ascorbic acid and 2 ml of purified water) to each tube and vortex.
  • the tubes were incubated in a 37 °C water bath for 2 hours.
  • the absorbance was measured at 825 nm with a spectrophotometer.
  • Shodex guard column, Shodex 805 column and Shodex 804 column in series were mounted on calibrated HPLC system.
  • the purge valve of pump (by turning it counter clockwise) was opened and flow rate was set to 3-5ml/min.
  • the vacuum degasser was flushed with lOmM PBS for ten minutes.
  • the flow rate was then set to l.Oml/min and the reference cell of detector purged for 40 minutes with 10 mM phosphate buffer saline solution until a stable baseline is obtained which can be viewed on the chromatographic software.
  • samples of meningococcal polysaccharide where then filtered 0.2 pm syringe filter in HPLC glass vials following to the injection of 50 pl of the sample on HPLC.
  • results The percentage purity for serotypes was identified to be 90% for serotype A, 93% for serotype C, 96% for serotype Y, 90% for serotype W -135 and 91% for serotype X.
  • the Kavg for all samples analysed was identified to be 0.15, 0.27, 0.18, 0.16 and 0.20 for serotypes A, C, Y, W-135 and X respectively (Fig 1-5).
  • results - The protein content within the polysaccharide was identified to be 0.27% for serotype A, 0.34% for serotype C, 2.75% for serotype Y, 1.55% for serotype W-135 and 0.36% for serotype X.
  • Example 5 Estimation of the Nucleic acid content in Meningococcal polysaccharide samples.
  • a Img/ml solution of polysaccharide was prepared in Lal reagent water and use it as sample
  • the nucleic acid content in polysaccharide was identified to be 0.19% and 0.15% for serotype A and X respectively.
  • serotype C, Y and W - 135 the nucleic acid content was identified to be 0.23%, 1.18% and 1.02% respectively
  • the meningitis serogroup strains were first cultivated in a bioreactor at Separate fermentation runs for 12-20 hours to produce meningococcal A, C, Y, W, and X polysaccharides.
  • CDAP l-cyano-4- dimethylaminopyridine tetrafluoroborate
  • the carrier protein i.e., rCRM was simultaneously activated using Carbodiimide reagent such as ED AC which helps in coupling reactions as a catalyst (protein and ED AC ration 1:1).
  • the reaction was carried out with an optimum reaction time of 16-24 hours.
  • the optical density of ACYWX batches was achieved between 3.01 and 10.4 in aerobic conditions and feed addition as per requirement, with pH ranging from 6.5-7.4, temperature ranging from 35-37 °C, and RPM of 50-220.
  • PS polysaccharides
  • Total polysaccharide content was identified to be 70.8 pg/ml for serotype A, 81.1 pg/ml for serotype C, 87.9 pg/ml for serotype Y, 69.4 pg/ml for serotype W- 135pg/ml and 75.6 pg/ml for serotype X.
  • Example 8 Protein Concentration In Active Raw Material Of CRM And Pneumococcal CRM Conjugate Vaccine By BCA Protein Assay Kit
  • BSA Bovine Serum Albumin
  • the protein content was identified to be 132.1 pg/mL for serotype A, 51.9 pg/mL for serotype C, 248.0 pg/mL for serotype Y, 31.0 pg/mL for serotype W-135 and 82.7 pg/mL for serotype X.
  • IM Sodium Phosphate buffer was prepared in 1000ml.
  • Sodium azide 0.05% and 5mg of Salmon DNA in 5 ml of purified water was also prepared. All the solutions prepared for use are vacuum filtered using Nylon 6, 6 membrane 0.20/0.22/0.45 pm, depending upon the nature of solution to be filtered (filtration not necessary if the solution is prepared in 0.2p filtered purified water). For organic solvents filtration not necessary if the solution is HPLC grade. Set the flow rate to 0.5mL/min. Equilibrate the system and column (TSK gel G6000) with 0.1M sodium phosphate solution until a stable baseline for RID is obtained.
  • results The percentage purity was identified to be 100% for serotype A and X, 99% for serotype Y, 93% for serotype C and 84% for serotype W-135.
  • the Ka Vg was identified to be 0.33 for serotype A, 0.26 for serotype C, 0.10 for serotype Y, 0.19 for serotype W- 135 and 0.27 for serotype X (Fig 6- 10).
  • Example 10 Determination of Free carrier protein in Meningococcal polysaccharide and CRM197 conjugate by high performance size exclusion chromatography
  • IM Sodium Phosphate buffer was prepared in 1000ml.
  • Sodium azide 0.05% and 1 mg/mL of p- Amino benzoic Acid Stock was also prepared.
  • 1 mg/mL of the Aminobenzoic Acid Stock solution was diluted to 1/10 with purified water. All the solutions prepared for use are vacuum filtered using Nylon 6,6 membrane 0.20 pm or 0.22 pm , depending upon the nature of solution to be filtered. For organic solvents 0.45pm filters should be used.
  • the percentage of free carrier protein was identified to be 2.21% and 2.52% for serotype A and X, 1.85% for serotype Y, 2.22% for serotype C and 0.53% for serotype W -135
  • endotoxin was 0.75 EU/pG of polysaccharide, therefore sample was diluted to 1 pg/ml using lai reagent water to its maximum valid dilution (MVD),. MVD for Ipg/ml purified polysaccharide sample was calculated to be 12 therefore it was diluted further to 12 fold, lysate sensitivity was identified to be 0.06 EU. Product positive control was prepared, by diluting the test samples according to MVD/2 value with LAL Reagent Water. 0.2 ml of 4 standard endotoxin was added to 0.2 ml of this dilution.
  • LAL reagent water was taken as the negative control for the assay which was prepared by adding 100 pl of LRW in reaction tube and add 100 pl of 2 standard endotoxin.
  • test sample Following the addition of lysate, the tubes were incubated at 37+ 1°C for 60 + 2 minutes in a tube heating block. After 60 + 2 minutes of incubation each reaction tube was examined for gelation. A positive reaction was indicated by a firm gel that remains intact momentarily when tube is inverted at 180°. If clot formed in the test sample, it meant that it contains endotoxin equal to or greater than 0.06 EU/ml. If no clot formed, it meant that the test sample contains endotoxin less than 0.06 EU/ml.
  • Example 12- Process of preparation of mutated factor H binding proteins (fHbp) of A02 (SEQ ID NO: 1), A42 (SEQ ID NO: 2), A46 (SEQ ID NO: 3), B44 (SEQ ID NO: 4) and B107 (SEQ ID NO: 5) of N. meningitidis serogroup B
  • the induction for all the five fHbp’s was performed (at an OD600nm between 0.6-0.8 using ImM IPTG (Sigma, USA) at 37°C, 250 rpm for 4 hours in the incubator shaker. Before induction, an uninduced sample was withdrawn and kept at the same conditions as described for induction. After induction, the total cell pellet was centrifuged at 6000rpm for 20 mins and the pellet was stored and supernatant discarded. The mutated protein were purified by Ni NT A chromatography.
  • the purified fHbp proteins from both the subfamilies were desalted and brought in phosphate buffer saline using PD 10 columns and analysed using SDS PAGE and Western blot for the purified fHbp (Fig 11 - 12).
  • Purified Bulk of conjugate is formulated along with other purified bulk conjugate in to desired doses form using appropriate buffer, Aluminum compound adjuvant, suitable excipients and preservative.
  • Individual conjugates of ACYWX along with fHbp shall be formulated by adsorbing on alum to develop a fully liquid hexavalent vaccine. Formulation activity is performed at required temperature for desired time. Once tested and approved, formulated vaccine is filled of desired dose volume of either 0.5-1 ml ampules. The labelled vials were stored at about 2-8°C for optimum shelf life. The single dose formulation was without any preservative whereas multi dose formulation contained suitable stabilizer and preservative.
  • MenHexa vaccine was formulated using six serotypes of Neisseria meningitis out of which five i.e, A, C, Y, W, X were glycoconjugates and one serotype i.e., Serogroup B was a recombinant fHBP protein which was further composed of two subfamilies A and B.
  • serotypes were mixed wherein 25pg per dose of polysaccharides of serotypes A, C, Y, W and X were taken and 120 pg (60 pg subfamily A and 60 pg subfamily B) per dose of fhbp protein of serotype B was taken. All these serotypes were mixed with 250 pg per dose of adjuvant, 20 mg Sucrose per dose, 0.2 mg PS-80 per dose, 2 PE (2 mg per dose) and Phosphate buffer saline at 6.8 pH.
  • the hexavalent composition comprising fHbp’s of Neisseria meningitidis serogroup B and conjugate of serogroups A, C, Y, W and X with CRM197 were 1 administered into animal models.
  • the study involved female BALB/c mice for vaccine testing. Mice were selected due to their relevance in immunological research, sourced from Hylasco Biotechnology, and included nulliparous, non-pregnant females aged 9-10 weeks. Before the study, the mice underwent veterinary examinations to ensure good health.
  • mice were housed in groups of up to five per cage in polypropylene cages with stainless steel grills, under controlled environmental conditions, including a temperature range of 19-25°C, humidity of 30-70%, and a 12-hour light/dark cycle.
  • the animals were provided with ad libitum 'Altromin' pelleted rodent feed and reverse osmosis water, both tested for contaminants.
  • the test vaccine was administered subcutaneously, as it is a standard method for vaccine delivery in mice. According to WHO guidelines, the maximum human dose was adjusted for mice, with a total volume of 0.5 mL per mouse divided into two 0.25 mL injections at different sites. Group G2 received a smaller dose of 0.1 mL.
  • the injections were administered on days 0 and 14, using sterile stainless steel needles (26G) and appropriate syringes. Buffer saline was administered similarly for the control group. This design and dosing were aimed at ensuring an appropriate immune response while adhering to established animal care protocols.
  • Indirect ELISA was performed. Plates were coated with serogroups and then the samples of animal models were added and incubated. The plates were then washed with wash buffer and then with blocking buffer. The plates were incubated for 1 hour at RT with secondary antibody (anti mouse conjugated with horseradish peroxidase (HRP). After the incubation was complete, the plates were washed and the TMB substrate (3,3',5,5'-Tetramethylbenzidine). After 10 mins, H2SO4 was added to end the reaction and the absorbance of the respective plates was read. The QC serum prepared was given an arbitrary value and used as a standard in each plate. The standard of each plate was utilized to generate a standard ELISA curve facilitating in the estimating of IgG concentrations of the corresponding samples/groups.
  • HRP horseradish peroxidase
  • Phosphate buffer saline [10X]: Weigh 2.6 gram (gm) KH2PO4, 11.5 gm Na2HPO4 and 87.1 gm NaCl in 500-6000ml Milli Q water (MQW). Adjust pH at 7.3 ⁇ 0.1 with HC1 or NaOH and make up volume up to 1000ml. It is advisable to filter the buffer through 0.2p filter assembly before use. After the buffer is made it can be kept at room temperature (RT) [25 ⁇ 2°C] for one month, however discard it, if any clumps, aggregation or any growth or contamination is observed.
  • RT room temperature
  • Wash Buffer comprises IX PBS, pH 7.3+0.1 containing 0.1 % Tween 20.
  • the 100 ml wash buffer contains 0.1 ml [lOOpl] Tween 20 and 99.9 ml of IX PBS. Mix gently. Prepare as required and to be made fresh while performing the ELISA. Volume to be made as per requirement.
  • Blocking Buffer/Assay buffer comprises IX PBS, pH 7.3+0.1, 0.1% tween 20 and 5% fetal bovine serum (FBS).
  • the 100ml blocking buffer/assay buffer will have 5 ml FBS and 0.1 ml [lOOpl] Tween 20 solution to 94.9 ml IX PBS. Prepare as required and to be made fresh while performing the ELISA. Volume to be made as per requirement.
  • the coating antigen will be respective polysaccharide [PS] for which the ELISA is to be performed. Whichever antigen is to be coated on the 96 well plate.
  • PS polysaccharide
  • the coating antigen will be respective polysaccharide [PS] for which the ELISA is to be performed. Whichever antigen is to be coated on the 96 well plate.
  • meningitidis PS of respective serogroup from 1 mg/ml stock prepared in MQW and stored at -200C) in 9.9 ml coating buffer. Prepare fresh every day.
  • Substrate Readymade 3, 3 ',5, 5 '-Tetramethylbenzidine (TMB) [Cat T0440, Sigma], which was cooled to room temperature before adding to the plate during the last incubation step.
  • TMB 5 '-Tetramethylbenzidine
  • Quality control (QC) sera composition The QC sera is to be prepared by pooling approx. 30pl or more and equal volume of serum from each mice from the groups which are assumed to have generated high IgG concentrations. After pooling of the serum, designate appropriate name to the QC sera and assign an arbitrary value such as 6000 EU/ml or 5000 EU/ml for the evaluation and quantification of anti-meningococcal IgG concentrations in the test sera samples. The QC sera was stored at -20°C. For each serogroup such as ABCYWX QC sera to be optimized for predilutions of both primary (l°Ab) and secondary (2°Ab) antibodies.
  • the primary (l°Ab) antibodies are serum samples and secondary (2°Ab) antibody is the commercially available antibody A4416 from Sigma.
  • Coating 50 pl mHSA (from 1 mg/ml stock prepared in MQW and stored at - 20°C) and 50 pl N. meningitidis PS of respective serogroup (from 1 mg/ml stock prepared in MQW and stored at -20°C) were mixed in 9.9 ml coating buffer and were prepared fresh every day. Afterwards, 100 pl coating buffer was added per well of the 96-well ELISA plate having 500ng of polysaccharide.
  • the predilutions and combination of l°Ab and 2°Ab gave OD values between 1.5+0.5, however more preferably an OD of 2 was considered for each of the serogroups i.e. ACYWX for the QC sera to be used in the successive ELISA experiments. Also each time ELISA was performed for each of the serogroups i.e. ACYWX, the optimized QC sera had to be put in duplicate along with the test samples (in duplicate). After ELISA, QC sera OD values were mapped along with test sample OD values obtained, in the combitats software, which followed a four parametric logistic curve model (4PL) for the estimation IgG of all the samples in the study design.
  • 4PL parametric logistic curve model
  • step B3 After blocking was over from step 3, the plates were washed thrice as mentioned in step B3. 200 pl of pre-diluted QC sera were added in the respective well of the row A. 100 pl assay buffer was added in all the other wells. 6 two-fold serial dilutions of QC sera were performed in the respective wells by adding transferring 100 pl from row A to row B and so on till row G for the QC sera samples. 100 pl discarded from last serum dilution well used so that each well had 100 pl diluted serum with buffer in it. The plates were put for at RT with 2 hours of incubation time.
  • Secondary antibody [Anti-Mouse IgG (whole molecule)-Peroxidase antibody, A4416] was prepared in assay buffer and 100 pl/well was added with 60 mins incubation at RT. ELISA plates were covered and placed in a dark place.
  • step B3 The plates were washed as mentioned in step B3 and 100 pl of substrate solution (TMB)/well was added, followed by incubating the plate for 10+1 min at RT in a dark place to protect the ELISA reaction, blue color developed after the reaction of HRP with substrate.
  • TMB substrate solution
  • the reaction is stopped by adding 50 pl of 2 M H2SO4 per well. The color changed from blue to Yellow.
  • the test sample may have the l°Ab predilution as half of the QC sera dilution whereas for 2°Ab, the predilution may be the same as optimized for the QC sera for each serogroup of ACYWX.
  • the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.
  • the present invention provides the composition, process of preparation of hexavalent combination vaccine.
  • the present invention provides a novel, specific and industrially advantageous composition of hexavalent combination vaccine that are capable of complete prophylaxis against meningococcal diseases. These compositions shall be fulfilling the unmet medical need for Meningitis prophylaxis and treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne un vaccin combiné comprenant un sérogroupe hexavalent de Neisseria meningitidis pour la prévention et la prophylaxie d'une infection provoquée par des bactéries méningococciques. La présente invention concerne un vaccin combiné hexavalent comprenant des sérogroupes A, B, C, Y, W, X de Neisseria meningitidis. La présente invention concerne un vaccin combiné hexavalent comprenant un conjugué polysaccharide-protéine de sérogroupes A, C, Y, W, X et des protéines de sérogroupe B de Neisseria meningitidis. La présente invention concerne plus précisément un vaccin combiné hexavalent comprenant un conjugué polysaccharide-protéine de sérogroupes A, C, Y, W, X et des protéines de liaison au facteur H mutées (fHbp) de sérogroupe B de Neisseria meningitidis. La présente invention concerne en outre la composition, l'utilisation et le procédé de préparation d'un vaccin combiné hexavalent.
PCT/IN2024/052447 2024-04-26 2024-12-31 Vaccin combiné hexavalent méningococcique Pending WO2025224732A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN202421033292 2024-04-26
IN202421033292 2024-04-26

Publications (1)

Publication Number Publication Date
WO2025224732A1 true WO2025224732A1 (fr) 2025-10-30

Family

ID=97489561

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2024/052447 Pending WO2025224732A1 (fr) 2024-04-26 2024-12-31 Vaccin combiné hexavalent méningococcique

Country Status (1)

Country Link
WO (1) WO2025224732A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007102797A2 (fr) * 2005-12-22 2007-09-13 Sanofi Pasteur, Inc. Conjugues de polysaccharides-proteines derivatises meningococciques multivalents et vaccin
US8574597B2 (en) * 2006-12-22 2013-11-05 Wyeth Llc Immunogenic compositions for the prevention and treatment of meningococcal disease
WO2017036136A1 (fr) * 2015-08-31 2017-03-09 成都欧林生物科技股份有限公司 Procédé d'activation d'un vaccin conjugué à base de polysaccharides des groupes a et c de neisseria meningitidis
US20200247911A1 (en) * 2017-05-17 2020-08-06 Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. Purification of Bacterial Polysaccharides
IN202321021121A (fr) * 2023-03-24 2024-04-19

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007102797A2 (fr) * 2005-12-22 2007-09-13 Sanofi Pasteur, Inc. Conjugues de polysaccharides-proteines derivatises meningococciques multivalents et vaccin
US8574597B2 (en) * 2006-12-22 2013-11-05 Wyeth Llc Immunogenic compositions for the prevention and treatment of meningococcal disease
WO2017036136A1 (fr) * 2015-08-31 2017-03-09 成都欧林生物科技股份有限公司 Procédé d'activation d'un vaccin conjugué à base de polysaccharides des groupes a et c de neisseria meningitidis
US20200247911A1 (en) * 2017-05-17 2020-08-06 Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. Purification of Bacterial Polysaccharides
IN202321021121A (fr) * 2023-03-24 2024-04-19

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAJAN MATTHEW, MARCHEVSKY NATALIE, SINCLAIR GEMMA, O’BRIEN KATIE, JEFFERIES KIMBERLEY, OWINO NELLY, HALLIS BASSAM, GOLDBLATT DAVID: "A Randomized Trial Assessing the Immunogenicity and Reactogenicity of Two Hexavalent Infant Vaccines Concomitantly Administered With Group B Meningococcal Vaccine", PEDIATRICS INFECTIOUS DISEASE JOURNAL, vol. 42, no. 1, 1 January 2023 (2023-01-01), US, pages 66 - 73, XP093369816, ISSN: 0891-3668, DOI: 10.1097/INF.0000000000003753 *

Similar Documents

Publication Publication Date Title
JP7318034B2 (ja) 多価肺炎球菌多糖体－タンパク質複合体組成物
TWI815860B (zh) 多價肺炎球菌多醣-蛋白質共軛物組成物
CN108430500B (zh) 用于针对肠外致病性大肠杆菌的免疫保护的方法和组合物
CN101785857B (zh) 一种新的肺炎球菌结合疫苗及其制备方法
TWI864052B (zh) 包含多價肺炎球菌多醣-蛋白共軛物的免疫組成物、醫藥組成物及其用途
JP2022513562A (ja) 肺炎連鎖球菌の精製された莢膜多糖
EP4512419A1 (fr) Composant de vaccin conjugué de polysaccharide pneumococcique polyvalent et son application
JP2012140456A (ja) 髄膜炎菌由来の誘導体化多糖類−タンパク質の多価コンジュゲートおよびワクチン
CN116688109A (zh) 多糖蛋白缀合物
WO2025224732A1 (fr) Vaccin combiné hexavalent méningococcique
US20210369833A1 (en) Bacterial polysaccharide-conjugated carrier proteins and use thereof
CN101443454A (zh) 多价脑膜炎球菌衍生化的多糖-蛋白质缀合物和疫苗
US20240066110A1 (en) Immunogenic Composition Comprising Multivalent Streptococcus Pneumoniae Polysaccharide-Protein Conjugates
KR20250103717A (ko) 수막염균의 돌연변이된 인자 H 결합 단백질(fHbp), 그의 조성물 및 그의 용도
WO2025021712A1 (fr) Composition immunogène
CN116898960A (zh) 细菌多糖蛋白缀合物及其应用
EA048146B1 (ru) Композиции поливалентной пневмококковой вакцины, содержащие конъюгаты полисахарид-белок
HK1204574B (en) Multivalent meningococcal derivatized polysaccharide-protein conjugates and corresponding vaccines

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24937066

Country of ref document: EP

Kind code of ref document: A1