Classifications

• • 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
  • A61K39/099—Bordetella
• • 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
  • A61K39/0016—Combination vaccines based on diphtheria-tetanus-pertussis
  • A61K39/0018—Combination vaccines based on acellular diphtheria-tetanus-pertussis
• • 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
  • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
• • 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
  • A61K39/08—Clostridium, e.g. Clostridium tetani
• • 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
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • 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
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • 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
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
• • 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present disclosure relates to acellular pertussis vaccines and methods of using the same.
• Pertussis or whooping cough is an acute and highly contagious respiratory disease caused primarily by Bordetella pertussis.
• Bordetella pertussis Before the broad implementation of immunization programs, pertussis was highly endemic. Evidence suggests that almost all children became infected with B. pertussis before they reached adulthood, with most of them suffering some degree of clinical disease, and that high circulation of the bacterium provided natural boosting of infection-acquired immunity, which estimates suggest lasted from 7-10 to 20 years (Wendelboe et al, Pediatr Infect Dis J, 2005;24: S58-S61).
• Vaccination has been the most effective strategy to reduce the number of cases of pertussis (Halperin, N Engl J Med. 2005;353: 1615-7).
• the initial pertussis vaccines included killed whole cells of B. pertussis (wP) that were chemically detoxified and formulated with diphtheria and tetanus antigens. Since the 1990s, wP vaccines have been replaced in many countries by acellular pertussis vaccines.
• Acellular pertussis (aP) vaccines induce relatively fewer side-effects compared to wP vaccines, which are associated with a high risk for fever, reactogenicity at the injection site and, to a lesser extent, convulsions.
• acellular vaccines are typically based on the following virulence factors: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial agglutinogen 2 and fimbria! agglutinogen 3 (FIM2/3 or FIM). While some acellular vaccines contain only PT and FHA or PT alone, it is generally believed that acellular pertussis vaccines containing PT, FI-IA, PRN, and FTM2/3 components are the most effective aP vaccines currently available.
• PT pertussis toxin
• FHA filamentous hemagglutinin
• PRN pertactin
• FIM2/3 fimbrial agglutinogen 2
• FIM2/3 or FIM fimbrial agglutinogen 3
• Tdap acellular pertussis-containing vaccines
• Tdap vaccination was associated with a decreased incidence of clinical illness (i.e., cough greater than 21 days) and of increases in anti-pertussis antibody levels over the 1-year observation period, for a vaccine efficacy estimate of 92% against laboratory- confirmed pertussis (Ward et al, Clin Infect Dis 2006;43: 151-57).
• the effectiveness of Tdap vaccination in pertussis control was also shown in several observational studies in non outbreak settings in the US and Australia, demonstrating a significant impact of adolescent Tdap vaccination on disease incidence; one study estimated an effectiveness of 85.4% against laboratory confirmed pertussis (Skoff et al, Arch Pediatr Adolesc Med.
• Thl/Thl7 bias of the immune response observed after wP vaccination may be associated with longer duration of protection and faster clearance of infection (i.e., stronger initial protection) than with the Th2 bias observed after aP vaccination.
• Thl/Thl7 versus Th2 responses induced by primary vaccination with wP and aP, respectively are maintained following aP booster vaccination, even years after the initial primary vaccination, suggesting that the priming vaccination influences the Thl/Thl7 versus Th2 bias following aP booster vaccination (Bancroft et al., Cell Immunol, 2016, 304-305:35-43).
• the key role of Thl/Thl7 effector cells in immunity against B. pertussis has been confirmed in the murine model of infection, further strengthening this hypothesis (Ross et al., PLoS Pathog. 2013;9(4): el003264).
• the present inventors have developed new, modified acellular pertussis (aP) booster vaccines comprising a toll-like receptor (TLR) agonist. More specifically, the present inventors surprisingly found that administering an aP booster vaccine with a TLR4 and/or a TLR9 agonist can reorient a Th2-biased immune response, induced by a previously administered aP vaccine, towards a Thl -biased immune response. Without intending to be bound by any theory, it appears that the repolarization of T helper cells and shift in Thl/Th2 balance induced by the modified aP booster vaccine is associated with accelerated B. pertussis clearance.
• a first aspect of this disclosure is directed to an aP booster vaccine, comprising a tetanus toxoid, a diphtheria toxoid, a detoxified pertussis toxin (typically a genetically- modified pertussis toxin), filamentous hemagglutinin, pertactin, fimbriae types 2 and 3, a TLR agonist, and an aluminum salt, wherein at least the TLR agonist is formulated with an aluminum salt (Aspect 1).
• Another aspect is directed to a method of inducing an immune response in a human subject who has previously been exposed to B. pertussis antigens, the method comprising administering to the subject an aP booster vaccine, wherein the aP booster vaccine comprises a tetanus toxoid, a diphtheria toxoid, a detoxified pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae types 2 and 3, at least one TLR agonist, and an aluminum salt, wherein at least the TLR agonist is formulated with an aluminum salt, and wherein administering the aP booster vaccine reorients a Th2-biased immune response, induced by previous exposure to B.
• the aP booster vaccine comprises a tetanus toxoid, a diphtheria toxoid, a detoxified pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae types 2
• Aspect 2 Also covered by Aspect 2 is the use of the aP booster vaccine for reorienting a Th2-biased immune response toward a Thl -biased or Thl/Thl7-biased immune response in a human subject who has been previously exposed to B. pertussis antigens, typically via an aP priming vaccine.
• the human subject has previously received an acellular pertussis vaccine (also referred to herein as an aP priming vaccine) prior to administering the aP booster vaccine, which aP priming vaccine induces a Th2-biased immune response.
• an acellular pertussis vaccine also referred to herein as an aP priming vaccine
• the human subject may have been previously exposed to B. pertussis antigens by receiving a wP vaccine or through a natural infection with B. pertussis.
• aP priming vaccine typically receives an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ), the non-TLR agonist containing aP booster vaccine boosts the Th2-biased immune response induced by the aP priming vaccine.
• a TLR agonist e.g., ADACEL ®
• the modified, TLR agonist containing aP booster vaccine described herein unexpectedly reorients the Th2-biased immune response induced by the aP priming vaccine towards a Thl-biased immune response or a Thl/Thl7-biased immune response.
• the Thl-biased immune response is characterized by one or more of decreased IL-5 production, increased IFN-g production, or a lower IgGl/IgG2a ratio, as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• a TLR agonist e.g., ADACEL ®
• the Thl -biased immune response is characterized by decreased IL- 5 production and/or a lower IgGl/IgG2a ratio, as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• a Thl/Thl7-biased response is characterized by increased IL-17 production and one or more of decreased IL-5 production or a lower IgGl/IgG2a ratio.
• a Thl/Thl7-biased response is characterized by increased IL-17 production and one or more of decreased IL-5 production or a lower IgGl/IgG2a ratio, as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• a TLR agonist e.g., ADACEL ®
• the TLR agonist is a TLR4 agonist.
• the TLR4 agonist is an agonist of human TLR4.
• the TLR4 agonist is E6020.
• the TLR agonist is a TLR9 agonist.
• the TLR9 agonist is an agonist of human TLR9.
• the TLR9 agonist is a CpG oligonucleotide.
• the CpG oligonucleotide is a Class A, Class B, Class C, or Class P CpG oligonucleotide.
• the CpG oligonucleotide is CpG1018, having the nucleotide sequence of SEQ ID NO: 1 in which all the nucleotides in SEQ ID NO: 1 are linked with a phosphorothioate linkage.
• the tetanus toxoid is present in an amount of 8-12 Lf/mL, optionally 9-11 Lf/mL, or optionally 10 Lf/mL.
• the diphtheria toxoid is present in an amount of 3-8 Lf/mL, optionally, 3-6 Lf/mL, or optionally 4-5 Lf/mL.
• the detoxified pertussis toxin is a genetically detoxified pertussis toxin (gdPT).
• the gdPT comprises a mutation at R9.
• the gdPT comprises a R9K mutation and an E129G mutation.
• the gdPT is present in an amount of 4-30 pg/mL, optionally 16-24 pg/mL, optionally 18-22 pg/mL, or optionally 20 pg/mL.
• the filamentous hemagglutinin is present in an amount of 5-15 pg/mL, optionally 8-12 pg/mL, or optionally 10 pg/mL.
• the pertactin is present in an amount of 5- 15 pg/mL, optionally 8-12 pg/mL, or optionally 10 pg/mL.
• the fimbriae types 2 and 3 are present in an amount of 10-20 pg/mL, optionally 14-16 pg/mL or optionally 15 pg/mL.
• the TLR4 agonist such as E6020, is present in an amount of no more than 10 pg/mL, optionally 0.5-5 pg/mL, or optionally no more than 2 pg/mL.
• the TLR9 agonist such as CpG1018, is present in an amount of 250-750 pg/mL, optionally 400-600 pg/mL, or optionally 500 pg/mL.
• the aP booster vaccine comprises tetanus toxoid in an amount of about 8-12 Lf/mL and optionally 9-11 Lf/mL; diphtheria toxoid in an amount of about 3-8 Lf/mL and optionally 3-6 Lf/mL; genetically-detoxified pertussis toxin in an amount of about 16-24 pg/mL and optionally 18-22 pg/mL; filamentous hemagglutinin in an amount of about 5-15 pg/mL and optionally 8-12 pg/mL; pertactin in an amount of about 5-15 pg/mL and optionally 8-12 pg/mL; fimbriae types 2 and 3 in an amount from about 10-20 pg/mL and optionally 14-16 pg/mL; aluminum hydroxide (AIOOH) in an amount of about 0.25-0.75 mg/mL and optionally 0.6-0.7 mg
• AIOOH aluminum hydroxide
• the aP booster vaccine comprises tetanus toxoid in an amount of 10 Lf/mL; diphtheria toxoid in an amount of 4-5 Lf/mL; genetically- detoxified pertussis toxin in an amount of 20 pg/mL; filamentous hemagglutinin in an amount of 10 pg/mL; pertactin in an amount of 10 pg/mL; fimbriae types 2 and 3 in an amount 15 pg/mL; aluminum hydroxide (AIOOH) in an amount of 0.66 mg/mL; and a TLR4 agonist, such as E6020, in an amount of no more than 2 pg/mL.
• the aP booster vaccine may contain a TLR9 agonist, such as CpG1018, in an amount of 500 pg/mL.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises tetanus toxoid in an amount of 4-6 Lf, optionally 4.5-5.5 Lf, or optionally 5 Lf per 0.5 mL dose.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises diphtheria toxoid in an amount of 1-4 Lf, optionally 1.5-3 Lf, or optionally 2-2.5 Lf.
• the aP booster vaccine is in a unit dose form for administration to a human subject which is typically per 0.5 mL dose and comprises genetically-detoxified pertussis toxin in an amount of 2-12 pg, optionally 8-12 pg, or optionally 10 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises filamentous hemagglutinin in an amount of 2.5-7.5 pg, optionally 4-6 pg, or optionally 5 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises pertactin is present in an amount of 2.5-7.5 pg, optionally 4-6 pg, or optionally 5 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises fimbriae types 2 and 3 in an amount of 5-10 pg, optionally 7-8 pg or optionally 7.5 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the TLR4 agonist, such as E6020, in an amount of no more than 5 pg, optionally 0.25-2.5 pg, or optionally no more than 1 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the TLR9 agonist, such as CpG1018, in an amount of 125-375 pg, optionally 200-300 pg, or optionally 250 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: tetanus toxoid in an amount of about 4-6 Lf and optionally 4.5-5.5 Lf; diphtheria toxoid in an amount of about 1-4 Lf and optionally 1.5-3 Lf; genetically-detoxified pertussis toxin in an amount of about 2-12 pg and optionally 8-12 pg; filamentous hemagglutinin in an amount of about 2.5 -7.5 pg and optionally 4-6 pg; pertactin in an amount of about 2.5 -7.5 pg and optionally 4-6 pg, fimbriae types 2 and 3 in an amount of about 5-10 pg and optionally 7-8 pg; aluminum hydroxide (AIOOH) in an amount of about 0.125-0.375 mg and optionally 0.3-0.35 mg; and a TL4
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: tetanus toxoid in an amount of 5 Lf, diphtheria toxoid in an amount of 2-3 Lf, genetically-detoxified pertussis toxin in an amount of 10 pg, filamentous hemagglutinin in an amount of 5 pg, pertactin in an amount of 5 pg, fimbriae types 2 and 3 in an amount from about 7.5 pg, aluminum hydroxide (AIOOH) in an amount of 0.33 mg, and a TLR4 agonist, such as E6020, in an amount of no more than 1 pg.
• the aP booster vaccine may contain a TLR9 agonist, such as CpG1018, in an amount of 250 pg.
• the aP booster vaccine further comprises one or more of the following antigens: Haemophilus influenzae type-b oligosaccharide or polysaccharide conjugate (Hib), hepatitis B virus surface antigen (HBsAg) and/or inactivated polio virus types 1, 2 and 3 (IPV).
• Hib Haemophilus influenzae type-b oligosaccharide or polysaccharide conjugate
• HBsAg hepatitis B virus surface antigen
• IPV inactivated polio virus types 1, 2 and 3
• the aP booster vaccine further comprises a tris-buffered saline.
• the aP booster vaccine has an aluminum concentration of 0.25-0.75 mg/mL, optionally 0.6-0.7 mg/mL, or optionally 0.66 mg/mL.
• the aP booster vaccine is in a unit dose form for administration to a human subject and contains aluminum in an amount of 0.125-0.375 mg, optionally 0.3-0.35 mg, or optionally 0.33 mg.
• At least one of the tetanus toxoid, the diphtheria toxoid, and the detoxified pertussis toxin is adsorbed to the aluminum salt.
• the tetanus toxoid, the diphtheria toxoid, the detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3 are adsorbed to the aluminum salt.
• the TLR4 agonist or TLR9 agonist is formulated with the aluminum salt.
• all of the bacterial antigens in the aP booster vaccine and the TLR4 or TLR9 agonist are formulated with the aluminum salt.
• the aluminum salt is an aluminum hydroxide. In certain embodiments of Aspects 1 and 2, the aluminum salt is an aluminum phosphate.
• the aP booster vaccine further comprises a Haemophilus influenzae type-b saccharide (Hib) conjugate, a hepatitis B virus surface antigen (HBsAg) and/or an inactivated polio virus (IPV).
• Hib Haemophilus influenzae type-b saccharide conjugate
• HBsAg hepatitis B virus surface antigen
• IPV inactivated polio virus
• the human subject is 4 years of age or older when the aP booster vaccine is administered. [046] In certain embodiments of Aspect 2, the human subject is 10 years of age or older when the aP booster vaccine is administered.
• the Thl -biased immune response is characterized by one or more of decreased IL-5 production, increased IFN-g production, increased IL-17 production, or a lower IgGl/IgG2a ratio, as compared to the acellular pertussis vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• the Thl-biased immune response is characterized by one or more of decreased IL-5 production or a lower IgGl/IgG2a ratio, as compared to the acellular pertussis vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• the aP priming vaccine comprises a tetanus toxoid, a diphtheria toxoid, a detoxified pertussis toxin, filamentous hemagglutinin, and pertactin, with the proviso that the aP priming vaccine does not contain a TLR agonist.
• the aP priming vaccine includes but is not limited to one or more doses of DAPTACEL ® , INFANRIX ® , INFANRIX-HEXA ® , PENTACEL ® , QUADRACEL ® , KINRIX ® , PEDIARIX ® , or VAXELIS ® .
• the aP priming vaccine comprises DAPTACEL ® .
• the aP priming vaccine comprises INFANRIX ® or INFANRIX-HEXA ® .
• the aP priming vaccine comprises PENTACEL ® .
• the aP priming vaccine comprises KINRIX ® or PEDIARIX ® .
• the aP priming vaccine comprises VAXELIS ® .
• Figures 1A-B show a comparison of genetically-detoxified PT (gdPT) immunogenicity with chemically-detoxified PT (PTxd) at eliciting a PT specific antibody response.
• Figure 1A shows Anti-PT, IgGl and IgG2a antibody titers measured by ELISA after 2 immunizations.
• Figure IB shows Anti-PT neutralizing titers as measured by CHO assay after 2 immunizations. Mice were immunized two times at day 0 and 21. Mice were bled 17 days after the second immunization (Day 38).
• Figures 2A-B show a comparison of the boostability of a PTxd-primed response with PTxd or gdPT.
• Figure 2A shows the ELISA PT-specific IgGl and IgG2a response after priming with PTxd or gdPT followed by boosting with PTxd or gdPT.
• Figure 2B shows the PT neutralizing titers after priming with PTxd or gdPT followed by boosting with PTxd or gdPT, as measured by CHO assay. Mice were immunized three times at day 0, 21 and 42. Mice were bled 17 days after the second immunization and 8 days after the third immunization.
• Figure 3 shows the evaluation of absence of immunological interference by gdPT with the IgGl and IgG2a responses induced against FIM antigens after 2 immunizations.
• Figures 4A-B show the evaluation of immunological interference by other Tdap antigens on gdPT-induced anti-PT IgGl and IgG2a responses and neutralizing antibody responses against PT antigens after 3 immunizations.
• Figure 4A shows Anti-PT, IgGl and IgG2a antibody titers as measured by ELISA after 3 immunizations.
• Figure 4B shows Anti- PT neutralizing titers as measured by CHO assay after 3 immunizations.
• Figure 5 shows the ability of the modified Tdap (gdPT + E6020-A100H) and the modified Tdap (gdPT + CpG1018-AlOOH) formulations to down-modulate a DTaP-induced Th2 immune memory response using a long prime-boost schedule, by measuring cytokine (IL-5, IFN-g, and IL-17) levels. Cytokine levels were measured by Fluorospot assay.
• Figures 6A-L show the levels of different antigen-specific IgGl and IgG2a induced in mice after different prime/boost schedules.
• Naive adult CD1 mice were immunized (primed) i.m. with DTwP or DTaP.
• DTwP-primed mice were boosted with DTwP vaccine and DTaP-primed mice were boosted with modified TdaP vaccines (mTdaP-AlOOH, mTdaP-CpG-AIOOH or mTdaP-E6020-A100H).
• FHA- and FIM2,3-, PRN-, PT-, DT-, and TT-specific IgGl and IgG2a antibody responses were assessed by a modified ELISA technique (MSD) in sera collected 42 days post boost (D84) and again at 42 days later (D126) after a second boost at D84 using a long prime-boost schedule.
• MSD modified ELISA technique
• the numbers above the IgGl and IgG2a bars in Figures 6A-L represent the IgGl/IgG2a ratio with lower numbers representing lower ratios and vice versa.
• Lower IgGl/IgG2a ratios were observed in mice boosted with modified Tdap vaccines containing a TLR agonist (CpG or E6020).
• Figures 6Aand 6B show the IgGl and IgG2a results and ratios for FHA at D84 and D126 respectively.
• Figures 6C and 6D show the IgGl and IgG2a results and ratios for FIM at D84 and D126 respectively.
• Figures 6E and 6F show the IgGl and IgG2a results and ratios for PRN at D84 and D126 respectively.
• Figures 6G and 6H show the IgGl and IgG2a results and ratios for gdPT at D84 and D126 respectively.
• Figures 61 and 6J show the IgGl and IgG2a results and ratios for DT at D84 and D126 respectively.
• Figures 6K and 6L show the IgGl and IgG2a results and ratios for TT at D84 and D126 respectively.
• Figure 7 depicts the mouse adoptive transfer model used for evaluating the new Tdap boost vaccine formulations.
• Figure 8A shows the IgG responses for DTaP/Tdap (prime/boost), DTwP/Tdap (prime/boost), or DTwP/DTwP (prime/boost) at several time points after boost.
• Figure 8B shows accelerated and higher anti- PT, PRN, FHA, and FIM2,3 IgG titers of the modified Tdap (gdPT + E6020-A100H) boost and the modified Tdap (gdPT + CpG1018-A100H) boost as compared to Tdap boost response in adoptive transferred mice, following a DTaP priming vaccine.
• the P- value ⁇ 0.05 is indicated as follows: * DTaP/Tdap (PRIME/BOOST) versus DTwP/Tdap (PRIME/BOOST); # DTaP/Tdap (PRIME/BOOST) versus DTwP/DTwP (PRIME/BOOST); t DTwP/Tdap (PRIME/BOOST) versus DTwP/DTwP (PRIME/BOOST).
• Figures 9A-B show that boosting with the modified Tdap (gdPT + E6020-A100H) and the modified Tdap (gdPT + CpG1018-AlOOH) formulations provides early and/or accelerated bacterial clearance after intranasal challenge with B pertussis.
• Figure 9A shows results of clearance after priming with DTaP or DTwP followed by a Tdap or DTwP boost.
• Figure 9B shows results of clearance after priming with DTaP followed by a Tdap, modified Tdap (gdPT + E6020-A100H) or modified Tdap (gdPT + CpG1018) boost.
• the P-value ⁇ 0.05 is indicated as follows: * DTaP/Tdap (PRIME/BOOST) vs DTaP/mTdap-CPG-ALOOH (PRIME/BOOST); ⁇ DTaP/Tdap
• Figures 10A-B depict a mouse intranasal challenge assay (INCA) using a short immunization schedule ( Figure 10A) and a long schedule ( Figure 10B).
• Figure IOC shows that DTaP/Tdap (prime/boost) and DTwP/DTwP (prime/boost) protect mice against Bordetella pertussis lung colonization following intranasal challenge in the INCA short model.
• FIG. 11 shows that the modified Tdap (gdPT + E6020-A100H) booster significantly accelerates B. pertussis clearance as compared to the Tdap booster in the mouse intranasal challenge assay (INCA) using a short prime-boost schedule.
• Figures 12A-B show that the modified Tdap (gdPT + E6020-A100H) and the modified Tdap (gdPT + CpG1018-AlOOH) booster vaccines are able to protect DTaP primed mice against disease and colonization of the lower respiratory tract using a long prime-boost schedule.
• Figure 12A shows the reduction in all vaccinated groups 3 days post challenge with baseline bacterial load reached at day 7 for all treatment groups.
• Figure 12B shows that all acellular pertussis dosing schedules had similar kinetics as the DTwP/DTwP (prime/boost) dosing schedule.
• Figures 13A-B show that boosting a DTaP priming immunization with a modified Tdap (gdPT + E6020-A100H) formulation induces IL-17 production in an E6020 dose dependent manner, as measured by a fluorospot assay.
• the splenocytes from CD1 mice immunized with DTaP and boosted twice with mTdap-E6020-A100H (DO and D21) were isolated and re-stimulated in vitro with PTx ( Figure 13A) or a pool of pertussis antigens consisting of PTx, PRN, and FIM ( Figure 13B).
• FIG. 14 shows the thermal profiles of modified Tdap formulations: mTdap (gdPT + AIOOH), mTdap (gdPT + E6020-A100H), and mTdap (gdPT + CpG-AlOOH), showing the first derivative of intrinsic fluorescence emission ratio (350nm/330nm).
• Thermal transition (Tm) of mTdap (gdPT + AIOOH) is 74.6 °C
• mTdap (gdPT + E6020-A100H) is 74.2 °C
• mTdap (gdPT + CpG- AIOOH) is 77.0 °C.
• Pertussis vaccines are typically based on the following virulence factors: detoxified pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial agglutinogen 2 and fimbria! agglutinogen 3 (F1M2/3 or F1M). While some acellular pertussis vaccines contain only PT and FHA or PT, FHA and PRN, it is generally believed that acellular pertussis vaccines containing PT, FHA, PRN, and FIM2/3 components are the most effective aP vaccines currently available. Typically acellular pertussis vaccines are formulated with diphtheria toxoid and tetanus toxoid.
• wP refers to a whole cell B. pertussis vaccine.
• whole cell pertussis vaccines include whole cells of B. pertussis that have been chemically detoxified and formulated with diphtheria toxoid and tetanus toxoid.
• DTwP refers to a wP indicated in the prevention of diphtheria, tetanus and pertussis in infants as a first vaccination and in children as a booster.
• a DTwP is to D.T.COQ/D.T.P., which was marketed by Sanofi Pasteur and contains diphtheria toxoid and tetanus toxoid, B. pertussis inactivated by heat in the presence of thiomersal, and aluminum phosphate.
• DTaP refers to an aP indicated for active immunization against diphtheria, tetanus and pertussis in infants and children.
• the DTaP is administered as a five-dose series in infants and children 6 weeks through 6 years of age or a four-series dose series in infants and children 6 weeks through 2-4 years of age.
• Examples of DTaP include, but are not limited to, DAPTACEL ® , PENTACEL ® , and INFANRIX ® or INFANRIX-HEXA ® .
• DAPTACEL ® for example, is marketed by Sanofi Pasteur and contains diphtheria toxoid and tetanus toxoid, the following acellular pertussis antigens. PT (chemically detoxified), FHA, PRN, and FIM2/3, as well as aluminum phosphate. Typically, the DTaP contains increased amounts of diphtheria toxoid and PT as compared to the Tdap.
• Tdap refers to an aP indicated for active booster immunization against tetanus, diphtheria and pertussis. Typically, the Tdap is administered as a single dose in individuals 10 years of age and older. Examples of Tdap include, but are not limited to, ABAC EL ® and BOOSTRJX ® .
• ADACEL ® for example, is marketed by Sanofi Pasteur and contains diphtheria toxoid and tetanus toxoid and the following acellular pertussis antigens: PT (chemically detoxified), FHA, PRN, and FTM2/3, as well as aluminum phosphate.
• the Tdap contains reduced amounts of diphtheria toxoid and PT as compared to the DTaP.
• ‘modified Tdap” or“mTdap” refers to a modified version of a Tdap vaccine comprising diphtheria toxoid and tetanus toxoid and the following acellular pertussis antigens: genetically modified FT, FHA, PEN, and F1M2/3.
• the modified Tdap is different from Tdap at least because the modified Tdap contains a TLR agonist (for example a TLR4 agonist (e.g., E6020) or a TLR9 agonist (e.g., CpGlOlB)).
• the modified Tdap also optionally contains genetically-detoxified FT (gdPT) instead of chemically -detoxified PT (PTdx) or aluminum hydroxide instead of aluminum phosphate.
• the mTdap contains a TLR agonist (for example a TLR4 agonist (e.g., E6020) or a TLR9 agonist (e.g. CpGlOlB)), geneticall -detoxified PT (gdPT), and aluminum hydroxide.
• booster or“booster vaccine” refers to a vaccine administered following a priming vaccine.
• the booster vaccine contains antigens that were included in the priming vaccine so that the immune system has already been exposed to such antigens prior to administration of the booster vaccine.
• “priming vaccine” refers to one or more doses of a vaccine m a vaccination schedule that are administered before a booster vaccine and induce a primary immune response and immunological memory.
• CD4 T helper cell responses to antigens can be classified based on the cytokines they produce.
• Type 1 helper T cells preferentially produce inflammatory cytokines, such as IFN-g, IL-2, TNF-a, and TNF-b. Thl cells activate macrophages and are typically associated with cell-mediated immune responses and phagocyte-dependent protective responses (e.g., opsonizing antibodies).
• Type 2 helper cells (Th2), on the other hand, preferentially produce cytokines, such as IL-4, IL-5, IL-10, and IL-13. Th2 cells activate B cells and are typically associated with antibody-mediated immune responses.
• aP priming vaccines in mice induce predominantly IgGl antibodies, but also IgG2 and IgG4, with the proportion of IgG4 increasing after booster vaccinations, reflective of a Th2-biased response (Stenger et at., Vaccine, 2010, 28:6637-46 and Brummelman et at., Vaccine, 2015, 33: 1483-19) whereas wP priming vaccines in mice induce predominantly IgG2 antibodies, as well as IgGl and IgG3, (Raeven et al, J Proteome Res, 2015, 14:2929-42), consistent with a Thl-biased response.
• the modified aP booster vaccines described in this application are able to reorient a Th2-biased immune response induced by a previously administered aP vaccine towards a Thl- or mixed Thl/Thl7-biased immune response.
• a Thl-biased immune response induced by a previously administered aP vaccine towards a Thl- or mixed Thl/Thl7-biased immune response.
• one of skill in the art is able to measure cytokine profiles and antibody isotypes using conventional techniques to readily determine if an aP vaccine induces a Thl-biased or Th2-biased response.
• a Th2-biased immune response induced by an aP vaccine in mice is associated with increased IL-5 levels and/or an increased IgGl/IgG2a ratio
• a Thl- biased immune response is typically associated with one or more of decreased IL-5 levels, increased IFN-g levels, or a reduced IgGl/IgG2a ratio.
• the ability of an aP booster vaccine as described herein to reorient a Th2-biased immune response induced by a previously administered aP vaccine towards a Thl-biased immune response indicates a reduction in IL-5 levels and/or a reduction in the IgGl/IgG2a ratio as compared to the immune response induced by previously administered aP vaccine or an aP booster vaccine that does not contain a TLR agonist.
• the Thl 7 response is measured by the production of IL- 17.
• the tetanus toxoid is produced from Clostridium tentani, a Gram-positive, rod shaped, spore-forming, bacillus bacteria. Tetanus toxoid is a protein of about 150 kDa and consists of two subunits (about 100 kDa and about 50 kDa) linked by a sulfide bond.
• the tetanus toxoid is typically detoxified with formaldehyde and can be purified from culture filtrates using known methods, such as ammonium sulfate precipitation and/or chromatography techniques, as disclosed, for example, in WO 1996/025425.
• Clostridium tentani can be grown in any suitable growth medium, including, for example, Mueller-Miller casamino acid medium without beef heart infusion (Mueller et al., J Bacteriol, 1954, 67(3):271-277) or a Latham medium derived from bovine casein.
• the tetanus toxoid may also be inactivated by recombinant genetic means.
• the amount of tetanus toxoid can be expressed as an “Lf’ unit (i.e., limit of flocculation or flocculating unit), which is defined as the amount of toxoid that when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture.
• Lf limit of flocculation or flocculating unit
• the amount of tetanus toxoid in a composition can be readily determined by comparing the composition to a reference material calibrated with reference reagents in a flocculation assay. Diphtheria Toxoid
• the diphtheria toxoid is an ADP-ribosylating exotoxin produced by Corynebacterium diphtheriae, a Gram positive, non-sporing aerobic bacterium. Like tetanus toxoid, the diphtheria toxoid is detoxified, typically using formaldehyde, to yield a toxoid that is not toxic but is still antigenic.
• C. diphtheriae can be grown in any suitable growth medium, such as modified Mueller’s growth medium (Stainer, DW, In: Manclark CR, editor, Proceedings of an informal consultation of the WHO requirements for diphtheria, tetanus, pertussis and combined vaccines , U.S.
• diphtheria toxin can be purified using conventional techniques, such as ammonium sulfate fractionation and detoxified either before or after purification using standard techniques, such as formaldehyde treatment.
• the amount of diphtheria toxoid can be expressed as an “Lf’ unit.
• the amount of diphtheria toxoid in a composition can be readily determined by comparing the composition to a reference material calibrated with reference reagents in a flocculation assay.
• Pertussis toxin is a secreted protein exotoxin and an important virulence factor produced exclusively by B. pertussis.
• Pertussis toxin is composed of five subunits, named SI, S2, S3, S4 (x2) and S5 that are encoded by five genes organized into an operon of approximately 3200 base pairs. Expression of the five genes is regulated by a promoter located upstream of the S 1 encoding gene. Activation of the toxin promoter is under control of Bordetella virulence gene (bvg) system, which regulates not only the expression of pertussis toxin, but other known virulence factors such as FHA and PRN.
• bvg Bordetella virulence gene
• Pertussis toxin is a protein of about 105 kDa with an A/B configuration.
• the A domain composed of the SI subunit, is responsible for the ADP-ribosylating activity of the protein. It blocks the binding of G protein (guanine nucleotide-binding protein) to G protein- coupled receptor (GPCR) on the host cell membrane thus interfering with signal transduction and resulting in many of the biologic effects associated with PT activity such as histamine- sensitization, leukocytosis and alteration in insulin secretion.
• the B oligomer is a pentameric ring composed of subunits S2, S3, S4, and S5, associated in the ratio 1 : 1:2: 1 and is responsible for the binding to the receptor on eukaryotic cells. It binds to various (but mostly unidentified) gly coconjugate molecules on the surface of target cells.
• the pertussis toxin used in current acellular vaccines is typically chemically detoxified.
• the detoxified pertussis toxin is typically genetically modified to reduce enzymatic activity and/or toxicity.
• Many constructs containing genetic modifications of pertussis toxin have been engineered to reduce enzymatic activity and/or toxicity of the protein while preserving its immunogenicity and protective properties, including, for example, mutant pertussis toxin having a mutation at amino acid 129 of the SI subunit of the pertussis toxin, such as the E129G mutant or the R9K/E129G double mutant. See e.g., U.S. Patent Nos.
• the genetically detoxified pertussis toxin contains a mutation at amino acid 129 of the SI subunit of pertussis toxin.
• the mutation is an E129G mutation.
• the genetically detoxified pertussis toxin contains an R9K mutation and an E129G.
• a genetically detoxified pertussis toxin is preferred, it is also possible to use a chemically detoxified pertussis toxin in place of the genetically detoxified pertussis toxin.
• Chemical detoxification can, for instance, be performed by any of a variety of conventional chemical detoxification methods, such as treatment with formaldehyde, hydrogen peroxide, tetranitromethane, or glutaraldehyde. See e.g., U.S. Patent No. 5,877,298.
• Pertactin is a 69 kDa outer membrane protein originally identified from B. bronchiseptica (Montaraz, J.A. et al. Infect immim. 1985;161:581-582). It was shown to be a protective antigen against B. bronchiseptica and was subsequently identified in both B. pertussis and B. parapertussis.
• the 69 kDa protein binds directly to eukaryotic cells (Leimnger, E. et al, Proc Natl Acad Sci USA 1991:88:345-349) and natural infection with B. pertussis induces an anti-pertactin humoral response (Thomas, M.G. et al. 1. Infect.
• Pertactin also induces a cell-mediated immune response (Petersen, J.W. et al, Infect. immun. 1992;60:4563-70; De Magistns, T. et al., J. Exp. Med. 1988; 168: 1351- 1362, Seddon, P.C. et al, Serodiagnosis Immunother. Inf. Dis. 1990;3:337-43).
• Vaccination with whole-cell or acellular vaccines induces anti-pertactin antibodies (Edwards. K.M. et al, Pediair. Res.
• Pertactin protects mice against aerosol challenge with B. pertussis (Roberts, M. et al. Vaccine 1992;10:43-48) and in combination with FHA, protects in the intracerebral challenge test against B. pertussis (Novotny, P. et a!., J. Infect. Dis. 1991 , i 64: 1 14-22). Passive transfer of polyclonal or monoclonal anti-pertactin antibodies also protects mice against aerosol challenge (Shahin, R. D. et al, J. Exp. Med. 1990;171:63-73).
• Filamentous haemagglutinin is a large (220 kDa) non-toxic polypeptide which mediates attachment of B. pertussis to ciliated cells of the upper respiratory tract during bacterial colonization (Tuomanen, E. and Weiss, A. J. Infect. Dis., 1985;152: 1 18-25).
• Vaccination with whole-cell or acellular pertussis vaccines generates anti-FHA antibodies and acellular vaccines containing FHA also induce a cell mediated immune response to FHA (Gearing, A. et al, FEMS Microbial. Immunol. 1989;47:205-12; Thomas, M.G. et al., 1. Infect. Dis. 1989;160:838-45; Di Tommaso, A. et al, Infect. Immun. 1991;59:3313-15; Tomoda, T. et al, J. Infect. Dis. 1992;166:908-10).
• Serotypes of B. pertussis are defined by their agglutinating fimbriae.
• the WHO recommends that whole-cell vaccines include types 1, 2 and 3 agglutinogens (Aggs) since they are not cross-protective (Robinson, A. et al., Vaccine 1985;3: 1 1 -22).
• Agg 1 is non- fimbrial and is found on all B. pertussis strains while the serotype 2 and 3 Aggs are fimbrial.
• Natural infection or immunization with whole-cell or acellular vaccines induces anti-Agg antibodies (Thomas, M.G. et al, J Infect. Dis. 1989;160:838-45, Edwards, K.M.
• a specific cell-mediated immune response can be generated in mice by Agg 2 and Agg 3 after aerosol infection (Petersen, J.W. et al., immun. 1992,60:4563-70).
• Aggs 2 and 3 are protective in mice against respiratory challenge and human colostrum containing anti-agglutinogens will also protect in this assay (Oda, M. et al, infect immun. 1985;47:441-45; Robinson, A. et al, Develop. Biol. Stand. 1985;61 : 165-72, Robinson, A. et al. Vaccine 1989;7:321 -24).
• the aP booster vaccines described herein include a toll-like receptor (TLR) agonist.
• TLR toll-like receptor
• the TLR agonist is a compound that can agonize or activate TLRs.
• TLRs are an important component of the host’s pathogen sensing mechanism (Janeway et al, Annu. Rev. Immunol. 2002;20: 197-216); Akira et al, Nat Rev Immunol. 2004;4:499-511).
• TLRs are typically classified into two families based on their localization: TLRs 1, 2, and 4-6 are expressed on the cell surface and sense bacterial cell wall components whereas TLRs 3 and 7-9 are expressed in endosomes and sense viral or bacterial nucleic acids (Kawasaki et al, Front Immunol. 2014:5:461). The molecular structures recognized by TLRs have been evolutionarily conserved and are expressed by a wide variety of infectious microorganisms (Janeway et al, Amur Rev. Immunol. 2002;20: 197-216); Akira et al, Nat Rev Immunol. 2004;4:499-511).
• the innate immune response elicited by TLR activation is characterized by the production of pro-inflammatory cytokines, chemokines, type I interferons and anti microbial peptides.
• This innate response promotes and modulates the adaptive immune system.
• a common result is the expansion of antigen-specific B cells that produce high affinity antibodies and of cytotoxic T cells including long-lasting memory cells that protect against subsequent infection through enhanced cytotoxic function targeting the effector phase (Wille-Reece et al, J Exp Med. 2006;203: 1249-58); Xiao et al, J Immunol. 2013;190:5866- 73).
• TLR signaling appears to play an important role in many aspects of the innate immune response.
• the aP booster vaccine is surprisingly able to shift a Th2 biased immune response, established by a previously administered aP vaccine, to a Thl- biased immune response.
• the TLR agonist is an agonist of a human TLR.
• the TLR agonist is a TLR4 agonist and preferably an agonist of human TLR4.
• the TLR4 agonist is E6020, a synthetic phospholipid dimer that mimics the physicochemical and biological properties of the natural lipid A derived from Gram-negative bacteria (Ishizaka et al, Expert Rev Vaccines. 2007;(5):773-84).
• E6020 is dodecanoic acid, (1R,6R, 22R, 27R)-l,27-dihexyl-9,19-dihydroxy-9,19-dioxido-14- oxo-6,22-bis[(l,3-dioxotetradecyl)amino]-4,8,10, 18, 20, 24-hexaoxa-13,15-diaza-9,19- dephosphaheptacosane-l,27-diyl ester, disodium salt (C83Hi58N40i9P2Na2).
• the chemical synthesis of E6020 is a reproducible and well-controlled manufacturing process yielding a highly pure chemical compound.
• E6020 has the following chemical structure:
• E6020 interacts with TLR4 and has been evaluated as an adjuvant in preclinical studies, combined with emulsions, liposomes or aluminum salts. E6020 has been reported to enhance IgG2a, which in mice is associated with Thl activation. E6020 has also been shown to enhance granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6 and TNF-a in human peripheral blood mononuclear cells (PBMCs) and mouse spleen (Ishizaka et al, Expert Rev Vaccines. 2007;(5):773-84).
• GM-CSF granulocyte-macrophage colony-stimulating factor
• IL-1 IL-1
• IL-6 TNF-a
• PBMCs peripheral blood mononuclear cells
• mouse spleen Ishizaka et al, Expert Rev Vaccines. 2007;(5):773-84.
• the TLR agonist is a TLR9 agonist and preferably an agonist of human TLR9.
• the TLR9 agonist may be a CpG oligodeoxynucleotide (“ODN”).
• ODN CpG oligodeoxynucleotide
• a“CpG oligonucleotide” or“CpG ODN” is a single stranded DNA molecule that contains at least one central unmethylated CG dinucleotide embedded within specific flanking regions. CpG ODNs are present at high frequency in bacterial DNA and possess an immunostimulatory effect.
• CpG ODNs have been categorized into 4 distinct classes based on differences in structure and the nature of the immune response they induce. Although each class contains at least one central unmethylated CG dinucleotide plus flanking regions, they differ in structure and immunological activity.
• Class B ODNs (also referred to as“K” type) contain from one to five CpG motifs typically on a phosphorothioate backbone. Phosphorothioate is a non-naturally occurring intemucleoside linking group that replaces the phosphodiester linkage found in naturally occurring DNA and enhances resistance to nuclease digestion and substantially prolongs in vivo half-life.
• Class B ODNs trigger plasmacytoid dendritic cells to differentiate and produce TNFa and stimulate B cells to proliferate and secrete IgM.
• Class A ODNs (also referred to as“D” type) have a phosphodiester core flanked by phosphorothioate terminal nucleotides. They include a single CpG motif flanked by palindromic sequences that are able to form stem-loop structures. Class A ODN also have poly G motifs at the 3’ and 5’ ends that promote concatamer formation. Class A ODNs trigger plasmacytoid dendritic cells to mature and secrete IFNa but have no effect on B cells. Class C ODNs resemble Class B in that they are composed entirely of phosphorothioate nucleotides but resemble Class A in containing palindromic CpG motifs that can form stem loop structures or dimers.
• Class C ODNs stimulate B cells to secrete IL-6 and plasmacytoid dendritic cells to produce IFNa.
• Class P CpG ODNs are highly ordered structures containing double palindromes that can form hairpins at their GC-rich 3’ ends as well as concatamerize due to the presence of the 5’ palindromes.
• the CpG ODN used in the aP booster vaccines is a Class B CpG ODN.
• the CpG ODN used in the aP booster vaccines is a Class A CpG ODN.
• the CpG ODN used in the aP booster vaccines is a Class C CpG ODN.
• the CpG ODN used in the aP booster vaccines is a Class P CpG ODN.
• the CpG ODN contains at least one phosphorothioate linkage. In certain embodiments, all the nucleotides in the CpG ODN are linked with a phosphorothioate linkage. In certain embodiments, the CpG ODN contains 1-5 CG dinucleotides. In certain embodiments, the CpG ODN contains 1 CG dinucleotide. In certain embodiments, the CpG ODN contains 2 CG dinucleotides. In certain embodiments, the CpG ODN contains 3 CG dinucleotides. In certain embodiments, the CpG ODN contains 4 CG dinucleotides. In certain embodiments, the CpG ODN contains 5 CG dinucleotides. In certain embodiments, the CpG ODN is 18-28 nucleotides in length.
• the CpG ODN is ISS1018 (Higgins et al, Exp Rev Vaccines, 2007;6(5):747-59), a 22-mer oligonucleotide having the following nucleotide sequence: 5’- TGACTGTGAACGTTCGAGATGA-3’ (SEQ ID NO: 1). All of the nucleotide bases in ISS1018 are linked with phosphorothioate linkages. As used herein,“CpG1018” is used interchangeably with ISS1018.
• Adjuvants such as aluminum salts
• Aluminum salts that can be used as adjuvants include, but are not limited to, aluminum hydroxide/oxyhydroxide (AIOOH), aluminum phosphate (AIPO4), aluminum hydroxyphosphate sulfate (AAHS) and/or potassium aluminum sulfate. These aluminum salts have a long history of use in vaccines.
• one or more of the tetanus toxoid, the diphtheria toxoid, and the acellular B. pertussis antigens of the aP booster vaccine can be adsorbed to the aluminum salt.
• all of the vaccine antigens in the aP booster vaccine are adsorbed to the aluminum salt.
• the tetanus toxoid, the diphtheria toxoid, PT, FHA, PT, and FIM2,3 are adsorbed to AIOOH.
• the tetanus toxoid, the diphtheria toxoid, PT, FHA, PT, and FIM2,3 are adsorbed to AIPO4.
• one or more of the vaccine antigens in the aP booster vaccine are adsorbed to AIOOH and one or more of the vaccine antigens in the aP booster vaccine are adsorbed to AIPO4.
• the TLR agonist is formulated with the aluminum salt.
• the TLR4 agonist, such as E6020 is formulated with AIOOH.
• the TLR4 agonist, such as E6020 is formulated with AIPCE.
• the TLR9 agonist such as the CpG ODN (e.g., CpG1018)
• the TLR9 agonist is formulated with AIOOH.
• the TLR9 agonist such as the CpG ODN (e.g., CpG1018)
• the aP booster vaccine comprises a tetanus toxoid, a diphtheria toxoid, gdPT, FHA, PT, FIM2,3, and a TLR4 agonist (e.g., E6020) and each of the tetanus toxoid, the diphtheria toxoid, gdPT, FHA, PT, FIM2,3, is adsorbed to AIOOH, and the TLR4 agonist (e.g., E6020) is formulated with AIOOH.
• a TLR4 agonist e.g., E6020
• the aP booster vaccine comprises a tetanus toxoid, a diphtheria toxoid, gdPT, FHA, PT, FIM2,3, and a TLR9 agonist (e.g., CpG ODN, such as CpG1018) and each of the tetanus toxoid, the diphtheria toxoid, gdPT, FHA, PT, FIM2,3, is adsorbed to AIOOH, and the TLR9 agonist (e.g., CpG ODN, such as CpG1018) is formulated with AIOOH.
• a TLR9 agonist e.g., CpG ODN, such as CpG101
• the aP booster vaccine includes both AIOOH and A1P04 and antigens in the vaccine may be adsorbed to one or both of these aluminum salts.
• the aP booster vaccine can contain one or more additional antigens, including, but not limited to a Haemophilus influenzae type-b saccharide (Hib) conjugate, a hepatitis B virus surface antigen (HBsAg) and/or an inactivated polio virus (IPV).
• Hib Haemophilus influenzae type-b saccharide
• HBsAg hepatitis B virus surface antigen
• IPV inactivated polio virus
• Hib vaccines are typically formulated using Hib conjugated to a carrier protein to enhance its immunogenicity, especially in children.
• the carrier protein is tetanus toxoid, diphtheria toxoid, H. influenza protein D, or an outer membrane protein complex from serogroup B. Meningococcus.
• the carrier protein is tetanus toxoid, diphtheria toxoid, H. influenza protein D, or an outer membrane protein complex from serogroup B. Meningococcus.
• any appropriate carrier protein can be used.
• Methods of making Hib conjugates are known in the art.
• PENTACEL ® contains H. influenzae type b capsular polysaccharide (polyribosyl-ribitol-278 phosphate [PRP]) covalently bound to tetanus toxoid.
• PRP polyribosyl-ribitol-278 phosphate
• HBV Hepatitis B virus
• the infectious HBV virion has a spherical, double-shelled structure, consisting of a lipid envelope containing HBsAg that surrounds an inner nucleocapsid composed of hepatitis B core antigen (HBcAg) complexed with virally encoded polymerase and the viral DNA genome.
• HBsAg is a polypeptide that typically has a length of 226 amino acids and a molecular weight of about 24 kDa.
• HBV vaccines typically contain HBsAg. Thus, methods of making HBsAg and vaccines comprising HBsAg are well known in the art.
• the HBsAg is typically adsorbed to an aluminum salt (e.g., AIOOH or AlPCri)
• Poliomyelitis is a disease caused by any one of three types of polio virus: poliovirus Type 1 (e.g., Mahoney strain), poliovirus Type 2 (e.g., MEF-1 strain), and poliovirus Type 3 (e.g., Saukett strain). Polioviruses can be grown in cell culture using known techniques, followed by purification of virions using techniques, such as ultrafiltration, diafiltration, and chromatography. Next, the virions are inactivated using, for example, formaldehyde. Typically, each type of poliovirus is grown individually, purified from the cell culture, and inactivated before combining them to produce a trivalent poliovirus composition. Typically, the inactivated poliovirus is not adsorbed onto an aluminum salt prior to formulating the vaccine. However, the inactivated poliovirus may become adsorbed onto any aluminum salt in the vaccine that is not adsorbed to another vaccine antigen.
• poliovirus Type 1 e.g., Mahoney strain
• the aP booster vaccine is an immunogenic composition that includes one or more antigens— but not all antigens— which are derived from or homologous to, antigens from B. pertussis and other pathogens (e.g., Corynebacterium diphtheria, Clostridium tetani, etc.). Such a vaccine is substantially free of intact pathogenic particles or the lysate of such particles.
• the aP booster vaccine can be prepared from at least partially purified, or substantially purified, immunogenic polypeptides from a pathogen of interest or their analogs. Methods of obtaining an antigen or antigens in the vaccine include standard purification techniques, recombinant production, or chemical synthesis.
• the one or more antigens are formulated into a unit dose of an aP booster vaccine.
• A“unit dose” as used herein refers to an amount of vaccine that is administered to a subject in a single administration. Typically, this amount is present in a volume of 0.1-2 milliliters, e.g., 0.2-1 milliliters, and typically 0.5 milliliters. The indicated amounts may, thus, for instance, be present at a concentration of micrograms per 0.5 milliliters bulk vaccine. In certain embodiments a (single) unit dose thus equals 0.5 milliliters.
• the aP booster vaccine comprises a tetanus toxoid, a diphtheria toxoid, and the following acellular B. pertussis antigens: detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae Types 2 and 3.
• the aP booster vaccine also contains a TLR agonist, such as a TLR4 (e.g., E6020) or a TLR9 agonist (e.g., CpG1018) and an aluminum salt, such as AIOOH or AIPO4.
• the acellular pertussis antigens are typically prepared by isolation from B. pertussis cultures grown in liquid culture medium. Any liquid culture medium known in the art for cultivating Bordetella cells may be used.
• a complex medium is used.
• a“complex medium” refers to a medium that contains peptone digests or extracts of plant or animal-origin. Examples of complex media suitable for use with the present methods include e.g., Homibrook’s medium, Cohen-Wheeler medium, B2 Medium, or other similar liquid culture media.
• a modified Stainer & Scholte medium that also includes dimethyl beta-cyclodextrin and casamino acids is another example suitable for use.
• Pertussis toxin, filamentous hemagglutinin, and pertactin are typically isolated separately from the supernatant culture medium. Fimbriae types 2 and 3 are typically extracted and co-purified from the bacterial cells.
• the pertussis antigens can be purified from the supernatant and/or bacterial cells using any conventional methods, including, for example, sequential filtration, salt-precipitation, ultrafiltration and chromatography.
• the tetanus toxoid is typically present in the aP booster vaccine in an amount from about 8-12 limit of flocculation (Lf)/mL. In certain embodiments, the TT is present in an amount of 9-11 Lf/mL. In certain embodiments, the TT is present in an amount of 10 Lf/mL. As measured per unit dose form, where a unit dose is 0.5 mL, the TT is typically present in an amount of about 4-6 Lf. In certain embodiments of the 0.5 mL dose form, the TT is present in an amount of 4.5-5.5 Lf. In certain embodiments of the 0.5 mL dose form, the TT is present in an amount of 5 Lf. In the aP booster vaccine, TT is typically adsorbed to an aluminum salt. Typically, TT is adsorbed onto AIOOH. In other embodiments, TT may be adsorbed onto AIPO4.
• the diphtheria toxoid is typically present in the aP booster vaccine in an amount from about 3-8 Lf/mL.
• the DT is present in an amount of 3-6 Lf/mL.
• the DT is present in an amount of 4-5 Lf/mL.
• the DT is present in an amount of 4 Lf/mL.
• the DT is typically present in an amount of about 1 5-4 Lf. In certain embodiments of the 0.5 mL dose form, the DT is present in an amount of 1 5-3 Lf.
• the DT is present in an amount of 2 2.5 Lf. In certain embodiments of the 0.5 mL dose form, the DT is present in an amount of 2 Lf.
• DT is typically adsorbed to an aluminum salt. Typically, DT is adsorbed onto AIOOH. In other embodiments, DT may be adsorbed onto AIPCL.
• the detoxified pertussis toxin (PT) is typically present in the aP booster vaccine in an amount from about 4-30 pg/mL.
• PT is chemically detoxified PT and is present in an amount of 4-10 pg/mL.
• the PT is genetically- detoxified PT (gdPT) and is present in an amount of about 16-24 pg/mL. In certain embodiments, the gdPT and is present in an amount of 18-22 pg/mL. In certain embodiments, the gdPT is present in an amount of 20 pg/mL.
• the PT is typically present in an amount of about 2-15 pg. In certain embodiments of the 0.5 mL dose form, the PT is present in an amount of 2-5 pg. In certain embodiments of the 0.5 mL dose form, the PT is gdPT and is present in an amount of 8-12 pg. In certain embodiments of the 0.5 mL dose form, the gdPT is present in an amount of 9-11 pg. In certain embodiments of the 0.5 mL dose form, the gdPT is present in an amount of 10 pg.
• the PT is present in an amount ranging from 2 50 pg, 5-40 pg, 10-30 pg, or 20-25 pg per unit dose.
• PT is typically adsorbed to an aluminum salt.
• PT is adsorbed onto AIOOH.
• PT is adsorbed onto AIPO4.
• the filamentous hemagglutinin (FHA) is typically present in the aP booster vaccine in an amount from about 5-15 pg/mL. In certain embodiments, the FHA is present in an amount of 8-12 pg/mL. In certain embodiments, the FHA is present in an amount of 10 pg/mL. As measured per unit dose form, where a unit dose is 0.5 mL, the FHA is typically present in an amount of about 2.5 to 7.5 pg. In certain embodiments of the 0.5 mL dose form, the FHA is present in an amount of 4-6 pg. In certain embodiments of the 0.5 mL dose form, the FHA is present in an amount of 5 pg.
• the FHA is present in an amount ranging from 2-50 pg, 5-40 pg, 10-30 pg, or 20-25 pg per unit dose.
• FHA is typically adsorbed to an aluminum salt.
• FHA is adsorbed onto AIOOH.
• FHA is adsorbed onto AIPO4.
• the pertactin (PRN) is typically present in the aP booster vaccine in an amount from about 5-15 pg/mL. In certain embodiments, the PRN is present in an amount of 8-12 pg/mL. In certain embodiments, the PRN is present in an amount of 10 pg/mL.
• the PRN is typically present in an amount of about 2.5 to 7.5 pg. In certain embodiments of the 0.5 mL dose form, the PRN is present in an amount of 4-6 pg. In certain embodiments of the 0.5 mL dose form, the PRN is present in an amount of 5 pg. In other embodiments, the PRN is present in an amount ranging from 0.5- 100 pg, 1-50 pg, 2-20 pg, 3-30 pg, or 5-20 pg per unit dose. In the aP booster vaccine, PRN is typically adsorbed to an aluminum salt. In certain embodiments, PRN is adsorbed onto AIOOH. In certain embodiments, PRN is adsorbed onto AIPCL.
• the fimbriae types 2 and 3 are typically present in the aP booster vaccine in an amount from about 10-20 pg/mL.
• the FIM2,3 is present in an amount of 14-16 pg/mL.
• the FIM2,3 is present in an amount of 15 pg/mL.
• the weight ratio of FIM 2 to FIM 3 is from about 1:3 to about 3: 1, e.g., from about 1 :1 to about 3: 1, e.g., from about 1.5: 1 to about 2: 1.
• the FIM2,3 is typically present in an amount of about 5-10 pg.
• the FIM2,3 is present in an amount of 7-8 pg. In certain embodiments of the 0.5 mL dose form, the FIM2,3 is present in an amount of 7.5 pg. In other embodiments, FIM2/3 is present in an amount ranging from 1-100 pg per unit dose, such as 3-50 pg, or 3-30 pg per unit dose.
• FIM2,3 are typically adsorbed to an aluminum salt. In certain embodiments, FIM2,3 is adsorbed onto AIOOH. In certain embodiments, FIM2,3 is adsorbed onto AIPO4.
• the aP booster vaccine includes an aluminum salt, such as AIOOH or AIPO4, which is used to adsorb one or more of the vaccine antigens and/or to formulate the TLR agonist.
• the aluminum salt is present in an amount from about 0.25-0.75 mg/mL, 0.25-0.35 mg/mL, or 0.6-0.7 mg/mL. In certain embodiments, the aluminum salt is present in an amount of 0.66 mg/mL. As measured per unit dose form, where a unit dose is 0.5 mL, the aluminum salt is typically present in an amount of 0.125- 0.375 mg, 0.125-0.175 mg, or 0.3-0.35 mg. In certain embodiments, the 0.5 mL unit dose form contains 0.33 mg of aluminum salt. In certain embodiments, the aluminum salt is AIOOH. In other embodiments, the aluminum salt is AIPO4.
• the TLR4 agonist is E6020
• it can be present in an amount of no more than 10 pg/ml.
• E6020 is present in an amount of 0.5-5 pg/ml.
• E6020 is present in an amount of no more than 2 pg/ml.
• E6020 is typically present in an amount of no more than 5 pg.
• E6020 is present in an amount of about 0.25-2.5 pg.
• E6020 is present in an amount of no more than 1 pg.
• E6020 is formulated with an aluminum salt.
• the aluminum salt is AIOOH.
• the aluminum salt may be AIPCE.
• the TLR9 agonist is CpG1018
• it can be present in an amount of about 250-750 pg/ml.
• CpG1018 is present in an amount of 400-600 pg/ml.
• CpG1018 is present in an amount of 500 pg/ml.
• CpG1018 is typically present in an amount of about 125-375 pg.
• CpG1018 is present in an amount of 200-300 pg. In certain embodiments of the 0.5 mL dose form, CpG1018 is present in an amount of 250 pg.
• CpG1018 is formulated with an aluminum salt.
• the aluminum salt is AIOOH.
• the aluminum salt may be AIPO4.
• the aP booster vaccine comprises TT in an amount of 8-12 Lf/mL, DT in an amount of 3-8 Lf/mL, gdPT in an amount of 16-24 pg/mL, FHA in an amount of 5-15 pg/mL, PRN in an amount of 5-15 pg/mL, FIM2,3 in an amount from about 10-20 pg/mL, AIOOH in an amount of 0.25-0.75 mg/mL, and a TLR4 agonist, such as E6020, in an amount of no more than 10 pg/ml.
• the aP booster vaccine comprises TT in an amount of 8-12 Lf/mL, DT in an amount of 3-8 Lf/mL, gdPT in an amount of 16-24 pg/mL, FHA in an amount of 5-15 pg/mL, PRN in an amount of 5-15 pg/mL, FIM2,3 in an amount from about 10-20 pg/mL, AIOOH in an amount of 0.25-0.75 mg/mL, and a TLR9 agonist, such as CpG1018, in an amount of 250-750 pg/ml.
• the aP booster vaccine comprises TT in an amount of 9-11 Lf/mL, DT in an amount of 4-6 Lf/mL, gdPT in an amount of 18-22 pg/mL, FHA in an amount of 8-12 pg/mL, PRN in an amount of 8-12 pg/mL, FIM2,3 in an amount of 14-16 pg/mL, AIOOH in an amount of 0.6-0.7 mg/mL, and a TLR4 agonist, such as E6020, in an amount of 0.5-5 pg/ml.
• the aP booster vaccine comprises TT in an amount of 9-11 Lf/mL, DT in an amount of 4-6 Lf/mL, gdPT in an amount of 18-22 pg/mL, FHA in an amount of 8-12 mg/mL, PRN in an amount of 8-12 pg/mL.
• FIM2,3 in an amount of 14-16 pg/mL.
• AIOOH in an amount of 0.6-0.7 mg/mL
• a TLR9 agonist such as CpG1018, in an amount of 400-600 pg/ml.
• the aP booster vaccine comprises the following components at the indicated concentrations, as set forth in Table 1 :
• the diphtheria toxoid is present in an amount of 4 Lf/mL. In certain embodiments of the aP booster vaccine set forth in Table 1, the diphtheria toxoid is present in an amount of 5 Lf/mL.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: TT in an amount of 4-6 Lf, DT in an amount of 1.5-4 Lf, gdPT in an amount of 8-12 pg, FHA in an amount of 2.5-7.5 pg, PRN in an amount of 2.5-7.5 pg, FIM2,3 in an amount from about 5-10 pg, AIOOH in an amount of 0.125-0.375 mg, and a TLR4 agonist, such as E6020, in an amount of no more than 5 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: TT in an amount of 4-6 Lf, DT in an amount of 1.5-4 Lf, gdPT in an amount of 8-12 pg, FHA in an amount of 2.5-7.5 pg, PRN in an amount of 2.5-7.5 pg, FIM2,3 in an amount from about 5-10 pg, AIOOH in an amount of 0.125-0.375 mg, and a TLR9 agonist, such as CpG1018, in an amount of 125-375 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: TT in an amount of 4.5-5.5 Lf, DT in an amount of 1.5-3 Lf, gdPT in an amount of 9-11 pg, FHA in an amount of 4-6 pg, PRN in an amount of 4-6 pg. FIM2,3 in an amount from about 7-8 pg, AIOOH in an amount of 0.3-0.35 mg, and a TLR4 agonist, such as E6020, in an amount of 0.25-2.5 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose: TT in an amount of 4.5-5.5 Lf, DT in an amount of 1.5-3 Lf, gdPT in an amount of 9-11 pg, FHA in an amount of 4-6 pg, PRN in an amount of 4-6 pg, FIM2,3 in an amount from about 7-8 pg, AIOOH in an amount of 0.3-0.35 mg, and a TLR9 agonist, such as CpG1018, in an amount of 200-300 pg.
• the aP booster vaccine is in a unit dose form for administration to a human subject and comprises the following components per 0.5 mL dose, as set forth in Table 2:
• the diphtheria toxoid is present in an amount of 2 Lf. In certain embodiments of the aP booster vaccine set forth in Table 1, the diphtheria toxoid is present in an amount of 2.5 Lf.
• the aP booster vaccine is formulated to contain antigens other than tetanus toxoid, diphtheria toxoid, or Bordetella antigens.
• the aP booster vaccine comprises one or more of the following: Haemophilus influenzae type-b oligosaccharide or polysaccharide (Hib) conjugate, a hepatitis B virus surface antigen (HBsAg) and/or an inactivated polio virus (IPV).
• the aP booster vaccine described herein may be formulated as an injectable, liquid solution or emulsion.
• the tetanus toxoid, diphtheria toxoid, and Bordetella antigens may be mixed with pharmaceutically acceptable excipients which are compatible with the antigens.
• excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof.
• the aP booster vaccine may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
• the aP booster vaccine will be formulated in aqueous form.
• the components of the aP booster vaccine will be diluted with Tris-buffered saline to give the desired final concentrations.
• the diluent for formulation can be water for injection.
• the aP booster vaccines described herein are suitable for administration to a human subject.
• one aspect is directed to a method of inducing an immune response in a human subject, the method comprising administering to the human subject an aP booster vaccine as described herein.
• the aP booster vaccine for use in inducing an immune response in a human subject and/or for reorienting a Th2-biased immune response towards a Th-1 biased immune response or a Thl/Thl7-biased immune response in a human subject.
• the human subject has received either a wP vaccine, no pertussis vaccine, or an aP priming vaccine prior to administering the aP booster vaccine, which aP priming vaccine induces a Th2-biased immune response.
• a human subject who has received an aP priming vaccine receives an aP booster vaccine that does not contain a TLR agonist (e.g., AD AC EL ® )
• the non-TLR agonist containing aP booster vaccine boosts the Th2-biased immune response induced by the aP priming vaccine, maintaining the Th2 bias induced by aP priming vaccine.
• the modified, TLR agonist containing aP booster vaccine described herein unexpectedly reorients or shifts the Th2-biased immune response induced by the aP priming vaccine towards a Thl -biased immune response or Thl/Thl7-biased immune response.
• the Thl -biased immune response is characterized by one or more of decreased IL-5 production, increased IFN-g production, or a lower IgGl/IgG2a ratio, as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• the Thl-biased immune response is characterized by decreased IL-5 production and/or a lower IgGl/IgG2a ratio, as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• the Thl/Thl7 biased immune response is characterized by increased IL-17 production and one or more of decreased IL-5 production and/or a lower IgGl/IgG2a ratio as compared to the immune response induced by the aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL ® ).
• a TLR agonist e.g., ADACEL ®
• the aP priming vaccine may be administered as a single dose or a series of multiple doses (e.g., 2, 3, 4, or 5 doses) prior to administering the aP booster vaccine.
• the aP priming vaccine is administered as a series of doses, especially for children.
• the aP priming vaccine is administered as a series of five doses in infants and children between 6 weeks and 6 years of age.
• the aP priming vaccine can also be administered as a series of four doses in infants and children between 6 weeks and 4 years of age.
• the primary immunization schedule for a child includes administering an aP priming vaccine at 2 months, 4 months, 6 months, 15-20 months, and 4-6 years of age.
• the aP booster vaccine is typically administered after the primary immunization schedule is complete. In certain embodiments, the aP booster vaccine is administered to a human subject who is 10 years of age or older. In certain embodiments, the aP booster vaccine is administered to a human subject who is 4 years of age or older.
• the aP vaccine booster is administered by intramuscular injection.
• the aP priming vaccine comprises a tetanus toxoid, a diphtheria toxoid, a detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and optionally FIM2,3 with the proviso that the aP priming vaccine does not contain a TLR agonist.
• the aP priming vaccine includes one or more doses of DAPTACEL ® , PENTACEL ® , QUADRACEL ® , INFANRIX ® , INFANRIX-HEXA ® , KINRIX ® , PEDIARIX ® , or VAXELIS ® .
• the aP priming vaccine comprises DAPTACEL ® .
• the aP priming vaccine comprises PENTACEL ® or QUADRACEL ® .
• the aP priming vaccine comprises INFANRIX ® or INFANRIX-HEXA ® .
• the aP priming vaccine comprises KINRIX ® or PEDIARIX ® .
• the aP priming vaccine comprises VAXELIS ® .
• Bordetella pertussis 18323 were grown on Bordet-Gengou agar (Difco) supplemented with 1% glycerol, 20% defibrinated sheep blood (Sanofi Pasteur, Alba La Romaine). After 24h at 36°C, colonies were transferred into 1% Casamino Acid (Difco) buffer and the optical density of the bacterial suspension was measured. 5 x 10 6 colony-forming units (CFU) were instilled intranasally in a volume of 30pl into mice anesthetized by intramuscular injection of Imalgen (ketamine 60mg/kg; Merial SAS) and Rompun (Xylaxine 4mg/kg; Bayer).
• Imalgen ketamine 60mg/kg
• Merial SAS Merial SAS
• Rompun Xylaxine 4mg/kg; Bayer
• mice were then euthanized by intraperitoneal injection of Dolethal (pentobarbital 180 mg/kg; Vetoquinol SA) 2 hours after infection for quantification of the initial numbers of viable B. pertussis CFUs in the lungs and at either days 3, 7, 14 and 21 or days 1, 2, 3, 7 and 14 for determination of bacterial colonization. Briefly, lungs homogenates were plated onto Bordet- Gengou agar plates and the number of CFUs was counted after 4 days of incubation at 36°C. The measure of protective efficacy was expressed as a ratio of the area under the clearance curve (AUC) between naive control and immunized mice.
• AUC area under the clearance curve
• the modified Tdap (gdPT + CpG1018-AlOOH) booster and the modified Tdap (gdPT + E6020-A100H) booster contained 10 Lf/ml TT, 4 Lf/ml DT, 20 pg/ml gdPT, 10 pg/ml PRN, 15 pg/ml FIM2/3, 10 pg/ml FHA and 0.66 mg/ml aluminum (AIOOH) with either 500 pg/ml CpG1018 (TLR9 agonist) or 10 pg/ml E6020 (TLR4 agonist).
• mice were primed intra-muscularly with l/5th of a human dose (50m1 in both hind legs) of the pediatric diphtheria-tetanus-acellular pertussis (aP) DAPTACEL ® vaccine (Sanofi Pasteur), containing 10pg chemically detoxified PT, 5pg FHA, 3pg PRN and 5pg FIM2,3 in addition to tetanus and diphtheria toxoids (referred to in the examples and figures as DTaP for short), or of the diphtheria-tetanus-whole bacterial-cell pertussis (wP) D.T.COQ/D.T.P vaccine (Sanofi Pasteur) containing >4 I.U.
• B. pertussis inactivated by heat in presence of thiomersal (referred to in the examples and figures as DTwP for short).
• Recipient mice were boosted intra-muscularly with l/5th of a human dose (50m1 in both hind legs) of the following formulations: pediatric diphtheria-tetanus-aP AD AC EL ® vaccine (Sanofi Pasteur) containing 2.5ug chemically detoxified PT, 5ug FHA, 3pg PRN and 5pg FIM2,3 in addition to tetanus and diphtheria toxoids (referred to in the examples and figures as Tdap for short); D.T.COQ/D.T.P vaccine (i.e., DTwP); or the modified Tdap booster formulations described above (referred to in the examples and figures as modified Tdap or mTdap for short).
• Splenic IFN-g, IL-5 or IL-17 cytokine secreting cells were detected using a FluoroSpot assay (ELISPOT employing fluorophore-labeled detection reagents). Briefly, the membrane of 96-well IPFL-bottomed microplates (Millipore) was pre- wetted for 30 seconds with 50 pL of 35% ethanol. Ethanol was then removed and each well was washed 3 times with sterile PBS.
• ELISPOT FluoroSpot assay
• Microplates were then coated by adding 100 pL/well of a rat anti-mouse IFN-g, a rat anti-mouse IL-5 or a rat anti-mouse IL-17 antibody solution at 10 pg/mL (PharMigen), and were incubated overnight at +4°C. On the following day, plates were washed 3 times with sterile PBS and then blocked for 2 h at +37°C with 200 pL of RPMI GSP 10% FBS.
• splenocytes/well were incubated with the pertussis antigens (PT 2.5 pg/ml; PRN 5pg/ml; FIM 5pg/ml; FHA 5 pg/mL) or concanavalin A (Con A, 2.5 pg/mL) as a positive control, in presence of murine IL-2 (10 U/mL). After incubation for 24 hours for IFNy and IL-17, and 48 hours for IL-5, the plates were washed 6 times with PBS supplemented with 0.05% BSA (200 pL/well).
• the plates were stored at +5°C ⁇ 3°C in the dark until reading. Each spot, corresponding to an IFN-g, IL-5 or IL-17 secreting cell was enumerated with an automatic ELISPOT fluorescent plate reader (Microvision). Results were expressed as number of IFN- g, IL-5 or IL-17 secreting cells per 106 splenocytes. The geometric mean and standard deviation were calculated for each group.
• Serum IgGl and IgG2a antibodies specific to pertussis antigens (FIM, PT, FHA, PRN), diphtheria toxoid and tetanus toxoid were titrated in a multiplex U-PLEX assay (Meso-Scale Diagnostics, Rockville, MD).
• the U-PLEX assay consists of 5 unique U-PLEX linkers that specifically bind to 5 individual spots on a 96-well U-PLEX plate.
• the biotin-based capture coupling mechanism involves a two-step process: (1) a linker is bound to a biotinylated antigen and (2) the linker- coupled antigen is bound to the plate.
• the serial dilution of serum sample, control and reference serum is added, a wash step performed, and the IgGl or IgG2a antibodies bind to coated antigen were detected using a SULFO-TAGTM labeled ant-IgGl or SULFO-TAGTM labeled ant-IgG2a.
• mice were immunized with a single 0.5 pg dose of PTxd, followed by two gdPT immunizations at 0.5 pg per dose, each 3 weeks apart (at days 0, 21, and 42). All formulations contained 0.066 mg AlPCfi in 100 pL injection volume. Blood samples were collected 17 days after the second immunization (Day 38) and 8 days after the third immunization (Day 50) for analysis of IgGl and IgG2a titers by a pertussis toxin specific ELISA and PT neutralization antibody titers.
• Vaccines containing gdPT at 2.0, 0.1 or 0.02 pg/dose were formulated in combination with the other Tdap vaccine antigens (1 Lf tetanus toxoid, 0.4 Lf diphtheria toxoid, 1 pg FHA, 1 pg PRN, 1.5 pg FIM2,3 and 66 pg aluminum (AIOOH) per mouse dose in 100 pL, or 1/5 Human Dose).
• a control Tdap vaccine containing 2 pg of chemically-detoxified PT was formulated with the same Tdap antigen concentrations, as a comparator.
• mice that received gdPT alone at 0.1 or 0.02 pg per dose in AIOOH.
• CD1 mice received three immunizations of vaccine formulation, three weeks apart and were sacrificed 7 days after the last immunization. Blood samples were collected 10 days after the second immunization ( Figure 3) and at the sacrifice for analysis of PT-specific IgGl and IgG2a titers and the PT neutralization antibody response ( Figures 4A-B).
• a modified Tdap vaccine containing gdPT (at 2 pg per dose and 3 immunizations per mouse) elicited higher PT neutralizing antibody titers than an otherwise identical control Tdap vaccine containing 2 pg per dose PTxd ( Figure 4B), although the gdPT-containing and PTxd-containing Tdap vaccines had comparable PT-specific IgGl and IgG2a titers ( Figure 4A). Moreover, the IgGl and IgG2a antibody titers against FIM were comparable between the gdPT-containing Tdap formulation and the PTxd-containing Tdap formulation ( Figure 3).
• gdPT was confirmed to be more immunogenic than PTdx in the Tdap formulation. Also, gdPT does not interfere with the immunogenicity of other Tdap antigens in the vaccine formulation.
• Example 2 TLR Adjuvants Re-orient a DTaP-Induced Th2 Immune Memory Response Towards Thl Using a Long Prime-Boost Schedule
• mice were boosted 6 weeks later (Day 42) with the modified Tdap (gdPT + E6020-A100H) formulation or the modified Tdap (gdPT + CpG1018-AlOOH) formulations.
• mice received two doses of the modified Tdap booster vaccine, the first dose at 6 weeks (Day 42) and the second dose at 12 weeks (Day 84).
• the potential of the modified Tdap boosting formulations to re-orient the immune memory response towards Thl was evaluated by measuring ex vivo splenic cytokine producing cells or cytokine production in supernatant after in vitro antigen re-stimulation.
• Antibody titers (IgGl and IgG2a) to tetanus toxoid (TT), diphtheria toxoid (DT), PT, FHA, PRN and FIM were also determined in sera collected 1, 3 and 6 weeks after the last immunization.
• mice As controls, one group of mice was immunized with DTwP vaccine (prime) followed by DTwP vaccine (boost) and another group with DTaP vaccine (prime) followed by a boost with a control modified Tdap vaccine formulation having gdPT-AlOOH (20 pg/ml gdPT) but without a TLR agonist, i.e., control modified Tdap (gdPT-AlOOH).
• the DTwP/DTwP (prime/boost) schedule induced a less Th2 -biased immune profile compared to DTaP/modified control Tdap (gdPT-AlOOH) (prime/boost) schedule, as evidenced by weaker IL-5 secretion (Figure 5) and a lower IgGl/IgG2a ratio ( Figures 6A-L).
• the modified Tdap (gdPT + E6020-A100H) or modified Tdap (gdPT + CpGIOl-AlOOH) boost formulations induced a significantly decreased IL-5 production as compared to the control modified Tdap (gdPT-AlOOH) boost vaccine (Figure 5).
• modified Tdap gdPT + E6020-A100H
• modified Tdap gdPT + CpG1018-AlOOH
• Example 3 Adoptive transfer model of pertussis immunity
• the modified Tdap (gdPT + E6020-A100H) formulation and the modified Tdap (gdPT + CpG1018-A100H) formulation were tested for their ability to reactivate a DTaP- induced immune memory response for Bordetella pertussis protection in the absence of circulating antibodies induced by DTaP vaccination.
• Recipient mice were boosted by DTwP (only on DTwP-priming background), Tdap, the modified Tdap (gdPT + E6020-A100H) formulation or the modified Tdap (gdPT + CpG1018-AlOOH) formulation.
• Tdap modified Tdap
• gdPT + E6020-A100H modified Tdap
• gdPT + CpG1018-AlOOH modified Tdap
• mice primed with DTaP and boosted with modified Tdap (gdPT + CpG1018-AlOOH) and modified Tdap (gdPT + E6020-A100H) boost showed accelerated and higher anti-PT, FHA, FIM IgG titers as compared to mice receiving a Tdap boost ( Figure 8B).
• Replacing chemically-detoxified PT by gdPT in the Tdap boost vaccine had no impact on B. pertussis lung clearance despite an enhanced PT-specific IgG antibody response (data not shown).
• mice demonstrated that the modified Tdap (gdPT + E6020-A100H) and modified Tdap (gdPT + CpG1018-A100H) booster formulations are able to improve the pertussis-specific recall antibody responses resulting in accelerated protection against bacteria colonization.
• mice Adult BALB/cByJ mice were purchased from Charles River (L’Arbresle, France) and kept under specific pathogen free conditions. Mice were used at 6-8 weeks of age. All animal experiments were carried out in accordance with Swiss and European guidelines and approved by the Geneva Veterinary Office.
• Adoptive transfer Spleens were harvested 42 days after prime or boost. Single cell suspensions were obtained by mechanical disruption of the organs and further processed for red blood cell lysis. 50xl0 6 splenocytes were transferred intravenously in a volume of IOOmI into naive recipient mice.
• B. pertussis challenge Streptomycin-resistant Bordetella pertussis Tahoma I derivative BPSM were grown on Bordet-Gengou agar (Difco) supplemented with 1% glycerol, 10% defibrinated sheep blood (Chemie Brunschwig AG) and 100 pg/ml streptomycin.
• CFU colony -forming units
• lungs homogenates were plated onto Bordet-Gengou agar plates and the number of CFUs was counted after 4 days of incubation at 37°C.
• the measure of protective efficacy was expressed as a ratio of the area under the clearance curve (AUC) between naive control and immunized mice.
• ELISA PT-, PRN-, FHA- and FIM2,3-specific antibody titers were determined by Ag-capture ELISAs in serum samples collected at indicated time-points. Briefly, 96-well plates (Nunc MaxiSorpTM; Thermo Fisher Scientific) were coated with PT (lpg/ml), PRN (5pg/ml), FHA (5pg/ml) or FIM2,3 (2pg/ml) in carbonate buffer, pH 9.6, overnight at 4°C.
• HRP horseradish peroxidase conjugated anti-mouse IgG, anti-mouse IgG2a (both from Invitrogen), and anti-IgGl (BD Pharmingen) were incubated for lh at 37°C and revealed with 2,2'-Azinobis [3-ethylbenzthiazoline-6-sulfonic acid] -diammonium salt (ABTS) substrate for lh.
• results in Figure 8 for IgG and IgGl are expressed as Logio of Ag-specific titers determined in reference to serial dilutions of a WHO/NIBSC reference reagent Bordetella pertussis anti-serum (NIBSC code : 97/642) and IgG2a in reference to serial dilutions of a titrated pool of hyperimmune sera from immunized mice.
• a mouse lung clearance model that reflects the clinical efficacy levels of licensed pertussis vaccines has been developed, implemented, and validated (Guiso N. et al, Vaccine. 1999;17:2366-76).
• the mouse Intranasal Challenge Assay (INCA) model is recommended by the WHO for non-clinical testing for registration of new vaccine candidates and known as a valid research model in vaccine research and development (World Health Organization. WHO Expert Committee on Biological Standardization. Recommendations to Assure the Quality Safety and Efficacy of Acellular Pertussis Vaccines. 2011;WHO/BS/2011.2158.)
• the WHO advisory group has agreed that the INCA is useful for assessing potential impacts of changing formulation and/or manufacturing process for pertussis vaccines.
• FIG. 10A shows a pictorial representation of a mouse Intranasal Challenge Assay (INCA) using a short schedule
• Figure 10B shows a pictorial representation of a mouse INCA using a long Schedule.
• INCA Intranasal Challenge Assay
• Figure IOC shows that a DTwP/DTwP (prime/boost) schedule and a DTaP/Tdap (prime/boost) schedule protect mice against Bordetella pertussis lung colonization following intranasal challenge in this model using a short schedule.
• mice were injected with 1/5 human dose of DTaP by intramuscular route and received a second injection two weeks later with Tdap, the modified Tdap (gdPT + E6020-A100H) vaccine, or with a control Tdap vaccine formulated with chemically- detoxified PT, AIOOH, and without a TLR agonist (control Tdap (PTxd-AlOOH)).
• Tdap modified Tdap
• a control Tdap vaccine formulated with chemically- detoxified PT, AIOOH, and without a TLR agonist (control Tdap (PTxd-AlOOH)
• a group that received two injections of DTwP primarye/boost was also included.
• Two control groups received either PBS (infection control), or E6020-A100H adjuvant alone to verify that aluminum salts and E6020 had no effect on colonization compared to the infection control.
• mice Two weeks following the second immunization, mice were challenged by instillation of a suspension of B. pertussis 18323 into the nostrils. Following challenge, eight mice from each group were sacrificed at 2 hours post-challenge, and at 3, 7, 14 and 21 days post challenge. Lungs were removed aseptically and homogenized individually to measure bacterial load. The mean CFU per lung was determined by counting the colonies grown on Bordet-Gengou agar plates after plating serial dilutions of the homogenate. Blood samples were also collected following challenge and WBC counts were performed.
• B. pertussis 18323 colonized and expanded in the lung of PBS control mice three days after the challenge before entering a clearance phase. Complete B. pertussis lung clearance took more than 21 days in the PBS control mice. There was no impact of the adjuvant alone on lung colonization and disease. Reduction in B. pertussis lung colonization was observed in all vaccinated groups 3 days post challenge with baseline bacterial load reached at 14 days post challenge for all formulations. DTwP caused the fastest B. pertussis lung clearance with baseline reached at 7 days post challenge.
• the modified Tdap (gdPT + E6020-A100H) formulation drastically reduced the bacterial load in the lungs when compared to the infection control (4 log at day 3 and 5 log at day 7) demonstrating its capacity to prevent colonization of the lower respiratory tract following intranasal challenge with B. pertussis 18323.
• the clearance profile was closer to that obtained for DTwP/DTwP (prime/boost) and farther from that obtained for DTaP/Tdap (prime/boost).
• the acellular pertussis vaccine control was a Tdap vaccine (control Tdap (PTxd-AlOOH)) containing chemically-detoxified PT but modified to contain the same doses of antigens and aluminum salt (AIOOH) as in the modified Tdap (gdPT + E6020-A100H) formulation to better appreciate the role of gdPT and TLR agonist in the new modified Tdap formulation.
• Groups of 40 mice were injected with 1/5 human dose of DTaP by intramuscular route and received a second injection six weeks later with the modified Tdap (gdPT + E6020- AIOOH) or with the control Tdap (PTxd-AlOOH).
• mice were challenged by instillation of a suspension of B. pertussis 18323 into the nostrils. Following challenge, eight mice from each group were sacrificed at 2 hours post-challenge, and at 1, 2, 3, 7, and 14 days post-challenge. Lungs were removed aseptically and homogenized individually to measure bacterial load. The mean CFU per lung was determined by counting the colonies grown on Bordet-Gengou agar plates after plating serial dilutions of the homogenate. Blood samples were also collected following challenge and WBC counts were performed.
• the modified Tdap (gdPT + E6020-A100H) formulation drastically reduced the bacterial load in the lungs (6 log reduction) as soon as day 1 when compared to the infection control.
• a significant difference (p value 0.007) was also observed between modified Tdap (gdPT + E6020-A100H) and the control Tdap (PTxd- AIOOH) at day 1 with a 7.7 fold reduction of CFU counts in lungs.
• the bacterial load for all vaccinated mice returned to base line by day 7, indicating no detectable bacteria in the lungs ( Figure 12B).
• IL-17 cytokines were measured in the following experiments.
• CD1 mice were primed with DTaP composition and boosted twice, 2 weeks apart on DO and D21 with mTdap-E6020-ALOOH with or without the TLR4 adjuvant in a dose-effect design.
• E6020 and AIOOH were present in the mTdap formulation in an amount of 10 pg and 66 pg, respectively.
• the mTdap antigen components were as follows per dose of 0.1 mL:
• Thl7 cytokines The production of Thl7 cytokines (IL-17) was measured in the supernatant of splenocytes after 3 days of in vitro stimulation with 2.5 pg/mL of PTx ( Figure 13A) or a pool of pertussis antigens (2.5 pg/mL PTx, 5 pg/mL PRN, 5 pg/mL FIM) using the fluorospot assay ( Figure 13B) and the MSD Uplex Kit.
• IL-17 secreting cell frequency was below the positive cutoff in the fluorospot assay after mTdap vaccine injection without TLR4 (below the line of 19 spots/10 6 cells).
• the mTdap-E6020 formulations induced increasing amounts of IL-17 secreting cells in a dose dependent manner from 0.1 pg to 4 pg/dose, when the splenocytes were re-stimulated with either PT toxoid (PTx) ( Figure 13 A) or a pool of pertussis antigens (Figure 13B).
• PTx PT toxoid
• Figure 13B a pool of pertussis antigens
• a mTdap boost vaccine containing 4 pg E6020/dose increased IL-17 secreting cell frequency above the positive cutoff in the fluorospot assay when re-stimulated with PTx ( Figure 13A)
• mTdap boost vaccines containing 0.5, 1, 4, and 10 pg E6020/dose increased IL-17 secreting cell frequency above the positive cutoff in the fluorospot assay when re-stimulated with a pool of pertussis antigens Figure 13B.
• cytokine secretion profiles measured by MSD were in agreement with cytokine secreting cell frequency measured by fluorospot (data not shown).
• the nanoDSF method was performed using the Prometheus NT.48 system (Nano Temper Technologies, Kunststoff, Germany). NanoDSF uses intrinsic fluorescence to evaluate changes in aromatic residues (fluorophores) within proteins in response to the changes in their local environment. The shift and intensity change in fluorescence emission is monitored, with a change in the intrinsic fluorescence indicating that the protein has unfolded. Thermal stability of protein is characterized using the melting temperature (Tm), which indicates the point at which half the protein is unfolded. In the nanoDSF method, this is determined by using the ratio of fluorescence recorded at 330 nm and 350 nm. This ratio has shown to be more sensitive in detecting Tm as compared to the use of a single wavelength. Samples were filled in capillary tubes without any further preparation and excited at 285 nm with 100% power output. The thermal profiles were recorded from 20 to 95 °C with 2 °C/min scan rate.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Mycology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Microbiology (AREA)
• Immunology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Virology (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=65628487

Family Applications (1)

Country Status (6)

Cited By (1)

Families Citing this family (1)

Citations (3)

Family Cites Families (2)

• 2019
  • 2019-11-29 JP JP2021531950A patent/JP7605739B2/ja active Active
  • 2019-11-29 EP EP19893265.9A patent/EP3890770A4/en active Pending
  • 2019-11-29 WO PCT/US2019/063840 patent/WO2020117618A1/en not_active Ceased
  • 2019-11-29 CN CN201980090686.9A patent/CN113395977A/zh active Pending
  • 2019-11-29 US US17/299,244 patent/US20220054615A1/en not_active Abandoned
  • 2019-12-04 TW TW108144399A patent/TWI877126B/zh active
• 2024
  • 2024-08-02 JP JP2024126656A patent/JP2024156862A/ja active Pending

Patent Citations (3)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events