Whooping cough booster vaccine

Abstract

The present disclosure is directed to modified acellular pertussis booster vaccines that include TLR agonists and methods of using same to induce immune responses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and relies on the filing date of European Patent Application No. 18306621.6, filed December 5, 2018, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on November 27, 2019, is named 0171_0016-PCT_SL and is 1 kilobyte in size.

[0003] Field The present disclosure relates to acellular pertussis vaccines and methods of using same. [Background technology]

[0004] Pertussis or whooping cough is an acute, highly infectious respiratory disease caused primarily by Bordetella pertussis. Before immunization became widely practiced, pertussis was highly endemic. Evidence suggests that almost all children are infected with pertussis before they become adults, most of whom suffer from some degree of clinical disease, and that vigorous circulation of the pathogen provides a natural boosting of infection-acquired immunity, which is estimated to last from 7 to 10 to 20 years (Non-Patent Document 1).

[0005] Vaccination has been the most effective strategy to reduce the number of whooping cough cases (Non-Patent Document 2). Early pertussis vaccines contained killed pertussis whole cells (wP) that were chemically detoxified and formulated with diphtheria and tetanus antigens. Since the 1990s, the wP vaccine has been replaced by acellular pertussis vaccines in many countries. Acellular pertussis (aP) vaccines cause relatively few side effects compared to the wP vaccine, which is associated with a high risk of fever, injection site reactogenicity, and (to a lesser extent) convulsions. Current acellular vaccines are typically based on the following virulence factors: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial agglutinogen 2 and fimbrial agglutinogen 3 (FIM2 / 3 or FIM). Although some acellular vaccines contain only PT and FHA or PT alone, acellular pertussis vaccines containing the PT, FHA, PRN, and FIM2 / 3 components are generally considered to be the most effective aP vaccines currently available.

[0006] Despite decades of vaccination, pertussis remains endemic worldwide, with localized epidemic peaks or outbreaks occurring every 2 to 5 years (typically every 3 or 4 years) without a consistent seasonal pattern (Non-Patent Document 3; Non-Patent Document 4). Despite the large variation in incidence between reported countries, the highest age-specific incidence, hospitalization, and complications of pertussis cases are still reported in infants under 1 year of age, primarily under 3 months of age, in each country. Infants too young to have completed the primary vaccine series account for the majority of pertussis-related complications, hospitalizations, and deaths (Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9). In recent epidemiological findings, adolescents and adults tend to show the second highest disease incidence and the highest incidence increase in countries with well-established vaccine schedules, such as the United States and the United Kingdom.

[0007] Accumulating evidence suggests that the observed resurgences and outbreaks probably result from the combined effects of multiple factors, such as awareness (Non-Patent Document 10), increasing sensitivity of laboratory confirmation methods (Non-Patent Document 11), incomplete schedules or suboptimal vaccine coverage (Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15), weakening and changes in the nature of vaccine-induced immunity accompanied by genotypic and phenotypic changes in the organism (Non-Patent Document 16; Non-Patent Document 17; Non-Patent Document 18).

[0008] To reduce the incidence of pertussis in children, aP booster vaccination at approximately 4-6 years of age has been implemented in many countries (Non-Patent Document 19; Non-Patent Document 20). The efficacy of a booster dose of acellular pertussis-containing vaccine (Tdap) has been evaluated in several settings. A randomized clinical trial conducted in the United States from 1997 to 1999 evaluated the efficacy of Tdap vaccine in adolescents and adults aged 15 to 65 years. The study found that Tdap vaccination was associated with a reduced incidence of clinical disease (i.e., coughing for more than 21 days) and increased anti-pertussis antibody levels over a 1-year observation period, for a vaccine efficacy estimate of 92% against laboratory-confirmed pertussis (Non-Patent Document 21). The efficacy of Tdap vaccination in suppressing pertussis has been shown in several observational studies in non-outbreak settings in the United States and Australia, which have demonstrated a significant impact of adolescent Tdap vaccination on disease incidence, with one study estimating efficacy of 85.4% against laboratory-confirmed pertussis (Non-Patent Document 22; Non-Patent Document 23; Non-Patent Document 24). However, several recent cases (suppression studies conducted in outbreak settings in the United States in groups primed primarily with acellular vaccine) have consistently estimated the efficacy of Tdap vaccination to be moderate, around 65% or less against laboratory-confirmed pertussis (Non-Patent Document 25; Non-Patent Document 26; Non-Patent Document 27; Non-Patent Document 28; Non-Patent Document 29). Despite the inherent methodological limitations present in most of these observational studies, the evidence of rapid waning of efficacy of all brands after the Tdap booster dose observed in the Wisconsin study is consistent with observations from a study conducted in an outbreak setting in Washington state, which estimated that the efficacy of Tdap vaccination waned rapidly from 75% in the first year after the booster dose to 42% after the second year (30; 31).Recent epidemiological trends and distribution of pertussis cases in the United States and Canada have been interpreted as supporting the idea that individuals primed with aP vaccine respond less robustly to pertussis booster vaccines and that their protection fades more rapidly than the previous group primed with wP vaccine (Non-Patent Document 32; Non-Patent Document 33). Furthermore, several studies analyzing data obtained in the 2010 and 2012 US outbreaks and the 2008-2012 Australian outbreaks suggest that the protection induced by the booster vaccine fades more quickly in individuals primed with aP vaccine than in those primed with wP vaccine (Non-Patent Document 34; Non-Patent Document 35; Non-Patent Document 36; Non-Patent Document 37; Non-Patent Document 38; Non-Patent Document 39; Non-Patent Document 40; Non-Patent Document 41). Although the results of these studies were challenged by some experts on methodological grounds, their conclusions were supported by economic observations reported by the CDC in the United States, which showed that waning of protection occurred earlier in the aP-primed group compared with previous observations in the wP-primed group (Non-Patent Document 42). In fact, comparison of immunogenicity results obtained in clinical studies on adolescents primed with aP or wP vaccines provides consistent evidence of lower humoral or cellular (B and Th1) immune responses one month after Tdap vaccine administration (Non-Patent Document 43; Non-Patent Document 44; Non-Patent Document 45; Non-Patent Document 46).

[0009] Modeling studies based on Swedish pertussis surveillance data have suggested a herd effect in pertussis epidemiology in the aP vaccine setting, while several other studies using surveillance data from the United States, Italy, and other countries have found that models that best fit the observed epidemiological trends tend to hypothesize that aP vaccines induce lower booster responses, earlier waning protection, and higher asymptomatic infections compared to wP vaccines (Non-Patent Documents 47; 48; 49).

[0010] Based on a study in non-human primates by Warfel et al., it has been suggested that the mechanisms of protection are probably different between the wP and aP vaccines (Non-Patent Document 50). This study found that the aP vaccine prevented clinical disease but not overt colonization to the same extent as the wP vaccine, allowing transmission to susceptible animals, although not in humans and in a non-statistically significant sample (Id.). A similar conclusion was reached from a study using a mouse model of whooping cough (Non-Patent Document 51). Studies performed in non-human primates have notably pointed to a different immune profile following wP vaccination compared to aP priming. In particular, a Th1 or mixed Th1 / Th17 response profile was associated with wP vaccine priming, whereas a Th2 profile was associated with aP vaccine priming. As a result, it was hypothesized that the Th1 / Th17 bias of immune responses observed after wP vaccination may be associated with a longer duration of protection and more rapid clearance of infection (i.e., stronger initial protection) than the Th2 bias observed after aP vaccination. Furthermore, it was observed that the initial Th1 / Th17 vs. Th2 responses induced by primary vaccination with wP and aP, respectively, were maintained after aP booster vaccination even several years after the initial primary vaccination, suggesting that priming vaccination affects the Th1 / Th17 vs. Th2 bias after aP booster vaccination (Non-Patent Document 52). The important role of Th1 / Th17 effector cells in immunity to Bordetella pertussis was confirmed in a mouse model of infection, further strengthening this hypothesis (Non-Patent Document 53). Although evidence in humans supporting this hypothesis has yet to be developed, several studies have suggested differences in T helper cell responses in humans vaccinated with wP or aP vaccines (Non-Patent Document 54). Furthermore, the Th2-dominant response to aP vaccine also appears to correspond to a distinct IgG isotype distribution: responses to aP vaccination yield primarily IgG1, but also IgG2 and IgG4, with increased IgG4 protection following booster vaccination.In contrast, the Th1-dominated response of the wP vaccine is primarily accompanied by IgG1 and IgG2 antibody responses (55; 56; 57).

[0011] It would be advantageous to provide an improved booster aP vaccine with longer lasting protection against Bordetella pertussis infection. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Wendelboe et al., Pediatr Infect Dis J, 2005;24:S58-S61 [Non-Patent Document 2] Halperin, N Engl J Med. 2005;353:1615~7 [Non-Patent Document 3] Edwards et al., Whooping Cough Vaccine. In Vaccines 6th ed. Plotkin S, Orenstein W, Offit P. eds. 6th ed. Philadelphia, Elsevier, 2012:447-92 [Non-Patent Document 4] JD Cherry, Pediatrics 2005;115:1422~27 [Non-Patent Document 5] Bisgard et al., Pediatr Infect Dis J. 2004;23:985~89 [Non-Patent Document 6] Haberling et al. Pediatr Infect Dis J. 2009;28(3):194~98 [Non-Patent Document 7] Public Health England, Health Protection Report. 2014;8(17) [Non-Patent Document 8] Centers for Disease Control and Prevention, MMWR 2012;61(28):517~22 [Non-Patent Document 9] Winter et al., J Pediatr. 2012;161:1091-96 [Non-Patent Document 10] Kaczmarek et al. MJA 2013;198:624~8 [Non-Patent Document 11] Tarr et al., Am J Epidemiol. 2013;178(2):309-18 [Non-Patent Document 12] Atwell et al. Pediatrics 2013;132:624~30 [Non-Patent Document 13] Quinn et al., Pediatrics 2014;133(3):e513~9 [Non-Patent Document 14] Glanz et al., JAMA Pediatr. 2013;167(11);1060-64 [Non-Patent Document 15] Imdad et al. Pediatrics 2013;132:37~43 [Non-Patent Document 16] Lam et al., Australia Emerg Infect Dis. 2014;20:626-33 [Non-Patent Document 17] Pawloski et al. Clin Vaccine Immunol 2014;21(2):119~25 [Non-Patent Document 18] Martin et al., Clin Infect Dis. 2014;60(2):223-27) [Non-Patent Document 19] Zepp et al., Lancet Infect Dis, 2011;11(7):557-70 [Non-Patent Document 20] Clark et al., NASN Sch Nurse, 2012;27(6):297-300 [Non-Patent Document 21] Ward et al., Clin Infect Dis 2006;43:151-57 [Non-Patent Document 22] Skoff et al. Arch Pediatr Adolesc Med. 2012;166(4):344~49 [Non-Patent Document 23] Rank et al., Pediatr Infect Dis J. 2009;28(2):152~53 [Non-Patent Document 24] Quinn et al. Bull World Health Organ 2011;89:666~674 [Non-Patent Document 25] Acosta et al., Washington State, 2012. IDWeek 2013 Meeting of the Infectious Diseases Society of America. Poster 139 [Non-Patent Document 26] Wei et al. Clinical Infectious Diseases 2010;51(3):315~21 [Non-Patent Document 27] Baxter et al., BMJ. 2013;347:f4249 [Non-Patent Document 28] Liko et al. N Engl J Med. 2013 Feb 7;368(6):581~82 [Non-Patent Document 29] Klein et al., Pediatrics. 2016;137(3):e20153326 [Non-Patent Document 30] Koepke et al., J Infect Dis. 2014;210(6);942-53 [Non-Patent Document 31] Acosta et al. IDWeek 2013 Meeting of the Infectious Diseases Society of America. Poster 139 [Non-Patent Document 32] A. Acosta, Advisory Committee on Immunization Practices. Summary Report June 19~20, 2013 [Non-Patent Document 33] Chambers et al. CCDR. 2014;40(3):31~41 [Non-Patent Document 34] Liko et al. N Engl J Med. 2013 Feb 7;368(6):581~82 [Non-Patent Document 35] Smallridge et al., Infect Dis. 2014;209(12):1981~88 [Non-Patent Document 36] Witt et al., Clin Infect Dis. 2012;54(12):1730~35 [Non-Patent Document 37] Klein et al. N Engl J Med. 2012;367(11):1012~19 [Non-Patent Document 38] Tartof et al., Pediatrics. 2013;131(4):e1047~52 [Non-Patent Document 39] Sheridan et al., JAMA. 2012;308(5):454~56 [Non-Patent Document 40] Witt et al., Clin Infect Dis. 2013;May;56(9):1248~54 [Non-Patent Document 41] Klein et al., Pediatrics. 2013;131(6):e1716~22 [Non-Patent Document 42] A. Acosta, Advisory Committee on Immunization Practices. Summary Report June 19~20, 2013 [Non-Patent Document 43] Marshall et al. Clinical and Vaccine Immunology. 2014;21(11):1560~64

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[0013] The present inventors have developed a novel modified acellular pertussis (aP) booster vaccine that contains a Toll-like receptor (TLR) agonist. More specifically, the present inventors have surprisingly found that the aP booster vaccine with TLR4 and / or TLR9 agonist can reorient the Th2-biased immune response induced by the previously administered aP vaccine toward a Th1-biased immune response. Without intending to be bound by any theory, the re-biasing of T helper cells and the shift in Th1 / Th2 balance induced by the modified aP booster vaccine appear to be associated with accelerated clearance of Bordetella pertussis. [Means for solving the problem]

[0014] A first aspect of the present disclosure is directed to an aP booster vaccine comprising tetanus toxoid, diphtheria toxoid, 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 the aluminum salt (Aspect 1).

[0015] Another embodiment is directed to a method of inducing an immune response in a human subject previously exposed to a Bordetella pertussis antigen, comprising administering to the subject an aP booster vaccine, the aP booster vaccine comprising tetanus toxoid, diphtheria toxoid, detoxified pertussis toxin, filamentous hemagglutinin, pertactin, fimbria types 2 and 3, at least one TLR agonist, and an aluminum salt, wherein the at least one TLR agonist is formulated with the aluminum salt, and wherein administering the aP booster vaccine redirects a Th2-dominated immune response induced by the previous exposure to a Bordetella pertussis antigen toward a Th1-dominated immune response or a Th1 / Th17-dominated immune response in the human subject (Embodiment 2). The use of an aP booster vaccine to redirect a Th2-dominated immune response towards a Th1-dominated or Th1 / Th17-dominated immune response in a human subject that has previously been exposed to a Bordetella pertussis antigen, typically by an aP priming vaccine, is also covered by embodiment 2.

[0016] Typically, in the context of embodiment 2, the human subject has previously received an acellular pertussis vaccine (also referred to herein as an aP priming vaccine) before administering the aP booster vaccine, in which case the aP priming vaccine induces a Th2-dominant immune response. Alternatively, the human subject may have previously been exposed to Bordetella pertussis antigens by receiving a wP vaccine or by natural infection with Bordetella pertussis. Typically, when a human subject receiving an aP priming vaccine receives an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL®), the aP booster vaccine containing the non-TLR agonist boosts the Th2-dominant immune response induced by the aP priming vaccine. In contrast, the modified aP booster vaccine containing a TLR agonist described herein unexpectedly redirects the Th2-dominated immune response induced by the aP priming vaccine towards a Th1-dominated or Th1 / Th17-dominated immune response.

[0017] In certain embodiments of aspect 2, the Th1-dominated immune response is characterized by one or more of decreased IL-5 protection, increased IFN-γ protection, or a lower IgG1 / IgG2a ratio when compared to an immune response induced by an aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL®). In certain embodiments of aspect 2, the Th1-dominated immune response is characterized by decreased IL-5 protection and / or a lower IgG1 / IgG2a ratio when compared to an immune response induced by an aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL®). In certain embodiments of aspect 2, the Th1 / Th17-dominated response is characterized by increased IL-17 protection and one or more of decreased IL-5 protection or a lower IgG1 / IgG2a ratio. In certain embodiments of aspect 2, the Th1 / Th17 dominated response is characterized by one or more of increased IL-17 protection and decreased IL-5 protection or a lower IgG1 / IgG2a ratio when compared to an immune response induced by an aP priming or booster vaccine that does not contain a TLR agonist (e.g., ADACEL®).

[0018] In certain embodiments of aspects 1 and 2, the TLR agonist is a TLR4 agonist. Preferably, the TLR4 agonist is an agonist of human TLR4. In certain embodiments, the TLR4 agonist is E6020.

[0019] In other embodiments of aspects 1 and 2, the TLR agonist is a TLR9 agonist.Preferably, the TLR9 agonist is an agonist of human TLR9.In certain embodiments, the TLR9 agonist is a CpG oligonucleotide.In certain embodiments, the CpG oligonucleotide is a class A, class B, class C or class P CpG oligonucleotide. In certain embodiments, the CpG oligonucleotide is CpG1018 having the nucleotide sequence of SEQ ID NO:1, where all nucleotides of SEQ ID NO:1 are linked with phosphorothioate linkages.

[0020] In certain embodiments of aspects 1 and 2, the tetanus toxoid is present in an amount of 8-12 Lf / mL, optionally 9-11 Lf / mL, or optionally 10 Lf / mL.

[0021] In certain embodiments of aspects 1 and 2, the diphtheria toxoid is present in an amount of 3-8 Lf / mL, optionally 3-6 Lf / mL, or optionally 4-5 Lf / mL.

[0022] In certain embodiments of aspects 1 and 2, the detoxified pertussis toxin is a genetically detoxified pertussis toxin (gdPT). In certain embodiments, the gdPT comprises a mutation in R9. In certain embodiments, gdPT comprises an R9K mutation and an E129G mutation. In certain embodiments, gdPT is present in an amount of 4-30 μg / mL, optionally 16-24 μg / mL, optionally 18-22 μg / mL, or optionally 20 μg / mL.

[0023] In certain embodiments of aspects 1 and 2, the filamentous hemagglutinin is present in an amount of 5-15 μg / mL, optionally 8-12 μg / mL, or optionally 10 μg / mL.

[0024] In certain embodiments of aspects 1 and 2, pertactin is present in an amount of 5-15 μg / mL, optionally 8-12 μg / mL, or optionally 10 μg / mL.

[0025] In certain embodiments of aspects 1 and 2, fimbriae types 2 and 3 are present in an amount of 10-20 μg / mL, optionally 14-16 μg / mL, or optionally 15 μg / mL.

[0026] In certain embodiments of aspects 1 and 2, the TLR4 agonist, such as E6020, is present in an amount of 10 μg / mL or less, optionally 0.5-5 μg / mL, or optionally 2 μg / mL or less.

[0027] In certain embodiments of aspects 1 and 2, the TLR9 agonist, such as CpG1018, is present in an amount of 250-750 μg / mL, optionally 400-600 μg / mL, or optionally 500 μg / mL.

[0028] In certain embodiments of aspects 1 and 2, the aP booster vaccine comprises tetanus toxoid in an amount of about 8-12 Lf / mL, optionally 9-11 Lf / mL; diphtheria toxoid in an amount of about 3-8 Lf / mL, optionally 3-6 Lf / mL; genetically detoxified pertussis toxin in an amount of about 16-24 μg / mL, optionally 18-22 μg / mL; filamentous hemagglutinin in an amount of about 5-15 μg / mL, optionally 8-12 μg / mL; and a TLR4 agonist, such as E6020, in an amount of 10 μg / mL or less and optionally 0.5-5 μg / mL or optionally 1-2 μg / mL. Alternatively, the aP booster vaccine may comprise, instead of the TLR4 agonist, a TLR9 agonist, such as CpG1018, in an amount of 250-750 μg / mL, optionally 400-600 μg / mL.

[0029] In certain embodiments of aspects 1 and 2, 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 μg / mL; filamentous hemagglutinin in an amount of 10 μg / mL; pertactin in an amount of 10 μg / mL; fimbriae types 2 and 3 in an amount of 15 μg / mL; aluminum hydroxide (AlOOH) in an amount of 0.66 mg / mL; and a TLR4 agonist, such as E6020, in an amount of 2 μg / mL or less. Alternatively, the aP booster vaccine may comprise, instead of the TLR4 agonist, a TLR9 agonist, such as CpG1018, in an amount of 500 μg / mL.

[0030] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage 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.

[0031] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage 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.

[0032] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject, typically per 0.5 mL dose, and comprises genetically detoxified pertussis toxin in an amount of 2-12 μg, optionally 8-12 μg, or optionally 10 μg.

[0033] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and comprises filamentous hemagglutinin in an amount of 2.5-7.5 μg, optionally 4-6 μg, or optionally 5 μg.

[0034] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and comprises pertactin present in an amount of 2.5-7.5 μg, optionally 4-6 μg, or optionally 5 μg.

[0035] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and comprises fimbriae types 2 and 3 in an amount of 5-10 μg, optionally 7-8 μg, or optionally 7.5 μg.

[0036] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and comprises a TLR4 agonist, such as E6020, in an amount of 5 μg or less, optionally 0.25-2.5 μg, or optionally 1 μg or less.

[0037] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and comprises a TLR9 agonist, such as CpG1018, in an amount of 125-375 μg, optionally 200-300 μg, or optionally 250 μg.

[0038] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject, comprising the following components per 0.5 mL dose: tetanus toxoid in an amount of about 4-6 Lf, optionally 4.5-5.5 Lf; diphtheria toxoid in an amount of about 1-4 Lf, optionally 1.5-3 Lf; genetically detoxified pertussis toxin in an amount of about 2-12 μg, optionally 8-12 μg; filamentous hemagglutinin in an amount of about 2.5-7.5 μg, optionally 4-6 μg; pertactin in an amount of about 5-10 μg, optionally 7-8 μg; aluminum hydroxide (AlOOH) in an amount of about 0.125-0.375 mg, optionally 0.3-0.35 mg; and a TLR4 agonist, such as E6020, in an amount of 5 μg or less, optionally 0.25-2.5 μg. Alternatively, the aP booster vaccine may comprise, instead of the TLR4 agonist, a TLR9 agonist, such as CpG1018, in an amount of 125-375 μg, optionally 200-300 μg.

[0039] In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage form for administration to a human subject and includes 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 μg; filamentous hemagglutinin in an amount of 5 μg; pertactin in an amount of 5 μg / mL; fimbriae types 2 and 3 in an amount of about 7.5 μg; aluminum hydroxide (AlOOH) in an amount of 0.33 mg; and a TLR4 agonist, such as E6020, in an amount of 1 μg / mL or less. Alternatively, the aP booster vaccine may include a TLR9 agonist, such as CpG1018, in an amount of 250 μg, instead of the TLR4 agonist.

[0040] In certain embodiments of aspects 1 and 2, 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 poliovirus types 1, 2, and 3 (IPV).

[0041] In certain embodiments of aspects 1 and 2, the aP booster vaccine further comprises Tris-buffered saline.

[0042] In certain embodiments of aspects 1 and 2, 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. In certain embodiments of aspects 1 and 2, the aP booster vaccine is in a unit dosage 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.

[0043] In certain embodiments of aspects 1 and 2, at least one of tetanus toxoid, diphtheria toxoid, and detoxified pertussis toxin is adsorbed to an aluminum salt. In certain embodiments, tetanus toxoid, diphtheria toxoid, detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae type 2 and 3 are adsorbed to an aluminum salt. In certain embodiments, the TLR4 agonist or TLR9 agonist is formulated with an aluminum salt. In certain embodiments, all of the bacterial antigens in the aP booster vaccine and the TLR4 or TLR9 agonist are formulated with an aluminum salt.

[0044] In certain embodiments of aspects 1 and 2, the aluminum salt is aluminum hydroxide. In certain embodiments of aspects 1 and 2, the aluminum salt is aluminum phosphate.

[0045] In certain embodiments of aspects 1 and 2, 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 poliovirus (IPV). Includes.

[0046] In certain embodiments of aspect 2, the human subject is 4 years of age or older when the aP booster vaccine is administered.

[0047] In certain embodiments of aspect 2, the human subject is 10 years of age or older when the aP booster vaccine is administered.

[0048] In certain embodiments of aspect 2, the Th1-dominated immune response is characterized by one or more of decreased IL-5 protection, increased IFN-γ protection, increased IL-17 protection, or a lower IgG1 / IgG2a ratio when compared to an acellular pertussis vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL®). In certain embodiments, the Th1-dominated immune response is characterized by one or more of decreased IL-5 protection or a lower IgG1 / IgG2a ratio when compared to an acellular pertussis vaccine or an aP booster vaccine that does not contain a TLR agonist (e.g., ADACEL®).

[0049] In certain embodiments of aspect 2, the aP priming vaccine comprises tetanus toxoid, diphtheria toxoid, detoxified pertussis toxin, filamentous hemagglutinin, and pertactin, with the proviso that the aP priming vaccine does not contain a TLR agonist. In certain embodiments, the aP priming vaccine comprises one or more doses of, but not limited to, DAPTACEL®, INFANRIX®, INFANRIX-HEXA®, PENTACEL®, QUADRACEL®, KINRIX®, PEDIARIX®, or VAXELIS®. In certain embodiments, the aP priming vaccine comprises DAPTACEL®. In certain embodiments, the aP priming vaccine comprises INFANRIX® or INFANRIX-HEXA®. In certain embodiments, the aP priming vaccine comprises PENTACEL®. In certain embodiments, the aP priming vaccine comprises KINRIX® or PEDIARIX®. In certain embodiments, the aP priming vaccine comprises VAXELIS®.

[0050] Further scope of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. The detailed description and specific examples, while illustrating some preferred aspects of the present disclosure, are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. [Brief description of the drawings]

[0051] [Figure 1]Figures 1A-B show a comparison of genetically detoxified PT (gdPT) immunogenicity with chemically detoxified PT (PTxd) in eliciting PT-specific antibody responses. Figure 1A shows anti-PT, IgG1 and IgG2a antibody titers measured by ELISA after two immunizations. Figure 1B shows anti-PT neutralization titers as measured by CHO assay after two immunizations. Mice were immunized twice, on days 0 and 21. Mice were bled 17 days after the second immunization (day 38). [Diagram 2] 2A-B show a comparison of the boosting potential of PTxd-primed responses with PTxd or gdPT. FIG. 2A shows ELISA PT-specific IgG1 and IgGa2 responses after priming with PTxd or gdPT followed by boosting with PTxd or gdPT. FIG. 2B shows PT neutralization titers as measured by CHO assay after priming with PTxd or gdPT followed by boosting with PTxd or gdPT. Mice were immunized three times, on days 0, 21 and 42. Mice were bled 17 days after the second immunization and 8 days after the third immunization. [Diagram 3] FIG. 3 shows an assessment of the absence of immunological interference by gdPT on IgG1 and IgG2a responses induced against FIM antigens after two immunizations. [Figure 4] Figures 4A-B show an evaluation of immunological interference by other Tdap antigens on gdPT-induced anti-PT IgG1 and IgG2a responses as well as neutralizing antibody responses after three immunizations. Figure 4A shows anti-PT, IgG1 and IgG2a antibody titers as measured by ELISA after three immunizations. Figure 4B shows anti-PT neutralizing titers as measured by CHO assay after three immunizations. [Diagram 5]Figure 5 shows the ability of modified Tdap (gdPT+E6020-AlOOH) and modified Tdap (gdPT+CpG1018-AlOOH) formulations to down-regulate DTaP-induced Th2 immune memory responses using an extended prime-boost schedule, as measured by cytokine (IL-5, IFN-γ, and IL-17) levels. Cytokine levels were measured by fluorospot assay. [Figure 6-1]6A-L show the levels of different antigen-specific IgG1 and IgG2a induced in mice after different prime / boost schedules. Naive adult CD1 mice were immunized (primed) with intramuscular injection of DTwP or DTaP. Forty-two days later (D42), DTwP-primed mice were boosted with DTwP vaccine and DTaP-primed mice were boosted with modified TdaP vaccine (mTdaP-AlOOH, mTdaP-CpG-AlOOH or mTdaP-E6020-AlOOH). Using an extended prime-boost schedule, FHA-specific and FIM2,3-specific, PRN-specific, PT-specific, DT-specific and TT-specific IgG1 and IgG2a antibody responses were assessed by modified ELISA (MSD) in sera collected 42 days after the boost (D84) and again 42 days after the second boost at D84 (D126). The numbers above the IgG1 and IgG2a bars in Figures 6A-L represent the IgG1 / IgG2a ratios, with smaller numbers representing lower ratios or vice versa. Lower IgG1 / IgG2a ratios were observed in mice boosted with modified Tdap vaccines containing TLR agonists (CpG or E6020). Figures 6A and 6B show the IgG1 and IgG2a results and the ratios at D84 and D126 for FHA, respectively. Figures 6C and 6D show IgG1 and IgG2a results, and the ratios at D84 and D126 for FIM, respectively. Figures 6E and 6F show IgG1 and IgG2a results, and the ratios at D84 and D126 for PRN, respectively. Figures 6G and 6H show IgG1 and IgG2a results, and the ratios at D84 and D126 for gdPT, respectively. Figures 6I and 6J show IgG1 and IgG2a results, and the ratios at D84 and D126 for DT, respectively. Figures 6K and 6L show IgG1 and IgG2a results, and the ratios at D84 and D126 for TT, respectively. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3]Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 6-5] Continued from Figure 6-4. [Figure 6-6] Continued from Figure 6-5. [Figure 7] FIG. 7 depicts the mouse adoptive transfer model used to evaluate the novel Tdap boost vaccine formulation. [Figure 8A] Figures 8A-B show the kinetics of PT-, PRN-, FHA-, and FIM2,3-specific IgG antibodies in a mouse adoptive transfer model as measured by mean Log10 IgG titers ± SEM of pooled sera (n=4, 6-7 mice per pool). Figure 8A shows the IgG response to 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 modified Tdap (gdPT+E6020-AlOOH) boost and modified Tdap (gdPT+CpG1018-AlOOH) boost in adoptive transfer mice after DTaP priming vaccine compared to the response to Tdap boost. P values ​​<0.05 in Figure 8A indicate: * DTaP / Tdap (prime / boost) vs. DTwP / Tdap (prime / boost); # DTaP / Tdap (prime / boost) vs. DTwP / DTwP (prime / boost); ‡ DTwP / Tdap (prime / boost) vs. DTwP / DTwP (prime / boost). [Figure 8B]Figures 8A-B show the kinetics of PT-, PRN-, FHA-, and FIM2,3-specific IgG antibodies in a mouse adoptive transfer model as measured by mean Log10 IgG titers ± SEM of pooled sera (n=4, 6-7 mice per pool). Figure 8A shows the IgG response to 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 modified Tdap (gdPT+E6020-AlOOH) boost and modified Tdap (gdPT+CpG1018-AlOOH) boost in adoptive transfer mice after DTaP priming vaccine compared to the response to Tdap boost. P values ​​<0.05 in Figure 8A indicate: * DTaP / Tdap (prime / boost) vs. DTwP / Tdap (prime / boost); # DTaP / Tdap (prime / boost) vs. DTwP / DTwP (prime / boost); ‡ DTwP / Tdap (prime / boost) vs. DTwP / DTwP (prime / boost). [Figure 9A]9A-B show that boosting with modified Tdap (gdPT+E6020-AlOOH) and modified Tdap (gdPT+CpG1018-AlOOH) formulations results in early and / or accelerated bacterial clearance after intranasal challenge with Bordetella pertussis. FIG. 9A shows clearance results after priming with DTaP or DTwP followed by a Tdap or DTwP boost. FIG. 9B shows clearance results after priming with DTap followed by a Tdap, modified Tdap (gdPT+E6020-AlOOH) or modified Tdap (gdPT+CpG1018) boost. FIG. 9A and B show the Log10 values ​​± SEM of the number of CFU per lung at the indicated time points for n=3-4 mice per group. <0.05 in Figure 9B indicates: * DTaP / Tdap (prime / boost) vs. DTaP / mTdap-CPG-AlOOH (prime / boost); § DTaP / Tdap (prime / boost) vs. DTaP / mTdap-CPG-AlOOH (prime / boost); + DTaP / Tdap (prime / boost) vs. mTdap-E6020-AlOOH (prime only); $ DTaP / Tdap (prime / boost) vs. mTdap-CPG-AlOOH (prime only); # DTaP / mTdap-CPG-AlOOH (prime / boost) vs. DTaP / mTdap-E6020-AlOOH (prime / boost). Individual mice tested (4 per group per time point). Statistical test: 2-way ANOVA. Symbols indicate time points where significance was found. [Figure 9B]9A-B show that boosting with modified Tdap (gdPT+E6020-AlOOH) and modified Tdap (gdPT+CpG1018-AlOOH) formulations results in early and / or accelerated bacterial clearance after intranasal challenge with Bordetella pertussis. FIG. 9A shows clearance results after priming with DTaP or DTwP followed by a Tdap or DTwP boost. FIG. 9B shows clearance results after priming with DTap followed by a Tdap, modified Tdap (gdPT+E6020-AlOOH) or modified Tdap (gdPT+CpG1018) boost. FIG. 9A and B show the Log10 values ​​± SEM of the number of CFU per lung at the indicated time points for n=3-4 mice per group. <0.05 in Figure 9B indicates: * DTaP / Tdap (prime / boost) vs. DTaP / mTdap-CPG-AlOOH (prime / boost); § DTaP / Tdap (prime / boost) vs. DTaP / mTdap-CPG-AlOOH (prime / boost); + DTaP / Tdap (prime / boost) vs. mTdap-E6020-AlOOH (prime only); $ DTaP / Tdap (prime / boost) vs. mTdap-CPG-AlOOH (prime only); # DTaP / mTdap-CPG-AlOOH (prime / boost) vs. DTaP / mTdap-E6020-AlOOH (prime / boost). Individual mice tested (4 per group per time point). Statistical test: 2-way ANOVA. Symbols indicate time points where significance was found. [Figure 10A] 10A-B depict mouse intranasal challenge assays (INCA) using a short-term immunization schedule (FIG. 10A) and a long-term schedule (FIG. 10B). [Figure 10B] 10A-B depict mouse intranasal challenge assays (INCA) using a short-term immunization schedule (FIG. 10A) and a long-term schedule (FIG. 10B). [Figure 10C]FIG. 10C shows that DTaP / Tdap (prime / boost) and DTwP / DTwP (prime / boost) prevent B. pertussis lung colonization of mice following intranasal challenge in the INCA acute model. [Figure 11] FIG. 11 shows that using an acute prime-boost schedule, the modified Tdap(gdPT+E6020-AlOOH) booster significantly accelerates B. pertussis clearance compared to the Tdap booster in a mouse intranasal challenge assay (INCA). [Figure 12A] Figures 12A-B show that using an extended prime-boost schedule, modified Tdap (gdPT+E6020-AlOOH) and modified Tdap (gdPT+CpG1018-AlOOH) booster vaccines can prevent disease and colonization of the lower respiratory tract in DTaP-primed mice. Figure 12A shows reductions in all vaccinated groups 3 days after challenge, with all treatment groups reaching baseline bacterial loads by day 7. Figure 12B shows that all acellular pertussis dosing schedules had similar kinetics to the DTwP / DTwP (prime / boost) dosing schedule. [Figure 12B] Figures 12A-B show that using an extended prime-boost schedule, modified Tdap (gdPT+E6020-AlOOH) and modified Tdap (gdPT+CpG1018-AlOOH) booster vaccines can prevent disease and colonization of the lower respiratory tract in DTaP-primed mice. Figure 12A shows reductions in all vaccinated groups 3 days after challenge, with all treatment groups reaching baseline bacterial loads by day 7. Figure 12B shows that all acellular pertussis dosing schedules had similar kinetics to the DTwP / DTwP (prime / boost) dosing schedule. [Figure 13A]13A-B show that boosting a DTaP priming immunization with a modified Tdap (gdPT+E6020-AlOOH) formulation induces IL-17 production in an E6020 dose-dependent manner, as measured by fluorospot assay. Splenocytes from CD1 mice immunized with DTaP and boosted twice (D0 and D21) with mTdap-E6020-AlOOH were isolated and restimulated in vitro with PTx (FIG. 13A) or a pool of pertussis antigens consisting of PTx, PRN, and FIM (FIG. 13B). [Figure 13B] 13A-B show that boosting a DTaP priming immunization with a modified Tdap (gdPT+E6020-AlOOH) formulation induces IL-17 production in an E6020 dose-dependent manner, as measured by fluorospot assay. Splenocytes from CD1 mice immunized with DTaP and boosted twice (D0 and D21) with mTdap-E6020-AlOOH were isolated and restimulated in vitro with PTx (FIG. 13A) or a pool of pertussis antigens consisting of PTx, PRN, and FIM (FIG. 13B). [Figure 14] 14 shows the temperature profiles of modified Tdap formulations: mTdap(gdPT+AlOOH), mTdap(gdPT+E6020-AlOOH), and mTdap(gdPT+CpG-AlOOH) showing the first derivative of the autofluorescence emission ratio (350 nm / 330 nm). The temperature transition (Tm) of mTdap(gdPT+AlOOH) is 74.6 degrees; mTdap(gdPT+E6020-AlOOH) is 74.2°C; and mTdap(gdPT+CpG-AlOOH) is 77.0°C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The following description of various preferred embodiments is merely exemplary in nature and is in no way intended to limit the disclosure, its application, or uses.

[0053] When used throughout, ranges are used as shorthand for describing any and all values ​​within the range. Any value within the range can be selected as the end point of the range. Furthermore, all references cited herein are incorporated herein by reference in their entirety. In the event of any discrepancy between the description of this disclosure and the description of the reference cited, this disclosure shall prevail.

[0054] As used herein, "aP" refers to acellular Bordetella pertussis vaccine. Current acellular Bordetella pertussis vaccines are typically based on the following virulence factors: detoxified pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), fimbrial agglutinogen 2 and fimbrial agglutinogen 3 (FIM2 / 3 or FIM). Although some acellular pertussis vaccines contain only PT and FHA or PT, FHA, and PRN, acellular pertussis vaccines containing PT, FHA, PRN, and FIM2 / 3 components are generally considered to be the most effective aP vaccines currently available. Typically, acellular pertussis vaccines are formulated with diphtheria toxoid and tetanus toxoid.

[0055] As used herein, "wP" refers to whole cell pertussis vaccine. Typically, whole cell pertussis vaccines contain whole cells of Bordetella pertussis that have been chemically detoxified and formulated with diphtheria and tetanus toxoids.

[0056] As used herein, "DTwP" refers to the wP indicated for the prevention of diphtheria, tetanus, and whooping cough in infants as a primary vaccination and in children as a booster. One example of a DTwP is for DTCOQ / DTP, which is marketed by Sanofi Pasteur and contains diphtheria and tetanus toxoids, Bordetella pertussis heat-inactivated in the presence of thiomersal, and aluminum phosphate.

[0057] As used herein, "DTaP" refers to aP indicated for active immunization against diphtheria, tetanus, and whooping cough in infants and children. Typically, DTaP is administered as a series of five doses in infants and children aged 6 weeks to 6 years, or as a series of four doses in infants and children aged 6 weeks to 2-4 years. Examples of DTaP include, but are not limited to, DAPTAPE, PENTACEL, and INFANRIX or INFANRIX-HEXA, DAPTAPE, marketed by, for example, Sanofi Pasteur, and containing diphtheria and tetanus toxoids and the following acellular pertussis antigens: PT (chemically detoxified), FHA, PRN, and FIM2 / 3, and aluminum phosphate. Typically, DTaP contains increased amounts of diphtheria toxoid and PT compared to Tdap.

[0058] As used herein, "Tdap" refers to aP indicated for active booster immunization against tetanus, diphtheria, and whooping cough. Typically, Tdap is administered as a single dose in individuals aged 10 years or older. Examples of TdapP include, but are not limited to, ADACEL® and BOOSTRIX®, ADACEL®, marketed by, for example, Sanofi Pasteur, and containing diphtheria and tetanus toxoids and the following acellular pertussis antigens: PT (chemically detoxified), FHA, PRN, and FIM2 / 3, and aluminum phosphate. Typically, Tdap contains reduced amounts of diphtheria toxoid and PT compared to DTaP.

[0059] As used herein, "modified Tdap" or "mTdap" refers to a modified version of the Tdap vaccine that includes diphtheria and tetanus toxoids and the following acellular pertussis antigens: genetically modified PT, FHA, PRN, and FIM2 / 3. Modified Tdap differs from Tdap at least because modified Tdap includes a TLR agonist, such as a TLR4 agonist (e.g., E6020) or a TLR9 agonist (e.g., CpG1018). Modified Tdap also optionally includes genetically detoxified PT (gdPT) instead of chemically detoxified PT (PTdx), or aluminum hydroxide instead of aluminum phosphate. In certain embodiments, mTdap comprises a TLR agonist (e.g., a TLR4 agonist (e.g., E6020) or a TLR9 agonist (e.g., CpG1018)), genetically detoxified PT (gdPT), and aluminum hydroxide.

[0060] As used herein, "booster" or "booster vaccine" refers to a vaccine administered after a priming vaccine. The booster vaccine contains antigens that were included in the priming vaccine, such that the immune system has already been exposed to such antigens prior to administration of the booster vaccine.

[0061] As used herein, a "priming vaccine" refers to one or more doses of a vaccine in a vaccination schedule that is administered before a booster vaccine to induce a primary immune response and immunological memory.

[0062] Th1 / Th2 immune response CD4 T helper cells that respond to antigens can be classified based on the cytokines they produce. Type 1 helper T cells (Th1) preferentially produce inflammatory cytokines such as IFN-γ, IL-2, TNF-α, and TNF-β. Th1 cells activate macrophages and are typically associated with cell-mediated immune responses and phagocyte-dependent defense responses (e.g., antibody opsonization). On the other hand, type 2 helper cells (Th2) preferentially produce cytokines such as IL-4, IL-5, IL-10, and IL-13. Th2 cells activate B cells and are typically associated with antigen-mediated immune responses.

[0063] Studies in children have shown that wP priming vaccines induce predominantly Th1-dominated responses, whereas aP priming vaccines induce predominantly Th2-dominated responses. Ryan et al., Immunology, 1998, 93:1-10; Ausiello et al., Infect Immun, 1997, 65:2168-74. In addition to inducing distinct cytokine profiles, aP-primed vaccines induced predominantly IgG1 antibodies in mice, but also IgG2 and IgG4, with the proportion of IgG4 increasing after booster vaccination, reflecting a Th2-dominated response (Stenger et al., Vaccine, 2010, 28:6637-46 and Brummelman et al., Vaccine, 2015, 33:1483-19), whereas wP-primed vaccines induced predominantly IgG2 antibodies in mice, but also IgG1 and IgG3 (Raeven et al., J Proteome Res, 2015, 14:2929-42), consistent with a Th1-dominated response.

[0064] As disclosed herein, the modified aP booster vaccine described in this application can redirect the Th2-dominated immune response induced by the previously administered aP vaccine to a Th1 or mixed Th1 / Th17-dominated immune response. As reflected in the relevant literature, such as the literature cited in the previous section, a person skilled in the art can easily identify whether the aP vaccine induces a Th1-dominated or Th2-dominated response by measuring cytokine profile and antibody isotype using conventional techniques. Typically, the Th2-dominated immune response induced by the aP vaccine in mice is associated with an increase in IL-5 levels and / or an increase in the IgG1 / IgG2a ratio, while the Th1-dominated immune response is typically associated with one or more of a decrease in IL-5 levels, an increase in IFN-γ levels, or a decrease in the IgG1 / IgG2a ratio. For example, the ability of the aP booster vaccine described herein to redirect a Th2-dominated immune response induced by a previously administered aP vaccine toward a Th1-dominated immune response shows reduced IL-5 levels and / or reduced IgG1 / IgG2a ratios compared to immune responses induced by a previously administered aP vaccine or aP booster vaccine that does not contain a TLR agonist. Th17 responses are measured by the production of IL-17.

[0065] Tetanus toxoid Tetanus toxoid is produced from Clostridium tetani, a gram-positive rod-shaped spore-forming bacillus. Tetanus toxoid is a protein of about 150 kDa, consisting of two subunits (about 100 kDa and about 50 kDa) linked by sulfide bonds. Tetanus toxoid is typically detoxified with formaldehyde and can be purified from the culture filtrate using known methods, such as ammonium sulfate precipitation and / or chromatographic techniques, as disclosed in WO1996 / 025425, for example. C. tetani can be grown in any suitable growth medium, such as Mueller-Miller casamino acids medium without beef heart infusion (Mueller et al., J Bacteriol, 1954, 67(3):271-277) or Latham's medium derived from bovine casein. Tetanus toxoid has also been inactivated by genetic means by recombinant gene transfer.

[0066] The amount of tetanus toxoid can be expressed as "Lf" units (i.e., limits of agglutination or agglutination units), which is defined as the amount of toxoid that produces an optimally agglutinating mixture when mixed with 1 international unit of antitoxin. See Module 1 of The immunological basis for immunization series from WHO (Galazka). The amount of tetanus toxoid in a composition can be readily determined by comparing the composition to a reference material calibrated with a reference reagent in an agglutination assay.

[0067] Diphtheria toxoid Diphtheria toxoid is an ADP-ribosylating exotoxin produced by the gram-positive, non-spore-forming, aerobic bacterium Corynebacterium diphtheriae. Like tetanus toxoid, diphtheria toxoid is typically detoxified using formaldehyde to obtain a non-toxic, but still antigenic, toxoid. Corynebacterium diphtheriae can be grown in any suitable growth medium, such as modified Mueller growth medium (Stainer, DW, In: Manclark CR, editor, Proceedings of an informal consultation of the WHO requirements for diphtheria, tetanus, pertussis and combined vaccines, US Public Health Service, Bethesda, MD. DHHS 91-1174, 1991, 7-11) or Fenton medium or Linggoud and Fenton medium, which may be supplemented with bovine extract. Diphtheria toxin can be purified using conventional techniques, such as ammonium sulfate fractionation, and can be detoxified using standard techniques, such as formaldehyde treatment, either before or after purification.

[0068] Similar to tetanus toxoid, the amount of diphtheria toxoid can be expressed in "Lf" units. The amount of diphtheria toxoid in a composition can be readily determined by comparing the composition to a reference material calibrated with a reference reagent in an agglutination assay.

[0069] Pertussis toxin Pertussis toxin (PT) is a secreted protein exotoxin and an important virulence factor produced exclusively by Bordetella pertussis. Pertussis toxin is composed of five subunits, designated S1, S2, S3, S4 (x2), and S5, encoded by five genes organized in an operon of approximately 3200 base pairs. Expression of the five genes is regulated by a promoter located upstream of the gene encoding S1. Activation of the toxin promoter is under the control of the Bordetella virulence gene (bvg) system, which regulates the expression of pertussis toxin as well as other known virulence factors, such as FHA and PRN.

[0070] Pertussis toxin is a protein of about 105 kDa with an A / B configuration. The A domain, composed of the S1 subunit, is responsible for the ADP-ribosylating activity of the protein. It prevents the binding of G proteins (guanine nucleotide-binding proteins) to G protein-coupled receptors (GPCRs) on the host cell membrane, thus disrupting signal transduction and resulting in many biological effects associated with PT activity, such as histamine sensitization, leukocytosis, and altered insulin secretion. The B oligomer is a pentameric ring composed of subunits S2, S3, S4, and S5 associated in a 1:1:2:1 ratio, and is responsible for binding to receptors on eukaryotic cells. It binds to various (but largely unidentified) glycoconjugate molecules on the surface of target cells.

[0071] The pertussis toxin used in current acellular vaccines is typically chemically detoxified. In the aP booster vaccine described herein, the detoxified pertussis toxin is typically genetically modified to reduce enzymatic activity and / or toxicity. Many constructs that contain genetic modifications of pertussis toxin have been engineered to reduce the enzymatic activity and / or toxicity of the protein while retaining its immunogenic and protective properties, including, for example, mutant pertussis toxin with a mutation at amino acid 129 of the S1 subunit of pertussis toxin, such as the E129G mutant or the R9K / E129G double mutant. See, for example, U.S. Patent Nos. 5,433,945, 7,144,576, 7,666,436, and 7,427,404. Thus, in certain embodiments, the genetically detoxified pertussis toxin contains a mutation at amino acid 129 of the S1 subunit of pertussis toxin. In certain embodiments, the mutation is an E129G mutation. In certain embodiments, the genetically detoxified pertussis toxin comprises an R9K mutation and an E129G mutation. Although genetically detoxified pertussis toxin is preferred, it is also possible to use chemically detoxified pertussis toxin instead of genetically detoxified pertussis toxin. Chemical detoxification can be carried out, for example, by any of a variety of conventional chemical detoxification methods, such as treatment with formaldehyde, hydrogen peroxide, tetranitromethane, or glutaraldehyde. See, for example, U.S. Patent No. 5,877,298.

[0072] Pertactin Pertactin is a 69 kDa outer membrane protein originally identified from B. bronchiseptica (Montaraz, JA et al. Infect. Immun. 1985;161:581-582). It was found 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 (Leininger, E. et al. Proc Natl Acad Sci USA 1991;88:345-349), and natural infection with B. pertussis induces an anti-pertactin human response (Thomas, MG et al. J. Infect. Dis. 1989;159:211-18). Pertactin also induces cell-mediated immune responses (Petersen, JW et al., Infect. Immun. 1992;60:4563-70; De Magistris, T. et al., J. Exp. Med. 1988;168:1351-1362; Seddon, PC et al., Serodiagnosis Immunother. Inf. Dis. 1990;3:337-43). Vaccination with whole-cell or acellular vaccines induces anti-pertactin antibodies (Edwards, KM et al., Pediatr. Res. 1992;31:91A; Podda, A. et al., Vaccine 1991;9:741-45), and acellular vaccines induce pertactin cell-mediated immunity (Podda, A. et al., Vaccine 1991;9:741-45). Pertactin protects mice against aerosol challenge with Bordetella pertussis (Roberts, M. et al., Vaccine 1992;10:43-48) and, in combination with FHA, protects against Bordetella pertussis in an intracerebral challenge assay (Novotny, P. et al., J. Infect. Dis. 1991;164:114-22). Passive transfer of polyclonal or monoclonal anti-pertactin antibodies also protects mice against aerosol challenge (Shahin, RD et al., J. Exp. Med. 1990;171:63-73).

[0073] Filamentous hemagglutinin Filamentous hemagglutinin (FHA) is a large (220 kDa), non-toxic polypeptide that mediates adhesion of Bordetella pertussis to ciliated cells of the upper respiratory tract during bacterial colonization (Tuomanen, E. and Weiss, A., J. Infect. Dis., 1985;152:118-25). Vaccination with whole cell or acellular pertussis vaccines generates anti-FHA antibodies, and acellular vaccines containing FHA also induce cell-mediated immune responses to FHA (Gearing, A. et al., FEMS Microbial. Immunol. 1989;47:205-12; Thomas, MG et al., J. 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).

[0074] Fimbriae types 2 and 3 Serotypes of Bordetella pertussis are determined by their agglutinating pili. The WHO recommends that whole cell vaccines contain types 1, 2, and 3 agglutinogens (Agg) because they are not cross-protective (Robinson, A. et al., Vaccine 1985;3:11-22). Agg1 is nonfimbria and found in all Bordetella pertussis strains, whereas Agg of serotypes 2 and 3 are fimbria. Natural infection or immunization with whole cell or acellular vaccines induces anti-Agg antibodies (Thomas, MG et al., J. Infect. Dis. 1989;160:838-45; Edwards, KM et al., Pediatr. Res. 1992;31:91A). Following aerosol infection, Agg2 and Agg3 can generate specific cell-mediated immune responses in mice (Petersen, JW, et al., Immun. 1992;60:4563-70). Agg2 and 3 are protective in mice against respiratory challenge infection, and human colostrum containing antiagglutinogens will also be protective 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).

[0075] TLR agonists The aP booster vaccine described herein includes a Toll-like receptor (TLR) agonist. A TLR agonist is a compound that can stimulate or activate a TLR. TLRs are key components in the host's pathogen detection mechanism (Janeway et al., Annu. Rev. Immunol. 2002;20:197-216); Akira et al., Nat Rev Immunol. 2004;4:499-511). Typically, TLRs are classified into two families based on their location: TLR1, 2, and 4-6 are expressed on the cell surface and sense bacterial cell wall components, while TLR3 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 are evolutionarily conserved and expressed by a variety of infectious microorganisms (Janeway et al., Annu. 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 proinflammatory cytokines, chemokines, type I interferons, and antimicrobial peptides. This innate response promotes and regulates the adaptive immune system. The common outcome is the proliferation of antigen-specific B cells that produce high-affinity antibodies and cytotoxic T cells, including long-lasting memory cells that protect against subsequent infections by enhancing cytotoxic function to target 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 a key role in various aspects of the innate immune response.

[0076] Surprisingly, by including a TLR agonist, the aP booster vaccine is able to shift the Th2 dominated immune response established by the previously administered aP vaccine towards a Th1 dominated immune response. Preferably, the TLR agonist is an agonist of a human TLR.

[0077] In certain embodiments, the TLR agonist is a TLR4 agonist, preferably an agonist of human TLR4. In one embodiment, the TLR4 agonist is E6020, a synthetic phospholipid dimer that mimics the physicochemical and biological properties of natural lipid A from gram-negative bacteria (Ishizaka et al., Expert Rev Vaccines. 2007;(5):773-84). E6020 is the disodium salt of dodecanoic acid (1R,6R,22R,27R)-1,27-dihexyl-9,19-dihydroxy-9,19-dioxide-14-oxo-6,22-bis[(1,3-dioxotetradecyl)amino]-4,8,10,18,20,24-hexaoxa-13,15-diaza-9,19-diphosphaheptacosane-1,27-diyl ester (C 83 H 158 N4O 19 P2Na2). The chemical synthesis of E6020 is a reproducible and well-controlled manufacturing process that results in a highly pure compound. E6020 has the following chemical structure:

[0078] [ka]

[0079] 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 is associated with Th1 activation in mice. E6020 is also known to enhance granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, and TNF-α in human peripheral blood mononuclear cells (PBMCs) and mouse spleens (Ishizaka et al., Expert Rev Vaccines. 2007;(5):773-84).

[0080] In other embodiments, the TLR agonist is a TLR9 agonist, preferably an agonist of human TLR9.For example, the TLR9 agonist can be a CpG oligodeoxynucleotide ("ODN").As used herein, "CpG oligonucleotide" or "CpG ODN" is a single-stranded DNA molecule that contains at least one central unmethylated CG dinucleotide that is incorporated within specific flanking regions.CpG ODN is frequently present in bacterial DNA and has immune stimulating effects.

[0081] In humans, CpG ODNs have been classified into four different classes based on differences in structure and the nature of the immune response they induce. Each class contains at least one central unmethylated CG dinucleotide and adjacent regions, which differ in structure and immunological activity. Class B ODNs (also called "K" type) typically contain one to five CpG motifs in a phosphorothioate backbone. Phosphorothioates are non-naturally occurring internucleotide linkages that replace the phosphodiester bonds found in naturally occurring DNA, enhancing resistance to nuclease digestion and substantially extending half-life in vivo. Class B ODNs act to induce plasmacytoid dendritic cells to differentiate and produce TNFα, as well as stimulate B cells to proliferate and secrete IgM.

[0082] Class A ODNs (also called "D" type) have a phosphodiester core flanked by phosphorothioate terminal nucleotides. They contain a single CpG motif flanked by palindromic sequences that can form stem-loop structures. Class A ODNs also have poly-G motifs at the 3' and 5' ends that promote concatemer formation. Class A ODNs act on plasmacytoid dendritic cells to mature and secrete IFNα, but have no effect on B cells. Class C ODNs are similar to class B, which are composed entirely of phosphorothioate nucleotides, but are similar to class A in that they contain palindromic CpG motifs that can form stem-loop structures or dimers. Class C ODNs stimulate B cells to secrete IL-6, as well as plasmacytoid dendritic cells to produce IFNα. Class P CpG ODNs are highly ordered structures that contain double palindrome and can form hairpins at their GC-rich 3' ends as well as concatamerize due to the presence of a 5' palindrome.

[0083] In certain embodiments, the CpG ODN used in the aP booster vaccine is a class B CpG ODN. In certain embodiments, the CpG ODN used in the aP booster vaccine is a class A CpG ODN. In certain embodiments, the CpG ODN used in the aP booster vaccine is a class C CpG ODN. In certain embodiments, the CpG ODN used in the aP booster vaccine is a class P CpG ODN.

[0084] In certain embodiments, the CpG ODN comprises at least one phosphorothioate bond. In certain embodiments, all nucleotides in the CpG ODN are linked by phosphorothioate bonds. In certain embodiments, the CpG ODN comprises 1-5 CG dinucleotides. In certain embodiments, the CpG ODN comprises one CG dinucleotide. In certain embodiments, the CpG ODN comprises two CG dinucleotides. In certain embodiments, the CpG ODN comprises three CG dinucleotides. In certain embodiments, the CpG ODN comprises four CG dinucleotides. In certain embodiments, the CpG ODN comprises five CG dinucleotides. In certain embodiments, the CpG ODN is 18-28 nucleotides in length.

[0085] In one embodiment, 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.

[0086] Aluminum salts Adjuvants such as aluminum salts have been used to enhance immune responses to various antigens. Aluminum salts that can be used as adjuvants include, but are not limited to, aluminum hydroxide / oxyhydroxide (AlOOH), aluminum phosphate (AlPO4), aluminum hydroxyphosphate sulfate (AAHS), and / or aluminum potassium sulfate. These aluminum salts have been used in vaccines for many years.

[0087] As discussed elsewhere in this application, one or more of the tetanus toxoid, diphtheria toxoid, and acellular Bordetella pertussis antigens of the aP booster vaccine can be adsorbed to an aluminum salt. In certain embodiments, all of the vaccine antigens in the aP booster vaccine are adsorbed to an aluminum salt. For example, in one embodiment, the tetanus toxoid, diphtheria toxoid, PT, FHA, PT, and FIM2,3 are adsorbed to AlOOH. In another embodiment, the tetanus toxoid, diphtheria toxoid, PT, FHA, PT, and FIM2,3 are adsorbed to AlPO4. In yet another embodiment, one or more of the vaccine antigens in the aP booster vaccine are adsorbed to AlOOH, and one or more of the vaccine antigens in the aP booster vaccine are adsorbed to AlPO4.

[0088] In certain embodiments, the TLR agonist is formulated with aluminum salt.Typically, the TLR4 agonist, such as E6020, is formulated with AlOOH.In other embodiments, the TLR4 agonist, such as E6020, is formulated with AlPO4.Typically, the TLR9 agonist, such as CpG ODN (e.g., CpG1018), is formulated with AlOOH.In other embodiments, the TLR9 agonist, such as CpG ODN (e.g., CpG1018), is formulated with AlPO4.

[0089] In certain embodiments, the aP booster vaccine comprises tetanus toxoid, diphtheria toxoid, gdPT, FHA, PT, FIM2,3, and a TLR4 agonist (e.g., E6020), wherein each of the tetanus toxoid, diphtheria toxoid, gdPT, FHA, PT, FIM2,3 is adsorbed onto AlOOH, and the TLR4 agonist (e.g., E6020) is formulated with AlOOH. In certain embodiments, the aP booster vaccine comprises tetanus toxoid, diphtheria toxoid, gdPT, FHA, PT, FIM2,3, and a TLR9 agonist (e.g., a CpG ODN, such as CpG1018), wherein each of the tetanus toxoid, diphtheria toxoid, gdPT, FHA, PT, FIM2,3 is adsorbed onto AlOOH, and the TLR9 agonist (e.g., a CpG ODN, such as CpG1018) is formulated with AlOOH.

[0090] In certain embodiments, the aP booster vaccine contains both AlOOH and AlPO4, and the antigen in the vaccine is adsorbed to one or both of these aluminum salts.

[0091] Methods for adsorbing diphtheria toxoid, tetanus toxoid, and pertussis antigens to aluminum salts, such as AlOOH and AlPO4, are known in the art.

[0092] Other antigens In addition to tetanus toxoid, diphtheria toxoid, and acellular Bordetella pertussis antigens, the aP booster vaccine can include one or more additional antigens, such as, but not limited to, Haemophilus influenzae type b saccharide (Hib) conjugate, Hepatitis B surface antigen (HBsAg), and / or inactivated poliovirus (IPV).

[0093] Haemophilus influenzae type b causes bacterial meningitis. Hib vaccines are typically formulated using Hib conjugated to a carrier protein to enhance its immunogenicity, especially in children. Typically, the carrier protein is tetanus toxoid, diphtheria toxoid, Haemophilus influenzae protein D, or an outer membrane protein complex derived from serogroup B meningococcus. However, any suitable carrier protein can be used. Methods for making Hib conjugates are known in the art. For example, PENTACEL® contains Haemophilus influenzae type b capsular polysaccharide (polyribosyl-ribitol-278 phosphate [PRP]) covalently bound to tetanus toxoid. Hib conjugates are typically adsorbed to an aluminum salt (e.g., AlOOH or AlPO4).

[0094] Hepatitis B virus (HBV) causes viral hepatitis, a potentially life-threatening liver infection. Infectious HBV virions have a spherical double-core structure and consist of a lipid envelope containing HBsAg surrounding an inner nucleocapsid composed of the hepatitis B core antigen (HBcAg) complexed with a 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 for making HBsAg and vaccines containing HBsAg are known in the art. HBsAg is typically adsorbed to an aluminum salt (e.g., AlOOH or AlPO4).

[0095] Acute poliomyelitis is a disease caused by any of three types of poliovirus; poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-1 strain), and poliovirus type 3 (e.g., Saukett strain). Poliovirus can be grown in cell culture using known techniques, followed by purification of the virions using techniques such as ultrafiltration, diafiltration, and chromatography. The virions are then inactivated, for example, using formaldehyde. Typically, each type of poliovirus is grown separately, purified from cell culture, and inactivated before combining them to create a trivalent poliovirus composition. Typically, the inactivated poliovirus is not adsorbed onto an aluminum salt before formulating the vaccine. However, the inactivated poliovirus can become adsorbed onto any aluminum salt in the vaccine that is not adsorbed to another vaccine antigen.

[0096] Acellular pertussis booster vaccine The aP booster vaccine is an immunogenic composition that includes one or more antigens derived from or homologous to antigens from Bordetella pertussis and other pathogens (e.g., Corynebacterium diphtheriae, Clostridium tetani, etc.), but not all of the antigens. Such vaccines are substantially free of intact pathogenic particles or lysates of such particles. Thus, the aP booster vaccine can be prepared from at least partially purified or substantially purified immunogenic polypeptides or analogs thereof from the pathogen of interest. Methods for obtaining the antigen(s) in the vaccine include standard purification techniques, recombinant production, or chemical synthesis.

[0097] In various embodiments, one or more antigens are formulated into a single dose of aP booster vaccine. "Single dose" as used herein refers to the amount of vaccine administered to a subject in a single administration. Typically, this amount is present in a volume of 0.1 to 2 milliliters, for example, 0.2 to 1 milliliter, typically 0.5 milliliters. Thus, the indicated amount may be present, for example, in a concentration of milligrams per 0.5 milliliters of bulk vaccine. Thus, in certain embodiments, a (single) unit dose is equal to 0.5 milliliters.

[0098] The aP booster vaccine described herein includes tetanus toxoid, diphtheria toxoid, and the following acellular Bordetella 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 AlOOH or AlPO4.

[0099] Acellular pertussis antigens are typically prepared by isolation from Bordetella pertussis cultures grown in liquid culture medium. Any liquid culture medium known in the art for culturing Bordetella cells can be used. In various embodiments, a complex medium is used. As used herein, "complex medium" refers to a medium containing peptone digests or extracts of plant or animal origin. Examples of complex media suitable for use in the present method include, for example, Hornibrook medium, Cohen-Wheeler medium, B2 medium, or other similar liquid culture media. Modified Stainer & Scholte medium, which also contains dimethyl beta-cyclodextrin and casamino acids, is another example suitable for use. Stainer et al., J Gen Microbiol, 1970, 63:211-20. Pertussis toxin, filamentous hemagglutinin, and pertactin are typically isolated separately from the culture medium supernatant. Pili types 2 and 3 are typically extracted and co-purified from bacterial cells. Pertussis antigens may be purified from the supernatant and / or bacterial cells using any conventional method including, for example, sequential filtration, salting out precipitation, ultrafiltration and chromatography.

[0100] Tetanus toxoid (TT) is present in the aP booster vaccine in an amount of about 8-12 limit flocculation (Lf) / mL. In certain embodiments, TT is present in an amount of 9-11 Lf / mL. In certain embodiments, TT is present in an amount of 10 Lf / mL. Measured per unit dosage form, where the unit dose is 0.5 mL, TT is typically present in an amount of about 4-6 Lf. In certain embodiments for a 0.5 mL dosage form, TT is present in an amount of 4.5-5.5 Lf. In certain embodiments for a 0.5 mL dosage form, TT is present in an amount of 5 Lf. In aP booster vaccine, TT is typically adsorbed onto an aluminum salt. Typically, TT is adsorbed onto AlOOH. In other embodiments, TT may be adsorbed onto AlPO4.

[0101] Diphtheria toxoid (DT) is present in the aP booster vaccine in an amount of about 3-8 Lf / mL. In certain embodiments, DT is present in an amount of 3-6 Lf / mL. In certain embodiments, DT is present in an amount of 4-5 Lf / mL. In certain embodiments, DT is present in an amount of 4 Lf / mL. Measured per unit dosage form, where the unit dose is 0.5 mL, DT is typically present in an amount of about 1.5-4 Lf. In certain embodiments for a 0.5 mL dosage form, DT is present in an amount of 1.5-3 Lf. In certain embodiments for a 0.5 mL dosage form, DT is present in an amount of 2-2.5 Lf. In certain embodiments for a 0.5 mL dosage form, DT is present in an amount of 2 Lf. In aP booster vaccine, DT is typically adsorbed onto an aluminum salt. Typically, DT is adsorbed onto AlOOH. In other embodiments, DT may be adsorbed onto AlPO4.

[0102] Detoxified pertussis toxin (PT) is typically present in the aP booster vaccine in an amount of about 4-30 μg / mL. In certain embodiments, PT is chemically detoxified PT and is present in an amount of 4-10 μg / mL. In certain embodiments, PT is genetically detoxified PT (gdPT) and is present in an amount of about 16-24 μg / mL. In certain embodiments, gdPT is present in an amount of 18-22 μg / mL. In certain embodiments, gdPT is present in an amount of 20 μg / mL. Measured per unit dosage form, where the unit dose is 0.5 mL, PT is typically present in an amount of about 2-15 μg. In certain embodiments for 0.5 mL dosage forms, PT is present in an amount of 2-5 μg. In certain embodiments for 0.5 mL dosage forms, PT is gdPT and is present in an amount of 8-12 μg. In certain embodiments for a 0.5 mL dosage form, gdPT is present in an amount of 9-11 μg. In certain embodiments for a 0.5 mL dosage form, gdPT is present in an amount of ∼10 μg. In other embodiments, PT is present at 2-50 μg, 5-40 μg, 10-30 μg, or 20-25 μg per unit dose. In aP booster vaccines, PT is typically adsorbed onto an aluminum salt. In certain embodiments, PT is adsorbed onto AlOOH. In certain embodiments, PT is adsorbed onto AlPO4.

[0103] Filamentous hemagglutinin (FHA) is typically present in the aP booster vaccine in an amount of about 5-15 μg / mL. In certain embodiments, FHA is present in an amount of 8-12 μg / mL. In certain embodiments, FHA is present in an amount of 10 μg / mL. Measured per unit dosage form, where the unit dose is 0.5 mL, FHA is typically present in an amount of about 2.5-7.5 μg. In certain embodiments for a 0.5 mL dosage form, FHA is present in an amount of 4-6 μg. In certain embodiments for a 0.5 mL dosage form, FHA is present in an amount of 5 μg. In other embodiments, FHA is present in an amount of 2-50 μg, 5-40 μg, 10-30 μg, or 20-25 μg per unit dose. In aP booster vaccines, FHA is typically adsorbed onto an aluminum salt. In certain embodiments, FHA is adsorbed onto AlOOH. In one particular embodiment, the FHA is adsorbed onto AlPO4.

[0104] Pertactin (PRN) is typically present in the aP booster vaccine in an amount of about 5-15 μg / mL. In certain embodiments, PRN is present in an amount of 8-12 μg / mL. In certain embodiments, PRN is present in an amount of 10 μg / mL. Measured per unit dosage form, where the unit dose is 0.5 mL, PRN is typically present in an amount of about 2.5-7.5 μg. In certain embodiments for a 0.5 mL dosage form, PRN is present in an amount of 4-6 μg. In certain embodiments for a 0.5 mL dosage form, PRN is present in an amount of 5 μg. In other embodiments, PRN is present in an amount of 0.1-100 μg, 1-50 μg, 2-20 μg, 3-30 μg, or 5-20 μg per unit dose. In the aP booster vaccine, PRN is typically adsorbed onto an aluminum salt. In certain embodiments, PRN is adsorbed onto AlOOH. In one particular embodiment, PRN is adsorbed onto AlPO4.

[0105] Fimbrial types 2 and 3 (FIM2,3) are typically present in the aP booster vaccine in an amount of about 10-20 μg / mL. In certain embodiments, FIM2,3 are present in an amount of 14-16 μg / mL. In certain embodiments, FIM2,3 are present in an amount of 15 μg / mL. In certain embodiments, the weight ratio of FIM 2 to FIM 3 is about 1:3 to about 3:1, such as about 1:1 to about 3:1, such as about 1.5:1 to about 2:1. Measured per unit dosage form with a unit dose of 0.5 mL, FIM2,3 are typically present in an amount of about 5-10 μg. In certain embodiments for a 0.5 mL dosage form, FIM2,3 are present in an amount of 7-8 μg. In certain embodiments for a 0.5 mL dosage form, FIM2,3 are present in an amount of 7.5 μg. In other embodiments, FIM2 / 3 is present in an amount ranging from 1 to 100 μg per unit dose, for example, 3 to 50 μg per unit dose, or 3 to 30 μg per unit dose. In aP booster vaccines, FIM2,3 is typically adsorbed onto an aluminum salt. In certain embodiments, FIM2,3 is adsorbed onto AlOOH. In certain embodiments, FIM2,3 is adsorbed onto AlPO4.

[0106] Typically, the aP booster vaccine includes an aluminum salt, such as AlOOH or AlPO4, used to adsorb one or more of the vaccine antigens and / or to formulate the TLR agonist. In certain embodiments, the aluminum salt is present in an amount of 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. Measured per unit dosage form with a unit dose of 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, a 0.5 mL unit dosage form contains 0.33 mg of aluminum salt. In certain embodiments, the aluminum salt is AlOOH. In other embodiments, the aluminum salt is AlPO4.

[0107] When the TLR4 agonist is E6020, the TLR4 agonist may be present in an amount of 10 μg / ml or less. In certain embodiments, E6020 is present in an amount of 0.5-5 μg / ml. In certain embodiments, E6020 is present in an amount of 2 μg / ml or less. Measured per unit dosage form with a unit dose of 0.5 mL, E6020 is typically present in an amount of 5 μg or less. In certain embodiments for a 0.5 mL dosage form, E6020 is present in an amount of about 0.25-2.5 μg. In certain embodiments for a 0.5 mL dosage form, E6020 is present in an amount of 1 μg or less.

[0108] In certain embodiments, E6020 is formulated with an aluminum salt. Typically, the aluminum salt is AlOOH. In other embodiments, the aluminum salt may be AlPO4.

[0109] When the TLR9 agonist is CpG1018, the TLR9 agonist may be present in an amount of about 250-750 μg / ml. In certain embodiments, CpG1018 is present in an amount of 400-600 μg / ml. In certain embodiments, CpG1018 is present in an amount of 500 μg / ml. Measured per unit dosage form with a unit dose of 0.5 mL, CpG1018 is typically present in an amount of about 125-375 μg. Measured per unit dosage form with a unit dose of 0.5 mL, CpG1018 is present in an amount of 200-300 μg. In certain embodiments for a 0.5 mL dosage form, CpG1018 is present in an amount of 250 μg.

[0110] In certain embodiments, CpG1018 is formulated with an aluminum salt. Typically, the aluminum salt is AlOOH. In other embodiments, the aluminum salt can be AlPO4.

[0111] In certain embodiments, 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 μg / mL, FHA in an amount of 5-15 μg / mL, PRN in an amount of 5-15 μg / mL, FIM2,3 in an amount of about 10-20 μg / mL, AlOOH in an amount of 0.25-0.75 mg / mL, and a TLR4 agonist, such as E6020, in an amount of 10 μg / ml or less.

[0112] In certain embodiments, 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 μg / mL, FHA in an amount of 5-15 μg / mL, PRN in an amount of 5-15 μg / mL, FIM2,3 in an amount of about 10-20 μg / mL, AlOOH in an amount of 0.25-0.75 mg / mL, and a TLR9 agonist, such as CpG1018, in an amount of 250-750 μg / ml.

[0113] In certain embodiments, 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 μg / mL, FHA in an amount of 8-12 μg / mL, PRN in an amount of 8-12 μg / mL, FIM2,3 in an amount of 14-16 μg / mL, AlOOH in an amount of 0.6-0.7 mg / mL, and a TLR4 agonist, such as E6020, in an amount of 0.5-5 μg / ml.

[0114] In certain embodiments, 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 μg / mL, FHA in an amount of 8-12 μg / mL, PRN in an amount of 8-12 μg / mL, FIM2,3 in an amount of 14-16 μg / mL, AlOOH in an amount of 0.6-0.7 mg / mL, and a TLR9 agonist, such as CpG1018, in an amount of 400-600 μg / ml.

[0115] In certain embodiments, the aP booster vaccine comprises the following components in the amounts indicated, as set forth in Table 1:

[0116] [Table 1]

[0117] In certain embodiments of the aP booster vaccines set forth in Table 1, the diphtheria toxoid is present in an amount of 4 Lf / mL. In certain embodiments of the aP booster vaccines set forth in Table 1, the diphtheria toxoid is present in an amount of 5 Lf / mL.

[0118] In certain embodiments, the aP booster vaccine is in a unit dosage 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 μg, FHA in an amount of 2.5-7.5 μg, PRN in an amount of 2.5-7.5 μg, FIM2,3 in an amount of about 5-10 μg, AlOOH in an amount of 0.125-0.375 mg, and a TLR4 agonist, e.g., E6020, in an amount of 5 μg or less.

[0119] In certain embodiments, the aP booster vaccine is in a unit dosage 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 μg, FHA in an amount of 2.5-7.5 μg, PRN in an amount of 2.5-7.5 μg, FIM2,3 in an amount of about 5-10 μg, AlOOH in an amount of 0.125-0.375 mg, and a TLR9 agonist, such as CpG1018, in an amount of 125-375 μg.

[0120] In certain embodiments, the aP booster vaccine is in a unit dosage 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 μg, FHA in an amount of 4-6 μg, PRN in an amount of 4-6 μg, FIM2,3 in an amount of about 7-8 μg, AlOOH in an amount of 0.3-0.35 mg, and a TLR4 agonist, e.g., E6020, in an amount of 0.25-2.5 μg.

[0121] In certain embodiments, the aP booster vaccine is in a unit dosage 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 μg, FHA in an amount of 4-6 μg, PRN in an amount of 4-6 μg, FIM2,3 in an amount of about 7-8 μg, AlOOH in an amount of 0.3-0.35 mg, and a TLR9 agonist, such as CpG1018, in an amount of 200-300 μg.

[0122] In certain embodiments, the aP booster vaccine is in a unit dosage form for administration to a human subject and contains the following components per 0.5 mL dose, as set forth in Table 2:

[0123] [Table 2]

[0124] In certain embodiments of the aP booster vaccines set forth in Table 1, the diphtheria toxoid is present in an amount of 2 Lf. In certain embodiments of the aP booster vaccines set forth in Table 1, the diphtheria toxoid is present in an amount of 2.5 Lf.

[0125] In certain embodiments, the aP booster vaccine is formulated to contain an antigen other than a tetanus toxoid, a diphtheria toxoid, or a Bordetella antigen. For example, in certain embodiments, the aP booster vaccine comprises one or more of the following: Haemophilus influenzae type b oligosaccharide or polysaccharide (Hib) conjugate, Hepatitis B surface antigen (HBsAg), and / or inactivated poliovirus (IPV).

[0126] The aP booster vaccine described herein can be formulated as an injectable solution or emulsion.For example, tetanus toxoid, diphtheria toxoid, and Bordetella antigen can be mixed with pharma- ceutically acceptable excipients that are compatible with these antigens.Such excipients can include water, saline, dextrose, glycerol, ethanol, and combinations thereof.The aP booster vaccine can further include auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance its effectiveness.

[0127] Typically, the aP booster vaccine will be formulated in an aqueous form. Typically, the components of the aP booster vaccine will be diluted in Tris-buffered saline to obtain the desired final concentration. Alternatively, the diluent for the formulation may be water for injection.

[0128] How to administer the aP booster vaccine The aP booster vaccine described herein is suitable for administration to human subjects.Therefore, one embodiment relates to a method for inducing immune response in human subjects, comprising administering the aP booster vaccine described herein to human subjects.Also described is an aP booster vaccine for use in inducing immune response in human subjects and / or redirecting the Th2-dominated immune response in human subjects to Th-1-dominated immune response or Th1 / Th17-dominated immune response.

[0129] Typically, human subjects are administered with wP vaccine without pertussis vaccine, or with aP priming vaccine that induces Th2-dominant immune response before administering aP booster vaccine.Typically, when aP booster vaccine that does not contain TLR agonist (e.g., ADACEL®) is administered to human subjects that have been administered with aP priming vaccine, the non-TLR agonist-containing aP booster vaccine boosts the Th2-dominant immune response induced by aP priming vaccine, and sustains the Th2-dominant immune response induced by aP priming vaccine.In contrast, the TLR agonist-containing aP booster vaccine described herein unexpectedly redirects or shifts the Th2-dominant immune response induced by aP priming vaccine to Th1-dominant immune response or Th1 / Th17-dominant immune response.

[0130] In certain embodiments, a Th1-dominated immune response is characterized by one or more of decreased IL-5 production, increased IFN-γ production, or a lower IgG1 / IgG2a ratio compared to an immune response induced by an aP priming or booster vaccine that does not contain a TLR agonist (e.g., ADACEL®). In certain embodiments, a Th1-dominated immune response is characterized by decreased IL-5 production and / or a lower IgG1 / IgG2a ratio compared to an immune response induced by an aP priming or booster vaccine that does not contain a TLR agonist (e.g., ADACEL®).

[0131] In certain embodiments, a Th1 / TH17 dominated immune response is characterized by one or more of increased IL-17 production, decreased IL-5 production and / or a lower IgG1 / IgG2a ratio compared to an immune response induced by an aP priming or booster vaccine that does not contain a TLR agonist (e.g., ADACEL®).

[0132] The aP priming vaccine can be administered as a single dose or a series of multiple doses (e.g., 2, 3, 4, or 5 doses) prior to administration of the aP booster vaccine. Typically, the aP priming vaccine is administered as a series of doses, particularly for children. For example, in certain embodiments, the aP priming vaccine is administered as a series of 5 doses for infants and children between 6 weeks and 6 years of age. For infants and children between 6 weeks and 4 years of age, the aP priming vaccine can also be administered as a series of 4 doses. Typically, the primary immunization schedule for children includes administration of the aP priming vaccine at 2 months of age, 4 months of age, 6 months of age, 15-20 months of age, and 4-6 years of age.

[0133] aP booster vaccine is typically administered after the completion of the primary immunization schedule.In certain embodiments, aP booster vaccine is administered to human subjects who are 10 years old or older.In certain embodiments, aP booster vaccine is administered to human subjects who are 4 years old or older.

[0134] Typically, the aP vaccine booster is administered by intramuscular injection.

[0135] In certain embodiments, the aP priming vaccine comprises tetanus toxoid, diphtheria toxoid, detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and optionally FIM2,3, with the proviso that the aP priming vaccine does not contain a TLR agonist.

[0136] In certain embodiments, the aP priming vaccine comprises one or more doses of DAPTACEL®, PENTACEL®, QUADRACEL®, INFANRIX®, INFANRIX-HEXA®, KINRIX®, PEDIARIX®, or VAXELIS®. In certain embodiments, the aP priming vaccine comprises DAPTACEL®. In certain embodiments, the aP priming vaccine comprises PENTACEL® or QUADRACEL®. In certain embodiments, the aP priming vaccine comprises INFANRIX® or INFANRIX-HEXA®. In certain embodiments, the aP priming vaccine comprises KINRIX® or PEDIARIX®. In certain embodiments, the aP priming vaccine comprises VAXELIS®. EXAMPLES

[0137] Materials and Methods – General Bordetella pertussis challenge B. pertussis 18323 was grown on Bordet-Jung agar (Difco) supplemented with 1% glycerol and 20% defibrinated sheep blood (Sanofi Pasteur, Alba La Romaine). After 24 h at 36°C, colonies were transferred to 1% casamino acids (Difco) buffer and the optical density of the bacterial suspension was measured. Mice anesthetized by intramuscular injection of Imalgen (ketamine 60 mg / kg; Merial SAS) and Rompun (xylazine 4 mg / kg; Bayer) were cultured with 5 × 10 10 10 10 20 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 15 20 25 30 35 40 45 50 55 60 65 70 75 8 ... 6Colony forming units (CFU) were instilled intranasally. Mice were then euthanized by intraperitoneal injection of Dolethal (pentobarbital 180 mg / kg; Vetoquinol SA) 2 hours after infection for quantification of the initial number of live B. pertussis CFU in the lungs, and either on days 3, 7, 14, and 21 or on days 1, 2, 3, 7, and 14 for assessment of bacterial colonization. Briefly, lung homogenates were plated on Bordet-Jung agar plates and the number of CFU was counted after 4 days of incubation at 36°C. A measure of disease prevention was expressed as the ratio of the area under the clearance curve (AUC) between naive controls and immunized mice.

[0138] Adjuvant Formulations (all examples) The modified Tdap(gdPT+CpG1018-AlOOH) and modified Tdap(gdPT+E6020-AlOOH) boosters (for mice) contained 10 Lf / ml TT, 4 Lf / ml DT, 20 μg / ml gdPT, 10 μg / ml PRN, 15 μg / ml FIM2 / 3, 10 μg / ml FHA, and 0.66 mg / ml aluminum (AlOOH) with either 500 μg / ml CpG1018 (TLR9 ​​agonist) or 10 μg / ml E6020 (TLR4 agonist).

[0139] Antigens, Adjuvants and Immunizations (All Examples) Mice were primed with an intramuscular injection of 1 / 5 of a human dose (50 μl into both hind legs) of either the pediatric diphtheria-tetanus-acellular pertussis (aP) DAPTACEL® vaccine (Sanofi Pasteur) (abbreviated as DTaP in this example and figures), which contains, in addition to tetanus and diphtheria toxoids, 10 μg chemically detoxified PT, 5 μg FHA, 3 μg PRN and 5 μg FIM2,3, or the diphtheria-tetanus-whole bacterial-cellular pertussis (wP) DTCOQ / DTP vaccine (Sanofi Pasteur) (abbreviated as DTwP in this example and figures), which contains ≧4 I.U. of Bordetella pertussis heat-inactivated in the presence of thiomersal. Recipient mice were boosted with 1 / 5 the human dose (50 μl in both hind legs) of the following formulations: Pediatric Diphtheria-Tetanus-aP ADACEL® Vaccine (Sanofi Pasteur), containing tetanus and diphtheria toxoids plus 2.5 μg chemically detoxified PT, 5 μg FHA, 3 μg PRN and 5 μg FIM2,3 (abbreviated as Tdap in this example and figures); DTCOQ / DTP vaccine (i.e., DTwP); or the modified Tdap booster formulation described above (abbreviated as modified Tdap or mTdap in this example and figures).

[0140] FluoroSpot (all): FluoroSpot assay (ELISPOT utilizing fluorophore-labeled detection reagents) was used to detect splenic IFN-γ, IL-5 or IL-17 cytokine secreting cells. Briefly, 96-well IPFL bottom microplates (Millipore) were pre-wetted with 50 μL of 35% ethanol for 30 seconds. The ethanol was then removed and each well was washed three times with sterile PBS. The microplates were then coated by adding 100 μL / well of rat anti-mouse IFN-γ, rat anti-mouse IL-5 or rat anti-mouse IL-17 antibody solution (PharMigen) at 10 μg / mL and incubated overnight at +4°C. The next day, the plates were washed three times with sterile PBS and then incubated with 200 μL of RPMI GSPβ 10% FBS at +37°C for 2 hours. After washing the plates, 10 freshly isolated splenocytes were added to the wells. 6 Plates per well were incubated with pertussis antigens (PT 2.5 μg / ml; PRN 5 μg / ml; FIM 5 μg / ml; FHA 5 μg / ml) or concanavalin A (ConA, 2.5 μg / ml) as a positive control in the presence of mouse IL-2 (10 U / mL). After 24 h of incubation for IFNγ and IL-17 and 48 h for IL-5, plates were washed six times with PBS supplemented with 0.05% BSA (200 μL / well). After washing, 100 μL / well of biotinylated anti-mouse IFN-γ (2 μg / mL) or anti-mouse IL-5 (1 μg / mL) or anti-mouse IL-17 (1 μg / mL) antibodies were added for 2 h at room temperature in the dark. Plates were then washed three times with PBS-BSA 0.05% (200 μL / well). Then, 100 μL / well of 1 μg / mL streptavidin-PE in PBS-BSA 0.05% was incubated for 1 h at room temperature in the dark. The plates were washed another six times with PBS-BSA 0.05% (200 μL / well). The plates were stored in the dark at +5° C.±3° C. until reading. Each spot corresponding to IFN-γ, IL-5 or IL-17 secreting cells was counted in an automated ELISPOT fluorescent plate reader (Microvision). Results were analyzed using 10 spleen cells. 6The results were expressed as the number of IFN-γ, IL-5 or IL-17 secreting cells per individual. Geometric means and standard deviations were calculated for each group.

[0141] Antibody quantification Serum IgG1 and IgG2a antibodies specific for pertussis antigens (FIM, PT, FHA, PRN), diphtheria toxoid, and tetanus toxoid were titrated with a multiplex U-PLEX assay (Meso-Scale Diagnostics, Rockville, MD).

[0142] The U-PLEX assay consists of five unique U-PLEX linkers that specifically bind to five individual spots on a 96-well U-PLEX plate. The biotin-based capture linkage mechanism involves a two-step process: (1) the linker is bound to the biotinylated antigen, and (2) the linker-linked antigen is bound to the plate. Serial dilutions of serum samples, controls and reference sera were added, a washing step was performed, and IgG1 or IgG2a antibodies bound to the coated antigen were detected using SULFO-TAG™-labeled anti-IgG1 or SULFO-TAG™-labeled anti-IgG2a. EXAMPLES

[0143] Comparison of immunogenicity of genetically detoxified (gdPT) with chemically detoxified PT (PTxd) A study was conducted to compare the immunogenicity of genetically detoxified pertussis toxin (gdPT) with that of chemically detoxified PT (PTxd). Groups of naive CD1 mice were immunized with 2.5, 0.5, 0.1 or 0.02 μg per mouse dose of gdPT or PTxd, two times, each 3 weeks apart (on days 0 and 21). In addition, to evaluate the ability of gdPT to boost responses primed by PTxd, one group of mice was immunized with a single 0.5 μg dose of PTxd, followed by two gdPT immunizations at 0.5 μg per dose, each 3 weeks apart (on days 0, 21 and 42). All formulations contained 0.066 mg AlPO4 in a 100 μL injection volume. Blood samples were taken 17 days after the second immunization (day 38) and 8 days after the third immunization (day 50) for analysis of IgG1 and IgG2a titers by pertussis toxin-specific ELISA and PT neutralizing antibody titers.

[0144] Formulations containing gdPT induce strong and dose-dependent anti-PT specific IgG1 and IgG2a responses, especially at low doses, as well as higher PT neutralizing antibody titers, compared to formulations of PTxd (Figure 1A-B). Both gdPT and PTxd can similarly boost PT specific IgG1 and IgG2a responses primed with PTxd (Figure 2A). However, priming mice with PTxd and boosting with gdPT resulted in higher PT neutralizing antibody titers than boosting with PTxd (Figure 2B).

[0145] Next, studies were conducted to assess whether the replacement of PTxd with gdPT in the Tdap vaccine would have an impact on the immunogenicity of other vaccine components. To address this possibility, the immunogenicity of a control Tdap vaccine formulation containing PTxd was compared to that of a vaccine formulation containing gdPT. Vaccines containing 2.0, 0.1, or 0.02 μg / dose of gdPT were formulated in combination with other Tdap vaccine antigens (1 Lf tetanus toxoid, 0.4 Lf diphtheria toxoid, 1 μg FHA, 1 μg PRN, 1.5 μg FIM2,3, and 66 μg aluminum (AlOOH) per mouse dose in 100 μL, or 1 / 5 human dose). A control Tdap vaccine containing 2 μg of chemically detoxified PT was formulated as a comparison control at the same Tdap antigen concentration. The study also included groups of mice receiving gdPT alone at 0.1 or 0.02 μg per dose in AlOOH. CD1 mice received three immunizations of the vaccine formulation, spaced 3 weeks apart, and were sacrificed 7 days after the last immunization. Blood samples were taken 10 days after the second immunization (Figure 3) and at sacrifice for analysis of PT-specific IgG1 and IgG2a titers and PT-neutralizing antibody responses (Figures 4A-B).

[0146] The modified Tdap vaccine containing gdPT (2 μg per dose and 3 immunizations per mouse) elicited higher PT neutralizing antibody titers than an otherwise identical control Tdap vaccine containing 2 μg PTxd per dose (Figure 4B), whereas the gdPT- and PTxd-containing Tdap vaccines had comparable PT-specific IgG1 and IgG2a titers (Figure 4A). Furthermore, IgG1 and IgG2a antibody titers against FIM were comparable between the gdPT- and PTxd-containing Tdap formulations (Figure 3). Similar IgG1 and IgG2a profiles were observed for other vaccine antigens (i.e., tetanus toxoid, diphtheria toxoid, FHA, and PRN). Thus, gdPT did not affect the antibody responses induced by other vaccine antigens (Figure 3). However, the presence of other Tdap vaccine antigens appeared to reduce the PT-neutralizing antibody titers induced by low doses of gdPT (Fig. 4A, B). Specifically, the mean log PT-neutralizing titers induced by 0.1 μg gdPT 7 days after the third immunization were significantly higher (0.7-fold) than those with Tdap antigens in the absence of other Tdap antigens (Fig. 4B), whereas no differences were observed in PT-specific IgG1 and IgG2a titers (Fig. 4A).

[0147] In conclusion, gdPT was found to be more immunogenic than PTdx in Tdap formulations, and gdPT did not interfere with the immunogenicity of other Tdap antigens in the vaccine formulation. EXAMPLES

[0148] TLR adjuvants reorient DTaP-induced Th2 immune memory responses toward Th1 using an extended prime-boost schedule A long-term immunization schedule was applied to test the ability of modified Tdap formulations to boost and re-bias previously established Th2-dominated memory responses. In this study, groups of eight CD1 mice were primed by intramuscular injection of DTaP vaccine to establish Th2-dominated memory responses. In the first study, mice were boosted 6 weeks later (day 42) with modified Tdap (gdPT+E6020-AlOOH) or modified Tdap (gdPT+CpG1018-AlOOH) formulations. In the second study, mice received two doses of 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 formulation to re-orient immune memory responses toward Th1 was evaluated by measuring ex vivo splenic cytokine-producing cells or cytokine production in the supernatant after in vitro antigen restimulation. Antibody titers (IgG1 and IgG2a) against tetanus toxoid (TT), diphtheria toxoid (DT), PT, FHA, PRN and FIM were also determined in sera taken 1, 3 and 6 days after the last immunization. As controls, one group of mice was immunized with DTwP vaccine (prime) followed by DTwP vaccine (boost) and another group was immunized with DTaP vaccine (prime) followed by a boost with a control modified Tdap vaccine formulation with gdPT-AlOOH (20 μg / ml gdPT) but without a TLR agonist, i.e., control modified Tdap (gdPT-AlOOH).

[0149] The DTwP / DTwP (prime / boost) schedule induced a less Th2-dominant immune profile as evidenced by weaker IL-5 secretion (Figure 5) and lower IgG1 / IgG2a ratios (Figure 6A-L) compared to the DTaP / modified control Tdap(gdPT-AlOOH) (prime / boost) schedule. The modified Tdap(gdPT+E6020-AlOOH) or modified Tdap(gdPT+CpG101-AlOOH) boost formulations induced significantly reduced IL-5 production compared to the control modified Tdap(gdPT-AlOOH) boost vaccine (Figure 5). However, none of these novel modified Tdap formulations containing TLR agonist adjuvants altered the IFN-γ levels observed with the control modified Tdap(gdPT-AlOOH) (Figure 5). Consistent with the overall cytokine balance towards a less Th2-dominated response, lower IgG1 / IgG2a ratios were observed in mice boosted with modified Tdap (gdPT+E6020-AlOOH) or modified Tdap (gdPT+CpG1018-AlOOH) vaccines (Figure 6A-L). After immunization with mTdap-CpG-AlOOH, a statistically significant decrease in the IgG1 / IgG2a ratio was observed after one boost for anti-FHA and two boosts for anti-FIM compared to mice immunized with mTdap-AlOOH without a TLR agonist (Figure 6A and 6D). After immunization with mTdap-E6020-AlOOH, a statistically significant decrease in the IgG1 / IgG2a ratio was observed after one boost for anti-FHA compared to mice immunized with mTdap-AlOOH without a TLR agonist (Figure 6A). In mice primed with DTaP, no statistically significant differences were observed in IL-17 secretion after boosting with modified Tdap(gdPT+E6020-AlOOH) or modified Tdap(gdPT+CpG1018-AlOOH) formulations compared to the control modified Tdap(gdPT-AlOOH) boost vaccine containing no TLR agonist (Figure 5).

[0150] In conclusion, the modified Tdap(gdPT+E6020-AlOOH) and modified Tdap(gdPT+CpG1018-AlOOH) vaccines were able to modify the helper T cell response to a less Th2-dominant response in DTaP-primed mice, thus achieving a repolarization of the helper T cell response and a shift in the Th1 / Th2 balance. EXAMPLES

[0151] Adoptive transfer model of pertussis immunity Modified Tdap(gdPT+E6020-AlOOH) and modified Tdap(gdPT+CpG1018-AlOOH) formulations were tested for their ability to reactivate DTaP-induced immune memory responses for B. pertussis protection in the absence of circulating antibodies induced by DTaP vaccination.

[0152] One difference between humans and mice is the longevity of serum antibodies induced by DTaP priming. While these antibodies decay rapidly in humans, their long-term antibody persistence in mice interferes with the readout of booster responses and therefore with the evaluation of modified Tdap booster formulations. To address this difference, we used a mouse dual transfer model to eliminate circulating antibodies induced by DTaP vaccination (Gavillet et al. 2015 Vaccine), as summarized in Figure 7. In this setting, adult BALB / c mice were primed once with DTwP or DTaP, and 6 weeks later their splenocytes were harvested and transferred (50 × 10 6 Recipient mice were boosted with DTwP (only in the DTwP-primed background), Tdap, modified Tdap (gdPT+E6020-AlOOH) or modified Tdap (gdPT+CpG1018-AlOOH) formulations. Six weeks later, splenocytes from boosted mice were harvested and transferred (50 × 10 6 One week after transfer, the double adoptive transfer recipient mice were 6Mice were challenged with live Bordetella pertussis and sacrificed at 0, 7, 10, 14 or 21 days post-challenge. Bacterial counts in the lungs of sacrificed mice were measured as an index of effective immune recall responses in the absence of circulating antigen-specific antibodies induced by vaccination (Figure 9A-B). Vaccine-induced responses were also monitored by detection of PT-, PRN-, FHA- and FIM2,3-specific IgG antibody responses in sera collected 7, 14, 21, 28 and 42 days after boost (Figure 8A-B). Accelerated and higher vaccine antigen IgG titers are characteristic of recall antibody responses in adoptively transferred mice.

[0153] In the absence of circulating antibodies upon B. pertussis challenge, memory cells in DTwP / DTwP (prime / boost) mice led to early B. pertussis control and facilitated bacterial clearance from the lungs (Figure 9A). This was due, at least in part, to the rapid generation of antibodies specific for pertussis vaccine antigens such as PT and PRN (Figure 8A). In contrast, DTaP priming / Tdap boosting did not accelerate bacterial clearance in the lungs compared to naive control animals (Figure 9A-B). In this adoptive transfer model, mice primed with DTap and boosted with modified Tdap (gdPT+CpG1018-AlOOH) and modified Tdap (gdPT+E6020-AlOOH) boosts showed higher anti-PT, FHA, and FIM IgG titers that were accelerated compared to mice that received a Tdap boost (Figure 8B).

[0154] Substitution of the chemically detoxified PT with gdPT in the Tdap-boosted vaccine did not affect pulmonary clearance of B. pertussis despite the enhancement of PT-specific IgG antibody responses (data not shown). Nevertheless, a strong reactivation of antigen-specific memory responses was observed for the modified Tdap(gdPT+CpG1018-AlOOH) vaccine. Early bacterial control and accelerated bacterial clearance were recorded in the DTaP-primed / modified Tdap(gdPT+CpG1018-AlOOH)-boosted group with kinetics similar to those of the DTwP-primed / DTwP-boosted group (Figure 9A-B). Similarly, rapid generation of antibodies specific for PT and FHA vaccine antigens was observed with the modified Tdap(gdPT+CpG1018-AlOOH) formulation (Figure 8B). The modified Tdap(gdPT+E6020-AlOOH) study suggested early reactivation of pertussis-specific antibody responses against some aP antigens, e.g., PT and FHA (Figure 8B). However, less impact on early B. pertussis control was observed with the modified Tdap(gdPT+E6020-AlOOH) boost compared to the modified Tdap(gdPT+CpG1018-AlOOH) boost (Figure 9B). In conclusion, this adoptive transfer experiment in mice demonstrated that the modified Tdap(gdPT+E6020-AlOOH) and modified Tdap(gdPT+CpG1018-AlOOH) booster formulations can improve pertussis-specific recall antibody responses, resulting in accelerated protection against bacterial colonization.

[0155] Materials and Methods for Example 3 Mice: Adult BALB / cByJ mice were purchased from Charles River (L'Arbresle, France) and housed under specific pathogen-free conditions. Mice were used at 6–8 weeks of age. All animal experiments were performed in accordance with Swiss and European guidelines and approved by the Geneva Veterinary Office.

[0156] Adoptive transfer: Spleens were harvested 42 days after priming or boosting. Single cell suspensions were obtained by mechanical disruption of the organ and further processed for red blood cell lysis. 50×106 Splenocytes were transferred intravenously into naive recipient mice in a volume of 100 μl.

[0157] B. pertussis challenge: Streptomycin-resistant B. pertussis Tahoma I derivative BPSM was grown on Bordet-Jung agar (Difco) supplemented with 1% glycerol, 10% defibrinated sheep blood (Chemie Brunschwig AG) and 100 μg / ml streptomycin. After 24 h at 37°C, colonies were transferred to PBS and the optical density of the bacterial suspension was measured. Mice anesthetized by intraperitoneal injection of Ketasol (100 mg / kg; Graeub) and Rompun (10 mg / kg; Bayer) were cultured with 1x10 bacteria in a volume of 20 μl. 6 Colony forming units (CFU) were instilled intranasally. Mice were sacrificed 3 hours after infection for quantification of the initial number of live B. pertussis CFU in the lungs, and on days 7, 10, 14, and 21 for assessment of bacterial colonization. Briefly, lung homogenates were plated on Bordet-Jung agar plates, and the number of CFU was counted after 4 days of incubation at 37°C. A measure of disease prevention was expressed as the ratio of the area under the clearance curve (AUC) between naive controls and immunized mice.

[0158] ELISA: PT-, PRN-, FHA- and FIM2,3-specific antibody titers were determined by Ag capture ELISA in serum samples taken at the indicated time points. Briefly, 96-well plates (Nunc MaxiSorp™; Thermo Fisher Scientific) were coated overnight at 4°C with PT (1 μg / ml), PRN (5 μg / ml), FHA (5 μg / ml) or FIM2,3 (2 μg / ml) in carbonate buffer, pH 9.6. After saturation for 1 h at 37°C with PBS, 0.05% Tween 20 and 1% BSA (Sigma), wells were incubated for 1 h at 37°C with two-fold serial dilutions of individual or pooled mouse sera. Secondary horseradish peroxidase (HRP)-conjugated anti-mouse IgG, anti-mouse IgG2a (both from Invitrogen), and anti-IgG1 (BD Pharmingen) were incubated for 1 h at 37° C. and revealed with 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) substrate for 1 h. The optical density of each well was measured at 405 nm and the data was analyzed with SoftMax Pro software. The results are shown in FIG. 8, with the Log of Ag-specific titers determined against serial dilutions of WHO / NIBSC standard reagent Bordetella pertussis antiserum (NIBSC code: 97 / 642) for IgG and IgG1, and against serial dilutions of a titered pool of hyperimmune sera from immunized mice for IgG2a. 10 It is expressed as:

[0159] Statistical analysis: Values ​​are expressed as mean ± SEM. Statistical analysis was performed using unpaired t-test or one-way ANOVA followed by Tukey's multiple comparisons when more than two groups of mice were tested. All analyses were performed using GraphPrism software. Differences with p>0.05 were considered not significant. EXAMPLES

[0160] Mouse intranasal challenge model A mouse lung clearance model that reflects the clinical efficacy level of licensed pertussis vaccines has been developed, implanted, and evaluated (Guiso N. et al., Vaccine. 1999;17:2366-76). This mouse intranasal challenge assay (INCA) model is recommended by WHO for non-clinical trials for registration of new vaccine candidates and is known as a valid research model for 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). In addition, the WHO Advisory Panel has recognized that INCA is useful for assessing the potential impact of changing the formulation and / or manufacturing process of pertussis vaccines.

[0161] In this model, after intranasal challenge with a sublethal dose, bacteria adhere to the ciliated epithelium of the trachea and invade macrophages in the lungs. The disease persists for 4-8 weeks, accompanied by leukocytosis and immunosuppression. Rapid clearance of infection is observed after rechallenge of convalescent mice. The INCA model has been proven to identify acellular pertussis vaccines that have shown different efficacy in clinical trials (Guiso N. et al. 1999).

[0162] The INCA mouse model was used to evaluate whether novel modified Tdap booster vaccines, such as modified Tdap (gdPT+CpG1018-AlOOH) and modified Tdap (gdPT+E6020-AlOOH) vaccines, elicit short-term protection levels equal to or higher than Tdap control boost vaccines in mice initially immunized with DTaP. Figure 10A shows a pictorial representation of a mouse intranasal challenge assay (INCA) using a short-term schedule, and Figure 10B shows a pictorial representation of a mouse INCA using a long-term schedule. Figure 10C shows that a DTwP / DTwP (prime / boost) schedule and a DTaP / Tdap (prime / boost) schedule prevent B. pertussis lung colonization of mice after intranasal challenge in this model using a short-term schedule.

[0163] Mouse intranasal challenge assay using a short prime-boost schedule A 2-weekly short-term immunization schedule was applied as described in the WHO harmonized protocol (WHO Expert Committee on Biological Standardization, 2011). The readouts for this study were reduction in bacterial burden in the lungs (CFU / lung) as well as leukocytosis based on white blood cell (WCB) counts in the blood.

[0164] Groups of 40 mice were injected with 1 / 5 human dose of DTaP by intramuscular route and 2 weeks later received a second injection of Tdap, modified Tdap (gdPT+E6020-AlOOH) vaccine or a control Tdap vaccine formulated with chemically detoxified PT, AlOOH and no TLR agonist (control Tdap (PTxd-AlOOH)). A group was also included that received two injections (prime / boost) of DTwP. Two control groups received either PBS (infected control) or E6020-AlOOH adjuvant alone to verify that aluminum salts and E6020 do not affect colonization compared to infected control.

[0165] Two weeks after the second immunization, mice were challenged by instillation of a suspension of B. pertussis 18323 into the nares. After challenge, eight mice from each group were sacrificed 2 hours and 3, 7, 14, and 21 days after challenge. Lungs were aseptically removed and individually homogenized to measure bacterial burden. The average CFU per lung was determined by counting colonies grown on Bordet-Jung agar plates after plating serial dilutions of the homogenate. Blood samples were also taken after challenge and WBC counts were performed.

[0166] As shown in FIG. 11, B. pertussis 18323 colonized and expanded in the lungs of PBS control mice 3 days after challenge before entering the clearance phase. Complete B. pertussis lung clearance took longer than 21 days in PBS control mice. Adjuvant alone had no effect on lung colonization and disease. Reduction in B. pertussis lung colonization was observed in all vaccinated groups 3 days after challenge, with all formulations reaching baseline bacterial loads 14 days after challenge. DTwP produced the fastest B. pertussis lung clearance, reaching baseline 7 days after challenge. Results showed that the modified Tdap(gdPT+E6020-AlOOH) formulation significantly reduced bacterial load in the lungs (4 logs on day 3 and 5 logs on day 7) compared to infected controls, demonstrating its ability to prevent colonization of the lower respiratory tract after intranasal challenge with B. pertussis 18323. Notably, the clearance profile was closer to that obtained for DTwP / DTwP (prime / boost) and more distant from that obtained for DTaP / Tdap (prime / boost). The difference in bacterial load obtained on day 7 between the modified Tdap (gdPT+E6020-AlOOH) formulation boost and the Tdap (containing chemically detoxified PT) boost was significant (p-value<0.0001). All vaccines were protective against disease (FIG. 11) and prevented leukocytosis. This study demonstrated that the modified Tdap (gdPT+E6020-AlOOH) formulation significantly accelerated Bordetella pertussis clearance compared to Tdap (ADACEL®) in mouse INCA using a short prime-boost schedule.

[0167] Mouse intranasal challenge assay using an extended prime-boost schedule To conduct a study to confirm the efficacy of the novel modified Tdap formulation in mice with a previously established immunological background with aP priming, the ability of the modified Tdap(gdPT+E6020-AlOOH) booster to prevent lung colonization and disease with Bordetella pertussis using an extended prime-boost schedule as discussed above was evaluated. See FIG. 10B. In this study, the acellular pertussis vaccine control was a Tdap vaccine (control Tdap(PTxd-AlOOH)) modified to contain chemically detoxified PT but the same dose of antigen and aluminum salt (AlOOH) as in the modified Tdap(gdPT+E6020-AlOOH) formulation to better understand the role of gdPT and TLR agonists in the novel modified Tdap formulation.

[0168] Groups of 40 mice were injected with 1 / 5 human dose of DTaP by intramuscular route and 6 weeks later received a second injection with modified Tdap (gdPT+E6020-AlOOH) or with control Tdap (PTxd-AlOOH). Groups primed with DTwP and boosted with DTwP were also included. Six weeks after the second immunization, mice were challenged by instillation into the nares with a suspension of Bordetella pertussis 18323. After challenge, eight mice from each group were sacrificed 2 hours and 1, 2, 3, 7, and 14 days after challenge. Lungs were aseptically removed and individually homogenized to measure bacterial load. The average CFU per lung was determined by counting colonies grown on Bordet-Jung agar plates after plating serial dilutions of the homogenate. Blood samples were also taken after challenge and WBC counts were performed.

[0169] As shown in Figure 12B, the modified Tdap(gdPT+E6020-AlOOH) formulation significantly reduced the bacterial burden in the lungs (6 log reduction) as early as day 1 compared to infected controls. A significant difference (p value=0.007) was also observed between modified Tdap(gdPT+E6020-AlOOH) and control Tdap(PTxd-AlOOH) with a 7.7-fold reduction in CFU counts in the lungs on day 1. The bacterial burden of all vaccinated mice returned to baseline by day 7 indicating no detectable bacteria in the lungs (Figure 12B).

[0170] This study demonstrated that the modified Tdap (gdPT+E6020-AlOOH) formulation was more effective at preventing lung colonization (at least during the early stages of infection) in an extended prime-boost schedule than Tdap controls that did not contain gdPT or a TLR agonist, suggesting that this effect is due to the gdPT and / or TLR agonist contained in the modified Tdap formulation. EXAMPLES

[0171] Th17-secreting cells measured by fluorospot in splenocytes To study the ability of TLR adjuvants to re-bias immune responses from Th2 towards more Th-17 responses, IL-17 cytokines were measured in the following experiment. CD1 mice were primed with the DTaP composition and boosted twice, 2 weeks apart, at DO and D21 with mTdap-E6020-AlOOH with or without TLR4 adjuvant in a dose-effect design. E6020 and AlOOH were present in the mTdap formulation at amounts of 10 μg and 66 μg, respectively. The mTdap antigen components were as follows per 0.1 mL dose:

[0172] [Table 3]

[0173] Blood samples were taken from mice on D20 (200 μL) and D35 (bleeding) for all groups under anesthesia (Imalgen / Rompun 80 mg / kg ketamine and 16 mg / kg quirilazine, 100 μL / 10 g or isoflurane). Cellular immune responses in splenocytes were analyzed by ex vivo fluorospot assay to measure IL-17 secreting cells and by MSD assay to measure secretion of IL-17. The fluorospot technique was described above. After 3 days of in vitro stimulation with PTx 2.5 μg / mL (Figure 13A) or a pool of pertussis antigens (2.5 μg / mL PTx, 5 μg / mL PRN, 5 μg / mL FIM) (Figure 13B), the production of Th17 cytokines (IL-17) was measured in the supernatants of splenocytes using the fluorospot assay and the MSD Uplex kit.

[0174] As shown in Figures 13A-B, the frequency of IL-17-secreting cells was below the positive cutoff in the fluorospot assay (19 spots / 10) after injection of the mTdap vaccine that does not contain TLR4. 6 mTdap-E6020 formulations induced a dose-dependent increase in the amount of IL-17-secreting cells from 0.1 μg to 4 μg / dose when splenocytes were restimulated with PT toxoid (PTx) (FIG. 13A) or a pool of pertussis antigens (FIG. 13B). The mTdap boosted vaccine containing 4 μg E6020 / dose increased the frequency of IL-17-secreting cells above the positivity cutoff in the fluorospot assay when restimulated with PTx (FIG. 13A), whereas the mTdap boosted vaccines containing 0.5, 1, 4, and 10 μg E6020 / dose increased the frequency of IL-17-secreting cells above the positivity cutoff in the fluorospot assay when restimulated with a pool of pertussis antigens (FIG. 13B).

[0175] In general, the cytokine secretion profiles measured by MSD were consistent with the cytokine-secreting cell frequencies measured by Fluorospot (data not shown). mTdap formulations containing E6020 induced IL-17 secretion in a dose-effect manner, with maximal efficacy achieved at 0.5 μg-4 μg / dose. EXAMPLES

[0176] thermal stability NanoDSF was performed using a Prometheus NT.48 system (Nano Temper Technologies, Munich, Germany). NanoDSF uses autofluorescence to assess changes in aromatic residues (fluorophores) in proteins due to changes in the local environment. The change in autofluorescence, which indicates that the protein is unfolded, is used to monitor the shift in fluorescence emission and the intensity change. The melting temperature (Tm), which indicates the point at which half of the protein is unfolded, is used to characterize the thermal stability of the protein. In the nanoDSF method, this is determined by using the ratio of the fluorescence recorded at 330 nm and 350 nm. This ratio has been shown to be more sensitive in detecting the Tm compared to the use of a single wavelength. The sample was loaded into a capillary without any further preparation and excited at 285 nm using 100% power. The temperature profile was recorded from 20 to 95 °C with a scan rate of 2 °C / min.

[0177] The tertiary structure and thermal stability of the different vaccine formulations can be assessed by nanoDSF to assess whether any differences in conformation can be detected when the vaccine is formulated with different adjuvants. For all vaccine formulations, one temperature transition was detected. Figure 14. When the mTdap vaccine (gdPT) was adsorbed to AlOOH (mTdap-AlOOH), the mTdap vaccine had a temperature transition of 74.6°C. Similarly, when the mTdap-AlOOH formulation contained E6020 (mTdap E6020-AlOOH), the temperature transition was 74.2°C. When the mTdap-AlOOH formulation contained CpG (mTdap CpG-AlOOH), there was a small increase in the temperature transition, which was detected at 77.0°C.

[0178] While one or more exemplary embodiments have been described herein, it will be understood by those skilled in the art that various changes in form and detail may be made to such embodiments without departing from the spirit and scope of the inventive concept as defined by the following claims.

Claims

1. An acellular pertussis (aP) booster vaccine for use in redirecting a Th2-dominated immune response toward a Th1-dominated or Th1 / Th17-dominated immune response in a human subject previously exposed to a Bordetella pertussis antigen, wherein said exposure to the Bordetella pertussis antigen has induced a Th2-dominated immune response in said human subject, comprising tetanus toxoid, diphtheria toxoid, detoxified pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae types 2 and 3, at least one Toll-like receptor (TLR) agonist, and an aluminum salt, wherein the at least one TLR agonist is formulated with the aluminum salt. The aP booster vaccine.

2. 2. The aP booster vaccine for use according to claim 1, wherein the TLR agonist is a TLR4 agonist and / or a TLR9 agonist.

3. 3. The aP booster vaccine for use according to claim 2, wherein the TLR4 agonist comprises E6020.

4. 3. The aP booster vaccine for use according to claim 2, wherein the TLR9 agonist comprises CpG1018.

5. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the tetanus toxoid is present in an amount of 8 to 12 Lf / mL.

6. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the diphtheria toxoid is present in an amount of 3 to 8 Lf / mL.

7. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the detoxified pertussis toxin is a genetically detoxified pertussis toxin and is present in an amount of 16 to 24 μg / mL.

8. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the filamentous hemagglutinin is present in an amount of 5 to 15 μg / mL.

9. The aP booster vaccine for use according to any one of claims 1 to 4, wherein pertactin is present in an amount of 5 to 15 μg / mL.

10. The aP booster vaccine for use according to any one of claims 1 to 4, wherein fimbriae types 2 and 3 are present in an amount of 10 to 20 μg / mL.

11. The aP booster vaccine for use according to any one of claims 2 to 4, wherein the TLR4 agonist is present in an amount of 10 μg / mL or less.

12. The aP booster vaccine for use according to any one of claims 2 to 4, wherein the TLR9 agonist is present in an amount of 250 to 750 μg / mL.

13. The aP booster vaccine for use according to any one of claims 1 to 4, further comprising Tris-buffered saline.

14. The aP booster vaccine for use according to any one of claims 1 to 4, having an aluminium concentration of 0.5 to 0.75 mg / mL.

15. 5. The aP booster vaccine for use according to any one of claims 1 to 4, wherein at least one of the tetanus toxoid, diphtheria toxoid and detoxified pertussis toxin is adsorbed to an aluminium salt, and the detoxified pertussis toxin is a genetically detoxified pertussis toxin.

16. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the aluminium salt is aluminium hydroxide or aluminium phosphate.

17. 5. An aP booster vaccine for use according to any one of claims 1 to 4, wherein the tetanus toxoid is present in an amount of 9-11 Lf / mL, the diphtheria toxoid is present in an amount of 3-8 Lf / mL, the detoxified pertussis toxin is genetically detoxified pertussis toxin and is present in an amount of 16-24 μg / mL, the filamentous hemagglutinin is present in an amount of 5-15 μg / mL, the pertactin is present in an amount of 5-15 μg / mL, the fimbriae types 2 and 3 are present in an amount of 10-20 μg / mL, the aluminium salt is aluminium hydroxide and is present in a concentration of 0.25-0.75 mg / mL, and the TLR agonist is a TLR4 agonist and / or a TLR9 agonist.

18. 18. The aP booster vaccine for use according to claim 17, wherein the TLR4 agonist comprises E6020 and is present in an amount of 2 μg / mL or less, or the TLR9 agonist comprises CpG1018 and is present in an amount of 400-600 μg / mL.

19. 19. An aP booster vaccine for use according to claim 18, wherein the tetanus toxoid is present in an amount of 10 Lf / mL, the diphtheria toxoid is present in an amount of 4-5 Lf / mL, the detoxified pertussis toxin is genetically detoxified pertussis toxin and is present in an amount of 20 μg / mL, the filamentous hemagglutinin is present in an amount of 10 μg / mL, the pertactin is present in an amount of 10 μg / mL, the fimbriae types 2 and 3 are present in an amount of 15 μg / mL, the aluminium salt is aluminium hydroxide and is present in a concentration of 0.66 mg / mL, and the TLR agonist is a TLR4 agonist and / or a TLR9 agonist.

20. 20. The aP booster vaccine for use according to claim 19, wherein the TLR4 agonist comprises E6020 and is present in an amount of 0.5-5 μg / mL, or the TLR9 agonist comprises CpG1018 and is present in an amount of 500 μg / mL.

21. 21. The aP booster vaccine for use according to claim 20, in a 0.5 mL unit dosage form for administration to a human subject, wherein tetanus toxoid is present in an amount of 5 Lf, diphtheria toxoid is present in an amount of 2-2.5 Lf, genetically detoxified pertussis toxin is present in an amount of 10 μg, filamentous hemagglutinin is present in an amount of 5 μg, pertactin is present in an amount of 5 μg, fimbriae types 2 and 3 are present in an amount of 7.5 μg / mL, aluminum hydroxide is present in a concentration of 0.33 mg, E6020 is present in an amount of 0.25-2.5 μg, or CpG1018 is present in an amount of 250 μg.

22. The aP booster vaccine for use according to any one of claims 1 to 4, wherein the detoxified pertussis toxin is a genetically detoxified pertussis toxin and comprises the R9K and E129G mutations.

23. The aP booster vaccine for use according to any one of claims 1 to 4, further comprising Haemophilus influenzae type b glycoconjugate, Hepatitis B virus surface antigen, and / or inactivated poliovirus.

24. The method of claim 1, wherein a human subject previously exposed to a Bordetella pertussis antigen has received an acellular pertussis (aP) priming vaccine.

25. The aP booster vaccine of any one of claims 1 to 4, wherein the human subject is aged 4 years or older.

26. The aP booster vaccine of any one of claims 1 to 4, wherein the human subject is aged 10 years or older.

27. The aP booster vaccine of any one of claims 1 to 4, wherein the Th1 dominated immune response is characterized by one or more of decreased IL-5 protection or a lower IgG1 / IgG2a ratio when compared to a Th2 dominated immune response induced by an aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist, and the Th1 / Th17 dominated response is characterized by one or more of increased IL-17 protection and decreased IL-5 protection or a lower IgG1 / IgG2a ratio when compared to a Th2 dominated immune response induced by an aP priming vaccine or an aP booster vaccine that does not contain a TLR agonist.

28. 25. The aP booster vaccine of claim 24, wherein the aP priming vaccine comprises tetanus toxoid, diphtheria toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3, but does not contain a TLR agonist.

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