Bbvac: A Live Vaccine Candidate That Provides Long-Lasting Anamnestic and Th17-Mediated Immunity against the Three Classical Bordetella spp

Abstract

Acute pathogens such as Bordetella pertussis can cause severe disease but are ultimately cleared by the immune response. This has led to the accepted paradigm that convalescent immunity is optimal and therefore broadly accepted as the "gold standard" against which vaccine candidates should be compared. However, successful pathogens like B. pertussis have evolved multiple mechanisms for suppressing and evading host immunity, raising the possibility that disruption of these mechanisms could result in substantially stronger or better immunity. Current acellular B. pertussis vaccines, delivered in a 5-dose regimen, induce only short-term immunity against disease and even less against colonization and transmission. Importantly, they provide modest protection against other Bordetella species that cause substantial human disease. A universal vaccine that protects against the three classical Bordetella spp. could decrease the burden of whooping cough-like disease in humans and other animals. Our recent work demonstrated that Bordetella spp. suppress host inflammatory responses and that disrupting the regulation of immunosuppressive mechanisms can allow the host to generate substantially stronger sterilizing immunity against the three classical Bordetella spp. Here, we identify immune parameters impacted by Bordetella species immunomodulation, including the generation of robust Th17 and B cell memory responses. Disrupting immunomodulation augmented the immune response, providing strong protection against the prototypes of all three classical Bordetella spp. as well as recent clinical isolates. Importantly, the protection in mice lasted for at least 15 months and was associated with recruitment of high numbers of B and T cells in the lungs as well as enhanced Th17 mucosal responses and persistently high titers of antibodies. These findings demonstrate that disrupting bacterial immunomodulatory pathways can generate much stronger and more protective immune responses to infection, with important implications for the development of better vaccines. IMPORTANCE Infectious diseases are a major cause of morbidity and mortality in the United States, accounting for over 40 million hospitalizations since 1998. Therefore, novel vaccine strategies are imperative, which can be improved with a better understanding of the mechanisms that bacteria utilize to suppress host immunity, a key mechanism for establishing colonization. Bordetella spp., the causative agents of whooping cough, suppress host immunity, which allows for persistent colonization. We discovered a regulator of a bacterial immunosuppressive pathway, which, when mutated in Bordetella spp., allows for rapid clearance of infection and subsequent generation of protective immunity for at least 15 months. After infection with the mutant strain, mice exhibited sterilizing immunity against the three classical Bordetella spp., suggesting that the immune response can be both stronger and cross-protective. This work presents a strategy for vaccine development that can be applied to other immunomodulatory pathogens.

Keywords: Bordetella; Bordetella bronchiseptica; Bordetella parapertussis; Bordetella pertussis; antibodies; immunity; mucosal immunity; mucosal vaccines; vaccine.

FIG 1

Bbvac confers sterilizing immunity against infection with the 3 classical Bordetella spp. C57BL/6J mice were intranasally inoculated with PBS (black) or PBS containing 5 × 10⁵ CFU of B. bronchiseptica (blue), Nobivac (green), or Bbvac (red). Another group was intraperitoneally vaccinated with 1/5 of Adacel (purple). Ninety days later, mice were intranasally challenged with PBS containing 5 × 10⁵ CFU of B. bronchiseptica (A), B. pertussis (B), or B. parapertussis (C). Mice were euthanized 7 days postinoculation, and colonies were enumerated in the respiratory tract. Two-way ANOVA, Dunnett's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error of the mean. n = 4 per condition.

FIG 2

Bbvac confers robust protection against clinical isolates of B. bronchiseptica. C57BL/6J mice were intranasally vaccinated with PBS or PBS containing 5 × 10⁵ CFU of Bbvac. Three (A) and 7 (B) months after vaccination, mice were intranasally challenged with PBS containing 5 × 10⁵ CFU of different clinical isolates of B. bronchiseptica. Mice were euthanized 7 days postinoculation, and colonies were enumerated in the respiratory tract. Two-way ANOVA, Dunnett's multiple-comparison test. ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error of the mean. n = 4 per strain and condition.

FIG 3

Bbvac confers robust protection for at least 15 months postvaccination. C57BL/6J mice were intranasally vaccinated with PBS (black) or PBS containing 5 × 10⁵ CFU of Bbvac (red). Seven and 15 months after vaccination, mice were intranasally challenged with PBS containing 5 × 10⁵ CFU of B. bronchiseptica (A, B, and C), B. pertussis (D, E, and F), or B. parapertussis (G, H, and I). Mice were euthanized 7 days postinoculation, and colonies were enumerated in the respiratory tract. Two-way ANOVA, Dunnett's multiple-comparison test. ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error of the mean. n = 4 per strain and condition.

FIG 4

Bbvac promotes α-bordetellae antibodies for at least 15 months. C57BL/6J mice were intranasally vaccinated with PBS (black) or PBS containing 5 × 10⁵ CFU of Bbvac (colors). Three, 7, and 15 months after vaccination, mice were intranasally challenged with PBS containing 5 × 10⁵ CFU of B. bronchiseptica (A), B. pertussis (B), or B. parapertussis (C). Mice were euthanized 7 days postinoculation, and sera were collected for analysis. Antibody titers were determined to be reciprocal of the lowest dilution in which an OD of 0.1 was obtained. Mann-Whitney test was performed. ****, P < 0.0001. Error bars represent standard error of the mean. n = 4 per strain and condition.

FIG 5

Bbvac-induced antibodies confer some protection against infection with B. bronchiseptica and B. pertussis. Serum from mice that were naive (α-naïve), inoculated with B. bronchiseptica (α-BB), inoculated with B. pertussis (α-BP), vaccinated with Nobivac (α-Nobivac), vaccinated with Adacel (α-Adacel), or vaccinated with Bbvac (α-Bbvac) was transferred to naive C57BL/6J mice. Three hours later, mice were intranasally challenged with B. bronchiseptica (A) or B. pertussis (B), and mice were euthanized 7 days postinoculation to enumerate colonies in the respiratory tract. Two-way ANOVA, Dunnett's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error of the mean. n = 4 to 9 per strain and condition.

FIG 6

Bbvac promotes IL-17⁺CD4⁺ T cells. C57BL/6J were either vaccinated with PBS, vaccinated with the currently available vaccines Nobivac and Adacel, previously infected with B. bronchiseptica, or vaccinated with Bbvac. Three months postvaccination, mice were intranasally challenged with B. bronchiseptica (A and D), B. pertussis (B and E), or B. parapertussis (C and F). Mice were euthanized 7 days postchallenge to enumerate and characterize the CD4⁺ T cells in the lungs. Two-way ANOVA, Dunnett's multiple-comparison test. *, P < 0.05. Error bars represent standard error of the mean. n = 4 per strain and condition.

FIG 7

Bbvac promotes long-lasting IL-17⁺CD4⁺ T cell memory. C57BL/6J mice were vaccinated with PBS or vaccinated with Bbvac. Fifteen months postvaccination, mice were intranasally challenged with B. bronchiseptica (A and D), B. pertussis (B and E), or B. parapertussis (C and F). Mice were euthanized 7 days postinfection to enumerate and characterize the CD4⁺ T cells in the lungs. Two-way ANOVA, Dunnett's multiple-comparison test. *, P < 0.05. Error bars represent standard error of the mean. n = 4 per strain and condition.