Despite high global vaccine coverage, whooping cough, also known as pertussis, caused by the gram-negative obligate human pathogen Bordetella pertussis (Bp), is resurging worldwide. The inactivated whole cell vaccines (wPV), introduced in the 1940s, were extremely effective in preventing severe disease, controlling the bacterial burden in the entire respiratory tract, and preventing transmission. Because of severe reactogenicity and negative public perceptions regarding safety, wPV were discontinued and acellular subunit (1 to 5 protein components) vaccines adjuvanted with alum (aPV) were introduced in many countries. While safer and effective in disease prevention, these aPVs elicit poor and short-lived immunity and fail to prevent infection [1, 2]. Here, we discuss reasons for pertussis resurgence, bacterial evolution, and limitations of current pertussis vaccines. We also propose new directions to fill existing research gaps and accelerate the development of more effective vaccines.
Vaccines are a 20 th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and has contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next generation vaccine that induces protective immunity without the adverse side effects of the whole cell vaccine (wPV).Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvest and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.
IntroductionResurgence of pertussis, caused by Bordetella pertussis, necessitates novel vaccines and vaccination strategies to combat this disease. Alum-adjuvanted acellular pertussis vaccines (aPV) delivered intramuscularly reduce bacterial numbers in the lungs of immunized animals and humans, but do not reduce nasal colonization. Thus, aPV-immunized individuals are sources of community transmission. We showed previously that modification of a commercial aPV (Boostrix) by addition of the Th1/17 polarizing adjuvant Bordetella Colonization Factor A (BcfA) attenuated Th2 responses elicited by alum and accelerated clearance of B. pertussis from mouse lungs. Here we tested whether a heterologous immunization strategy with systemic priming and mucosal booster (prime-pull) would reduce nasal colonization.MethodsAdult male and female mice were immunized intramuscularly (i.m.) with aPV or aPV/BcfA and boosted either i.m. or intranasally (i.n.) with the same formulation. Tissue-resident memory (TRM) responses in the respiratory tract were quantified by flow cytometry, and mucosal and systemic antibodies were quantified by ELISA. Immunized and naïve mice were challenged i.n. with Bordetella pertussis and bacterial load in the nose and lungs enumerated at days 1-14 post-challenge.ResultsWe show that prime-pull immunization with Boostrix plus BcfA (aPV/BcfA) generated IFNγ+ and IL-17+ CD4+ lung resident memory T cells (TRM), and CD4+IL-17+ TRM in the nose. In contrast, aPV alone delivered by the same route generated IL-5+ CD4+ resident memory T cells in the lungs and nose. Importantly, nasal colonization was only reduced in mice immunized with aPV/BcfA by the prime-pull regimen.ConclusionsThese results suggest that TH17 polarized TRM generated by aPV/BcfA may reduce nasal colonization thereby preventing pertussis transmission and subsequent resurgence.
Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-β-1,6-N-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and released during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps.
Bordetella pertussis is the causative agent of whooping cough, a resurging vaccine-preventable disease. This highly contagious disease is most severe in infants and young children. Current alum-adjuvanted acellular pertussis vaccines (aPV) prevent severe disease but do not prevent nasal carriage and subsequent person-to-person pathogen transmission. The Th1/2 skewed immune response induced by aPV is one potential explanation for this failure. We are testing the hypothesis that modification of current aPV by the addition of an adjuvant, BcfA, that skews immune responses towards the more protective Th1/17 phenotype will improve protection. We also hypothesize that changing the delivery route from intramuscular (i.m) to intranasal (i.n) administration will generate mucosal immunity and further improve protection. In the established mouse model of B. pertussis infection, we found that i.m. immunization of mice with an experimental aPV containing BcfA and alum as the adjuvants significantly reduced bacterial numbers in the lungs compared to an alum-adjuvanted vaccine alone. Further, i.n administration of the BcfA+alum containing vaccine provided better protection against colonization of the trachea and lungs than i.m. immunization. Alum-induced Th2 cytokine responses were reduced by addition of BcfA, and were further reduced by i.n. vaccine delivery. Thus, a revised aPV that includes BcfA, administered i.n, may improve protection against B. pertussis infection compared with current formulations and delivery method.
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