Semester

Summer

Date of Graduation

2019

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

F. Heath Damron

Committee Co-Chair

Slawomir Lukomski

Committee Member

Slawomir Lukomski

Committee Member

Gordon P. Meares

Committee Member

Rosana Schafer

Committee Member

Michael D. Schaller

Abstract

Pertussis is a human respiratory disease, primarily caused by the Gram-negative pathogen Bordetella pertussis. The infection is most severe and can be life-threatening in young children and infants where it manifests as a series of paroxysmal coughs. The disease is more commonly known as whooping cough, due to the whoop omitted during a massive inspiratory effort to bring air back into the lungs. Pertussis is a toxin-mediated disease that persists due to an early release of toxins that allow that bacteria to evade the cells of the innate immune response. The inhibition of the host response continues as toxin activity disrupts the signaling generating an adaptive immune response. Fortunately, two generations of vaccines have been developed to combat the spread of the highly contagious disease. The whole-cell pertussis (wP) vaccine was implemented in the 1950s and was successful in controlling cases. However, due to adverse side effects associated with the immunization, wP vaccines were replaced with acellular protein (aP) vaccines absorbed to an alum adjuvant. The second generation aP vaccines induced an immune response capable of neutralizing key virulence factors contained in the vaccine, thereby controlling the symptoms of the disease. Unfortunately, there has been a re-emergence of pertussis incidence in countries using the aP vaccines. Included in this resurgence is an increased number of adolescents and adults with unrecognized pertussis, leading to the potential of adult and adolescent reservoirs of the infection. We reasoned that a pertussis vaccine capable of inducing sterilizing immunity, rather than controlling the symptoms, would decrease the spread of pertussis. In this work, we propose two directions towards this goal. The first was through the neutralization of a key virulence factor not included in any current vaccine formulations, adenylate cyclase toxin (ACT). This was accomplished through the optimization of a mouse immunization and challenge model. In initial experiments using a neutrophil reporter mouse, NeCre luc, we visualized the spatiotemporal localization of neutrophils after B. pertussis challenge in wP or aP immunized mice with a vaccine dose known to be protective. In these experiments wP immunized mice elicited an exaggerated immune response, that persisted after clearance of the pathogen, raising concerns to the relevance of the high vaccine dose in characterizing the immune response. In subsequent experiments, we addressed these concerns, by titrating the concentration of a Diphtheria-Tetanus-acellular Pertussis (DTaP) subunit vaccine to a dose that did not induce a protective response in a murine model. In defining a sub-optimal protective dose for mice, the additive effects of potential antigens could be evaluated, that were previously masked by the higher concentrations of vaccine. This model was confirmed through the addition of an ACT toxoid, RTX. The inclusion of RTX into a DTaP vaccine produced an antibody response that neutralized the toxin ­in vitro, and upon challenge with B. pertussis reduced the bacterial burden compared to mice only receiving the DTaP vaccine. We continued in the direction of promoting a vaccine-induced immune response capable of clearing B. pertussis from the respiratory tract in future experiments. We hypothesized that early neutralization of the bacterial toxins through the generation of a mucosal immune response in the respiratory tract would increase the clearance of the pathogen. This was tested using a mucosal, intranasally (IN) administered DTaP vaccine. Administration of DTaP through the IN route induced a B. pertussis­-specific systemic IgG response, and mucosal IgA response, capable of neutralizing the toxins at the site of infection. Furthermore, IN vaccinated mice reduced the bacterial burden to the same levels of intraperitoneal immunized mice. The work completed in this dissertation demonstrates the protective effects of the antigen RTX when incorporated into a current DTaP vaccine. We then altered the route of administration to an intranasal delivered vaccine, demonstrating the effectiveness of inducing a local mucosal response against B. pertussis infection. Together these findings establish a foundation for potential vaccine strategies to suppress the toxin-mediated dysregulation of the host response and lead to the more efficient clearance of B. pertussis from the respiratory tract.

Embargo Reason

Publication Pending

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