Author ORCID Identifier

https://orcid.org/0000-0002-9734-6424

Semester

Fall

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

F. Heath Damron

Committee Member

Slawomir Lukomski

Committee Member

Jennifer Franko

Committee Member

Matthew Dietz

Committee Member

Jamie Potter

Abstract

Bordetella pertussis is a Gram-negative obligate aerobe that causes a respiratory disease known as pertussis or whooping cough. Pertussis is most severe in younger children, especially infants but the bacteria has been known to colonize adult populations. Before the introduction of the whole-cell pertussis (wP; wP-DTP) vaccine reported numbers of pertussis cases within the US routinely topped 100,000 cases per year. However, with the widespread usage of the wP vaccine case numbers began dropping and reached a low of less than 5,000 cases per year in the late 1970’s and early 80’s. It appeared that B. pertussis was heading towards eradication, but the wP vaccine proved to be highly reactogenic due to remnant endotoxin from the manufacturing process. This reactogenicity caused fear in wP vaccination both abroad and within the US which led to an increase in cases during the late 1980s in the US. Thankfully, work was already being done on the development of an acellular pertussis (aP) vaccine to replace the reactogenic wP. In 1996, the first aP vaccine, combined with diphtheria and tetanus toxoid (DTaP), was licensed for use within the US. These vaccines proved to be less reactogenic as measured by adverse effects but with that came a different immune response to aP vaccination versus wP vaccination. Coinciding with the introduction of DTaP vaccination was a steady increase in pertussis case numbers within the US peaking at over 50,000 in 2012. The theories on this increase include genomic differences in emerging clinical isolates (ECIs) of B. pertussis, rapid waning immunity of DTaP induced immunity, and the humoral Th2 skewed immune response by DTaP vaccination. Each theory has its own merits and we hypothesized that the increase observed in pertussis cases is a combination of all three theories and we propose a potential solution to this increase by using a novel mRNA-DTP vaccine. We first wanted to evaluate if genomic differences between ECIs and historic isolates were causing different levels of pathogenesis and innate immune response in a naïve mouse model. To begin, we cultured 15 different isolates of B. pertussis in a liquid medium and extracted RNA from end log-phase growth of the bacteria. RNAseq was performed and we observed many differences in the transcriptomic profile of ECIs compared to historic isolates in virulence factors including ptxA (pertussis toxin; PT), fhaB (filamentous hemagglutinin; FHA), prn (pertactin; PRN), sphB1 (serine protease homolog of Bordetella 1; SphB1) and brkA (Bordetella resistant to killing genetic locus A; BrkA). However, when the experiment was repeated and end log-phased growth had its proteomic profiles analyzed we observed a major reduction in these differences especially for BrkA, SphB1, and FHA. Next, we intranasally (IN) challenged mice with 15 different isolates of B. pertussis (13 ECIs and 2 historic isolates) to assay markers of disease burden including leukocytosis and bacterial burden. All test isolates had similar levels of bacterial burden, as quantified by CFU, in the lung, trachea, and nasal lavage 3 days post-challenge. We did observe a slight increase in white blood cell and neutrophil counts in the blood of mice challenged with ECIs compared to historic isolates. To complete the study, we chose four representative isolates including one historical (UT25Sm1; UT25) and three ECIs (D420, H762, and I762) to observe differences in host immune response in the lungs of challenged mice. Briefly, mice were IN challenged as before and lungs were harvested 3 days post-challenge. RNA was extracted from the lungs and RNAseq was performed which revealed no major differences in host gene activation and repression in challenged mice. Even though there were large differences in the transcriptomic profiles of ECIs, there was no difference in the host genomic response or bacterial burden upon challenge in mice between ECIs and historic isolates. There was a slight increase in leukocytosis in ECI challenged mice. These data suggest that genomic differences in ECIs are not a major contributor to increased pathogenesis leading to an increase in reported pertussis cases. We then continued the previous study by introducing a vaccination component to evaluate if ECIs were better at evading vaccine-mediated immunity. Mice were primed and boosted with 1/80th human dose of either DTaP or wP vaccine and IN challenged with one of the four representative isolates used previously. We observed that B. pertussis specific IgG antibodies induced by both DTaP and wP vaccinations bound to all tested isolates similarly. Vaccinated mice were then IN challenged with either UT25Sm1 (historic) or an ECI (D420, H762, or I762) and we observed that both vaccines controlled the induction of proinflammatory cytokines regardless of which challenge isolate was used. We next investigated if PT produced by ECIs was able to evade neutralizing antibodies and induce leukocytosis. Again, DTaP was able to reduce leukocytosis regardless of the challenge isolate. ECIs did not induce more white blood cells or neutrophils on days 3 and 7 post-challenge compared to the historic isolate. Lastly, we investigated if ECIs challenge would lead to increased bacterial burden post-challenge. Bacterial clearance of ECIs was comparable to the historic isolate control in the lung, trachea, and nasal lavage 3 days post-challenge. Interestingly, while the burden was not increased more than the historic isolate, it appeared that ECIs were able to persist longer in the lungs in mice. All these data taken together suggest ECIs are not able to evade aP vaccine-mediated immunity in mice. After evaluating the role ECIs may have in the rise in pertussis cases, we wanted to develop a standardized aerosol challenge protocol due to the inconsistencies in this challenge method in the field. The protocol was developed using commercially available nebulizers, dosing chamber, and dose controller to remove any difference in laboratory designed challenge systems. We observed that this aerosol protocol was reproducible and deposited near identical numbers of bacteria from challenge to challenge. Along the same line, we developed a streptomycin resistant ECI (D420Sm1) from the baboon challenge model challenge isolate (D420). This lab adapted isolate has a single nucleotide polymorphism which confers streptomycin resistance. D420Sm1 has near identical growth characteristics and pathogenesis to the parent D420 isolate. Lastly, we aimed to evaluate a potential solution to the rise in pertussis cases with a novel mRNA-DTP vaccine. To begin, we sought to optimize the PtxA-mRNA antigen for future inclusion in the mRNA-DTP vaccine. Mice were vaccinated with either a control vaccine (DTaP, wP, gPT), an experimental recombinant PT protein (Protein) vaccine, or one of two mRNA vaccines (mRNA-C210 or mRNA-C180). Post-boost, no vaccine induced IgG antibody titers specific to any B oligomer of PT. Both the mRNA-C210 and mRNA-C180 induced IgG antibodies to PT and PtxA. Mice were then challenged with purified PT and leukocytosis was evaluated 3 days later. Again, both mRNA vaccines reduced leukocytosis and neutrophilia compared to the mock vaccinated group and similar to DTaP. The mRNA-C180 antigen was selected due to the higher IgG antibody titers specific to PT holotoxin and this antigen was included in the study formulation of mRNA-DTP. Also, we utilized the coughing rat model of pertussis to add a coughing parameter to the study. Briefly, Sprague-Dawley rats were vaccinated with 1/10th human dose of DTaP or wP, or 10 μg of mRNA-DTP. Serological analysis via ELISA revealed that mRNA-DTP was immunogenic in rats and induced comparable titers of PRN-specific, FHA-specific, and B. pertussis-specific IgG antibodies to DTaP and wP vaccination. mRNA-DTP did induce PT-specific IgG antibodies however they were lower than DTaP vaccinated rats. Interestingly, in previous experiments in mice the mRNA-DTP vaccine was able to skew towards Th1 subclass of IgG antibodies however in rats the subclass was skewed towards Th2 IgG1 subclass. Even so, bacterial burden in respiratory tissues were reduced for all vaccine groups and an even further reduction was observed in mRNA-DTP compared to DTaP vaccination. Moreover, mRNA-DTP vaccination reduced bacterial burden to the lower limit of detection in the nasal lavage by day 9 post-challenge. Next, we aimed to evaluate if mRNA-DTP vaccination would reduce coughing in rats comparable to the DTaP group because DTaP vaccination is known to reduce disease manifestations. We observed that coughing began on day 4 post-challenge and for MVC remained for the duration of the experiment. All vaccine groups were able to limit coughing compared to MVC however mRNA-DTP was able to completely abrogate the coughing phenotype and only a single cough was recorded during the study. This was comparable to the non-challenged group which had only three recorded coughs unrelated to B. pertussis challenge. These data suggest that mRNA-DTP was able to protect rats from bacterial burden and pertussis manifestations comparable to currently licensed DTaP and wP vaccines. The work presented in this thesis suggests that genomic differences in ECIs compared to historic isolates is not a major factor in the recent increase in pertussis cases. Further, it provides a standardized protocol for aerosol challenge and the adaptation of an ECI to be used in laboratory studies. The main finding in this thesis is that a novel mRNA-DTP vaccine was shown to be a potential solution to the increase in pertussis cases.

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