Eman Barahim

Date of Award


Document Type

Master's Thesis - Open Access

Degree Name

Master of Science


Microbiology and Immunology

First Advisor

Paul Arnaboldi, Ph.D.

Second Advisor

Chandra Shekhar Bakshi, Ph.D.

Third Advisor

Dana Mordue, Ph.D.


Pseudomonas aeruginosa is a facultative anaerobic bacterium that is the most common type of Gram-negative bacteria causing nosocomial pneumonia in the United States. P. aeruginosa is an opportunistic pathogen ubiquitously found in the atmosphere that can colonize both plants and animals. It is known to cause a wide range of severe, persistent, and fatal infections ranging from acute and chronic pneumonia, skin and soft tissue infections to urinary tract infections, especially in immune-compromised individuals. Patients with cystic fibrosis are at high risk because of deficiencies in mucosal immunity and phagocytosis. Ultimately, Pseudomonas infections can be lethal. There is a very high level of antibiotic resistance in P. aeruginosa leading to many attempts to design a vaccine. However, no licensed vaccine has been approved or is commercially available. In this study we utilized the tobacco mosaic virus platform delivery system to develop a vaccine against P. aeruginosa. The vaccine consists of the tip protein of the Pseudomonas type 3 secretion system, PcrV, conjugated to the coat protein of recombinant TMV. We vaccinated mice by administering 20µg of vaccine conjugate (TMV-PcrV) or 10µg of unconjugated rPcrV in PBS by intranasal droplet (IN) to anesthetized mice. In the first experiment mice were vaccinated on days 0, 14, and 28. In the second experiment, 2ug of dicyclic-GMP adjuvant was included in the vaccine groups and were administered on days 0, 21, and 35. Vaccinated mice were divided into two groups of equal numbers, each challenged IN in a model of acute pneumonia with lethal doses of P. aeruginosa Boston 41510 (1x107 or 1x108 CFU) seven days after the final boost. Mice were monitored daily for morbidity and mortality. Strong total antibody response was detected in mice vaccinated with both TMV-PcrV vaccine and TMV-PcrV + di-c-GMP adjuvant compared to unvaccinated mice and xii mice vaccinated with unconjugated rPcrV. All mice that were vaccinated without adjuvant that were challenged with 1x108 CFU of P. aeruginosa succumbed to infection, though a 2-day right shift in mean-time-to death was observed in TMV-PcrV vaccinated mice compared to rPcrV vaccinated. TMV-PcrV vaccinated mice challenged with 1x107 CFU had an 80% survival rate and regained their body weight by day 2.5, while all other mice succumbed. Challenge results with mice vaccinated in the presence of adjuvant enhanced the antibody production for both rPcrV and TMV-PcrV, consequently enhancing the survival rate. These results demonstrated the potential of TMV as a protein subunit vaccine delivery system for P. aeruginosa antigens. Collectively, TMV conjugation induced a strong humoral immune response and enhanced vaccine-mediated protection against P. aeruginosa in an acute pneumonia model, providing proof of principle for a TMV-mediated vaccine against this antibiotic-resistant nosocomial pathogen.