NYMC Student Theses and Dissertations

Date of Award

6-10-2025

Document Type

Doctoral Dissertation - Restricted (NYMC/Touro only) Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Paul M Arnaboldi

Second Advisor

Chandra Shekhar Bakshi

Abstract

Pseudomonas aeruginosa (PA) is a gram-negative bacterium that is ubiquitous in the environment and is often resistant to multiple antibiotics. In healthcare settings, PA is one of the most common causes of ventilator associated pneumonia and several other types of mucosal and systemic infections, in part through its ability to form biofilms on medical devices and to persist in harsh environments. As an alternative to antibiotics, vaccination against PA can protect those at risk of morbidity and mortality. Intranasal vaccination can protect against localized respiratory PA infections in addition to systemic PA infections.

We designed a PA vaccine using tobacco mosaic virus (TMV), a plant virus that is harmless to humans, as a platform to present recombinant whole protein antigens. PcrV, the tip protein of PA’s type 3 secretion system (T3SS), was chemically conjugated to TMV and evaluated as the primary vaccine antigen. Curdlan (c), a β-glucan exopolysaccharide, was used as an adjuvant. The vaccine was given to mice intranasally to assess protection against a mouse model of acute PA pneumonia. We examined humoral and cellular immune responses elicited by the vaccine to determine whether curdlan enhanced the protective efficacy of the TMV-PcrV vaccine, to identify immune correlates of protection, and to elucidate the effector functions of vaccine-induced antibodies.

TMV-PcrV+c, the adjuvanted vaccine, was 56% protective against a 22.5 LD50 dose of PA. The TMV platform significantly enhanced production of localized IgA in the lungs compared to the unconjugated recombinant PcrV protein subunit vaccines. Systemically, serum anti-PcrV IgG1 was primarily associated with survival after a PA challenge, identifying IgG1 as a potential correlate of protection. We also found that serum antibodies could neutralize the release of toxic exoenzymes secreted from the T3SS.

PA infections are primarily cleared by phagocytes, as PA is an extracellular bacterium. Macrophages and neutrophils are activated and recruited to the site of infection by TH1 and TH17 signaling, respectively. Interferon gamma, the prototypical TH1 cytokine, and interleukin-17, the prototypical TH17 cytokine, were secreted by lung and spleen cells from vaccinated mice in response to restimulation with rPcrV. Cells from mice given vaccines containing curdlan were more responsive than cells from mice given unadjuvanted vaccines. Neutrophils were quickly recruited to the lungs after PA infection, but in mice that received TMV-conjugate and curdlan-containing vaccines, they declined more rapidly than in unvaccinated mice and mice that were vaccinated with unconjugated PcrV. Blockade of interleukin-17 during PA challenge enhanced protection of vaccinated mice. This suggests that excessive or prolonged infiltration of neutrophils can lead to acute tissue damage, morbidity, and mortality. In contrast, vaccinated mice recruited macrophages to the site of infection more quickly than unvaccinated mice. Moreover, interferon gamma blockade during PA challenge delayed clearance of PA from infected mice, suggesting that recruiting macrophages to the site of infection is beneficial for PA clearance and host survival.

We also tested four additional vaccine antigens: OprF, OprI, ExoAt, and FliC. Serum antibodies raised against these antigens could bind to native PA proteins and the vaccines elicited antibody responses. However, these vaccines were not protective against a lethal dose of PA. Despite the lack of in vivo protection, anti-ExoAt serum profoundly neutralized ExoA-mediated apoptosis of mouse cells when analyzed in vitro, suggesting a potential therapeutic use of antibodies targeting ExoA for high-risk patients with PA infections.

Overall, these results suggest that both humoral and cellular immunity contribute to vaccine-mediated protection against PA. The adjuvanted TMV-PcrV+c vaccine was more protective than its unadjuvanted counterpart. Vaccine-induced antibodies do not appear to be bactericidal, but they do appear to have a neutralizing function, which can prevent tissue damage that would otherwise be induced by PA’s secreted toxins. Vaccination also enhances T cell responses and appropriate recruitment of phagocytes to the site of infection to engulf and kill PA. Together, TMV-PcrV+c vaccine-mediated humoral and cellular responses can clear PA infections while limiting acute tissue damage derived from host and pathogen factors to ultimately improve morbidity and mortality after PA infections.

Keywords

Pseudomonas aeruginosa, mucosal vaccine, mouse model, neutralizing antibodies, IL-17, Th17, flow cytometry, antigen stimulation, phagocytosis

Disciplines

Immunology of Infectious Disease | Immunoprophylaxis and Therapy

Download

Available for download on Friday, May 01, 2026

Share

COinS