Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A

Abstract

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.

Intranasal Immunization Strategy To Impede Pilin-Mediated Binding ofPseudomonas aeruginosato Airway Epithelial Cells

“…immunization with ntPEpilin-PAK was considered crucial to generation of an optimal protective immune response against oropharyngeal P. aeruginosa challenge, because nasally induced IgA antibody secreting cells, based upon their complement of surface adhesion molecule, should traffic back to tissues that include salivary glands and the respiratory tract while systemically injected antigen would not induce this outcome (30). Since PE has been shown to act as a potent virulence factor for P. aeruginosa infection, efforts to induce a neutralizing immunization against this toxin have been extensive (8,17,18,21). It is important to note that immunization with ntPEpilinPAK resulted in neutralizing salivary immune responses, although it is unclear whether sIgA or exudates of IgG are more critical.…”

Protective Effect of DNA Vaccine Encoding Pseudomonas Exotoxin A and PcrV against Acute Pulmonary P. aeruginosa Infection

“…The toxA and pcrV genes were PCR-amplified from genomic DNA of PAO1 [17], [50], respectively labeled by a Human influenza hemagglutinin (HA)-tag sequence and a His-tag sequence. Gene toxA -HA was mutated following previous methods [51], namely toxA m -HA.…”

Dual-Function Vaccine for Pseudomonas aeruginosa : Characterization of Chimeric Exotoxin A-Pilin Protein

“…Controlling P. aeruginosa infections, or better yet, preventing them, has thus become a critical unmet medical need in the care of CF patients (2). To address this, a number of vaccine approaches have been explored, many focused on outer membrane constituents (35,40,46), some focused on toxins (7,14,20,34) and some focused on a combination approach (7,(9)(10)(11)(12)28).…”