A Live-Attenuated Pseudomonas aeruginosa Vaccine Elicits Outer Membrane Protein-Specific Active and Passive Protection against Corneal Infection

Abstract

Pseudomonas aeruginosa can cause sight-threatening corneal infections in humans, particularly those who wear contact lenses. We have previously shown that a live-attenuated P. aeruginosa vaccine given intranasally protected mice against acute lethal pneumonia in a lipopolysaccharide (LPS) serogroup-specific manner. In the current study, we evaluated the protective and therapeutic efficacies, as well as the target antigens, of this vaccine in a murine corneal infection model. C3H/HeN mice were nasally immunized with the vaccine (an aroA deletion mutant of strain PAO1, designated PAO1⌬aroA) or with Escherichia coli as a control and were challenged 3 weeks later by inoculating the scratch-injured cornea with P. aeruginosa. For passive prophylaxis and therapy, we utilized a serum raised in rabbits nasally immunized with PAO1⌬aroA or E. coli. Outcome measures included corneal pathology scores and, in some experiments, reductions in total and internalized bacterial CFU. We found that both active and passive immunization reduced corneal pathology scores after challenge with a variety of P. aeruginosa strains, including several serogroup-heterologous strains. Even when given therapeutically starting as late as 24 h after infection, the rabbit antiserum to PAO1⌬aroA was effective at reducing corneal pathology scores. Immunotherapy of established infections also reduced the numbers of total and internalized corneal P. aeruginosa bacteria. Experiments using absorbed sera showed that the protective antibodies are specific to outer membrane proteins. Thus, live-attenuated P. aeruginosa vaccines delivered nasally protect against corneal infections in mice and potentially can be used to prepare passive therapy reagents for the treatment of established P. aeruginosa corneal infections caused by diverse LPS serogroups.

A Cleavage-potentiated Fragment of Tear Lacritin Is Bactericidal

“…Lacritin and truncations were then assayed in equimolar (2 M) amounts in the presence of mid-log E. coli, P. aeruginosa, or S. epidermidis. P. aeruginosa is an eye pathogen often responsible for keratitis in contact lens wear (41). S. epidermidis is a common cause of conjunctivitis and keratitis and is abundant in blepharitis (42,43), an eyelid inflammation associated with slightly altered tear composition, including selectively less lacritin (44).…”

Assessment of immunogenicity of alginate microparticle containing Brucella melitensis 16M oligo polysaccharide tetanus toxoid conjugate in mouse

“…In this phase, EDAC causes covalence link between ADH-OPS to protein carrier as an amide link between hydrazide polysaccharide with carboxyl groups of TT [ZAIDI et al, 2006, KASPER et al, 1996 .…”

“…Pure alcohol at -20°C or less effectively resulted in LPS precipitation. Then, supernatant was thrown away, and LPS precipitate remained at the tube bottom [ZAIDI et al, 2006, KASPER et al, 1996 .…”

“…Extracted LPS was dissolved in Acid Acetic 2 % and the tube was put in autoclave at 120 °C for 10 minutes. Then, dialysis in distilled water, precipitation, and gel filtration chromatography were done [ZAIDI et al, 2006, KASPER et al, 1996 .…”

“…In this phase, in order to discharge the impurities, supernatant was collected and a dialysis bag was used with cut off 10 Kd in cool water (for 24 hours, and three times replacement, and every time 4 liters of distilled water) for dialysis to take place and therefore, impurities such as phenol and ethanol were completely discharged [ARENAS-GAMBOA et al, 2011, ZAIDI et al, 2006, KASPER et al, 1996 .…”