Active Immunization with Pneumolysin versus 23-Valent Polysaccharide Vaccine for Streptococcus pneumoniae Keratitis.

PURPOSE: The purpose of this study was to determine whether active immunization against pneumolysin (PLY), or polysaccharide capsule, protects against the corneal damage associated with Streptococcus pneumoniae keratitis. METHODS: New Zealand White rabbits were actively immunized with Freund's adjuvant mixed with pneumolysin toxoid (ψPLY), Pneumovax 23 (PPSV23; Merck, Whitehouse Station, NJ), or phosphate-buffered saline (PBS), before corneal infection with 105 colony-forming units (CFU) of S. pneumoniae. Serotype-specific rabbit polyclonal antisera or mock antisera were passively administered to rabbits before either intravenous infection with 10¹¹ CFU S. pneumoniae or corneal infection with 105 CFU of S. pneumoniae. RESULTS: After active immunization, clinical scores of corneas of the rabbits immunized with ψPLY and Freund's adjuvant were significantly lower than scores of the rabbits that were mock immunized with PBS and Freund's adjuvant or with PPSV23 and Freund's adjuvant at 48 hours after infection (P ≤ 0.0010), whereas rabbits immunized with PPSV23 and Freund's adjuvant failed to show differences in clinical scores compared with those in mock-immunized rabbits (P = 1.00) at 24 and 48 hours after infection. Antisera from rabbits actively immunized with PPSV23 and Freund's adjuvant were nonopsonizing. Bacterial loads recovered from infected corneas were higher for the ψPLY- and PPSV23-immunized rabbits after infection with WU2, when compared with the mock-immunized rabbits (P ≤ 0.007). Conversely, after infection with K1443, the ψPLY-immunized rabbits had lower bacterial loads than the control rabbits (P = 0.0008). Quantitation of IgG, IgA, and IgM in the sera of ψPLY-immunized rabbits showed high concentrations of PLY-specific IgG. Furthermore, anti-PLY IgG purified from ψPLY-immunized rabbits neutralized the cytolytic effects of PLY on human corneal epithelial cells. Passive administration of serotype-specific antisera capable of opsonizing and killing S. pneumoniae protected against pneumococcal bacteremia (P ≤ 0.05), but not against keratitis (P ≥ 0.476). CONCLUSIONS: Active immunization with pneumococcal capsular polysaccharide and Freund's adjuvant fails to produce opsonizing antibodies, and passive administration of serotype specific opsonizing antibodies offers no protection against pneumococcal keratitis in the rabbit, whereas active immunization with the conserved protein virulence factor PLY and Freund's adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.

"Boom" and "Bust" cycles in virus growth suggest multiple selective forces in influenza a evolution.

BACKGROUND: Influenza A virus evolution in humans is driven at least in part by mutations allowing the virus to escape antibody neutralization. Little is known about the evolution of influenza in birds, a major reservoir of influenza A. METHODS: Neutralizing polyclonal antiserum was raised in chicken against reassortant influenza virus, CalX, bearing the hemagglutinin (HA) and neuraminidase (NA) of A/California/7/2004 [H3N2]. CalX was serially passaged in the presence of anti-CalX polyclonal IgY to derive viruses capable of growth in the presence of antibody. RESULTS: Polyclonal chicken antibody neutralized both HA activity and infection by CalX, but had no effect on a strain bearing an earlier human H3 and an irrelevant neuraminidase (A/Memphis/71-Bellamy/42 [H3N1]). Surprisingly, most of the antibody-resistant viruses were still at least partially sensitive to neutralization of HA activity and viral infection. Although mutant HA genes bearing changes that might affect antibody neutralization were identified, the vast majority of HA sequences obtained were identical to wild type, and no individual mutant sequence was found in more than one passage, suggesting that those mutations that were observed did not confer sufficient selective advantage to come to dominate the population. Different passages yielded infectious foci of varying size and plaques of varying size and morphology. Yields of infectious virus and relative frequency of different morphologies changed markedly from passage to passage. Sequences of bulk, uncloned PCR products from antibody-resistant passages indicated changes in the PB2 and PA proteins with respect to the wild type virus. CONCLUSIONS: Each antibody-selected passage consisted of a variety of different cocirculating populations, rather than pure populations of virus able to escape antibody by changes in antibody epitopes. The ability to escape antibody is apparently due to changes in genes encoding the viral polymerase complex, probably resulting in more robust viral replication, allowing the few virus particles not completely neutralized by antibody to rapidly produce large numbers of progeny. Our data suggest that the relative success of an individual variant may depend on both its own gain and loss of fitness, as well as that of its cocirculating variants.

Cholesterol as treatment for pneumococcal keratitis: cholesterol-specific inhibition of pneumolysin in the cornea.

PURPOSE: The purpose of this study was to determine whether cholesterol, the host cell receptor for pneumolysin of Streptococcus pneumoniae, could effectively treat pneumococcal keratitis. METHODS: New Zealand White rabbits were intrastromally injected with 10(5) colony-forming units (CFUs) of S. pneumoniae D39. Corneas were treated with topical drops of 1% cholesterol every 2 hours beginning 25 hours after infection and were examined by slit lamp microscopy 24, 36, and 48 hours after infection. Rabbits were killed, and CFUs were recovered from the corneas after the final slit lamp examination (SLE). Minimal inhibitory concentration (MIC) assays of cholesterol against bacteria were performed. Specific inhibition of pneumolysin by cholesterol in the rabbit cornea was tested by intrastromal injection of pneumolysin with or without cholesterol and was compared with cholesterol inhibition of pneumolysin in vitro using hemolysis assays with rabbit erythrocytes. RESULTS: Corneas treated with cholesterol had significantly lower SLE scores 48 hours after infection than corneas treated with vehicle (P = 0.0015). Treated corneas also had significantly less log(10) CFUs than vehicle-treated corneas (P = 0.0006). Cholesterol at a 1% concentration was bactericidal to bacteria in vitro, and lower concentrations of cholesterol were partially inhibitory in a concentration-dependent manner. Cholesterol also specifically inhibited 1 mug pneumolysin in vivo and up to 50 ng pneumolysin in vitro. CONCLUSIONS: Topical cholesterol is an effective treatment for S. pneumoniae keratitis. Cholesterol not only inhibits pneumolysin, it is also bactericidal.