Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations

The UspA2 protein from the bacterium Moraxella catarrhalis is a potential v accine candidate for preventing human diseases caused by this organism. Bef ore a vaccine can be administered parentally, the level of endotoxin must b e reduced as much as possible, However, in this case the endotoxin was very tightly complexed with the UspA2 protein and could not be dissociated with Triton X-100. It was found that it dissociated from the protein with the z witterionic detergents Zwittergent 3-12 and Zwittergent 3-14. The endotoxin could then be separated from the protein by either ion-exchange or gel fil tration chromatography. Using the limulus amoebocyte lysate assay for quant itation, the endotoxin was reduced approximately 20 000-fold. The removal o f residual endotoxin from UspA2 preparations had no detrimental effect on t he immunological properties of the protein. Mouse antisera raised against U spA2 prior to, and following endotoxin reduction exhibited comparable antib ody and bactericidal titers against the tested strains. Further, mice immun ized with both preparations, followed by pulmonary challenge with either a homologous or a heterologous isolate, exhibited comparable levels of cleara nce. (C) 2001 Elsevier Science B.V. All rights reserved.