Identification and cloning potentially protective antigens of Coxiella burnetii using sera from mice experimentally infected with Nine Mile phase I.

Abstract

Coxiella burnetii is an obligate intracellular bacterium that causes acute Q fever and occasional chronic infections in humans. To determine the immunodominant antigens during infection with C. burnetii, sera from mice experimentally infected with Nine Mile phase I were tested by immunoblotting. The mouse sera recognized antigens with a variety of molecular weights, including proteins of 14, 22, 28, 34, and 60 kDa as immunodominant antigens. In order to clone potential protective antigens, a genomic DNA library of Nine Mile phase I was constructed in the expression vector Lambda ZAP Express and screened with sera from mice that recovered from C. burnetii infection. A total of 102 immunoreactive clones with various signal intensities were identified from about 8,000 plaques. These clones were purified and expressed in the excised plasmid pBK-CMV. The proteins expressed by these recombinant plasmids were analyzed by SDS-PAGE and immunoblotting. Fifty-four clones expressed immunoreactive proteins of molecular masses ranging from approximately 14 to 60 kDa. Sequence analysis and BLAST search of the recently completed genome sequence identified a variety of novel immunoreactive proteins. These proteins are logical vaccine candidates for testing protective activity against C. burnetii challenge. We established a sublethal challenge model in BALB/c mice with protection from the development of severe splenomegaly as an indicator of vaccinogenic activity. Further characterization of these proteins will provide essential information for developing novel, specific diagnostic reagents and potential subunit vaccine candidates against C. burnetii infection.