Identification and characterization of putative secreted antigens from Babesia microti

Abstract

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.

FIG. 1.

Relationship between BMN1-21 and other BMN1 proteins. Relationships were determined with the MegAlign program from DNAStar, Inc. Alignment was made with the CLUSTAL W program in MegAlign. Branching order was determined by using the Jotun-Hein method in MegAlign. Branch length represents the average distance between sequence pairs, while units at the bottom indicate the numbers of substitution events.

FIG. 2.

Identification of protein bands for mass spectrometry. (A) Silver-stained 4 to 12% acrylamide gel with a lysate of B. microti isolated from infected RBCs. (B) Immunoblot of an identical gel that was probed with sera from B. microti-infected patients. The band of interest is denoted with an arrow; both variants of BMN1-21 [BM11-5(a) and BM11-51(b)] were isolated from this band. MW, molecular weight, in thousands.

FIG. 3.

Alignment of the two putative peptides encoded by the two BMN1-21 sequences. The peptide regions of each variant that were identified by MS sequencing are shown in boxes. The peptide residues that differed between the two variants are shaded.

FIG. 4.

Reactivity of Babesia clones in ELISAs. The reactivity of recombinant antigens against sera from confirmed Babesia-positive and -negative patients or donors is shown. Cutoff values for each assay were determined from the mean for the random donor population plus three standard deviations of the mean. Bars indicate the mean optical density (O.D.) at 450 nm plus 1 standard error of the mean. term, terminus.