Serological Expression Cloning of Novel Immunoreactive Antigens of Babesia microti

Abstract

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.

Identification and Characterization of Putative Secreted Antigens fromBabesia microti

“…One of the 27 clones isolated contains a probable ORF that is nearly identical to the previously described BMN1-10 sequence (17). Ten single nucleotide polymorphisms relative to the originally identified BMN1-10 sequence result in six amino acid changes in the putative peptide sequence.…”

“…The longest clone is 3,524 nucleotides long, defining an ORF encoding 913 aa; however, the composite sequence of BMN1-15, including the overlap of isolated clones, is 4,723 nucleotides long and defines an ORF encoding 1,312 aa. This new BMN1-15 sequence adds a significant amount of sequence data to both the N and C termini of the originally reported BMN1-15 sequence (17). Some of the clones (BM26 and BM45) may define separate genomic copies of the BMN1-15 sequence or may indicate the presence of mixed isolates in the material that was used for library construction, as they show significant differences in the N terminus and in the coding or noncoding sequence, respectively.…”

“…Ten single nucleotide polymorphisms relative to the originally identified BMN1-10 sequence result in six amino acid changes in the putative peptide sequence. Sequence analysis of the putative polypeptide predicts an uncleavable Nterminal signal sequence and identifies the protein as a type II membrane protein (17). It is likely that there are multiple copies of BMN1-10 within the library because of the differences detected between the new and original BMN1-10 sequences.…”

“…The library was plated on 11 large petri plates (150 by 15 mm) containing Luria-Bertani medium at a concentration of approximately 20,000 PFU (total number of plaques screened, 2.2 ϫ 10 5 ). The plaques were lifted and thereby transferred to nitrocellulose filters and then were processed by established protocols (17) with the adsorbed SCID sera as the primary antibodies (17). Seventy plaques were picked upon the first screening of the library.…”

“…and then ligated to the lambda ZAP II vector (Stratagene). The ligation mixture was packaged with Gigapack III Gold packaging extract (Stratagene) (17).…”