Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein, Cp-P34, unique to Cryptosporidium

Abstract

Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.

Keywords: Cryptosporidium; MORN; P34; Sporozoite; Surface antigen; VHH; gp900.