Antibody Responses to the Immunodominant Cryptosporidium gp15 Antigen and gp15 Polymorphisms in a Case–Control Study of Cryptosporidiosis in Children in Bangladesh

Abstract

Although Cryptospridium hominis is the dominant Cryptosporidium species infecting humans, immune responses to cognate antigens in C. hominis-infected persons have not been reported. We investigated antibody responses to the immunodominant gp15 antigen from C. hominis and C. parvum, in C. hominis-infected Bangladeshi children less than five years of age with diarrhea (cases) and uninfected children with diarrhea (controls). We also investigated polymorphisms in the C. hominis gp15 sequence from cases. Serum IgG responses to gp15 from both species were significantly greater in cases than controls. In spite of polymorphisms in the gp15 sequence, there was a significant correlation between antibody levels to gp15 from both species, indicating cross-reactivity to conserved epitopes. Cases with acute diarrhea had a significantly greater serum IgA response to gp15 compared with those with persistent diarrhea, suggesting that this response may be associated with protection from prolonged disease. These findings support further investigation of gp15 as a vaccine candidate.

Systemic Antibody Responses to the Immunodominant p23 Antigen and p23 Polymorphisms in Children with Cryptosporidiosis in Bangladesh

“…43,46 There were significant correlations in the initial, follow-up, and change between the IgG, IgA, and IgM (except for IgM follow-up) antibody levels to p23 with those to gp15 in cases over the three-week follow-up period (Table 3). In addition, there were significant correlations in antibody levels (except for IgG follow-up and change) to p23 with those to C. parvum lysate at the initial, follow-up, and change between them (Table 3).…”

“…Serum IgG, IgA, and IgM responses to p23 were assessed by ELISA using rp23 as antigen as described. 43 Briefly, 96-well microtiter plates (Nunc, Rochester, NY) were coated with 0.1 mg of rp23 per well, overnight at 4 C. The plates were washed three times with 0.05% Tween 20 in 20 mM sodium phosphate buffer, pH 7.0, 150 mM NaCl (phosphate-buffered saline [PBS]) by using an automated plate washer (BioTek-Elx50, Winooski, VT). Non-specific binding was blocked with 0.25% bovine serum albumin (BSA) in PBS for 2 hours at 37 C. Serum diluted 1:100 in 0.25% PBS-BSA was added and plates were incubated for 1 hour at 37 C.…”

“…The effect of such normalization on inter-plate and intraplate variability in ELISAs using the same serum samples was validated previously by using mixed effects regression models that indicated that the normalized values represent the original measures and are invariant to plate performance. 43 Therefore, results presented are expressed in normalized ELISA units.…”

“…41 Most human infections, particularly in resource-poor countries, are caused by C. hominis. Genes encoding other surface-associated Cryptosporidium proteins including gp40 42,43 and Muc4 and 5 44 are highly polymorphic among clinical isolates of different species and subtypes. If p23 is to be considered as a vaccine candidate, it is essential to investigate polymorphisms in the gene that encodes it among clinical isolates in different geographic areas.…”

“…The present study is part of a prospective case-control study to evaluate the clinical, epidemiologic, and immunologic features of cryptosporidiosis in children with diarrhea coming to 46 and C. hominis 43 and to gp15, 43 another immunodominant Cryptosporidium antigen in the same cohort of children.…”