Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies

Abstract

Surface proteins of schistosomes are exposed to host tissues and thus present as potential candidate molecules for the development of new intervention strategies. Herein, we have identified a new tegumental protein of Schistosoma mansoni, termed Sm29. In silico analysis revealed a signal peptide, three glycosylation sites and a transmembrane region on Sm29 amino acid sequence. Sm29 transcription in mammalian developmental stages cDNA libraries of S. mansoni was verified by PCR using specific primers for Sm29 nucleotide sequence and it revealed the presence of transcripts in schistosomula and adult worm stages of the parasite. Sm29 (40-169) fragment was produced in Escherichia coli and purified by affinity chromatography to be used in the immunological assays. Confocal microscopy confirmed bioinformatic studies, revealing that Sm29 is a membrane-bound protein localized on the tegument of S. mansoni adult worm. ELISA was performed using rSm29 protein to investigate the antibody isotype profile to Sm29 in sera of patients living in endemic areas for schistosomiasis. IgG1 and IgG3 subclass antibodies to rSm29 were predominant in sera of individuals naturally resistant to infection and resistant to re-infection whereas low levels of IgM, IgA or IgE were measured. Since, IgG1 and IgG3 are involved in parasite killing and in protective immunity the findings reported here suggest the use of Sm29 as a potential candidate vaccine against schistosomiasis.

Fig. 1

In silico analysis of Sm29 amino acid sequence. Sm29 is composed by a signal peptide of 26 amino acids (grey bar), with the signal cleavage site between Ser26 and Val27 (*). Three glycosylation sites are present in the mature sequence on the Thr39, Thr132 and Thr133 (circles). The transmembrane helix is found from the Leu169 to the Leu191 (box). Five promiscuous HLA binding peptides predicted in Sm29 are underlined. (Peptides CNGLTVDNTGKIPSV and GLTVDNTGKIPSVPI are overlapped).

Fig. 2

PCR analysis of cDNA libraries using specific primers for the complete sequence of Sm29. 1, ladder (bp); 2, positive control; 3, negative control; 4, egg cDNA library; 5, lung-stage cDNA library; 6, adult worm cDNA library; 7, cercariae cDNA library.

Fig. 3

SDS-PAGE and Western blot analysis of the recombinant Sm29–6xHIS fusion protein. (a) Coomassie blue stained SDS-12% PAGE profile of induced E.coli expressing the pET21a-Sm29 construct; 1, ladder (kD); 2, E. coli lysate expressing Sm29. (b) Comassie blue stained SDS-12% PAGE profile of the purified Sm29–6xHIS fusion protein. (c) Western blot analysis of the purified protein using anti6xHIS antibody. Arrow indicates the rSm29.

Fig. 4

Immunolocalization of Sm29 on S. mansoni tegument. Fluorescence confocal microscopy images (Fluor) and corresponding differential interface contrast (DIC) images of male adult worm of S. mansoni are shown. Polyclonal anti-Sm29 and secondary antibody coupled to FITC were used for fluorescence detection of Sm29 on male adult worm sections. Serum from naive mice was used as negative control. Flavoidin and Rodamin were used for the actin localization. The Sm29 localization is represented in (a) and the negative control is shown in (b).

Fig. 5

Isotype profile of schistosomiasis patients’ sera to rSm29 or SWAP. Analysis of (a) IgG and (b) IgA antibody responses in sera of infected patients (INF), naturally resistant individuals (NR), susceptible to reinfection individuals (SR), resistant to reinfection individuals (RR) and noninfected individuals (NI). Results are expressed as means of individual measurement. Error bars indicate SD of the means. #Statistically significant compared to the noninfected group (P < 0·05). *Statistically significant compared to the infected group. **Statistically significant compared to the susceptible to reinfection group.

Fig. 6

Analysis of IgG subclass responses to rSm29 (A) or SWAP (B). IgG subclass levels in sera of infected patients (INF), natural resistant individuals (NR), susceptible to reinfection individuals (SR), resistant to reinfection individuals (RR) and noninfected individuals (NI) were determined. Results are expressed as means of individual measurements. Error bars indicate SD of the means. #Statistically significant compared to the noninfected group (NI). *Statistically significant compared to the infected group (I). **Statistically significant compared to the susceptible to reinfection group (SR).