Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ (SslE)

Abstract

Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.

Keywords: IgA; SslE; YghJ; enterotoxigenic Escherichia coli; immunogenicity; protein glycosylation; vaccine development.

Figure 1

Western blots of non-glycosylated (nYghJ) and glycosylated (gYghJ) YghJ. (A, B) shows blots where YghJ was detected by targeting the 3xFLAG peptide that trail YghJ. In (C), the blots were incubated with serum from volunteer EV01 before (d0) and 10 days after (d10) dose ingestion, followed by detection of any bound IgA, IgG, or IgM antibodies. The marker (M) band sizes are listed on the left-hand side. Arrows indicate expected sizes of YghJ. The blots were based on denatured (A, C) and native gels (B).

Figure 2

Anti-YghJ IgA antibody level changes in serum and lavage from before and 10 days after dose ingestion. Graphs show changes in serum IgA targeting non-glycosylated YghJ (A) and glycosylated YghJ (B) from before (day 0) and 10 days after ingesting ETEC, as well as the corresponding fold changes in serum (C). The figures also show changes in lavage IgA targeting non-glycosylated YghJ (D) and glycosylated YghJ (E) from before (day 0) and 10 days after ingesting ETEC, as well as the corresponding fold changes in lavage (F). Grey lines (in A, B, D, E) and open circles (in C, F) represent volunteers who did not develop diarrhea. Correspondingly, black lines and filled circles represent volunteers who developed diarrhea. Line in boxes represents median values and boxes the values between 25th and 75th percentiles. IgA levels are expressed as arbitrary units (AU) for serum and normalized arbitrary units (nAU) for lavage.

Figure 3

Glycosylated epitope specificity in serum and lavage. Figures show anti-gYghJ antibody levels in serum after pre-incubation with buffer or nYghJ, or gYghJ (A) and anti-gYghJ antibody levels in lavage after pre-incubation with buffer or nYghJ, or gYghJ (B). Graph (C) shows the proportion of anti-gYghJ-specific antibodies out of total anti-nYghJ and anti-gYghJ specific IgA antibodies in serum and lavage. Grey lines and open circles represent volunteers who did not develop diarrhea. Correspondingly, black lines and filled circles represent volunteers who developed diarrhea. Line in the boxes represents median values and boxes the values between 25th and 75th percentiles. The upper and lower whiskers limit 95% of measured values. IgA levels are expressed as AU for serum and normalized arbitrary units (nAU) for lavage.

Figure 4

Proportion of glycosylation specific anti-YghJ IgA in lavage and serum samples. Open circles represent volunteers who did not develop diarrhea and filled circles represent volunteers who developed diarrhea.