Identification of the Entamoeba histolytica galactose-inhibitable lectin epitopes recognized by human immunoglobulin A antibodies following cure of amebic liver abscess

Abstract

Immunity to Entamoeba species intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolytica intestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolytica trophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P < 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.

FIG. 1.

Recombinant protein fragments produced from restriction enzyme digestion of LC3 DNA. Restriction enzyme sites are indicated, and fragments are represented by their amino acid sequence numbers. Seven recombinant LC3 protein fragments (A through G) were produced.

FIG. 2.

ELISA OD results for serum anti-LC3 IgA (a) and IgG (b) antibodies using purified LC3 antigen (horizontal bars, means). Study groups include seronegative uninfected controls; asymptomatic, seropositive subjects with concurrent E. histolytica infection as determined by fecal PCR; subjects recently cured of ALA with and without infection (determined by PCR); and ALA subjects free of infection by culture criteria 1 year after cure. There were substantially higher IgA and IgG antibody levels in recently cured ALA subjects with or without infection and seropositive subjects with asymptomatic intestinal infection than in controls and ALA subjects studied 1 year after treatment (P < 0.05 for each).

FIG. 3.

Recognition of LC3 epitopes by human anti-LC3 IgA and IgG antibodies and by anti-LC3 murine IgA monoclonal antibodies. By analysis of serum IgA and IgG antibody recognition of LC3 protein fragments, we found that IgA antibodies from ALA subjects (infected or uninfected) and from seropositive subjects with asymptomatic E. histolytica infection all recognized epitopes 3 and 7 (gray ovals). Only anti-LC3 IgG antibodies recognized epitope 6 (white oval), and recognition was lost only 1 year after cure of ALA. Anti-LC3 murine IgA monoclonal antibodies recognized epitopes 1, 2, 4, 5, and 6 (asterisks) and not epitopes 2 and 7.

FIG. 4.

Recognition by pooled human intestinal and serum anti-LC3 IgA antibodies of synthetic peptides based on the amino acid sequences of LC3 epitopes 3 and 7. Ten overlapping peptides were synthesized based on the sequence of epitope 3 and 2 peptides were synthesized based on epitope 7. Intestinal (triangles) and serum (circles) IgA antibodies were obtained from subjects cured of ALA with concurrent infection (black); gray symbols, seropositive, asymptomatic PCR-positive subjects; open symbols, seronegative PCR-negative control subjects. Peptides 2, 9, 11, and 12 were recognized by intestinal and serum IgA antibodies from all subjects, compared to the cutoff point established by seronegative controls. Peptides 2, 9, and 11 were recognized by serum IgA antibodies from ALA subjects and fecal and serum IgA antibodies from asymptomatic infected seropositive subjects.