VH3 gene usage in neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit

Abstract

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V kappa 1 family, in which the closest V kappa germ line gene was 02/012 or L5, with the J kappa 1, J kappa 2, J kappa 4, or J kappa 5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

FIG. 1.

Deduced amino acid sequences for genes coding for heavy-chain variable regions of human anti-E. histolytica Fab fragments. FR, framework regions. The dashes and dots indicate deletions and identical residues, respectively.

FIG. 2.

Deduced amino acid sequences for genes coding for light-chain variable regions of human anti-E. histolytica Fab fragments. FR, framework regions. The dashes and dots indicate deletions and identical residues, respectively.

FIG. 3.

Western immunoblot analysis of reactivities of human monoclonal antibody Fab fragments with trophozoites of E. histolytica HM-1:IMSS. Cell lysates were subjected to SDS-PAGE in a 7.5% polyacrylamide gel under nonreducing conditions and transferred to polyvinylidene difluoride membranes. The protein bands in lane 1 were stained with Coomassie brilliant blue. Lane 2, CP33; lane 3, CP33-H/L-CP17; lane 4, CP33-H/L-LA22; lane 5, CP33-H/L-CP26; lane 6, E.coli lysates (vector control); lane 7, plasma from a patient with an amebic liver abscess. The preparations in lanes 2 to 7 were treated with HRP-conjugated sheep antibody to human IgG F(ab′)₂. The numbers on the left indicate the molecular masses of size markers.

FIG. 4.

Dot immunoblot analysis of reactivities of human monoclonal antibody Fab fragments with an affinity-purified 260-kDa lectin and with rLecA. One microgram of the 260-kDa lectin and 1 μg of rLecA were blotted onto a nitrocellulose membrane. The spots were treated as follows: line 1, CP33; line 2, CP33-H/L-CP17; line 3, CP33-H/L-LA22; line 4, CP33-H/L-CP26; line 5, control human Fab; line 6, IgG purified from plasma of a patient with an amebic liver abscess; line 7, anti-260-kDa lectin rabbit antibody. Then the preparations were treated with HRP-conjugated sheep antibody to human IgG F(ab′)₂ (lines 1 to 6) or HRP-conjugated goat antibody to rabbit IgG (line 7).