Our findings demonstrate that neutralizing human antibodies to amebic adherence and erythrophagocytosis can be prepared from an immunoglobulin gene library derived from an asymptomatic cyst passer infected with E. histolytica
but not with E. dispar
. The epitope recognized by CP33 was located in a cysteine-rich domain of the heavy subunit of the Gal/GalNAc lectin. To date, seven different mouse monoclonal antibodies specific for nonoverlapping epitopes on the cysteine-rich domain of the heavy subunit of lectin have been identified (28
). Three of the seven murine antibodies inhibited amebic adherence to target cells, but two enhanced adherence by causing a marked increase in the galactose-binding activity of the lectin. Since the human antibodies prepared in this study had an inhibitory effect on amebic adherence to CHO cells, these antibodies must recognize an adherence-inhibiting epitope in the cysteine-rich domain (28
). This conclusion is also supported by the fact that CP33 did not react with E. dispar
, as previously it has been shown that the adherence-inhibiting epitopes are E. histolytica
). Demonstration of the ability of the Fabs to inhibit erythrophagocytosis indicates involvement of the Gal/GalNAc lectin in this process for the first time. Erythrophagocytosis is of interest as it is a characteristic property that distinguishes E. histolytica
from the nonpathogenic parasite E. dispar.
In a previous study, the immunogobulin gene library derived from a patient with an amebic liver abscess was screened by the methods used in the present study. The positive rate of the first screening was 0.054% (27 of 5 × 104
of clones were positive), which is 5.7-fold higher than the rate (0.0095%) observed in this study (9
). However, there was only one positive clone in the second screening by IFA with intact trophozoites. This suggests that the proportion of antibodies recognizing the trophozoite surface in the symptomatic patient with a liver abscess was smaller than the proportion in the asymptomatic cyst passer, even though the anti-E. histolytica
antibody titer was higher in the symptomatic patient.
In the present study, when the heavy or light chain of CP33 was recombined with genes from the two libraries and rescreened, the positive rates were higher in the LA library. This may have been due to the symptomatic patient with the amebic liver abscess having a high antibody titer. However, the relative values were decreased to 2- or 4-fold from 5.7-fold. We concluded from these results that asymptomatic cyst passers have a high ratio of antibodies recognizing the adherence-inhibiting epitope of the heavy-subunit lectin compared with the ratio in symptomatic patients. These adherence-inhibiting antibodies may help prevent the invasion of trophozoites into tissues in cyst passers, although no information was obtained in this study concerning antibodies to other adherence-inhibiting epitopes.
When the heavy chain of CP33 was combined with the light chains from the libraries, the positive rates for screening by colony blotting were 10- to 20-fold higher than the positive rates for screening of combinations of light chains of CP33 with heavy chains. This suggests that the heavy chain is more important for the binding of antibodies to the lectin. Indeed, the fact that the V-segment gene sequence of CP33-H contains many somatic mutations and the fact that no gene homologous with CP33-H has been reported are in accord with the observation that CP33 is reactive specifically with E. histolytica
. Heavy-chain dominance in determining antigen binding has been demonstrated for antibodies to gp120 and to the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) (6
). In contrast, it has been reported that DNA binding activity is determined by the light chains in human anti-double-stranded DNA IgG Fab clones (41
The present study revealed that all the most similar V-segment germ lines of the cloned heavy chain belonged to the VH3 family. The VH3 gene family, with 22 functional genes, is the largest of the seven families (VH1 to VH7) and comprises about one-half of the expressed VH repertoire in adult peripheral B cells (13
). However, biases in gene family usage of the heavy-chain variable region have been reported in a number of diseases. For example, restricted VH3 germ line gene usage was observed in intravenous immunoglobulin-bound Fabs from a patient with thrombocytopenia that had progressed to systemic lupus erythematosus (31
). It is known that VH3 antibodies are also important for defense against a variety of bacteria (1
) and viruses (2
). Although VH3 antibodies bind to HIV-1 gp120 in HIV-infected late-stage patients, VH3 gene family expression is reduced compared with the expression in healthy donors, but the other two main VH gene families, VH1 and VH4, show no significant variation in expression (14
). When the gene usage of another neutralizing anti-E. histolytica
lectin Fab, LA-01, which had previously been prepared from an LA library (9
), was examined, it was found that the most similar germ lines of LA-01 were VH3-30, D1-26, and JH6c for the heavy chain and Vκ02/012 and Jκ5 for the light chain. This observation also supports the preferential usage of the VH3 gene family for the adherence-inhibiting epitope of the lectin. To our knowledge, this report is the first report demonstrating that VH3 antibodies are important in defense against parasitic infections.
In contrast to heavy-chain gene usage, the light-chain gene repertoire of human antibodies to HIV does not exhibit a family bias (15
). In the present study, all 14 light chains from both libraries belonged to the Vκ1 family, in which the closest Vκ germ lines were 02/012 and L5, in spite of the selection of clones showing different patterns after restriction endonuclease digestion. This finding demonstrates that a limited repertoire of light-chain genes is required to create a functional binding site with the heavy chain of CP33. This is in accord with previous reports that some heavy chains prefer to pair with similar light-chain variable regions to form high-affinity binders (3
In the present study, we found that there was only one amino acid residue that was different in CDR3 when CP33-L and L-CP17 were compared. One of the advantages of recombinant antibody technology is possible modification of the original antibody gene. By introduction of synthetic genetic variability in CDR3, which is an important region in antigen binding, it may be possible to increase the affinity and/or neutralizing activity of the antibody.
Whereas the advantage of a phage display system for the preparation of human antibodies has recently been demonstrated (5
), the present study shows that screening by colony blotting and chain shuffling of cloned genes may be a useful way to find genes of immunoglobulins with high affinity to pathogens. Total analysis of antibody genes for the amebic lectin, including other adherence-inhibiting epitopes on the heavy and intermediate subunits of the lectin, should be helpful not only for understanding the mechanism of protective immunity but also for development of immunoprophylaxis against invasive amebiasis.