Shiga toxin-induced apoptosis is more efficiently inhibited by dimeric recombinant hybrid-IgG/IgA immunoglobulins than by the parental IgG monoclonal antibodies

Abstract

Abbreviations: CHO, Chinese hamster ovary; J chain, joining chain; SIgA, secretory IgA; Stx1, Shiga toxin 1; Stx1B, B subunit of Stx1.Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli strains such as O157:H7 and Shigella dysenteriae. To prevent entry of Stx1 from the mucosal surface, an immunoglobulin A (IgA) specific for Stx1 would be useful. Due to the difficulty of producing IgA monoclonal antibodies (mAb) against the binding subunit of Stx1 (Stx1B) in mice, we took advantage of recombinant technology that combines the heavy chain variable region from Stx1B-specific IgG1 mAb and the Fc region from IgA. The resulting hybrid IgG/IgA was stably expressed in Chinese hamster ovary cells as a dimeric hybrid IgG/IgA. We separated the dimeric hybrid IgG/IgA from the monomeric one by sizeexclusion chromatography. The dimer fraction, confirmed by immunoblot analyses, was used for toxin neutralization assays. The dimeric IgG/IgA was shown to neutralize Stx1 toxicity toward Vero cells by assaying their viability. To compare the relative effectiveness of the dimeric hybrid IgG/IgA and parental IgG1 mAb, Stx1-induced apoptosis was examined using 2 different cell lines, Ramos and Vero cells. The hybrid IgG/IgA inhibited apoptosis more efficiently than the parental IgG1 mAb in both cases. The results indicated that the use of high affinity binding sites as variable regions of IgA would increase the utility of IgA specific for virulence factors.

Prevention of Shiga toxin 1-caused colon injury by plant-derived recombinant IgA

“…Our results demonstrated that the structural platform of IgA is not inferior as to toxin neutralization. This has also been shown through a comparison between the recombinant hyIgA expressed in CHO cells and the original IgG1 mAb D11C6 29 . It is still possible that the utility of IgA depends on the target antigens and the origin of the antibody genes.…”

“…As to the hyIgA-plantibody, the cost was $33. In contrast, as to the hyIgA produced in Chinese Hamster Ovary cells 29 , the cost was $293 in our previous experiments. These figures indicated a practical advantage of the IgA-plantibody for testing the in vivo efficacy.…”

“…Stx1 holotoxin and a recombinant Stx1B were prepared as described previously 13 , 49 ; cloning vectors pGEM5zf/T LHCB1.1 , pGEM5zf/T LHCB1.3 and pGEM5zf/P LHCB as described previously 18 ; Agrobacterium tumefaciens ( Rhizobium radiobacter ) strain GV3101 as described previously 50 ; and Stx1B-specific IgG1 mAb (D11C6) as described previously 13 , 29 . A. thaliana ecotype Col-0 was obtained from Arabidopsis Biological Resource Center (Columbus, OH, USA).…”

Strategies to avoid shiga toxin effects

“…Imai et al (this issue) compared the effectiveness of the hybrid IgG/IgA and parenteral IgG1 by observing apoptosis inhibition using different cells lines. 26 This study demonstrated that the hybrid IgG/IgA inhibits apoptosis more efficiently than the parenteral IgG on Ramos cells and the sane was observed in Vero cells. This study highlights the potential expansion of IgA repertoire by using high affinity binding sites as variable regions of IgA.…”

Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA

“…The dimeric hybrid-IgG/IgA expressed in Chinese hamster ovary cells together with mouse J chain efficiently neutralized Stx1 toxicity in vitro. The neutralizing activity of the dimeric one was stronger than that of the recombinant monomeric IgA and the original IgG mAb2324. We then established transgenic Arabidopsis thaliana expressing the dimeric hybrid-IgG/IgA, and found that a leaf extract was capable of neutralizing Stx1 toxicity in vitro 25.…”