Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

Nakanishi, Koichi; Narimatsu, Sanshiro; Ichikawa, Shiori; Tobisawa, Yuki; Kurohane, Kohta; Niwa, Yasuo; Kobayashi, Haruo; Imai, Yumiko

doi:10.1371/journal.pone.0080712

Cited by 16 publications

(24 citation statements)

References 46 publications

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“…Recently, we succeeded in producing a dimeric hybrid-IgG/IgA in a plant system involving Arabidopsis thaliana. 14) In this plant, there are also monomers, and several incomplete IgA and fragments. Although plantibodies are able to neutralize toxins, we could not determine the relative efficacy of intact dIgA as to the toxin neutralization.…”

Section: Discussionmentioning

confidence: 99%

Stable Expression and Characterization of Monomeric and Dimeric Recombinant Hybrid-IgG/IgA Immunoglobulins Specific for Shiga Toxin

Iwata

Kurohane

Nakanishi

et al. 2014

Biological & Pharmaceutical Bulletin

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Antigen-specific immunoglobulin A (IgA) may be useful for preventing infectious diseases through passive immunization on the mucosal surface. We previously established mouse IgA and IgG monoclonal antibodies (mAbs) specific for the binding subunit of Shiga toxin 1 (Stx1B). We also developed a recombinant hybrid-IgG/IgA, in which variable regions from the IgG mAb were present. The binding activity of recombinant hybrid-IgG/IgA was verified by transient expression. Aiming at a constant supply, we established Chinese hamster ovary cells stably expressing monomeric or dimeric hybrid-IgG/IgA. The cDNAs encoding heavy and light chains were co-expressed for the monomeric hybrid-IgG/IgA, while those encoding heavy, light, and joining chains were co-expressed for the dimeric one. Serum-free culture supernatants of the cloned transfectants were subjected to size-exclusion chromatography. The elution patterns showed that the binding to immobilized Stx1B and the immunoblot signals of assembled immunoglobulins were correlated. In the transfectant for the dimeric hybrid-IgG/IgA, both monomers and dimers were observed. Size-exclusion chromatography enabled us to prepare a sample of the dimeric hybrid-IgG/IgA devoid of the monomeric one. The monomeric and dimeric forms of hybrid-IgG/IgA were prepared from the respective transfectants to examine the neutralization of Stx1. After pretreatment with monomeric or dimeric hybrid-IgG/IgA, the cytotoxicity of Stx1 toward Vero cells was abolished. Furthermore, the dimeric form was more than 10-fold more effective than the monomeric one in terms of toxin neutralization. These results suggest that the tetravalent feature of the binding sites of the dimeric hybrid-IgG/IgA contributes to the efficacy of toxin neutralization.Key words recombinant immunoglobulin A; Shiga toxin; neutralization; dimer Shiga toxin 1 (Stx1) is a virulence factor of enterohaemorrhagic Escherichia coli (EHEC) strains such as O157 : H7 and Shigella dysenteriae.1,2) It comprises one A subunit and five B subunits.3) After binding through B subunits (Stx1B), which are cell-binding subunits, Stx1 holotoxins are endocytosed. 4)The A subunit, which is translocated into the cytoplasm, induces cytotoxicity through dysfunction of ribosomes and inhibition of protein synthesis. 5)Immunoglobulin A (IgA) plays a role in the humoral arm of the adaptive immunity on the mucosal surface.6) It is produced by B cells after class switch of heavy (H) chains from IgM to IgA. IgA-producing B cells also produce joining (J) chains that covalently join IgA monomers to form dimeric IgA (dIgA).7) On the mucosal epithelium, the dimeric IgA is transported by polymeric immunoglobulin receptors (pIgR) from the basolateral to the apical side. Dimer formation is required for the binding to pIgR.6) On the mucosal surface, pIgR is cleaved by proteases, and the complex of dIgA and the ectodomain of pIgR is secreted onto the mucosal surface. The ectodomain of pIgR is termed a secretory component (SC) in a secretory IgA (SIgA). SIgA prevents invasion by ...

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Section: Discussionmentioning

confidence: 99%

Stable Expression and Characterization of Monomeric and Dimeric Recombinant Hybrid-IgG/IgA Immunoglobulins Specific for Shiga Toxin

Iwata

Kurohane

Nakanishi

et al. 2014

Biological & Pharmaceutical Bulletin

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“…In this experiment, we also performed comparison with the dimer hybrid-IgG/IgA transgenic A. thaliana (dimer Tg) expressing H, L and J chains but no SC, which had been established previously25. Under reducing conditions, α, κ and J chains were detected in the secretory Tg as well as in the dimer Tg25, whereas SC was only detected in the secretory Tg (Fig.…”

Section: Resultsmentioning

confidence: 87%

“…We have previously established A. thaliana producing dimeric hyIgA where IgA heavy and light chains are expressed under a CAB promoter while J chain is expressed under a 35S promoter25. In contrast, the current S-hyIgA is expressed only using two CAB promoters in the absence of 35S promoter.…”

Section: Discussionmentioning

confidence: 99%

“…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. In this transgenic plant, the transcription of the H and L chain genes is controlled by a chlorophyll a/b -binding protein (CAB) promoter derived from A. thaliana , whereas the J chain gene is controlled by the cauliflower mosaic virus 35S promoter.…”

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

Nakanishi

Morikane

Ichikawa

et al. 2017

Sci Rep

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Shiga toxin is a major virulence factor of food-poisoning caused by Escherichia coli such as O157:H7. Secretory immunoglobulin (Ig) A (SIgA) is supposed to prevent infection of the mucosal surface and is a candidate agent for oral immunotherapy. We previously established a recombinant monoclonal antibody (mAb) consisting of variable regions from a mouse IgG mAb specific for the binding subunit of Shiga toxin 1 (Stx1) and the Fc region of mouse IgA. Here we produced a secretory form of the recombinant IgA (S-hyIgA) with transgenic Arabidopsis thaliana plant. All the S-hyIgA cDNAs (heavy, light, J chain and secretory component) were expressed under the control of a bidirectional promoter of a chlorophyll a/b-binding protein of A. thaliana without using a viral promoter. The plant-based S-hyIgA exhibited antigen binding, and was modified with plant-specific N-linked sugar chains. The Ig heavy chain and secretory components were observed in an intracellular protein body-like structure of the transgenic leaves on immuno-electron microscopy. An extract of the transgenic leaves neutralized the cytotoxicity of Stx1 toward butyrate-treated Caco-2 cells, a human colon carcinoma cell line. These results will contribute to the development of edible therapeutic antibodies such as those for the treatment of mucosal infection.

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“…After blotting onto a PVDF membrane (Millipore), the hybrid IgG/ IgA was detected with HRP-goat anti-mouse IgA (1:1,000) using a chemiluminescence reagent (West Pico; Thermo Scientific Pierce) with a luminescent image analyzer, LAS-3000 (FUJI Photo Film, Tokyo, Japan). 15 Fractions containing dimeric hybrid IgG/IgA were pooled and concentrated, and the buffer was changed to sterile PBS by means of a Vivaspin to remove NaN 3 , followed by sterilization by membrane filtration.…”

Section: Separation Of Dimeric Hybrid Igg/igamentioning

confidence: 99%

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

et al. 2014

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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.

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Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

Cited by 16 publications

References 46 publications

Stable Expression and Characterization of Monomeric and Dimeric Recombinant Hybrid-IgG/IgA Immunoglobulins Specific for Shiga Toxin

Stable Expression and Characterization of Monomeric and Dimeric Recombinant Hybrid-IgG/IgA Immunoglobulins Specific for Shiga Toxin

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

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

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