A Heterodimeric Antibody Fragment for Passive Immunotherapy Against Norovirus Infection

2020 ◽  
Vol 222 (3) ◽  
pp. 470-478 ◽  
Author(s):  
Yoshikazu Yuki ◽  
Shiho Kurokawa ◽  
Shintaro Sato ◽  
Ai Sasou ◽  
Naomi Matsumoto ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Antibody Fragment ◽  
Induced Pluripotent Stem Cell ◽  
Oral Immunotherapy ◽  
Intestinal Epithelial ◽  
Passive Immunotherapy ◽  
Human Noroviruses ◽  
Heavy Chain Antibody ◽  
Induced Pluripotent ◽  
Acid Stable

Abstract Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons.

scholarly journals Development of Antibody-Fragment–Producing Rice for Neutralization of Human Norovirus

2021 ◽  
Vol 12 ◽  
Author(s):  
Ai Sasou ◽  
Yoshikazu Yuki ◽  
Shiho Kurokawa ◽  
Shintaro Sato ◽  
Yuki Goda ◽  
...  
Keyword(s):  
Storage Proteins ◽  
Antibody Fragment ◽  
Induced Pluripotent Stem Cell ◽  
The Elderly ◽  
Oral Immunotherapy ◽  
Rice Seed ◽  
Human Norovirus ◽  
Neutralizing Activity ◽  
High Yields ◽  
Overexpression System

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.

scholarly journals Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells

2020 ◽  
Author(s):  
Aafke W. F. Janssen ◽  
Loes P. M. Duivenvoorde ◽  
Deborah Rijkers ◽  
Rosalie Nijssen ◽  
Ad A. C. M. Peijnenburg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
Cyp Enzymes ◽  
Intestinal Organoids ◽  
Induced Pluripotent

AbstractHuman intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by β-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

scholarly journals A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

2018 ◽  
Vol 10 (1) ◽  
pp. 314-328 ◽  
Author(s):  
Yu Takahashi ◽  
Shintaro Sato ◽  
Yosuke Kurashima ◽  
Tomohisa Yamamoto ◽  
Shiho Kurokawa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Culture System ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Intestinal Epithelial ◽  
Induced Pluripotent
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