Oral antibodies that interfere with gastrointestinal targets and can be manufactured at scale are needed. Here we show that a single-gene-encoded monomeric immunoglobulin A (IgA)-like antibody, composed of camelid variable single domain antibodies (VHH) fused to IgA Fc (mVHH-IgA), prevents infection by enterotoxigenic Escherichia coli (F4-ETEC) in piglets. The mVHH-IgA can be produced in soybean seeds or secreted from the yeast Pichia pastoris, freeze- or spray-dried and orally delivered within food.
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Gagniere, J. et al. World J. Gastroenterol. 22, 501–518 (2016).
Katsanos, K. H. & Papadakis, K. A. Gut Liver 11, 455–463 (2017).
Förster, B., Chung, P. K., Crobach, M. J. T. & Kuijper, E. J. Front. Microbiol. 9, 1382 (2018).
Kelly, D., Yang, L. & Pei, Z. Diseases 6, 109 (2018).
Brandtzaeg, P. Front. Immunol. 4, 222 (2013).
Corthésy, B. Curr. Pharm. Biotechnol. 4, 51–67 (2003).
Moor, K. et al. Nature 544, 498–502 (2017).
Strugnell, R. A. & Wijburg, O. L. C. Nat. Rev. Microbiol. 8, 656–667 (2010).
Reinhart, D. & Kunert, R. Biotechnol. Lett. 37, 241–251 (2015).
Virdi, V. et al. Proc. Natl Acad. Sci. USA 110, 11809–11814 (2013).
Fairbrother, J. M., Nadeau, É. & Gyles, C. L. Anim. Health Res. Rev. 6, 17–39 (2005).
Guerrant, R. L., Steiner, T. S., Lima, A. A. & Bobak, D. A. J. Infect. Dis. 179, S331–S337 (1999).
Smart, R. D., Blum, M. & Wesseler, J. J. Agric. Econ. 68, 182–198 (2017).
Ciofalo, V., Barton, N., Kreps, J., Coats, I. & Shanahan, D. Regul. Toxicol. Pharmacol. 45, 1–8 (2006).
Raveendran, S. et al. Food Technol. Biotechnol. 56, 16–30 (2018).
Moonens, K. et al. Vet. Res. 46, 14 (2015).
Muyldermans, S. Annu. Rev. Biochem. 82, 775–797 (2013).
Karimi, M., Inzé, D. & Depicker, A. Trends Plant Sci. 7, 193–195 (2002).
Paz, M. M., Martinez, J. C., Kalvig, A. B., Fonger, T. M. & Wang, K. Plant Cell Rep. 25, 206–213 (2006).
De Meyer, T. et al. Plant Biotechnol. J. 13, 938–947 (2015).
Näätsaari, L. et al. PLoS ONE 7, e39720 (2012).
Jacobs, P. P., Geysens, S., Vervecken, W., Contreras, R. & Callewaert, N. Nat. Protoc. 4, 58–70 (2009).
Stals, I. et al. FEMS Microbiol. Lett. 303, 9–17 (2010).
Goetstouwers, T. et al. PLoS ONE 9, e105013 (2014).
Ericsson, U. B., Hallberg, B. M., DeTitta, G. T., Dekker, N. & Nordlund, P. Anal. Biochem. 357, 289–298 (2006).
We thank H. De Greve for advice with the anti-F4-ETEC VHHs and sharing purified antigen. We thank E. Van Lerberge and J. Nolf for overall technical support, particularly for upscaling seeds, antibody expression analysis and protein analysis. We thank S. Brabant for help with blood sampling and analysis of the seroconversion, R. Cooman for animal caretaking and management of the stables and U. Van Nguyen for performing the villous adhesion assay. We thank K. Wang of Iowa State University for the soybean transformation, J. Haustraete of the Protein Service facility of VIB for large-scale production of Pichia cultures and D. De Paepe and K. Coudyser of ILVO food pilot for lyophilization of Pichia spent medium. For access to weaned piglets we acknowledge the farms of K. Devolder and ILVO, and thank their resident animal husbandry teams. We are grateful to S. Millet for feed consultation and access to large industrial mixers for feed formulation at ILVO, and T. Moravec of the Institute of Experimental Botany Prague, J. Mar Björnsson of ORF Genetics and J. Van Huylenbroeck of ILVO for kindly leasing additional greenhouse space for scaling up soybean seeds. The authors would finally like to thank M. Vuylsteke and V. Storme for help with the statistical analysis and A. Bleys for help with editing the manuscript. This work was supported by an IWT-innovation fellowship (No. IM-140851) awarded to V.V., co-sponsored by AVEVE Biochem, AVEVE Group. J.P. received a PhD stipend from the Research Foundation Flanders (FWO project grant No. G0C9714N). B.L. has been supported by the European Research Council’s consolidator grant awarded to N.C. (No. ERC-2013-CoG-616966). Overall we would like to acknowledge institutional funding and support from Ghent University and VIB.
E.V. is an employee of AVEVE Biochem. V.V., B.L., N.C. and A.D. are inventors on one or more patent applications related to the inventions reported in this publication. Research in the author’s laboratories has been sponsored in part by the AVEVE Group.
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(a) Representative immunoblots of V2A produced in Arabidopsis (At-V2A) and soybean (Gm-V2A), in which endoglycosidase T (Endo T) treatment leads to a mobility shift. A proteolytic fragment of < 30 kDa is prominent in both plant species. (b) Pichia-based expression of V2A (Pp-V2A) and V3A (Pp-V3A) depicted in representative immunoblots, in which Endo T treatment shows a marked mobility shift due to removal of glycans. The immunoblots were repeated at least thrice with similar results.
Supplementary Figure 2 The four batches of freeze-dried Pichia secreted mVHH-IgA produced for formulating the Pichia mVHH-IgA feed contained similar proportions of V2A and V3A.
(a) Immunoblot analysis of 10% w/v resuspended solutions from the four freeze-dried batches, pre-treated with Endo H, shows similar band intensity. The volume loaded in each well corresponds to 1.2 mg of freeze-dried material. (b) ELISA titration curve depicting identical endpoint titers for each of the four batches of Pichia produced mVHH-IgA in binding the immobilised antigen FaeGac. The slope of V2A and V3A curves are different as the KD of the VHH V2 and V3 is quiet different (0.1 µM and 7.7 µM). The analysis was repeated at least twice with similar results.
Supplementary Figure 3 The proportion of mVHH-IgA in the different feed formulations fed to the piglets.
Equal part mix of mVHH-IgA V2A and V3A in the final feed formulations either secreted from Pichia (Pichia mVHH-IgA feed), or produced in soybean (soybean mVHH-IgA feed) or Arabidopsis (Arabidopsis mVHH-IgA feed) were analyzed as compared to negative control feed without mVHH-IgA. (a) Immunoblot of 10% (w/v) feed suspension extracts which were pre-treated with Endo H. The volume of feed extract loaded corresponds to 1.2 mg of each feed formulation. The densitometric analysis on the ~ 37 kDa band, using the purified Endo H treated Pichia-secreted V2A dilution series as reference standards, corresponded to be 0.0011% for Pichia feed (3.3 mg in 300 gram daily feed), 0.0025% for soybean feed (7.5 mg in 300 gram daily feed) and 0.0013% for Arabidopsis feed (3.9 mg in 300 gram daily feed). (b, c) ELISA titration curves of the 10% (w/v) suspensions of all the experimental feed formulations containing mVHH-IgA (b) without Endo H pre-treatment and (c) with Endo H pre-treatment. In the latter case, the yeast N-glycan mediated inhibition of mVHH-IgA detectability in ELISA is eliminated, resulting in the curves and the endpoint titers as very similar. These analysis were repeated at least twice with similar results.
Supplementary Figure 4 Drying of the Pichia-secreted mVHH-IgAs can be scaled up with spray-drying technology.
(a) Purified V2A in spray drying buffer or in PBS (phosphate buffer saline) shows identical melting curve (first of two melting transition > 65 °C) in thermal shift assay (b) A pilot-scale spray-dried mixture of Pichia secreted V2A and V3A (equal proportion), showed identical binding activity, before and after drying, to the immobilized antigen in ELISA. Maltodextrin (10%) was used as carrier in the spray-drying run, and also incorporated as negative control in the ELISA. These analysis were repeated twice. (c) Picture of the pilot-scale spray drying installation used, showing bottom 2/3rd of drying chamber and the cyclone (upper right). (d) Sample of the dried powder containing Pichia-secreted mVHH-IgAs.
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Virdi, V., Palaci, J., Laukens, B. et al. Yeast-secreted, dried and food-admixed monomeric IgA prevents gastrointestinal infection in a piglet model. Nat Biotechnol 37, 527–530 (2019). https://doi.org/10.1038/s41587-019-0070-x