In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter.

Charlotte Vanmarsenille,Freddy Haesebrouck,Gholamreza Hassanzadeh-Ghassabeh,Gabriela Garcia Rodriguez,Charlotte Yvanoff,Frank Pasmans,Jean-Pierre Hernalsteens,Jelle Elseviers,Edo Martens,Henri De Greve,Ann Depicker,An Martel

doi:10.1371/journal.pone.0204222

Abstract

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds.

Highlights

The construction and expression of chimeric IgY and IgA antibodies is described. These were transiently expressed in Nicotiana benthamiana leaves and stably in A. thaliana seeds, under the control of the seed-specific β-phaseolin promoter. We show that these chimeric antibodies recognise their respective target antigens and the Campylobacter bacteria
C. jejuni KC40 [33] was grown on Nutrient Broth Nr.2 solidified with 1.5% agar (NB2, CM0067; Thermo Fisher Scientific) under microaerobic conditions (Oxoid CampyGen, Thermo Fisher Scientific) for 48 hours at 42 ̊C
Nb5 and Nb23 are directed against the major outer membrane protein (MOMP) and in a previous study it was shown that these nanobodies have a broad host specificity, interacting with multiple Campylobacter strains [27]

Summary

Introduction

Seed extract containing anti-flagellin chimeric antibodies (± 50 μg/ml) (S1 Table) was pre-incubated with equal volumes of C. jejuni KC40 (OD660 0.3) for 1 hour at room temperature. Like in the results of the transient expression, no clear differences in band pattern were observed after Coomassie Blue staining, between the extract of wild-type A. thaliana seeds and the ones transformed with any of the six chimeric antibody constructs.

Results

Conclusion