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

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.

Phage display was used for the isolation of Campylobacter-specific nanobodies from this immune library. The phage library was panned twice against the purified flagellins (0.1 µg per well) and uncoated wells. Serial tenfold dilution of binding phages (from undiluted up to 10 -5 ) from the second panning were eluted and used to infect of E. coli TG1 (Figure A in S2 File).
As can be seen from this figure, 100-fold more colonies were obtained after infection with phages eluted from the flagellin-coated well than from the uncoated well (negative control). wells and non-coated wells was at least 3 were further analysed. axis represents the colony number. The orange line represents the ratio of 3.
If the ratio was equal or larger than three clones were amplified from the TTCCGGCTCGTATG-3') and Nucleotide sequences were obtained for 13 clones (clon Nb2Flag33, Nb2Flag42, Nb2Flag45 Nb2Flag80, Nb2Flag81 and Nb2Flag82 The nanobodies were grouped into three sequence families sequences of the 13 clones ( was equal or larger than three, the nanobody-encoding genes from from the pHEN4 vector using the primers MP57 ) and GIII (5'-CCACAGACAGCCCTCATAG-Nucleotide sequences were obtained for 13 clones (clones Nb2Flag8, Nb2Flag24 Nb2Flag45, Nb2Flag46, Nb2Flag59, Nb2Flag66 Nb2Flag82) and these were aligned.
The nanobodies were grouped into three sequence families, on the basis of the To introduce a C-terminal histidine tag (His-tag), the nanobodies Nb2Flag8, Nb2Flag24 and Nb2Flag67 were cloned in the pHEN6c vector (Serge Muyldermans, personal communication), a derivative of the pHEN6 vector (Conrath et al., 2001).
The nanobody sequences were PCR-amplified using the In-Fusion primers IF-NB1 (5′-TGGCCCAGGTGC AGCTGCAGGAGTCTGGAG-3′ carrying the PstI site) and IF-NB2 (5′-TGAGGAGAC GGTGACCTGGGTCC-3′ carrying the BstEII site). The PCR fragments were introduced in the pHEN6c, digested with PstI and BstEII, using the In-Fusion® HD Cloning Kit (Takara Bio USA, Inc). The resulting constructs were transformed into CaCl 2 -competent E. coli DH5α (Dagert and Ehrlich, 1979) and transformants were selected on LB-agar plates supplemented with 100 µg/ml carbenicillin. Colonies were screened by PCR with the primers FP24 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and RP24 (5′-AGCGGATAACAATTT CACACAGGA-3′). PCR-positive colonies were sequenced to confirm that the nanobodies were correct and carried the C-terminal His-tag. Footnote: The pHEN6 vector is equivalent to the pHEN4 vector, except that the hemagglutinin tag and gene III were replaced by a His-tag to allow detection and purification (Conrath et al., 2001).
However, cloning of VHH genes in the pHEN6 resulted in an out of reading frame His-tag.
The pHEN6c (Serge Muyldermans, personal communication) is a derivative of pHEN6 in which the reading frame with the His-tag is restored. Both plasmids pHEN6 and pHEN6c carry the ampicillin resistance gene.
The pHEN6c (with lower case "c") is different from the pHEN6C (with capital "C") described in Conrath et al. (2011). The pHEN6C is the pHEN6 with a chloramphenicol resistance gene instead of the ampicillin resistance gene.