Production of Polyclonal Mixes of Full-Size IgG Antibodies in Plants Using a Two-Viral-Vector System

Plants are cost-effective bioreactors to produce recombinant proteins for many different biotechnological applications, and plant virus-derived vectors are frequently used to improve yield and shorten production times. Antibodies, the products of vertebrate immune systems, display unsurpassed features in terms of affinity, selectivity, and avidity for their cognate antigens. However, antibody size and heteromultimeric structures make their expression in plants difficult, particularly when employing viral vectors, which typically exhibit cargo restrictions. Here, we have worked on a dual vector system based on potato virus X (PVX) and tobacco mosaic virus (TMV) to produce polyclonal mixes of recombinant antibodies in Nicotiana benthamiana plants. In this system, the heavy and light chains of the different antibodies that compose the polyclonal mix are expressed, respectively, through defective PVX and TMV vectors. Importantly, to avoid antibody chain shuffling in the polyclonal mix, each pair of PVX-TMV vectors that express the cognate heavy and light chains of each monoclonal antibody is inoculated into the plant tissue using a single Agrobacterium tumefaciens clone co-transformed with two mini-binary plasmids with compatible replication origins, in which the viral vectors are cloned. Both viral vectors lack their coat proteins, which contributes to better coupling of the cell-to-cell movement of the two vectors. In addition, the recombinant antibodies are exported to the apoplast of infiltrated tissues from where the polyclonal mix can be easily extracted and directly used for many applications with no further purification. Using this dual vector system, we have successfully produced inN. benthamiana a polyclonal mix of recombinant antibodies that efficiently bind the nucleocapsid protein and two independent epitopes of the spike protein from SARS-CoV-2.