Significance: Sialic acid (Sia) residues are essential monosaccharides in mammals and confer multiple biological functions. Their precise generation is important for both structure–function studies and biotechnological applications. We describe a unique technology that enables the controlled generation of protein sialylation in Nicotiana benthamiana . The plant engineering approach relies on a combination of endogenous glycan deconstruction and the introduction of human sialylation capabilities. An arrangement of transgenic and transient expression modules resulted in the targeted synthesis of Sia structures in three different linkage types, reaching a polymerization degree exceeding 40 residues (polySia). Importantly, the obtained functional activities of polySia point to novel biotherapeutic applications. Our results highlight the exceptional flexibility of the plant-based expression platform for engineering complex posttranslational protein modifications.

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineeredNicotiana benthamianaplants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA’s structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.

Summary: Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (∆XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ∆XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ∆XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ∆XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.

Glyco‐modulation of therapeutic proteins produced in plants has shown great success. Plant‐based expression platforms for tailored human‐like N‐glycosylation are based on the overexpression of foreign genes. However, drawbacks such as protein miss targeting, interference with endogenous glycosyltransferases, or with plant development hamper the widespread use of the technology. Here a technique that facilitates the generation of recombinant proteins with targeted N‐glycosylation at high homogeneity is described. It is focused on the synthesis of human‐type β1,4‐galactosylation by the overexpression of the human β1,4‐galactosyltransferase (GalT) in Nicotiana benthamiana. A GalT construct that targets the enzyme to the required late Golgi compartment (^STGalT) is transiently co‐expressed with two pharmaceutically relevant glycoproteins. The impact of eight promoters driving the expression of ^STGalT is evaluated by mass spectrometry (MS) ‐based analyses. It is shown that five promoters (amongst them high expressors) induce aberrant non‐human glycosylation. In contrast, three promoters, considered as moderately active, regulate gene expression to levels leading to an improved efficiency of di‐galactosylation (and subsequent sialylation) on the reporter proteins. The results point to the importance of promoter choice for optimizing glycan engineering processes.