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Summary: Microalgae are considered as attractive expression systems for the production of biologics. As photosynthetic unicellular organisms, they do not require costly and complex media for growing and are able to secrete proteins and perform protein glycosylation. Some biologics have been successfully produced in the green microalgae Chlamydomonas reinhardtii. However, post‐translational modifications like glycosylation of these Chlamydomonas‐made biologics have poorly been investigated so far. Therefore, in this study, we report on the first structural investigation of glycans linked to human erythropoietin (hEPO) expressed in a wild‐type C. reinhardtii strain and mutants impaired in key Golgi glycosyltransferases. The glycoproteomic analysis of recombinant hEPO (rhEPO) expressed in the wild‐type strain demonstrated that the three N‐glycosylation sites are 100% glycosylated with mature N‐glycans containing four to five mannose residues and carrying core xylose, core fucose and O‐methyl groups. Moreover, expression in C. reinhardtii insertional mutants defective in xylosyltransferases A and B and fucosyltransferase resulted in drastic decreases of core xylosylation and core fucosylation of glycans N‐linked to the rhEPOs, thus demonstrating that this strategy offers perspectives for humanizing the N‐glycosylation of the Chlamydomonas‐made biologics. [ABSTRACT FROM AUTHOR]

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies., Competing Interests: Declaration of Competing Interest JC and JB were founders of AlbaJuna Therapeutics. AB, JB, JC and FC were employees of AlbaJuna therapeutics. Other authors, no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is required for the infection of human cells. It is the main target that elicits neutralizing antibodies and also a major component of diagnostic kits. The large demand for this protein has led to the use of plants as a production platform. However, it is necessary to determine the N -glycan structures of an RBD to investigate its efficacy and functionality as a vaccine candidate or diagnostic reagent. Here, we analyzed the N -glycan profile of the RBD produced in rice callus. Of the two potential N -glycan acceptor sites, we found that one was not utilized and the other contained a mixture of complex-type N -glycans. This differs from the heterogeneous mixture of N -glycans found when an RBD is expressed in other hosts, including Nicotiana benthamiana . By comparing the glycosylation profiles of different hosts, we can select platforms that produce RBDs with the most beneficial N -glycan structures for different applications.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development., Competing Interests: Authors MS and ARo were employed by company Diamante SB srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Santoni, Gutierrez-Valdes, Pivotto, Zanichelli, Rosa, Sobrino-Mengual, Balieu, Lerouge, Bardor, Cecchetto, Compri, Mazzariol, Ritala and Avesani.)

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N -glycosylation consensus sites as well as numerous mucin-type O -glycosylation sites. As a consequence, O - and N -glycosylations of this viral protein have received particular attention. Glycans N -linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N -glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana ( N. benthamiana ) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N- glycosylation sites are dominated by complex plant N -glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.