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The 2022 global outbreaks of monkeypox virus (MPXV) and increased human-to-human transmission calls for the urgent development of countermeasures to protect people who cannot benefit from vaccination. Here, we describe the development of glycovariants of 7D11, a neutralizing monoclonal IgG antibody (mAb) directed against the L1 transmembrane protein of the related vaccinia virus, in a plant-based system as a potential therapeutic against the current MPVX outbreak. Our results indicated that 7D11 mAb quickly accumulates to high levels within a week after gene introduction to plants. Plant-produced 7D11 mAb assembled correctly into the tetrameric IgG structure and can be easily purified to homogeneity. 7D11 mAb exhibited a largely homogeneous N-glycosylation profile, with or without plant-specific xylose and fucose residues, depending on the expression host, namely wild-type or glycoengineered plants. Plant-made 7D11 retained specific binding to its antigen and displayed a strong neutralization activity against MPXV, as least as potent as the reported activity against vaccinia virus. Our study highlights the utility of anti-L1 mAbs as MPXV therapeutics, and the use of glycoengineered plants to develop mAb glycovariants for potentially enhancing the efficacy of mAbs to combat ever-emerging/re-emerging viral diseases. [ABSTRACT FROM AUTHOR]

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody–viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Summary: This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan‐Hu‐1 RBD of SARS‐CoV‐2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human‐like, highly homogenous N‐linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well‐characterized mAbs, suggesting that it is a Class 4 RBD‐binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS‐CoV‐2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety‐friendly alternative platform for generating mAbs to address the evolving SARS‐CoV‐2 crisis. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a public health crisis over the last two years. Monoclonal antibody (mAb)-based therapeutics against the spike (S) protein have been shown to be effective treatments for SARS-CoV-2 infection, especially the original viral strain. However, the current mAbs produced in mammalian cells are expensive and might be unaffordable for many. Furthermore, the emergence of variants of concern demands the development of strategies to prevent mutant escape from mAb treatment. Using a cocktail of mAbs that bind to complementary neutralizing epitopes is one such strategy. In this study, we use Nicotiana benthamiana plants in an effort to expedite the development of efficacious and affordable antibody cocktails against SARS-CoV-2. We show that two mAbs can be highly expressed in plants and are correctly assembled into IgG molecules. Moreover, they retain target epitope recognition and, more importantly, neutralize multiple SARS-CoV-2 variants. We also show that one plant-made mAb has neutralizing synergy with other mAbs that we developed in hybridomas. This is the first report of a plant-made mAb to be assessed as a potential component of a SARS-CoV-2 neutralizing cocktail. This work may offer a strategy for using plants to quickly develop mAb cocktail-based therapeutics against emerging viral diseases with high efficacy and low costs. [ABSTRACT FROM AUTHOR]

COVID-19, Omicron, Monoclonal antibody (mAb), plant-made antibody, SARS-CoV-2, neutralization synergy Empirically determined percent neutralization values for individual mAbs were then analysed in SynergyFinder.org using four models (Figure 1d) to calculate a predicted percent neutralization, which represents the percent neutralization of the cocktail assuming there is no synergistic interaction between the mAbs. Keywords: SARS-CoV-2; COVID-19; Omicron; Monoclonal antibody (mAb); plant-made antibody; neutralization synergy EN SARS-CoV-2 COVID-19 Omicron Monoclonal antibody (mAb) plant-made antibody neutralization synergy 463 465 3 02/23/23 20230301 NES 230301 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections continue to persist around the globe, amid ongoing emergences of variants that are immune evasive. [Extracted from the article]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has caused more than 4.5 million deaths worldwide. Severe and fatal cases of COVID-19 are often associated with increased proinflammatory cytokine levels including interleukin 6 (IL-6) and acute respiratory distress syndrome. In this study, we explored the feasibility of using plants to produce an anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) and examined its utility in reducing IL-6 signaling in an in vitro model, which simulates IL-6 induction during SARS-CoV-2 infection. The anti-IL6R mAb (IL6RmAb) was quickly expressed and correctly assembled in Nicotiana benthamiana leaves. Plant-produced IL6RmAb (pIL6RmAb) could be enriched to homogeneity by a simple purification scheme. Furthermore, pIL6RmAb was shown to effectively inhibit IL-6 signaling in a cell-based model system. Notably, pIL6RmAb also suppressed IL-6 signaling that was induced by the exposure of human peripheral blood mononuclear cells to the spike protein of SARS-CoV-2. This is the first report of a plant-made anti-IL-6R mAb and its activity against SARS-CoV-2-related cytokine signaling. This study demonstrates the capacity of plants for producing functionally active mAbs that block cytokine signaling and implies their potential efficacy to curb cytokine storm in COVID-19 patients. [ABSTRACT FROM AUTHOR]

The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification. [ABSTRACT FROM AUTHOR]