Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

A Highly Expressing, Soluble, and Stable Plant-Made IgG Fusion Vaccine Strategy Enhances Antigen Immunogenicity in Mice Without Adjuvant.

Therapeutics based on fusing a protein of interest to the IgG Fc domain have been enormously successful, though fewer studies have investigated the vaccine potential of IgG fusions. In this study, we systematically compared the key properties of seven different plant-made human IgG1 fusion vaccine candidates using Zika virus (ZIKV) envelope domain III (ZE3) as a model antigen. Complement protein C1q binding of the IgG fusions was enhanced by: 1) antigen fusion to the IgG N-terminus; 2) removal of the IgG light chain or Fab regions; 3) addition of hexamer-inducing mutations in the IgG Fc; 4) adding a self-binding epitope tag to create recombinant immune complexes (RIC); or 5) producing IgG fusions in plants that lack plant-specific ß1,2-linked xylose and α1,3-linked fucose N-linked glycans. We also characterized the expression, solubility, and stability of the IgG fusions. By optimizing immune complex formation, a potently immunogenic vaccine candidate with improved solubility and high stability was produced at 1.5 mg IgG fusion per g leaf fresh weight. In mice, the IgG fusions elicited high titers of Zika-specific antibodies which neutralized ZIKV using only two doses without adjuvant, reaching up to 150-fold higher antibody titers than ZE3 antigen alone. We anticipate these findings will be broadly applicable to the creation of other vaccines and antibody-based therapeutics.

Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity.

Zika virus (ZIKV) reemergence poses a significant health threat especially due to its risks to fetal development, necessitating safe and effective vaccines that can protect pregnant women. Zika envelope domain III (ZE3) has been identified as a safe and effective vaccine candidate, however it is poorly immunogenic. We previously showed that plant-made recombinant immune complex (RIC) vaccines are a robust platform to improve the immunogenicity of weak antigens. In this study, we altered the antigen fusion site on the RIC platform to accommodate N-terminal fusion to the IgG heavy chain (N-RIC), and thus a wider range of antigens, with a resulting 40% improvement in RIC expression over the normal C-terminal fusion (C-RIC). Both types of RICs containing ZE3 were efficiently assembled in plants and purified to >95% homogeneity with a simple one-step purification. Both ZE3 RICs strongly bound complement receptor C1q and elicited strong ZE3-specific antibody titers that correlated with ZIKV neutralization. When either N-RIC or C-RIC was codelivered with plant-produced hepatitis B core (HBc) virus-like particles (VLP) displaying ZE3, the combination elicited 5-fold greater antibody titers (>1,000,000) and more strongly neutralized ZIKV than either RICs or VLPs alone, after only two doses without adjuvant. These findings demonstrate that antigens that require a free N-terminus for optimal antigen display can now be used with the RIC system, and that plant-made RICs and VLPs are highly effective vaccines targeting ZE3. Thus, the RIC platform can be more generally applied to a wider variety of antigens.

Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis.

BACKGROUND: Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of C. perfringens produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. METHODS: We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. RESULTS: Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry.

High Level Production of Monoclonal Antibodies Using an Optimized Plant Expression System.

Biopharmaceuticals are a large and fast-growing sector of the total pharmaceutical market with antibody-based therapeutics accounting for over 100 billion USD in sales yearly. Mammalian cells are traditionally used for monoclonal antibody production, however plant-based expression systems have significant advantages. In this work, we showcase recent advances made in plant transient expression systems using optimized geminiviral vectors that can efficiently produce heteromultimeric proteins. Two, three, or four fluorescent proteins were coexpressed simultaneously, reaching high yields of 3-5 g/kg leaf fresh weight or ~50% total soluble protein. As a proof-of-concept for this system, various antibodies were produced using the optimized vectors with special focus given to the creation and production of a chimeric broadly neutralizing anti-flavivirus antibody. The variable regions of this murine antibody, 2A10G6, were codon optimized and fused to a human IgG1. Analysis of the chimeric antibody showed that it was efficiently expressed in plants at 1.5 g of antibody/kilogram of leaf tissue, can be purified to near homogeneity by a simple one-step purification process, retains its ability to recognize the Zika virus envelope protein, and potently neutralizes Zika virus. Two other monoclonal antibodies were produced at similar levels (1.2-1.4 g/kg). This technology will be a versatile tool for the production of a wide spectrum of pharmaceutical multi-protein complexes in a fast, powerful, and cost-effective way.