Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines

PLOS ONE RESEARCH ARTICLE Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines Jordan Demone ID1‡, Mariam Maltseva2‡, Maryam Nourimand1, Mina Nasr-Sharif1, Yannick Galipeau2, Emilio I. Alarcon2,3, Marc-André Langlois ID2,4*, Allyson M. MacLean ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biology, University of Ottawa, Ottawa, Ontario, Canada, 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada, 3 BEaTS Research, Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Ontario, Canada, 4 University of Ottawa Centre for Infection, Immunity and Inflammation (CI3), Ottawa, Ontario, Canada ‡ JD and MM are joint first authors to this work. * (AMM); (MAL) Abstract OPEN ACCESS Citation: Demone J, Maltseva M, Nourimand M, Nasr-Sharif M, Galipeau Y, Alarcon EI, et al. (2022) Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines. PLoS ONE 17(12): e0277668. https://doi.org/10.1371/journal. pone.0277668 Editor: Etsuro Ito, Waseda University: Waseda Daigaku, JAPAN Received: August 4, 2022 Accepted: November 1, 2022 Published: December 14, 2022 Copyright: © 2022 Demone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: This study was funded in part by a COVID-19 Rapid Response grant by the Canadian Institute of Health Research (CIHR, #VR2 – 172722) and by a grant supplement by the Canadian Immunity Task Force (CITF) to MAL. MM holds a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) and YG Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits highaffinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms. Introduction Agroinfiltration is a well-established method used frequently in plant biology in which a strain of the Gram-negative alpha-Proteobacterial species Agrobacterium tumefaciens is ‘injected’ with a needleless syringe into plant leaves. This bacterial plant pathogen then employs a specialized secretion system to genetically transform plant host nuclei by transferring genes of interest into recipient host cells with a high degree of efficiency. The transient transformation of Nicotiana benthamiana via agroinfiltration is one of the most rapid methods to efficiently express recombinant proteins in any eukaryotic system [1]. Unlike traditional stably transformed transgenic plants that may require many months to generate, we can transiently transform the leaves of N. benthamiana to (co-)express one or multiple proteins simultaneously, PLOS ONE | https://doi.org/10.1371/journal.pone.0277668 December 14, 2022 1 / 17 PLOS ONE holds a Canadian Institute of Health Research (CIHR) Frederick Banting and Charles Best graduate scholarship (CGS-M). Production of RBD was supported financially by the National Research Council Canada Pandemic Response Challenge program. Greenhouse management was supported by funding from the University of Ottawa Faculty of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Plant-based production of SARS-CoV-2 antigen observing high levels of protein expression in 3–4 days. This system is highly responsive and flexible. It is well-suited for supporting the rapid development of viral antigen-based diagnostic tests, such as serological assays to detect antibodies in blood, and vaccines against diseases such as COVID-19 in real time where the occurrence of viral variants represent an ever-evolving target. In this work, we describe the development of a rapid and flexible agroinfiltration-based platform for the production of recombinant SARS-CoV-2 Spike Receptor-Binding Domain (RBD) in the plant Nicotiana benthamiana. This platform is simple, massively scalable, costeffective and easily adaptable to reflect rapid changes in circulating viral sequences. The RBD of the viral spike is the region primarily involved in binding to the cell surface receptor of the virus, the angiotensin converting enzyme 2 (ACE2) receptor [2]. Most neutralizing antibodies against SARS-CoV-2 are directed to RBD [3–5]. A number of studies have already demonstrated the feasibility of plant-based RBD antigen development: histidine-tagged RBD has been expressed at levels as high as 8 ug/g leaf biomass [6], and similar RBD antigens exhibit effective neutralizing responses in mice [7] and non-human primates [8]. The system we describe here utilizes Agrobacterium tumefaciens-mediated transient transformation of N. benthamiana (Fig 1), to enable the biosynthesis of high-quality SARS-CoV-2 RBD antigen in planta. This accessible and cost-effective process requires approximately only six weeks, encompassing seed germination to delivery of the tandem-purified antigen. We demonstrate in this study that plant expressed RBD displays similar biochemical, structural and antigenic properties as RBD produced in classical mammalian cell expression systems, thereby indicating its suitability for use in diagnostic tests. Aside from diagnostics assays, SARS-CoV-2 antigens can also be utilized in adjuvanted subunit vaccines [9–11], as well as unadjuvanted vaccines when used as a booster shot [12]. While most SARS-CoV-2 vaccines focus on intramuscular delivery systems, development of a subunit nasal spray vaccine offers a complementary method to increase mucosal immunity to SARS-CoV-2 variants and provide vaccine-hesitant and needle-phobic individuals with alternative immunization options [13]. Prot (...truncated)