Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates

Abstract

The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.

Keywords: COVID-19; Fc fusion protein; Nicotiana benthamiana; SARS-CoV-2; plant-produced recombinant protein; receptor-binding domain; subunit vaccine.

FIGURE 1

Diagrammatic representation showing the T-DNA of plant expression vector pBYR2e-SARS-CoV-2 RBD-Fc and the overview of transient expression in N. benthamiana plants. RB and LB, left and right borders of the T-DNA used in Agrobacterium DNA delivery into plant cells; Pin II 3’, the terminator from potato proteinase inhibitor II gene; P19, the RNA silencing suppressor from Tomato Bushy Stunt Virus (TBSV); P35s, 35s promoter from Cauliflower Mosaic Virus (CaMV); P35s × 2, 35s promoter from CaMV with duplicated enhancer; Ext3’ FL, 3’ region of tobacco extension gene; Rb7 5’ del, tobacco RB7 promoter; SIR, short intergenic region of Bean Yellow Dwarf Virus (BeYDV); LIR, long intergenic region of BeYDV; C2/C1, Rep/RepA gene from BeYDV encoding for replication initiation protein (Rep) and RepA.

FIGURE 2

Expression profiles of plant-produced SARS-CoV-2 RBD-Fc fusion protein. Phenotype of leaf infiltrated with Agrobacterium control (1) and Agrobacterium containing pBYR2e-SARS-CoV-2 RBD-Fc (2) after 4 dpi (A). SDS-PAGE analysis of plant-produced SARS-CoV-2 RBD-Fc fusion protein stained with Coomassie staining (B) and western blot of plant-produced SARS-CoV-2 RBD-Fc fusion protein probed with anti-human gamma-HRP conjugate antibody (C). Lane 1 and 2, purified plant-produced SARS-CoV-2 RBD-Fc fusion protein under reducing and non-reducing condition, respectively.

FIGURE 3

Binding activity of the plant-produced SARS-CoV-2 RBD-Fc with the commercial angiotensin-converting enzyme 2 (ACE2 proteins) derived from HEK293 and CHO cells analyzed by ELISA. PBS was used as negative control. Data are presented as mean ± standard deviation (SD) of triplicates in each sample dilution.

FIGURE 4

Immunogenic studies in mice. Schematic representation of immunization protocol and sample collection. Groups of mice (five mice per each group) were intramuscularly immunized with 10 μg of SARS-CoV-2 RBD-Fc fusion protein alone or with alum adjuvant, followed by booster dose at 21 days after first immunization. Mice sera were collected on day 0 (pre-bleed) and day 14 post-immunization (A). Titers of SARS-CoV-2 RBD-specific total IgG (B), IgG1 (C), and IgG2a (D) in the immunized sera collected on day 0, 14, and 35 were analyzed by indirect ELISA using Sf9-produced SARS-CoV-2 RBD-His as the capture antigen. Potent neutralizing antibody titers in mice sera were tested by in vitro microneutralization assay using Vero E6 cell line and live SARS-CoV-2 (E). The functional profiles of SARS-CoV-2 RBD-specific T-cell responses expressing in mouse splenocytes immunized with plant-produced SARS-CoV-2 RBD-Fc adjuvanted with alum were determined by mouse ELISpot assay (F). Data presented as mean ± SD of the endpoint titers in each mice vaccination group (n = 5). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

FIGURE 5

Immunogenic studies in non-human primates (Macaca fascicularis). Experimental design of immunogenicity studies in non-human primates. Thirteen juvenile-adult non-human primates were separated into 3 groups; Control group was immunized with PBS adjuvanted by alum (n = 3) and two experimental groups were immunized with 25 and 50 μg SARS-CoV-2 RBD-Fc along with alum adjuvant (n = 5 per group). All non-human primates were intramuscularly injected either with PBS or plant-produced RBD-Fc for 3 weeks interval (on day 0 and 21). The sera were collected on day 0 and 14 after each boost (A). Serum specific IgG response in non-human primates were determined by ELISA (B). Virus neutralizing titer of RBD immunized non-human primate sera against live SARS-CoV-2 were evaluated (C). The functional profiles of SARS-CoV-2 RBD-specific T-cell responses expressing in non-human primate peripheral blood mononuclear cells immunized with plant-produced RBD-Fc adjuvanted with alum on day 14 after second immunization (D). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.