Production of a Highly Immunogenic Antigen from SARS-CoV-2 by Covalent Coupling of the Receptor Binding Domain of Spike Protein to a Multimeric Carrier

Abstract

Since the discovery of SARS-CoV-2, several antigens have been proposed to be part of COVID-19 vaccines. The receptor binding domain (RBD) of Spike protein is one of the promising candidates to develop effective vaccines since it can induce potent neutralizing antibodies. We previously reported the production of RBD in Pichia pastoris and showed it is structurally identical to the protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer construct with the purpose of increasing RBD immunogenicity. We produced multimeric particles by a transpeptidation reaction between the RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric protein of 170 kDa. We vaccinated mice with two doses 30 days apart, and then we measured humoral immune response. When the number of RBD copies coupled to BLS was high (6-7 RBD molecules per BLS decamer, in average), the immune response was significantly better than that elicited by RBD alone or even by RBD-BLS comprising low number of RBD copies (1-2 RBD molecules per BLS decamer). Remarkably, the construct with high number of RBD copies induced high IgG titers with high neutralizing capacity. Furthermore, a superior immune response was observed when Al(OH)3 adjuvant was added to this formulation, exhibiting a higher titer of neutralizing antibodies. Altogether our results suggest that RBD covalent coupled to BLS forming a multimer-particle shows an advantageous architecture to the antigen-presentation to the immune system which enhances immune responses. This new antigen should be considered a potent candidate for a protein-based vaccine.

Trimeric receptor-binding domain of SARS-CoV-2 acts as a potent inhibitor of ACE2 receptor-mediated viral entry

“…Conversely, an engineered trimeric ACE2 has been shown to bind three RBD proteins and inhibit SARS-CoV-2 infection (Guo et al, 2020;Xiao et al, 2021). An engineered RBD multimer construct coupled with an immunogenic carrier protein also induced superior immunogenicity as a multimer, than as a monomer, further suggesting the benefits of utilizing RBD multimers in SARS-CoV-2 research (Berguer et al, 2021). Tagged recombinant SARS-COV-2 RBD protein is commonly used as a tool in SARS-CoV-2 research.…”

Development and characterization of a multimeric recombinant protein using the spike protein receptor binding domain as an antigen to induce SARS‐CoV‐2 neutralization

“…This is because immunogens with a more multimeric structure elicit a stronger humoral response in mice. 56 Furthermore, proteins based on total protein S may have difficulty stabilizing the prefusion conformation and fail to generate adequate antibodies. 57 The sera produced by mice immunized with AS03 were capable of neutralizing SARS-CoV-2 virus strain Wuhan infection in Vero cells up to a dilution of 1:2560, whereas other strains required higher concentrations.…”

Development and characterization of a multimeric recombinant protein based on the spike protein receptor binding domain of SARS-CoV-2 that can neutralize virus infection

“…Therefore, production of antibodies that recognize the trimeric structure may be more effective in inhibiting this binding. This is related to the fact that immunogens with a more multimeric structure elicit a more robust humoral response in mice [52]. In addition, proteins based on total protein S may exhibit some difficulties in stabilizing the prefusion conformation [53] and may fail to generate adequate antibodies.…”