Dataset Information

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

ABSTRACT: Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is important to meet the challenge of producing a high amount of recombinant RBD (rRBD). For this reason, in the present study, we developed a process based on Sf9 insect cells to improve rRBD yield. rRBD was recovered from the supernatant of infected cells and easily purified by metal ion affinity chromatography, with a yield of 82% and purity higher than 95%. Expressed under a novel chimeric promoter (polh-pSeL), the yield of rRBD after purification was 21.1 ± 3.7 mg/L, which is the highest performance described in Sf9 cell lines. Finally, rRBD was successfully used in an assay to detect specific antibodies in COVID-19 serum samples. The efficient strategy herein described has the potential to produce high-quality rRBD in Sf9 cell line for diagnostic purpose.

SUBMITTER: Poodts J

PROVIDER: S-EPMC9785345 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Poodts Joaquín J Smith Ignacio I Birenbaum Joaquín Manuel JM Rodriguez María Sol MS Montero Luciano L Wolman Federico Javier FJ Marfía Juan Ignacio JI Valdez Silvina Noemí SN Alonso Leonardo Gabriel LG Targovnik Alexandra Marisa AM Miranda María Victoria MV

Viruses 20221215 12

Insect cell-baculovirus expression vector system is one of the most established platforms to produce biological products, and it plays a fundamental role in the context of COVID-19 emergency, providing recombinant proteins for treatment, diagnosis, and prevention. SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host's cellular receptor. As RBD is required for many applications, in the context of pandemic it is im ...[more]

PMID: 36560798

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Project description:BackgroundThere is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials.MethodologyIn this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies' specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response.ResultThe outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response.ConclusionThe produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

Dataset Information

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Publications

Improved Expression of SARS-CoV-2 Spike RBD Using the Insect Cell-Baculovirus System.

Similar Datasets

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