Abstract
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.
Highlights
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the genus Betacoronavirus in the family Coronaviridae and is genetically close to the 2003 outbreak of SARS-CoV and CoV isolated from bats [1, 2]
The expression in silkworm larvae was confirmed by SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining and Western blotting with His-probe, which showed that, compared to the previously reported SARS2/SNF+knob+TEV-H8STREPH6 [20], the introduction of the proline mutation and fusion of cartilage matrix protein (CMP) greatly improved the secretory expression in silkworm serum (Figure 1D)
Time-course analysis of SARS2/SNFPP+CMP+TEV-H8STREPH6 expression in silkworm larvae after baculovirus infection showed that S protein in serum was highest at 5 days post-infection (Figure 1E), but high mortality was observed at 5 dpi
Summary
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the genus Betacoronavirus in the family Coronaviridae and is genetically close to the 2003 outbreak of SARS-CoV and CoV isolated from bats [1, 2]. SARS-CoV-2 is responsible for the symptoms called “Coronavirus disease 2019” (COVID-19), which causes a high fever and severe pneumonia in humans. The elderly, diabetics, or people with respiratory or cardiac disease are prone to severe disease [3]. The COVID-19 cluster was initially discovered in a local seafood market in Wuhan, China. SARS-CoV-2 spread globally due to its high infectivity, causing a pandemic. As of July 19, 2021, the World Health Organization (WHO) announced 189,921,964 confirmed cases and 4,088,281 deaths globally by the spread of SARS-CoV-2
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