DEVELOPMENT OF MULTIEPITOPE PEPTIDE-BASED COVID-19 VACCINE CANDIDATE BASED ON SARS-COV-2 SPIKE AND NSP3 FUSION PROTEIN

• Main Author: Yamahoki, Nicholas
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/65121
• Institution: Institut Teknologi Bandung

Vaccination is an effective method to end pandemics, including the Coronavirus disease-2019 (Covid-19) which has caused a multidimensional crisis around the world in recent years. Evolution of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) virus which is characterized by the emergence of new variants has a potential to cause a decrease in the effectiveness of the vaccines that have been developed at this time. On the other hand, it is known that a multiepitope peptide-based subunit vaccine developed with a reverse vaccinology approach has the potential to induce both humoral and cellular immune responses, and may overcome vaccine failure due to viral genetic mutations. In the previous study, a multiepitope Covid-19 vaccine candidate has been designed and characterized based on SARS-CoV-2 Spike and NSP3 fusion protein in silico. However, the vaccine candidate fusion protein has not been expressed and tested for its antigenicity in vitro. Therefore, in this study, expression of the designed vaccine candidate fusion protein was carried out and the antigenicity test of the vaccine candidate was carried out using serum of Covid-19 positive patients. In this study, the gene encoding the vaccine candidate fusion protein was cloned into pET-23a(+) plasmid and transformed into E. coli BL21 (DE3) cells. Vaccine candidate fusion protein expression in E. coli BL21 (DE3) was carried out at 16°C with variations in incubation duration (6, 8, and 24 hours) and in IPTG concentrations (0, 0.05, 0.1, 0.25, and 0.5 mM). Protein purification was carried out using a Ni-NTA column. Furthermore, the suspected band of vaccine candidate fusion protein on SDS-PAGE gel was analyzed using ImageJ and confirmed by Western blotting. Antigenicity of the vaccine candidate fusion protein was tested using indirect ELISA against Covid-19 specific antibodies in the serum of Covid-19 positive confirmed patients (n=3). The results showed that the gene encoding the vaccine candidate fusion protein was confirmed by PCR and DNA sequencing. Incubation at 16°C for 24 hours with the addition of 0.05 mM IPTG resulted in the highest concentration of vaccine candidate fusion protein significantly (p<0.05), although this optimal condition did not increase the solubility of the fusion protein. The vaccine candidate fusion protein was successfully obtained by purification and was confirmed by Western blotting. Furthermore, the ELISA result showed that the vaccine candidate can bind to antibodies of Covid-19 positive patients (n=3). Thus, it can be concluded that the designed multiepitope Covid-19 vaccine candidate fusion protein can be expressed and can be recognized by antibodies of Covid-19 patient. Further research is needed to examine the immunogenicity of the vaccine candidate.