Development of nanotechnological vaccine against COVID-19 disease

Project no. 22333 · Status: Kapanmış (Yayın bekleniyor) · 2021

Summary

The coronavirus disease 2019 (COVID-19), declared a pandemic by the World Health Organization on March 11, 2020, continues to affect the world negatively. The fifty vaccines approved for human use are sufficient to induce an immune response. However, immunity only lasts for an average of six months before needing booster doses. In addition, variants of concern, such as the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.617.2 (delta) variant, are more contagious than the first Wuhan strain shows that the effectiveness of vaccines should be increased. This thesis aimed to develop a DNA vaccine against the SARS-CoV-2 delta variant, determine the immunogenicity of the vaccine in BALB/c mice, and develop its nanotechnological formulation.The soluble spike protein (STDSpike) of the SARS-CoV-2 delta variant, containing six proline substitutions, two alanine substitutions, and a T4 fibritin folding region, was designed in silico to generate vaccine antigen with a stable structure in the trimeric prefusion state. The STDSpike gene was cloned into the pVAX1 vector to generate the pSTDSpike DNA vaccine. The STDSpike protein was determined by Western blot and immunofluorescent antibody staining test in HEK293T cells. To determine the immunogenicity of the vaccine, BALB/c mice were immunized three times with naked pSTDSpike, either intramuscularly (IM) or intradermally, using an electroporation device (ID+EP). While Western blot, recombinant ELISA, and competitive ELISA tests were performed to determine the humoral immune response, the cellular immune response was determined by flow cytometry, cytokine ELISA, and MTT tests in restimulated splenocytes. In addition, pSTDSpike:Liposome complexes were formed using a commercial liposome kit containing L-α-phosphatidylcholine, stearylamine and cholesterol to prepare nanotechnological formulations. DNA protection, serum stability, in vitro toxicity, and transfection efficiency of the complexes were determined.The STDSpike protein was produced at high levels in HEK293T cells. The pSTDSpike vaccine induced high titers of antigen-specific IgG antibodies in BALB/c mice. The IgG titers of pSTDSpike administered by ID+EP reached ~52.000, while IM routes reached ~21.000. Both pSTDSpike IM and ID+EP revealed strong neutralizing activity with inhibitions of 87.3% (P<0.0001) and 81.8% (P<0.0001). The percentages of IFN-γ secreting CD8+ T cells were ~10-fold (p=0.0014) and ~15-fold (p<0.0001) higher, respectively, when mice vaccinated with pSTDSpike IM and pSTDSpike ID+EP were compared with control groups. The percentages of CD4+ T cells that secrete IFN-γ were 2-fold higher than control groups. The IFN-γ level of the pSTDSpike ID+EP vaccine group was 3.5 times higher than pSTDSpike IM group (p<0.0001). Among the generated pSTDSpike:Liposome complexes, two successful candidates, 1:100 and 1:200 complexes, were selected for in vitro testing and cell culture studies. These complexes, which have a spherical structure, were determined to have dimensions of 143 nm and 116 nm, pDI values of 0.25 and 0.17, and zeta potentials of 45.4 and 47.2, respectively. The complexes were determined to protect the pSTDSpike vaccine against DNase I and serum-mediated degradation. However, the complexes showed less than 70% viability and failed to perform efficient transfection in HEK293T cells.In conclusion, the pSTDSpike DNA vaccine induced strong cellular and humoral immune responses when administered by IM or ID+EP. The pSTDSpike vaccine may be a promising candidate for protecting against SARS-CoV-2.

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