Development of Adenovirus-Based Covid-19 Vaccine Candidate in Indonesia

Abstract

Covid-19 pandemic has struck worldwide by end of 2019 and the use of various vaccine platforms was one of the main strategies to end this. To meet the needs for vaccine technology equality among many countries, we developed adenovirus-based Covid-19 vaccine candidate in Indonesia. SARS-CoV-2 Spike gene (S) was constructed into pAdEasy vector. The recombinant serotype 5 Adenovirus (AdV_S) genome was transfected into AD293 cells to produce recombinant adenovirus. Characterization using PCR confirmed the presence of spike gene. Transgene expression analysis showed the expression of S protein in AdV_S infected AD293 and A549 cells. Optimization of viral production showed the highest titer was obtained at MOI of 0.1 and 1 at 4 days. The in vivo study was performed by injecting Balb/c mice with 3.5 × 10⁷ ifu of purified adenovirus. The result showed that S1-specific IgG was increased up to 56 days after single-dose administration of AdV_S. Interestingly, significant increase of S1 glycoprotein-specific IFN-γ ELISpot was observed in AdV_S treated Balb/c mice. In conclusion, the AdV_S vaccine candidate was successfully produced at laboratory scale, immunogenic, and did not cause severe inflammation in Balb/c mice. This study serves as initial step towards manufacturing of adenovirus-based vaccine in Indonesia.

Keywords: AdV_S; Adenovirus; Covid-19; SARS-CoV-2; Vaccine; hAd5.

Fig. 1

Characterization of recombinant shuttle plasmid and adenoviral genome. A, B pShuttle_S was characterized using 1% agarose gel electrophoresis in comparison to pShuttle (A) and cut with HindIII and SalI (B). C, D pAdeasy_S was characterized by restriction analysis using PacI (C) and characterized by PCR using primer specific for spike gene (D)

Fig. 2

Characterization of recombinant adenovirus. A AdV_S produced from round 1, 2 and 3 amplifications were analyzed by PCR using primer specific for spike gene. B Transgene expression of AD293 and A549 cells infected with AdV_lacZ and AdV_S were analyzed by Western blot using antibody specific for spike and beta-actin. The depicted results were representatives from three independent experiments

Fig. 3

Optimization of recombinant adenovirus production. A AdV_S production was analyzed upon infection at MOI of 0.1, 1, 2, and 5 after 4 days of infection. B AdV_S production was measured upon infection at MOI of 0.1 and 1 for 2–8 days. Adenoviral titer was measured using Adeasy Viral Titer Kit. Data plotted was mean ± SEM from three independent experiments

Fig. 4

Immunogenicity and cellular immune responses of AdV_S in mice. A IgG titer measured before vaccination and every 2 weeks until week 8 after vaccination using S1-specific ELISA. Data shown were mean ± SEM from 5 animals per group. B IL-4 and IFN-γ ELISpot assays performed using ex vivo splenocytes obtained at week 8 after vaccination. Data shown were mean ± SEM from 5 animals per group. *p < 0.05, ***p < 0.001, two-way anova with Bonferroni posttest