Fig. 1.

Serum antibody response against RBD of the SARS-CoV-2 S protein in ELISA assay. (a) The experimental design of each AAV-vaccine vector. Vectors contains a CAG promoter (purple), secretory signal peptide (sp, light pink), various antigen (gradual yellow), and ploy A (pA, blue) that flanked with two ITR elements. The RBDs were linked with linker sequence in AAV-2xRBD and trimeric AAV-3xRBD (Supplementary Sequence). (b) The scheme of mice immunization. Mice were either mock immunized via intramuscular in caudal thigh muscle with AAV-tPA or PBS, or were vaccinated with AAV-RBD(wt), AAV-RBD(max), AAV-2xRBD, and AAV-3xRBD. A total of 54 mice were randomly divided into 6 groups that received PBS, AAV-tPA, AAV-RBD(wt), AAV-RBD(max), AAV-2xRBD, and AAV-3xRBD, respectively. The 9 mice in each AAV group were further divided into 3 groups that received 10⁹, 10¹⁰, and 10¹¹ genomes, respectively. Time points of prime vaccination and serum sample collection are indicated by purple and red arrows, respectively. (c) Sera were collected at day 9 and were tested at indicated dilution for IgG against RBD by ELISA. The group of mice received 10¹¹ genomes was showed in the figure. (d) Sera were collected at day 16 and were tested at different dilution for IgG against RBD by ELISA. The group of mice received 10¹¹ genomes was showed in the figure. (e) Sera were collected at day 30 and were tested at indicated dilution for IgG against RBD by ELISA. The group of mice received 10¹¹ genomes was showed in the figure. (f,g) Gradient dilution of the sera collected on 30 days and corresponding specific IgG level were curved using ELISA. The groups of mice immunized with AAV-2xRBD (f) and AAV-3xRBD (g) upon 10¹¹ genomes were analyzed in the figures. (h) Gradient dilution of the sera collected at 180 days and corresponding specific IgG level were curved using ELISA. The groups of mice immunized with AAV-3xRBD upon 10¹¹ genomes were analyzed, n = 2 in each groups at day 180 sera. (i-j) The dosage effects on IgG titer were measured using ELISA, sera were collected on 16 days. (j) The SARS-CoV-2 pseudovirus was employed to assess the neutralization ability of infection by the sera at day 30 immunized with AAV-3xRBD vaccine or AAV-tPA upon 10¹¹ genomes. (k) The canine sera were collected at day 0, 9, 16, and 30, and then tested at dilution of 1 : 100 for IgG against RBD. Note that two dogs were received with 5x10¹⁰ (pink) and 5x10¹¹ (blue) viral genomes of AAV-3xRBD vaccine, respectively, and one dog was injected with PBS (brown) as a control (l) The canine sera were collected at day 30, and then tested at indicated dilution for IgG against RBD. n = 1 dog in (k) and (l). (m) The SARS-CoV-2 pseudovirus was employed to assess the neutralization ability of infection by the mice sera at day 30 immunized with AAV-3xRBD vaccine or AAV-tPA upon 10¹¹ genomes. (n) Assessment of immune responses to AAV vectors were tested against the Cap protein of AAV8 using ELISA. Sera on 30 days from mice vaccinated with AAV-3xRBD (10¹¹genomes) and PBS were shown. n = 3 mice per calculated point for all panels. *p < 0.05, **p < 0.01 and ***p < 0.001, for statistics, see Supplementary Table 1-11.