Potency, toxicity and protection evaluation of PastoCoAd candidate vaccines: Novel preclinical mix and match rAd5 S, rAd5 RBD-N and SOBERANA dimeric-RBD protein

Abstract

Despite substantial efforts, no effective treatment has been discovered for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. Therefore, vaccination to reach herd immunity is the ultimate solution to control the coronavirus disease 2019 (COVID-19) pandemic. This study aimed to evaluate the potency, toxicity, and protection of candidate PastoCoAd vaccines as novel mix and match of recombinant adenovirus type 5 (rAd5) containing the full-length spike protein (rAd5-S), rAd5 containing the receptor-binding domain of S protein and nucleoprotein (rAd5 RBD-N), and SOBERANA dimeric RBD protein of SARS-CoV-2. Three vaccine candidates were developed against SARS-CoV-2 using adenoviral vectors, including the prime-boost (rAd5-S/rAd5 RBD-N), heterologous prime-boost (rAd5-S/ SOBERANA vaccine), and prime only (mixture of rAd5-S and rAd5 RBD-N). The rAd5-S and rAd5 RBD-N were produced with a Cytomegalovirus promoter and the human tissue plasminogen activator (tPA) leader sequence. The immunogenicity of vaccine candidates was also evaluated in mouse, rabbit, and hamster models and protection was evaluated in a hamster model. Following the injection of vaccine candidates, no significant toxicity was observed in the tissues of animal models. The immunogenicity studies of mice, rabbits, and hamsters showed that responses of total IgG antibodies were significantly higher with the prime-only and heterologous prime-boost vaccines as compared to the other groups (P < 0.009). Virus neutralizing antibodies were detected, and the level of cytokines related to humoral and cellular immunity increased significantly in all vaccinated models. A high cellular immunity response was found in the vaccinated groups compared to the controls. On the other hand, the vaccine challenge test showed that the virus titers significantly decreased in the pharynx and lung tissues of vaccinated hamsters compared to the control group. These successful findings suggest the safety and protection produced by the heterologous prime-boost vaccine (adenovector/ SOBERANA RBD), as well as a single dose of adenovector vaccine in animal models.

Keywords: Adenovector; COVID-19; Iran; Protection; Safety; Vaccine.

Fig. 1

Graphical abstract of animal studies for evaluating the safety and efficacy of PastoCoAd vaccines.

Fig. 2

Repeated dose toxicity. The results showed no significant toxicity in the liver, muscles, kidney, lung, brain and heart tissues.

Fig. 3

Humoral immune responses in vaccinated BALB/C mice. Serum samples were tested by ELISA assay at 0 (A, B), 14 (C, E), 28 (D, F), 42 (C, E), and 56 (C) days after the inoculation of rAd5-S or RBD-N, along with SOBERANA as a booster dose. The S-specific antibody and N-total antibody levels were also assessed, and the subclasses of IgG1 and IgG2 antibodies were measured. Each dot in the graphs represents replicate samples in the mouse group (P < 0.05, one-way ANOVA test). A 95% CI was considered for all tests. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5-S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 4

The results of viral neutralization test 50% in mouse, rabbit and hamster has been shown. Four week after injection of vaccine, blood samples were collected. VNT has been done in Vero cell lines. In each well 100 TCID50% of the SARS-CoV-2 was added and 4 replicated in each dilution was mentioned. A: mice, B: rabbit and C: hamster. AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5-S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 5

The cytokine and CTL assays. The levels of IL-6 (A), IL-2 (B), and TNF-α (C) were examined, using the ELISA kit in mice on days 28 and 42. The IL-5 (E) and IFNγ (F) levels were examined by the ELISpot kit on day 28 after immunization. The evaluation of CD8 T cell response (D) in the vaccinated mice was determined by the granzyme B assay for the groups control, heterologous prime-boost (rAd5 S/RBD SOBERANA), and prime only (rAd5-S/rAd5 RBD-N mixture). Each dot in the graphs represents replicate samples in the mouse group (P < 0.05, one-way ANOVA test). A 95% CI was considered for all tests. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 6

Humoral immune responses in vaccinated rabbits. Serum samples were examined by ELISA assay on days 0 (A, B), 14, 28, 42 (C, D) and 56 (C) after immunization with rAd5-S or RBD-N, along with SOBERANA as a booster dose. The S-specific antibody and N-total antibody levels were also assessed. Each dot in the graphs represents replicate samples in the rabbit group (P < 0.05, one-way ANOVA test). A 95% CI was considered for all tests. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 7

Humoral immune responses in hamsters. Serum samples were examined by ELISA assay on days 0 (A), 14, 28 and 42 (B) after immunization with rAd5-S or RBD-N, along with SOBERANA as a booster dose. The S-specific antibody levels was assessed. Each dot in the graphs represents replicate samples in the hamster group (P < 0.05, one-way ANOVA test). A 95% CI was considered for all tests. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 8

Protection against SARS-CoV-2 viral replication in hamsters immunized with Ad5-based vaccines. The hamsters were intramuscularly immunized with a one-dose regimen (mixture of rAd5 S and rAd5 RBD-N) and a two-dose regimen (rAd5-S/rAd5 RBD-N and rAd5-S/booster SOBERANA). An empty Ad5 vector was used for the negative control group. The hamsters received an intranasal inoculation with 5 × 10⁵ TCID₅₀ of SARS-CoV-2 strain. In the prime-only group, the viral challenge was performed at 28 days after vaccination, while in the two-dose group, the challenge was performed at 42 days after vaccination. Throat swab samples were taken daily after inoculation, and the viral load was calculated as TCID50%/mL. Comparisons were made between the two groups by one-way ANOVA test (P < 0.05). (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 9

On days 4 and 7 after the viral challenge, the lung and tracheal tissues were isolated and scored for the presence and severity of alveolitis, alveolar damage, alveolar edema, alveolar hemorrhage, type II pneumocyte hyperplasia, bronchitis, bronchiolitis, and peribronchial or perivascular cuffing. The sum of scores is presented as the sum of lower respiratory tract parameters. A 95% CI was considered for all tests. (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).

Fig. 10

Immunolocalization of S1 protein in the bronchial epithelium and the lungs of hamsters following the viral challenge. Immunoreactivity and lack of immunoreactivity to S1 protein were observed in the control and vaccinated groups, respectively (100 × ma).

Fig. 11

The picture shows analysis of body weight loss in different vaccine group (normalized to 100% value). The values were compared using one-way ANOVA followed by a Tukey’s multiple comparison test. The AD5 S&N group was significant with weak evidence (P-value 0.0259). (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Error bars represent mean ± SD. AD5 (rAd5 empty), AD5 S/N (rAd5-S/rAd5 RBD-N), AD5 S/SO (rAd5 S/SOBERANA), AD5 S&N (rAd5-S & rAd5 RBD-N mix).