Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates

Abstract

ABSTRACTCOVID-19 vaccines are being developed urgently worldwide. Here, we constructed two adenovirus vectored COVID-19 vaccine candidates of Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length gene of SARS-CoV-2 spike protein. The immunogenicity of two vaccines was individually evaluated in mice. Specific immune responses were observed by priming in a dose-dependent manner, and stronger responses were obtained by boosting. Furthermore, five rhesus macaques were primed with 5 × 10⁹ PFU Sad23L-nCoV-S, followed by boosting with 5 × 10⁹ PFU Ad49L-nCoV-S at 4-week interval. Both mice and macaques well tolerated the vaccine inoculations without detectable clinical or pathologic changes. In macaques, prime-boost regimen induced high titers of 10^3.16 anti-S, 10^2.75 anti-RBD binding antibody and 10^2.38 pseudovirus neutralizing antibody (pNAb) at 2 months, while pNAb decreased gradually to 10^1.45 at 7 months post-priming. Robust T-cell response of IFN-γ (712.6 SFCs/10⁶ cells), IL-2 (334 SFCs/10⁶ cells) and intracellular IFN-γ in CD4⁺/CD8⁺ T cell (0.39%/0.55%) to S peptides were detected in vaccinated macaques. It was concluded that prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S can safely elicit strong immunity in animals in preparation of clinical phase 1/2 trials.

Keywords: COVID-19 vaccines; human adenovirus 49 vector; mice and non-human primates; prime-boost vaccination; simian adenovirus 23 vector.

Figure 1.

Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. (B) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. (C) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. (D) Seroprevalence of neutralizing antibody (AdNAb) to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.

Figure 2.

Specific antibody and T-cell response of C57BL/6 mice inoculated with a single shot of Sad23L-nCoV-S or Ad49L-nCoV-S vaccine at three different doses. C57BL/6 mice (n = 5/group) were immunized by a single dose of 10⁷, 10⁸ or 10⁹ PFU Sad23L-nCoV-S or Ad49L-nCoV-S vaccine. Mice sera and splenocytes were collected for measurement of antibody level and T-cell response 4 weeks post-immunization. (A–B) S binding antibody (S-BAb) titers were obtained by ELISA. (C) S-BAb was compared with Sad23L-nCoV-S and Ad49L-nCoV-S in 10⁹ PFU. (D–E) Neutralizing antibody (NAb) titers were obtained by pseudovirus-based neutralization test (pVNT). (F) pVNT was compared two vectors at the same doses of 10⁹ PFU. (G–H) IFN-γ secreting T-cell response (spot forming cells [SFCs]/million cells) of splenocytes to S peptides from Sad23L-nCoV-S or Ad49L-nCoV-S immunized mice was measured by ELISpot, respectively. (I) IFN-γ secreting T-cell response was compared two vectors at 10⁹ PFU. Data is shown as mean ± SEM (standard errors of means). P values are analysed by one-way ANOVA with twofold Bonferroni adjustment. Statistically significant differences are shown with asterisks (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).

Figure 3.

Specific humoral and cellular immune response of C57BL/6 and BALB/c mice to prime-boost immunization with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) C57BL/6 and BALB/c mice (n = 5/group) were prime immunized with a dose of 10⁹ PFU Sad23L-nCoV-S vaccine and boosted with a dose of 10⁹ PFU Ad49L-nCoV-S vaccine at 4 week interval. Sera and splenocytes were collected from vaccinated or control mice for measurement of antibody and T-cell responses 4 weeks after both prime only and boosting immunizations. (B–C) Anti-S-BAb and RBD-BAb titers determined by ELISA. (D–E) NAb titers measured by sVNT and pVNT. (F) IFN-γ secreting T-cell response (SFCs/million cells) to S peptides, S or RBD protein measured by ELISpot. (G) Frequency of IFN-γ or TNF-α expressing CD4⁺ and CD8⁺ T-cell response to S peptides determined by ICS. Data are shown as a mean ± SEM. P values are analysed with one-way ANOVA and two-tailed t test. Statistically significant differences are shown with asterisks (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).

Figure 4.

Antibody reactivity of Rhesus macaques to prime-boost vaccination with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Five rhesus macaques were prime immunized with 5 × 10⁹ PFU of Sad23L-nCoV-S vaccine and boosted with 5 × 10⁹ PFU of Ad49L-nCoV-S vaccine at 4 week interval. Blood samples were collected weekly from immunized or sham control macaques. Three macaques first immunized with 5 × 10⁹ PFU of Sad23L-GFP viruses and boosted with 5 × 10⁹ PFU of Ad49L-GFP viruses were used as sham controls. Convalescent serum samples from 25 asymptomatic, 14 mild and 9 severe COVID-19 infected patients were taken as positive controls. (B–C) S-BAb and RBD-BAb titers were tested by ELISA. (D–E) NAb titers were measured by sVNT and pVNT. (F–G) Correlation between RBD-BAb and sVNT or S-BAb and pVNT titers were compared using Spearman nonparametric correlation, respectively.

Figure 5.

Specific T-cell response of PBMCs from rhesus macaques immunized with Sad23L-nCoV-S and Ad49L-nCoV-S vaccines or sham controls by prime-boost vaccination regimen. (A–C) IFN-γ or (D–F) IL-2 secreting T-cell response (SFCs/million cells) to S peptides, S or RBD protein was measured by ELISpot. (G–J) Frequency of intracellular IFN-γ expressing CD4⁺/CD8⁺ T-cell response to S peptides was determined by ICS, respectively. Data are shown as mean ± SEM. P-values are calculated with two-tailed t test. Statistically significant differences are shown with asterisks (*, P < 0.05; **, P < 0.01 and ***, P < 0.001).

Figure 6.

Duration of antibody response of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines in mice and Rhesus macaques. (A) RBD-BAb and (B) pNAb were measured for 29 weeks in a single dose of Sad23L-nCoV-S (Prime 1) or Ad49L-nCoV-S (Prime 2), or prime-boost (Sad23L-nCoV-S and Ad49L-nCoV-S) vaccinated C57BL/6 and BALB/c mice. (C) RBD-BAb and (D) pNAb were measured for 29 weeks in prime-boost (Sad23L-nCoV-S/Ad49L-nCoV-S) vaccinated or sham (Sad23L-GFP/Ad49L-GFP) rhesus macaques. Mean Ab titers are presented in each group.

Figure 7.

AdNAb responses to Ad vectors. Serum AdNAb titers to Sad23L and Ad49L vectors were measured in macaques (A) immunized by prime-boost inoculation or in C57BL/6 and BALB/c mice (B) 4 weeks post prime only or prime-boost vaccination with two vaccines or vectorial controls.