Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity

Abstract

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.

Keywords: AAV vectors; Antigen structure design; Capsid engineering; SARS-CoV-2; Vaccine.

Graphical abstract

Figure 1

Optimizations on the Serotype of Vector. (A) The luminescence images of injected animals. BALB/c female mice (6–8 weeks old) were injected intramuscularly (i.m.) with 8 × 10⁹ VG of AAV5-Luc, AAV6-Luc or AAV8-Luc. The intensity of luminescence at injected muscle (B) and liver (C) at intended time point, n = 5. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Bartlett's test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

The design and in vitro characterization of AAV6-based vaccines. (A) Schematic of SARS-CoV-2 RBD antigens. (B) Three-dimensional structure of WT-RBD and S663V-RBD, red small dots indicate the mutation site at the 663 residue. (C) The morphology of WT-RBD or S663V-RBD was visualized by the transmission electron microscopy with a negative stain. (D) Virion purity was assessed by silver stain. (E) Western blot of transgene expression in cells cultures and lysates of HEK293T infected with WT-RBD or S663V-RBD with β-ME treatment or not. (F) The transgene expression kinetics. BALB/c female mice (6–8 weeks old) were injected intramuscularly (i.m.) with 1 × 10¹⁰ VG of WT-Luc or S663V-Luc, and the transgene expression were detected at intended time point, n = 4. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Two-tailed Student's t tests. ∗P < 0.05.

Figure 3

Single dose of AAV-based vaccines elicited a robust balance immune response over time. (A) Schematic of dose regimen and the principle of the enzyme-linked immunosorbent assays (ELISA). BALB/c female mice (6–8 weeks old) were injected intramuscularly (i.m.) with one dose (1 × 10¹¹ VG) of WT-RBD or S663V-RBD. Negative control mice were administered the same dose of WT-GFP or S663V-GFP. Positive control mice were injected intraperitoneally (i.p.) with two-dose of BBIBP-CorV using half adult dose (2 μg/dose) on Days 0 and 21. Sera were collected at the intended time for assessing SARS-CoV-2 RBD-specific binding antibody titers, n = 6. (B) The endpoint titer of IgG, IgG1 and IgG2a. (C) RBD-specific binding of antibodies in serial serum dilutions from mice on Day 70. (D) IFNγ spot-forming units (SFU) of splenocytes stimulated with MHC class I RBD-immunodominant peptide. Data are presented as mean ± SEM. Statistical significance was determined by two-way ANOVA with Dunnett's multiple comparisons test (B) or one-way ANOVA with Šídák's multiple comparisons test (D). ns: not significant (P > 0.05), ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4

Single dose of AAV-based vaccines provides a potent protection of multiple pseudoviruses over time. (A) Schematic of determination principle of pseudovirus neutralizing antibody titer. (B)‒(D) The 50% inhibitory dilution (IC₅₀) against WT strain, C.37 (Lambda) and B.1.617.2 (Delta) and the corresponding relative luminescence unit (RLU) in serial serum dilutions from mice on Day 70, respectively. Data are presented as mean ± SEM. Statistical significance was determined by two–way ANOVA with Dunnett's multiple comparisons test. ns: not significant (P > 0.05), ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, n = 5.

Figure 5

Single dose of AAV-based vaccines strong Germinal Center (GC) B cell responses, RBD-specific polyfunctional T-cell responses and a strong central memory T-cell response. (A) The scheme of experiments. (B) The gate strategy of GC B cell. (C) GC response in Ipsilateral draining lymph nodes of the injection site. (D) The central memory T cells responses of splenocytes. (E) RBD-specific polyfunctional CD4⁺ and CD8⁺ T cells responses of splenocytes. (F) The concentrations of cytokines in splenocytes culture supernatant. Data are presented as mean ± SEM. Statistical significance was determined by one–way ANOVA with Dunnett's multiple comparisons test (C– F). ns: not significant (P > 0.05), ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, n = 6.

Figure 6

Intramuscular administration of singleton modification AAV overcomes pre-existing immunity. (A) The scheme of experiments. Mice were inoculated with 5 × 10¹⁰ VG of WT-GFP or S663V-GFP at same site 14 days before vaccination with 1 × 10¹¹ VG of WT-RBD or S663V-RBD, n = 4. (B) The activity and level of neutralization antibody against wild type AAV6 capsid. (C) The endpoint titer of IgG, IgG1 and IgG2a under pre-existing NAbs against vector. Data are presented as mean ± SEM. Statistical significance was determined by two–way ANOVA with Tukey's multiple comparisons test (C). ∗P < 0.05 and ∗∗P < 0.01.

Figure 7

The storage stability of AAV6-based vaccines. The changes of transduction efficiency after storage for intended time at 2–8 °C. In vitro assay, HEK293T cells were infected (MOI 20,000) with WT-GFP or S663V-GFP. (A) After 48 h, images were collected by confocal microscope. (B), (C) Transduction efficiency changes were analyzed by FACS, and this experiment was repeated three times independently. (D) The changes of transduction efficiency in vivo. BALB/c female mice (6–8 weeks old) were injected intramuscularly (i.m.) with 1 × 10⁹ VG of WT-Luc or S663V-Luc, and the transgene expression were detected after 7 days, n = 3. Scale bars equal 100 μm.