A tandem-repeat dimeric RBD protein-based covid-19 vaccine zf2001 protects mice and nonhuman primates

Abstract

Safe, efficacious, and deployable vaccines are urgently needed to control COVID-19 in the large-scale vaccination campaigns. We report here the preclinical studies of an approved protein subunit vaccine against COVID-19, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and non-human primates, and elicited balanced T_H1/T_H2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 μg or 50 μg ZF2001 prevented infection with SARS-CoV-2 in lung, trachea, and bronchi, with milder lung lesions. No evidence of disease enhancement was observed in both animal models. ZF2001 has been approved for emergency use in China, Uzbekistan, Indonesia, and Columbia. The high safety, immunogenicity, and protection efficacy in both mice and NHPs found in this preclinical study was consistent with the results in human clinical trials.

Keywords: Covid-19; antibody; immune response; subunit protein vaccine; zf2001.

Figure 1.

Humoral immune responses to ZF2001 vaccination in BALB/c mice. (A) schematic diagram of SARS-CoV-2 RBD dimer protein. Two copies of RBD from R319 to K537 are connected as tandem-repeat dimer. SP, signal peptide. (B) Time courses of ZF2001 vaccine immunization and sampling in groups of 6–8-week-old female BALB/c mice (n = 5) vaccinated with 10 μg ZF2001 or placebo. (C) Enzyme-linked immunosorbent assay (ELISA) show serum IgG against SARS-CoV-2 RBD. The dashed line indicates the limit of detection. Data are geometric mean with 95% CI. (D) SARS-CoV-2 (HB01 strain) neutralization assay shows the 50% neutralization titre. The dashed line indicates the limit of detection. Data are geometric mean with 95% CI.

Figure 2.

Protective efficacy of ZF2001 vaccine in mice. (A) Time course of immunization, sampling, viral challenge and measurement. Two groups of C57BL/6 mice (n = 21) were immunized with two doses of 10 μg ZF2001 vaccine or placebo, 3 weeks apart. Serum samples were collected after both one and two doses as indicated. Mice were then transduced with 8 × 10⁹ vp of Ad5-hACE2 via intranasal (i.n.) route. Each group of mice were further split into three groups (n = 6-7), with the former two groups infected with high-dose (5 × 10⁵ TCID₅₀) SARS-CoV-2 and the latter group infected with low-dose (1 × 10⁵ TCID₅₀) SARS-CoV-2. Lung tissues were harvested at either 3 or 5 DPI for the two groups with high-dose virus challenge and 3 DPI for the group with the low-dose virus challenge. (B) ELISA shows serum IgG against SARS-CoV-2 RBD. Data are geometric mean with 95% CI. P-values were analyzed with unpaired t-test (****, P < 0.0001). The dashed line indicates the limit of detection. (C) SARS-CoV-2 (HB01 strain) neutralization assay shows the 50% neutralization titre. Data are geometric mean with 95% CI. P-values were analyzed with unpaired t-test (*, P < 0.05; ****, P < 0.0001). The dashed line indicates the limit of detection. (D–E). SARS-CoV-2 titration from lung tissues by qRT-PCR probing virus gRNA (D) and sgRNA (E). Data are means ± SEM. The dashed lines indicate the limit of detection. (F–G) Protective correlation of NAb titre with SARS-CoV-2 gRNA (F) or sgRNA (G), calculated with Spearman correlation in GraphPad Prism 9.0.

Figure 3.

Immunofluorescence and histopathology analyses of lung tissues in mice. (A) Immunofluorescence analysis of lung tissue section stained with anti-SARS-CoV-2 nucleoprotein (N) antibody. Green: SARS-CoV-2 N protein; Blue: DAPI. Scale bar in low magnifications images, 90 μm. Scale bar in high magnifications images, 30 μm. (B) Typical histopathology images of lung tissues section shown by haematoxylin-eosin staining. Both low magnifications and high magnifications are shown, highlighted by boxes. Scale bar in low magnifications images, 625 μm. Scale bar in high magnifications images, 100 μm.

Figure 4.

Humoral and cellular immune responses to ZF2001 vaccination in cynomolgus macaques. (A) Time course of immunization and sampling. Groups of cynomolgus macaques (n = 10) were immunized with four doses of placebo, the 25 μg vaccine and the 50 μg vaccine, respectively. Serum samples were collected at indicated time points post vaccination. Six macaques in each group were euthanized for spleen harvest at day 3 after the last vaccination. (B) ELISA shows serum IgG against SARS-CoV-2 RBD. Data are geometric mean with 95% CI. P-values were analyzed with unpaired t test (ns, not significant). (C) SARS-CoV-2 (HB01 strain) neutralization assay shows the 50% neutralization titre. Data are geometric mean with 95% CI. P-values were analyzed with unpaired t test (ns, not significant). The dashed line indicates the limit of detection. (D) Splenic IFN-γ, IL-2 and IL-4 ELISPOT responses to SARS-CoV-2 RBD antigen. SFCs: Spot-forming cells. Data are geometric mean with 95% CI. P-values were analyzed with one-way ANOVA (ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Figure 5.

Humoral immune responses to ZF2001 vaccination in rhesus macaques. (A) Time course of immunization, sampling, viral challenge and measurement. Groups of rhesus macaques (n = 3) were immunized with two doses of placebo, the 25 μg vaccine and the 50 μg vaccine. Serum samples were collected at indicated time points post vaccination. At 14 post the second immunization, macaques were challenged with 1 × 10⁶ TCID₅₀ SARS-CoV-2 (20SF107 strain) via intratracheal route. Macaques were euthanized at 7 DPI for tissue harvest. (B) ELISA shows serum IgG against SARS-CoV-2 RBD. Data are geometric mean with 95% CI. (C) SARS-CoV-2 neutralization assay shows the 50% neutralization titre. Data are geometric mean with 95% CI. The dashed line indicates the limit of detection.

Figure 6.

Viral loads and clinical signs in rhesus macaques challenged with SARS-CoV-2 after vaccination. (A) SARS-CoV-2 titration from lung tissues by qRT-PCR probing virus gRNA. Tissue samples: 7 lung lobes (4 sites for each lobe) of three macaques in each group (84 samples per group); tracheas of three macaques in each group; left and right bronchi of three macaques in each group. (B) Histopathology scores of overall lung lesions and pulmonary alveolar congestion. (C) Typical histopathology images of lung tissues section shown by haematoxylin-eosin staining. Scale bar, 100 μm.