A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS

Abstract

Vaccines are urgently needed to control the ongoing pandemic COVID-19 and previously emerging MERS/SARS caused by coronavirus (CoV) infections. The CoV spike receptor-binding domain (RBD) is an attractive vaccine target but is undermined by limited immunogenicity. We describe a dimeric form of MERS-CoV RBD that overcomes this limitation. The RBD-dimer significantly increased neutralizing antibody (NAb) titers compared to conventional monomeric form and protected mice against MERS-CoV infection. Crystal structure showed RBD-dimer fully exposed dual receptor-binding motifs, the major target for NAbs. Structure-guided design further yielded a stable version of RBD-dimer as a tandem repeat single-chain (RBD-sc-dimer) which retained the vaccine potency. We generalized this strategy to design vaccines against COVID-19 and SARS, achieving 10- to 100-fold enhancement of NAb titers. RBD-sc-dimers in pilot scale production yielded high yields, supporting their scalability for further clinical development. The framework of immunogen design can be universally applied to other beta-CoV vaccines to counter emerging threats.

Keywords: COVID-19; MERS; MERS-CoV; SARS; SARS-CoV; SARS-CoV-2; betacoronavirus; coronavirus; receptor-binding domain (RBD); vaccine.

Graphical abstract

Figure 1

Dimeric Form of MERS-CoV RBD Enhances Immunogenicity and Is Protective in Mice (A) Left: analytical gel filtration profile of baculo-derived MERS-CoV RBD (E367-Y606) protein with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curves are shown. The proposed peaks of RBD-dimer and RBD-monomer are indicated with arrows. Right: SDS-PAGE migration profiles of RBD-dimer and -monomer proteins at non-reduced and reduced conditions. (B) Time course of MERS vaccine immunization, viral challenge and measurement. Groups of 6- to 8-week-old female BALB/c mice (n = 6) were vaccinated with three doses of 3, 10, or 30 μg immunogen with adjuvant of AddaVax or Alum in 3-week intervals. PBS with and without adjuvant were given as controls. Serum samples were collected 14 days after last immunization. Mice were then transduced with 2.5 × 10⁸ PFU of Ad5-hCD26 via i.n. route followed by infection with 5 × 10⁵ PFU of MERS-CoV via i.n. route. Lung tissues were harvested and split for virus titer detection (n = 3) and pathological examination (n = 3), respectively. (C) Enzyme-linked immunosorbent assay (ELISA) assay shows the RBD specific IgG titers. (D) MERS-CoV pseudovirus neutralization assay shows the NT₉₀. (E) MERS-CoV (EMC2012 strain) neutralization assay shows the NT₅₀. (F) Virus titers in lung. (G) Histology of lung sections (100-fold magnification). The lung injuries are marked as slight, mild, and severe, according to the degree of interstitial pneumonia. Black bar represents 100 μm. The values shown in (C)–(F) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P-values were analyzed with one-way ANOVA (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001).

Figure 2

Structural Characterization of MERS-CoV RBD-Dimer and Structure-Guided Design of RBD-SC-Dimer as a MERS Vaccine (A) A cartoon representation of the MERS-CoV RBD-dimer structure. Two RBD protomers are arranged in axial symmetry and are colored in hot pink and cyan, respectively. Intra- and inter-molecular disulfide bonds are numbered. Dashed loops represent the N- and C-terminal residues that are invisible in the electron density maps. The cysteine residue C603 is highlighted. The light yellow ellipses represent the regions of RBM. (B) The schematic representation of MERS-CoV RBD-sc-dimer. RBDs are truncated at C-terminal residue N602 and connected as tandem repeat to form a single chain dimer (SP, signal peptide). (C) Analytical gel filtration profile of MERS-CoV RBD-sc-dimer protein with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curve is shown. Non-reduced and reduced SDS-PAGE migration profiles of the pooled samples are shown. (D) Ultracentrifugation sedimentation profiles of MERS-CoV RBD-sc-dimer. (E) Representative BIAcore diagrams of MERS-CoV RBD-sc-dimer and RBD-monomer bound to hCD26 protein. The K_D value was calculated by the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. (F and G) Groups of 6- to 8-week-old female BALB/c mice (n = 6) were immunized with a 10-μg dose of RBD-sc-dimer and HEK293T-derived disulfide-linked RBD-dimer, respectively, with AddaVax as adjuvant. PBS formulated with adjuvant was given as control. A three-dose vaccination regimen was performed. Serum samples were collected after each immunization (19 days after 1^st immunization, 14 days after 2^nd immunization, and 14 days after 3^rd immunization) to evaluate the humoral response dynamics. ELISA assay shows the MERS-CoV RBD-specific IgG titers in (F) and MERS-CoV pseudovirus neutralization assay shows the NT₉₀ in (G). The values shown in (F) and (G) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P values were analyzed with one-way ANOVA (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001). See also Figure S1 and Table S1.

Figure S1

MERS-CoV RBD-Dimer Fully Exposes Dual RBMs for Its Receptor hCD26, Related to Figure 2 The complex structure of hCD26/MERS-CoV-RBD (PDB: 4KR0) are docked onto MERS-CoV RBD dimer, showing the complete exposure of dual RBMs. Two RBD protomers are shown as surface and colored in hotpink and cyan, respectively. Two hCD26 are shown as cartoon and colored in yellow.

Figure S2

Amino Acid Alignment of 19 RBDs from Beta-CoV Genera, Related to Figures 3 and 4 The sequences are aligned based on a MUSCLE alignment. Conserved residues are highlighted in red. To construct the RBD-sc-dimer, the start and stop residues are highlighted by text in green boxes with arrow mark. The highly conserved C603 position of MERS-CoV S protein is highlighted by text in red with arrow mark. The sequence alignment was generated with Clustal X and ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/index.php) (Robert and Gouet, 2014).

Figure 3

Design and Assessment of RBD-SC-Dimer as a Vaccine against SARS-CoV-2 (A) A schematic diagram of SARS-CoV-2 RBD-sc-dimer. Two SARS-CoV-2 RBD (R319-K537) were dimerized as tandem repeat (SP, signal peptide). Analytical gel filtration of SARS-CoV-2 RBD-sc-dimer protein was performed with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curve is shown. Non-reduced and reduced SDS-PAGE migration profiles of the pooled samples are shown. (B) Ultracentrifugation sedimentation profiles of SARS-CoV-2 RBD-sc-dimer. (C) Representative BIAcore diagrams of SARS-CoV-2 RBD-sc-dimer and monomer bound to hACE2 protein. The K_D value was calculated by the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. (D and E) Groups of BALB/c mice were immunized with a 10-μg dose of SARS-CoV-2 RBD-sc-dimer and conventional RBD-monomer, respectively, with AddaVax as adjuvant. PBS formulated with adjuvant was given as control. A three-dose vaccination regimen was performed. Serum samples were collected after each immunization (19 days after 1^st immunization, 14 days after 2^nd immunization, and 14 days after 3^rd immunization) to evaluate the humoral response dynamics. ELISA assay shows the SARS-CoV-2 RBD specific IgG titers in (D) and SARS-CoV-2 pseudovirus neutralization assay shows the NT₉₀ in (E). The values shown in (D) and (E) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P-values were analyzed with one-way ANOVA (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001). (F) Evaluation of neutralization activity with live SARS-CoV-2. The serum samples of RBD-sc-dimer, RBD-monomer, and control groups from the second immunization were serially diluted and mixed with 100 50% tissue culture infectious dose (TCID₅₀) SARS-CoV-2 (2020XN4276 strain). The mixtures were added into Vero cells and cytopathic effects (CPE) could be observed 72 h post infection. The NT₅₀ was calculated as reciprocal of serum dilution required for 50% neutralization of viral infection. See also Figures S2 and S3.

Figure S3

Characterization of the Cellular Immune Response for COVID-19 Vaccine, Related to Figure 3 Fourty-five after the last vaccination, the splenocytes were isolated from mice vaccinated with SARS-CoV-2 RBD-sc-dimer (plus AddaVax™ adjuvant) and PBS (plus AddaVax™ adjuvant), respectively. (A) ELISPOT assay was performed to evaluate the ability of splenocytes to secrete IFN-γ following stimulation with different concentrations of peptide pool of SARS-CoV-2 RBD (2 μg/mL, 10 μg/mL and 50 μg/mL). Spot-forming cells (SFCs) per million cells are shown. (B) An ICS assay was conducted to quantify the proportion of CD8+ and CD4+ T cells producing key cytokines (IFN-γ, IL-2, TNF-α and IL-4) following stimulation with 10 μg/mL peptide pool (SARS-CoV-2 RBD). Shown are the frequencies of respective cytokine-producing cells. The values are the mean ± SEM. P-values were analyzed with unpaired t test (ns, p > 0.05).

Figure 4

Design and Assessment of RBD-SC-Dimer as Vaccine against SARS-CoV (A) A schematic diagram of SARS-CoV RBD-sc-dimer. Two SARS-CoV RBD (R306-Q523) were dimerized as tandem repeat (SP, signal peptide). Analytical gel filtration of SARS-CoV RBD-sc-dimer proteins was performed with HiLoad 16/600 Superdex 200 pg. The 280-nm absorbance curve is shown. Non-reduced and reduced SDS-PAGE migration profiles of the pooled samples are shown. (B) Ultracentrifugation sedimentation profiles of SARS-CoV RBD-sc-dimer. (C) Representative BIAcore diagrams of SARS-CoV RBD-sc-dimer and monomer bound to hACE2 protein. The K_D value was calculated by the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. (D and E) Groups of BALB/c mice were immunized with 10 μg dose of SARS-CoV RBD-sc-dimer and conventional RBD-monomer, respectively, with AddaVax as adjuvant. PBS formulated with adjuvant was given as control. A three-dose vaccination regimen was performed. Serum samples were collected after each immunization (19 days after 1^st immunization, 14 days after 2^nd immunization, and 14 days after 3^rd immunization) to evaluate the humoral response dynamics. ELISA assay shows the SARS-CoV RBD specific IgG titers in (D) and SARS-CoV pseudovirus neutralization assay shows the NT₉₀ in (E). The values shown in (D) and (E) are the mean ± SEM. The horizontal dashed line indicates the limit of detection. P-values were analyzed with one-way ANOVA (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001). See also Figure S2.

Figure 5

Pilot Scale Production of RBD-SC-Dimers of MERS-CoV and SARS-CoV-2 (A) RBD-sc-dimers were produced in industry-standard CHO cell system in GMP grade manufacturing. The immunogen yields and purities for vaccine stock solution are shown. (B) Non-reduced SDS-PAGE migration profile of increasing amounts of GMP grade RBD-sc-dimers are shown.