MERS-CoV RBD-mRNA Presents Better Immunogenicity and Protection than the Spike-mRNA

Abstract

Pathogenic Middle East respiratory syndrome CoV (MERS-CoV), first identified in Saudi Arabia in 2012, continues to pose a threat to public health. The trimeric spike (S) protein of MERS-CoV binds to the cellular receptor through the receptor-binding domain (RBD) in the S1 subunit to initiate virus entry and infection. Therefore, both the S protein and its RBD are targets for the development of MERS-CoV vaccines. Nevertheless, a direct comparison of the immune efficiency of S- and RBD-based MERS-CoV vaccines has not been made. Here, we compared two mRNA vaccines, respectively, targeting the S (S-mRNA) and RBD (RBD-mRNA) of MERS-CoV for their durable immunogenicity, neutralizing activity, and protective efficacy in a mouse model. Both mRNAs encapsulated with lipid nanoparticles (LNPs) maintained strong stability at various temperatures during the detection period. LNP-encapsulated RBD-mRNA elicited significantly higher and more durable antibodies than LNP-encapsulated S-mRNA, maintaining stronger and broadly neutralizing activity against the MERS-CoV original strain, as well as multiple variants containing key mutations within the RBD region. Importantly, RBD-mRNA provided durable protective efficacy against MERS-CoV infection in middle-aged mice, and this protection was associated positively with serum neutralizing antibody titers. Overall, this study identifies RBD-mRNA as an effective vaccine against MERS-CoV, with great potential for further development.

Keywords: MERS-CoV; coronavirus; neutralizing activity; protective efficacy; receptor-binding domain; spike.

Figure 1

The design and characterization of MERS-CoV S-mRNA and RBD-mRNA vaccines. (A) The design, synthesis, and formulation of mRNAs. The full-length S protein of MERS-CoV contains the S1 and S2 subunits with its original signal peptide (SP) on the N-terminus, whereas the S2 subunit includes the transmembrane domain (TM) and cytoplasmic tail (CP), among other components. Each mRNA encodes the tPA signal peptide and a truncated S protein (i.e., excluding TM and CP regions), or its RBD fragment of MERS-CoV, with a C-terminal foldon (Fd) trimeric motif and a His₆ tag. The synthesized mRNAs, capped at the N-terminus and tailed (with a Poly(A) sequence) at C-terminus, were encapsulated with lipid nanoparticles (LNPs) using the NanoAssemblr Ignite nanoparticle formulation system instrument to form mRNA-LNPs. (B) The determination of the stability of LNP-encapsulated S-mRNA or RBD-mRNA by measurement of the particle sizes using DynaPro NanoStar II Light Scattering Detector (DLS, WYATT Technology). Each mRNA was, respectively, stored at 4 °C, 25 °C, and 37 °C for 24, 48, 72, and 120 h, followed by the measurement of the particle sizes (hydrodynamic diameter: nm). LNP (without encapsulation with mRNA) was included as a control. The data in (B) indicates the mean ± standard deviation of the mean (s.e.m) of triplicate wells. (C) The agarose gel electrophoresis of RNAs extracted from the LNP-encapsulated mRNA samples (after LNPs). RNA samples without formulation with LNPs (before LNPs) were added as controls. Each RNA sample was loaded with 1 μg per well. The RNA ladder is shown on the left. The experiments were repeated once, with similar results.

Figure 2

The measurement of MERS-CoV S or RBD-specific antibody responses induced by the MERS-CoV mRNA vaccines. (A) Immunization schedules and antibody testing. B6 mice were injected with S-mRNA, RBD-mRNA, or LNP control 3 times at 3-week intervals. Sera were collected 10 days post-last dose or once every other month for up to 5 months to test for total IgG antibody (Ab) response, IgG subtype Abs (IgG1 and IgG2c), or neutralizing Abs. The measurement of IgG antibody (B,C) and its subtypes (IgG1 and IgG2c) (D–G) specific to MERS-CoV RBD (B,D,F) or S (C,E,G) protein by ELISA. Sera collected 10 days post-last dose were used for this testing. The dashed lines indicate the detection limit. The evaluation of durable IgG Abs specific to MERS-CoV RBD (H) or S (I) protein by ELISA. Sera collected from 1, 3, and 5 months post-last dose were used for this testing. The ELISA plates were coated with RBD or S protein of MERS-CoV, and the Ab titer was presented as the mean ± s.e.m. of five mice in each group. Ordinary one-way ANOVA (Tukey’s multiple comparison test) was used to compare statistical significance among different groups. ** and **** indicate p < 0.01 and p < 0.0001, respectively. The experiments were repeated once, with similar results.

Figure 3

The evaluation of neutralizing antibodies induced by the MERS-CoV mRNA vaccines. Mouse sera collected 10 days post-last immunization of S-mRNA, RBD-mRNA, or LNP control were tested by the pseudovirus neutralization assay for broadly neutralizing antibodies (Abs) against the pseudotyped MERS-CoV original strain (MERS-WT) (A) or its variant strains, including MERS-L506F (B), MERS-L507P (C), MERS-A520S (D), MERS-E536K (E), and MERS-D537E (F), which contain key mutations in the RBD region. The dashed lines indicate the detection limit. (G) The evaluation of durable neutralizing Abs against pseudotyped MERS-WT strain by pseudovirus neutralization assay. Sera collected from 1, 3, and 5 months post-last immunization were used for this testing. The 50% neutralizing Ab titer (NT₅₀) was determined based on the serum dilution at which 50% of the pseudotyped virus was neutralized. The neutralizing Ab titer is presented as the mean ± s.e.m. of five mice in each group. Ordinary one-way ANOVA (Tukey’s multiple comparison test) was used to compare statistical significance among different groups. *, *** and **** indicate p < 0.05, p < 0.001 and p < 0.0001, respectively. The experiments were repeated once, with similar results.

Figure 4

The evaluation of protective efficacy of the MERS-CoV mRNA vaccines. (A) Ten months post-last immunization of S-mRNA, RBD-mRNA, or LNP control, mice were intranasally (i.n.) transduced with Adenovirus 5 (Ad5)-hDPP4 vector, and five days later, they were i.n. challenged with MERS-CoV original strain (EMC2012, 10⁵ plaque-forming unit (PFU)/50 μL/mouse), followed by the testing of viral titers in the lungs 3 days later. (B) The evaluation of viral titers (PFU/mL) in the lungs of challenged mice by plaque assay. (C) The evaluation of serum neutralizing antibodies of mice before challenge. Sera collected before challenge were tested for neutralizing antibody (Ab) by pseudovirus neutralization assay against the pseudotyped MERS-CoV original strain (MERS-WT). The 50% neutralizing Ab titer (NT₅₀) was determined based on the serum dilution at which 50% of the pseudotyped virus was neutralized. The data in (B,C) indicates the individual values of five mice in each group (mouse No. 1–5). The dashed line in (C) indicates the detection limit. Ordinary one-way ANOVA (Tukey’s multiple comparison test) was used to compare statistical significance among different groups. * indicates p < 0.05. The experiments were repeated once, with similar results.