Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a betacoronavirus that causes severe respiratory illness in humans. There are no licensed vaccines against MERS-CoV and only a few candidates in phase I clinical trials. Here, we develop MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Two-component nanoparticles displaying spike (S)-derived antigens induce neutralizing responses and protect mice against challenge with mouse-adapted MERS-CoV. Epitope mapping reveals the dominant responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle elicits antibodies targeting multiple non-overlapping epitopes in the RBD. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.

Keywords: CP: Immunology; EMPEM; MERS-CoV; NTD; RBD; nanoparticle; spike; vaccine.

Figure 1.. Production and characterization of nanoparticle immunogens

(A) Schematics of MERS-CoV nanoparticle immunogen assembly. (B) Size exclusion chromatograms of MERS-CoV nanoparticle components (dotted lines) and assembled nanoparticles (solid lines) on a Superose 6 10/300 GL. (C) Left, raw micrographs and right, 2D class averages of negatively stained SEC-purified nanoparticles. For S-2P-T33_dn10, class averages of the nanoparticle core and displayed S-2P trimer are shown on top and bottom, respectively. Scale bar, 50 nm. (D) Binding of left, antigen-bearing components prior to assembly and right, SEC-purified nanoparticle immunogens to mAbs G2 (NTD-specific), S41 (RBD-specific), 4C2 (RBD-specific), and CV-30 (SARS-CoV-2 S RBD-specific; negative control).

Figure 2.. Antibody responses elicited by MERS-CoV nanoparticle immunogens in mice

(A) Study design and groups. Groups of 10 BALB/c mice were immunized at weeks 0 and 4, and serum was obtained at weeks 0, 2, 6, and 8. (B) Serum antibody binding titers against vaccine-matched (EMC) MERS-CoV S-2P, measured by ELISA. (C) Vaccine-elicited neutralizing activity against VSV pseudotyped with closely related MERS-CoV EMC, London variant, Kenya variant, or South Korea variant spikes. The mouse immunization study was performed twice, and representative data from one study are shown. (D) Left, Domain-based antigen study design and groups. Groups of 10 BALB/c mice were immunized at weeks 0 and 4, and bled at weeks 0, 2, 6, and 8. Right, Serum antibody binding titers against vaccine-matched (EMC) MERS-CoV S-2P, measured by ELISA. (E) Vaccine-elicited neutralizing activity against VSV pseudotyped with closely related MERS-CoV EMC, London variant, Kenya variant, or South Korea variant spikes. Groups were compared using Kruskal-Wallis followed by Dunn’s multiple comparisons. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.1. Only significant differences are shown; see Supplemental Data for all statistical comparisons.

Figure 3.. Epitope mapping of vaccine-elicited antibodies

(A) Serum competition of study groups with hDPP4 (top) and G2 (bottom) against MERS-CoV S-2P. (B) Composite 3D model of MERS S-2P with no bound Fabs and representative 2D class averages. Scale bar, 27 nm. (C to G) Composite 3D models and representative 2D class averages of ns-EMPEM analysis depicting Fab-bound MERS S-2P spikes from week 8 sera from mice immunized with S-2P (C), S-2P-T33_dn10 (D) S-2P-I53–50 (E), NTD-I53–50 (F), and RBD-I53–50 (G). In (G), a composite 3D model is shown to visualize Fabs bound to a “three-down” MERS-CoV Spike, while a separate 3D reconstruction is shown to visualize Fabs bound to an RBD in the “up” conformation.

Fig. 4.. Protection against challenge with mouse-adapted MERS-CoV

(A) Challenge study design and groups. Groups of 288/330^+/+ mice were immunized at weeks 0 and 4, serum was obtained at weeks 0, 2, and 6, and the mice were challenged at week 15. (B) Serum antibody binding titers against vaccine-matched (EMC) MERS-CoV S-2P, measured by ELISA. (C) Vaccine-elicited neutralizing activity against VSV pseudotyped with closely related MERS-CoV EMC, London variant, Kenya variant, or South Korea variant spikes. Groups were compared using Kruskal-Wallis followed by Dunn’s multiple comparisons. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.1. (D) Changes in body weight after maM35c4 challenge. (E) Lung congestion score. Data analyzed with two-way ANOVA or mixed model followed by Sidak’s multiple comparison. ****, p < 0.0001; *** p < 0.001; ** p < 0.01. (F) Viral titers in the lungs of challenged mice from (3 dpi: n = 5 for bare I53–50, S-2P, S-2P-T33_dn10, and S-2P-I53–50; n = 6 for RBD-I53–50. 5 dpi: n = 4 for bare I53–50; n = 5 for S-2P and S-2P-I53–50; n = 6 for S-2P-T33_dn10 and RBD-I53–50. Data were analyzed with two-way ANOVA or mixed model followed by Dunnett’s multiple comparison. ****, p < 0.0001. (G) Viral titers in nasal turbinates of challenged mice. n = 5 for bare I53–50, S-2P, S-2P-T33_dn10, and S-2P-I53–50; n = 6 for RBD-I53–50. Data analyzed with one-way ANOVA with Geisser-Greenhouse correction followed by Dunnett’s multiple comparison. In panels (B), (C), (E), (F), and (G), only significant differences are shown; see Supplemental Data for all statistical comparisons.