Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera

Abstract

Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV_S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines.

Fig. 1. Design and characterization of CoV-S-2P displayed on I53-dn5.

a Computer-generated models of prefusion-stabilized spike trimers (S-2P) from SARS-1, SARS-2, and MERS, and their homotypic display on the icosahedral I53-dn5 nanoparticle displaying 20 trimers. b Trace profiles of S-2P_dn5B trimer and _dn5 nanocage purification by size exclusion chromatography. c ELISA comparing binding of antibodies specific for SARS-1, SARS-2, or MERS_S-2P to soluble trimer (triangles) or dn5 assembly (circles) respectively. d Representative images of CoV-S-2P_dn5 at 50,000× magnification and 2D class averages. Scale bars correspond to 100 nm (representative images) and 20 nm (2D class averages). All experiments were performed at least twice, each repeat with similar results.

Fig. 2. Assembly of SARS-1_S-2P on dn5 elicits potent cross-neutralizing antibodies.

a–f Groups of 10 female BALB/cJ were immunized at weeks 0 and 3 with 10 µg of SARS-1_S-2P as a soluble trimer or displayed on I53_dn5 particles, MERS_S-2P trimer or H1 trimer displayed on I53_dn5 nanoparticles with SAS adjuvant and bled at week 5 for serology. Control mice were immunized with H1_dn5. a–c Sera were screened for binding by ELISA to SARS-1_, SARS-2_, and MERS_S-2P. d–f Serum was then assessed for its capacity to neutralize SARS-1, SARS-2, and MERS pseudotyped viruses. g–i To plot the potency of neutralizing antibodies, correlation plots of binding (x-axis) to neutralization (y-axis) where the slope (neutralization/binding) indicates the ratio of neutralizing to binding antibody titers were generated. a–f Boxes and horizontal bars denote the interquartile range (IQR) and medians, respectively. Whisker endpoints are equal to the minimum and maximum values. Statistical analysis was performed using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 3. SARS_dn5 elicits two distinct antibody populations targeting the S1 domain of MERS_S-2P.

a–c To elucidate cross-reactive domain specificity, SARS-1_dn5 sera was depleted with MERS_S-2P and its domains, S1, SS, and RBD, then screened for residual binding to a SARS-1_S-2P, b SARS-2_S-2P, and c MERS_S-2P. d–g Mice immunized with SARS-1_dn5 were terminally bled and serum was pooled, IgG-purified and digested to Fabs. Immunocomplexes of SARS-1_dn5-elicited Fabs bound to MERS_S-2P were imaged with negative stain EM. d, e Squares are magnified views of Fabs bound within the image. Scale bars correspond to 100 nm (representative image) and 20 nm (2D classes). Arrows point to Fabs bound to the top or side of MERS_S-2P. f, g 3D map reconstruction was generated from NSEM and overlayed with structures of MERS_S-2P and MERS-specific mAb G2. a–c Boxes and horizontal bars denote the IQR and medians, respectively. Whisker endpoints are equal to the minimum and maximum values. Circles denote each individual animal. Statistical analysis was performed using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons. **P < 0.01, ***P < 0.001, ****P < 0.0001. EMPEM was performed once.

Fig. 4. b-mosaic particles elicit broad antibody responses.

a Groups of 10 female BALB/cJ mice were immunized twice with β-CoV mosaic_I53_dn5, MERS_I53_dn5, SARS-1_I53_dn5, SARS-2_I53_dn5, HKU1_I53_dn5, or OC43_I53_dn5 to compare antibody responses elicited from co-display and monotypic display of each spike. Control mice were immunized with H1_I53_dn5. Mice were bled at week 5 for serology. b–h Sera were screened by ELISA for IgG binding to each strain. Boxes and horizontal bars denote the IQR and medians, respectively. Whisker endpoints are equal to the minimum and maximum values. Circles denote each individual animal. Immune profiling in BALB/cJ performed once.

Fig. 5. β-CoV mosaic particles protect against lethal MERS-CoV challenge.

288/330^+/+ mice were immunized twice with 10 µg of the specified nanoparticle or soluble trimer. Control mice were immunized with H1_dn5. 4 weeks post-boost, mice were challenged with a lethal dose, 5 × 10⁵ plaque-forming units (p.f.u.) of maM35c4 MERS-CoV. a Following challenge, mice were monitored for weight loss. b, c 5 days post challenge, b lung discoloration (scored as: 0 = no discoloration, 4 = severe discoloration in all lobes) and c lung viral titers were assessed. All groups were compared with H1_dn5 control mice by Kruskal–Wallis analysis of variance (ANOVA) with Dunn’s multiple comparisons test; in (a), the comparison was made at each day post-challenge. *P < 0.05, **P < 0.01. Data depict mean ± s.d. in (a, b) or GMT ± geometric s.d. in (c). In (c), the dotted line represents the assay limit of detection. Challenge experiments were performed once.