Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses

Abstract

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.

Fig. 1. Preparation of multiviral Quartet Nanocages.

a, Plug-and-Display vaccine assembly of mosaic and Quartet Nanocages. Genetic fusion of SpyCatcher003 (dark blue) with mi3 (purple) allows efficient multimerization of single or Quartet RBDs linked to SpyTag (cyan) through spontaneous isopeptide bond formation (marked in red). Only some antigens are shown in the schematic for clarity. b, Phylogenetic tree of sarbecoviruses used in this study, based on RBD sequence. c, Genetic organization of the multiviral Quartet-SpyTag, indicating the viral origin of RBDs, N-linked glycosylation sites and tag location. d, Analysis of Quartet-SpyTag with SDS–PAGE/Coomassie staining, with or without PNGase F deglycosylation. A representative gel from two independent experiments. Molecular weight markers are in kDa. e, Coupling of RBD Quartet to SpyCatcher003-mi3 Nanocage at different molar Nanocage:antigen ratios, analysed by SDS–PAGE/Coomassie. A representative gel from two independent experiments. Molecular weight markers are in kDa. Source data

Fig. 2. Broad immune response from immunization with Quartet Nanocages.

a, Schematic of antigens for this set of immunizations, comparing uncoupled proteins or proteins coupled to the SpyCatcher003-mi3 nanocage. b, Procedure for immunization and sampling. c, ELISA for post-boost serum IgG binding to different sarbecovirus RBDs is shown as the area under the curve (AUC) of a serial dilution. Sera are from mice immunized with uncoupled SARS2 Wuhan RBD (orange), uncoupled Quartet-SpyTag (yellow), SARS2 Wuhan RBD coupled to SpyCatcher003-mi3 (green) or Quartet-SpyTag coupled to SpyCatcher003-mi3 (blue). Solid rectangles under samples indicate ELISA against a component of that vaccine (matched). Striped rectangles indicate ELISA against an antigen absent in that vaccine (mismatched). Each dot represents one animal. The mean is denoted by a bar, shown ±1 s.d.; n = 6. Significance was calculated with an ANOVA test using Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001; other comparisons were non-significant. Source data

Fig. 3. Comparison of immunization with Mosaic or Quartet Nanocages.

a, Schematic of antigens for this set of immunizations. b, Validation of the Alternate Quartet by SDS–PAGE with Coomassie staining, shown ±PNGase F deglycosylation. A representative gel from two independent experiments. Molecular weight markers are in kDa. c, DLS of SpyCatcher003-mi3 alone (uncoupled nanocage) or each immunogen. The mean hydrodynamic radius (R_H) is shown ±1 s.d., derived from 20 scans of the sample. Uncoupled Nanocage is shown in black, with the other particles coloured as in the table inset. d, ELISA for post-boost serum IgG as the AUC of serial dilution, from mice immunized with Homotypic SARS2 Nanocages (dark blue), Mosaic-4 (green), Mosaic-8 (light blue), SpyTag-Quartet Nanocage (pink), Dual Quartet Nanocage (purple) or Uncoupled Quartet (grey). Squares indicate ELISA against a component of that vaccine (matched) while crosses indicate ELISA against an antigen absent in that vaccine (mismatched). Responses are shown to the set of sarbecovirus RBDs, with SpyTag-MBP as a negative control. The mean is shown ±1 s.d.; n = 6. Individual data points and statistics are shown in Supplementary Figs. 10 and 11. Source data

Fig. 4. Neutralization induced by Quartet immunogens.

a, Neutralization of Wuhan SARS2 virus by boosted mouse sera. Mice were primed and boosted with Uncoupled RBD (orange), Uncoupled Quartet (yellow), Homotypic Nanocage (green) or Quartet Nanocage (blue). Each dot represents one animal, showing the serum dilution giving 50% inhibition of infection (ID₅₀). b, Neutralization of Delta SARS2 virus by boosted mouse sera, as in a. c, Neutralization of SARS1 pseudovirus (mismatched) by post-boost mouse sera, after immunization with different Quartet and Mosaic immunogens. Dashed horizontal lines represent the limit of detection. The mean is denoted by a bar + 1 s.d.; n = 6. Significance was calculated with an ANOVA test, followed by Tukey’s multiple comparison post hoc test of ID₅₀ values converted to log₁₀ scale. *P < 0.05, **P < 0.01, ***P < 0.001; other comparisons were non-significant. Source data

Fig. 5. Quartet immunization induces broad antibodies even after a preprimed SARS2 response.

a, Summary of timeline and antigens for this set of immunizations. b, ELISA for serum IgG to SARS2 Wuhan RBD presented as the AUC of a serial dilution. All mice were primed with Wuhan SARS2 Spike, before boosting with Wuhan SARS2 Spike protein (light green), Homotypic Nanocage (pink), Mosaic-8 (dark blue), SpyTag-Quartet Nanocage (red), Dual Quartet Nanocage (orange), Quartet Nanocage with SARS1 RBD replacing SARS2 (purple) or Dual Quartet Nanocage with SARS1 RBD replacing SARS2 (cyan). Solid rectangles under samples indicate ELISA against a component of that vaccine (matched). Striped rectangles indicate ELISA against an antigen absent in that vaccine (mismatched). Each dot represents one animal. The mean is denoted by a bar ±1 s.d.; n = 6. c, ELISA for serum IgG to other sarbecovirus RBDs, as for b, with each dot representing one animal and the mean being denoted by a bar ±1 s.d.; n = 6. Significance was calculated with an ANOVA test using Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001; other comparisons were non-significant. Source data