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[Preprint]. 2023 Feb 24:2023.02.24.529520.
doi: 10.1101/2023.02.24.529520.

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology

Rory A Hills  1   2 , Tiong Kit Tan  3 , Alexander A Cohen  4 , Jennifer R Keeffe  4 , Anthony H Keeble  1   2 , Priyanthi N P Gnanapragasam  4 , Kaya N Storm  4 , Michelle L Hill  5 , Sai Liu  5 , Javier Gilbert-Jaramillo  5 , Madeeha Afzal  5 , Amy Napier  5 , William S James  5 , Pamela J Bjorkman  4 , Alain R Townsend  3   6 , Mark Howarth  1   2
Affiliations

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology

Rory A Hills et al. bioRxiv. .

Update in

Abstract

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.

Keywords: Covid-19; bioconjugation; nanobiotechnology; outbreak pathogens; synthetic biology; zoonotic.

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Conflict of interest statement

Competing interests: M.H. is an inventor on a patent on spontaneous amide bond formation (EP2534484) and a SpyBiotech co-founder and shareholder. M.H. and A.H.K. are inventors on a patent on SpyTag003:SpyCatcher003 (UK Intellectual Property Office 1706430.4). P.J.B. and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the methodology to generate cross-reactive antibodies using mosaic nanoparticles. P.J.B., and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the monoclonal antibodies elicited by vaccination with Mosaic nanoparticles described in this work. P.J.B., A.A.C. and J.R.K. are inventors on a US patent application filed by the California Institute of Technology that covers the methods of isolating cross-reactive antibodies by vaccination with mosaic nanoparticles. All other authors have no competing interests to declare.

Figures

Fig. 1
Fig. 1. Preparation of Multiviral Quartet.
(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. (E) Coupling of RBD Quartet to SpyCatcher003-mi3 nanocage at different molar nanocage:antigen ratios, analyzed by SDS-PAGE/Coomassie.
Fig. 2
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 RBD (orange), uncoupled Quartet-SpyTag (yellow), SARS2 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. * p < 0.05, ** p < 0.01, *** p < 0.001; other comparisons were non-significant.
Fig. 3
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. (C) DLS of SpyCatcher003-mi3 alone (uncoupled nanocage) or each immunogen. The mean hydrodynamic radius (RH) is shown ± 1 s.d., derived from 20 scans of the sample. Uncoupled Nanocage is shown in black, with the other particles colored as in (A). (D) ELISA for post-boost serum IgG as the area under the curve of serial dilution, from mice immunized with Homotypic SARS2 Nanocages (pink), Mosaic-4 (purple), Mosaic-8 (blue), SpyTag-Quartet Nanocage (red), Dual Quartet Nanocage (orange), or Uncoupled Quartet (yellow). Filled circles indicate ELISA against a component of that vaccine (matched) while empty circles 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 denoted by a circle, shown ± 1 s.d., n = 6. Individual data points and statistics are shown in Fig. S6 and S7.
Fig. 4
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. * p < 0.05, ** p < 0.01, *** p < 0.001; other comparisons were non-significant.
Fig. 5
Fig. 5. Quartet immunization induces broad antibodies even after a pre-primed SARS2 response.
(A) Summary of timeline and antigens for this set of immunizations. (B) ELISA for serum IgG to SARS2 RBD presented as the area under the curve 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). * p < 0.05, ** p < 0.01, *** p < 0.001; other comparisons were non-significant.
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