Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation

Abstract

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.

Keywords: SpyTag; bioconjugation; nanoparticle; nanotechnology; vaccines.

Figure 1

Spy‐nanoassembly for different antigen symmetries. A) Schematic of reactive protein/peptide pair. Lysine on SpyCatcher003 (dark blue) forms a spontaneous amide bond with aspartic acid of SpyTag003 (cyan). Residues forming the isopeptide bond are colored as red spheres, based on PDB ID: 4MLI. B) Plug‐and‐display vaccine assembly. Genetic fusion of a monomeric antigen (yellow) to SpyTag003 (cyan) allows simple efficient reaction (isopeptide bond in red) on nanoparticles (purple) fused to SpyCatcher003 (dark blue). For clarity, only a portion of SpyCatcher003 sites are shown. C) Typical symmetries of multimeric protein antigens (arrow indicates relative subunit orientation). D) Matching of nanoparticle and antigen symmetry. VLP‐display of multimeric antigen (in orange) may proceed to different display densities. Coupling of multimeric antigens could destabilize the antigen (misfolding in magenta) or bridge VLPs, leading to aggregation.

Figure 2

SpyCatcher003 enhanced VLP stability and decreased pre‐existing sero‐reactivity. A) SpyCatcher‐mi3 and SpyCatcher003‐mi3 were heated for 60 min at the indicated temperature, cooled to 4 °C, aggregates were removed by centrifugation, and the fraction of soluble protein determined. Mean±s.d., n=3. B) SpyCatcher‐mi3 or SpyCatcher003‐mi3 were subjected to the indicated number of freeze‐thaw cycles and the insoluble fraction was removed by centrifugation. VLP in the supernatant was analyzed by SDS‐PAGE. Mean is marked by a horizontal line; two‐tailed Student's t‐test, n=3. C) Pre‐existing sero‐reactivity to S. pyogenes antigens. Antibody titer of serum from 52 people against the positive control Spy0469, SpyTag:SpyCatcher, or SpyTag003:SpyCatcher003. Each person is shown as a dot, with the mean in orange. D) Summary of immune responses in (C). E) Pairwise comparison of titer. The person with increased titer against SpyTag003:SpyCatcher003 is indicated in orange (Wilcoxon matched‐pairs signed rank test, n=52). F–G) Structural model (PDB ID: 2X5P) of the CnaB2 domain and sequence alignment with SpyTag/SpyCatcher pairs. Substitutions from CnaB2 are magenta (SpyCatcher) or orange (SpyCatcher003). Substitutions in SpyTag003 are cyan. The dotted line indicates a region not resolved in CnaB2.

Figure 3

SpyCatcher003‐mi3 particles allowed efficient display of antigens with different sizes and symmetries. A) Structures of the antigens with varying symmetry. Each antigen is shown in two orientations with space‐filling model, with each chain in a different color. A model of SpyCatcher003‐mi3 is presented at the same scale. PDB IDs: RBD: 6W41, OspC: 1G5Z, NDV HN: 3T1E, HA: 4UO0, NA: 2HTY (with tetramerization domain: 1USE), streptavidin: 1SWB, β‐galactosidase: 6DRV. Red spheres indicate where the reactive peptide is fused. B) Coupling of model antigens at different molar VLP:antigen ratios. Samples were reacted for 16 h at 4 °C, insoluble aggregates removed by centrifugation, and supernatant loaded on reducing SDS‐PAGE before Coomassie staining. C) Quantification of VLP:antigen reactions. The intensity of each band from gel densitometry was divided by the intensity of the band of VLP alone at 2 μM. Bars represent mean+s.d., n=3. D) Antigens with cyclic symmetry maintained good VLP solubility. Soluble fraction of antigens conjugated at 1:1 molar ratio was determined by densitometry (mean±s.d., n=3). Some error bars are too small to be visible.

Figure 4

Particle size‐distribution after VLP decoration of antigens of various symmetry. A) Hydrodynamic radius for SpyCatcher003‐mi3 particles, with or without conjugation to antigens (mean±s.d.; for H3 Vic n=2 independent experiments; for all other samples n=3). B) TEM of negative‐stained antigen‐decorated SpyCatcher003‐mi3 VLPs. Scale‐bar: 50 nm.

Figure 5

Display of trimeric or tetrameric influenza antigens on SpyCatcher003‐mi3 enhanced antibody titre. A) Immunization schedule. B) Antibody titer against H3 Vic from mice primed and boosted with free H3 Vic, H3 Vic displayed on SpyCatcher003‐mi3 (H3 Vic:VLP), or Vic/361 influenza virus. Each dot represents one mouse and the mean is indicated in orange. n=6. Sera were assayed against plasma membrane‐bound H3 Vic. Serum from mice immunized with N1:VLP was a negative control. C) As for (B) with H3 Aichi. D) As for (B) with N1. Serum from immunizing with H3 Aichi:VLP was a negative control. E) Schematic of microneutralization assay. Neutralization potency is quantified through the decrease in GFP in cells incubated with virus pre‐mixed with serum. F) Schematic of NA inhibition assay. Inhibition of NA catalytic activity is quantified through decrease of lectin‐HRP binding to immobilized fetuin in the presence of NA‐neutralizing antibodies. G) VLP‐display enhanced virus‐neutralizing activity from H3 Vic immunization. Microneutralization assay on serum following each immunization route, tested against A/Victoria/361/2011 influenza. Serum from immunizing with N1:VLP was a negative control. n=6, except N1:VLP n=5. H) Microneutralization assay as in (G) but for H3 Aichi and A/Aichi/2/1968 virus. I) NA inhibition assay for mice immunized with free N1, N1 displayed on VLP, or matching S‐FLU. Serum from immunizing with H3 Aichi:VLP was a negative control. n=6, except H3 Aichi:VLP n=4. Dashed lines indicate limit of detection (1:40 dilution). Significance was analyzed using log₁₀‐transformed data and one‐way ANOVA with Bonferroni correction. n.s.=not significant.