Induction of neutralising antibodies against conserved rhinovirus capsid protein VP4 is dependent on presentation of VP4 peptide immunogens in a virus-like conformation

Fig 1. Regions of RV VP4 are highly conserved within and between RV species. Conservation of VP4 residues 1-45 between RV species A (A), RV species B (B), RV species C (C) and species A,B,C (D) was analysed using the WebLogo online server. Alignments were based on sequences from 77 RV-A serotypes, from 26 RV-B serotypes and 26 RV-C genotypes. All sequences were retrieved from NCBI.

Fig 2. Different lengths of VP4 peptides can be effectively displayed on mi3 VLP via SpyTag/SpyCatcher. (A) Conjugation of SpyVLP with different SpyTag-VP4 peptides, including non-myristoylated (Non myr) and myristoylated (Myr) VP4 peptides, spanning amino acids 1-15, 1-30 and 1-45 from the N-terminus. Reactions were performed at 22 °C overnight and analysed using SDS–PAGE with Coomassie staining. Similar reactions have been repeated multiple times during subsequent preparation of material for immunisations. (B-C) Dynamic light scattering (DLS) characterisation of SpyVLP alone and conjugates 1-15-VP4-SpyVLP, 1-30-VP4-SpyVLP and 1-45-VP4-SpyVLP with or without myristoylation. The expected hydrodynamic diameter for SpyVLP is 37 nm.

Fig 3. Sera from mice immunised with 1-15-VP4 peptides conjugated to SpyVLP neutralise RV-A16. (A) Schematic diagram of mouse immunisation schedule. (B) VNT was used to assess the ability of terminal bleed immune sera to neutralise RV-A16 infections in HeLa H1 cells. Serum dilutions spanned from 1:2 to 1:32. Sera were from mice immunised with peptides of different lengths (1-15, 1-30, 1-45, or vector alone) presented on SpyVLP at two different doses (SpyVLP-0.5 and SpyVLP-5) or presented on KLH. For these samples, each dot represents a different mouse (n = 5). Control samples included a PBS negative control (-) and a previously characterised anti-VP4 neutralising sheep sera (+), each dot for these control sera represents a technical replicate (n = 3). CPE was quantified by crystal violet staining and read at OD560. Each point corresponds to a single vaccinated mouse from n = 5 per vaccination group. Data are shown as mean ± 1 SD. Neutralisation is represented by a solid black line at OD 0.7. Statistical analysis was performed using two-way ANOVA, and post-hoc analysis was performed using Sidak’s pairwise comparison with a significance threshold of p = 0.05. Comparisons were between…

Fig 4. Neutralising sera from mice immunised with 1-15-VP4 immunogens do not react with 1-45-VP4 peptides. ELISAs using the VP4-1-45 peptide as a capture were used to assess the reactivity of immune sera. (A-L) Sera corresponding to pre-bleed, post-prime, post-boost 1 and post-boost 2 were used at a dilution of 1:160 in ELISA with VP4-1-45 peptide as the capture antigen. Sera from each group were used, SpyVLP-0.5 (A), SpyVLP-5 (B), KLH (C), 1-15-VP4-SpyVLP-0.5 (D), 1-15-VP4-SpyVLP-5 (E), 1-15-VP4-KLH (F), 1-30-VP4-SpyVLP-0.5 (G), 1-30-VP4-SpyVLP-5 (H), 1-30-VP4-KLH (I), 1-45-VP4-SpyVLP-0.5 (J), 1-45-VP4-SpyVLP-5 (K), 1-45 VP4-KLH (L). (M) Summary of endpoint (post boost 2) ELISA data in panels A-L. Data were normalised to positive and negative controls. Statistical analysis was performed to compare VP4-specific and vector-only sera using two-way ANOVA, and post-hoc analysis was performed using Sidak’s pairwise comparison with a significance threshold of p = 0.05. **p < 0.05, ***p < 0.005, ****p < 0.0005.

Fig 5. VP4 capture peptides are able to react with anti-VP4 positive control sera. Peptides VP4-1-15, VP4-1-30, and VP4-1-45 are functional as capture antigens in ELISA when used with anti-VP4 neutralising positive control sera. Wells were coated with capture peptides corresponding to either 1-15-VP4 (red), 1-30-VP4 (blue) or 1-45-VP4 (yellow). ELISAs were performed with a dilution range of 1:320 to 1:2,560. Each point corresponds to a technical replicate. Absorbance for all ELISAs was read at OD450. All ELISAs were performed using an anti-mouse IgG secondary antibody.

Fig 6. Peptide length and antigen display system influence the site of binding of VP4-specific antibodies. (A-L) ELISA was used to determine the reactivity of sera with capture antigens of different peptide lengths. Wells were coated with capture peptides corresponding to either 1-15-VP4 (red), 1-30-VP4 (blue) or 1-45-VP4 (yellow). ELISAs were performed for each serum group against each capture peptide using a dilution range of 1:80 to 1:640. Sera groups included SpyVLP-0.5 (A), SpyVLP-5 (B), KLH (C), 1-15-VP4-SpyVLP-0.5 (D), 1-15-VP4-SpyVLP-5 (E), 1-15-VP4-KLH (F), 1-30-VP4-SpyVLP-0.5 (G), 1-30-VP4-SpyVLP-5 (H), 1-30-VP4-KLH (I), 1-45-VP4-SpyVLP-0.5 (J), 1-45-VP4-SpyVLP-5 (K), 1-45-VP4-KLH (L). Absorbance was read at OD450. All ELISAs were performed using an anti-mouse IgG secondary antibody. Each point corresponds to a mouse (n = 5 per vaccination group). Data are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA, and post-hoc analysis was performed using Dunnett’s test using the corresponding mock vaccination as the control mean. Significance threshold was set at p = 0.05. *p < 0.05, ***p < 0.0005, ****p < 0.00005. (M) Heat map visualisation of epitope…

Fig 7. Lack of peptide reactivity in 1-15-VP4-SpyVLP neutralising sera is not due to a mismatched isotype of detection antibody. ELISAs were performed for each serum group with different isotypes of mouse secondary antibodies (IgA, IgM, IgG1, IgG2a, IgG2b, IgG3). Peptide 1-15-VP4 was used as a capture antigen for all sera raised against 1-15-VP4 peptides. Peptide 1-30-VP4 was used as a capture antigen for all sera raised against 1-30-VP4 peptides. Peptide 1-45-VP4 was used as the capture antigen for all sera raised against the 1-45-VP4 peptide. ELISAs were performed for each sera group at a 1:160 dilution, sera group is labelled on the left, 1-15-VP4-SpyVLP-0.5 (i), 1-30 VP4-SpyVLP-0.5 (ii), 1-45 VP4-SpyVLP-0.5 (iii), 1-15-VP4-SpyVLP-5 (iv), 1-30 VP4-SpyVLP-5 (v), 1-45 VP4-SpyVLP-5 (vi), 1-15 VP4-KLH (vii), 1-30 VP4-KLH (viii), 1-45 VP4-KLH (ix). Absorbance was read at OD450. Each row corresponds to a single mouse (n = 5 per vaccination group). Each column corresponds to a different secondary antibody isotype.

Fig 8. Neutralising 1-15-VP4-SpyVLP sera only react with 1-15-VP4 peptide when conjugated to a display system. ELISAs were performed to assess if conjugation of 1-15-VP4 peptide to DogCatcher-MBP affected its reactivity with sera from 1-15-VP4-SpyVLP-5, 1-30-VP4-SpyVLP-5 or 1-45-VP4-SpyVLP-5 immunised mice. Wells were coated with equimolar concentrations of either unconjugated or conjugated 1-15-VP4 solution to act as capture antigens. ELISAs were carried out using a 1:160 dilution with 1-15-VP4-SpyVLP-5 (red), 1-30-VP4-SpyVLP-5 (blue) or 1-45-VP4-SpyVLP-5 (yellow) or vector only as a negative control (white). An IgG secondary antibody was used. Background absorbance is represented by a black dashed line. Absorbance was read at OD450. Each point corresponds to a mouse (n = 5 per vaccination group). Data is shown as mean ± SD. Statistical analysis was performed using one-way ANOVA, and post-hoc analysis was performed using Dunnett’s test using the corresponding mock vaccination as the control mean. Significance threshold was set at p = 0.05. *p < 0.05, ***p < 0.0005.

Fig 9. Structural analyses indicate that residues 1-15 adopt different conformations in 1-15-VP4, 1-30-VP4 and 1-45-VP4. (A) Cartoon representations of the structural models for peptide monomers predicted using AlphaFold 3. Residues corresponding to VP4 regions and tags are colour-coded as indicated in the legend. The mean pLDDT scores for VP4 residues only and for all residues are indicated above the models. (B) Spectral overlay of 1H,15N HMQCs for the backbone amides of 1-15-VP4 and 1-30-VP4 with cross peaks labelled with the corresponding residue. (C) Per-residue secondary structure propensities (SSP) for 1-15-VP4 and 1-30-VP4 peptides. Positive values indicate β-strand propensity and negative values indicate α-helical propensity. SSP values of +1 or -1 would correspond to the presence of a completely ordered β-strand or α-helix, respectively.