Streptococcus pyogenes pharyngitis elicits diverse antibody responses to key vaccine antigens influenced by the imprint of past infections

Abstract

Knowledge gaps regarding human immunity to Streptococcus pyogenes have impeded vaccine development. To address these gaps and evaluate vaccine candidates, we established a human challenge model of S. pyogenes pharyngitis. Here, we analyse antibody responses in serum and saliva against 19 antigens to identify characteristics distinguishing 19 participants who developed pharyngitis and 6 who did not. We show that pharyngitis elicits serum IgG responses to key vaccine antigens and a muted mucosal IgA response, whereas IgG responses are minimal and IgA responses more pronounced in participants without pharyngitis. Serum IgG responses to pharyngitis in adult participants resemble those in children and are inversely correlated with the magnitude of pre-existing responses. While a straightforward correlate of protection is not evident, baseline antibody signatures distinguish clinical and immunological outcomes following experimental challenge. This highlights the influence of a complex humoral imprint from previous exposure, relevant for interpreting immunogenicity in forthcoming vaccine trials.

Fig. 1. Experimental human S. pyogenes pharyngitis is not associated with new serum opsonophagocytic responses or inhibition of adherence by saliva.

A Schematic overview of challenge study timelines and samples analysed in this study. Created in BioRender. Hill, D. (2023) BioRender.com/g84v832. B Bacterial adherence to Detroit 562 cells in the presence of saliva collected pre-challenge and 1 week after pharyngitis diagnosis (n = 19) or discharge without pharyngitis (n = 6). Bacterial burden determined by CFU of recovered bacteria and % adherence determined relative to bacteria-only controls. Responses for each group are shown as median + range, with within-group comparisons performed using two-sided paired Wilcoxon signed-rank tests and between-group comparisons using two-sided Mann–Whitney tests, p values are shown following false discovery rate (FDR)-adjustment for multiple comparisons. C Serum opsonophagocytic killing assay results from pre-challenge and one-month post-challenge samples (n = 25), highlighting results from two participants with a detectable opsonophagocytic response (SN013, SN010; mean and standard error of three technical replicates shown), and grouping the remainder of non-responders by clinical pharyngitis outcome (blue = non-pharyngitis, n = 5; red = pharyngitis, n = 18). Opsonic index determined by the linear regression of the two dilutions closest to 50% killing. Text descriptions below describe clinical and infection parameters for SN013 and SN010.

Fig. 2. Experimental pharyngeal challenge with Streptococcus pyogenes induces mucosal and systemic antibody responses against major vaccine antigens.

A Schematic overview of the time-points and antigens studied to detect IgG from serum and IgA saliva. Antigens were grouped into two categories; Main included leading vaccine candidates and key virulence factors, and Additional included peptides or domains of proteins included among Main Ags (M protein, SpyCEP, T antigen). Created in BioRender. Hill, D. (2023) BioRender.com/m87o308. B Heatmap showing the relative abundance of IgA and IgG responses pre and post-challenge at indicated time-points in pharyngitis and non-pharyngitis participants for 11 main antigens. ELISA titres are shown with responses scaled across IgA and IgG separately. Pharyngitis outcome indicated as coloured bar (red = pharyngitis, blue = no pharyngitis). C Heatmap showing the median fold-change observed relative to baseline at 1 week for IgA, and 1 month for IgG with associated p values from two-sided paired Wilcoxon signed-rank test after FDR-adjustment for 11 main antigens and six additional peptide or vaccine antigens. (*P_adjusted < 0.05, **P_adjusted < 0.01). D The percentage of pharyngitis and non-pharyngitis participants that showed a 25% increase in antibodies post-challenge, with 1 week for IgA and 1 month for IgG. Numbers indicate how many participants responded to each antigen. E A tally of the number of antigens to which participants showed a 25% increase in IgA and IgG (shown in D), with participants split by pharyngitis outcome. P value determined from two-sided Mann–Whitney U-test. F Representative examples of antigens that showed significant change after challenge (as shown in C) expressed in ELISA arbitrary units (arb. units). Within-group comparisons were performed using a two-sided paired Wilcoxon signed-rank test, and groups were compared using a two-sided Mann–Whitney test, all FDR-adjusted for multiple comparisons. G Spearman correlation of fold-change in saliva IgA at 1 week with fold-change in serum IgG at 1 week and 1 month post-challenge. All comparisons were p > 0.1.

Fig. 3. Antibody responses induced by experimental pharyngeal challenge with Streptococcus pyogenes are maintained for at least 3 months.

A Schematic overview of time-points and antigens measured by a multiplex bead-based assay for IgG antibodies in sera. Created in BioRender. Hill, D. (2023) BioRender.com/m17l045. B IgG responses for 6 antigens at indicated time-points with the cohort split into those who showed a 25% increase in IgG at 1-month post-challenge (responders) and those that did not (non-responders). For Responders, each dot represents an individual, and lines connect paired samples. The violin plots summarise the distribution (median+range) of responses across time-points for each group, with colour corresponding to time-point. The analysis included 22 participants with samples available from all four time-points. P values were determined for within-group paired sample comparisons using Friedman’s test followed by a two-sided Wilcoxon signed-rank test with FDR-adjustments, and for between-group comparisons using Kruskal–Wallis and Dunn’s multiple comparison testing.

Fig. 4. Antibody responses to experimental human Streptococcus pyogenes pharyngitis in healthy adults are similar to natural responses in children.

Serum IgG concentrations measured by multiplex bead-based assay to 6 antigens among challenge participants pre- and 1-month post-challenge was compared to healthy children and children >1 month after acute pharyngitis (aged between 5 and 14 years). P values were determined using Dunn’s multiple comparison testing or Wilcoxon signed-rank test for paired data, both two-sided.

Fig. 5. Pre-challenge baseline titres negatively correlate with post-challenge antibody responses.

A Correlation between IgG responses to 6 antigens measured by multiplex bead-based assay in samples from pre-challenge with those at 1-month post-infection (pharyngitis n = 17, no pharyngitis n = 6). B Correlation between anti-ScpA and anti-M75 IgA responses measured by ELISA from pre-challenge and 1-week post-challenge (pharyngitis n = 19, no pharyngitis n = 6). Coefficient and p value were determined using Spearman’s method (two-sided) with a linear regression line included to indicate linear trends. Each dot represents an individual participant with the pharyngitis group shown in red and non-pharyngitis in blue.

Fig. 6. Serological signatures differ by clinical outcome following experimental pharyngeal human challenge with S. pyogenes and are shaped by infection.

A Non-metric multidimensional scaling (MDS) was performed using IgG responses to 19 antigens for 23 individuals. Each point represents a sample, with a shape denoting time-point and coloured to indicate pharyngitis outcome (left), paired samples (centre), or responses to 2 or more of the dominant antigens SpyCEP, SLO, ScpA or GAC. B Violin plots of first MDS dimension values pre-challenge (left), or at 1 month with pre-challenge values subtracted (right), between pharyngitis and non-pharyngitis groups. P value determined by two-sided Mann–Whitney U-test. C Two-sided Spearman correlation analysis between MDS dimension 1 and IgG responses, with only antigens with significant correlation shown (p < 0.01 after FDR-adjustment). Antigens with results from ELISA assays are shown in black font, and multiplex bead-based assays are shown in gold font.

Fig. 7. Baseline antibody titres are associated with clinical pharyngitis outcome following experimental human challenge with Streptococcus pyogenes.

A Schematic overview of methods and variables included in correlation analysis and sparse partial least squares discriminant analysis (sPLS-DA). B Heatmap of correlation coefficients between various clinical variables and pre-challenge antibody levels. Antigens shown are those that showed significant two-sided Spearman correlation for one or more clinical variables. Asterisks denote unadjusted p values *<0.05, **<0.01. C Samples distributed across two dimensions from sPLS-DA. D Contributions of top 5 variables to sPLS-DA dimension 1, with variables in red higher in the pharyngitis group and variables in blue higher in the non-pharyngitis group. E Pre-challenge antibody responses against the top five variables from sPLS-DA, differentiating between participants who subsequently developed pharyngitis (red) and those who did not (blue). P value determined using the two-sided Mann–Whitney test. Antigens with results from ELISA assays are shown in black (IgG) and blue (IgA) text, and multiplex bead-based assays (IgG) are shown in gold text.