Methodologies for Interrogating Streptococcus pyogenes

The Network uses a harmonised suite of laboratory platforms to define the mechanisms of , combining longitudinal natural infection cohorts with .

Antibody & Serology T Cell & Cellular

Harmonised Cross-Network Assay Workflow (iSpy-LIFE × iSpy-EXPLORE)

Click any stage to inspect platform specifications

Stage 01 • Inputs 11 Countries

Cohorts & Human Challenge

Longitudinal school surveillance, RHD clinical cohorts, and controlled human infection (CHIM) biospecimens.

Stage 02 • Humoral IgG / IgA / FcR

Serology & Pan-Proteomics

High-density PhIP-Seq, BAMA, ADI microarrays, quantitative mass spectrometry, and OPKA killing kinetics.

Stage 03 • Cellular Single-Cell

T Cells & Tonsil Organoids

Spectral flow AIM gating, CITE-seq clonotyping, 3D germinal centre organoids, and ~200-protein IEDB megapools.

Stage 04 • Synthesis CoP Matrix

Correlates of Protection

Integrating humoral and cellular datasets to define protective vaccine targets free of autoimmune cross-reactivity.

01. Antibody & Serological Platforms

Humoral Repertoire • Systems Serology • Pan-Proteomics

Repertoire Interrogation IgG1-4 • sIgA • FcγR

High-Resolution Serological Profiling

OPKA 4PL Hill Curve & BAMA Multiplex y = B + (T - B) / [1 + (x / EC₅₀)⁻ᴴ]

Using high-resolution and multiplex bead-based immunoassays (), we map antibody repertoires following both natural infection and experimental vaccination. Alongside binding titres, we measure functional activity including antibody-induced opsonophagocytosis and killing ().

Systems Serology & Phage Display

Systems Antigenomics LC-MS/MS • GAC Glycans

Quantitative Proteomic Discovery

LC-MS/MS Pan-Proteome & GAC Epitope Spectrum m/z Abundance • ΔG Binding

Integrating structural biology with quantitative mass spectrometry, we define conserved targets across the S. pyogenes pan-proteome and glycan variants. Identifying protective epitopes directly from human antibody repertoires guides our design of multivalent nanoparticle immunogens.

Structural & Immuno-Proteomics

Methodologies are supported by key partners, including Antigen Discovery Inc. (), to deploy bespoke, high-density antigen microarrays.

02. T Cell & Cellular Platforms

Single-Cell CITE-seq • 3D Tonsil Organoids • IEDB Megapools

Single-Cell Phenotyping UMAP • TCR

High-Dimensional Response Tracking

CITE-seq UMAP & AIM⁺ Gate OX40⁺CD40L⁺

Using spectral flow cytometry, assays, and single-cell sequencing (), we track both conventional and unconventional T cell responses across blood and tonsillar tissue to characterise the cellular functions associated with durable protection.

Single-Cell Multi-Omics

Ex Vivo Modelling GC Tfh • IL-21

Tonsil Organoid Systems

3D Germinal Centre Organoid Synapse PD-1⁺CXCR5⁺

Because the upper respiratory mucosa is a primary site of infection, we stimulate donor-derived tonsil organoids and populations with conserved S. pyogenes antigens to examine how mucosal T and B cell immunity matures across the life course.

Mucosal & Germinal Centres

Systematic Epitope Mapping ~200 ORFs • HLA-II

Comprehensive Antigen Profiling

IEDB Megapool HLA-II Promiscuity Matrix IC₅₀ < 50 nM

We systematically map T cell epitopes across ~200 selected S. pyogenes proteins representing diverse global strains. Using peptide megapools and the Immune Epitope Database (), we define immunodominant regions required for broadly protective vaccines without autoimmune cross-reactivity.

Cross-Strain Epitope Discovery