Jacob Glanville

Neutralizing the VSG Repertoire

Trypanosoma brucei and the Variable Surface Glycoprotein (VSG)

Stijlemans B, Caljon G, Natesan SKA, Saerens D, et al. (2011) High Affinity Nanobodies against the Trypanosome brucei VSG Are Potent Trypanolytic Agents that Block Endocytosis. PLoS Pathog 7(6): e1002072. doi:10.1371/journal.ppat.1002072

Anti-VSG antibodies neutralize T Brucei

Immunodominance & cryptic epitopes

① If bnMabs can be made, why don’t we all make them?

② If in HA and HIV, why not VSG?

Hypothesis: There exist conserved epitopes in the VSG repertoire that cannot escape antibody recognition.

Aim 1: Identify conserved candidate broadly neutralizing epitopes from high-throughput primary and tertiary structure analysis of the VSG repertoire

A) Sequence many peopleB) Map variation to structureC) Identify putative conserved epitopes

• Trypanosomes from 100-1000 patients• 5’RACE cDNA amplification• SafeSeqS protocol • Multiplex barcoding (5-50 samples/run)• Miseq (>5M 250x2 paired-end reads)• Profile Hidden Markov Models• Structural modeling

Estimating recovery10 samples pooled per run such that each patient sample is sequenced to an approximate depth of 500,000 250x2 paired end reads. Given an assumed mean minor VSG frequency of 1e-4 and dominant VSG at 99%, we can expect to recover ~100 unique active VSGs per patient, and ~1,000 active VSGs per sequencing run, at 50x coverage. Given 200 patients over 20 sequencing runs, a database of 20,000 VSGs could be recovered for analysis.

Aim 2: Recover cross-reactive anti-VSG antibodies from human patients.

A) Generate protein panel of VSGsB) Test serum of many infected peopleC) Single cell isolate B-cells and test monoclonal VDG reactivity profilesD) Epitope mapping

1800 patients -> 1 primary candidate -> 30,000 single cells -> 2 bnAbs

Significance

① Database for analyzing VSG Diversity dynamics② Reagents for analyzing VSG antigens③ Practical outcome for vaccine design④ Fundamental implications for immune evasion

Take-away

Pathogens use immunodominance and antigenic drift to rig the B-cell activation race so that the losers always win.