Recombinant Anti-HIV ENV VHH Single Domain Antibody (PNBL-020)

sdAbs binding to gp120 (bound via the D7324 antibody to epoxybeads) or to gp140 (directly immobilized on epoxybeads) were detected via a peroxidase labeled anti-c-myc Ab. Histograms correspond to the three main families of sdAbs selected. JM3, JM4 and JM5 bind both Env forms while JM7 only binds to the cross-linked Env.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.

Each sdAb was tested in a single-round neutralization assay as described in the "Materials and Methods" section using TZM-bl cells as target cells. Pseudovirions (100 TCID50) carrying various envelope glycoproteins from different HIV-1 subtypes were pre-incubated in 96-well plates for 1 h at 37°C with purified sdAbs at 10 µM.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.

10 µg/ml of sdAbs, X5 and b12 were added to immobilized Env as indicated in the "Materials and Methods". After 1 h, phage-sdAbs were added at subsaturating concentration. Bound phage-sdAbs were revealed using peroxidase labeled anti-M13 antibody.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.

10 µg/ml of sdAbs, X5 and b12 were added to immobilized Env as indicated in the "Materials and Methods". After 1 h, phage-sdAbs were added at subsaturating concentration. Bound phage-sdAbs were revealed using peroxidase labeled anti-M13 antibody. Competition was done on gp140-S-S-M64U1.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.

sdAbs were immobilized on sensorchip CM5. Using a Biacore 3000 instrument, Env was injected at various concentrations on different channels. Uncomplexed gp140 and gp140 + M48U1 complex were run over immobilized JM4, JM5 and JM7.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.

sdAb was assessed for its ability to bind WT Env (pink line), CD4BS mutant D368R (orange line), CoRBS mutant I420R (blue line) and triple mutant I423M, N425K and G431E (green line), in the presence of miniCD4 M48U1, except for JM2.

Matz, J., Kessler, P., Bouchet, J., Combes, O., Ramos, O. H. P., Barin, F., ... & Chames, P. (2013). Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120. Journal of virology, 87(2), 1137-1149.