Structural Insights into HIV-1 Neutralization by Broadly Neutralizing Antibodies PG9 and PG16 Robert Pejchal, Laura M. Walker, Robyn L. Stanfield, Wayne C. Koff, Sanjay K. Phogat, Pascal Poignard, Dennis R. Burton and Ian A. Wilson Session 05: Novel Monoclonals and Structural Insights AIDS Vaccine 2010, Atlanta wilson@scripps.edu Title
PG9 and PG16 recognize a V2/V3 epitope on the HIV-1 Env trimer PG9 neutralizes 87% of a panel of 82 viruses (M. Seaman, unpublished data) IC 50 ~< 0.1 ug/ml Novel epitope centered on V1/V2 stem, V2 and V3 (especially for PG16) No structural information for epitope, but can localize near apex of trimer Cryo-ET reconstruction density and modeling based on Liu et al. 2008 Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW; Protocol G Principal Investigators, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR. (2009) Science 326:285-9 Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target
PG16 crystal structure reveals a novel hammerhead subdomain for its 28-residue CDR H3 R. Pejchal, L. M. Walker, R. L. Stanfield, S. K. Phogat, W. C. Koff, P. Poignard, D. R. Burton & I. A. Wilson (2010). PNAS 107:11483-88 Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1
Crystal structure of the antigen-binding fragment (Fab) of antibody PG16 Pancera, M. et al. & P.D. Kwong 2010. J. Virol. 84:8098-8110
PG16 crystal structure reveals a novel, sulfated hammerhead subdomain for its 28-residue CDR H3 Hammerhead subdomain protrudes 20Å from combining site Sulfated tyrosine (TyrH100H) provides 10-fold boost in neutralization potency (PG9 hypersulfated) R327A (and bridging sheet residues) knockdown suggests involvement of a CoRbs element R. Pejchal, L. M. Walker, R. L. Stanfield, S. K. Phogat, W. C. Koff, P. Poignard, D. R. Burton & I. A. Wilson (2010). PNAS 107:11483-88 Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1
Structure of 412d with YU2 gp120 (core+v3) Huang CC, Lam SN, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewley CA, Kwong PD. Science 317:1930-34 (2007)
Variable loop in CDR H3 determines fine specificity of PG9 and PG16 * Tyrosine sandwich stalk stabilizes protrusion of H3 hammerhead 7-residue H3 region varies between PG9 and PG16; forms anti-parallel beta hairpin H3 swap experiment switches PG9 and PG16 neutralization and binding specificity R. Pejchal, L. M. Walker, R. L. Stanfield, S. K. Phogat, W. C. Koff, P. Poignard, D. R. Burton & I. A. Wilson (2010). PNAS 107:11483-88 Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1
Effect of CDR H3 mutation on PG9 neutralization and binding to DU422 gp120 5 Tyr residues in PG9 CDR H3: 100A, 100E, 100G, 100H (sulfated in PG16), and 100K Mutation of 100G and 100H more disruptive than 100A, 100E, and 100K R. Pejchal, L. M. Walker, R. L. Stanfield, S. K. Phogat, W. C. Koff, P. Poignard, D. R. Burton & I. A. Wilson (2010). PNAS 107:11483-88 Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1
Sulfoforms of PG9 CDR H3 Tyr->Phe mutants identified by ESI-MS Purified Tyr->Phe mutants were fractionated by Mono S and eluted fractions were analyzed by ESI- MS Rob Pejchal et al. P04.59 LB Doubly sulfated forms were not detected for 100G and 100H mutants supporting 100G and 100H as natural sulfated sites
PG9 homology model Tys H100H Tys H100G Homology model composed of PG9 V L (PDB:3MUH) and PG16 V H (PDB:3MUG) V H V L Tyrosine sulfation based on mass spec data for Tyr->Phe mutants The two sulfated residues are in close proximity Robert Pejchal
Revisiting the role of tyrosine sulfation in neutralization potency Co-expression of human tyrosyl protein sulfotransferase promotes hypersulfation (up to quintuple), suggesting all five Tyr in the CDR H3 are capable of being sulfated PG9-S2 achieves optimal neutralization potency >10X higher than hyposulfated forms Rob Pejchal et al. P04.59 LB
Sensitivity of PG9 and PG16 neutralization to substitutions eliminating N-linked glycosylation sites * * * N156 and N160 in V1/V2 stem and V2 are important for forming the PG9/16 epitope K. Doores & D. R. Burton (2010). J. Virol, 84:10310-21 Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16
Neutralization activity of MAbs against JR-CSF pseudoviruses using glycosidase inhibitors Expression of trimer in the presence of kifunensine blocks neutralization K. Doores & D. R. Burton (2010). J. Virol, 84:10310-21 Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16
wilson@scripps.edu Acknowledgements Wilson Lab Robert Pejchal Yuanzi Hua Dennis Burton Laura Walker Katie Doores Robyn Stanfield IAVI Sanjay Phogat Wayne Koff Peter Kwong, VRC Crystallization Robot Marc Elsliger David Marciano Henry Tien Rob Pejchal et al. P04.59 LB Rob Pejchal et al. P04.59 LB Rob Pejchal et al. P04.59 LB Funding: IAVI, NIH, NIAID NIGMS, Skaggs Institute for Chemical Biology
IAVI Neutralizing Antibody Consortium
CCR5 N-term binding: results Best Solution Docking energy -15.48 kcal/mol Priyamvada Acharya Huang CC, Lam SN, A, Bewley CA, Kwong PD, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewley CA, Kwong PD. Science 317:1930-34(2007)
CCR5 N-terminus versus 412d Arg 327 STY10 (CCR5) vs STY 104 (412d) STY14 (CCR5) vs STY 107 (412d) Precise chemical mimicry found at energetically important sulfated tyrosine residues: confirmed by mutagenesis
Structure of 412d with YU2 gp120 (core+v3) CD4 gp120 412d heavy chain V3 loop Buried STY 107 412d light chain Huang CC, Lam SN, A, Bewley CA, Kwong PD, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewley CA, Kwong PD. Science 317:1930-34(2007)
Acknowledgements Ian Wilson Rob Pejchal Yuanzi Hua Robyn Stanfield Dennis Burton Laura Walker Katie Doores IAVI NAC Pascal Poignard IAVI Sanjay Phogat Wayne Koff Peter Kwong Funding NIH NIAID IAVI
Neutralization of glycan mixed trimer viruses by PG9, PG16, and 2G12 Cells transfected with various proportions of JR-FL E168K (suboptimal) and JR-FL E168K N189A (optimal) show only one optimal monomer is required per trimer to reach maximum neutralization Consistent with previous data demonstrating binding of PG9/16 to a single gp120 monomer K. Doores & D. R. Burton (2010). J. Virol, 84:10310-21 Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16
Neutralization activity of MAbs against 92RW020 and SF162 K160N pseudoviruses when made in the presence of glycosidase inhibitors K. Doores & D. R. Burton (2010). J. Virol, Variable Loop Glycan Dependency of the Broad and Potent HIV-1-Neutralizing Antibodies PG9 and PG16