Isolation of a Broadly Neutralizing Antibody with Low Somatic Mutation from a Chronically Infected HIV-1 Patient Amanda Fabra García, Carolina Beltrán Pavez, Alberto Merino Mansilla, Cristina Xufré, Isabel Crespo, Josep M Gatell, Felipe García, Eloísa Yuste, Víctor Sánchez Merino. AIDS research group: Retrovirology and Viral Immunopathology Laboratory. IDIBAPS/Hospital Clínic. Barcelona The AIDS Immunopathology Unit. National Center of Microbiology. National Institute of Health Carlos III (ISCIII)
Broadly Neutralizing Antibodies (BnAbs) against HIV-1 BnAbs are capable to neutralize diferent HIV-1 isolates from differents clades. Unmutated Have high SHM Common Between Ancestor 10% and 50% of individuals, depending upon the but definition are not broad of (Germnline) potency and breadth, develop a broadly neutralizing capacity against diverse HIV-1 strains. Burton, D. R., & Hangartner, L. (2016). Annual rev Time of immunol). BnAbs are able to PREVENT and CONTROL infection in animal models. Somatic Hypermutation (SHM) (Gautam R, Nature 2016 ) bnabs mature to Certain neutralizing antibodies are able to CONTROL recognize infection multiple in Clinical viral Trials. (Caskey, Nature 2016; Scheid, Nature 2016; Bar, N Engl J Med 2016) variants Dead-end antibodies do not evolve They cannot tolerate viral escape mutations ANTIBODY BREAD/POTENCY 0-2 year 2-4 years Moore P et al. Immunol Rev 2017
Broadly Neutralizing Antibodies Characteristics V1/V2 CD4bs V3 gp120 gp41 gp120- gp41 interface V1/V2 CD4 V3 N-glycans binding-site MPER region epitopes directed High Long SHM gp120-gp41 HCDR3 (Somatic Autoreactivity long HCDR3 (heavy interface High Mutation) chain complementarity-determining (PGT151, High 35022, SHM SHM 8ANC195) region 3) (b12, VRC01, VRC07, (2G12, (PG16,PG9, 10-1074, 3BNC117, (10E8, 4E10, PGDM1400) PGT121, PGV04) 2F5) PGT128, PGT135) MPER region Viral membrane HIV-1 envelope glycoprotein (Env) is the sole target of bnabs
Usually bnabs have long HCDR3. High SOMATIC HYPERMUTATION (SHM). BnAbs have long maduration periods with the antigen Difficult to reproduce through conventional vaccination High SHM: Anti-Antibody responses
Vector-mediated gene transfer: long-term systemic production of bnabs rhesus monkeys study SHM SHM Distance from germline (High SHM) correlates with the magnitude of the anti-antibody response Martinez-Navio, et al. Molecular Therapy 2016
Our work focus Viral load 100% Non-neutralizing antibodies until 50% Broadly Neutralizing Antibodies(bNAbs) T CD4 + cells CD4 T cells 100% Autologous Neutralizing Antibodies Adapted from Euler et al, Front Immunol. 2012
Main objetive Isolation and characterization of antibodies against HIV-1 Env in the patients previously described
Isolation of individual Env-specific B cells We have adapted to the laboratory a method described in 2008 by Dr. Nussenzweig's group
Sorting of Env-specific B cells 293F co-transfection: + GFP Env+ HIV-1 63,5% 293F B cells PBMCS from patients with broadly neutralizing response CD19+ IgG+ Cytometry Plataform IDIBAPS In colaboration with Dr. Cristina Xufré CD19+ IgG+ Klein et al,. Science 2012; 209: 1469-1479 FACS sorting doblets
Env+ IgG+ Gating Strategy Env-GFP+ IgG-APC+ CD19-PerCP Cy5.5+ CD19+
Cell Doublets Amnis Imaging Flow Cytometers - EMD Millipore
Antibody production CD19+ IgG+ Cell doublets sorting Single B-cell Variable region-igg RT-PCR IgH Ig Ig Cloning VH DH JH V J V J 1 HC Constant region 1 Human LC Constant region 1 Human LC Constant region 1 Human Nussenzweig s plasmids H + L co-transfection (293F cell) Ab purification https://en.wikipedia.org/
We have found 3 gp120 +IgGs from chronic patients rgp120 AC10.0 ELISA
We have got one Broadly Neutralizing Antibody The antibody A7-572.006 is capable to neutralize 5 virus from the minipanel Virus Minipanel Virus Subtype Tier Tropism J G F1 F2 92UG024 (D) VSV H - - - D CM244 AE 2 K NL43(B) CCR5 CM 224(AE) AC10 B 2 CCR5 B AC10(B) A2 V1 191(A) 92BR025(C) 92UG024 D 2 C CXCR4 A1 NL4.3 B 1A CXCR4 92BR025 C 1B CCR5 VI191 A 2 CCR5 IC 50 N 0.1 O Medina-Ramírez M et al., J virol. (2011);12:5804-5813
Epitope mapping (in progress) Recognizes rgp120 (MPER negative) Recognizes a conformational epitope (SDS-PAGE negative) Does not recognizes the CD4bs RSC3 RSC3 P363N RSC3 G367R A 7 V R C 0 1 4 E 1 0 4 4 4 3 3 3 2 2 2 1 1 1 0 1 0 3 1 0 2 1 0 1 1 0 0 1 0-1 1 0-2 Dra. Nuria Gonzalez 1 0-3 A7-572.006 0 1 0 1 1 0 0 1 0-1 1 0-2 1 0-3 1 0-4 Concentration (µg/ml) 1 0-5 0 1 0 1 1 0 0 1 0-1 1 0-2 1 0-3 1 0-4 1 0-5
% Infectivity Patient 572.006 cart 572-006 patient 1000 100 10 VSV CM244 AC10 92UG024 NL4-3 92BR025 V1 191 1 1000 100 Purified IgG g/ml 10 1 Patient on ART Low viral load 4.5 years A7-572.006 isolation Serum crosses 4 subtypes
A7-572-006 genetic characterization 3BNC117 VRC01 Known bnabs A7-572.006 V-gene hypermutation: 7.9 and 6.7 % for the heavy and light chains respectively (average chronic babs 21% and 15%) Infant bnabs displayed low levels of somatic mutations (2.0%-6.6%) Simonich, Cassandra A., et al. Cell 166.1 (2016): 77-87. A7-572-006 Close to the germ line: low Somatic Hypermutation
Conclusions We have isolated a new bnab with a low level of somatic hypermutation, A7-572.006, from a patient with chronic HIV-1 infection. Isolation of bnabs with low hypermutation is feasible and they could be very interesting for their potential low ability to induce anti-antibody responses
Amanda Fabra García Eloísa Yuste Víctor Sánchez-Merino Alberto Merino Acknowledgment Nuria González José Alcamí Clinic Researchers: Josep Mª Gatell Felipe García Cytometry plataform: Cristina Xufré Isabel Crespo Collaborators: Anke Schultz Andreas Meyerhans