Isolation of a Broadly Neutralizing Antibody with Low Somatic Mutation from a Chronically Infected HIV-1 Patient
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1 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)
2 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
3 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, , 3BNC117, (10E8, 4E10, PGDM1400) PGT121, PGV04) 2F5) PGT128, PGT135) MPER region Viral membrane HIV-1 envelope glycoprotein (Env) is the sole target of bnabs
4 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
5 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
6 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
7 Main objetive Isolation and characterization of antibodies against HIV-1 Env in the patients previously described
8 Isolation of individual Env-specific B cells We have adapted to the laboratory a method described in 2008 by Dr. Nussenzweig's group
9 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: FACS sorting doblets
10 Env+ IgG+ Gating Strategy Env-GFP+ IgG-APC+ CD19-PerCP Cy5.5+ CD19+
11 Cell Doublets Amnis Imaging Flow Cytometers - EMD Millipore
12 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
13 We have found 3 gp120 +IgGs from chronic patients rgp120 AC10.0 ELISA
14 We have got one Broadly Neutralizing Antibody The antibody A 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:
15 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 E Dra. Nuria Gonzalez A Concentration (µg/ml)
16 % Infectivity Patient cart patient VSV CM244 AC10 92UG024 NL4-3 92BR025 V Purified IgG g/ml 10 1 Patient on ART Low viral load 4.5 years A isolation Serum crosses 4 subtypes
17 A genetic characterization 3BNC117 VRC01 Known bnabs A 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 (2016): A Close to the germ line: low Somatic Hypermutation
18 Conclusions We have isolated a new bnab with a low level of somatic hypermutation, A , 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
19 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
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