1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch

  1. Zijun Wang
  2. Christopher O Barnes
  3. Rajeev Gautam
  4. Julio C Cetrulo Lorenzi
  5. Christian T Mayer
  6. Thiago Y Oliveira
  7. Victor Ramos
  8. Melissa Cipolla
  9. Kristie M Gordon
  10. Harry B Gristick
  11. Anthony P West
  12. Yoshiaki Nishimura
  13. Henna Raina
  14. Michael S Seaman
  15. Anna Gazumyan
  16. Malcolm Martin
  17. Pamela J Bjorkman
  18. Michel C Nussenzweig
  19. Amelia Escolano  Is a corresponding author
  1. Laboratory of Molecular Immunology, The Rockefeller University, United States
  2. Division of Biology and Biological Engineering, California Institute of Technology, United States
  3. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
  4. Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, United States
  5. Howard Hughes Medical Institute. The Rockefeller University, United States
Research Article
Cite this article as: eLife 2020;9:e61991 doi: 10.7554/eLife.61991
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Broadly neutralizing antibody isolated from a SHIVAD8-infected rhesus macaque.

(A) FACS plot showing germinal center B cells that bind to YU2 gp140-F and BG505 SOSIP from a lymph node collected from macaque CE8J at week 115 after SHIVAD8 infection. The gate shows the sorting window. The B cell carrying the isolated bNAb (Ab1485) is highlighted in red. (B) Graph shows the binding of several monoclonal antibodies isolated from macaque CE8J to BG505 SOSIP (Antibodies were tested for binding to BG505 two or three times. Graph shows data from a representative ELISA). Data is shown as area under the ELISA curve (AUC). (C and D) Table shows the neutralization activity of Ab1485 and other human V3-glycan bNAbs determined in TZM-bl assays against a panel of 42 multi clade tier 1B and tier two pseudoviruses (C) and replication-competent SHIVAD8 (D).

Figure 1—figure supplement 1
Antibodies isolated from a SHIVAD8-infected rhesus macaque.

(A) FACS plots show the gating strategy used to isolate single germinal center B cells that bind to YU2 gp140-F and BG505 SOSIP from a lymph node sample collected from the elite neutralizer macaque CE8J. The sorted population of B cells is highlighted in red. (B) Pie chart shows the clonal analysis of antibodies cloned from macaque CE8J. Expanded clones are represented in colored slices while singles are shown in white. The number of analyzed sequences is shown in the middle of the pie chart. (C) Graph shows the number of nucleotide mutations in the VH, VL, and VK genes of the antibodies shown in B. (D) Length of CDRH3 for antibodies shown in B and C. The red dot corresponds to Ab 1485. (E) Neutralization activity of Ab1538, Ab1542, and Ab1485 determined in TZM-bl assays against a panel of 7 tier 1B and tier two pseudoviruses. (F) Sorting strategy used in an attempt to isolate Env-specific B cells from a PBMC sample collected from macaque CE8J at week 38 post-SHIVAD8 infection.

Mapping of Ab1485 binding to Env.

(A) Description of Ab1485. (B) Representative ELISA graph showing binding of Ab1485 to BG505 in competition with antibodies that target the V3-glycan epitope (10–1074), the CD4- binding site (3BNC117), the gp120-gp41 interface (8ANC195), the apex (PG9) or the fusion peptide (VRC34) and in the absence of a competing antibody (n = 3). (C) Table shows the neutralization activity of Ab1485 determined in TZM-bl assays against a JRCSF pseudovirus and a series of JRCSF mutants that affect the binding of human bNAbs to the interface (N611D), the CD4-binding site (T278A+ A281T), the apex (N160K), the MPER (F673L), and the fusion peptide (N88Q).

Figure 3 with 2 supplements
Cryo-EM reconstruction of the Ab1485-BG505 complex reveals a distinct Env-binding orientation relative to human bNAbs.

(A) Cryo-EM map of the BG505 SOSIP.664 trimer bound to three Ab1485 (purple shades) and three 8ANC195 (orange shades) Fabs. Densities for glycans are colored in dark teal. (B) Cartoon depiction of the modeled complex with a close-up view of the Ab1485 Fab – gp120 interface. Conserved regions of the V3-epitope are highlighted. (C) Cartoon and stick representation of the Ab1485 CDRH3 recognition of the N332gp120-glycan. Reconstructed cryo-EM map shown as a mesh, contoured at three sigma. (D–F) Comparison of Ab1485’s (purple) Env-binding orientation to (D) PGT128 (teal, PDB 5ACO), (E) BG18 (orange, PDB 6CH7), and (F) 10–1074 (blue, PDB 5T3Z).

Figure 3—figure supplement 1
Cryo-EM data processing and validation.

(A) Representative micrograph and 2D class averages shown for Ab1485-BG505-8ANC195 complex data. (B) Angular distribution, (C) Gold-standard Fourier shell correlation plot, with dashed lines showing resolutions at 0.5 and 0.143. (D) Local resolution as determined by ResMap depicted on side and top views of reconstructed volume. (E) Representative density contoured at three sigma for different regions of the complex.

Figure 3—figure supplement 2
Binding assays and structural comparison to V1//V3 antibodies raised in animals.

(A–C). SPR sensorgrams for binding of Ab1485 Fab to immobilized BG505 (A), RC1 (B), or RC1-glycan KO 324GAIA327 (C). Sensorgrams traces are shown in colors; the fits to a 1:1 binding model are shown in black. (D) Comparison of Ab1485 (purple shades) Env-binding orientation to antibodies 874NHP (PDB: 6ORN; orange), 897NHP (PDB: 6ORO; green), and 43A2rabbit (PDB:6VO0; blue). Comparison with the predominant V1/V3 binding orientation observed after repeated challenge with SHIVBG505 is also shown (modeled Fab (olive)) based on cryo-EM density (EMD-20396).

Molecular details of Ab1485-gp120 interactions.

(A) Sequence alignment of mature Ab1485 heavy chain with germline VH4-2*01M. Paratope residues are highlighted. (B–D) Comparison of the paratope CDR loops and FWRs involved in epitope recognition for (B) Ab1485, (C) PGT128, and (D) 10–1074. (E) Stick representation of interactions between Ab1485 (purple) and either the GDIR peptide motif (red) or V1 loop (green). Potential H-bonds defined are shown as black dashes.

Figure 5 with 1 supplement
Ab1485 protects macaques against a high dose intrarectal challenge with SHIVAD8.

(A) Diagrammatic representation of the regimen used to assess the protective efficacy of Ab1485. Macaques were administered with Ab1485 at a dose of 10 mg kg−1 and challenged one day later with 1000 TCID50 of SHIVAD8 intrarectally (I.R.) (B) Longitudinal analysis of plasma viral loads in two control macaques (FZH and JG7) receiving no Ab and four macaques (DH18, DH27, DH29, and DHAP) infused with Ab1485 24 hr prior to SHIVAD8 challenge. (C) Serum neutralizing antibody titers in macaques receiving Ab1485. The IC50 titers are color coded: 1:21–99 in green; 1:100–999 in yellow and ≥1:1000 in red.

Figure 5—figure supplement 1
Characterization of Ab1485.

(A) ELISA graphs showing binding of Ab1485 to lipopolysaccharide (LPS), Keyhole limpet hemocyanin (KLH), Insulin, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Antibody ED38 (Barouch et al., 2013; Nishimura et al., 2017) and antibody mGO53 (Barouch et al., 2013) were used as positive and negative controls, respectively (n = 1). (B) Graph shows the antibody concentrations of Ab1485-macaque-LS and the control 10–1074 in the serum of mice that carry a null mutation for the mouse neonatal Fc receptor (FcRn) and a transgene for the human FcRn at different time points after intravenous infusion of the antibodies (Representative experiment. n = 2).

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
strain, strain background (Mus musculus Female)B6. Cg-Fcgrttm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ mice (FcRn -/- hFcRn)The Jackson Laboratory0049197–8 weeks of age
cell line (human)HEK293-6ENational Research Council of CanadaRRID:CVCL_HF20
Cell line (human)Expi293F cellsInvitrogenA14527
cell line
(CHO)
CHO Flp-InTM cellsInvitrogenR75807Chung et al., 2014
PMID:24767177
Recombinant proteinAvi-tagged BG505 SOSIPKindly provided by Dr. Rogier W. Sanders and Dr. Marit van Gils.Sok et al., 2013
PMID:25422458
Recombinant proteinAvi-tagged YU2-gp140 fold-onKindly provided by Dr. R. Wyatt
Recombinant proteinAvi-tagged Hepatitis B surface antigenProtein Specialistshbs-875
Recombinant proteinAvi-tagged RC1Escolano et al., 2019
PMID:31142836
Recombinant proteinAvi-tagged RC1_KOEscolano et al., 2019
PMID:31142836
Recombinant proteinBG505 SOSIP.664 trimerSchoofs et al., 2019
PMID:31126879
Commercial assay or kitZombie NIR Fixable Viability KitBiolegend77184(1:400)
Commercial assay or kitTCL lysis bufferQiagen1031576
Commercial assay or kitRNAClean XPBeckman CounterA63987
antibodyanti CD3-APC-eFluor 780 (mouse monoclonal)Invitrogen47-0037-41(1:200)
antibodyanti CD14-APC-eFluor 780 (mouse monoclonal)Invitrogen47-0149-42(1:200)
antibodyanti CD38 FITCStem Cell60131FI(1:200)
antibodyanti CD16-APC-eFluor 780 (mouse monoclonal)Invitrogen47-0168-41(1:200)
antibodyanti CD8-APC-eFluor 780 (mouse monoclonal)Invitrogen47-0086-42(1:200)
antibodyanti CD20-PE-Cy7 (mouse monoclonal)BD Biosciences335793(1:200)
antibodyHRP-conjugated anti-human IgG (Fc) CH2 Domain antibody (mouse monoclonal)Bio-RadMCA647P(1:5000)
software, algorithmWinNonlin 6.3Certara Software
software, algorithmMacVector v.17.0.2MacVector
software, algorithmFlowJo v.10.6.1Becton Dickinson
software, algorithmFCS EXPRESSDe Novo
software, algorithmGraphPad Prism 7GraphPad
software, algorithmSeqIOBiopython
software, algorithmcutadapt v.2.3cutadapt
Software, algorithmChange-O toolkit v.0.4.5PMID:26069265
software, algorithmSerialEM v3.7RRID:SCR_017293Mastronarde, 2005
PMID:16182563
software, algorithmcryoSPARCv2.14RRID:SCR_016501Punjani et al., 2017
PMID:28165473
software, algorithmRelion v3.0RRID:SCR_016274Zivanov et al., 2018
PMID:30412051
software, algorithmUCSF Chimera v1.13RRID:SCR_004097Goddard et al., 2007, PMID:16963278
software, algorithmPhenix v1.17RRID:SCR_014224Afonine, et al, ACTA D, 2018,
PMID:29872004
software, algorithmCoot v0.8.9RRID:SCR_014222Emsley et al., 2010,
PMID:20383002
software, algorithmPyMOL v1.8.2.1Schrodinger, IncRRID:SCR_000305https://pymol.org/2/
software, algorithmBiacore T200 Evaluation Software v3.2Cytiva

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