Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

  1. Lynn Morrisa,b,f
  1. aCentre for HIV and STIs, National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg, South Africa
  2. bFaculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
  3. cVaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
  4. dInstitute of Infectious Disease and Molecular Medicine and Division of Medical Virology, University of Cape Town and National Health Laboratory Service, Cape Town, South Africa
  5. eSouth African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa
  6. fCentre for the AIDS Programme of Research in South Africa, University of KwaZulu-Natal, Durban, South Africa
  7. gDepartment of Epidemiology, Columbia University, New York, New York, USA
  1. F. Kirchhoff, Editor
  1. Ulm University Medical Center
  1. FIG 1
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    FIG 1

    Resurfaced RHPA gp120 antigens bind differentially to CAP257 CD4bs plasma antibodies. (A) Surface view of a modeled RHPA gp120 core showing the location of N/C-terminal truncations; deleted V1, V2, or V3 loops; resurfacing mutations; and the N276D/T278A, N279D, and R456W escape mutations, relative to the CD4 binding site. Hypervariable regions α2, β14, V4, and V5 were not resurfaced. (B) Comparison of the abilities of longitudinal donor CAP257 plasma samples to bind to the newly designed gp120 core sorting antigens RHPA-RC and RHPA-ADW by ELISAs and neutralization of the heterologous tier 2 strain RHPA or the RHPA-ADW mutant variant resistant to CAP257 CD4bs antibodies. The lack of binding to RSC3 is shown in gray. Absorbance readings are plotted on the left y axis, neutralization titers are plotted on the right y axis, and time course postinfection is plotted on the x axis. The vertical dotted line indicates the 122-week time point at which the adsorption experiments described below for panel C were performed. (C) Adsorption of CAP257 peak CD4bs neutralizing titers by the positive gp120 antigen RHPA-RC but not the negative gp120 antigen RHPA-ADW or uncoated beads. Neutralization of four HIV-1 strains by donor CAP257 plasma that had been adsorbed with blank beads or beads coated with the positive gp120 antigen RHPA-RC or the negative gp120 antigen RHPA-ADW is plotted on the y axis as percent inhibition. Serial dilutions of the adsorbed plasma are indicated on the x axis.

  2. FIG 2
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    FIG 2

    Isolation of an early N276 glycan-dependent CD4bs neutralizing antibody. (A) Sort plot showing the percentage of identified memory B cells that were able to bind the positive sorting antigen RHPA-RC (fluorescence plotted on the y axis) but not to the negative sorting antigen RHPA-ADW (fluorescence plotted on the x axis). (B) Properties of the CAP257-RH lineage members, defined by the International Immunogenetics database (IMGT) and numbered according to Kabat numbering. (C) Comparison of the binding of CAP257-RH1 or HJ16 to wild-type RHPA core gp120 (no symbols) as well as to the positive gp120 sorting antigen RHPA-RC or the negative gp120 sorting antigen RHPA-ADW by ELISAs. Absorbance is plotted on the y axis versus the antibody concentration on the x axis. (D) Neutralization of RHPA and select mutants with various levels of resistance to CAP257 CD4bs plasma antibodies by either donor CAP257 plasma at 107 weeks postinfection or the CAP257-RH1 monoclonal antibody isolated at the same time point. Percent neutralization is plotted on the y axis, and the dilution is plotted on the x axis. (E) ELISA binding of an anti-HIV immunoglobulin pool (positive control) or monoclonal antibody CAP257-RH1 to 11 clade C gp120 proteins. (F) Neutralization of seven heterologous viruses sensitive to early CAP257 CD4bs antibodies by either donor CAP257 plasma at 107 weeks postinfection (p.i.) or monoclonal antibody CAP257-RH1. Increasing potency is indicated by warmer colors.

  3. FIG 3
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    FIG 3

    Crystal structure of CAP257-RH1 bound to the N276 glycan in RHPA gp120. (A) Overview of the CAP257-RH1 Fab and RHPA core gp120 cocrystal structure shown in a cartoon view. The truncated N/C termini as well as the V3 loop stem are labeled. Glycans are shown in stick view with green semitransparent surfaces. The N276 glycan is indicated by its lighter shade. (B) Surface view of the HIV-1 Env trimer (PDB accession no. 4TVP) with monoclonal antibody CAP257-RH1 docked into its epitope (by superimposition). The location of the viral membrane is indicated. (C) Surface view of RHPA gp120. Loop D and V5 are indicated, and the N276 glycan (shown in stick-and-surface view) is outlined. (D) Surface view of RHPA gp120 with the CD4 binding footprint. Loop D, V5, and the N276 glycan are indicated as described above for panel C. The CAP257-RH1 CDRs are shown as thick blue cartoon loops. For clarity, the protein chain is indicated in subscript (heavy chain, light chain, or gp120).

  4. FIG 4
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    FIG 4

    Analysis of the N276 glycan bound by neutralizing antibodies. (A) CAP257-RH1 shown in cartoon view, as described in the legend of Fig. 3, interacting with the N276 glycan (green sticks). The 2Fo − Fc electron density map (contoured at 1 σ) of the N276 glycan is shown with black mesh. Additional mannose moieties added to the glycan termini are shown in lime green, and the glycan arms are labeled. A schematic of the glycan is also shown at the bottom, and sugars with visible density or those modeled onto the structure are labeled. (B) Comparison of the orientations of the N276 glycan bound by 8ANC195 (with Fab heavy and light chains in peach and red, respectively), CAP257-RH1 (with Fab heavy and light chains in blue and cyan, respectively), or VRC01-like antibodies (with Fab heavy and light chains in purple and pink, respectively). The Fab-bound glycan complexes (PDB accession no. 4P9H and 4JKP) were superimposed onto the HIV-1 Env trimer structure reported under PDB accession no. 4TVP and are shown in light gray (gp120) and dark gray (gp41) surface views. (C, left) Surface view of the N276 glycan bound by CAP257-RH1 (colored as for panel A). (Right) HJ16 cocrystal structure (PDB accession no. 4YE4) superimposed onto CAP257-RH1-bound gp120 showing compatibility with this N276 glycan orientation.

  5. FIG 5
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    FIG 5

    Atomic details of the CAP257-RH1 interaction with RHPA core gp120. (A) Polar contacts between CAP257-RH1 and the CD4bs/N276 glycan. The CAP257-RH1 CDRs are shown in various shades of blue, while the CD4b binding loop, loop D, and the N276 glycan are shown in gray, purple, and green, respectively. Potential hydrogen bonds are shown as black dotted lines. (B) Paratope mapping of the CAP257-RH1 heavy chain by alanine scanning mutagenesis. Mutations are grouped by CDR loop using different shades of blue. Percent inhibition is plotted on the y axis, and the antibody concentration is plotted on the x axis. (C) Paratope mapping of the CAP257-RH1 light chain, as described above for panel B. (D) Atomic details of the interaction between CAP257-RH1 CDR-L1 (cyan) and the V5 loop of RHPA (red). Polar interactions are indicated as described above for panel A. (E) Neutralization of various RHPA alanine mutants, plotted as described above for panel B.

  6. FIG 6
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    FIG 6

    CAP257-RH1 neutralization is constrained by V5 glycosylation. (A) Comparison of the relative levels of penetration into the recessed CD4bs for various CD4bs neutralizing antibodies (crystal structures determined previously) superimposed onto the RHPA crystal structure, shown in surface view. The antibodies are grouped as CDR-H3 dominated or VH gene restricted and ordered according to increasing neutralization breadth. N276 glycan-dependent antibodies are indicated. For all antibodies, the light chains are shown in black, while the heavy chains are shown in various colors. (B) Comparison of the binding angles for the CD4bs antibodies shown in panel A relative to RHPA V5, with the CAP257-RH1-bound N276 glycan shown as sticks with a semitransparent surface. All antibodies are shown in cartoon view, with the heavy chains in white and the light chains colored according to neutralization breadth, as described above for panel A. Previously reported neutralization breadth estimates are shown. (C) Zoomed view of the glycan-free RHPA V5 loop shown as sticks with a transparent surface, with amino acids 460 to 464 being labeled. (D) Neutralization of RHPA and glycosylated V5 mutants by VRC01, HJ16, and CAP257-RH1. Percent inhibition is plotted on the y axis, and the antibody concentration is plotted on the x axis.

  7. FIG 7
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    FIG 7

    Rare glycan-free V5 sequences from CAP257 reveal a hole in the glycan shield. (A) Deep sequencing of autologous V5 loop sequences in donor CAP257, with the number of glycans on the y axis and weeks postinfection on the x axis. The relative frequencies of the number of glycans in V5 at a particular time point are represented by the size of the spheres, where larger spheres indicate a greater relative number of sequences at that particular time point that have the indicated number of glycosylation sequons in V5. The dotted vertical line indicates the time point at which CAP257-RH1 was isolated. (B) Relative number of glycan-free autologous V5 loops plotted as a percentage of the total sequences (y axis) over time (x axis). (C) Neutralization titers (plotted on the y axis) of the second wave of broadly neutralizing antibodies from donor CAP257 plasma (as described in reference 36), which target the CD4bs, over time (x axis). (D, left) The HIV-1 Env trimer is shown in a solvent-accessible surface representation (gray). The approximate location of the viral membrane is indicated. (Right) The glycan shield is modeled and displayed as the solvent-accessible surface of NAG2MAN9 glycan coverage based on two probe sizes of 1.4 Å (solvent radius) (top) and 10.0 Å (the estimated steric footprint of an antibody-combining region) (bottom), using the structure reported under PDB accession no. 4TVP with an additional glycosylation site modeled at residue 241. The solvent-accessible protein surface of V5 comprises a largely exposed region upon the deletion of the commonly glycosylated site at residue 462 (right).

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  1. Accepted manuscript posted online 31 August 2016, doi: 10.1128/JVI.01357-16 J. Virol. vol. 90 no. 22 10220-10235
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