Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

doi:10.1016/j.cell.2021.04.042

. 2021 May 27;184(11):2955-2972.e25.

doi: 10.1016/j.cell.2021.04.042. Epub 2021 May 20.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

Wilton B Williams¹, R Ryan Meyerhoff², R J Edwards², Hui Li³, Kartik Manne⁴, Nathan I Nicely⁴, Rory Henderson², Ye Zhou⁵, Katarzyna Janowska⁴, Katayoun Mansouri⁴, Sophie Gobeil⁴, Tyler Evangelous⁴, Bhavna Hora⁴, Madison Berry⁴, A Yousef Abuahmad⁴, Jordan Sprenz⁴, Margaret Deyton⁴, Victoria Stalls⁴, Megan Kopp⁴, Allen L Hsu⁶, Mario J Borgnia⁶, Guillaume B E Stewart-Jones⁷, Matthew S Lee⁸, Naomi Bronkema⁹, M Anthony Moody¹⁰, Kevin Wiehe², Todd Bradley⁴, S Munir Alam², Robert J Parks⁴, Andrew Foulger⁴, Thomas Oguin⁴, Gregory D Sempowski², Mattia Bonsignori², Celia C LaBranche¹¹, David C Montefiori¹², Michael Seaman¹³, Sampa Santra¹³, John Perfect¹⁴, Joseph R Francica⁷, Geoffrey M Lynn¹⁵, Baptiste Aussedat¹⁶, William E Walkowicz¹⁶, Richard Laga¹⁷, Garnett Kelsoe¹⁸, Kevin O Saunders¹⁹, Daniela Fera⁹, Peter D Kwong⁷, Robert A Seder⁷, Alberto Bartesaghi²⁰, George M Shaw³, Priyamvada Acharya²¹, Barton F Haynes²²

Affiliations

¹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA. Electronic address: wilton.williams@duke.edu.
² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA.
³ Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁴ Duke Human Vaccine Institute, Durham, NC 27710, USA.
⁵ Department of Computer Science, Duke University, Durham, NC 27708, USA.
⁶ Genome Integrity and Structural Biology Laboratory, NIEHS, NIH, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
⁷ Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA.
⁸ Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
⁹ Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, PA 19081, USA.
¹⁰ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA; Department of Pediatrics, Duke University, Durham, NC 27710, USA.
¹¹ Department of Surgery, Duke University, Durham, NC 27710, USA.
¹² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA.
¹³ Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
¹⁴ Department of Medicine, Duke University, Durham, NC 27710, USA.
¹⁵ Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA; Avidea Technologies, Inc., Baltimore, MD, USA.
¹⁶ Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
¹⁷ Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic.
¹⁸ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA.
¹⁹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA.
²⁰ Department of Computer Science, Duke University, Durham, NC 27708, USA; Department of Biochemistry, Duke University, Durham, NC 27705, USA; Department of Electrical and Computer Engineering, Duke University, Durham, NC 27708, USA.
²¹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA. Electronic address: priyamvada.acharya@duke.edu.
²² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA. Electronic address: hayne002@mc.duke.edu.

PMID: 34019795
PMCID: PMC8135257
DOI: 10.1016/j.cell.2021.04.042

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

Wilton B Williams et al. Cell. 2021.

. 2021 May 27;184(11):2955-2972.e25.

doi: 10.1016/j.cell.2021.04.042. Epub 2021 May 20.

Authors

Affiliations

¹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA. Electronic address: wilton.williams@duke.edu.
² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA.
³ Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁴ Duke Human Vaccine Institute, Durham, NC 27710, USA.
⁵ Department of Computer Science, Duke University, Durham, NC 27708, USA.
⁶ Genome Integrity and Structural Biology Laboratory, NIEHS, NIH, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
⁷ Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA.
⁸ Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA.
⁹ Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, PA 19081, USA.
¹⁰ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA; Department of Pediatrics, Duke University, Durham, NC 27710, USA.
¹¹ Department of Surgery, Duke University, Durham, NC 27710, USA.
¹² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA.
¹³ Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
¹⁴ Department of Medicine, Duke University, Durham, NC 27710, USA.
¹⁵ Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892, USA; Avidea Technologies, Inc., Baltimore, MD, USA.
¹⁶ Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
¹⁷ Institute of Macromolecular Chemistry, Czech Academy of Sciences, Prague, Czech Republic.
¹⁸ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA.
¹⁹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA.
²⁰ Department of Computer Science, Duke University, Durham, NC 27708, USA; Department of Biochemistry, Duke University, Durham, NC 27705, USA; Department of Electrical and Computer Engineering, Duke University, Durham, NC 27708, USA.
²¹ Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Surgery, Duke University, Durham, NC 27710, USA. Electronic address: priyamvada.acharya@duke.edu.
²² Duke Human Vaccine Institute, Durham, NC 27710, USA; Department of Medicine, Duke University, Durham, NC 27710, USA; Department of Immunology, Duke University, Durham, NC 27710, USA. Electronic address: hayne002@mc.duke.edu.

PMID: 34019795
PMCID: PMC8135257
DOI: 10.1016/j.cell.2021.04.042

Abstract

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (V_H) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without V_H-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM⁺IgD⁺CD27⁺, thus suggesting that they originated from a pool of antigen-experienced IgM⁺ or marginal zone B cells.

Keywords: FDG Abs; Fab dimerization; HIV-1 Env glycans; IgM-memory B cells; SARS-CoV-2 spike glycans; glycan-dependent Ab binding; marginal zone B cells; natural Abs.

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Conflict of interest statement

Declaration of interests B.A. and W.E.W. are co-founders of Chemitope Technologies, and G.M.L. is a founder of Avidea Technologies that now commercially produce peptides used in our HIV-1 vaccination regimen. The remaining authors declare no competing interests.

Figures

**Figure 1**
Characteristics of HIV-1 vaccine-induced Env glycan-reactive neutralizing B cell lineage, DH717 (A) Schedule of Man₉-V3 immunizations in RMs. (B) Plasma IgG at weeks (WK) post infection from four immunized RMs were tested in ELISA for binding to Man₉-V3. Data shown are representative from multiple assays and binding titer was reported as log area under the curve (AUC). Vertical dotted lines indicate immunization time points. (C) Representative flow cytometry sort plot of HIV-1 Env-reactive memory B cells detected at WK 26 and 73. Phylogram of V_H genes isolated from a clonal lineage of DH717 Abs found at WK 26 and 73. (D) NSEM 2D class averages for DH717.1–DH717.4, and HIV-1 bnAbs 2G12 and VRC01, are labeled I or Y according to shape. (E and F) Monomeric (E) and dimeric (F) Fabs are also shown. (G) Crystal structure of DH717.1 Fab monomer (V_H and V_λ in dark and light teal, respectively) bound to Man₉-V3. The terminal glycan-moieties of Man₉-V3 (gray sticks) bound a pocket formed by the CDRH1-3 loops (dark red, red and orange) of the DH717.1 Fab. Inset shows a zoomed-in view of glycan binding. (H) Crystal structure of DH717.1 dimer with dark teal or pink V_H, and light teal or pink V_λ. Inset shows a zoomed-in view of the inter-Fab disulfide (yellow spheres). (I) NSEM of cysteine to serine DH717.1 mutant (C74S). (J) DH717.1 wild-type and C74S mutant IgGs were tested for neutralization of *env*-pseudotyped HIV-1 bearing Envs with heterogeneous [−Kif] or Man₉-enriched [+Kif] glycans in TZM-bl cells in a single assay. Neutralization titers were reported as IC₅₀ in μg/mL. These Abs were also tested in a SPR assay for binding soluble stabilized forms of recombinant HIV-1 Env trimers (K); shown are SPR binding curves for each Ab. See also Figure S1 and Data S1, S3, and S4.

**Figure 2**
The ontogeny and dynamics of HIV-1 vaccine-induced DH717 FDG Abs (A) Absolute counts of unique V_H reads for blood-derived DH717 lineage members at longitudinal time points (weeks). ^∗p < 0.0001 (non-parametric Wilcoxon test) for increase in the distribution of estimated mutation frequencies at week 105 compared to week 145. The arrows indicate immunization time points with the initial Man₉-V3 monomer (black arrow), Man₉-V3 multimer (red arrow), nanoparticle (green arrow), and the first of five recombinant HIV-1 Env trimers (blue arrow). (B–D) Recombinant mAbs bearing the week 0 (DH717.5_NGS and DH717.6_NGS), near-germline (DH717UCA), and post-vaccination (DH717.1-DH717.4) Ab sequences were tested in ELISA for binding soluble stabilized form of recombinant HIV-1 Env trimer (B), Man₉-V3 (C), and heat-killed yeast antigens (D). CH65 was used as a negative control mAb, whereas biotinylated trimer-specific mAbs (see Figure S1), and peptide (DH1013)- and Env glycan (2G12)-specific mAbs were used as positive controls. ELISA data shown are averaged from 3–5 separate assays for each mAb tested; error bars represent SEM. Binding was measured at OD_450nm. (E) Binding of DH717 mAbs to individual glycans in a luminex binding assay, where glycan reactivity was reported as MFI after background subtraction. (F) Top panel: UMAP visualization of 23 transcriptionally unique immune cell clusters from peripheral blood. Bottom panel: clonally related DH717 B cells (shown in red dots within the rectangle outlines) detected among B cell clusters. (G) Heatmap summary of gene enrichment analysis across the 23 clusters; gene upregulation is shown in red compared to gene downregulation shown in blue. Shown are the top three genes identified for the DH717 B cell clusters 0 and 4. (H) Representative sort plots demonstrating IgM⁺IgD⁺CD27⁺ phenotype of a DH717 lineage B cell (blue dot) among antigen-specific B cells that were sorted (gray dots). The IgM sort plot displayed only the number of events from the IgD⁺CD27⁺ population as well as DH717.3 B cell. The contour plot (left) was used to establish the analysis gates. See also Data S5 and STAR Methods.

**Figure S1**
Characterization of HIV-1 vaccine-induced FDG Ab lineage, DH717, related to Figure 1 (A) DH717 mAbs as well as biotinylated (b) control mAbs were tested in ELISA for binding to soluble recombinant HIV-1 Env trimer (CH848 strain) in a single ELISA. Binding titers were reported as Log AUC. Control mAbs targeted different epitopes on HIV-1 Env: both Env glycan and peptide – PGT121, PGT125 and PGT128; linear peptide (V3 loop) reactive – 19B; glycan-only reactive – 2G12; co-receptor binding site – 17B; and CD4 binding site – DH493). (B) Binding titers of DH717 and control mAbs with heat-killed yeast antigens *Candida albicans* or *Cryptococcus neoformans*. MAbs were tested in standard diluent or diluent spiked with 0.5M D-mannose. Binding data were representative of duplicate ELISAs. (C) Kifunensine [Kif] enriches Man₉-glycans on recombinant HIV-1 Envs (Saunders et al., 2017a) (see STAR Methods). DH717 mAbs were tested for neutralization against a multi-clade panel of *env*-pseudotyped HIV-1 bearing Envs with heterogeneous [(-)Kif] or Man₉-enriched glycans [(+)Kif]. Neutralization was tested in TZM-bl cells in a single experiment and titers are reported as IC₅₀ in μg/ml. (D) Negative stain 3D reconstruction of DH717 I-shaped Ab. Map shown as transparent surface. Atomic model of two DH717.1 Fabs shown as ribbon diagrams, fit as rigid bodies into the 20-Å resolution NSEM map using UCSF Chimera’s automatic fitmap function. As fit, the V_H cysteine at residue 74 pointed toward one another (arrow) and the terminal sulfurs were 3.5 Å apart. (E) View of DH717.1 monomer crystal structure showed contact with aspartate, V_H residue 34 (D34), and the man-9 ligand (green); electron density shown as mesh. (F) V_H sequence alignment of DH717.1-4 with the inferred near-germline unmutated common ancestor (UCA). DH717.1, 0.2, and 0.4 have cysteine at residue 74, whereas DH717.3 and the UCA do not. (G) Close up views of DH717.1 dimer crystal structure, labeled by region. (H) Size exclusion chromatography (SEC) profile of DH717.1 C74S IgG. The SEC runs were performed on a Superose 6 Increase 10/300 column in running buffer composed of 10mM HEPES, pH 7.3, 150mM NaCl, 5% Glycerol. (I) Immobilization of DH717.1 wild-type (black lines) and DH717.1 C74S (gray lines) on anti-Fc surface. At least three technical repeats are shown for each. The Abs were captured on a CM5 chip by flowing 200 nM of the Ab over a flow cell immobilized with ~9000 RU of anti-human Fc Ab. Following this immobilization, binding was measured by flowing over 200 nM solution of Env in the running buffer HBS-EP+ that is composed of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20. Env binding was shown in Figure 1K.

**Figure 3**
Characteristics of HIV-1 Env glycan-reactive neutralizing Abs in the B cell repertoire of naive humans (A) Repertoire analysis of blood-derived Env glycan-reactive B cells in nine HIV-1 naive individuals. Enriched B cells were studied via different sort strategies listed that used fluorophore-labeled Man₉-V3 or soluble stabilized recombinant HIV-1 Env trimers. (B) Immunogenetics of candidate Env glycan-reactive B cells (N = 37) that were isolated from individuals listed in (A). (C) Repertoire of Env glycan-reactive B cells with IgM⁺IgD⁺CD27⁺ phenotype among six individuals. Flow cytometry data were analyzed from B cells sorted via strategies listed in (A); strategy “c” was used for LKP04. (D–F) NSEM 2D class averages of DH1005 Ab with I-shaped Ab conformations denoted by “I” in image. 2D class averages of DH1005-SOSIP complex shows Fab-dimer binding to soluble stabilized recombinant HIV-1 Env trimer. Fc domain is not visible here because it lies outside the circular mask used during class averaging. Shown also are 2D class averages of DH1009 (E) and DH1010 (F) that displayed I-shaped Abs. (G) Seven Env glycan-reactive mAbs were tested in ELISA for binding Man₉-V3 and non-glycosylated aglycone V3 peptide and heat-killed yeast antigens. Data shown are from a representative ELISA. Reference mAbs included peptide-reactive DH1013, Env glycan-reactive DH717.2 and 2G12, trimer-reactive PGT151, and negative control CH65 Abs. (H) Only DH1005 neutralized multi-clade *env*-pseudotyped HIV-1 bearing Envs with Man₉-enriched glycans [Kif]. Neutralization titers are representative of two assays in TZM-bl cells, and neutralization titers were reported as IC₅₀ in μg/mL. Control mAbs were 2G12 and CH65. See also Data S3 and STAR Methods.

**Figure 4**
Characteristics of HIV-1 Env glycan-reactive neutralizing B cell lineage, DH898, elicited by SHIV infection in RMs (A) Phylogram and immunogenetics of DH898 neutralizing Abs isolated from a pathogenic SHIV-infected RM (6163). (B) DH898 mAbs were tested for neutralization of *env*-pseudotyped HIV-1 bearing an autologous Env from SHIV infection. MAbs were tested for neutralizing HIV-1 bearing wild-type and glycan-deleted mutant Envs in TZM-bl cells. Data shown are from a representative assay and neutralization titers are IC₅₀ in μg/mL. (C) DH898 mAbs were tested for binding in ELISA to Man₉-V3 (black bars) and non-glycosylated aglycone V3 peptide (gray bars). Binding titer was reported as log AUC. Control Abs tested included peptide-reactive DH1013, Env glycan-reactive 2G12, and negative-control anti-influenza CH65 mAbs. Data shown are representative of four assays. (D) NSEM of DH898.1 IgG showing a mixture of I-shaped (I) and Y-shaped (Y) Abs. (E) Segmented cryo-EM map of DH898.1 Fabs bound to a soluble stabilized HIV-1 Env trimer showing a single Fab-dimer bound to the trimer. Colors are as in Figure 1. (F) Zoomed-in view of the Fab-dimer showing a gap between the two Fabs. (G) A fitted model showing the Fab-dimer binding to high mannose glycans at Env positions 332, 392, and 396. (H) Atomic model of the Fab-dimer showing a gap between the Fabs (arrow) and an interface between the Fv domains (star). (I) The beta-sandwich view of the interface demonstrating that the beta- strands of the two V_H domains are angled relative to one another. (J) Schematic diagram of the Fab-dimer interface showing a patch of three hydrophobic or aromatic residues (green) surrounded by small hydrophilic residues (orange) and a complementary pair of charged residues (blue +, red −). See also Figures S2 and S3, Data S3 and S4, and Videos S1 and S2.

**Figure S2**
Characterization of neutralizing FDG Ab lineage (DH898) elicited by SHIV infection in RMs, related to Figure 4 DH898 mAbs were elicited by pathogenic SHIV (CH848TF) infection in RM6163 (see STAR Methods). (A) DH898 mAbs were tested in ELISA for binding to Man₉-V3, and heat-killed yeast antigens *Candida albicans* or *Cryptococcus neoformans*, in the absence (black bars) or presence (white bars) of 1M D-mannose. Control mAbs are reactive to Env glycan (DH501 and 2G12), Env peptide (DH1013) or influenza (CH65). Binding titers were reported as Log AUC. Data shown were from a single ELISA that were in agreement with an independent experiment of DH898 mAbs binding to Man₉-V3 and yeast antigens in the absence or presence of 0.5M D-mannose. (B) DH898 mAbs were tested in ELISA for binding soluble stabilized recombinant HIV-1 trimers, CH848 10.17 DS.SOSIP wild-type and mutant trimers, in a single experiment. Mutations in CH848 10.17 DS.SOSIP trimers included deletions of potential N-linked glycan sites in the V3 (N301A_N332A) or V1 (N133D_N138T) regions. Mutations also included amino acid insertions that filled glycan holes on the trimer (D230N_H289N). Binding levels were measured at OD_450nm. CH848 10.17 SOSIP trimers were captured using PGT151. Biotinylated (B) 2G12, PGT128 (V3 glycan bnAb) and CH65 mAbs were tested as control Abs. (C) Binding levels of DH898 mAbs to CH848TF SOSIPv4.1 in one ELISA, representative of at least two independent experiments. CH848TF SOSIP trimer was captured using anti-AVI mAb. 2G12, PGT151 and CH65 were tested as control Abs. (D) NSEM class averages for DH898 mAbs with lineage member indicated at upper left of each panel. At the lower left corner, each class average was identified as Y-shaped (Y), I-shaped (I), or ambiguous (A). (E) Sequence analysis of key residues within the Fab-dimer interface shows three hydrophobic or aromatic residues in the interface, similar to 2G12. V_H interface residues were numbered according to standard Kabat numbering (Wu and Kabat, 1970). Amino acids at each position were indicated by their one-letter code and colored according to Taylor (Taylor, 1997), with polar groups orange, hydrophobic and aromatic groups in shades of green to yellow, positively charged groups blue and negatively charged groups red. Residues that are rare for a particular position, i.e., ≤ 1% in the abYsis database (Swindells et al., 2017), are indicated with an asterisk. Bottom row indicates VRC01 as a non-Fab-dimerized negative control. (F) Bar graph indicating the fraction of I-shaped Abs for wild-type (blue bars), strengthening mutants (green bars), or disrupting mutants (red bars) in DH898 mAbs. Mutations were engineered in the Ab V_H genes. The % I-shaped was estimated by the fraction of particle images that sorted into I-shaped classes as indicated in each of the NSEM 2D class averages shown in G. (G) NSEM 2D class averages from of DH898.4 wild-type (WT), strengthening double mutations T19I and T70F, and disrupting double mutations R64D and F68D. The class averages shown represented the average of ~8,000 to 20,000 individual particle images classified and averaged into ten classes, arranged from the most populated class at the top left to the least populated at the bottom right, and marked as I-shaped (I), Y-shaped (Y), or ambiguous (A). (H) DH898.4 wild-type and mutant mAbs were tested in ELISA for binding to HIV-1 CH848TF SOSIP trimer. MAbs were tested in technical replicates within a single ELISA and binding levels measured at OD_450nm; error bars represent standard error of the mean. (I) DH898.4 wild-type and mutant mAbs weres tested for neutralization against autologous (CH848TF) HIV-1 strain bearing Env with heterogeneous glycoforms as well as heterologous HIV-1 isolates bearing Envs Man₉-enriched glycans [Kif] in TZM-bl cells. These data were generated in a single neutralization assay and titers were reported as IC₅₀ in μg/ml.

**Figure S3**
DH898.1 complex cryo-EM data processing summary and structural details, related to Figure 4 (A) Representative micrograph; (B) Power spectrum of micrograph, and fitted contrast transfer function; (C) Picked particles in white circles; and (D) Representative 2D class averages. (E) *Ab initio* volumes that showed three particle populations: free trimer, Fab-dimer bound to glycans near the CD4-binding site (bs), and Fab-dimer bound to glycans near the base of the V3 loop (left to right) seen in top and side views (top to bottom). Dashed line in side view indicated viral membrane location. (F) Top and side view of free soluble stabilized recombinant HIV-1 Env trimer (SOSIP) colored by local resolution. Mesh surface indicates mask used for FSC calculation. Eye indicates viewing direction in side view. (G) HIV-1 Env SOSIP with Fab-dimer bound to glycans near the CD4bs. (H) HIV-1 Env SOSIP with Fab-dimer bound to glycans near the base of the V3 loop. (I) Gold standard Fourier Shell Correlation (FSC) curves for F-H and J-K indicating global resolution ranging from 3.9 – 7.0 Å. (J) Local refinement of Fab-dimer from CD4bs particle set. (K) Local refinement of Fab-dimer only from V3-glycan bound particle set. (L) Segmented cryo-EM map showing Fab-dimer bound near the CD4bs. Gold-standard FSC resolution is indicated below each map. (M) Local refinement of the Fab-dimer bound near to the CD4bs of a HIV-1 Env SOSIP. (N) Close-up view of the epitope near the CD4bs with a HIV-1 Env SOSIP and Fab-dimer models shown as ribbons; glycans shown as sticks; and the cryo-EM map shown as a transparent surface. (O) (left) Local refined, density modified map of the DH898.1 Fab dimer with cryo-EM reconstruction shown as blue mesh and underlying fitted model in cartoon representation, and (right) Zoomed-in view of the Fab dimer interface with select interfacial residues shown as sticks. (P) Views of the Fab dimer interface rotated 90° clockwise (left) or counter-clockwise (right) relate to the view shown in panel O. (Q) Zoomed-in views of Env regions in the apo structure.

**Figure 5**
Characteristics of HIV-1 Env glycan-reactive bnAb B cell lineage, DH851, elicited by SHIV infection in RMs (A) Phylogram and immunogenetics of DH851 bnAbs isolated from a pathogenic SHIV-infected RM (6163). (B) DH851 mAbs were tested for neutralization of *env*-pseudotyped HIV-1 bearing Envs with difficult-to-neutralize autologous and heterologous strains. Data shown are representative of multiple neutralization assays tested in TZM-bl cells with different Ab lots, and neutralization titers were reported as IC₅₀ in μg/mL. (C) DH851 mAbs were tested in ELISA for binding to Man₉-V3 (black bars) and non-glycosylated aglycone V3 peptide (gray bars). Reference Abs tested included peptide-reactive DH1013, Env glycan-reactive 2G12, and negative control anti-influenza CH65 mAbs. Data shown are representative of four assays and binding titers were reported as Log AUC. (D and E) DH851 and 2G12 mAbs were tested for blocking each other via competition ELISA. Data show percent blocking of biotinylated (b) DH851 and 2G12 binding to soluble recombinant HIV-1 Env monomeric protein by competing non-biotinylated mAbs at varying concentrations. 2G12 blocking was representative of three experiments, but DH851 blocking data shown were from a single experiment. (F) NSEM of DH851.3 showing a mix of Y-shaped (Y) and I-shaped (I) Abs. (G) Segmented cryo-EM map of DH851.3 bound to soluble stabilized recombinant HIV-1 Env trimer showing six Fab domains arranged in three Fab-dimer pairs and binding to the trimer with branched glycans well resolved. Trimer, gray; glycans, black; Fab V_H, teal or dark pink; Fab V_λ, cyan or light pink. (H) View enlarged, turned 40°, and at a higher contour level shows a single Fab-dimer with a gap in the middle indicating that the two Fabs sit side-by-side and are not V_H domain-swapped. (I) Zoomed-in view of the map shown as transparent surface with the fitted model shown in cartoon representation and glycans shown as sticks. (J) Atomic model of the Fab-dimer showing a gap between the Fabs (arrow) and an interface between the Fv domains (star). (K) The beta-sandwich view of the interface (starred region in J, turned 90°) showed the beta-strands of the two V_H as nearly parallel. Pink beta-strands were labeled A–E. (L) Schematic diagram of the Fab-dimer interface showing a patch of four hydrophobic or aromatic residues (green) surrounded by small hydrophilic residues (orange). Beta strands in the schematic correspond to the pink strands in (K) and were labeled A– E accordingly. See also Figures S4 and S5 and Data S2, S3, and S4.

**Figure S4**
Characterization of FDG bnAb lineage (DH851) elicited by SHIV infection in RMs, related to Figure 5 DH851 mAbs were elicited by pathogenic SHIV (CH848TF) infection in RM6163 (see STAR Methods). (A) DH851 mAbs were tested for neutralization against *env*-pseudotyped HIV-1 bearing Envs with Man₉-enriched glycans [(+) Kif] and heterogeneous glycoforms [(-) Kif]. Neutralization was tested in TZM-bl cells and titers reported as IC₅₀ in μg/ml. (B) Summary of neutralization profile for DH851 and 2G12 bnAbs that were tested against 119 difficult-to-neutralize multi-clade HIV-1 strains in TZM-bl cells (Seaman et al., 2010). (C) DH851 neutralization epitopes mapped on a SHIV bearing wild-type or mutant HIV-1 Ce1176-strain Envs. Mutant Envs had deletion of potential N-linked glycosylation sites that constituted the Env glycan-containing bnAb epitope targeted by glycan-only Abs as well as Abs that bound both Env glycans and peptide (GDIR motif) (Kong et al., 2013). While DH851 mAbs also neutralized HIV-1 Ce1176 strain, a mutation in the GDIR motif (D325N) did not impact the neutralization sensitivity of HIV-1 Ce1176 (not shown). (D) DH851 mAbs bearing sequences for clonally-related V_H genes from week 0 (DH851.5, DH851.6 and DH851.8), computationally-inferred near-germline unmutated common ancestor (DH851UCA), and post-infection (DH851.1-DH851.4) Abs were tested in ELISA for binding to soluble recombinant HIV-1 Env trimer (SOSIP) of autologous CH848TF strain in triplicate experiments. Biotinylated (B) PGT151 (Env trimer reactive) and CH65 (influenza reactive) mAbs were tested as controls. Binding levels were measured at OD_450nm. (E) DH851 mAbs were tested in ELISA for binding to CH848 10.17 DS.SOSIP in the presence (+) or absence (-) of 0.05-0.5M D-mannose, compared with control mAbs 2G12, PGT151 and CH65. Data shown were from a single experiment that was in agreement with two additional experiments of DH851 mAbs binding to CH848 10.17 gp120 ± D-mannose. (F) DH851 mAbs were tested in ELISA for binding to Man₉-V3 and non-glycosylated aglycone V3 peptide in triplicate experiments; error bars represent standard error of the mean. DH1013 (peptide reactive), 2G12 (Env glycan reactive) and CH65 mAbs were tested as control mAbs. (G) DH851 mAbs were tested in ELISA for binding to heat-killed yeast antigens, *Candida albicans* or *Cryptococcus neoformans* in triplicate experiments; error bars represent standard error of the mean. 2G12 and CH65 were tested as control mAbs. (H) NSEM 2D class averages of DH851 mAbs displaying I- and Y-shaped Abs. (I) Sequence analysis of key residues within the Fab-dimer interface. (I) Bar graph indicating the fraction of I-shaped Abs for wild-type (blue bars) and strengthening mutants (green bars) in DH851 mAbs. Mutations were engineered in the Ab V_H genes. The % I-shaped was estimated by the fraction of particle images that sorted into I-shaped classes as indicated in each of the NSEM 2D class averages from DH851.2 wild-type (WT) and strengthening double mutations A19T and T21L. The class averages shown represented images classified and averaged into five classes, and marked as I-shaped (I) or Y-shaped (Y).

**Figure S5**
DH851.3 complex cryo-EM data processing summary and structural details, related to Figure 5 (A) Representative micrograph; (B) Power spectrum of micrograph, and fitted contrast transfer function; (C) Picked particles in white circles; and (D) Representative 2D class averages. (E) *Ab initio* volume of DH851, bound to a soluble stabilized HIV-1 Env trimer (SOSIP) seen in top and side view. Dashed line in side view indicated viral membrane location. (F) Refined 3D map, without filtering or sharpening and colored by local resolution from 5.5 to 12.5 Å, blue to red. (G) Map after local filtering and B-factor sharpening. Fab constant domains are noisy and at this contour level mostly disappear and are only seen as small, disconnected blobs, but greater details of the complex can be seen. (H) Gold standard Fourier Shell Correlation (FSC) curves indicated global resolution ranging from 5.6 to 8.7 Å. (I) 2D class average shows view of Fab in complex with a HIV-1 Env SOSIP looking down the central axis. Red box shows region where the DH851.3 Fab dimer contacts the adjacent SOSIP protomer. (J) Fitted model shown as ribbon diagram with cryo-EM map shown as transparent volume. (K) Close up of contact seen in panel (I) showed that it may involve the framework region 3 of the light chain, and Env residues around 426-431 or 105-112. (L) View of the DH851.3 Fab dimer bound to Env glycans. Cryo-EM reconstruction was shown as a blue mesh, with underlying fitted coordinates in cartoon and stick representation. (M) and (N) Zoomed-in views of the Fab dimer interface shown within the dotted rectangle in the left panel.

**Figure 6**
Glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike (A) FDG mAbs were tested for binding recombinant SARS-CoV-2 Spike (S) in ELISA. Ab binding was assessed in the absence (−) or presence (+) of 1M D-mannose. Binding Ab titers were reported as Log AUC. Controls were SARS-CoV-1 RBD (D001) and influenza HA (CH65) reactive mAbs. Data shown are from a representative assay. (B) Cryo-EM reconstruction of 2G12 in complex with recombinant SARS-CoV-2 S. The cryo-EM map was colored by chain. SARS-CoV-2 S chains were colored salmon, green, and blue. The 2G12 chains are colored dark gray and orange for the heavy chains (HC), and yellow and dark pink for the light chains (LC). (C) Top: schematic showing domain organization of SARS-CoV-2 S, with positions of N-linked glycosylation sequons numbered and shown as branches. Bottom: zoomed-in view of domain-swapped, dimerized 2G12 Fab interacting with SARS-CoV-2 S. The structure was colored by chain as in (B), with 2G12 and SARS-CoV-2 S shown in cartoon representation and the interacting glycans in surface representation. (D) Binding of 2G12 Fab dimer to (top) unmutated and (bottom) N709A mutant recombinant S proteins, measured by SPR using single-cycle kinetics. The black lines show the data and the red lines show the fit of the data to a 1:1 Langmuir binding model. (E) Binding of the unmutated and N709A mutant S proteins to a panel of FDG Abs measured by SPR; data shown as a heatmap for Log AUC binding. See also Figures S6 and S7 and Data S4.

**Figure S6**
Characterization of glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike protein, related to Figure 6 (A) FDG, SARS-CoV-1 RBD (D001), and influenza HA (CH65) mAbs were tested in ELISA for binding to recombinant SARS-CoV-2 Spike (S) protein. mAb binding was assessed in the absence (-) or presence (+) of D-mannose [1M] to determine if free high mannose can outcompete glycans on the S protein for binding. Binding Ab titers reported as Log AUC were shown in Figure 1. (B) FDG mAbs were tested in ELISA for binding to a set of commercially available constructs expressing the SARS-CoV-2 S1 and S2 extracellular domain (left), S2 domain (middle), and the receptor binding domain (right). Black and white bars show binding in the absence and presence of D-mannose [1M], respectively. Binding Ab titers were reported Log AUC. (C) We tested 2G12 mAb for binding to (from top to bottom) soluble stabilized recombinant HIV-1 Env trimer (CH505TF SOSIP), Man₉-V3 and non-glycosylated aglycone V3 peptide, and recombinant SARS-CoV-2 spike ectodomain. HIV-1 CH505TF SOSIP was captured using mouse anti-AVI-tag mAb, whereas SARS-CoV-2 ectodomain and peptides (Man₉-V3 and Aglycone) were captured using streptavidin. Blue and red symbols indicated binding in the absence and presence of D-mannose [1M], respectively. Binding was measured at OD_450nm. All ELISAs (A-C) were done using BSA-based buffers (see STAR Methods). Data shown are from a representative assay. (D) Size-exclusion chromatogram of protein A affinity purified 2G12 IgG. (E) NSEM 2D class averages of (top) 2G12 IgG dimer, (bottom) 2G12 IgG monomer and (right) 2G12 Fab obtained by digesting 2G12 IgG monomer with papain. (F) SPR sensorgrams showing binding of 2G12 IgG dimer (red line) and 2G12 IgG monomer (black line) to the SARS-CoV-2 S protein. (G) We tested FDG mAbs for binding to the unmutated SARS-CoV-2 S and the N709-glycan deleted variant. Binding was assessed by SPR by capturing the unmutated spike and the N709-glycan deleted spike on flow cells 2 and 4 of a streptavidin coated (SA) chip, and flowing over a 200 nM solution of each Ab simultaneously over all four flow cells. Flow cells 1 and 3 were used as reference flow cells for flow cells 2 and 4, respectively. Buffer blanks were run in a similar manner and the sensorgrams were double-referenced by first subtracting the signal from the reference flow cell and then subtracting the reference-corrected buffer blank. CR3022 IgG and ACE-2 tagged with a mouse-Fc region were used as controls. (H) Binding of 2G12 to spike proteins of SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green). The SARS-CoV-2/2G12 complex is shown with the spike in red and the bound 2G12 as a transparent pink surface, with the glycans contacting 2G12 shown as sticks and the respective Asn residues as spheres. Similar glycosylation was observed for the SARS-CoV and MERS-CoV S proteins (Asn residues shown as spheres). SPR binding data shown for SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green) S proteins. The data shown are representative of three independent experiments, and for each dataset the graph showed three technical replicates. IgGs were captured on flow cells of a CM5 chip immobilized with human Anti-Fc antibody (8000RU). 200 nM solution of the SARS-CoV-2 spike was flowed over the flow cells. The surface was regenerated between injections by flowing over 3M MgCl2 solution for 10 s with flow rate of 100μl/min.

**Figure S7**
Cryo-EM data processing details for SARS-CoV-2 S protein complex with 2G12, related to Figure 6 (A) Representative micrograph. (B) CTF fit (C) Representative 2D class averages. (D) Maps for (left) unliganded and (right) 2G12-bound S obtained after 3 D classification. (E-G) Refined maps for SARS-CoV-2 S protein bound to (E) 1-2G12, (F) 2-2G12-, and (G) 2-2G12 (with partial occupancy at the third binding site) Fab2 molecules. Red arrow in (G) points to disordered 2G12 Fab2 bound at the third binding site. (H) (Left) Map combining all particles and focusing refinement on the region within the masks that is shown as a gray mesh overlaid on the final refined map shown as a gray surface. (Right) Fourier shell correlation curves. (I) (Left) Cryo-EM reconstruction of 2G12 bound to the SARS-CoV-2 spike colored by local resolution. (Right) Zoomed-in view showing the cryo-EM reconstruction of the bound 2G12 Fab. (J) (Left) Two distinct states were resolved from the cryo-EM data by heterogeneous classification. Density for the two observed states were shown in green and gray. (Right) Cartoon representation of the SARS-CoV-2 S-protein (bright green, bright orange, blue) and the two 2G12 orientations. The axis of rotation hinged around glycan 709 is represented by a gray cylinder. (K) Zoomed-in view of (from left to right) a region in the S2 domain with map shown as blue mesh and fitted model shown as sticks; glycan 709; glycan 801; glycan 717 bound to 2G12. While not in direct contact with the bound antibody, the HR1 helix may play an indirect role in the binding by stabilizing glycan 717 via a stacking interaction with residues N925 and Q926.

**Figure 7**
Polyreactivity profile of FDG Abs (A) Indirect immunofluorescence assay testing reactivity of FDG Abs in HEp-2 cells. Each mAb was tested at 50 and 25 μg/mL in duplicate reactions; a representative of the replicate data was reported. Positivity scores were determined relative to RM positive (DH1037) and negative (DH570.30) control mAbs. Staining patterns were identified using the Zeus Scientific pattern guide. (B) Images of representative mAbs staining of HEp-2 cells. Ab reactivity pattern in HEP-2 cells were described in (A). (C) Nine autoantigens were tested for reactivity by FDG Abs using a commercially available AtheNA Multi-Lyte ANA kit (Zeus Scientific). Serially diluted mAbs were tested for binding and the data analyzed using an AtheNA software. The dash lines represented the positivity score (121 units), which was consistent across independent experiments. 4E10 and CH65 represented positive and negative control mAbs, respectively. Data shown were for representative FDG mAbs that were reactive with HEp-2 cells (A and B). See also STAR Methods.

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1. Adams P.D., Afonine P.V., Bunkóczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010;66:213–221. - PMC - PubMed
1. Alam S.M., Liao H.X., Tomaras G.D., Bonsignori M., Tsao C.Y., Hwang K.K., Chen H., Lloyd K.E., Bowman C., Sutherland L., et al. Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion. J. Virol. 2013;87:1554–1568. - PMC - PubMed
1. Alam S.M., Aussedat B., Vohra Y., Meyerhoff R.R., Cale E.M., Walkowicz W.E., Radakovich N.A., Anasti K., Armand L., Parks R., et al. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide. Sci. Transl. Med. 2017;9:eaai7521. - PMC - PubMed
1. Alexander J., del Guercio M.F., Maewal A., Qiao L., Fikes J., Chesnut R.W., Paulson J., Bundle D.R., DeFrees S., Sette A. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J. Immunol. 2000;164:1625–1633. - PubMed
1. Andrabi R., Voss J.E., Liang C.H., Briney B., McCoy L.E., Wu C.Y., Wong C.H., Poignard P., Burton D.R. Identification of Common Features in Prototype Broadly Neutralizing Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design. Immunity. 2015;43:959–973. - PMC - PubMed

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[1] Adams P.D., Afonine P.V., Bunkóczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010;66:213–221. - PMC - PubMed

[2] Adams P.D., Afonine P.V., Bunkóczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 2010;66:213–221. - PMC - PubMed

[3] Alam S.M., Liao H.X., Tomaras G.D., Bonsignori M., Tsao C.Y., Hwang K.K., Chen H., Lloyd K.E., Bowman C., Sutherland L., et al. Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion. J. Virol. 2013;87:1554–1568. - PMC - PubMed

[4] Alam S.M., Liao H.X., Tomaras G.D., Bonsignori M., Tsao C.Y., Hwang K.K., Chen H., Lloyd K.E., Bowman C., Sutherland L., et al. Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion. J. Virol. 2013;87:1554–1568. - PMC - PubMed

[5] Alam S.M., Aussedat B., Vohra Y., Meyerhoff R.R., Cale E.M., Walkowicz W.E., Radakovich N.A., Anasti K., Armand L., Parks R., et al. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide. Sci. Transl. Med. 2017;9:eaai7521. - PMC - PubMed

[6] Alam S.M., Aussedat B., Vohra Y., Meyerhoff R.R., Cale E.M., Walkowicz W.E., Radakovich N.A., Anasti K., Armand L., Parks R., et al. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide. Sci. Transl. Med. 2017;9:eaai7521. - PMC - PubMed

[7] Alexander J., del Guercio M.F., Maewal A., Qiao L., Fikes J., Chesnut R.W., Paulson J., Bundle D.R., DeFrees S., Sette A. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J. Immunol. 2000;164:1625–1633. - PubMed

[8] Alexander J., del Guercio M.F., Maewal A., Qiao L., Fikes J., Chesnut R.W., Paulson J., Bundle D.R., DeFrees S., Sette A. Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses. J. Immunol. 2000;164:1625–1633. - PubMed

[9] Andrabi R., Voss J.E., Liang C.H., Briney B., McCoy L.E., Wu C.Y., Wong C.H., Poignard P., Burton D.R. Identification of Common Features in Prototype Broadly Neutralizing Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design. Immunity. 2015;43:959–973. - PMC - PubMed

[10] Andrabi R., Voss J.E., Liang C.H., Briney B., McCoy L.E., Wu C.Y., Wong C.H., Poignard P., Burton D.R. Identification of Common Features in Prototype Broadly Neutralizing Antibodies to HIV Envelope V2 Apex to Facilitate Vaccine Design. Immunity. 2015;43:959–973. - PMC - PubMed

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Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

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Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

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