Volume 44, Issue 5, Pages 1215-1226 (May 2016) Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch  Daniel T. MacLeod, Nancy M. Choi, Bryan Briney, Fernando Garces, Lorena S. Ver, Elise Landais, Ben Murrell, Terri Wrin, William Kilembe, Chi-Hui Liang, Alejandra Ramos, Chaoran B. Bian, Lalinda Wickramasinghe, Leopold Kong, Kemal Eren, Chung-Yi Wu, Chi- Huey Wong Matt A. Price, Jill Gilmour, Pat Fast, Anatoli Kamali, Eduard J. Sanders, Omu Anzala, Susan Allen, Eric Hunter, Etienne Karita, William Kilembe, Shabir Lakhi, Mubiana Inambao, Vinodh Edward, Linda-Gail Bekker, Sergei L. Kosakovsky Pond, Ian A. Wilson, Dennis R. Burton, Pascal Poignard Daniel T. MacLeod, Nancy M. Choi, Bryan Briney, Fernando Garces, Lorena S. Ver, Elise Landais, Ben Murrell, Terri Wrin, William Kilembe, Chi-Hui Liang, Alejandra Ramos, Chaoran B. Bian, Lalinda Wickramasinghe, Leopold Kong, Kemal Eren, Chung-Yi Wu, Chi-Huey Wong Matt A. Price, Jill Gilmour, Pat Fast, Anatoli Kamali, Eduard J. Sanders, Omu Anzala, Susan Allen, Eric Hunter, Etienne Karita, William Kilembe, Shabir Lakhi, Mubiana Inambao, Vinodh Edward, Linda-Gail Bekker, Sergei L. Kosakovsky Pond, Ian A. Wilson, Dennis R. Burton, Pascal Poignard  Immunity  Volume 44, Issue 5, Pages 1215-1226 (May 2016) DOI: 10.1016/j.immuni.2016.04.016 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Development of N332-Glycan-Specific Neutralizing Serum Response in Donor PC76 (A) Neutralization assays were performed with serum isolated from donor PC76 at various times post-infection, and multiple HIV-1 pseudoviruses were used. Neutralization inhibitory dilution 50 (ID50) values calculated as 1/(serum dilution at 50% inhibition of infection). Samples for which 50% neutralization was not reached at a dilution of 1/50, were determined as having no neutralization. The percent of viruses neutralized and ID50 geometric mean values are shown for each subtype and overall values at the bottom. (B) Neutralization assays were performed at the time points indicated; 92BR020 WT or N332A pseudovirus with PC76 donor serum was used. (C) Serum from donor PC76 at 33 mpi was depleted of Abs binding to the proteins listed on the x axis. This depleted serum was used in neutralization assays with the eight pseudoviruses listed. Experiments were performed in duplicate, and the means were used to calculate ID50 values. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 PCDN mAb Gene Usage, Evolutionary Tree, and Relative Maturation (A) PCDN HC and LC V- and J-gene usage, CDR3 length and sequences, and percent mutation for nt or aa sequences are tabulated for the 12 mAbs isolated at 16 to 38 mpi. The number in each mAb name corresponds to the isolation time point. (B) The evolutionary distance between the mAbs is illustrated as a phylogenetic tree for both HC and LC nt sequences. Each color corresponds to a time point reflected in the mAb name. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Binding and Neutralization of PCDN mAbs (A) PCDN Abs were tested by ELISA for binding to rgp120, and EC50 values (μg/mL) are listed. If 50% binding was not reached at 100 μg/mL of Ab, the Abs were considered non-binding (>100). (B) A heterologous panel of 37 pseudoviruses was tested for neutralization by PCDN mAbs. The percent neutralization and geometric mean of IC50 values (μg/mL) are shown for each HIV-1 virus subtype; overall values are at the bottom. All samples for which 50% neutralization was not reached at 100 μg/mL were determined as having no neutralization. The geometric mean was calculated only with viruses that were neutralized. (C) A heterologous virus panel of 110 pseudoviruses was tested for neutralization by the 3 most mature mAbs and the percentage of viruses neutralized is shown for each subtype of HIV-1 virus; overall values are listed at the bottom of the table. (D) Neutralization assays were performed with PCDN Abs and the indicated pseudoviruses produced in HEK293T cells in the presence of kifunensine or swainsonine or in HEK293S cells. Neutralization IC50 values were calculated and presented as a fold increase relative to neutralization of WT pseudoviruses produced in untreated cells. (E) Glycan array binding by PCDN mAbs and PGT128 for Man9GlcNAc2 and Man8. ELISA and neutralization experiments were performed in triplicate, and the means were used to calculate EC50 and IC50 values. Glycan array was performed in duplicate, and values are from a representative experiment. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Autologous Virus Neutralization and Development (A) 76 autologous virus clones from 8 time points were tested for neutralization by PCDN mAbs. The IC50 values (μg/mL) are represented in the color gradient. All samples for which 50% neutralization was not reached at 50 μg/mL were determined as having no neutralization (gray boxes). Neutralization matrix was performed once. (B) Sequences of Env from 323 to 339 by HXB2 numbering of 76 of the virus clones are shown. MRCA (most recent common ancestor) is an inferred earliest ancestral sequence. The residues or PNGSs that change over time are shaded in blue, yellow, or pink. The number of isolated autologous virus clones with each sequence at each time point is shown on the right. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Autologous Virus Mutagenesis and Mapping (A) The dominant autologous viruses from three critical time points were tested for neutralization by all PCDN mAbs. The unmutated virus is indicated as WT for each time point. Neutralization assays with pseudoviruses expressing Env with the specified mutations were performed in comparison to WT. (B) PNGSs on the 10 mpi virus were mutated and tested for neutralization in comparison to the WT virus. (C) Residues from 322 to 336 were mutated to Ala in the 10 mpi virus and tested for neutralization in comparison to WT. The numbers show IC50 values (μg/mL) and are shaded for potency as indicated in the color gradient bar. Neutralization experiments were performed in triplicate, and the means were used to calculate IC50 values. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 NGS of the PCDN mAb Lineage (A) The frequency of sequences from the PCDN lineage relative to the entire repertoire is shown in pink with circles for each time point. The neutralization score at each time point is indicated in blue with squares. (B) The phylogenetic tree of PCDN Ab HC sequences from six different time points shaded in the colors as indicated. The relative evolutionary distance is indicated as a bar at the bottom. Collapsed sequences are shown in circles in sizes relative to the number of sequences. The PCDN mAb sequences are indicated in black with the Ab name. The neutralization breadth and IC50 values for autologous and heterologous viruses are illustrated as concentric circles on the right side of the tree after the dotted lines. The outside circle represents breadth; the center circle represents IC50 values; the color code is as depicted at the bottom right. (C) SHM was calculated for each time point either as divergence (number of aa changes compared to UCA; top panel) or as sum of the evolutionary distance (middle panel). The rate increase of evolutionary distance normalized by the rate of GL divergence is shown in the bottom panel. The boxes show the first and third quartile as the lower and upper limits of the box with a horizontal line for the median, and the end of the vertical lines indicate the minimum and maximum values. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Crystal Structures of PCDN-27A and PCDN-27B Fabs (A) The secondary structure of the PCDN-27A is illustrated in lavender with the CDR and FR regions highlighted in different colors. Beneath this, a superimposition of the PCDN-27A structure in lavender and the PCDN-27B structure in orange is shown. (B) The electrostatic potential of the two Fabs is illustrated. Red indicates a negatively charged surface, and blue indicates a positive charge. Rotated views, shown with a yellow circle, highlight the areas with the most change. Immunity 2016 44, 1215-1226DOI: (10.1016/j.immuni.2016.04.016) Copyright © 2016 Elsevier Inc. Terms and Conditions