Study Population

The subjects studied here were enrolled in Protocol C, a uniform vaccine-preparedness study developed and implemented by the International AIDS Vaccine Initiative (IAVI; http://www.iavi.org) that was carried out at multiple sites in Africa, including the Zambia-Emory HIV Research Project (ZEHRP, coded IDs Z1800M, Z1047M) and Projet San Francisco (coded IDs R774F, R53F, R1142F, R66M) (33, 34). Projet San Francisco subjects were associated with epidemiologically linked donors, while the two subjects from ZEHRP were not. These subjects were also included in our previous study (32).

Cell Line Authentication

Genetica Cell Line Testing was used to authenticate 293 and TZM-bl cell lines. 293T cells: 88.9% identity, 80.00% match to HEK 293T/17. Expi293F: 90.32% identity, 93.33% match to HEK-293 [293] (ATCC CRL-1573). TZM-bl: 100.00% identity, 100% match to HeLa (ATCC CCL-2). All lines were mycoplasma negative. CEM.NKr.CCR5 cells were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: CEM.NKr.CCR5 Cells from Dr. Alexandra Trkola (35–37).

Neutralization Assays

Generation of Env pseudoviruses and the experimental protocol for the TZM-bl neutralization assay have been described previously (9, 11, 38–49). As R66M began antiretroviral therapy at ~1.25 years post-infection, which can give a false impression of “neutralization” in the TZM-bl assay, IgG antibodies were purified from the 36.5-month plasma sample (Time Point 3) using a GE Healthcare Life Sciences Ab SpinTrap, according to the manufacturer's instructions (GE 28-4083-47). The concentration of the purified IgG was determined by ELISA and adjusted, and was then used in place of plasma in neutralization assays for this time point. In all other assays, plasma was used to evaluate polyclonal autologous neutralization. 2000 IU of each titered Env pseudovirus (in DMEM with ~3.5% (vol/vol) FBS (Hyclone, # SH30088.03) and 40 μg/mL DEAE-dextran) was mixed with five-fold serial dilutions (beginning at 1:100) of heat-inactivated plasma samples or purified polyclonal IgG and assayed for its inhibitory potential against the appropriate autologous Env pseudovirus using the TZM-bl indicator cell line, with luciferase as the readout. The average background luminescence from a series of uninfected wells was subtracted from each experimental well. All assays contained duplicate wells and were repeated at least once independently. For mAb neutralization, 2000 IU pseudovirus with 10 μg/ml of mAb and added to plated TZM-bl cells. Samples were run in triplicate, and when antibody yield was sufficient, this was duplicated. “Antibody only” wells were also included as a negative control to monitor for any potential cytotoxic activity of the mAb and/or elution buffer. HIV bnAb VRC01 was used as a positive control and anti-influenza HA mAb EM4C04 was used as a negative control, both at 10 μg/ml.

Granzyme B Activity Assay

A panel of potential HIV-negative effector cell donors at Emory University were screened for CD16 158V/F polymorphisms via flow cytometry, similar to a method previously described (50). Donor PBMC were stained with APC conjugated anti-human CD56 (BioLegend, #318309), PacBlue anti-human CD3 (BioLegend, #300330), and either FITC anti-human CD16 (Thermo, #MHCD1601) for total CD16, or a FITC anti-human CD16 158V-specific clone MEM-154 (Thermo, #MAI-19563) and analyzed on an LSRII (BD Bioscience) instrument and with FlowJo 10.4 software (Treestar). The resulting ratio of the mean fluorescence of FITC-positive, CD56, CD3, cells (MEM-154:Total) was used to eliminate 158F homozygous donors, and identify 158V/F or 158V/V donors for downstream use in the GzB assay. Two donors were selected (Donor 190, Donor 457), and PBMC were isolated via Ficoll centrifugation and cryopreserved in liquid nitrogen (90% FBS, 10% DMSO) until needed.

Assessment of GzB activity was performed as previously described (51). T/F Env gp120 proteins with a C-terminal 6XHis-tag were synthesized by GENEART (ThermoFisher). Genes were synthesized and inserted into pcDNA3.3 and transfected into FreeStyle™ 293 cells. Proteins were purified via 5 ml HisTrap™ FF column, linear gradient from 20 to 500 mM Imidazole in PBS, 500 mM NaCl at 6 days post-transfection, with a HiLoad 26/600 Superdex200 polishing step. Cryopreserved PBMC from uninfected donors were recovered in RPMI (10% FBS (Hyclone) Pen/strep/l-glut) the night before the assay. To ensure consistency across assays, effector cell viability was required to be >80% on the day of the assay to proceed. CEM.NKr.CCR5 cells were coated with 8 μg gp120/million cells at RT for 45 min. Dyes TFL4 and NFL1 were added according to the manufacturer's instructions for 15 min in a 37 degree C water bath (OncoImmunin, Inc. GranToxiLux PLUS! and NFL1). CEM.NKr.CCR5 target cells were washed 2x in complete RPMI media, then mixed with primary donor PBMC cells at a 30:1 Effector:Target ratio (25 μl 300,000 Effector cells; 25 μl 10,000 Target cells), along with 75 μl of GzB substrate, and incubated for 5 min at RT. Twenty five microliters of five-fold serially diluted plasma (beginning at a dilution of 1:200), 50 μg/ml HIV-Ig [NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from NABI and NHLBI (cat# 3957) (52)] positive control, or 50 μg/ml commercial IgG (SouthernBiotech, #0150-01) negative control were added, and incubated at RT for 15 min. Tubes were spun at 300 × g for 1 min, and incubated at 37°C, 5% CO2, for 1 h. Fixed cells (final concentration 1.85% formaldehyde, 10 min) were washed, then resuspended in buffer for flow cytometric analysis. Because fixation could result in loss of GzB signal, samples were immediately analyzed on an LSRII (BD Bioscience) instrument and with FlowJo 10.4 software (Treestar). Target cell only controls were run to ensure proper gating of targets vs. effector cells. Target with added effector cells in the absence of antibody (media only) were included to quantify background GzB signaling. Values that became negative after subtraction of background were normalized to 0%. Negative (commercial IgG) and positive (HIV-Ig) controls were included in every assay run. Assays were not valid unless the percentage of GzB positive cells in the positive control tube were >10%, after subtraction of background activity. Only results from valid assays are included. Example Granzyme B assay flow-plots are included in Supplementary Figure 1. To test for inherent differences across the gp120 proteins that could influence the assay, negative and positive control GzB activity was examined. There were no statistical differences between the T/F gp120 proteins in background (Target + Effector cell only samples), negative control (commercial IgG), or positive control (HIV-Ig) GzB activity (Supplementary Figures 2A–C). There was also no statistical difference in all experimental values between the two effector cell donors, as values obtained from each donor were not statistically different (Supplementary Figure 2D, Mann–whitney, p > 0.05) and were highly correlated (Supplementary Figure 2E, Spearman, p < 0.0001, r = 0.83). To measure GzB activity induced by mAbs, the protocol was repeated in an identical manner, with all corresponding positive and negative controls, but with experimental mAbs tested at five-fold serial dilutions beginning at 5 μg/ml.

B Cell Sorting

The same preparations of T/F Env gp120 proteins as used in the GzB assay, described in detail above, were used for single B cell sorting. Cryopreserved PBMC obtained from the sites in Rwanda and Zambia (~5–10 million cells/vial) were washed and resuspended in PBS + 2% FBS. After counting, cells were incubated with 8 μg of the autologous T/F gp120 per million cells and stained with: live/dead Aqua (Invitrogen, # L34957), anti-CD3 Pacific Blue (BD Pharmingen, # 558124), anti-CD19 BV650 (BioLegend, # 302237), anti-IgG FITC (BD Pharmingen, #555786), anti-His PE (Miltenyi Biotec, # 130-098-810), and single-cell sorted into 96-well plates containing 20 μl cell lysis buffer (Superscript III RT buffer, Tween, DTT, Invitrogen, #18080-044) (RNaseOUT, Invitrogen, #100000840). Sorts were carried out using a FACS Aria Cell Sorter and the following gating strategy: size, singlets, live, CD3-, CD19+, IgG+, gp120/His+. CD3+ gp120/His+ cells were monitored as positive controls for gp120 binding and detection.

Immunoglobulin heavy and light chain variable domain regions were PCR-amplified as previously described, using oligo-dT (ThermoScientific, #AM5730G) and SuperScriptIII (Invitrogen, #18080-044) (53). IgG-specific primer was also included in the RT reaction for individuals R53F, R774F, and Z1047M to increase recovery of heavy chain regions. Five microliters of the RT reaction was used to amplify heavy (IgG only), kappa, and lambda chain variable regions using high performance liquid chromatography (HPLC) purified primers as described in Liao et al. (53) and Phusion Hotstart II High Fidelity DNA Polymerase (Thermo Scientific, #F537S) for first round nested PCR. For the second round PCR reaction, 2.5 μl of first-round was used as a template. PCR amplified variable regions were gel purified (Qiagen, #28706) and combined with a CMV promoter containing DNA fragment, and the appropriate corresponding constant region DNA fragment (including a polyA tail) via overlapping PCR (53). Plasmids HV0024, HV0023, HV0025, and HV0026 were kindly provided by Dr. Larry (Huaxin) Liao at the Duke Human Vaccine Institute. The assembled full-length heavy- and light-chain segments were then cloned into pCR2.1TOPO-TA (ThermoFisher, # K4500-40) for long-term storage and mAb expression. VH and VL plasmid pairs were co-transfected at a 1:2 ratio into 6-well plates containing Expi293F cells (Thermo Fisher Scientific, Expi293 Expression System Kit, #A14635). Five to seven days post-transfection, mAbs were purified from the cell culture supernatant using Ab SpinTrap with Protein G Sepharose High Performance (GE Healthcare, # 28408347). The concentrations of the purified mAbs were quantified on an Octet RED96 using Anti-Human IgG Fc AHQ biosensors (ForteBio, #18-5001). mAb expression plasmids that could be stably amplified and resulted in transfections with yields >100 μg/ml were carried forward with antibody effector function characterizations.

PCR and Sequence Analysis of Immunoglobulin Variable Domains

The nucleotide sequence of each PCR amplicon was determined by Beckman Coulter Genomics or Sequetech using the following primers: H-R474 (5′- GCTGTGCCCCCAGAGGTG-3′) or K-R405 (5′-GACAGATGGTGCAGCCACAGTTCG-3′) or L-R400 (5′-CAGAGTGACCGAGGGGGCAGC-3′). These partial sequences were analyzed with NCBI IGBLAST for putative germline gene identification (https://www.ncbi.nlm.nih.gov/igblast/igblast.cgi) (Supplementary Table 1) (54). After assembly of the PCR fragments and cloning, plasmids were sequenced with the appropriate reverse primer, as well as CL-F681 (5′- TCTGGGTTCCAGGTTCCACTGGTGAC-3′). The resulting sequences were re-analyzed using IMGT vquest (http://imgt.org/IMGT_vquest/vquest) for germline gene verification, framework and CDR mapping, quantification of percent identity to germline, CDR H3 and L3 amino acid length, molecular mass, and pI (Supplementary Table 1) (55, 56). Circos diagrams were generated via the Circos online tool (http://mkweb.bcgsc.ca/tableviewer/) (57). CDRH2 grand average of hydropathy (GRAVY) scores were calculated using an online tool (http://www.gravy-calculator.de/). CDRH3 tyrosine sulfation sites were enumerated with the Sulfinator online tool (http://web.expasy.org/sulfinator/) (58). Sequences were uploaded to Genbank under the following accession numbers: MK269362–MK270058.

Assessment of Affinity and Epitope Binning Using Biolayer Interferometry (BLI)

Affinity assays were performed on an Octet RED96 (ForteBio, Inc, Menlo Park, CA) at 30°C, with 1,000 rpm agitation. Each mAb was immobilized on an Anti-hIgG Fc Capture (AHC) biosensor (Fortebio, # 18-5060) at a concentration of 25 μg/ml for 300 s, and a baseline reading was recorded for 60 s in kinetics buffer (PBS with 0.01% BSA, 0.02% Tween20, and 0.005% sodium azide). Sensors were then immersed in varying molarities of the corresponding autologous T/F gp120 protein for 300 s, then allowed to dissociate in kinetics buffer for 600 s. Sensors were regenerated before each kinetics assay. Human IgG at 25 μg/ml dipped into kinetics buffer was included on every assay plate for reference during analysis (SouthernBiotech, #0150-01). Molarity (range 5–0.06 μM), association time (range 300–600 s), and dissociation time (range 600–1,200 s) were varied as necessary to obtain appropriate X² and R² values. Anti-influenza HA antibody EM4C04 (kindly provided by Dr. Jens Wrammert, Emory University) and/or commercial IgG were included to survey for non-specific mAb binding against each gp120.

K_D values were calculated using ForteBio Data Analysis 9.0. After reference subtraction, Y-axis data was aligned to the 50–59.9 s of the baseline reading. For inter-step correction, data was aligned to the dissociation step. Data was then processed with Savitzky-Gola filtering, and curve fitting was performed using a 1:1 binding model. K_D values were averaged from ≥2 reads (average number of reads ≥3) with appropriate X² and R² (median X² = 0.11, range = 0.00–1.94; median R² = 0.99, range = 0.93–1.0) (Supplementary Table 3).

Binning (competition) assays were performed at 30 degrees C, with 1,000 rpm agitation. Synthesized gp120 (R66M T/F Env O20) at a concentration of 25 μg/ml was immobilized on an Anti-Penta-HIS (HIS1K, ForteBio, # 18-5120) biosensor for 300 s, and a baseline reading was recorded for 30 s in kinetics buffer. Sensors were then immersed in 0.66 μM primary (R66M-derived) mAb for 10 min. R66M-derived mAbs that resulted in a primary signal of <0.1 at 0.66 uM (lower affinity mAbs) were increased in molarity, as production allowed, to reach that threshold. mAbs that could not be produced at this concentration were not analyzed. After a second baseline reading for 30 s in kinetics buffer, sensors were immersed in 0.66 μM secondary antibody (“reference mAb”—VRC01, 3074, PGT121, R66M 7C12) (VRC01 mAb Heavy Chain Expression Vector and VRC01 mAb Light Chain Expression Vector was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: from Dr. John Mascola (cat# 12035 and 12036) (59, 60); 3074, kindly provided by Xiangpeng Kong, NYU School of Medicine; PGT121, PGT 121 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (PGT121) catalog #12343 (61) to assess reference mAb binding capacity in the presence of primary R66M mAb binding. Reference mAb as primary and secondary mAb was included as a positive control for competition (reduced reference binding). EM4C04 (anti-influenza mAb) primary and reference mAb secondary was used as an additional control for non-specific competition (peak reference binding). Binning values were calculated using ForteBio Data Analysis 9.0. R66M mAbs capable of reducing reference mAb binding to <50% of reference binding in the presence of EM4C04 primary (set at 100% binding), were considered competitors for that reference mAb.

Flow Cytometric Analysis of mAb Binding to Surface Env With and Without sCD4

293T cells were transfected with a plasmid encoding the T/F env gene isolated from R66M (the same plasmid utilized for generation of R66M Env pseudovirus) using Fugene HD (Promega). At 48-h after transfection, cells were washed with PBS, and briefly trypsinized. After washing again with PBS, cells were aliquoted and resuspended in flow buffer (PBS + 5% FBS), with or without 25 μg/ml recombinant human sCD4 (R&D Systems, 514-CD), and allowed to incubate for 30 min at RT. Cells were then incubated with 25 μg/ml polyclonal IgG (SouthernBiotech, #0150-01), 5 μg/ml PGT121, 10 μg/ml of CD4i mAb17b, or an R66M mAb at RT for 30 min. After washing with flow buffer, samples were then incubated with Goat Anti-Human IgG Fc biotin (SouthernBiotech, # 2014-08) at 1:200 for 20 min at RT. After washing with flow buffer, samples were incubated with 1:1,000 PE-conjugated streptavidin (BD Pharmingen, #554061) for 20 min at RT, while protected from light. Cells were then washed and fixed for 10 min (3.7% formaldehyde). The percentage of PE/Env positive cells were determined via quantification on an LSRII (BD Bioscience) instrument and with FlowJo 10.4 software (Treestar). All samples were analyzed on at least three independent occasions. PGT 121 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (PGT121) (61). 17b was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-HIV-1 gp120 Monoclonal (17b) from Dr. James E. Robinson (62–67).

Memory B Cells With VH1-69 Dominate the Early Anti-gp120 Response in Five of Six Individuals

To understand the early antibody responses to the autologous HIV-1 T/F Env gp120 at higher resolution, variable heavy (VH) and variable light (VL) gene segments were recovered from single IgG+ memory B cells sorted for reactivity with the autologous T/F Env gp120 protein using cryopreserved PBMC collected at 7.5 months after infection (Time Point 2). This approach was designed to capture a wide variety of Env-specific B cells that were activated and selected for survival during the first 7 months of HIV-1 infection, and had the capacity to contribute to the plasma nAb and ADCC functions observed at this time point (Figure ​(Figure1B).1B). A total of 352 Env-specific memory B cells were recovered, and the VH and VL germline gene usage and pairings present in each subject are illustrated in Figure ​Figure2.2. In the Circos plots, each colored ribbon indicates a unique VH/VL combination, and the width is proportional to frequency (57). Broad diversity of VH and VL usage and pairing was observed in each individual, supporting that a highly polyclonal antibody response against epitopes in gp120 had occurred during the first months of infection.

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Figure 2

Circos diagrams of VH and VL pairing networks at 7.5-months post-infection (time point 2). Germline genes were assigned to each VH and VL sequence from each gp120-reactive single B cell using NCBI IGBLAST and vquest analysis. Circos diagrams were utilized to illustrate the network of VH germline (shades of blue VH-1, yellow VH-2, shades of red VH-3, shades of green VH-4, purple VH-5, pink VH-6) with the paired VL κ (dark gray) or λ (light gray) germline from individuals infected by HIV-1 Subtype A (left column), Subtype C (center column), and Subtype A/C unique recombinants (right column). Band width is proportional to the number of paired sequences that contain those VH and VL pairs. The total number of paired sequences analyzed for each individual is shown.

Notably, VH1-derived B cells were commonly elicited in these individuals (Figure ​(Figure2,2, shades of blue), similar to what has been described in chronic HIV-1 infection, VH1-derived B cells (68–72). Specifically, VH1-69 was the dominant VH germline gene utilized by T/F gp120-specific memory B-cells in five of the six individuals studied (Figure ​(Figure2,2, royal blue). The frequency of VH1-69 B cells in those five individuals ranged from 21 to 51% (Figure ​(Figure3A).3A). Indeed, the VH1-69-lineage was not only dominant, but also over-utilized in all five individuals when compared to frequencies obtained from circulating B cells with successfully rearranged IGHV genes [reported range 3.1–9.7 73], or a previous analysis of eight HIV-1 vaccine volunteers pre-vaccination [reported range 0.7–8.2% IgG peripheral blood 74]. Therefore, we investigated in more detail the dominance of VH1-69 to understand the impact of this immunological phenomenon on the early antibody responses.

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Figure 3

VH1-69 is over-represented among anti-gp120 memory B cells at 7.5-months post-infection. (A) Percentages of VH1-69 germline usage (blue graph sections) vs. all other variable heavy-chain germline genes (gray graph sections) isolated from memory B cells reactive to autologous T/F gp120 in six individuals infected with Subtype A (left column), Subtype C (center column), and Subtype A/C unique recombinants (right column). The total number of heavy chain sequences analyzed for each individual is shown. (B) To determine whether these VH1-69 sequences maintained a hydrophobic complementarity-determining region-2 (CDRH2) associated with VH1-69, grand average hydropathy (GRAVY) scores (Y-axis) were calculated from non-VH1-69 (black symbols) and VH1-69 sequences (colored symbols) obtained from each individual (http://www.gravy-calculator.de), bars represent median values. Individual codes are listed below the X-axis. VH1-69 CDRH2 were significantly more hydrophobic (positive values) than the non-VH1-69 CDRH2 in every individual (Ordinary one-way ANOVA, Tukey's multiple comparisons test: R53F p = 0.018, all others p < 0.0001). Raw values are reported in Supplementary Table 1.

Pathogenically cross-reactive VH1-69 lineages likewise dominated a DNA prime/trivalent gp140 protein boost HIV vaccination regimen (74). And, hydrophobic interactions between VH1-69 CDRH2 and hydrophobic cavities within HIV-1 gp120 have also previously been observed (30). In the data collected here, grand average hydropathy (GRAVY) scores of isolated T/F gp120-specific VH1-69 CDHR2 amino acid sequences ranged from 0.34 to 2.7, with median GRAVY scores ranging from 1.28 to 1.89, indicating high hydrophobicity (Figure ​(Figure3B,3B, Supplementary Table 1). In contrast, the median CDRH2 GRAVY score for the corresponding non-VH1-69 T/F gp120-specific B cells for each individual was significantly lower, ranging from −1.16 to −0.46 (Ordinary one-way ANOVA, Tukey's multiple comparisons test: R53F p = 0.018, all others p < 0.0001). These observations reinforce the concept that high CDRH2 hydrophobicity is a unique and universal feature of the VH1-69 antibodies.

During influenza infection, the presence of a phenylalanine (F) or leucine (L) polymorphism at position 54 can strongly influence the frequency of VH1-69 utilization in anti-influenza antibodies, and their resulting functions (75). Though individuals' genomes were not sequenced to determine specific alleles within this study, this information was inferred from the isolated VH1-69 sequences. Within this group of individuals, VH1-69 containing 54F (associated with neutralization of influenza) comprised the majority of anti-gp120 VH1-69 sequences in five of the six individuals (Figure ​(Figure4,4, green). These findings reflect previous analyses that noted the 54L-homozygous genotype is rarely found in African populations, with 54F-homozygous and 54F/L-heterozygous alleles composing the vast majority of VH1-69 genotypes (2, 76). Interestingly, the 54L polymorphism was observed in the majority of VH1-69 sequences from one individual, R66M (73%, Figure ​Figure4,4, Supplementary Tables 1, 2), while it was at a much lower frequency of between 0 and 14% in the other individuals. VH1-69 sequences that contained an amino acid other than F or L at position 54 were isolated from three individuals: R774F, Z1800M, and R66M, further demonstrating the polymorphic nature of VH1-69 alleles.

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Figure 4

VH1-69 polymorphisms at amino acid 54 within complementarity-determining region-2 (CDRH2). The frequency of 51p1-like 54F (green), hv1263-like 54L (red), and other amino acids in six individuals infected with Subtype A (left column), Subtype C (center column), and Subtype A/C unique recombinants (right column) is illustrated. The 54F polymorphism was dominant except for R66M (bottom right), where 54L was present in the majority of the anti-gp120 VH1-69 sequences and R53F where only one VH1-69 sequence was present. A minority of VH1-69 sequences contained “other” amino acids (black) at this position in three individuals: R774F, Z1800M, and R66M. The total number of VH1-69 sequences analyzed for each individual is shown. Raw values are reported in Supplementary Table 2.

VH1-69 Utilizing mAbs Mediate ADCC Effector Functions but Are Poorly Neutralizing

In the context of influenza, “innate-like” VH1-69 lineage mAbs can be not only broadly reactive, but broadly neutralizing as well (77). To explore the potential antiviral functions of the dominant anti-gp120 VH1-69 antibody response in early HIV-1 infection, mAbs were produced by cloning VH and VL regions into expression vectors that provided a common IgG1 constant region (53). Thus, all mAbs had identical Fc regions, allowing us to attribute differences solely to the variable domains. Neutralizing activity was present against the autologous T/F Env in five of the six plasma samples contemporaneous to the antibody isolation (Figure ​(Figure1B).1B). We therefore quantified the neutralization capacity of 105 VH1-69 mAbs against virions pseudotyped with the corresponding autologous T/F Env in the same manner. It should be noted that only one VH1-69 mAb was regenerated from subject R53F, due to the genetics of the B cell response (Figure ​(Figure2).2). At 10 μg/ml, none of the mAbs demonstrated potent autologous neutralizing activity, when defined as the ability to reduce infectivity of the T/F Env to below 50% (Figure ​(Figure5A,5A, Supplementary Table 4). These VH1-69 antibodies are therefore probably not responsible for the observed plasma nAb activity.

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Figure 5

Effector function capabilities of VH1-69 antibodies. (A) A concentration of 10 μg/ml of each mAb was evaluated in the TZM-bl neutralization assay against the autologous T/F Env pseudovirus for each individual (labeled below the X-axis). Percent residual infectivity of the T/F Env pseudotyped virus for individual mAbs, relative to virus-only controls, is shown (Y-axis). Symbols represent the mean of triplicate wells, bars represent the median of all mAbs from each individual. (B) Each mAb was used in a five-fold dilution series of 5, 1, and 0.2 μg/ml against CEM.NKr.CCR5 cells coated with autologous T/F Env gp120. The peak percentage of cells positive for Granzyme B (GzB) activity over this dilution series, after subtraction of background activity (percentage of GzB positive cells in target + effector reactions), is shown on the Y-axis. Each symbol represents the mean of two separate assays, utilizing effector cells from two independent HIV-negative effector cell donors. The activity of each mAb against target cells coated with the autologous gp120 are labeled for each individual subject below the X-axis. “High” activity was defined as achieving GzB activity >10% (dashed line, Y-axis). Bars indicate the median value for each subject. Raw values are reported in Supplementary Table 4.

Additionally, ADCC activity was also present in the 7.5-month plasma samples in all individuals except R53F (Figure ​(Figure1B).1B). We therefore evaluated 94 VH1-69 mAbs (overlapping almost completely with the 105 analyzed for nAb activity) for their capacity to mediate ADCC functions in the same manner, using concentrations of 5, 1, and 0.2 μg/ml of each mAb against CEM.NKr.CCR5 cells coated with the autologous T/F Env gp120 (Supplementary Table 4). In contrast to the universally low neutralization capacity exhibited by the VH1-69 mAbs, 27 VH1-69 mAbs mediated high GzB activity, defined as having a peak of GzB positive cells >10% over the course of the dilution series (Figure ​(Figure5B,5B, Supplementary Table 4), a conservative threshold based on natural variation in “target + effector only” control values between patient-derived gp120s (Supplementary Figure 2). Furthermore, “high GzB” VH1-69 mAbs were present in the same five individuals that exhibited plasma ADCC activity (Figure ​(Figure1B).1B). The largest proportion of high ADCC-mediating VH1-69 mAbs was observed in Z1800M (56% of tested mAbs), while the highest magnitude was observed in a subset of R66M mAbs (more than 30% peak GzB signaling). As noted above, only one VH1-69 antibody was recovered and tested from subject R53F. Interestingly, R53F was also the only subject that did not have detectable plasma GzB activity at the 7.5-month time point (Figure ​(Figure1B).1B). Thus, some anti-gp120 VH1-69 mAbs exhibit high ADCC-mediated effector capabilities but poor neutralizing activity in early HIV-1 infection, based on the analysis of memory B cells in these six individuals. These VH1-69 antibodies could contribute to the GzB activity mediated by the contemporaneous plasma.

High ADCC Activity Is Associated With Shorter CDRH3 Regions and Higher Isoelectric Point in VH1-69 mAbs

Because all of the VH1-69 mAbs studied here have the same Fc region, any differences in the ability to mediate ADCC effector function such as GzB signaling must be attributable to the variable regions. Therefore, all of the VH1-69 mAbs shown in Figure ​Figure5B5B were divided into two groups: those with peak GzB positive cells >10% (after subtraction of background activity), and those below 10%, to search for defining features. When stratified in this manner, there was no statistical difference between the two groups in a number of genetic and functional features, including VH identity to germline, number of tyrosine sulfation sites within CDRH3 (30, 58), CDRH2 GRAVY score, kappa or lambda VL pairing, or binding affinity for the autologous T/F gp120 (Supplementary Figure 3, Supplementary Tables 1, 4). However, there was a significant difference between high and low ADCC VH1-69 antibodies in autologous neutralization capacity, with some of the low GzB antibodies capable of weak autologous neutralization (p = 0.0015, Supplementary Figure 3, Supplementary Table 4). It is important to note that, despite this difference, neither group of antibodies was capable of potent autologous neutralization at 10 μg/ml, which is a relatively high concentration (GzB <10% median = 97% infectivity; GzB >10% median = 106% infectivity).

In contrast, CDRH3 length did emerge as a defining feature of high Gzb activity, with antibodies capable of mediating high ADCC activity having statistically shorter CDRH3 amino acid length (p = 0.0007, Figure ​Figure6A,6A, Supplementary Tables 1, 2). However, the median CDRH3 length for these high GzB antibodies was not unusually short compared to the median length determined for rearranged human B cells (median CDRH3 length for GzB >10% = 14 AA vs. reported median value = 15 AA) (70). This observation suggests that long CDRH3 regions may be less conducive to antibody-Fc interactions as measured in this setting.

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Figure 6

Genetic signatures of VH1-69 mAbs capable of mediating high ADCC activity. (A) Amino acid length of mAb complementarity-determining region-3 (CDRH3) (Y-axis) of mAbs with GzB <10% are longer than those from mAbs that mediate high ADCC activity (Mann–Whitney, p = 0.0007). (B) Distribution of J1 (red), J2 (orange), J3 (yellow), J4 (green), J5 (blue), and J6 (purple) utilization of VH1-69 mAbs that mediated low ADCC activity compared to (C) J-region utilization in mAbs that mediated high ADCC activity revealed differential J-region usage between the two groups of mAbs (Chi-square test, p = 0.0004). Number of sequences analyzed and percentage of each J-region utilized is indicated. (D) A modest difference in isoelectric point (Y-axis) of the heavy chain of the antibodies able to mediate high ADCC vs. low ADCC also emerged (Mann–Whitney, p = 0.035). ^*p ≤ 0.05, ^***p ≤ 0.001.

To further investigate the association of CDRH3 length and high ADCC function, we focused on the impact of the J region, which is selected during VDJ rearrangement and contributes to the length of CDRH3. While there are six J_H regions in the human repertoire, it is striking that bnAbs commonly use the longest J region, J6, including b12, 3BNC117, 8ANC131, PGT145, PG9, PGT121, PGT151, and 2F5 (16). We therefore examined J region usage in the VH1-69 mAbs to gain a better understanding of how CDRH3 length was generated. Using the VH1-69 mAbs grouped by low or high GzB activity as shown in Figure ​Figure6A,6A, similar J1, J3, and J4 usage was found between the two groups (Figure ​(Figure6B6B low and Figure ​Figure6C6C high, Supplementary Tables 2, 4). Conversely, J6 was more frequently utilized by low GzB antibodies (22.0%) compared to the high GzB group (3.9%), the latter of which had higher J5 usage. None of the VH1-69 mAbs contained J2. The overall distribution of J chains was significantly different between the two groups of VH1-69 antibodies (Chi-square, p = 0.0004, J2 was excluded due to the zero frequency). Thus, the infrequent presence of J6 could contribute to shorter CDRH3 regions in this antibody population. There was also a significant difference in the calculated isoelectric point (pI) for the heavy chain of the antibodies able to mediate high GzB activity vs. those that did not (p = 0.035, Figure ​Figure6D,6D, Supplementary Table 2). The pI is the pH at which the antibody has no net charge, and can be estimated based on the amino acid sequence. pI can influence such parameters as tissue distribution and clearance in vivo. Interestingly, mAbs that mediated higher GzB activity exhibited a higher, closer to neutral, median isoelectric point (4.58 vs. 6.15). Thus, at a physiological pH, the high GzB mAbs would be less negatively charged than the low GzB mAbs, perhaps influencing their recognition of certain epitopes. Overall, this analysis revealed that VH1-69 antibodies that mediate higher GzB activity against target cells coated with the autologous T/F Env gp120 protein have unique features that include relatively shorter CDRH3 regions, associated with J chain preference, and a closer to neutral isoelectric point than those with low GzB activity.

We gratefully acknowledge the staff, interns, and participants of the Zambian and Rwandan cohorts. This work was facilitated by the Emory Vaccine Center Flow Core of the Center for AIDS Research at Emory University (P30AI050409). We extend our gratitude to Kiran Gill and Barbara Cervasi for their assistance in the flow core. Plasmids HV0024, HV0023, HV0025, and HV0026 were kindly provided by Dr. Larry (Huaxin) Liao, Duke University (53). Anti-V3 antibody 3074 was kindly provided by Dr. Xiangpeng Kong, NYU School of Medicine. Anti-influenza antibody EM4C04 was kindly provided by Dr. Jens Wrammert, Emory University. This work was funded in part by IAVI and made possible by the support of many donors, including United States Agency for International Development (USAID). The full list of IAVI donors is available at http://www.iavi.org. The contents of this manuscript are the responsibility of the authors and do not necessarily reflect the views of USAID or the US Government.