Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C–infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 μg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2–neutralizing V3 Abs and demonstrates partial reproduction of the human donor’s humoral immune response through nonhuman primate vaccination.

This article is featured in Top Reads, p.921

The HIV envelope glycoprotein (Env) mediates viral attachment to host cells and presents the only known target for neutralizing Abs (NAbs). Composed of a trimer of gp41 and gp120 heterodimers, the native conformation oscillates between a “closed” state 1 configuration largely immunogenically protected by a glycan shield, a transitional and partially relaxed state 2, and an “open” state 3 conformation induced by CD4 receptor binding (13). The preponderance of the poorly immunogenic state 1, coupled with sparse Env distribution on the viral surface that precludes extensive BCR crosslinking (4), hinders the generation of a controlling humoral response during infection. Virus strains are categorized into tiers designating sensitivity to HIV+ plasma neutralization, from tier 1A (very high), tier 1B (above average), tier 2 (moderate), to tier 3 (low) (5, 6). Abs capable of neutralizing autologous and heterologous tier 1 strains often appear within a few months after primary infection (5, 7). Due to low replication fidelity and recombination events that rapidly generate diverse viral swarms (8), these Abs are generally ineffective at controlling viral replication. In addition to the challenges facing natural control, vaccine efforts are confounded by the enormous diversity of circulating HIV strains (6, 9, 10) and the challenge of generating subunit vaccines that are faithful mimics of the native Env trimer (11).

Nevertheless, broadly NAbs (bNAbs) displaying near panneutralizing activity against globally representative virus panels have been identified in clinical subjects able to suppress viral replication and suggest the potential for inducing such protective Abs by vaccination (1217). In contrast to autologous NAbs, bNAbs typically bind conformational epitopes on closed state 1 Env, and most display high levels (19–25%) of V region nucleotide somatic hypermutation (SHM) driven by several years of virus-BCR interplay (1821). Numerous Ig lineages encoding bNAb precursors have been identified (16, 17, 22). Thus, a major focus of current vaccine efforts is to drive affinity maturation from known lineage precursors with the goal of generating Abs that neutralize >60% of heterologous multitier and multiclade viruses (2328). If achieved, and coupled with other preventive measures, elicitation of such Abs could enable widespread control of the pandemic (29, 30).

Preclinical vaccine development is largely dependent on nonhuman primate (NHP) models. Although making essential contributions, immune receptor germline gene disparity and the reduced pathogenic phenotype of SIV are limitations that can confound translatability of these models (31, 32). We previously evaluated whether viral Env variants circulating in two human donors demonstrating modest neutralization breadth against heterologous tier 2 viruses at 1.29 and 1.79 y after primary infection (33) could similarly produce breadth through vaccination in NHPs. Viral quasispecies circulating in these subjects prior to and concurrent with the development of neutralizing breadth were identified and Envs were first screened for immunogenicity in rabbits (34). The most promising candidate immunogen combinations, composed of two to six Env quasispecies, were then administered to macaques as adjuvanted recombinant gp140 trimeric protein in combination with full-length gp160 DNA expression plasmids. Transcutaneous delivery of DNA was used to facilitate the presence of native-like conformations within the epidermal layer of the skin by direct intracellular delivery into APC in the epidermis (3539). After three DNA + Protein coimmunizations, plasma from all NHPs in both immunization groups neutralized select heterologous tier 1 and tier 2 viruses (40).

In this study, we characterized both NAbs and nonneutralizing Abs generated from macaque vaccination using Env empirically curated from donor quasispecies. We show that macaque vaccination at least partially reproduced the humoral response observed in the Env donor by similarly focusing a V3- and CD4b-directed NAb response and elicited polyfunctional Abs with robust clade-specific neutralization comparable to those generated in small animals to date (25, 4143). This study informs translatability of NHP vaccination and the contribution of unconstrained trimers as immunogens, particularly the elicitation of potent and highly functional V3 Abs, albeit limited by clade-restricted breadth.

Rhesus macaques of Indian origin were cohoused at the Oregon National Primate Research Center, and all care and experimental protocols were approved by the institutional animal care and use committee at Oregon Health & Science University. Rhesus macaques were coimmunized four times on weeks 0, 4, 12, and 20 with 36 μg of gp160 DNA composed of six different Envs in expression plasmids delivered intradermally via gene gun (PowderMed) and i.m. with 50 μg of recombinant gp140 trimeric protein composed of two Envs and adjuvanted with 20% Adjuplex (Sigma-Aldrich). The Envs used as immunogens are as follows: 041504 G10a, 041504 G6a, 041504 F8, 101504 C6a, 101504 E5a, and 101504 H10 (DNA); 041504 F8 and 101504 C6a (protein) representing early breadth development in human patient; and VC10014 from 1.29 to 1.79 y postinfection. A schematic of the immunization scheme is shown in Fig. 1, and all animal studies are detailed in the vaccine study publications (34, 40).

To generate an Env probe, autologous F8 gp140 trimeric protein was expressed with an avidin tag (Blue Heron Biotech), conjugated to biotin (GeneCopoeia), and labeled with streptavidin/allophycocyanin (Invitrogen). Week 24 PBMCs from macaque 25257 were sorted with a three-laser FACSAria Cell Sorter (BD Biosciences) for Env-specific live CD3, CD14, CD20+, CD27+, and IgG+ single B cells as shown in Supplemental Fig. 1. Sorted B cells were stored at −80°C until RNA isolation using RNeasy Mini Kit (Qiagen) and cDNA synthesized with random hexamer primers using Sensiscript RT (Qiagen) according to the manufacturer’s directions. Cloning and expression of mAbs was performed as described previously (44, 45). In brief, variable regions on both IgH and IgL were amplified by two rounds of PCR using HotStar Taq Plus polymerase (Qiagen) using the primer sets listed in Supplemental Fig. 1B. Amplified products were visualized on a 2% agarose gel, and all ∼450-bp bands were purified and sequenced. Restriction sites were inserted via PCR using primers and cycling reactions as described (45), and VDJ (H chain) or VJ (L chain) sequences digested and ligated into the Abvec Igg1H, Iggκ1, or Iggλ2 expression vector (46), which was then used to transform XL10-Gold Ultracompetent Cells (Agilent). Correct plasmid inserts for all IgH and IgL were confirmed by sequencing prior cotransfection of 293F cells for Ab expression. Ab was purified from culture supernatants over a protein A-Sepharose column (GE Healthcare) as described (45).

Neutralization assays were performed in TZM-bl cells expressing luciferase under control of the HIV tat promoter as standardized by Wei et al. (47) using single cycle–competent pseudoviruses (48). In brief, 3-fold serial dilutions of Ab were incubated in with the indicated pseudovirus for 1 h in DMEM (Life Technologies) supplemented with 4.5 g/l d-glucose, 10% heat-inactivated FBS, 1% l-glutamine, 1% penicillin, and 1% streptomycin at 37°C and 5% CO2. Next, 1 × 104 cells per well were added in media further supplemented with 7.5 μg/ml DEAE-dextran to aid viral entry. After 48 h of coincubation, all but 40 μl of media was gently removed from each well and adherent cells were lysed by the addition of 60 μl of Bright-Glo (Promega) for 2 min. Eighty microliters was then transferred to black plates and viral infection determined as the relative level of luciferase activity measured on a Victor X Light Plate Reader (Perkin Elmer). All experimental samples were plated in duplicate and the percent neutralizing activity was calculated using the following formula: [Mean no Ab (i.e., virus and cells minus cells-only background) − Mean sample (i.e., sample value minus cells-only background)]/[Mean no Ab] × 100. Isotype and positive control Abs were included on all assay runs.

Competition neutralization assays were performed with SF162 pseudovirus as described above, except that prior to incubation with the known NAb, pseudovirus was incubated for 30 min with 0.05 μg/ml (∼IC50) O.2 or O.11.

O.1–O.58 were screened for linear epitopes using a clade B Env consensus peptide pool (AIDS Reagent catalog no. 12438). Flat-bottom 96-well plates were coated with 10 μg/ml peptide pool by incubating overnight at 4°C in 0.2 M H2CO3 buffer at pH 9.4. Ab binding was detected by ELISA (described below) using 10 μg/ml of each Ab. Those suspected of positive binding were then tested in wells coated with 2 μg/ml individual 15-mer peptides (catalog no. 9480), and linear epitope regions were inferred as shown in Supplemental Fig. 2. Subsequent ELISAs were performed using 4 μg/ml CD4 binding site (CD4bs) mimetic recombinant surface core (RSC-3) (12), V1/V2 scaffold protein (49), or SF162 gp41 for NAbs O.2 and O.11.

Assays were performed largely as described by Malherbe et al. (50). Recombinant monomeric gp140 (F8 and SF162) or gp120 (BaL and JRCSF) was coated on flat-bottom plates by incubating 0.5–1 μg/ml in 0.2 M H2CO3 buffer (pH 9.4) at 4°C overnight. Plates were then washed three times in binding buffer (PBS [pH 7.4] + 0.1% Triton X-100) and blocked with 150 μl of PBS containing 5% dried milk and 1% goat serum for 1 h at room temperature. Blocking buffer was discarded, and 3-fold serial dilutions of Ab were added to unwashed cells in 50 μl of binding buffer. After 1 h at room temperature, plates were washed three times and then incubated for 1 additional h with 50 μl of 1:5000 dilution of goat anti-human H and L chains, conjugated to HRP (Invitrogen). Plates were then washed five times, and bound Ab was visualized by the addition of 50 μl of tetramethylbenzidine (SouthernBiotech) for 10 min before stopping the reaction with 50 μl of 1 N H2SO4. OD was immediately quantified on a SoftMax Pro 5 microplate reader (Molecular Devices) at 450 nm.

Binding kinetics were measured with an OctetRed384 using anti-human IgG Fc capture biosensors (ForteBio) according to the manufacturer’s recommendations. In brief, 5 μg/ml of each mAb was used as ligand and incubated with 120 nm of the stabilized native-like HIV-1 Env trimers, F8.V3.N7 SOSIP or BG505 SOSIP analytes serially diluted 2-fold in PBS with 0.1% BSA and 0.02% Tween-20. Running parameters were as follows: baseline (60 s), ligand binding (180 s), baseline (60), association (800 s), dissociation (2000 s), followed by biosensor regeneration in 10 mM glycine buffer (pH 2.8). Assays were run with a plate temperature of 29°C and shake speed of 1000 rpm. Biosensors were regenerated no more than six times. Reaction constants were calculated when sensorgram displacement showed clear binding compared with analyte-only/ligand-only control wells (generally 0.2–1.5 nm) and where R2 ≥ 0.97. All results are representative of at least two independent runs.

Rhesus splenocytes were obtained from SHIV-naive macaques by passing fresh tissue through 75-μm filters. Target CD4 cells were enriched through MACS (Miltenyi) and cultured at 2 × 106 cells per milliliter in RPMI 1640 (Life Technologies) containing 15% macaque serum and 100 U/ml IL-2 (R15-100), 1.5 μg/ml staphylococcal enterotoxin B, 0.4 μg/ml anti-CD3, 1.5 μg/ml anti-CD28, and 1.5 μg/ml anti-CD49d. After 24 h, activated splenocytes were washed and resuspended in R15-100 and expanded for an additional 72 h before SHIV infection. Cells were divided into six-well plates at 4 × 106 cells milliliter with 200 ng/ml p27 SHIVBaL or SHIVSF162P3 and spinoculated at 1200 × g for 2 h at 25°C. Infected cultures from individual macaques were maintained at 2 × 106 to 4 × 106 cells milliliter in R15-100 for 7 d before use.

Ab binding to the surface of infected cells was measured by flow cytometry on infected splenocyte cultures originating from one macaque per assay. Infected cells were stained for viability and surface stained with 5 μg/ml test Ab followed by anti-human IgG Fc/PE prior to cell permeabilization and intracellular staining for p27/FITC. Cells were analyzed according to the gating scheme shown in Fig. 4D, and non-SHIV–reactive Deng-3 was used as an isotype control.

Determination of Ab-dependent cellular cytotoxicity (ADCC) was performed as described previously (51). In brief, CD4+CCR5+ NKR24 target cells that express luciferase under control of a tat-dependent promoter were infected with replication-competent SHIVSF162P3 or SHIVBaL (200 ng/ml p27) by spinoculation at 1200 × g for 2 h with 40 μg/ml polybrene. Three days postspinoculation, 1 × 104 target cells per well were coincubated with effector KHYG-1 NK cells at an E:T ratio of 10:1 with or without serial plasma or Ab dilutions in 200 μl of assay media (RPMI supplemented with 5 U/ml IL-2) in round-bottom 96-well plates at 37°C and 5% CO2. All Ab and plasma dilutions were plated in duplicate. Effector KHYG-1 NK cells expressing macaque or human CD16 were used for assays with plasma or cloned Ab, respectively. After 8 h of coincubation, each assay well was mixed by pipetting and 150 μl was transferred to black flat-bottom plates containing 50 μl of Bright-Glo (Promega) and incubated for 2 min at 25°C. Luminescence was measured on a Victor X Light Plate Reader (Perkin Elmer) and relative light units were normalized according to the following formula: [sample mean − background (mock-infected targets and effectors)]/[maximum (SHIV-infected targets and effectors, no mAb − background)] × 100. ADCC activity is reported as the percentage loss of relative light units.

The phagocytic potential of the NHP Abs was assessed by a method adapted from Ackerman et al. (52). Briefly, mAbs were 3-fold serially diluted to achieve a final concentration range of 3 nM–4 pM in 96-well tissue culture–treated microplates (CLS3596; Corning), followed by the addition of a homogeneous solution of a 1:20 ratio of effector THP-1 monocyte-like cells to target fluorescent Ag beads. The V3 glycan supersite targeting mAb 10–1074 was used as a positive control, and the negative control was mAb 10E8v4 that recognizes the membrane-proximal external region epitope not present on the gp120 Ag. Following a 4-h incubation at 37°C and 5% CO2, the sampled contents of each well were analyzed by flow cytometry, and Ab-dependent cellular phagocytosis (ADCP) results were reported as a phagocytosis score metric, defined as the product of the percentage of THP-1 monocytes that engulfed at least one fluorescent bead (the percentage of gated THP-1 cells with FITC signal above a threshold median fluorescence intensity) and the average number of beads engulfed (the median fluorescence intensity signal of the FITC + gated THP-1 cells). Fluorescent Ag beads were prepared by amine-coupling HIV-1SF162 gp120 protein (IT-001-0028p; Immune Technology) to carboxyl-functionalized low-intensity yellow fluorescent polystyrene beads (CFL-0852-2; Spherotech), and were confirmed to be properly functionalized by assessing anti–HIV-1 Ab staining alongside negative isotype controls. An immortalized monocyte-like cell line, THP-1 cells were purchased from American Type Culture Collection (TIB-202) and cultured and incubated in conditions consistent with manufacturer recommendations.

An individualized IGHV germline gene database from macaque 25257 PBMCs was produced from total mRNA (53). IgDiscover was used as an inference tool (54), and libraries for high-throughput sequencing were produced by 5′ multiplex PCR as previously described (55). Briefly, mRNA was reverse transcribed with IgM C region-specific primer containing a unique molecular identifier and a universal outer primer sequence. The cDNA was amplified using the universal 3′ primer and two leader/untranslated region primer sets, covering all gene families. Illumina indices and adapters were introduced by PCR prior to sequencing the library with Illumina’s MiSeq v3 Kit. The output library was analyzed with IgDiscover (v0.10a) to infer the germline gene IGHV alleles (54). The initial database used for the analysis was obtained from genomic DNA sequencing (56) with the addition of nonlocated (NL) alleles inferred from a larger set of macaques (53). The H chain sequences of the 58 isolated mAbs were assigned to the IGHV-individualized database from macaque 25257 and to the IMGT’s L chain database using IgBlast for analysis of SHM and CDR3 sequences. IGHJ sequences were identified using the IMGT rhesus macaque database.

Statistical analyses were performed using GraphPad Prism version 8.4.3. The specific tests are indicated for each figure in the legends. As O.19 and O.20 were considered to be identical mAbs cloned from two independent Env+ B cells, one of these was removed from all statistical comparisons (Figs. 4C, 5D, 5E, 6C, 6D) to avoid biasing phenotypic comparisons with a functional replicate.

Six rhesus macaques were coimmunized intradermally by gene gun with DNA plasmids expressing gp160 and i.m. by needle injection with gp140 recombinant clade B trimers formulated with Adjuplex adjuvant as reported previously (40). Immunogens were selected from Env quasispecies obtained from human study participant VC10014 first curated by phylogenic and sequence analysis and then screened in rabbits, where rabbit groups received four immunizations of either a single Env variant repeatedly (clonal), sequential administration of longitudinally derived Env variants from >5 y of infection (clonal or multiple), or repeated delivery of Env isolated immediately prior to and during early donor breadth (between 1.29 and 1.79 y post infection), (Fig. 1) (34, 57). The goal of the rabbit vaccine groups was to determine whether Envs from VC10014 emerging at particular time points during Env development were sufficient to elicit neutralizing breadth, or if immunizing with Envs representing the generation of greater Env heterogeneity over time during quasispecies evolution was required. Of the four groups tested, the early breadth rabbit vaccine group displayed the strongest plasma Ab binding and neutralization responses, and this immunization scheme was chosen for immunogenicity studies in rhesus macaques, as detailed in Malherbe 2014 (Env clones used for macaque immunizations are listed in 2Materials and Methods). After four coimmunizations with repeated delivery of early breadth Envs, macaque 25257 developed autologous Abs that neutralized tier 2 pseudoviruses with ID50 titers >600 and robust Env-specific T follicular helper cell responses in peripheral lymph nodes. Overall, this animal displayed the greatest plasma neutralization potency and breadth and was chosen for mAb isolation (40). Single IgG+ Env+ B cells were sorted from PBMCs collected 1 mo after the last immunization, and of 99 unique IgH-IgL pairings identified by PCR, 58 were successfully cloned and expressed as rmAbs (Supplemental Fig. 1).

FIGURE 1.

Schematic of Env immunogen curation and rhesus sample generation. Vanderbilt Center for AIDS Research cohort study participant VC10014 developed modest neutralization breadth <2 y after primary infection (33). Env immunogens cloned from VC10014 were previously curated in rabbits as described (34). In brief, 50 full-length env genes capable of producing infectious pseudovirus were cloned from VC10014 plasma collected at nine time points spanning almost 6 y following primary infection. The evolution of viral quasispecies driving emerging plasma breadth was analyzed in silico and select Env variants were tested for immunogenicity in four groups of rabbits by coimmunizing i.m. with trimeric gp140 and intradermally with gp160 plasmid DNA. Two of the rabbit groups were immunized with either a single Env clone or with six Envs present immediately prior to and concurrent with the development of VC10014 plasma breadth (Early Breadth group) using the same immunogens throughout. The other rabbit immunization groups were sequentially immunized with quasispecies’ Env diverging longitudinally with a total of four immunizations. Binding and neutralization titers were strongest in the Early Breadth group, and this vaccine scheme was then employed on six rhesus macaques as described in Hessell et al. (40). Following four immunizations, macaque 25257 displayed the highest binding and neutralization titers, including against heterologous tier 2 viruses, and robust levels of Env-responsive lymph node germinal center T follicular helper cells (∼0.2% of CD3+CD4+ICOS+PD-1hi) and was selected for Ab cloning.

FIGURE 1.

Schematic of Env immunogen curation and rhesus sample generation. Vanderbilt Center for AIDS Research cohort study participant VC10014 developed modest neutralization breadth <2 y after primary infection (33). Env immunogens cloned from VC10014 were previously curated in rabbits as described (34). In brief, 50 full-length env genes capable of producing infectious pseudovirus were cloned from VC10014 plasma collected at nine time points spanning almost 6 y following primary infection. The evolution of viral quasispecies driving emerging plasma breadth was analyzed in silico and select Env variants were tested for immunogenicity in four groups of rabbits by coimmunizing i.m. with trimeric gp140 and intradermally with gp160 plasmid DNA. Two of the rabbit groups were immunized with either a single Env clone or with six Envs present immediately prior to and concurrent with the development of VC10014 plasma breadth (Early Breadth group) using the same immunogens throughout. The other rabbit immunization groups were sequentially immunized with quasispecies’ Env diverging longitudinally with a total of four immunizations. Binding and neutralization titers were strongest in the Early Breadth group, and this vaccine scheme was then employed on six rhesus macaques as described in Hessell et al. (40). Following four immunizations, macaque 25257 displayed the highest binding and neutralization titers, including against heterologous tier 2 viruses, and robust levels of Env-responsive lymph node germinal center T follicular helper cells (∼0.2% of CD3+CD4+ICOS+PD-1hi) and was selected for Ab cloning.

Close modal

An initial screen for neutralizing activity against autologous tier 2 F8 and heterologous tier 1A SF162 pseudoviruses in CCR5+ CD4+ TZM-bl reporter cells identified a panel of 20 cloned mAbs for further screening (Fig. 2A). Eleven mAbs neutralized autologous F8 with an IC50 of 1.0 μg/ml or less. Fifteen mAbs neutralized SF162 with an IC50 of 1.0 μg/ml or less, and nine of those were IC50 <0.1 μg/ml (Fig. 2B). These 20 mAbs were then tested against a multitier and multiclade panel of 11 additional pseudoviruses and ranked according to breadth. Fourteen mAbs (O.1–O.14) displayed strong neutralizing activity against heterologous tier 1B and notably, albeit to a lesser extent, also neutralized at least one heterologous tier 2 clade B virus (Fig. 2B). The strongest neutralizer, O.1, neutralized 12 out of 13 (92%) of viruses tested with a median IC50 of 1.35 μg/ml, whereas O.2–O.20 displayed a wide range of breadth (8–77%) and potency (0.10–31.8 μg/ml) (Fig. 2C, 2D).

FIGURE 2.

Vaccine-elicited mAbs efficiently neutralize heterologous clade B pseudoviruses. (A) Abs cloned from macaque 25257 single IgG+ B cells collected 4 wk post–final immunization (week 24) were screened for neutralization against autologous F8 and heterologous tier 1A SF162 pseudoviruses using the standard TZM-bl reporter assay. The proportion of mAbs showing neutralization activity with IC50s ≤50 μg/ml against either virus is shown. (B) Heat map of IC50 values of mAbs tested against a panel of 13 pseudoviruses in the TZM-bl assay. Broadly neutralizing mAb VRC01 is shown as an intraassay reference control. All neutralization assays were performed with serial dilutions in duplicate and select mAbs were repeated twice to ensure accuracy. Cloned mAbs O.21–O.58 showed no activity against autologous F8 or heterologous SF162 or JRCSF and were not further tested. (C) Percentage breadth and (D) median IC50 value against all detected susceptible viruses shown in (B).

FIGURE 2.

Vaccine-elicited mAbs efficiently neutralize heterologous clade B pseudoviruses. (A) Abs cloned from macaque 25257 single IgG+ B cells collected 4 wk post–final immunization (week 24) were screened for neutralization against autologous F8 and heterologous tier 1A SF162 pseudoviruses using the standard TZM-bl reporter assay. The proportion of mAbs showing neutralization activity with IC50s ≤50 μg/ml against either virus is shown. (B) Heat map of IC50 values of mAbs tested against a panel of 13 pseudoviruses in the TZM-bl assay. Broadly neutralizing mAb VRC01 is shown as an intraassay reference control. All neutralization assays were performed with serial dilutions in duplicate and select mAbs were repeated twice to ensure accuracy. Cloned mAbs O.21–O.58 showed no activity against autologous F8 or heterologous SF162 or JRCSF and were not further tested. (C) Percentage breadth and (D) median IC50 value against all detected susceptible viruses shown in (B).

Close modal

All cloned mAbs (O.1–O.58) were screened for linear epitopes using a clade B Env 15mer peptide scan, and a majority (12 out of 20) of the neutralizing mAbs mapped to the V3 region. Sequences containing each linear epitope were inferred from overlap in peptide binding (Supplemental Fig. 2) and are presented in Fig. 3A. In addition to those targeting V3, neutralizing mAbs were directed against C1, C4, or gp41, whereas three nonneutralizing mAbs were mapped to C1 or C5. The remaining nonneutralizing mAbs were not further mapped.

FIGURE 3.

Potent NAbs target the CD4bs or V3 region. (A) All 58 mAbs were screened by ELISA for linear epitopes using a clade B consensus peptide pool. mAbs in positive wells were then screened against overlapping 15-mer consensus peptides to map the linear epitope as detailed in Supplemental Fig. 2. The remaining neutralizing mAbs O.2 and O.11 were scanned against gp41 and gp120, and epitopes for both were exclusive to gp120. (B) ELISA binding of VRC01, 2219, and O.2 to SF162 gp140 without blocking (solid lines) or after blocking with O.2 Fab′2. The complete competition binding panel is shown in Supplemental Fig. 3. (C) mAbs directed to the V3 region were tested for binding by ELISA against previously developed cradle (2219-like binding) and ladle (447-52D–like binding) mimotopes. For ELISAs against the cradle mimotope, a C4-binding mAb was used as a negative control.

FIGURE 3.

Potent NAbs target the CD4bs or V3 region. (A) All 58 mAbs were screened by ELISA for linear epitopes using a clade B consensus peptide pool. mAbs in positive wells were then screened against overlapping 15-mer consensus peptides to map the linear epitope as detailed in Supplemental Fig. 2. The remaining neutralizing mAbs O.2 and O.11 were scanned against gp41 and gp120, and epitopes for both were exclusive to gp120. (B) ELISA binding of VRC01, 2219, and O.2 to SF162 gp140 without blocking (solid lines) or after blocking with O.2 Fab′2. The complete competition binding panel is shown in Supplemental Fig. 3. (C) mAbs directed to the V3 region were tested for binding by ELISA against previously developed cradle (2219-like binding) and ladle (447-52D–like binding) mimotopes. For ELISAs against the cradle mimotope, a C4-binding mAb was used as a negative control.

Close modal

The two remaining neutralizing mAbs that do not target linear epitopes, O.2 and O.11, were then tested in competition binding assays by blocking gp140 with their respective F(ab′)2 regions prior to incubating with Abs specific for known neutralizing epitopes. All CD4bs targeting mAbs, including VRC01, N6, 3BNC117, and b12, showed reduced binding with O.2 block, strongly indicating at least partial overlap of epitopes (Fig. 3B, Supplemental Fig. 3A–C). However, O.2 did not bind the canonical VRC01 epitope expressed on resurfaced stabilized core-3 (RSC-3; Supplemental Fig. 3B) (12). The binding region of O.11 was not identified beyond gp120, but based on competition assays and its failure to bind V1/V2 scaffold proteins (49), it is unlikely to target V1/V2, V3, or the CD4bs (Supplemental Fig. 3A–C).

Balasubramanian et al. delineated two common approaches for Abs targeting the V3 crown, termed “cradle” or C-type, which characterizes mAb 2219 like binding, and “ladle” or l-type practiced by broadly neutralizing 447-52D. Their findings suggest that although most chronically infected individuals produce both V3 region in the cradle orientation (V3C)– and V3 ladle orientation (V3L)–specific NAbs with V3L comprising the predominant V3 response, many vaccine regimens in both humans and small animals favor elicitation of V3C Abs (58). Indeed, macaque 25257 was unique in its group (one out of six) with both V3C and V3L targeting Abs detected in plasma. In this study, we tested the V3 mAbs against V3C and V3L mimotopes and found that representatives from both types were obtained (Fig. 3C). Consistent with analysis in over 200 pseudoviruses that suggests V3 neutralization sensitivity is impacted by regional considerations, particularly epitope occlusion from extended V1/V2 loops and glycosylation in the V2 hypervariable region in addition to primary amino acid sequence (42, 59), the primary amino acid sequence of pseudovirus V3 crown region did not predict neutralization sensitivity to our cloned V3C or V3L mAbs (Supplemental Fig. 3D).

Binding profiles were explored for both neutralizing and nonneutralizing mAbs via ELISA to four clade B gp140s, and neutralizing mAbs displayed comparatively stronger and more broad binding than nonneutralizers. Neutralizing mAbs O.1–O.20 showed 100% binding with a median EC50 of 0.002 μg/ml whereas nonneutralizing mAbs O.21–O.58 had 67.1% binding and a median EC50 of 0.009 μg/ml (Fig. 4A). Binding kinetics to constrained SOSIP trimers, engineered to better mimic the native state 1 closed conformation, were examined for nine neutralizing mAbs representing each epitope target (Fig. 4B, 4C, Supplemental Fig. 4). The V3C mAbs (O.1 and O.3) displayed strong affinity for both autologous clade B (KD ≤ 1.5 × 10−10) and heterologous clade A BG505 (KD = 1.65 × 10−9 to 1.9 × 10−9) trimers. Of note, the CD4bs mAb O.2 also bound both analytes but with lower affinity (3.63 × 10−8 and 3.03 × 10−6, respectively). In contrast, V3L mAbs O.4 and O.10 only bound autologous SOSIP (Fig. 4B, 4C). Neutralizing mAbs O.15 and O.17 targeting C1 and C4, respectively, did not bind either constrained trimer (Fig. 4B, 4C).

FIGURE 4.

Neutralizing mAbs bind soluble stabilized trimers and infected rhesus PBMCs. (A) Heatmap of mAb EC50 values determined by ELISA against the indicated gp140 (F8 and SF162) or gp120 (BaL and JRCSF). (B) Neutralizing mAb-binding kinetics to autologous (F8.V3.N7) and clade A heterologous (BG505) SOSIP trimers measured by biolayer interferometry. Values were calculated from kinetic traces of serial dilutions starting at 120 nm of analyte shown in Supplemental Fig. 4 as described in the 2Materials and Methods. (C) Comparative biolayer interferometry traces showing each mAb with 60-nm analyte measured simultaneously. (D). mAb binding of infected cells categorized by neutralization sensitive virus (red bars) or neutralization resistant virus (gray bars). Rhesus splenocytes were CD4 enriched by MACS, then stimulated with PHA and spinoculated with SHIVBaL or SHIVSF162P3. Seven days postinfection, mAb surface binding was examined by incubating cells with each cloned mAb or an isotype control followed by flour-conjugated secondary mAb, then intracellular stained with anti-p27/FITC and analyzed by flow cytometry. The gating scheme is presented at left and the dotted line indicates median fluorescence intensity of the isotype control. Symbols are colored to indicate Env targeting of each mAb. Significance was evaluated with a paired t test.

FIGURE 4.

Neutralizing mAbs bind soluble stabilized trimers and infected rhesus PBMCs. (A) Heatmap of mAb EC50 values determined by ELISA against the indicated gp140 (F8 and SF162) or gp120 (BaL and JRCSF). (B) Neutralizing mAb-binding kinetics to autologous (F8.V3.N7) and clade A heterologous (BG505) SOSIP trimers measured by biolayer interferometry. Values were calculated from kinetic traces of serial dilutions starting at 120 nm of analyte shown in Supplemental Fig. 4 as described in the 2Materials and Methods. (C) Comparative biolayer interferometry traces showing each mAb with 60-nm analyte measured simultaneously. (D). mAb binding of infected cells categorized by neutralization sensitive virus (red bars) or neutralization resistant virus (gray bars). Rhesus splenocytes were CD4 enriched by MACS, then stimulated with PHA and spinoculated with SHIVBaL or SHIVSF162P3. Seven days postinfection, mAb surface binding was examined by incubating cells with each cloned mAb or an isotype control followed by flour-conjugated secondary mAb, then intracellular stained with anti-p27/FITC and analyzed by flow cytometry. The gating scheme is presented at left and the dotted line indicates median fluorescence intensity of the isotype control. Symbols are colored to indicate Env targeting of each mAb. Significance was evaluated with a paired t test.

Close modal

Although neutralizing soluble virus is critical, this interaction does not always predict Ab binding to infected cells, an important component for countering cell-to-cell-spread and potentiating Ab-mediated opsonization or cell lysis. To examine infected cell binding, rhesus splenocytes were enriched for CD4+ T cells and stimulated with staphylococcal enterotoxin B, α-CD3, α-CD28, and α-CD49d prior to spinoculation with SHIVSF162P3 or SHIVBaL. Seven days postchallenge, mAbs were tested for surface binding against p27+ cells by flow cytometry. Neutralizing mAbs were significantly more likely to bind the surface of SHIVBaL-infected cells (p < 0.0001) with the greatest binding observed with V3C Abs (Fig. 4D). To a lesser extent these mAbs also bound the surface of cells infected with SHIVSF162P3, although only O.1 neutralized this tier 2 virus. Somewhat surprisingly based on the absence of binding to SHIVBaL-infected cells, the CD4bs mAb O.2 displayed the greatest binding to cells infected with SHIVSF162P3 underscoring the unique interaction between this mAb and different Envs (Fig. 4D, red symbol).

To determine whether surface binding of infected cells predicted functional significance, ADCC assays were performed as described previously (51). In brief, CCR5-tropic NKR24 luciferase reporter cells were infected with SHIV prior to 8 h of coincubation with each mAb and CD16+ KHYG-1 effector cells. Vaccinated macaque 25257 plasma and that from another NHP that received the same vaccine regimen were tested against both SHIVBaL and SHIVSF162P3-infected cells with high ADCC activity observed against both viruses; indeed, possible cell lysis occurred in a range of ∼50% at 1:100 plasma dilution to >80% at 1:10 dilution for each (Fig. 5A, 5B, left panels). Against SHIVBaL, both V3C- and V3L-targeting mAbs showed strong ADCC activity, with at least some activity observed with mAbs binding CD4bs (O.2), C1 (O.15), and an unknown conformational epitope on gp120 (O.11; Fig. 5A, right panel). Unexpectedly, no mAbs mediated ADCC against SHIVSF162P3-infected cells (Fig. 5B, right panel). Although this result is consistent with the comparatively reduced binding to infected cells (Fig. 4D), high plasma ADCC activity indicates that our panel of cloned mAbs is not completely representative of the functional polyclonal Ab response elicited in 25257 following vaccination.

FIGURE 5.

Effector function correlates with neutralization potency. (A and B) ADCC of macaque plasma at the time of cloning (week 24, left) and of the indicated cloned mAbs (right). NKR24 luciferase reporter cells were infected with SHIVBaL (A) or SHIVSF162P3 (B) and incubated with CD16+ KHYG-1 effectors without mAb (background) or with serial dilutions of the indicated mAbs. KHYG-1 cells expressing rhesus or human CD16 were used for plasma and mAb assays, respectively. ADCC activity is depicted as the normalized loss of luminescence, and dotted lines indicate the intraassay threshold for activity (50% for plasma, 80% for mAbs). (C) ADCP of HIVSF162 gp120–coated beads. THP-1 monocytes were incubated with Ag-conjugated fluorescent beads without mAb (background) or with serial dilutions of the indicated mAbs. The phagocytosis score is shown as the percent bead+ cells multiplied by the median fluorescence intensity (percentage of phagocytosing × no. of internalized beads). (D and E) Correlations of mAb surface binding to infected cells, ADCC, and ADCP activity with neutralization IC50 to the corresponding pseudovirus. Gray shading designates the experimental cutoff where no activity was detected. (A–E) Individual mAb data are colored to indicate corresponding Env epitope.

FIGURE 5.

Effector function correlates with neutralization potency. (A and B) ADCC of macaque plasma at the time of cloning (week 24, left) and of the indicated cloned mAbs (right). NKR24 luciferase reporter cells were infected with SHIVBaL (A) or SHIVSF162P3 (B) and incubated with CD16+ KHYG-1 effectors without mAb (background) or with serial dilutions of the indicated mAbs. KHYG-1 cells expressing rhesus or human CD16 were used for plasma and mAb assays, respectively. ADCC activity is depicted as the normalized loss of luminescence, and dotted lines indicate the intraassay threshold for activity (50% for plasma, 80% for mAbs). (C) ADCP of HIVSF162 gp120–coated beads. THP-1 monocytes were incubated with Ag-conjugated fluorescent beads without mAb (background) or with serial dilutions of the indicated mAbs. The phagocytosis score is shown as the percent bead+ cells multiplied by the median fluorescence intensity (percentage of phagocytosing × no. of internalized beads). (D and E) Correlations of mAb surface binding to infected cells, ADCC, and ADCP activity with neutralization IC50 to the corresponding pseudovirus. Gray shading designates the experimental cutoff where no activity was detected. (A–E) Individual mAb data are colored to indicate corresponding Env epitope.

Close modal

Because an infected cell ADCP assay has yet to be developed, ADCP was performed by measuring THP-1 monocytes’ ability to internalize SF162-conjugated fluorescent beads (52). In contrast to ADCC, a wider range of ADCP activity was observed with V3C-targeting mAbs, whereas the only V3L mAb tested did not mediate ADCP (Fig. 5C). Interestingly, those targeting C4 displayed higher ADCP activity than V3-glycan targeting bNAb 10–1074. One third of mAbs (8 out of 24 tested) that did not neutralize SF162 did mediate ADCP (Fig. 5C, right panel). However, given the limitations of this assay, it should be noted that these results may not perfectly reflect ADCP activity in vivo.

Most neutralizing mAbs displayed potent activity against SHIVBaL (Fig. 2), and a strong correlation (p = 0.002) was found between neutralization potency (IC50) and the magnitude of infected cell surface binding (Fig. 5D). Consistently, mAbs O.1–O.20 showed little neutralization of SHIVSF162P3 coupled with comparatively lower binding (Figs. 2B, 4C). Another correlation was observed between neutralization potency and the degree of ADCC activity against SHIVBaL, with minimal or no activity seen against SHIVSF162P3 (Fig. 5D). Taken together, these observations indicate that neutralization potency may predict the level of ADCC activity, at least among NAbs binding open trimer conformations. Among all mAbs, ADCP was associated with neutralization, but neutralization potency did not correlate with the level of ADCP activity (Fig. 5E). This observation suggests that although neutralization activity indicates a greater likelihood that an mAb can also mediate ADCP compared with a nonneutralizer, neutralization potency does not predict the comparative magnitude of ADCP activity.

Although some IgHV orthologs between macaques and humans have been described (60, 61), macaques possess substantial interindividual IgHV allelic diversity (54). To ensure accurate allele identification of the O.1–O.58 mAbs, total RNA was isolated from macaque 25257 PBMCs and used to generate an individualized IgHV Ig gene database against which cloned sequences were queried. L chain allele assignments were limited to those inferred from the IMGT database, as the complete rhesus IgL allelic repertoire is incompletely characterized. The complete IgH and IgL pairing, CDR3 sequences, and percent nucleotide SHM are shown in Fig. 6. The 58 mAbs were encoded by 23 different IgHV alleles, of which the NAbs used five different alleles (Fig. 7A). Consistent with previous reports (60, 62), V3C-directed mAbs were encoded by an allele of the IgHV5 family, in this study defined as IgHV5-157, (Fig. 7B). Of these, four belonged to the same clonotype (Figs. 6, 7B), defined by identical V and J allele assignments, identical HCDR3 length, and at least 80% HCDR3 aa homology (63). Additionally, all four mAbs targeting C4 belonged to an IGHV4-NL_27*01_S9001/IGLV6-2*02 clonotype, whereas the remaining 50 mAbs had unique allele assignments within the panel (Figs. 6, 7B).

FIGURE 6.

Allele assignments, CDR3 regions, and SHM levels for O.1–O.58. IgH and IgL allele assignments, CDR3 regions, and SHM rates obtained by IgBlast assignment to an individualized IGHV database of 25257 and IMGT’s IGKV and IGLV databases. In L chain assignments, parenthesis indicate alternative assignments with the same homology percentage. Symbols denote mAbs belonging to the same clonotype.

FIGURE 6.

Allele assignments, CDR3 regions, and SHM levels for O.1–O.58. IgH and IgL allele assignments, CDR3 regions, and SHM rates obtained by IgBlast assignment to an individualized IGHV database of 25257 and IMGT’s IGKV and IGLV databases. In L chain assignments, parenthesis indicate alternative assignments with the same homology percentage. Symbols denote mAbs belonging to the same clonotype.

Close modal
FIGURE 7.

Gene assignments and V region SHM rates. (A) Alleles for the 58 cloned mAbs are shown with corresponding neutralizing activity against at least one of 13 tested pseudoviruses and (B) against their cognate epitopes. The number of mAbs belonging to each clonotype is shown as an insert, where unlabeled slices in the pie equal 1 mAb. Each clonotype is defined as having identical V and J allele assignments, identical HCDR3 length, and at least 80% HCDR3 aa homology. IgH gene assignments were determined using an individualized IgH germline database generated for macaque 25257 by IgDiscover. (C) Prevaccine “baseline” allele usage in macaque 25257 inferred from frequency of barcoded total RNA compared with mAb-encoding alleles. (D) Number of amino acids comprising the CDR-3 (CDR3) of IgH or (E) percent V region nucleotide SHM for each mAb categorized by neutralization activity and compared with a multiple t test (**p < 0.01). Statistical conclusions were not made for the IgL because CDR3 and SHM rates were approximated from the IMGT database instead of an individualized database.

FIGURE 7.

Gene assignments and V region SHM rates. (A) Alleles for the 58 cloned mAbs are shown with corresponding neutralizing activity against at least one of 13 tested pseudoviruses and (B) against their cognate epitopes. The number of mAbs belonging to each clonotype is shown as an insert, where unlabeled slices in the pie equal 1 mAb. Each clonotype is defined as having identical V and J allele assignments, identical HCDR3 length, and at least 80% HCDR3 aa homology. IgH gene assignments were determined using an individualized IgH germline database generated for macaque 25257 by IgDiscover. (C) Prevaccine “baseline” allele usage in macaque 25257 inferred from frequency of barcoded total RNA compared with mAb-encoding alleles. (D) Number of amino acids comprising the CDR-3 (CDR3) of IgH or (E) percent V region nucleotide SHM for each mAb categorized by neutralization activity and compared with a multiple t test (**p < 0.01). Statistical conclusions were not made for the IgL because CDR3 and SHM rates were approximated from the IMGT database instead of an individualized database.

Close modal

Baseline allele usage in 25257 prior to Env vaccination was inferred from total RNA barcoded by allele. The barcode frequency showed that despite the small mAb sample size, the proportion of Env-specific mAb-encoding alleles reflected baseline gene usage for many genes. The greatest differences were seen in IGHV4-NL_42*01_S3572 (encoding nonneutralizers) and IGHV5-157*01_S6479 (encoding V3C neutralizers), which comprised a small percentage of prevaccine transcripts (0.53 and 1.23%, respectively) but had substantially greater representation among alleles encoding cloned mAbs (10.3 and 20.7%, respectively; Fig. 7C), indicating vaccine-induced expansion of B cells using these alleles.

Notably, V3C-targeting mAbs exclusively paired with Igλ, with the most potent mAbs, O.1 and O.3, using IGLV1S7*01 (Fig. 6). These mAbs displayed relatively low SHM levels, on average 3.77% in the H chain. By contrast, the two V3L mAbs, O.4 and O.10, used IgHV4 and Igκ, pairing with IGKV2S14*01 and IGKV2S17*01, respectively (Fig. 6). O.4 displayed 8.4% SHM in the H chain, whereas that of the weaker neutralizer O.10 was 2.0%, indicating that affinity maturation may be more important for V3L-binding Abs than for those that target V3C. Overall, the neutralizing mAbs possessed significantly longer CDR3 sequences in H chains (p < 0.01), although SHM levels between these categories were comparable (Fig. 7D, 7E).

In this study, we report the cloning and characterization of neutralizing and nonneutralizing mAbs elicited in macaques isolated after four coimmunizations with native gp160 DNA and Adjuplex-adjuvanted gp140-unconstrained Envs. It has been difficult to generate potent and broad Abs following vaccination with HIV Env, whether using constrained or unconstrained trimeric Env (24, 64). By characterization of the resulting individual Abs expressed by macaque B cells, insights can be gained into the ability of different immunization strategies to elicit NAbs (25, 64). The strongest of the cloned neutralizing mAbs displayed encouraging potency and breadth at concentrations between 0.21 and 15.2 μg/ml. Several of these mAbs, particularly those targeting V3C, bound soluble native-like autologous and heterologous SOSIP trimers with high affinities and bound to the surface of primary SHIVBaL-infected splenocytes. Additionally, these mAbs were capable of mediating ADCC activity in a strain-dependent manner, and the comparative level of ADCC activity correlated to the signal magnitude of infected cell binding. These mAbs signify vaccine-induced clonal expansion from IGHV5-157 and provide insights into optimal IGHL pairing, with the strongest neutralizer pairing with IGLV1S1*01.

Our results reflect similar findings of a recent report that vaccine-elicited V3-directed Abs can neutralize a subset of tier 2 viruses (42). Prior to CD4 receptor binding, these difficult-to-neutralize viruses favor a state 1 closed conformation that renders the V3 apex largely inaccessible to Ab (2, 3). Although modeling suggests numerous point mutations or individual glycosylation events that disrupt the V1/V2 shield and expose the V3-binding pocket to NAbs (65), selective pressure maintains limited accessibility of the immunogenic V3 loop on tier 2–3 free virions. Hence, the majority of V3 Abs identified to date bind a transitional state 2 or open state 3 conformation induced upon virion engagement with CD4, are highly clade specific, and even with potent IC50s, neutralization thresholds are sometimes observed prior to achieving complete in vitro protection (42). Although structural analysis was outside the scope of this study, it is likely that the V3C- and V3L-targeting mAbs reported in this study are functionally limited by similar constraints. Nevertheless, V3-targeting Abs were associated with vaccine-induced protection in humans and reduced mother-to-child transmission (66, 67) and thus remain an important consideration of vaccine design. The potent V3C and V3L mAbs cloned in this study, particularly O.1 and O.4, suggest that immunogens that mimic Env in an open state 3 conformation could be used as a complement to closed state 1 trimers aimed at generating greater cross-clade neutralization breadth.

The degree to which the NHP immunogen selection approach used in this study (Fig. 1) recapitulated the clonotype diversity and breadth observed in the Env donor is of considerable interest. Immunized NHP and donor plasma binding and neutralizing titers were comparable, even if somewhat enhanced, in NHP vaccinees (33, 40, 57). Numerous Ab clones obtained longitudinally from the VC10014 donor were recently reported by Chukwuma et al. (68). Limited characterization of 14 neutralizing mAbs from this study participant revealed that these largely targeted V3, with one neutralizing mAb mapped to the CD4bs, a targeting profile that seems more than coincidental in its similarity to the mAbs we isolated from vaccinated macaques in this study. No single mAb neutralized more than 75% (9 out of 12) of autologous viral quasispecies, and the most potent individual mAbs neutralized 40% (4 out of 10) of a heterologous panel composed mostly of clade B viruses and comparable to the ones used in this study. In contrast, mixtures of the isolated mAbs neutralized 100% of the autologous and 70% of heterologous viruses in the respective panels, indicating that the encouraging plasma neutralization breadth in this patient is due primarily to the combined potency of a diverse polyclonal profile (68).

By comparison, the neutralizing mAb panel elicited through vaccination of NHPs and reported in this study displayed substantially greater heterologous tier 2 breadth, particularly O.1–O.14, whereas the strongest isolated neutralizers in both the human donor and immunized NHP were similarly V3 or CD4bs directed. Thus, the humoral response developed during human chronic infection was at least in part recapitulated through NHP vaccination with the Envs curated from VC10014 phylogeny. The promising Ab pool generated by this immunization scheme has the potential to be further expanded by the inclusion of native state 1–mimicking Envs that may favor epitopes with cross-clade breadth (69), and by the inclusion of immunogens designed to direct Ab elicitation and maturation to bNAb epitopes (25).

Another important step in evaluating translatability of NHP vaccine studies is the use of orthologous clonotypes between species and shared within the human population. Recent efforts have identified clonotypes capable of HIV-1 neutralization and shared within large segments of the human population (70). Among these are V3C-targeting Abs using the human VH5-51 gene, which are well characterized and responsive to both HIV infection and immunization (60, 62, 71). The rhesus macaque functional ortholog is VH5-157 (64), which encoded all V3C mAbs identified in this study. Preferential usage of IGHV5 for V3-directed mAbs suggest that germline-encoded residues play a role in binding the target epitope and that such sequences are conserved between humans and macaques. Functional differences between the V3C mAbs reported in this study further inform optimal use of this gene, with the two V3C mAbs with greatest breadth, O.1 and O.3 both paired with IGLV1S1*01 (Fig. 6).

As nonneutralizing Abs were a correlate of protection in the RV144 vaccine efficacy trial (66) and in some NHP vaccine studies (7275), efforts have been made to define the possible antiviral mechanisms of these Abs. Data in NHPs show them capable of reducing the number of viable T/F viruses (76, 77), and they have been shown to clear virus and mitigate infection in humanized mice in an Fc-dependent fashion (78). In this study, however, neutralizing activity and potency strongly correlated with both an Ab’s ability to bind the surface of infected cells (p ≤ 0.0001) or mediate ADCC and ADCP (p ≤ 0.0001 and p = 0.0003, respectively; Fig. 5D). Although exceptions were found, particularly with O.29, O.34, O.35, and O.40, which did not neutralize but could mediate ADCP at concentrations <0.1 μg/ml, these data are consistent with a model that polyclonal NAbs often bind with greater avidity than nonneutralizing Abs (33), and are thus favored for FcγR recognition in the absence of steric hindrance (79).

The report of tier 2 virus neutralization by V3-targeting mAbs with little SHM cloned from macaques vaccinated with unconstrained Env (42) opens the door for further investigations of polyclonal vaccine responses that include NAbs against different conformations of HIV-1. The low levels of SHM in the strong neutralizers we isolated suggest that a subset of tier 2 viruses present accessible V3 regions that can be targeted by vaccine-elicited NAbs. Without further sorting with constrained Env trimers, we cannot predict the range of additional BCRs induced in 25257 that may have targeted conformational epitopes. Because of the use of unstabilized recombinant gp140 trimers as probes for B cell sorting, we may not have captured BCRs targeting closed state 1 conformations in which the V3 region is less exposed. Thus, our mAbs may not be fully representative of the VC10014 donor plasma functional Ab responses that possessed ADCC activity against SHIVSF162P3 (Fig. 5B) and had binding Abs mapped to the membrane-proximal external region (40). Further studies with conformational probes such as CD4bs mimetics will complement the characterization of functional Ab responses. Additionally, the recent capability of linking BCR to Ag specificity through sequencing enables robust evaluation of BCR binding breadth prior to cloning (80) and will be an important tool for evaluating functional Ab responses moving forward.

In conclusion, the mAb panel reported in this study reveals differences in IGHV usage, binding, and effector functions between NAbs and nonneutralizing Abs elicited through NHP vaccination, supports a curated approach to immunogen selection, and demonstrates partial reproducibility of the Ab response observed in the human donor. These data underscore the value of the NHP model of HIV vaccination and highlight the contribution of the coimmunization strategy that together, support a rationale for further studies incorporating improved constrained trimers paired with DNA immunogens.

We thank Diane Kubitz and Jonathan Otsuji at The Scripps Research Institute Antibody Production Core Facility for assistance with Ab production, Christina Corbaci for assistance with figure design, and Madhubanti Basu and Michael Piepenbrink for technical assistance with early lineage analysis.

This work was funded by National Institute of Allergy and Infectious Diseases, National Institutes of Health R01 AI129801 (to A.J.H.), ONPRC P51 OD011092 (to N.L.H.), P51 OD011092-57S2 (to A.J.H.), and P01-AI104722 (to G.B.K.H.), as well as a Distinguished Professor Grant (2017-00968) from the Swedish Research Council (to G.B.K.H.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ADCC

    Ab-dependent cellular cytotoxicity

  •  
  • ADCP

    Ab-dependent cellular phagocytosis

  •  
  • bNAb

    broadly NAb

  •  
  • CD4bs

    CD4 binding site

  •  
  • Env

    envelope glycoprotein

  •  
  • NAb

    neutralizing Ab

  •  
  • NHP

    nonhuman primate

  •  
  • SHM

    somatic hypermutation

  •  
  • V3C

    V3 region in the cradle orientation

  •  
  • V3L

    V3 ladle orientation.

1
Munro
,
J. B.
,
J.
Gorman
,
X.
Ma
,
Z.
Zhou
,
J.
Arthos
,
D. R.
Burton
,
W. C.
Koff
,
J. R.
Courter
,
A. B.
Smith
III
,
P. D.
Kwong
, et al
.
2014
.
Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions.
Science
346
:
759
763
.
2
Alsahafi
,
N.
,
N.
Bakouche
,
M.
Kazemi
,
J.
Richard
,
S.
Ding
,
S.
Bhattacharyya
,
D.
Das
,
S. P.
Anand
,
J.
Prévost
,
W. D.
Tolbert
, et al
.
2019
.
An asymmetric opening of HIV-1 envelope mediates antibody-dependent cellular cytotoxicity.
Cell Host Microbe
25
:
578
587.e5
.
3
Lu
,
M.
,
X.
Ma
,
L. R.
Castillo-Menendez
,
J.
Gorman
,
N.
Alsahafi
,
U.
Ermel
,
D. S.
Terry
,
M.
Chambers
,
D.
Peng
,
B.
Zhang
, et al
.
2019
.
Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.
Nature
568
:
415
419
.
4
Zhu
,
P.
,
J.
Liu
,
J.
Bess
Jr.
,
E.
Chertova
,
J. D.
Lifson
,
H.
Grisé
,
G. A.
Ofek
,
K. A.
Taylor
,
K. H.
Roux
.
2006
.
Distribution and three-dimensional structure of AIDS virus envelope spikes.
Nature
441
:
847
852
.
5
Seaman
,
M. S.
,
H.
Janes
,
N.
Hawkins
,
L. E.
Grandpre
,
C.
Devoy
,
A.
Giri
,
R. T.
Coffey
,
L.
Harris
,
B.
Wood
,
M. G.
Daniels
, et al
.
2010
.
Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies.
J. Virol.
84
:
1439
1452
.
6
deCamp
,
A.
,
P.
Hraber
,
R. T.
Bailer
,
M. S.
Seaman
,
C.
Ochsenbauer
,
J.
Kappes
,
R.
Gottardo
,
P.
Edlefsen
,
S.
Self
,
H.
Tang
, et al
.
2014
.
Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies.
J. Virol.
88
:
2489
2507
.
7
Richman
,
D. D.
,
T.
Wrin
,
S. J.
Little
,
C. J.
Petropoulos
.
2003
.
Rapid evolution of the neutralizing antibody response to HIV type 1 infection.
Proc. Natl. Acad. Sci. USA
100
:
4144
4149
.
8
Song
,
H.
,
E. E.
Giorgi
,
V. V.
Ganusov
,
F.
Cai
,
G.
Athreya
,
H.
Yoon
,
O.
Carja
,
B.
Hora
,
P.
Hraber
,
E.
Romero-Severson
, et al
.
2018
.
Tracking HIV-1 recombination to resolve its contribution to HIV-1 evolution in natural infection.
Nat. Commun.
9
:
1928
1943
.
9
Bbosa
,
N.
,
P.
Kaleebu
,
D.
Ssemwanga
.
2019
.
HIV subtype diversity worldwide.
Curr. Opin. HIV AIDS
14
:
153
160
.
10
Hemelaar
,
J.
,
R.
Elangovan
,
J.
Yun
,
L.
Dickson-Tetteh
,
I.
Fleminger
,
S.
Kirtley
,
B.
Williams
,
E.
Gouws-Williams
,
P. D.
Ghys
;
WHO–UNAIDS Network for HIV Isolation Characterisation
.
2019
.
Global and regional molecular epidemiology of HIV-1, 1990-2015: a systematic review, global survey, and trend analysis.
Lancet Infect. Dis.
19
:
143
155
.
11
Torrents de la Peña
,
A.
,
K.
Rantalainen
,
C. A.
Cottrell
,
J. D.
Allen
,
M. J.
van Gils
,
J. L.
Torres
,
M.
Crispin
,
R. W.
Sanders
,
A. B.
Ward
.
2019
.
Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics.
PLoS Pathog.
15
:
e1007920
.
12
Wu
,
X.
,
Z. Y.
Yang
,
Y.
Li
,
C. M.
Hogerkorp
,
W. R.
Schief
,
M. S.
Seaman
,
T.
Zhou
,
S. D.
Schmidt
,
L.
Wu
,
L.
Xu
, et al
.
2010
.
Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1.
Science
329
:
856
861
.
13
Huang
,
J.
,
B. H.
Kang
,
E.
Ishida
,
T.
Zhou
,
T.
Griesman
,
Z.
Sheng
,
F.
Wu
,
N. A.
Doria-Rose
,
B.
Zhang
,
K.
McKee
, et al
.
2016
.
Identification of a CD4-binding-site antibody to HIV that evolved near-pan neutralization breadth.
Immunity
45
:
1108
1121
.
14
McCoy
,
L. E.
,
D. R.
Burton
.
2017
.
Identification and specificity of broadly neutralizing antibodies against HIV.
Immunol. Rev.
275
:
11
20
.
15
Pinto
,
D.
,
C.
Fenwick
,
C.
Caillat
,
C.
Silacci
,
S.
Guseva
,
F.
Dehez
,
C.
Chipot
,
S.
Barbieri
,
A.
Minola
,
D.
Jarrossay
, et al
.
2019
.
Structural basis for broad HIV-1 neutralization by the MPER-specific human broadly neutralizing antibody LN01.
Cell Host Microbe
26
:
623
637.e8
.
16
Zhang
,
L.
,
A.
Irimia
,
L.
He
,
E.
Landais
,
K.
Rantalainen
,
D. P.
Leaman
,
T.
Vollbrecht
,
A.
Stano
,
D. I.
Sands
,
A. S.
Kim
, et al
IAVI Protocol G Investigators
.
2019
.
An MPER antibody neutralizes HIV-1 using germline features shared among donors.
Nat. Commun.
10
:
5389
5404
.
17
Schommers
,
P.
,
H.
Gruell
,
M. E.
Abernathy
,
M. K.
Tran
,
A. S.
Dingens
,
H. B.
Gristick
,
C. O.
Barnes
,
T.
Schoofs
,
M.
Schlotz
,
K.
Vanshylla
, et al
.
2020
.
Restriction of HIV-1 escape by a highly broad and potent neutralizing antibody.
Cell
180
:
471
489.e22
.
18
Bhiman
,
J. N.
,
C.
Anthony
,
N. A.
Doria-Rose
,
O.
Karimanzira
,
C. A.
Schramm
,
T.
Khoza
,
D.
Kitchin
,
G.
Botha
,
J.
Gorman
,
N. J.
Garrett
, et al
.
2015
.
Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies.
Nat. Med.
21
:
1332
1336
.
19
Bonsignori
,
M.
,
E. F.
Kreider
,
D.
Fera
,
R. R.
Meyerhoff
,
T.
Bradley
,
K.
Wiehe
,
S. M.
Alam
,
B.
Aussedat
,
W. E.
Walkowicz
,
K. K.
Hwang
, et al
.
2017
.
Staged induction of HIV-1 glycan-dependent broadly neutralizing antibodies.
Sci. Transl. Med.
9
: eaai7514.
20
Bonsignori
,
M.
,
H. X.
Liao
,
F.
Gao
,
W. B.
Williams
,
S. M.
Alam
,
D. C.
Montefiori
,
B. F.
Haynes
.
2017
.
Antibody-virus co-evolution in HIV infection: paths for HIV vaccine development.
Immunol. Rev.
275
:
145
160
.
21
Pancera
,
M.
,
A.
Changela
,
P. D.
Kwong
.
2017
.
How HIV-1 entry mechanism and broadly neutralizing antibodies guide structure-based vaccine design.
Curr. Opin. HIV AIDS
12
:
229
240
.
22
Scheid
,
J. F.
,
H.
Mouquet
,
B.
Ueberheide
,
R.
Diskin
,
F.
Klein
,
T. Y.
Oliveira
,
J.
Pietzsch
,
D.
Fenyo
,
A.
Abadir
,
K.
Velinzon
, et al
.
2011
.
Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding.
Science
333
:
1633
1637
.
23
Scheepers
,
C.
,
R. K.
Shrestha
,
B. E.
Lambson
,
K. J.
Jackson
,
I. A.
Wright
,
D.
Naicker
,
M.
Goosen
,
L.
Berrie
,
A.
Ismail
,
N.
Garrett
, et al
.
2015
.
Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline Ig gene repertoire.
J. Immunol.
194
:
4371
4378
.
24
Kwong
,
P. D.
,
J. R.
Mascola
.
2018
.
HIV-1 vaccines based on antibody identification, B cell ontogeny, and epitope structure.
Immunity
48
:
855
871
.
25
Dubrovskaya
,
V.
,
K.
Tran
,
G.
Ozorowski
,
J.
Guenaga
,
R.
Wilson
,
S.
Bale
,
C. A.
Cottrell
,
H. L.
Turner
,
G.
Seabright
,
S.
O’Dell
, et al
.
2019
.
Vaccination with glycan-modified HIV NFL envelope trimer-liposomes elicits broadly neutralizing antibodies to multiple sites of vulnerability.
Immunity
51
:
915
929.e7
.
26
Steichen
,
J. M.
,
Y. C.
Lin
,
C.
Havenar-Daughton
,
S.
Pecetta
,
G.
Ozorowski
,
J. R.
Willis
,
L.
Toy
,
D.
Sok
,
A.
Liguori
,
S.
Kratochvil
, et al
.
2019
.
A generalized HIV vaccine design strategy for priming of broadly neutralizing antibody responses.
Science
366
: eaax4380.
27
Saunders
,
K. O.
,
K.
Wiehe
,
M.
Tian
,
P.
Acharya
,
T.
Bradley
,
S. M.
Alam
,
E. P.
Go
,
R.
Scearce
,
L.
Sutherland
,
R.
Henderson
, et al
.
2019
.
Targeted selection of HIV-specific antibody mutations by engineering B cell maturation.
Science
366
: eaay7199.
28
Kong
,
R.
,
H.
Duan
,
Z.
Sheng
,
K.
Xu
,
P.
Acharya
,
X.
Chen
,
C.
Cheng
,
A. S.
Dingens
,
J.
Gorman
,
M.
Sastry
, et al
NISC Comparative Sequencing Program
.
2019
.
Antibody lineages with vaccine-induced antigen-binding hotspots develop broad HIV neutralization.
Cell
178
:
567
584.e19
.
29
Long
,
E. F.
,
D. K.
Owens
.
2011
.
The cost-effectiveness of a modestly effective HIV vaccine in the United States.
Vaccine
29
:
6113
6124
.
30
Buchbinder
,
S. P.
,
A. Y.
Liu
.
2019
.
CROI 2019: advances in HIV prevention and plans to end the epidemic.
Top. Antivir. Med.
27
:
8
25
.
31
Garcia-Tellez
,
T.
,
N.
Huot
,
M. J.
Ploquin
,
P.
Rascle
,
B.
Jacquelin
,
M.
Müller-Trutwin
.
2016
.
Non-human primates in HIV research: achievements, limits and alternatives.
Infect. Genet. Evol.
46
:
324
332
.
32
Crowley
,
A. R.
,
M. E.
Ackerman
.
2019
.
Mind the gap: how interspecies variability in IgG and its receptors may complicate comparisons of human and non-human primate effector function.
Front. Immunol.
10
:
697
716
.
33
Sather
,
D. N.
,
J.
Armann
,
L. K.
Ching
,
A.
Mavrantoni
,
G.
Sellhorn
,
Z.
Caldwell
,
X.
Yu
,
B.
Wood
,
S.
Self
,
S.
Kalams
,
L.
Stamatatos
.
2009
.
Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection.
J. Virol.
83
:
757
769
.
34
Malherbe
,
D. C.
,
F.
Pissani
,
D. N.
Sather
,
B.
Guo
,
S.
Pandey
,
W. F.
Sutton
,
A. B.
Stuart
,
H.
Robins
,
B.
Park
,
S. J.
Krebs
, et al
.
2014
.
Envelope variants circulating as initial neutralization breadth developed in two HIV-infected subjects stimulate multiclade neutralizing antibodies in rabbits.
J. Virol.
88
:
12949
12967
.
35
Eisenbraun
,
M. D.
,
D. H.
Fuller
,
J. R.
Haynes
.
1993
.
Examination of parameters affecting the elicitation of humoral immune responses by particle bombardment-mediated genetic immunization.
DNA Cell Biol.
12
:
791
797
.
36
Condon
,
C.
,
S. C.
Watkins
,
C. M.
Celluzzi
,
K.
Thompson
,
L. D.
Falo
Jr
1996
.
DNA-based immunization by in vivo transfection of dendritic cells.
Nat. Med.
2
:
1122
1128
.
37
Falo
,
L. D.
 Jr
1999
.
Targeting the skin for genetic immunization.
Proc. Assoc. Am. Physicians
111
:
211
219
.
38
Doria-Rose
,
N. A.
,
C.
Ohlen
,
P.
Polacino
,
C. C.
Pierce
,
M. T.
Hensel
,
L.
Kuller
,
T.
Mulvania
,
D.
Anderson
,
P. D.
Greenberg
,
S. L.
Hu
,
N. L.
Haigwood
.
2003
.
Multigene DNA priming-boosting vaccines protect macaques from acute CD4+-T-cell depletion after simian-human immunodeficiency virus SHIV89.6p mucosal challenge.
J. Virol.
77
:
11563
11577
.
39
Lawson
,
L. B.
,
J. D.
Clements
,
L. C.
Freytag
.
2012
.
Mucosal immune responses induced by transcutaneous vaccines.
Curr. Top. Microbiol. Immunol.
354
:
19
37
.
40
Hessell
,
A. J.
,
D. C.
Malherbe
,
F.
Pissani
,
S.
McBurney
,
S. J.
Krebs
,
M.
Gomes
,
S.
Pandey
,
W. F.
Sutton
,
B. J.
Burwitz
,
M.
Gray
, et al
.
2016
.
Achieving potent autologous neutralizing antibody responses against tier 2 HIV-1 viruses by strategic selection of envelope immunogens.
J. Immunol.
196
:
3064
3078
.
41
Wang
,
Y.
,
S.
O’Dell
,
H. L.
Turner
,
C. I.
Chiang
,
L.
Lei
,
J.
Guenaga
,
R.
Wilson
,
P.
Martinez-Murillo
,
N.
Doria-Rose
,
A. B.
Ward
, et al
.
2017
.
HIV-1 cross-reactive primary virus neutralizing antibody response elicited by immunization in nonhuman primates.
J. Virol.
91
: e00910-17.
42
Han
,
Q.
,
J. A.
Jones
,
N. I.
Nicely
,
R. K.
Reed
,
X.
Shen
,
K.
Mansouri
,
M.
Louder
,
A. M.
Trama
,
S. M.
Alam
,
R. J.
Edwards
, et al
.
2019
.
Difficult-to-neutralize global HIV-1 isolates are neutralized by antibodies targeting open envelope conformations.
Nat. Commun.
10
:
2898
2913
.
43
van Diepen
,
M. T.
,
R.
Chapman
,
N.
Douglass
,
S.
Galant
,
P. L.
Moore
,
E.
Margolin
,
P.
Ximba
,
L.
Morris
,
E. P.
Rybicki
,
A. L.
Williamson
.
2019
.
Prime-boost immunizations with DNA, modified vaccinia virus ankara, and protein-based vaccines elicit robust HIV-1 tier 2 neutralizing antibodies against the CAP256 superinfecting virus.
J. Virol.
93
:
e02155-18
.
44
Wang
,
Y.
,
C.
Sundling
,
R.
Wilson
,
S.
O’Dell
,
Y.
Chen
,
K.
Dai
,
G. E.
Phad
,
J.
Zhu
,
Y.
Xiao
,
J. R.
Mascola
, et al
.
2016
.
High-resolution longitudinal study of HIV-1 env vaccine-elicited B cell responses to the virus primary receptor binding site reveals affinity maturation and clonal persistence.
J. Immunol.
196
:
3729
3743
.
45
Sundling
,
C.
,
G.
Phad
,
I.
Douagi
,
M.
Navis
,
G. B.
Karlsson Hedestam
.
2012
.
Isolation of antibody V(D)J sequences from single cell sorted rhesus macaque B cells.
J. Immunol. Methods
386
:
85
93
.
46
Tiller
,
T.
,
E.
Meffre
,
S.
Yurasov
,
M.
Tsuiji
,
M. C.
Nussenzweig
,
H.
Wardemann
.
2008
.
Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.
J. Immunol. Methods
329
:
112
124
.
47
Wei
,
X.
,
J. M.
Decker
,
S.
Wang
,
H.
Hui
,
J. C.
Kappes
,
X.
Wu
,
J. F.
Salazar-Gonzalez
,
M. G.
Salazar
,
J. M.
Kilby
,
M. S.
Saag
, et al
.
2003
.
Antibody neutralization and escape by HIV-1.
Nature
422
:
307
312
.
48
Mahalanabis
,
M.
,
P.
Jayaraman
,
T.
Miura
,
F.
Pereyra
,
E. M.
Chester
,
B.
Richardson
,
B.
Walker
,
N. L.
Haigwood
.
2009
.
Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers.
J. Virol.
83
:
662
672
.
49
Hessell
,
A. J.
,
R.
Powell
,
X.
Jiang
,
C.
Luo
,
S.
Weiss
,
V.
Dussupt
,
V.
Itri
,
A.
Fox
,
M. B.
Shapiro
,
S.
Pandey
, et al
.
2019
.
Multimeric epitope-scaffold HIV vaccines target V1V2 and differentially tune polyfunctional antibody responses.
Cell Rep.
28
:
877
895.e6
.
50
Malherbe
,
D. C.
,
N. A.
Doria-Rose
,
L.
Misher
,
T.
Beckett
,
W. B.
Puryear
,
J. T.
Schuman
,
Z.
Kraft
,
J.
O’Malley
,
M.
Mori
,
I.
Srivastava
, et al
.
2011
.
Sequential immunization with a subtype B HIV-1 envelope quasispecies partially mimics the in vivo development of neutralizing antibodies.
J. Virol.
85
:
5262
5274
.
51
Alpert
,
M. D.
,
L. N.
Heyer
,
D. E.
Williams
,
J. D.
Harvey
,
T.
Greenough
,
M.
Allhorn
,
D. T.
Evans
.
2012
.
A novel assay for antibody-dependent cell-mediated cytotoxicity against HIV-1- or SIV-infected cells reveals incomplete overlap with antibodies measured by neutralization and binding assays.
J. Virol.
86
:
12039
12052
.
52
Ackerman
,
M. E.
,
B.
Moldt
,
R. T.
Wyatt
,
A. S.
Dugast
,
E.
McAndrew
,
S.
Tsoukas
,
S.
Jost
,
C. T.
Berger
,
G.
Sciaranghella
,
Q.
Liu
, et al
.
2011
.
A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.
J. Immunol. Methods
366
:
8
19
.
53
Vázquez Bernat
,
N.
,
M.
Corcoran
,
I.
Nowak
,
M.
Kaduk
,
X.
Castro Dopico
,
S.
Narang
,
P.
Maisonasse
,
N.
Dereuddre-Bosquet
,
B.
Murrell
,
G. B.
Karlsson Hedestam
.
2020
.
Rhesus and cynomolgus IgH genotyping yields comprehensive databases of germline VDJ alleles for in-depth immunological studies.
Immunity.
In press.
54
Corcoran
,
M. M.
,
G. E.
Phad
,
N.
Vázquez Bernat
,
C.
Stahl-Hennig
,
N.
Sumida
,
M. A.
Persson
,
M.
Martin
,
G. B.
Karlsson Hedestam
.
2016
.
Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity.
Nat. Commun.
7
:
13642
13656
.
55
Vázquez Bernat
,
N.
,
M.
Corcoran
,
U.
Hardt
,
M.
Kaduk
,
G. E.
Phad
,
M.
Martin
,
G. B.
Karlsson Hedestam
.
2019
.
High-quality library preparation for NGS-based immunoglobulin germline gene inference and repertoire expression analysis.
Front. Immunol.
10
:
660
672
.
56
Cirelli
,
K. M.
,
D. G.
Carnathan
,
B.
Nogal
,
J. T.
Martin
,
O. L.
Rodriguez
,
A. A.
Upadhyay
,
C. A.
Enemuo
,
E. H.
Gebru
,
Y.
Choe
,
F.
Viviano
, et al
.
2019
.
Slow delivery immunization enhances HIV neutralizing antibody and germinal center responses via modulation of immunodominance. [Published erratum appears in 2020 Cell 180: 206.]
Cell
177
:
1153
1171.e28
.
57
Sather
,
D. N.
,
S.
Carbonetti
,
D. C.
Malherbe
,
F.
Pissani
,
A. B.
Stuart
,
A. J.
Hessell
,
M. D.
Gray
,
I.
Mikell
,
S. A.
Kalams
,
N. L.
Haigwood
,
L.
Stamatatos
.
2014
.
Emergence of broadly neutralizing antibodies and viral coevolution in two subjects during the early stages of infection with human immunodeficiency virus type 1.
J. Virol.
88
:
12968
12981
.
58
Balasubramanian
,
P.
,
R.
Kumar
,
C.
Williams
,
V.
Itri
,
S.
Wang
,
S.
Lu
,
A. J.
Hessell
,
N. L.
Haigwood
,
F.
Sinangil
,
K. W.
Higgins
, et al
.
2017
.
Differential induction of anti-V3 crown antibodies with cradle- and ladle-binding modes in response to HIV-1 envelope vaccination.
Vaccine
35
:
1464
1473
.
59
Wang
,
H.
,
A. A.
Cohen
,
R. P.
Galimidi
,
H. B.
Gristick
,
G. J.
Jensen
,
P. J.
Bjorkman
.
2016
.
Cryo-EM structure of a CD4-bound open HIV-1 envelope trimer reveals structural rearrangements of the gp120 V1V2 loop.
Proc. Natl. Acad. Sci. USA
113
:
E7151
E7158
.
60
Gorny
,
M. K.
,
X. H.
Wang
,
C.
Williams
,
B.
Volsky
,
K.
Revesz
,
B.
Witover
,
S.
Burda
,
M.
Urbanski
,
P.
Nyambi
,
C.
Krachmarov
, et al
.
2009
.
Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1.
Mol. Immunol.
46
:
917
926
.
61
Navis
,
M.
,
K.
Tran
,
S.
Bale
,
G. E.
Phad
,
J.
Guenaga
,
R.
Wilson
,
M.
Soldemo
,
K.
McKee
,
C.
Sundling
,
J.
Mascola
, et al
.
2014
.
HIV-1 receptor binding site-directed antibodies using a VH1-2 gene segment orthologue are activated by Env trimer immunization.
PLoS Pathog.
10
:
e1004337
.
62
Gorny
,
M. K.
,
J.
Sampson
,
H.
Li
,
X.
Jiang
,
M.
Totrov
,
X. H.
Wang
,
C.
Williams
,
T.
O’Neal
,
B.
Volsky
,
L.
Li
, et al
.
2011
.
Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.
PLoS One
6
: e27780.
63
Phad
,
G. E.
,
P.
Pushparaj
,
K.
Tran
,
V.
Dubrovskaya
,
M.
Àdori
,
P.
Martinez-Murillo
,
N.
Vázquez Bernat
,
S.
Singh
,
G.
Dionne
,
S.
O’Dell
, et al
.
2020
.
Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses.
J. Exp. Med.
217
: e20191155.
64
Phad
,
G. E.
,
N.
Vázquez Bernat
,
Y.
Feng
,
J.
Ingale
,
P. A.
Martinez Murillo
,
S.
O’Dell
,
Y.
Li
,
J. R.
Mascola
,
C.
Sundling
,
R. T.
Wyatt
,
G. B.
Karlsson Hedestam
.
2015
.
Diverse antibody genetic and recognition properties revealed following HIV-1 envelope glycoprotein immunization.
J. Immunol.
194
:
5903
5914
.
65
Zolla-Pazner
,
S.
,
S. S.
Cohen
,
D.
Boyd
,
X. P.
Kong
,
M.
Seaman
,
M.
Nussenzweig
,
F.
Klein
,
J.
Overbaugh
,
M.
Totrov
.
2015
.
Structure/function studies involving the V3 region of the HIV-1 envelope delineate multiple factors that affect neutralization sensitivity.
J. Virol.
90
:
636
649
.
66
Haynes
,
B. F.
,
P. B.
Gilbert
,
M. J.
McElrath
,
S.
Zolla-Pazner
,
G. D.
Tomaras
,
S. M.
Alam
,
D. T.
Evans
,
D. C.
Montefiori
,
C.
Karnasuta
,
R.
Sutthent
, et al
.
2012
.
Immune-correlates analysis of an HIV-1 vaccine efficacy trial.
N. Engl. J. Med.
366
:
1275
1286
.
67
Permar
,
S. R.
,
Y.
Fong
,
N.
Vandergrift
,
G. G.
Fouda
,
P.
Gilbert
,
R.
Parks
,
F. H.
Jaeger
,
J.
Pollara
,
A.
Martelli
,
B. E.
Liebl
, et al
.
2015
.
Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.
J. Clin. Invest.
125
:
2702
2706
.
68
Chukwuma
,
V. U.
,
N.
Kose
,
D. N.
Sather
,
G.
Sapparapu
,
R.
Falk
,
H.
King
,
V.
Singh
,
R.
Lampley
,
D. C.
Malherbe
,
N. T.
Ditto
, et al
.
2018
.
Increased breadth of HIV-1 neutralization achieved by diverse antibody clones each with limited neutralization breadth.
PLoS One
13
: e0209437.
69
Dubrovskaya
,
V.
,
J.
Guenaga
,
N.
de Val
,
R.
Wilson
,
Y.
Feng
,
A.
Movsesyan
,
G. B.
Karlsson Hedestam
,
A. B.
Ward
,
R. T.
Wyatt
.
2017
.
Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response.
PLoS Pathog.
13
: e1006614.
70
Setliff
,
I.
,
W. J.
McDonnell
,
N.
Raju
,
R. G.
Bombardi
,
A. A.
Murji
,
C.
Scheepers
,
R.
Ziki
,
C.
Mynhardt
,
B. E.
Shepherd
,
A. A.
Mamchak
, et al
.
2018
.
Multi-donor longitudinal antibody repertoire sequencing reveals the existence of public antibody clonotypes in HIV-1 infection.
Cell Host Microbe.
23
:
845
854.e6
.
71
Gorny
,
M. K.
,
C.
Williams
,
B.
Volsky
,
K.
Revesz
,
S.
Cohen
,
V. R.
Polonis
,
W. J.
Honnen
,
S. C.
Kayman
,
C.
Krachmarov
,
A.
Pinter
,
S.
Zolla-Pazner
.
2002
.
Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades.
J. Virol.
76
:
9035
9045
.
72
Barouch
,
D. H.
,
J.
Liu
,
H.
Li
,
L. F.
Maxfield
,
P.
Abbink
,
D. M.
Lynch
,
M. J.
Iampietro
,
A.
SanMiguel
,
M. S.
Seaman
,
G.
Ferrari
, et al
.
2012
.
Vaccine protection against acquisition of neutralization-resistant SIV challenges in rhesus monkeys.
Nature
482
:
89
93
.
73
Barouch
,
D. H.
,
J. B.
Whitney
,
B.
Moldt
,
F.
Klein
,
T. Y.
Oliveira
,
J.
Liu
,
K. E.
Stephenson
,
H. W.
Chang
,
K.
Shekhar
,
S.
Gupta
, et al
.
2013
.
Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys.
Nature
503
:
224
228
.
74
Barouch
,
D. H.
,
G.
Alter
,
T.
Broge
,
C.
Linde
,
M. E.
Ackerman
,
E. P.
Brown
,
E. N.
Borducchi
,
K. M.
Smith
,
J. P.
Nkolola
,
J.
Liu
, et al
.
2015
.
Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.
Science
349
:
320
324
.
75
Barouch
,
D. H.
,
F. L.
Tomaka
,
F.
Wegmann
,
D. J.
Stieh
,
G.
Alter
,
M. L.
Robb
,
N. L.
Michael
,
L.
Peter
,
J. P.
Nkolola
,
E. N.
Borducchi
, et al
.
2018
.
Evaluation of a mosaic HIV-1 vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase 1/2a clinical trial (APPROACH) and in rhesus monkeys (NHP 13-19).
Lancet
392
:
232
243
.
76
Santra
,
S.
,
G. D.
Tomaras
,
R.
Warrier
,
N. I.
Nicely
,
H. X.
Liao
,
J.
Pollara
,
P.
Liu
,
S. M.
Alam
,
R.
Zhang
,
S. L.
Cocklin
, et al
.
2015
.
Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques.
PLoS Pathog.
11
: e1005042.
77
Burton
,
D. R.
,
A. J.
Hessell
,
B. F.
Keele
,
P. J.
Klasse
,
T. A.
Ketas
,
B.
Moldt
,
D. C.
Dunlop
,
P.
Poignard
,
L. A.
Doyle
,
L.
Cavacini
, et al
.
2011
.
Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody.
Proc. Natl. Acad. Sci. USA
108
:
11181
11186
.
78
Horwitz
,
J. A.
,
Y.
Bar-On
,
C. L.
Lu
,
D.
Fera
,
A. A. K.
Lockhart
,
J. C. C.
Lorenzi
,
L.
Nogueira
,
J.
Golijanin
,
J. F.
Scheid
,
M. S.
Seaman
, et al
.
2017
.
Non-neutralizing antibodies alter the course of HIV-1 infection in vivo.
Cell
170
:
637
648.e10
.
79
von Bredow
,
B.
,
J. F.
Arias
,
L. N.
Heyer
,
B.
Moldt
,
K.
Le
,
J. E.
Robinson
,
S.
Zolla-Pazner
,
D. R.
Burton
,
D. T.
Evans
.
2016
.
Comparison of antibody-dependent cell-mediated cytotoxicity and virus neutralization by HIV-1 env-specific monoclonal antibodies.
J. Virol.
90
:
6127
6139
.
80
Setliff
,
I.
,
A. R.
Shiakolas
,
K. A.
Pilewski
,
A. A.
Murji
,
R. E.
Mapengo
,
K.
Janowska
,
S.
Richardson
,
C.
Oosthuysen
,
N.
Raju
,
L.
Ronsard
, et al
.
2019
.
High-throughput mapping of B cell receptor sequences to antigen specificity.
Cell
179
:
1636
1646.e15
.

The authors have no financial conflicts of interest.

This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

Supplementary data