The study was approved by the Institutional Ethics Committee of the National Institute for Research in Tuberculosis (NIRT IEC No: 2011001), and all experiments were performed in accordance with relevant guidelines and regulations. Sample collection was done after obtaining written informed consent from the study participants.

A follow-up blood sample was obtained from 5 of the 12 individuals identified to have broad cross-clade neutralization property in our previous study (1); follow-up sample could not be obtained from the remaining 7 individuals. The follow-up time point was 4 years after the initial screening, which was within 3 years from the time of initial diagnosis of HIV infection. Blood samples were collected in tri potassium-EDTA (K₃-EDTA) treated blood collection tubes (Ref#455036, Greiner Bio-One GmbH, Austria). Plasma was separated by centrifugation at 300 g and stored as 1 mL aliquots at −80°C. The plasma samples were heat inactivated at 56°C for 30 min before use.

CD4+ and CD8+ T-lymphocyte counts were determined on the day of sample collection, by flow cytometry as described earlier using a FACS count flow cytometer (Becton-Dickinson) (1).

Total HIV-1 viral load in cryopreserved plasma was estimated using the fully automated COBAS TaqMan 48 version 2.0 kit (Roche), and expressed as copies of HIV-1 RNA/ml of plasma.

293 T cells, which are human embryonic kidney cells engineered with the T-antigen from SV40 to support constant growth, were kindly gifted by Dr. Udaykumar Ranga (JNCASR, Bangalore). TZM-bl cells (Catalog Number 8129), also called JC53-BL, were obtained from the NIH AIDS Reagents Repository. This is a genetically engineered HeLa cell clone that expresses CD4, CXCR4, and CCR5 and contains Tat-responsive reporter genes for firefly luciferase and Escherichia coli β-galactosidase under the regulatory control of an HIV-1 Long Terminal Repeat (37, 38). Both cell lines were maintained in DMEM (Lonza, Cat# 12-604 F) containing 10% heat-inactivated fetal bovine serum (Lonza) and 50 µg gentamicin/mL (Sigma) in vented T-75 tissue culture flasks (NUNC). Cultures were incubated at 37°C in a humidified 5% CO₂–95% air environment. Cell monolayers were split 1:10 at confluence by treatment with 0.25% trypsin, 1 mM EDTA (Sigma Cat# T4049).

Pseudoviruses were obtained from the NIH AIDS Reagent Repository (www.aidsreagent.org). The pseudovirus panel used in this study is provided in Table S1 in Supplementary Material. Murine leukemia virus (MuLV) env plasmid (env-pseudotyped virus containing the Env of MuLV in the same backbone vector as all HIV-1 env-pseudotyped viruses, MuLV/pSG3Δenv) was kindly gifted by Dr. V. Raghavan (IISc, Bangalore). The MuLV Env-pseudotyped virus was used as negative control in the virus neutralization assays.

HEK293T cells were transiently transfected with two plasmids, one carrying the HIV-1 envelope (pEnv), and the other carrying the entire HIV-1 genome except the envelope (pSGΔenv), using the standard calcium phosphate protocol (39). Briefly, exponentially dividing 293 T cells were seeded at a concentration of 0.7 × 10⁶ cells in a T-25 tissue culture flask and incubated at 37°C in a humidified 5% CO₂–95% air environment overnight. On the following day, old medium was replaced with fresh medium, and cells were co-transfected with the rev/env expression plasmid (5 µg) and env-deficient HIV-1 backbone vector (pSG3ΔEnv) (10 µg) using the calcium phosphate method, and incubated for 5 h at 37°C in a humidified 5% CO₂–95% air environment. After 5 h, the entire medium was removed from the flask and fresh DMEM was added. Pseudovirus-containing culture supernatant was harvested 2 days later, filtered using a 0.45 µm pore size filter, and stored as 1 mL stocks with 20% FBS at −80°C.

The 50% tissue culture infectious dose (TCID₅₀) was determined in TZM-bl cells as described by Montefiori (40). After 2 days of infection, the culture medium was removed from the wells. 100 µL of Britelite Plus reagent (Perkin Elmer) was added to each well, and the plate was incubated at room temperature (RT) for 2 min to allow complete cell lysis. The contents of the wells were mixed by pipettor action (at least two strokes) and 100 µL of cell suspension was transferred to corresponding wells of a 96-well black (opaque) plate. The plate was read immediately in a luminometer and TCID₅₀ was calculated using “TCID” macro (https://www.hiv.lanl.gov/content/nab-reference-strains/htmL/home.htm).

The neutralization potential of the plasma samples was evaluated using the standard neutralization assay with 200 TCID₅₀ pseudoviruses as described earlier (1). In order to determine the neutralization potency (ID₅₀ value), serial dilutions of the plasma samples ranging from 1:20 to 1:43,740 were used. Anti-glycan neutralization activity was evaluated using Du156WT, Du156N160K, Du156N332A, JR-FLWT, and JR-FL E168K pseudoviruses. Further, polyclonal IgG was eluted from the plasma samples using tosylactivated MyOne Dynabeads coupled with 93IN101-gp145 trimer, and neutralization (IC₅₀) and cell-surface binding (EC₅₀) ability of the samples were determined against a tier-2 pseudovirus, JR-FL WT, and its mutant, JR-FL E168K.

Paramagnetic polystyrene, tosylactivated MyOne Dynabeads (Life Technologies) were coupled with recombinant gp145 protein of Indie-C1 which is an Indian HIV-1 reference strain. The gp145 of Indie-C1 is codon optimized and well-studied (41). Briefly, 0.5 mg of protein was coupled with 2.50 mg of tosylactivated magnetic, i.e., MyOne Dynabeads. Coupling was done at room temperature in a total volume of 1.25 ml in coupling buffer (0.1 M sodium borate buffer [pH 9.5] containing 1 M ammonium sulfate) with gentle rocking for 48 h. The Dynabeads and bound protein were separated from the coupling buffer using a magnet and resuspended in 5 ml of blocking buffer (PBS [pH 7.4] containing 0.5% [wt/vol] bovine serum albumin [BSA] and 0.05% Tween 20) for an additional 48 h. The blocking buffer was removed by aspiration, and the protein-coupled Dynabeads were washed two times with 5 ml of wash buffer (PBS [pH 7.4] containing 0.1% BSA and 0.05% Tween 20). The beads were then resuspended in 0.5 ml of storage buffer (PBS [pH 7.4] supplemented with 0.1% BSA, 0.05% Tween 20, and 0.02% sodium azide + protease inhibitors) and stored at 4°C. On the following day, the protein-coupled beads were washed three times with DMEM containing 10% fetal bovine serum, and incubated in fresh DMEM containing 10% FBS at RT for 30 min to block nonspecific binding to the beads. All the follow-up samples were diluted 1:5 in DMEM containing 10% FBS, and 1 ml of the diluted plasma was incubated with 0.5 ml of beads at RT for 1 h. This step was repeated three times for the better removal of antibodies. Beads coupled with gp145-specific antibodies were stored in PBS containing 0.2% BSA and 0.02% sodium azide at 4°C. The protein-coupled beads were washed three times with PBS containing 500 mM NaCl and once with PBS, and the antibodies were eluted by decreasing the pH in a stepwise manner. First, the beads were mixed with 100 mM glycine-HCl elution buffer (pH 2.7) for 30 s. The acid-eluted solution containing IgG was quickly removed and placed in a separate tube containing 0.5 ml of 1 M Tris (pH 9.0) buffer so as to attain a pH of 7.0 to 7.4. This process was repeated three times. The eluted IgG was diluted in DMEM and concentrated over a 30-kDa Centricon plus filter (Millipore Corp.). Subsequently, the same procedure was performed on the beads at an elution pH of 2.2 to recover any IgG resistant to elution at pH 2.7. The IgG fractions recovered by both acid elution steps were combined and the concentration of the combined IgG fractions was measured using nanodrop technology (Thermofisher). The eluted IgG was characterized using cell-surface binding assay and virus neutralization assay.

Human immunodeficiency virus type 1 envelope glycoprotein (Env) expression on the cell surface was achieved as previously described (42). Briefly, 293 T cells were transiently co-transfected with codon-optimized JR-FL wild-type or JR-FL E168K mutant Env-encoding pSVIII plasmid with a pctat plasmid at 3:1 ratio using the calcium phosphate method (1). Cells were harvested 48 h post-transfection, washed thrice with FACS buffer (PBS with 2% FBS), stained with monoclonal neutralizing antibodies or polyclonal IgG eluted from BCN samples, and incubated for 1 h at 37°C at concentrations ranging from 10 µg/ml to 0.0001 µg/ml. After washing, the cells were stained with a PE-conjugated mouse anti-human IgG secondary antibody (1:100 dilutions, BD Bioscience) for 30 min. Cells were again washed and resuspended in 0.5% paraformaldehyde (in PBS). Cell-surface binding of HIV-1 Env-specific antibodies was analyzed by flow cytometry (FACS Verse/Canto analyzer II, BD Bioscience and FlowJo software version 10.0.6, Tree Star Inc.), and binding curves were generated by plotting the mean fluorescence intensity of antigen binding as a function of antibody concentration. Known monoclonal neutralizing antibodies (MAbs) VRC01, PG9, 2G12, and 4E10 that bind specifically to the CD4BS, N160-glycan, N332-glycan, and MPER regions of the HIV-1 envelope, respectively, were used as positive controls.

ELISA was performed with 93IN101 gp145 recombinant [cytoplasmic tail (CT) of 93IN101 gp160 completely deleted for the expression of gp145 protein] trimer and dimer proteins that express intact conformational epitopes targeted by the major classes of bNAbs, namely, CD4BS, N160-V1/V2 glycan, N332- V3 glycan and MPER (41), as well as HXB2 gp120 monomer and NL4-3 gp41 monomer proteins (kindly gifted by Dr. V. S. Kalyanaraman, ABL Inc). BCN samples were tested at a dilution of 1:200 following the protocol described earlier (1). HHP was used as the negative control.

ELISA was performed with recombinant re-stabilized core 3 WT (RSC3) protein that is employed widely for the identification of CD4BS antibodies, a double mutant of the RSC3 protein which contains a deletion at position 371 and a substitution at position 363 (Proline substituted with Asparagine), resulting in addition of an N-linked glycan on the β15 strand near the CD4-binding loop (RSC3Δ371I/P363N), and CD4OD protein which comprises of the outer domain of the CD4 molecule that is based on the HIV-1 HXB2 sequence but with better exposure of the highly conserved CD4BS leading to better identification of CD4BS-specific antibodies that could not be identified using RSC3 protein (43–45). BCN samples were tested at a dilution of 1:200 as described previously (1). HHP was used as the negative control.

Blood samples were collected from 23 healthy HIV negative individuals in tri potassium-EDTA (K₃-EDTA) treated blood collection tubes (Ref#455036, Greiner Bio-One GmbH, Austria). Plasma was separated by centrifugation at 300 g for 10 min. Plasma were pooled and stored as 1 mL aliquots at −80°C until use. This plasma was used as the negative control for all our experiments. The plasma was heat inactivated at 56°C for 30 min before use.

All statistical analyses were performed using Graphpad prism 5.0. Experiments were performed two or more times and values obtained from three replicate samples were averaged in each experiment. Statistical significance was tested using t-test. Differences were considered significant at p < 0.05.

Follow-up samples were obtained from five broad cross-clade neutralizers (four males and one female, aged between 35 and 45 years). Four of these individuals had CD4+ T cell counts >350 cells/mm³ (median 489 cells/mm³) at the time of follow-up. Only one had a lower CD4+ T cell count of 204 cells/mm³. Two of the five individuals (NAB033 and NAB059) continued to be ART-naïve till the time of the present study, while the remaining three were started on antiretroviral therapy during the intervening period. Demographic details and clinical profile of the study subjects are provided in Table 1.

Since the plasma samples of the study subjects were tested previously for neutralization against tier-1, tier-2, and tier-3 pseudoviruses (1), the follow-up samples were directly tested with a panel of 6 tier-3 pseudoviruses at varying dilutions. All five samples exhibited strong neutralization potency against the tested viruses. A heat map of the log neutralization titer of the 5 samples is shown in Figure 1; the dendrogram at the top of the figure shows three clusters of two, three and one viruses, with the one on the right (PVO-4) being most sensitive to neutralization, those in the middle (251-18, 253-11, and 278-50) being moderately sensitive to neutralization and those on the left (257-31 and 33-7) being less sensitive to neutralization with the tested samples (Table 2). Results of the neutralization titer analysis of the plasma samples are represented in graphical format in Figure 2. The 50% inhibitory dilution (ID₅₀) values were calculated using a dose-response curve fit with non-linear function in Graph Pad Prism, v. 5.0. The Geometric mean titer (GMT) was calculated for each sample against all the tier-3 pseudoviruses (Figure 2; Table 2). Four of the 5 samples (NAB001, NAB033, NAB063, and NAB065) neutralized all the tier-3 pseudoviruses with a GMT of 1,678; 1,803; 1,565; and 1,159, respectively. The fifth sample (NAB059) showed an extraordinarily high neutralization potency with a GMT of 7190, prompting us to categorize this individual as an elite neutralizer. Further, we compared first time point neutralization potency (1) with that seen during follow-up, and found that the neutralization potency of 3 of the 5 follow-up samples had increased significantly over time (Table 2; Figure S1 in Supplementary Material). Interestingly, two of these individuals (NAB033 and NAB059) continued to maintain stable CD4 counts and remained ART-naïve.

PepScan ELISA was performed with linear overlapping HIV-1 gp160 peptides to identify if newer antibody responses were generated in any of the BCN samples. Samples were considered reactive if the sample absorbance was two-fold more than that of the negative control (HHP) (Figure 3; Table S3 in Supplementary Material). Interestingly, samples NAB033 and NAB059, showed newer antibody responses with binding reactivity spread across the regions C2, V4, C4, C5, HR2, and CT of the envelope protein. We also found that samples NAB063 and NAB065 acquired additional binding antibody response to V4, V5, C5 and C3, C4, V4 regions of HIV-1 Env protein. As in the earlier time point, all the second time point samples also bound strongly to the V3 loop (peptide nos. 68 to 70) and Immuno Dominant region of gp41 (peptide nos.140 to 143) (Figure 3). Sample NAB059 showed strong binding reactivity to the C2 region which comprises a part of the CD4BS, and also to the HR1 and HR2 regions. NAB033 showed binding reactivity to the HR2 region. The HR1 and HR2 regions are known to play a major role in virus-host cell fusion.

The plasma samples were tested in an ELISA with different forms of the HIV-1 envelope protein, viz. 93IN101 gp145 trimer and dimer, HXB2 gp120 monomer and NL4-3 gp41 monomer, and the results were compared with that obtained at the first time point (Figure 4; Table 3). Interestingly, the second time point sample of NAB059 showed a significant increase in the binding reactivity to all four conformational proteins as compared to the previous time point (p < 0.05). On the other hand, NAB033 showed a significant increase in binding to the NL4-3 gp41 monomer (p < 0.001) but not to the other forms of the envelope protein. There was no significant increase in the binding reactivity to the conformational envelope proteins over time in the remaining samples.

The samples were tested in an ELISA with the wild-type RSC3 and double mutant RSC3Δ371I/P363N CD4BS recombinant proteins, at a dilution of 1:200 to investigate changes in CD4BS specific antibody responses in the BCN samples. Three of the five samples which showed CD4BS specificity at the initial time point again showed CD4BS-specific antibody response as revealed by greater binding response to the RSC3 wild-type protein than to the RSC3 mutant (RSC3Δ371I/P363N) protein (Table 4). When these results were compared with the corresponding results of the first time point samples, it was found that the level of RSC3 binding antibodies had significantly increased with time in two of the three samples (NAB033 and NAB059) (p < 0.001) (Figure 5A).

The recombinant RSC3 protein is designed based on a HIV-1 subtype B strain (8) and has been widely used for the identification and isolation of several well-characterized CD4BS neutralizing antibodies like VRC01, VRC02, VRC03, etc. However, there is sufficient evidence to show that the CD4BS of Indian subtype C HIV-1 Env varies from that of subtype B due to differences at the molecular level (46, 47). Since all our samples were obtained from HIV-1 subtype C-infected individuals, we decided to use an additional HIV-1 envelope protein for identification of CD4BS antibodies. Saha et al. (43) designed the CD4OD protein (CD4OD) for the identification of CD4BS antibodies from HIV-1 subtype C-infected individuals. We performed ELISA with the CD4OD protein and found that four of the five BCN samples showed significantly higher (p < 0.01) binding reactivity to the CD4OD protein than to RSC3 (Figure 5B). In contrast, one sample (NAB059) showed significantly higher (p < 0.001) binding to the RSC3 protein than to the CD4OD protein.

Previously, we had identified four BCN plasma samples with neutralization specificities similar to the PG9/PG16 class of bNAbs (1). However, second time point sample could be collected only from one of the four individuals. This sample (NAB033) was tested in a neutralization assay with pseudoviruses DU156WT and DU156 N160K. Strong neutralization of the DU156 WT pseudovirus was observed (Figure 6A; Table 5) with a 6.7-fold lesser neutralization of the DU156 N160K mutant as compared to the wild type, indicating the persistence of significant amount of antibodies that target the N160 glycan of the HIV-1 Env in this sample.

Neutralization assay was also performed with JR-FL WT and JR-FL E168K mutant viruses. The PG9/PG16 series of bNAbs are unable to neutralize the JR-FL WT HIV-1 strain because the glycan at position N160 is hidden by the glutamic acid (E) present at position 168 in this virus. The PG9/PG16 bNAbs show strong neutralization of the JR-FL pseudovirus when the glutamic acid at position 168 is replaced with lysine (K) in the JR-FL back bone (JR-FL E168K) (3). It was observed that the plasma of NAB033 showed >10-fold stronger neutralization of the JR-FL E168K mutant than the JR-FL WT pseudovirus, providing further evidence to support the presence of significant amounts of PG9/PG16-like antibodies in this sample (Figure 6B; Table 6). Interestingly, we observed significant increase in the neutralization property specifically targeting the N160 glycan of the HIV-1 Env at the second time point in NAB033 as compared to the first time point (p < 0.001) (Figure S2 in Supplementary Material).

In the initial screening, we identified seven BCN samples showing neutralization specificity similar to that of the PGT series of bNAbs (1). However, we could collect second time point sample only from two of these individuals (NAB033 and NAB065). The plasma samples were tested in a neutralization assay with the DU156 WT and DU156 N332A mutant pseudoviruses generally used for this purpose (Figures 6A,C). It was observed that both samples showed significantly stronger neutralization of the DU156 WT pseudovirus than the DU156 N332A mutant; the neutralization activity of NAB033 and NAB065 being 13.6- and 9.2-fold lower with the DU156 N332A mutant as compared to DU156 WT virus (Table 6). Interestingly, both samples showed a significant increase in neutralization property specifically targeting the N332 glycan of the HIV-1 Env in these two samples when compared to the initial time point (p < 0.001) (Figure S2 in Supplementary Material).

To further characterize the neutralization property of HIV-1 Env-specific antibodies present in the samples, we eluted Env trimer specific (93IN101-gp145 trimer) polyclonal IgG from the plasma and performed cell-surface binding as well as neutralization assays with JR-FL WT and JR-FL E168K mutant Env protein and pseudoviruses, respectively. Analysis of the results of the cell-surface binding assay with eluted IgG showed that one of the samples (NAB033) showed stronger binding to the JR-FL E168K mutant (30.49-fold) than to the JR-FL WT Env (Figure 7; Table 7), similar to what was observed with the positive control mAb PG9, which also exhibited significantly stronger binding (142.43-fold) to JR-FL mutant than to JR-FL WT. We then compared the neutralization potency of the eluted IgG with that of known mAbs using HIV-1 JR-FL WT and single mutant JR-FL E168K pseudoviruses. We observed that the median inhibitory concentration (IC₅₀) of NAB033.2 with JR-FL E168K mutant was 12.4-fold lower (i.e., more potent neutralization) than the IC₅₀ with JR-FL WT, as was also seen with the mAb PG9 (Figure 7; Table 7). Collectively our results suggest strongly that the sample NAB033 contains potent PG9/PG16 like bNAbs.