Factors Associated with the Development of Cross-Reactive Neutralizing Antibodies during Human Immunodeficiency Virus Type 1 Infection^▿

Cohorts.

HIV⁺ subjects from two cohorts were used, the UW/CFAR and the Vanderbilt/CFAR cohorts. We examined subjects with CD4⁺ T-cell counts of ≥250 per μl without antiretroviral therapy and with no AIDS-defining illness during the period of observation. In the Vanderbilt cohort (indicated by the prefix VC), 53 plasma samples from 21 subjects (Fig. 1) were studied, and in the CFAR cohort (indicated by the prefix CC), 43 plasma samples from 18 subjects were studied (Fig. 2). The VC cohort includes subjects with known times of seroconversion and with times since infection ranging from less than 1 year to more than 20 years. The VC cohort consists of 3 African American females and 18 males, 7 of which are African American, 10 of which are Caucasian, and 1 of which is of Asian descent. Subjects VC10014, VC10028, and VC10067 have secondary infections with hepatitis C virus. Subject VC10042 has secondary infections with both hepatitis C and hepatitis B viruses. In contrast, the time of seroconversion is unknown for the CC cohort, and only the time of enrollment is known. Further demographic information for the CC cohort currently is unavailable. For each subject in both cohorts, a minimum of two samples collected at different time points during infection were analyzed. Each sample was heat inactivated at 54°C for 1 h and centrifuged at 17,000 × g for 10 min prior to use in any assay.

HIV-1 envelope-specific IgG titers.

Envelope-specific immunoglobulin G (IgG) titers were determined by enzyme-linked immunosorbent assay (ELISA). Fifty nanograms of soluble trimeric SF162 gp140 was absorbed onto each well of 96-well Immulon 2HB ELISA plates (Thermo) overnight in 0.1 M NaHCO₃, pH 9.4, at room temperature. After blocking the plate in phosphate-buffered saline (PBS), 10% dry milk, 0.3% Tween-20, plasma samples diluted 1:1,000 in 10% dry milk, 0.03% Tween-20 in PBS in triplicate were titrated fivefold to a maximum dilution of 1:15,560,000 and incubated for 1 h at 37°C. Plates were washed in a plate washer, and bound antibodies were detected at 37°C for 1 h with goat anti-human IgG-horseradish peroxidase (HRP) (Bio-Rad) diluted 1:3,000. Plates were developed with 50 μl 1-Step Ultra TMB-ELISA solution (Pierce) and stopped with 50 μl 1N H₂SO₄, and the absorption at 450 nm was read on a Versamaxx microplate reader (Molecular Devices).

Antibody binding avidity determination.

The binding avidity index of plasma antibodies to HIV Env was determined as previously described, with slight modifications (34). Fifty nanograms of soluble trimeric SF162 gp140 was absorbed onto each well of 96-well Immulon 2HB ELISA plates in 0.1 M sodium bicarbonate, pH 9.0, overnight at room temperature. After being blocked in PBS, 10% dry milk, 0.3% Tween-20 and washed in PBS, 10% dry milk, 0.03% Tween-20, a single plasma dilution of 1:500 was incubated in each well for 2 h at room temperature. After being washed in a plate washer, ammonium thiocyanate was added in concentrations ranging from 10 to 4 M (in PBS) and incubated for 20 min at room temperature with shaking. Plates were washed again, and antibodies still bound to SF162 gp140 trimers were detected with goat anti-human IgG-HRP (Bio-Rad) as described above. The avidity index is reported as the concentration of NH₄SCN required to remove 50% of antibodies bound to Env, which was calculated using a dose-response curve fit with nonlinear regression with GraphPad Prism 4.0 software (San Diego, CA).

Neutralization assays.

Neutralization assays were performed using Env pseudovirus variants in the TZM-bl cell-based assay as previously described (9, 33). Briefly, plasma samples at various dilutions were incubated for 90 min at 37°C in the presence of single-round-competent virions. The virus-plasma mixture was added for 72 h at 37°C to TZM-bl cells plated at a density of 3 × 10³ cells per well in a 96-well plate 24 h prior to inoculation. The cell supernatants were removed, and 100 μl of SteadyLite plus (Perkin Elmer) was added to each well. Plates were incubated for 20 min at room temperature with gentle shaking to lyse the cells. Seventy-five microliters of the lysate was transferred to a microtiter plate, and the cell-associated luciferase activity (luminescence) for each well was determined on a Fluoroscan luminometer (Thermo). The neutralization values reported here are the plasma dilutions at which viral entry was inhibited by 50% compared to that in the absence of plasma (IC₅₀). IC₅₀s were calculated using a dose-response curve fit with nonlinear regression with GraphPad Prism 4.0 software (San Diego, CA). A plasma sample was scored as displaying neutralizing activity against a particular virus if at least 50% inhibition of infection was recorded at the lowest plasma dilution tested (1:20) in at least two independent neutralization assays.

Plasmas were tested for neutralization activity against 17 clade B and 2 clade A heterologous isolates (Tables 1 and 2) (4, 17). Most of the clade B viruses tested are part of a panel of viruses created to evaluate the anti-HIV-neutralizing responses elicited during infection or during immunization (17). We define the percentage (0% to 100%) of isolates neutralized by that sample out of the 19 isolates tested as the breadth of the cross-NAb response of a plasma sample. To test plasmas for non-HIV-specific neutralization activity, all plasmas initially were screened for neutralizing activity against the murine leukemia virus (MLV) envelope pseudotyped into the HIV backbone (data not shown). Subjects were excluded for further analysis if their plasma neutralized the pseudotyped MLV by more than 20% at a 1:20 dilution. The analysis of the autologous NAbs was not performed due to the current unavailability of viral Env clones from either cohort.

Peptide competition.

Peptide competition neutralization assays were performed as described previously (9), with slight modifications, using the following peptides: clade B consensus V1 (LMNATNTTNSSSGEK and NSSSGEKMEKGEIKN at 1:1) and V2 (SFNITTSIRDKVQKE, QKEYALFYKLDVVPI, and VPIDNDNTSSYRLIS at 1:1:1), V3 (TRPNNNTRKSIHIGP, KSIHIGPGRAFYTTG, and TTGEIIGDIRQAHCN at 1:1:1), 2F5 (EQELLELDKWASLWN), 4E10 (NWFDITNWLWYIRKKK), and 4E10 scrambled (FTNDWINWLWYIRKKK). Briefly, peptides were added at a final concentration of 10 μg/ml of each peptide to serially diluted plasmas and incubated at 37°C for 90 min. At this concentration of peptide and a concentration of antibody of 20 μg/ml, we estimated the peptide to be in excess of antibody by approximately 40-fold. At the final peptide concentration used during the peptide competition assays, the 4E10 and 4E10 scramble peptides did not interfere with virus infectivity. Pseudovirus was added to the plasma-peptide mixture for 37°C for 90 min. The plasma-virus-peptide mixture was added to TZM-bl cells, plated at a density of 3 × 10³ cells/well, and incubated for 72 h at 37°C. After 72 h the medium was removed and the cells were lysed in 100 μl of SteadyLite (Perkin Elmer). The cell-associated luciferase activity was detected as described above. The percentage of neutralization at each plasma dilution (with or without peptide) was determined. At 70% neutralization in the absence of peptide (the linear portion of the neutralization curve), the percent reduction in neutralizing activity of plasma in the presence of peptide was determined.

Expression and purification of recombinant gp120.

SF162-derived gp120 was expressed by transiently transfecting 293EBNA (293E) cells with the pTT3 vector in Freestyle 293 expression medium (FS medium) (Invitrogen). High-density transfection (2 × 10⁷ cells/ml) of 293E cells was carried out by incubating the cells in 25 μg/ml of plasmid DNA and 50 μg/ml of linear 25-kDa polyethylenamine (Polysciences) for 4 h with shaking at 37°C, as previously described (1). Following incubation, the transfected cells were seeded into a 10-liter WAVE (GE Healthcare) bag containing 4.75 liters of fresh FS medium and incubated for 96 h before the first harvest. The WAVE settings for expression are 5% CO₂, 0.1 liter of airflow/min, and a 7° rocking angle, starting at 18 rpm for the first 24 h and then increasing to 23 rpm for the remainder of the expression.

Fresh Env supernatants were processed immediately following collection and spun at 500 × g for 10 min to pellet the cells. The concentration and buffer exchange of the cell supernatant was done using a tangential flow filtration system (Ultrasette Lab tangential flow device 30KD mwco sc) (Pall Life Sciences). Generally, the volume was reduced 20× and the buffer was exchanged into GNA loading buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, and 1 mM EGTA) for lectin affinity chromatography using GNA resin (Galanthus nivalis) (Vector laboratories). Following concentration and buffer exchange, the sample was centrifuged at 10,000 × g for 10 min to clarify the concentrated supernatant prior to chromatography. The sample was loaded at 3 ml/min onto a 30-ml GNA column packed in an XK26 column (GE Healthcare) equilibrated in GNA loading buffer using an ÄKTA 10/100 purifier (GE Healthcare). After being washed with approximately 5 to 8 column volumes, the column was eluted at 1 ml/min using GNA elution buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 0.75 M methyl-α-d-mannopyranoside, 1 mM EDTA, and 1 mM EGTA). Protein-containing fractions were pooled and subjected to DEAE anion-exchange chromatography. The sample was diluted fivefold in cold 20 mM Tris, pH 8.2, 1 mM EDTA, 1 mM EGTA in order to reduce the NaCl concentration to 20 mM before loading it onto the next column. The anion-exchange step was performed using a Tricorn 10/100 (GE Healthcare) column packed with 10 ml DEAE resin (GE Healthcare) equilibrated in DEAE buffer A (20 mM Tris, pH 8.0, 20 mM NaCl, 1 mM EDTA, 1 mM EGTA). The sample was loaded at 3 ml/min, and the flowthrough was collected. A linear NaCl gradient with DEAE buffer B (20 mM Tris, pH 8.0, 2 M NaCl, 1 mM EDTA, 1 mM EGTA) was run from 0 to 15% over 7.5 column volumes, and all fractions eluting from the column under 20 ms/cm were pooled with the flowthrough. The sample then was concentrated using 30-kDa Amicon ultracentrifugation concentrators (Millipore). This sample contains greater-than 95% pure gp120 according sodium dodecyl sulfate-polyacrylamide gel electrophoresis examination.

Adsorption of plasma antibodies to gp120.

Plasma anti-HIV Env antibodies were adsorbed on beads coated with HIV Env as previously described (18). Two different SF162 gp120 proteins were used, the wild-type (WT) and a CD4-BS mutant with a single amino acid substitution (D368R, based on the HXB2 Env numbering, which corresponds to position 364 in SF162). To generate the D368R mutant, mutagenesis was carried out using the primers SF162D364R (5′-CAATCCTCAGGAGGGCGCCCAGAAATTGTAATG-3′) and SF162D364R-r (5′-CATTACAATTTCTGGGCGCCCTCCTGAGGATTG-3′). The reaction mix was denatured at 95°C for 5 min, followed by 20 cycles of 95°C for 30 s, 55°C for 1 min, and 68°C for 7 min, followed by a final 10-min extension at 68°C. This amino acid substitution abrogates the binding of anti-CD4-BS antibodies to gp120 while retaining the ability to bind other MAbs (Fig. 3A).

The recombinant SF162 gp120 proteins were coupled to MyOne Dynabeads Tosylactivated (Invitrogen) by following the manufacturer's instructions. Briefly, 40 mg of magnetic beads were reacted with 2 mg protein ligand overnight at 37°C with gentle rotation. After collecting the beads on a magnet, the supernatant was removed and the beads were incubated overnight at 37°C in PBS, 0.5% bovine serum albumin (BSA), 0.05% Tween 20. The magnetic beads were washed twice with PBS, 0.1% BSA, 0.05% Tween 20 and stored at 4°C in the same buffer, with the addition of 0.02% sodium azide. Bead-coupled Env proteins were tested for antigenic integrity by flow cytometry using the known MAbs b12, 447-52D, 2G12, and IgG-CD4, followed by detection with goat anti-human IgG-fluorescein isothiocyanate secondary antibody (data not shown). Mock adsorption/elution experiments using several anti-HIV Env MAbs at a concentration of 10 μg/ml in naïve plasma were performed as a positive control (data not shown). Six hundred microliters of plasma diluted 1:20 in Dulbecco's modified Eagle's medium-10% fetal bovine serum were incubated with 300 μl Env protein-coupled beads at room temperature for 120 min with gentle rotation. The samples were placed on a magnet, and the beads were isolated. Up to four serial adsorptions were performed for each plasma.

The antibodies bound to the bead-coupled Env proteins were eluted in a series of increasingly acidic solutions as previously described (18). The beads from each serial adsorption were combined and incubated in 0.1 M glycine-HCl, pH 2.7, for 30 s with vortexing. The beads were collected by brief centrifugation and held in place by a magnet. The supernatant was removed and adjusted to pH 7.5 with 1 M Tris (pH 9.0). The process was repeated with the beads in 0.1 M glycine-HCl, pH 2.3, and then again in 0.1 M glycine-HCl at pH 1.7. The final supernatants were buffer exchanged in PBS and washed over a 30-kDa Amicon ultracentrifugation concentrator (Millipore). Antibody concentrations were determined by bicinchoninic acid assay (Pierce). The depleted plasmas and the antibodies that were eluted from gp120 were used for ELISA and neutralization assays as described above. Both BSA-coupled beads and blank beads were used as negative controls in all assays.

Statistical analysis.

Logarithmic transformation was used for viral load numbers as well as CD4⁺ and CD8⁺ T-cell numbers. Initially, we computed the within-subject variation among the sampled time points for each immunological factor, including neutralizing breadth. We then computed the between-subject variance for each immunological factor and compared the between-subject variance to the within-subject variance using the F test. All of the F tests concluded that the within-subject variations were much smaller than the between-subject variations (P ≤ 0.001). Therefore, for each immunological factor examined, the within-subject average was computed and used for further statistical analyses. To assess what host factors influence the development of broadly neutralizing antibiotic responses, a logistic regression analysis was carried out using time-invariant variables for both the response and the predictors. Specifically, the response was the average breadth of cross-neutralization for each subject. First, univariate models were fitted one at a time with each of the predictors, and then all the potential predictors were added to a multivariate logistic regression model using the Vanderbilt cohort only. A manual backward variable selection procedure was carried out to remove one variable at a time using the Wald test and a significance level of 5%.

Factors correlated with the breadth of NAb responses in HIV⁺ plasma.

A wide range of NAb responses were recorded in both cohorts (Tables 1 and 2). While several plasmas displayed moderate degrees of breadth, neutralizing 30 to 70% of isolates tested (e.g., VC10002 and CC1423), other plasma samples displayed very narrow breadths (e.g., VC20011 and CC1293), neutralizing less than 10% of the isolates tested. Several plasmas were identified that exhibited broad breadth, neutralizing over 75% of the isolates tested (e.g., VC10014 and CC1508). Only one patient (VC10042) was identified with plasma (collected two decades after infection) that displayed extremely broad breadth, neutralizing all isolates tested.

We investigated the potential relationship between the breadth of the plasma NAb responses and the time since infection, blood CD4⁺ and CD8⁺ T lymphocyte numbers, plasma viral load levels, relative titers of Env-specific IgG, and the binding avidity index of these antibodies. A univariate model analysis showed that all predictor variables (except CD4⁺ T-lymphocyte numbers) were significantly positively associated (P ≤ 0.001) with the breadth of the NAb responses (Table 1 for combined cohorts and Table 2 for the Vanderbilt cohort, for which the time of infection is known). A multivariate model analysis (possible only with the Vanderbilt cohort) indicated that only the time since infection (P = 0.001), antibody binding avidity index (P < 0.001), and plasma viral load levels (P = 0.002) had significant impacts on the breadth of the NAb response. Under the multivariate model, the estimated odds of cross-neutralization increased by 7% annually since infection (95% confidence interval [CI], 3 to 12%; P = 0.001); the estimated odds of cross-neutralization increased by 72%, with a 1-U increase in the Env-binding avidity index (95% CI, 44 to 106%; P ≤ 0.001); and the estimated odds of cross-neutralization increased by 75% (95% CI, 24 to 147%; P = 0.002), with a 1-U increase in the log10 viral load.

Continuous evolution of broad NAb responses.

The availability of longitudinal plasma samples from several subjects allowed us to examine whether and how the breadth of the NAb response evolves over time. For subject VC10014 (Fig. 1), the breadth of the NAb response increased from less than 30% to more than 75% during a 3-year period of observation. Similarly, an increase in breadth was recorded for subject CC1711 from 32% to more than 60% during a single year of observation and for subject CC1218 from 16 to 37% during a 3-year observation (Fig. 2). In the majority of subjects, however, the breadth of NAb responses did not change significantly over time. Despite this, plasma samples collected longitudinally from certain subjects displayed different neutralization profiles. For example, plasma collected from VC10067 at 14.72 years postinfection efficiently neutralized isolates BG1168.1 and Q259d2.17 and not isolate 7165.18, but plasma collected a few months later no longer neutralized the first two isolates and weakly neutralized the third isolate (Fig. 1). During that period, the breadth of cross-neutralizing activity did not change significantly (from 37 to 32%). Similarly, plasma from VC10076 collected 5.29 years postinfection efficiently neutralized isolate BG1168.1 but not isolates 422661 and RPHA4259.7; plasma collected a year later no longer neutralized the first isolate but neutralized the other two isolates (Fig. 1). During that period, the breadth of cross-NAb responses changed from 21 to 26%. Therefore, it appears that a continuous and rapid evolution of the broad NAb response takes place during HIV infection.

NAbs targeting the variable regions of Env do not contribute to neutralizing breadth.

Peptide competition neutralization experiments were performed using peptides derived from the first, second, and third variable regions of the consensus clade B gp120 sequence (V1, V2, and V3 loops, respectively) to define the contribution of antibodies to these regions of the HIV Env in the overall cross-neutralizing potential of plasmas. The initial screening was conducted with the SF162 virus, which is known to be susceptible to neutralization by antibodies that bind to the variable regions of Env (9, 33). Indeed, SF162 was neutralized by all plasma samples tested (Fig. 1 and 2). The results are summarized in Fig. 4, and representative experiments are shown in Fig. 3.

We found no evidence for the presence of cross-neutralizing anti-V1 or anti-V2 antibody against SF162 in these plasmas, irrespective of their overall breadth of cross-NAb responses (Fig. 4A and 3). In contrast, the majority of samples we studied contained V3-directed antibodies capable of neutralizing SF162. Interestingly, the anti-SF162-neutralizing activities of some plasmas with narrow neutralization breadths (CC1473, VC10060, and VC10066) were completely blocked by preincubating the plasmas with the consensus clade B V3-derived peptide, while the anti-SF162-neutralizing activities of plasmas with broad neutralizing potential (VC10042, CC1411, and CC1161) were unaffected by such treatment.

It is known, however, that most primary HIV-1 isolates are not susceptible to neutralization by anti-V3 antibodies (3, 17). To better assess the contribution of plasma anti-V3 antibodies in the overall cross-neutralizing activity of plasma, we performed V3 peptide competition experiments with several plasma samples exhibiting moderate to high breadth and several primary isolates: YU-2, JRFL, QH0692, TRO.11, 5768.4, and AC10.0.29. In all cases, the preincubation of plasmas with the consensus clade B V3 peptide had no effect on the overall neutralizing activities of plasmas against any of the isolates tested (Fig. 4B). Thus, plasma anti-V3 antibodies are not responsible for the neutralization of these primary isolates and contribute only minimally in defining the overall cross-neutralizing potential of plasmas collected from the HIV-infected subjects examined in these two cohorts.

NAbs targeting the MPER region contribute to the cross-neutralizing activity of certain HIV⁺ plasmas.

We next examined whether NAbs to the 2F5 and 4E10 epitopes, located in the membrane-proximal external region (MPER) of gp41, were present in these plasmas. These results are summarized in Fig. 6, and representative experiments are shown in Fig. 3. Here too we initially screened the plasma using SF612, since this virus is susceptible to MAb 2F5 and MAb 4E10 neutralization (19, 33). The preincubation of plasmas with a peptide representing the conserved 2F5 epitope did not decrease the anti-SF162-neutralizing activity of any of the plasmas (Fig. 6A). In contrast, the anti-SF162 neutralization activity of two longitudinal plasmas from subject VC10028 decreased by 29 and 38% when the plasmas were preincubated with a peptide representing the conserved E10 epitope (Fig. 6A and Fig. 3E), which is located in the MPER approximately four amino acids downstream from the 2F5 epitope.

It is possible that the 4E10-like antibodies present in plasma VC10028 were active only against SF162 and other neutralization-sensitive isolates but not against neutralization-resistant primary isolates and, thus, do not contribute in defining the overall cross-neutralizing potential of plasma VC10028. We therefore examined whether these antibodies also could neutralize primary isolates other than SF162. In contrast to what we recorded with the anti-V3 NAbs (Fig. 4B), the 4E10-like NAbs contribute in defining the overall cross-neutralizing activities of plasma VC10028. The preincubation of this plasma with the 4E10 peptide resulted in a reduction of the plasma's neutralizing activity against those primary isolates that are neutralizable by the individual plasmas (Fig. 6B) (YU-2 is not neutralized by plasma VC10028, and therefore the preincubation of this plasma with the 4E10 peptide had no effect on the entry of YU-2 into target cells). It appears that a small number of subjects infected with HIV will develop 4E10-like antibodies that are cross-neutralizing.

As expected, the preincubation of other plasmas with the 4E10 peptide had no effect on their antiviral activities (Fig. 6B), since they did not contain anti-4E10 antibodies. Additionally, the preincubation of plasma VC10028 with a 4E10 scrambled peptide did not reduce the neutralizing activity of this plasma (Fig. 5F). These observations and the fact that the 4E10 peptide reduced the neutralizing activity of plasma from only 1 out of 39 subjects examined (Fig. 5E and F and 6) are in support of the presence of 4E10-like plasma VC10028.

Contribution of anti-CD4-BS antibodies to the overall cross-neutralizing potentials of HIV⁺ plasmas.

The overall cross-neutralizing activities of several of the most broadly neutralizing plasmas (more than 75% breadth; for example, VC10042, VC20013, CC1508, or CC1161) in either cohort were unaffected by preincubation with peptides derived from the variable regions of Env or with the 2F5 and 4E10 epitope-derived peptides (Fig. 4 and 6). Recent studies suggested that broadly cross-reactive antibody responses in plasma could be due to NAbs that bind to the CD4-BS of the HIV Env (10, 18). To define the contribution of anti-CD4-BS antibodies in the overall cross-neutralizing potential of plasma samples examined here, we performed adsorption experiments using a variant of gp120 containing a mutation in the CD4-BS that prevents the binding of anti-CD4-BS antibodies (10, 18).

Plasma samples from seven subjects displaying various degrees of neutralizing breadth were investigated. From the Vanderbilt/CFAR cohort we analyzed plasma from subject VC10042 collected 21.5 years postinfection, which neutralizes 100% of isolates tested, and its neutralizing activity is not affected by preincubation with peptides; plasma from subject VC10002 collected 22.51 years postinfection, which neutralizes 32% of the isolates tested; plasma from subject VC10014 collected 3.59 years postinfection, which neutralizes 79% of isolates tested, and its anti-SF162 neutralizing activity is completely eliminated when preincubated with the consensus V3-derived peptide; plasma from VC20011 collected 1.45 years postinfection, which neutralizes only SF162 and, very weakly, 5768.4; and plasma from VC20013 collected 2.28 years postinfection, which neutralizes 63% of the isolates tested. From the UW/CFAR cohort we analyzed plasma from CC1161, which neutralizes 84% of isolates, and plasma CC1508, which neutralizes 95% of isolates tested.

All plasma samples were incubated with WT gp120 or the CD4-BS mutant gp120 coupled to magnetic beads, and the corresponding flowthroughs as well as the eluted antibodies were tested for anti-HIV neutralizing activity against four isolates. The results are summarized in Table 3, and selected experiments are shown in Fig. 5.

The CD4-BS mutant gp120 is not recognized by anti-CD4-BS antibodies, but it is recognized by antibodies directed to other regions of the HIV Env (Fig. 7). All plasma samples contained antibodies capable of recognizing the CD4-BS, as evidenced by the fact that the flowthrough from plasma adsorbed on the CD4-BS mutant gp120 contained antibodies capable of recognizing WT gp120 (Fig. 7B). In fact, very similar titers of anti-CD4-BS antibodies were present in plasmas with very different breadths of cross-NAb responses. In contrast, the adsorption of plasmas with WT gp120 was able to remove nearly all of the gp120-binding antibodies (Fig. 7B).

The flowthrough from WT gp120 of plasma VC10042 had no neutralizing activity against any of the isolates tested, while the flowthrough from the CD4-BS mutant gp120 retained most of this plasma's neutralizing activity (Fig. 5A and Table 3). The antibodies eluted from WT gp120 displayed cross-neutralizing activity, while those eluted from the CD4-BS mutant gp120 did not (Fig. 5B and Table 3). Therefore, the exceptionally broad cross-neutralizing activity of this plasma is due exclusively to anti-CD4-BS antibodies.

The flowthrough from either WT gp120 or the CD4-BS mutant gp120 of plasma VC10014 did not display any neutralizing activity against any of the viruses tested (Fig. 5C and Table 3). The antibodies eluted from either gp120 had similar neutralizing activities (Fig. 5D and Table 3). Thus, although the neutralizing activity of this plasma targets the gp120 subunit exclusively, as is the case for VC10042, it appears that the cross-reactive NAbs in this plasma do not target the CD4-BS.

The flowthrough from WT gp120 of plasma CC1161 displayed reduced neutralizing activity compared to that of the nonadsorbed plasma against YU-2 and QH0692 but not against JRFL and TRO.11 (Fig. 5E and Table 3). The flowthrough from the CD4-BS mutant gp120 neutralized all four isolates tested (Fig. 5E and Table 3). The antibodies eluted from WT gp120 neutralized all isolates, but those eluted from the CD4-BS gp120 neutralized TRO.11 and JRFL but not YU-2 or QH0692 (Fig. 5F and Table 3). These results suggest that anti-CD4BS NAbs partially contribute in the neutralization of YU-2 and QH0692 but not in the neutralization of JRFL and TRO.11 by this plasma. Thus, it appears that plasma from CC1161 neutralizes distinct primary isolates through multiple neutralization targets on Env.

The neutralizing activity of the flowthrough from WT gp120 of plasma VC10002 was only minimally reduced compared to that of the nondepleted plasma against all isolates tested (Fig. 5G and Table 3) (YU-2 was not susceptible to neutralization by this plasma). Similar observations were made for the flowthrough from the CD4-BS mutant gp120 (Fig. 5G and Table 3). The eluted antibodies from both WT gp120 and the CD4-BS mutant gp120 displayed cross-neutralizing activities of similar breadth and potencies (Fig. 5H and Table 3). Overall, these results suggest that anti-CD4-BS NAbs do not contribute significantly in defining the overall cross-neutralizing activity of this plasma. NAbs that bind to gp41 or the oligomeric Env potentially are responsible for the neutralizing potential of this plasma.

For plasmas CC1508 and VC20013, the flowthroughs from both WT gp120 and the CD4-BS mutant gp120 displayed only a minimal reduction in neutralization potency and breadth compared to that of the nonadsorbed plasma, and the antibodies eluted from WT gp120 or the CD4-BS mutant gp120 exhibited various degrees of neutralizing activity against one or two, but not all, of the primary isolates tested (Table 3). These results indicate that the anti-gp120 NAbs in plasmas CC1508 and VC20013 contribute only minimally to the overall neutralizing potential of this plasma. As seen with plasma VC10002, anti-gp41 antibodies may constitute the majority of the cross-reactive NAbs in this plasma. However, it is noteworthy in the case of VC20013 that the antibodies eluted from either WT gp120 or the CD4-BS mutant gp120 neutralized JRFL and not any of the other isolates tested. In contrast, in the case of CC1508 the antibodies eluted from either WT gp120 or the CD4-BS mutant gp120 neutralized QH0692 and TORNO but not JRFL. This suggests that in the first case, the plasma contains anti-gp120 antibodies that bind to epitopes on the JRFL Env that are not present or exposed on the other isolates, while in the second case the plasma contains antibodies with different epitope recognition properties that recognize epitopes on QH0692 and TRO.11 but not JRFL. Therefore, in some cases different antibodies present in plasma may mediate neutralization depending on the viral Env target.

As expected, the flowthrough of the gp120-eluted antibodies from plasma of VC20011 did not display any neutralizing activity against the isolates tested (Table 3), a consequence of the very narrow cross-neutralizing activity of this plasma.