Acute and chronic HIV-1 subjects.

A total of 209 samples (3 to 9 time points per subject) from 38 AHI subjects (plasma donors [ZeptoMetrix Corp. and SeraCare Life Sciences] and acute CHAVI 001 cohorts) and 10 chronic HIV-1 subjects from CHAVI 001 were examined. These cohorts have been previously described (15, 40, 44).

Plasma viral load.

Plasma viral RNA was measured by Quest Diagnostics (Lyndhurst, NJ) (HIV-1 RNA PCR Ultra). Plasma viral RNA for the quantification was also measured by in-house HIV-1 Gag real-time RT-PCR, as previously described (44).

Cells and replication-competent reporter viruses.

The 5.25.EGFP.Luc.M7 (M7-Luc) (provided by N. R. Landau, the Salk Institute for Biological Studies, La Jolla, CA) is a CEMx174 cell clone that was produced by retroviral vector transduction to express CCR5 (CD4 and CXCR4 are expressed naturally) and transfection to contain Tat-responsive luciferase (Luc) and green fluorescence protein (GFP) reporter genes. The cells were grown in RPMI 1640, supplemented with 12% fetal bovine serum (FBS), 50 μg/ml Geneticin (G418), 0.5 μg/ml puromycin, 1% Pen/strep, and 200 μg/ml hygromycin (8). Replication-competent virus stocks from infectious molecular clones (IMC) of Renilla luciferase (LucR) reporter viruses (designated NL-LucR.T2A-Env.ecto) (9) expressing envelope regions from lab-adapted NL4-3 or transmitted/founder viruses (CH040 and WITO) (19) were generated as described previously (9, 14). Briefly, proviral DNA was transfected into 293T cells by Fugene HD (Roche). Working stocks were amplified by passaging virus in human peripheral blood mononuclear cells (PBMCs) (American Red Cross). Virus supernatants were collected every 2 or 3 days and filtered through a 0.45-μm syringe filter, and titers were determined on TZM-bl cells (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from John C. Kappes, Xiaoyun Wu, and Tranzyme, Inc.). Wild-type HIV-1 MN was amplified by the H9 cell line (obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Robert Gallo).

HIV-1-specific binding antibody assay.

Plasma HIV-1-specific antibodies were measured by a custom HIV-1 binding antibody multiplex assay as previously described (44). HIV-specific Ab isotypes were detected with mouse-anti human IgG (Southern Biotech, Birmingham, AL), conjugated to phycoerythrin, at 4 μg/ml. Antibody measurements are acquired on a Bio-Plex instrument (Bio-Rad, Hercules, CA), and the readout is in mean fluorescent intensity (MFI) or μg/ml equivalents based on a 2F5 MAb (Polymune Scientific, Vienna, Austria) standard curve for gp41 IgG detection (5-PL curve fitting) using 21CFR part 11-compliant software. All assays were run under good clinical laboratory practice (GCLP)-compliant conditions, including tracking of positive controls by Levy-Jennings charts. Positivity criteria for antibody-antigen pairs were predetermined using a set of plasmas from 30 seronegative subjects (mean MFI + standard deviation [SD] of 3).

Quantitative IgG-HIV-1 virion immune complex assay.

IgG–HIV-1 virion (IC) were measured by protein G absorption. Briefly, plasma was centrifuged (10,000 × g) for 10 min to remove cellular debris and filtered in a 1.2-μm filter plate (Pall AcroPrep). The 150-μl filtered samples were loaded on each well in protein G HP MultiTrap 96-well plates (GE Healthcare, Inc.) and incubated 1 h with shaking. After incubation, the plate was centrifuged at 1,500 × g for 3 min. The flowthrough fraction was harvested. After washing to remove nonspecific bound material, the absorbed IgG-virion IC was lysed by 560 μl AVL lysis buffer from the QIAmp viral RNA minikit (Qiagen, Inc.) and was harvested by centrifuging 1,500 × g for 3 min. The viral RNA in the lysis buffer was purified with the QIAmp viral minikit according to the manufacturer's instructions. The viral RNA present in each fraction was measured by HIV-1 Gag real-time RT-PCR. Briefly, the one-step RT-PCR amplification reactions were performed in a MicroAmp optical 96-well plate (Applied Biosystems, Foster City, CA) in a 25-μl reaction mixture containing 1× one-step TaqMan RT-PCR master mix, 900 nM forward and reverse primers, 200 nM probe, and the template RNA in a final volume of 25 μl. Reverse transcription was performed at 48°C for 30 min and was followed by activation of TaqGold at 95°C for 10 min. Subsequently, 40 cycles of amplification were performed at 95°C for 15 s and 60°C for 1 min. Polyclonal HIV immunoglobulin (HIVIG) obtained from the NIH AIDS Research & Reference Reagent Program (ARRRP) and the HIV broadly neutralizing antibody 2G12 MAb (Polymune Scientific, Vienna, Austria) were utilized as positive controls. The negative controls, normal human serum (NHS) (Sigma), and virus-only control were utilized to establish the positivity cutoff. We utilized the mean ± 5 SD of the NHS negative control to calculate the positivity cutoff of 8.6% (NHS mean ± 5 SD).

Plasma IgG purification.

IgG was isolated from plasma using the protein G HP MultiTrap 96-well plates described above (GE Healthcare, Inc.). Briefly, plasma was 2-fold diluted with Tris-buffered saline (TBS) at pH 7.5, and 200 μl of diluted plasma was incubated in the plates for 1 h. Unbound fractions were removed by centrifugation at 1,500 × g for 3 min. Wells were then washed 3 times with 200 μl of TBS. IgG was eluted with 2.5% glacial acetic acid and immediately neutralized with 120 μl of 1 M Tris-HCl, pH 9.0. The eluted IgG fractions were concentrated and desalted using Amicon Ultra centrifugal filters (Millipore) with a 30,000 cutoff at 14,000 × g for 12 min. The IgG was then concentrated to the desired volume and assayed for protein concentration using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific).

Protein G capture of virus particles and infectious HIV-1 virions.

Purified plasma IgG or monoclonal IgG was mixed with 1 × 10⁷ RNA copies/ml NL-LucR.T2A viral stock at a final concentration of 10 μg/ml in 300 μl. The IgG and virion IC mixture were prepared in vitro and absorbed by a protein G MultiTrap 96-well plate as described in the measurement of endogenous IgG–HIV-1 virion IC described above. The virions in the flowthrough or captured fraction were measured by viral RNA with HIV-1 Gag real-time RT-PCR, and the 25-μl flowthrough or input was used to infect TZM-bl cells. Infection was measured by a firefly luciferase assay at 48 h postinfection as described previously (39). Briefly, 100 μl supernatant was removed, and 100 μl Britelite (Perkin Elmer) was added to each well. After 2-min incubation, the 150-μl lysis was used to measure HIV-1 replication as expressed as relative luciferase units (RLUs). Each sample was run in triplicate. The percentage of viral particles in the flowthrough or capture fraction was calculated as flowthrough or capture RNA/(flowthrough + capture) × 100%. The percentage of infectivity was calculated as flowthrough infectivity/input infectivity × 100%. The positive controls HIVIG and 2G12 MAb gave virus capture of 66.1 ± 3.7% at 0.83 mg/ml and 30.1 ± 1.4% at 10 μg/ml, respectively. The negative controls, NHS (Sigma), HIV-1⁻ IgG (purified IgG from HIV-1-negative donors [Sigma]), G8 MAb (an IgG monoclonal antibody derived from an Epstein-Barr virus (EBV)-transformed B cell, provided by Kwan-Ki Hwang, Duke University [23]), and virus-only controls were utilized to establish the positivity cutoff. The means ± 5 SD of G8 MAb and virus only are 4.43% and 6.35%, respectively. We utilized the mean ± 5 SD of the NHS-negative control to calculate the positivity cutoff of 8.6% (NHS mean ± 5 SD).

Infectious virus capture assay.

To measure the captured infectious IC, we adopted the Ig-virus capture assay described previously (44). Briefly, microplates (Nunc) were coated overnight at 4°C with mouse monoclonal anti-human IgG (Southern Biotech, Birmingham, AL) at a concentration of 1 μg/ml diluted in PBS. After coating and washing, coated plates were blocked for 2 h with PBS supplemented with 5% goat serum, 5% milk, 0.05% Tween. The indicated concentration of antibodies was mixed with the viral stock containing 5 × 10⁶ viral RNA and then centrifuged 90 min at 2,000 rpm. Then the mixture was centrifuged at 21,000 × g for 45 min at 4°C to remove the virus-free antibody (22), and the pellet was resuspended in the same volume of PBS. A total of 50 μl of the IC mixture was added to each coated well in triple wells for a 90-min incubation. Then, the wells were washed 4 times, and the indicator cell line (M7-luc or TZM-bl) was added. HIV-1 replication was assessed on day 5 after infection for M7-luc and on day 3 for TZM-bl. The infection was measured by the firefly luciferase assay and was expressed as RLU.

Antibody adsorption assay.

A total of 2.4 μg total-plasma-purified IgG was incubated with 0.36 × 10⁶ carboxylated fluorescent beads conjugated with 1.5 μg HIV-1 MN gp41 (ImmunoDiagnostics, Woburn, MA), gp120 protein, or a blank (Luminex Corp, Austin, TX) for 1.5 h. After incubating for 1.5 h, beads were centrifuged for 2 min at 8,000 × g, and the supernatant was harvested. The absorption was repeated another two times. The supernatant was used to make the IgG-virion IC mixture in vitro, and the infectious IgG-virion IC captured by coated anti-human IgG was measured by an M7-luc assay as described above. The HIV-1 anti-gp41 IgG depletion efficiency of absorbed supernatant was confirmed to be removed of HIV-1-specific antibodies by the HIV-1-specific binding antibody assay as described above.

SPR.

Surface plasmon resonance (SPR) binding assays were performed on a BIAcore 3000 (BIAcore, Inc., Piscataway, NJ) instrument at 25°C, and data analyses were performed using BIAevaluation 4.1 software (BIAcore) as previously described (1). HIV-1 MN gp41 and a control protein, HIV-1 p66, were coupled covalently on individual flow cells from 5,000 to 7,000 RU on a BIAcore CM5 sensor chip as described previously (1). IgG samples (100 μg/ml) were injected at 10 μl/min for 2 min and allowed to dissociate in running buffer (PBS [pH 7.4], 0.005% P20) for 10 min. Bound complexes were regenerated by 10 μl glycine-HCl, pH 2.0, at 50 μl/min. Rate constants for HIV-1-specific IgG binding were measured using a 1:1 Langmuir dissociation model (1). The off rate (or dissociation rate) expressed as 1/K_d in seconds is reported.

Single-genome amplification and sequence analysis of the HIV env gene.

Viral RNA from the flowthrough or captured fraction was extracted using the PureLink viral RNA/DNA minikit (Invitrogen, Carlsbad, CA) and reverse transcribed into cDNA using Superscript III (Invitrogen, Carlsbad, CA) and primer 1.R3.B3R 5′-ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3′ (nucleotides [nt] 9611 to 9639 in HXB2) in a 50-μl volume. Single-genome amplification (SGA) was used to obtain rev-vpu-env cassettes as previously described (19, 21). The first-round PCR was carried out with primer 07for7 5′-CAAATTAYAAAAATTCAAAATTTTCGGGTTTATTACAG-3′ (nt 4875 to 4912 in HXB2) and antisense primer 2.R3.B6R 5′-TGAAGCACTCAAGGCAAGCTTTATTGAGGC-3′ (nt 9608 to 9636 in HXB2); the second-round PCR was carried out with sense primer VIF1 5′-GGGTTTATTACAGGGACAGCAGAG-3′ (nt 4900 to 4923 in HXB2) and antisense primer LOW2C 5′-TGAGGCTTAAGCAGTGGGTTCC-3′ (nt 9591 to 9612 in HXB2). All SGA amplicons were sequenced directly by cycle sequencing and dye terminator methods using an ABI 3730xl genetic analyzer (Applied Biosystems, Foster City, CA). Individual sequences were assembled and edited using the Sequencher program 4.7 (Gene Codes, Ann Arbor, MI). The env sequences were aligned using CLUSTAL W (41), and manual adjustment for optimal alignment was done using MASE (10). A phylogenetic tree was constructed with complete env gene sequences using the neighbor-joining method (38) and the Kimura two-parameter model (20). Highlighter analysis was performed through the LANL database website (http://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html).

Quantity of HIV-1 antibodies in HIV-1 virion-IgG complexes during AHI.

To study the relationship between the concentration of anti-gp41 IgG and the percentage of viral load bound in the plasma of acutely infected patients, we assumed the following dynamics between antibodies and virions: A + V C. Antibodies A reversibly bind to free virions V to form an antibody-virion complex C. Since the binding is reversible, antibodies can dissociate from the complex. We assume that the binding kinetics are much faster than the changes in antibody concentration or viral load over time, leading to the following quasi-steady-state assumption: K_b·AV = K_d·C or C = K·AV, where K_b and K_d denote the binding and dissociation rate constants, respectively, and K_b/K_d = K_e, the (effective) affinity constant of the antibody studied. With V_T = V + C defining the total viral load, we obtain:

If more than one antibody binds to a virus, an analogous equilibrium expression can be derived:

where f denotes the total number of antibodies than can simultaneously bind a virion, K is the affinity of an antibody for one gp41 on the virion (i.e., the true affinity), and C_i(t) is the concentration of virions with i antibodies bound at time t.

Plasma IgG from AHI and HIV-1 chronically infected subjects capture infectious HIV-1 virions in vitro.

Since circulating plasma HIV-1 virions were opsonized by IgG in vivo, we also asked whether the purified antibody from plasma from the same subjects had the capacity to capture virions in vitro. Thus, in vitro ICs were made using infectious virus and purified AHI plasma IgG. The percentage of virus captured by purified plasma IgG was determined by separation of opsonized virions and nonopsonized virions using protein G columns. Both the percentage of virions bound and the infectious potential of IgG-bound virions were determined.

First, physical virions were measured in both the uncaptured (flowthrough) and the captured fraction (IgG bound) by real-time RT-PCR. The negative control G8 MAb and virus-only controls yielded capture of 3.5% and 2.9%, respectively (Fig. 4A and B). The positive controls included neutralizing polyclonal pooled IgG (HIVIG), the neutralizing antibody 2G12 MAb, and the nonneutralizing anti-gp41 monoclonal antibody 7B2 MAb (gp41 immunodominant region) (32). As shown in Fig. 4, these antibodies captured virions containing 2.77 × 10⁶ to 4.60 × 10⁶ copies of viral RNA at 10 μg/ml which accounted for 65.2%, 44.8%, and 71.2% of the input viral particles for HIVIG, 2G12 MAb, and 7B2 MAb, respectively. 7B2 MAb reflects a specificity of anti-gp41 antibody, which is among the first gp41 antibodies induced in AHI (44) and therefore serves as a good control to characterize the ability of this specificity of antibody to capture virions.

Open in a separate window

Fig. 4.

Plasma IgG from HIV-1 infection captures infectious or noninfectious HIV-1 viral particles. IgG-virion IC was prepared in vitro at final concentration of 10 μg/ml IgG, and IgG-virion IC mixture was absorbed by protein G. The viral RNA or infectivity in different fractions was measured by HIV-1 Gag real-time RT-PCR (Fig. 4A and B) or TZM-bl infection (Fig. 4C and D). The error bar represents the standard deviation. The percentage of virions (viral RNA) (Fig. 4A) or infectious viruses in each fraction (Fig. 4C) of the input was calculated as described in Materials and Methods and are shown here. vRNA, viral RNA.

We focused further characterization of plasma IgG capture of HIV-1 on three subjects (9075, PRB939, and C1-0586) that had detectable anti-gp41 IgG and detectable endogenous IgG-virion immune complexes (25.5% [25 days post-T₀], 44.5% [93 days post-T₀], and 55.3% [4 weeks postenrollment], respectively) and that represented two subjects from acute infection and one subject from chronic infection. Purified plasma IgG (10 μg/ml) from these three subjects were captured from 1.21 × 10⁶ to 3.67 × 10⁶ copies RNA of NL-LucR.T2A viral particles and accounted for 14.4%, 62.1%, and 69.1% of the input viral particles (Fig. 4A and B). Purified plasma IgG from subject PRB939 (93 days post-T₀) and subject C1-0586 (4 weeks poststudy) demonstrated a similar ability to capture NL-LucR.T2A as HIVIG and the anti-gp41 monoclonal antibody, 7B2 MAb. The in vitro immune complex results correspond with the in vivo endogenous IgG-virion IC results, in that the ranking of the highest to the lowest quantity of immune complexes was the same: C1-0586 > PRB939 > 9075 (see Table S1 in the supplemental material). These in vitro IC formation data support our findings that significant fractions of circulating plasma virions can be coated with IgG in vivo.

Next, we examined whether IgG ICs contained infectious virions. Due to the inherent difficulties in accurately assessing the infectious nature of virus in patient plasma, either due to a small percentage of virions being infectious (4) or due to limitations in the in vitro assays (33), we determined the capacity of IgG purified from plasma following HIV-1 transmission to capture exogenously added infectious virions. We first tested the amount of infectious virus that could be captured by known neutralizing antibodies with HIV-1 specificities known to mediate ADCC and ADCVI. 7B2 MAb targets the immunodominant region of gp41 and is nonneutralizing in the classic HIV-1 neutralization assays but represents antibodies that could have virus inhibitory potential by other mechanisms (17). We found that 77.6% of the infectious virus was captured by MAb 7B2 (Fig. 4C and D). Similarly, for HIVIG, a polyclonal mixture of pooled IgG known to mediate both neutralization and ADCC, we found that 68.9% of the infectious virus could be captured (Fig. 4C and D). MAb 2G12, a broadly neutralizing anti-HIV-1 human MAb reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120 (6, 46), selectively bound to virtually all infectious virions (Fig. 4). When 2G12 MAb-virion complexes were added to the column, there were no detectable infectious virions in the protein G-absorbed flowthrough as determined by TZM-bl infection; however, 55% of the input viral particles were detected in the flowthrough by measuring viral RNA. We confirmed that 2G12 MAb was not present in the flowthrough fraction (see Fig. S1A in the supplemental material), and the 2G12 flowthrough did not neutralize the infection (see Fig. S1B in the supplemental material). The purified plasma IgG from acute HIV-1 infection captured infectious virions at levels similar to those of MAb 7B2, which targets HIV-1 gp41 Env. Moreover, the rank order of infectious virus capture was similar to the results obtained by the virus capture assays that measured viral RNA: chronic C1-0586 > PRB939 > 9075. Plasma IgG from acute plasma subject PRB939 captured up to 51% infectious virus, similar to the levels captured by anti-gp41 nonneutralizing 7B2 MAb.

Nonselective binding of initial HIV-IgG to circulating plasma HIV-1 virions.

To address the question of whether the initial HIV-specific IgG selectively bound certain subpopulations of viral quasispecies, we obtained full-length HIV env gene sequences by SGA from the captured virions and compared the sequences to those of the uncaptured virions. Plasma samples collected 12 weeks postenrollment (the peak of the endogenous plasma IC) from subject C1-0358 were separated by the protein G column, and the env gene sequences from the captured and uncaptured (flowthrough) fractions were compared (23 and 20 env sequences from the uncaptured and the captured fractions, respectively). There was no distinction in virus sequence populations between the captured and uncaptured viruses by the phylogenetic tree and the highlighter plots (see Fig. S2A and B in the supplemental material). In some cases, the env sequences from the captured fraction were identical to the sequences from the flowthrough fraction. These results indicate that there are no genetic differences in the envelope sequences between the uncaptured (flowthrough) and IgG-captured viruses.

Anti-gp41 IgG is a component of plasma IgG-virion ICs.

Since the earliest detectable anti-Env IgG response after HIV-1 transmission targets gp41 and occurs close to the time of the first detectable endogenous HIV-1 IgG-virion IC, we investigated if this antibody specificity was a component of the initial IC (44). We found that the endogenous IgG-virion ICs were detected at the same time or later than the plasma anti-gp41 IgG in 76% (19/25) of IgG-virion IC⁺ subjects, and the two measurements had a corresponding increase in some subjects (Fig. 5A). The statuses of another 9 IgG-virion IC⁺ subjects were not able to be determined because the first available time point samples were both anti-gp41 antibody and IgG-virion IC positive. It is important to note that the presence of plasma IgG-virion IC arose before the appearance of anti-gp41 IgG in 24% of IgG-virion IC⁺ subjects (6/25). This indicates that natural cross-reactive antibodies to antigens present on HIV-1 virions or antibodies to host molecules that are incorporated in HIV-1 virions are likely a component of the specificities that comprise circulating immune complexes during AHI.

Open in a separate window

Fig. 5.

Circulating plasma IgG-virion immune complexes significantly correlate with the concentrations of anti-gp41 Env IgG during AHI. (A) IgG-virion IC and anti-gp41 IgG from one typical plasma donor subject (patient 9012) is presented here. (B) Levels of log10 anti-gp41 IgG (μg/ml) in plasma significantly correlate with the level of plasma IgG-HIV-1 virion IC (n = 8) (t = 4.88, P = 0.0001). Every log10 increase in anti-gp41 IgG correlates to a 6.2% increase in IgG-virion IC. Each color represents one individual subject.

Furthermore, we found that the complement component, C3d, also opsonized plasma virions in acute infection (see Fig. S3 in the supplemental material). For those samples collected within 30 days post-T₀ (except PRB939-09, 93 days post-T₀) and that had undetectable anti-gp120 plasma IgG in the study period (n = 8 subjects), we examined the correlation of gp41-specific antibodies with virion-IgG complexes. In these subjects, there was a significant positive correlation between anti-gp41 IgG (μg/ml) in plasma and endogenous IC (t = 4.88, P = 0.0001) (Fig. 5B). Furthermore, to determine whether the quality of anti-gp41 IgG was also associated with endogenous IgG-virion IC, we determined the dissociation rates (K_d) as one measure of avidity against Env gp41 of purified plasma IgG over time by surface plasmon resonance (SPR). In 4 AHI subjects, the increasing 1/K_d values suggested an increase in the avidity of anti-gp41 IgG to Env gp41 antigen and paralleled the plasma endogenous IgG-virion IC (Fig. 6A). IgG dissociation rates against HIV-1 MN gp41 were significantly correlated with the plasma endogenous IgG-virion IC (r² = 0.76, P < 0.01) (Fig. 6B; see also Fig. S4 in the supplemental material). This indicates that the quality (as measured by the off rate) of HIV-1-specific IgG may play a role in the ability of gp41-specific IgG antibodies to opsonize virions. These data strongly suggest that HIV-1-specific anti-gp41 IgG is a component of the plasma IgG-virion IC in vivo.

Open in a separate window

Fig. 6.

Endogenous plasma IgG-virion IC correlates with the dissociation rates (avidity maturation) of plasma anti-gp41 IgG. (A) The anti-gp41 IgG avidity (off rate,1/K_d in seconds) and the endogenous plasma IgG-virion IC are shown for a representative subject during acute infection. (B) Levels of IgG-virion IC and the anti-gp41 IgG dissociation rates significantly correlate (n = 4) (r² = 0.76, P < 0.01). Dissociation rates (s⁻¹) as a measure of avidity were calculated as described in Materials and Methods.

To further address the specificity of antibodies in the endogenous IgG-virion ICs, we measured the ability of purified plasma IgG from selected subjects, where endogenous IgG-virion ICs appeared at the same time or later than anti-gp41 IgG, to bind infectious virions over time. In subject PRB939, the purified plasma IgG from the later plasma time points (day 13 and day 93 post-T₀) captured infectious lab-adapted HIV-1 NL-LucR.T2A, whereas the earlier sample obtained day 11 post-T₀ that was negative for anti-gp41 IgG did not capture virus. In this subject, the levels of captured infectious NL-LucR.T2A corresponded with the increased levels of anti-gp41 IgG in plasma (Fig. 7A), and anti-gp41 was the only detectable specificity of antibody present in the plasma at this time point. We also assessed the ability of gp41 antibodies to capture virions expressing early transmitted/founder Env (19). The purified IgG from subject PRB939 (day 93 post-T₀) captured NL-LucR.T2A-WITO at a level equal to an about 1:1,000 dilution of plasma (Fig. 7B). Similar results were obtained with another transmitted/founder virus (CH040) (not shown). The capture of HIV-1 NL-LucR.T2A virions by purified plasma IgG from subject PRB939 was also confirmed by measuring HIV-1 viral RNA (3.4 ×10⁴ RNA copies/μg purified plasma IgG).

Open in a separate window

Fig. 7.

Purified plasma IgG from AHI (A, B) or chronic (C) subjects captures infectious HIV-1. A total of 50 μl IC mixture containing plasma IgG at 10 μg/ml and 5 × 10⁶ RNA copies viral particles was added to each mouse anti-human IgG-coated well after incubation in vitro for 1.5 h. The infection of captured IgG IC was measured by multiple-round M7-luc assay for NL-LucR.T2A at 5 days postinfection (A and C) or by TZM-bl at 3 days postinfection for NL-LucR.T2A-WITO (B). The infectivity was measured by firefly luciferase assay and expressed as relative luciferase units (RLU). The gray bar represents the positive or negative controls. The gray bar represents the clinical samples. The levels of anti-gp41 IgG of plasma samples (solid line) were plotted together with the in vitro capture. The cutoff values were 3× background. All runs were performed in triplicate.

Unlike plasma from AHI, plasma from chronic infection had persistently high levels of endogenous IgG-virion ICs (Fig. 3) and anti-gp41-specific IgG. To determine the amount of infectious virus capture by antibodies in chronic infection, IgG was purified from a series of time points (2 to 24 weeks poststudy) from patient C1-0586 and assessed for the ability to capture infectious HIV. The level of HIV infection ranged from 9.9 × 10⁴ to 1.5 × 10⁵ RLU (Fig. 7C), and the kinetics of the appearance of antibodies capable of binding to virions were similar to those of the plasma IgG-virion ICs (Fig. 7C).

In order to further confirm that anti-gp41 IgG in plasma was responsible for the formation of HIV-1 virion ICs seen in vivo and in vitro in AHI, the anti-gp41 IgG in the purified polyclonal IgG of subjects PRB939 (day 93 post-T₀) and 9075 (day 25 post-T₀) were depleted first by anti-gp41 protein-conjugated microspheres, before detection of IgG-virion immune complexes. Depletion of gp41- or gp120-specific antibodies was confirmed by a quantitative HIV-1 binding antibody assay (>99% depletion by gp41-conjugated beads and 90% depletion by gp120-conjugated beads, respectively). For AHI 9075 and PRB939, after anti-gp41 antibody depletion by gp41-conjugated beads, the captured infectious NL-LucR.T2A was decreased by 96% and 66.7%, respectively, compared to blank bead control; however, the depleted IgG by gp120-conjugated beads reduced the captured infection only by 30% and 19%, respectively (Fig. 8). These results demonstrate that the initial anti-gp41 IgG elicited by HIV-1 transmission was the predominant antibody coating HIV-1 virions. In contrast, the total purified IgG from chronic patient C1-0586 (4 weeks poststudy) captured virions that were competed out predominantly by gp120 Env and not gp41 protein (Fig. 8). The depletion by gp120-conjugated beads resulted in a 77.5% decrease in the captured HIV-1 infection, but the depletion by gp41 beads demonstrated only a minor 5.3% decline in infection (Fig. 8). Similar results were found in plasma IgG depletion by HIV-1 Env-conjugated beads in chronic subject C1-0645. The infection of wild-type HIV-1 MN captured by the purified IgG of C1-0586 week 4 declined 94% after absorbed by HIV-1 MN Env gp120-conjugated beads; however, only a 19% decrease in the infection was seen after gp41-conjugated bead depletion. These results show that anti-gp120 but not anti-gp41 antibodies comprise a majority of the antibodies in virion-IgG immune complexes in these two subjects with chronic HIV-1 infection. In contrast, gp41 antibodies are the primary mediators of IC formation during AHI.

Open in a separate window

Fig. 8.

Different specificities of antibodies in immune complexes in acute and chronic infection. Purified plasma IgG from AHI subjects 9075 (day 25 post-T₀) and PRB939 (day 93 post-T₀) or chronic subject C1-0586 (week 4 poststudy) were absorbed by Env gp41 or Env gp120-conjugated, or blank microsphere beads. Then the absorbed supernatant was used to prepare IgG-virion IC mixture in vitro. The infectious IC was captured by plates coated with anti-human IgG antibody and was measured for infection in the M7-luc infection assay as described above. The percentage of infection compared to the control blank bead is shown here. The results represent at least 3 experiments.

This work was supported by the National Institutes of Health (NIH/NIAID/DAIDS) Center for HIV/AIDS Vaccine Immunology Grant (U01 AI067854). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We are indebted to the CHAVI 001 clinical teams and patients for their numerous contributions that were central to this study. We also thank Kelly Soderberg and Jennifer Kirchherr for program support, Shelley Stewart, Judith T. Lucas, and Vicki C. Ashley for technical assistance, Hua-Xin Liao for HIV-1 gp140 envelope proteins, Cristine Cooper, SCHARP, for statistical support, and Michael Frank, Xiaoying Shen, Robert C. Schutte, and Genevieve Fouda for helpful discussions.