Involvement of FcγR I (CD64) in the Mechanism of HIV-1 Inhibition by Polyclonal IgG Purified from Infected Patients in Cultured Monocyte-Derived Macrophages

Vincent Holl, Stéphane Hemmerter, Renaud Burrer, Sylvie Schmidt, Alain Bohbot, Anne-Marie Aubertin and Christiane Moog
J Immunol November 15, 2004, 173 (10) 6274-6283; DOI: https://doi.org/10.4049/jimmunol.173.10.6274

Abstract

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcγRs in HIV-1 inhibition, F(ab′)2 and Fab of these Igs were produced. Results indicated that both F(ab′)2 and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcγRs Ab strengthen the participation of the FcγRs, and in particular of FcγRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.

Despite clinical improvement associated with multiple highly active antiretroviral therapies (HAART), current antiviral agents are not able to eradicate HIV-1 due to the persistence of latent virus reservoirs (1). Viral sanctuaries represent potential long-life persistant HIV-1 permissive cells that cannot be suppressed by HAART. Some of such identified HIV-1 reservoirs are resting memory CD4+ T lymphocytes, peripheral blood monocytes, macrophages, and dendritic cells (2, 3). Monocytes and monocyte-derived macrophages (MDM) constitute the cellular population where replicative virus is almost exclusively detected after HAART (4, 5). These cells are of particular importance in the pathogenesis of HIV-1 and their role in AIDS disease is well documented (6). Macrophages are productive sources of HIV-1. Once infected, they are capable of producing large amounts of both intracellular and extracellular HIV-1 without necessarily succumbing to the cytopathic effects of the productive viral infection (7, 8). These primary cells contribute also to viral persistence in tissues (9) and dissemination despite immune surveillance. HIV-1-infected macrophages have been proposed to be the “Trojan horse” for dissemination of infectious particles (9, 10, 11). HIV-1-infection of macrophages could also persist in tissue for extended periods of time (months) with large numbers of infectious particles contained within intracytoplasmic vacuoles (11). They have been implicated in the early stage of the disease (12) to play an important role in the initial transmission of HIV-1, in virus spreading and cell-to-cell transmission within lymphoid tissues (13) as well as in the late stage of the disease by causing brain damage (14). Ab neutralization and/or inhibition of HIV-1 infection in these primary mononuclear phagocytes may potentially block or at least reduce HIV-1 replication and dissemination.

Previous studies have shown that, when macrophages are used as target cells instead of the autologous blood lymphocytes, an increased efficiency, at least 100-fold, of the neutralizing titer was observed for plasma or sera from SIV-infected macaques or HIV-infected patients (15, 16). Ruppach et al. (16) have observed autologous neutralizing activities on primary human macrophages but not on lymphocytes when analyzing sera from early infected individuals (<12 mo after seroconversion). As neutralizing titer measured can considerably vary according to the experimental culture conditions used and is dependent on the level of virus replication and production (17), we reevaluated the variation of neutralizing titer using a new neutralization assay based on a single cycle of infection in macrophages, similar to that recently developed for PBMC (18). We showed a considerably higher neutralizing activity of IgG purified from late HIV-seropositive individuals when macrophages are used as target cells. The role of the FcγR on the human primary macrophage surface in the phagocytosis and the clearance of virus-IgG immune complexes was investigated.

Materials and Methods

Monoclonal Abs

Anti-HIV-1 human mAbs were used to study the mechanism of neutralization. IgG1b12 (directed against an epitope that overlaps the CD4 receptor-binding site on gp120) was obtained from Drs. D. Burton and P. Parren (The Scripps Research Institute, La Jolla, CA). IgG1κ human monoclonal 2F5 and 4E10 (both directed against a different epitope in the ectodomain of gp41) were provided by Dr. H. Katinger through the National Institute of Biological Standards and Control (NIBSC, Potters Bar, U.K.), Control Central Facility for AIDS Reagents and through Polymun Scientific (Vienna, Austria), respectively. Purified anti-human monoclonal IgG1κ against the different FcγR (CD16, clone 3G8; CD32, clone3D3; CD64, clone 10.1) were obtained from BD Biosciences/BD Pharmingen (San Diego, CA).

Cells and virus stocks

PBMC from normal donors seronegative for HIV-1 and hepatitis B and C were isolated from buffy coats or cytapheresis by Ficoll-Hypaque gradient centrifugation. Before HIV-1 infection, PBMC were activated in the presence of PHA (Sigma-Aldrich, St. Louis, MO) in RPMI 1640 cell medium culture containing antibiotics (penicillin at 100 IU/ml and streptomycin at 100 μg/ml), 2 mM l-glutamine, supplemented with 10% heat-inactivated FCS (Invitrogen Life Technologies, Carlsbad, CA). After 3 days of incubation with PHA, cells were washed and either used in neutralization assay for virus production or frozen for further use.

Monocytes are separated by countercurrent centrifugal elutriation of peripheral mononuclear cells obtained by cytapheresis as detailed elsewhere (19). Monocyte purity was determined by flow cytometry staining with fluorochrome Abs (BD Pharmingen, San Diego, CA) to CD3 (T cells), CD19 (B cells), CD14 (monocytes), and CD45 (leukocytes). Monocytes (109) from each HIV-seronegative donor were collected separately and either directly cultured at 1.7 × 106 cells/ml in AIM-V lymphocytes FCS-free medium with Glutamax 1×, 100 U/ml GM-CSF (PeproTech, Rocky Hill, NJ), or frozen with 90% of FCS at the final concentration of 50 × 106 cells/ml (by progressive lowering of temperature with an automate freeze substitution) for further use. Frozen monocytes are quickly thawed and washed with cold RPMI 1640 medium (1200 rpm for 10 min), then diluted at 3 × 106 cells/ml in AIM-V lymphocytes FCS-free medium with Glutamax 1×, 100 U/ml GM-CSF. Two hundred microliters of monocyte suspension were plated in 48-well plates (Costar 3548; Cambridge, MA) and used in a single cycle neutralization assay or 100 μl were plated in 96-well plates (Costar 3599) for multiple rounds of infection assay. Culture medium was changed after 3 days of culture and at day 5, macrophages were washed twice with RPMI 1640 at 37°C before infection.

HIV-1 strains Bx08 and BaL primary isolates (subtype B, HIV-1 R5 strains) were provided by Prof. H. Fleury (Laboratoire de Virologie, Hôpital Pellegrin, CHU de Bordeaux, France) and through the AIDS Research and Reference Reagent Program from Dr. S. Gartner (Department of Neurology, Johns Hopkins Hospital, Baltimore, MD) and Drs. M. Popovic and R. Gallo (Institute of Human Virology, University of Maryland Biotechnology Institute and Department of Microbiology, Baltimore, MD), respectively. Virus stocks obtained as previously described (17, 20) were concentrated by as much as 80-fold through a 100 kDa cut-off polyethersulfone filter (Centricon 80 Plus Biomax Filter; Millipore, Molsheim, France) for single cycle infection assay.

Preparation of purified F(ab′)2 or Fab

Polyclonal IgG were purified on protein A-Sepharose chromatography columns (Pharmacia, Peapack, NJ) as previously described (21). F(ab′)2 were obtained by pepsin (Sigma-Aldrich) digestion. Briefly, 10 μg/ml pepsin were added to each IgG sample in glycin buffer (0.1 M, pH 2.7) for 30 min at 37°C. The reaction was stopped after 30 min by increasing the pH to neutrality by the addition of Tris-HCl, pH 9. Fab parts of the IgG were obtained by papain digestion. Briefly, papain at 1 mg/ml was activated by incubation at 37°C for 1 h in a 0.05 M sodium phosphate buffer containing (0.01 M cystein, 0.02 M EDTA, and 0.13 M NaCl). Then 0.1 mg/ml activated papain was added to each IgG fraction in a buffer containing 0.01 M cystein, 0.002 M EDTA, 0.05 M sodium phosphate, and 0.1 M KCl. After 40 min at 37°C, the reaction was stopped by the addition of 0.02 M iodoacetamide. Digestion products corresponding to F(ab′)2 or Fab and Fc fragments were separated using a protein A-Sepharose column. These different products were dialyzed against PBS for 2 h at room temperature before being used for determination of a neutralizing titer. Efficiency of digestion and purification was checked on SDS-PAGE and Coomassie blue staining. The concentrations of IgG, F(ab′)2, Fab, and Fc were measured by the Bradford method. Note that each time, control without proteinase digestion had been run in parallel and we found neutralizing activity to be similar to initial IgG sample. These controls guaranteed that the loss of material generated by column purification, or/and dialysis steps was minor.

Neutralization assays

Multiple rounds neutralization assay.

Neutralizing titers were determined after multiple rounds of virus replication as previously described (21). Briefly, 25 μl of four 4-fold dilutions of virus in quadruplicate wells were incubated for 1 h at 37°C, with 25 μl of serial IgG (or corresponding F(ab′)2) dilutions, in a 96-well filtration plate (Durapor-Dv, 1.25-μm pore size; Millipore). For PBMC, 25 μl of PHA-stimulated cells (from a pool of five seronegative donors) at a concentration of 4 × 106 PBMC/ml were added to the Ab/virus mixture. For MDM (monocytes from several donors were used, each time separately), the same volume of Ab/virus mixture was added to the previously seeded 5-day-old MDM in a 96-well plate. After 24 h at 37°C, 100 μl of RPMI 1640 containing 10% FCS and 20 IU of IL-2 per milliliter for PBMC or 100 μl of AIM plus Glutamax 1× and GM-CSF for MDM were added to each well. Free virus and Abs were removed on day 4 by successive washing. Cells were then cultured in complete medium (200 μl) until day 7 postinfection, time at which p24 was measured in the supernatants by ELISA (Innotest; Innogenetics, Ghent, Belgium) to determine HIV-positive cultures. The viral titer (50% tissue culture infective dose (TCID50)) was determined in the presence (Vn) and in the absence (V0) of the serum (according to the Reed and Muench method). The neutralization titer was defined as the reciprocal of the serum dilution or as the concentration of protein (interpolated between successive dilutions or concentrations) that result in a 90% decrease of the viral titer (Vn/V0 = 0.1).

Single cycle neutralization assay.

Kinetics of HIV-1 replication in primary human PBMC or MDM were first performed by analyzing intracellular staining of p24 Ag at various time periods in the presence or in the absence of 10 μM AZT (Sigma-Aldrich). The p24-positive cells were only detected 18 h after infection and the addition of AZT before HIV infection totally abrogated the production of p24 in these cells (data not shown), demonstrating that p24 detected after 24 h corresponds to new viral p24 production. A linear dose response between virus input and the number of p24-positive cells was obtained. Thus, to compare the different neutralizing titer of IgG samples, we performed the neutralization assay within 24 h. Both viruses were used at concentrations of 3 and 6 μg of p24/ml allowing infection of 2% of PBMC and MDM, respectively, although these values vary slightly according to the virus stock concentration used.

Twenty-five microliters of various dilutions of polyclonal IgG purified from infected patients (21) or their corresponding Fab or F(ab′)2 were incubated for 1 h at 37°C with 25 μl of virus. Human PBMC (25 μl) at 20 × 106 cells/ml were added to the Ab/virus mixture in a 96-well plate (U-bottom, Costar 3599) and cultured for 24 h in RPMI 1640 10% FCS and 20 U/ml IL-2 (R&D Systems, Minneapolis, MN). For MDM, 50 μl of this Ab/virus mixture were added on 5-day-old MDM previously seeded in a 48-well plate.

To detect HIV-infected lymphocytes or MDM, intracellular staining of viral p24 Ag was performed and analyzed by flow cytometry. Cells were fixed and permeabilized using both Cytofix/Cytoperm and Perm/Wash kits (BD Biosciences/BD Pharmingen) according to the manufacturer and stained with a fluorescent anti-p24 mAb (FITC- or PE-anti-p24, clone KC57; Beckman Coulter/Immunotech, Hialeah, FL) used at a 1/160 dilution in Perm/Wash 1× solution added for 15 min at 4°C, as previously described (18). After washing in PBS with 3% FBS, PBMC or MDM were diluted in 300 μl of PBS or 250 μl of PBS containing 10 mM EDTA, respectively, before flow cytometry analysis (FACScan; BD Biosciences) with CellQuest software (BD Biosciences). The percentage of p24-positive cells in the different samples was determined by gating 20,000 events on a living cell population identified by forward- and side-scatter parameters. The living cell subsets were analyzed for p24 expression relative to background staining of uninfected cells. The p24 Ag-positive value was obtained after subtraction of background events in mock-infected cells (usually less than three positives per 20,000 events).

The percent of neutralization was defined as the reduction of p24-positive cells compared with control infected wells with no Ab. The neutralizing titer was defined either as the dilution of IgG sample or as the concentration of protein (interpolated between successive dilutions performed in triplicate) that allows a 90% decrease in the percentage of infected cells.

Competition experiments

Human polyclonal IgG from non-HIV-1-infected individuals or their corresponding Fc fragments or purified human anti-FcγR monoclonal IgG were incubated with polyclonal IgG from HIV-infected individuals or monoclonal neutralizing IgG before being processed as for the neutralization assay. Results are expressed as the percentage of p24-positive cells compared with infection without seronegative polyclonal IgG, corresponding Fc fragments, or purified human anti-FcγR monoclonal IgG.

Quantitation of β-chemokines by ELISA

The β-chemokines, such as MIP-1α, MIP-1β, and RANTES, were quantified in culture supernatants by using commercial ELISA duoset kits purchased from R&D Systems.

Results

Neutralizing titers of polyclonal and monoclonal IgG using primary PBMC or MDM as target cells

We first determined which subsets of cells are infected in vitro after one cycle of infection when PBMC or MDM are used as target cells. The major cell population detected positive for p24 staining was CD45RO+CD4+ T cells and differentiated macrophages, 24 h after the infection of PBMC and MDM, respectively (data not shown). As these cells represent the major infected cells in vivo, we further used PBMC and MDM in our neutralization protocols. Using the single cycle neutralization assay, we compared the neutralizing titer of polyclonal IgG samples obtained from a cohort of well-studied French HIV-infected patients (21) when PBMC or MDM were used as target cells (Fig. 1⇓ and Table I⇓). As shown in Fig. 1⇓, polyclonal IgG sample no. 8 is able to inhibit HIV replication in both HIV-infected peripheral blood lymphocytes and macrophages after a single round of infection. However, the concentration of Ab needed to reduce the proportion of p24 Ag-positive cells to 0.4% is much higher when PBMC are used as HIV-target cells (Fig. 1⇓).

FIGURE 1.
FIGURE 1.

Quantitation of HIV-1BaL-infected cells by flow cytometry in a single round neutralization assay. Mock- and HIV-1BaL-infected MDM (A) or PBMC (B) were stained for intracellular p24 Ag on day 1. Polyclonal IgG sample no. 8 (140 μg/ml for PBMC or 2 μg/ml for MDM) was incubated 1 h with HIV-1BaL before cell exposition. AZT (10 μM) was added to HIV-1-infected cells for inhibition of replication and gag p24 protein production. Each dot plot analysis of intracellular p24-Ag is representative for one of three experiments. Values represent here the percentage of p24-positive cells.

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Table I.

Neutralizing titers of human polyclonal IgG from HIV-1 patients and monoclonal IgG using PBMC or MDM as HIV-target cells in single cycle or multiple round infection assaysa

When PBMC were used as HIV-target cells, purified IgG samples displayed similar neutralizing titer whether standard multiple rounds of infection or one cycle of infection were used (Table I⇑). Different neutralization spectra could be identified (summarized in the legend of Table I⇑). We could not detect neutralizing activity in samples 2, 3, and 11 against virus Bx08, although theses samples contain high levels of viral particle-binding IgG.4 Two of these IgG samples, nos. 22 and 12 were able to inhibit virus BaL replication with low neutralizing titers of 1/15 and 1/12, respectively. The other IgG samples tested here can be divided into two different groups of neutralizing IgG: one group comprising sample nos. 8 and 44 that efficiently neutralized both viruses Bx08 and BaL, and the other group, with a medium neutralizing titer, encompassing the remaining IgG samples.

When MDM were used as HIV-target cells, we found a drastic increase of the neutralizing titer of these IgG samples against both BaL and Bx08 HIV-1 primary isolates. As depicted in Table I⇑, neutralizing titers of the different purified IgG increase to 100- to 2000-fold when macrophages were used as HIV-target cells. Moreover, polyclonal IgG, that were not found to neutralize viruses on PBMC, strongly inhibit virus replication in MDM. For example, purified IgG sample no. 11 has high neutralizing titer (dilution factor of 1/2000) on HIV-infected MDM whereas this sample has no detectable neutralizing activity when PBMC are used as target cells. When PBMC and MDM were obtained from the same donor, we also detected a higher neutralizing titer on HIV-infected MDM (Table II⇓), indicating that the difference in the neutralizing activity could not be attributed to the difference in susceptibility of the cell donors. When evaluating neutralization of HIV in multiple rounds of infection (Table I⇑), the inhibitory activity of IgG is further augmented as compared with single cycle infection assay in MDM. This increased neutralizing titer was not evidenced when PBMC were used as target cells (Table I⇑).

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Table II.

Neutralizing titer of human polyclonal IgG samples using PBMC or MDM obtained from four different donorsa

We have also compared the neutralizing titer of three mAbs, IgG1b12, 4E10, and 2F5, described to exhibit broad cross-neutralizing activity when PBMC are used as target cells. On PBMC, the neutralizing concentrations of mAbs were similar whether we used a multiple round infection assay that measured virus titer or a single cycle infection assay which quantified intracellular p24 production. When MDM were used as target cells, we again detect a drastic increase in neutralizing activity of mAbs. It is noteworthy that for 2F5 which recognized a conserved epitope on gp41, the concentration of mAb able to inhibit 90% infected cells is 4,000- to 12,000-fold lower on MDM vs PBMC, the neutralizing concentration of mAb IgG1b12 directed against the gp120-CD4 binding site is only 10- to 25-fold lower on MDM (Table I⇑). Purified IgG from the sera of seronegative individuals were included as HIV-negative control and did not have any neutralizing activity. Thus, a higher neutralizing activity is observed for both polyclonal IgG and mAbs when MDM instead of PBMC were used. Additional experiments were designed to investigate the mechanisms by which polyclonal and monoclonal IgG more efficiently neutralize HIV when MDM are used as target cells.

Neutralizing activity of corresponding Fab or F(ab′)2 of polyclonal IgG and IgA purified from HIV-infected patients

As MDM have the capacity to inhibit virus-IgG complexes via FcγR present at the cell surface of macrophages, we evaluated the ability of Fab or F(ab′)2 obtained from polyclonal IgG samples to neutralize HIV-1 primary isolate replication in these human primary cells. The concentration of polyclonal IgG or corresponding F(ab′)2 or Fab required to inhibit 90% of infected cells after single round infection of MDM vs PBMC are reported in Table III⇓. IgG inhibit HIV replication in MDM with much higher efficiency than their corresponding F(ab′)2 and Fab as up to a 100-fold lower concentration of these polyclonal IgG are able to decrease the number of infected cells by 90% (Fig. 2⇓ and Table III⇓). However, these fragments have neutralizing activities similar to IgG when PBMC are used in this single round neutralizing assay (Table III⇓), and, similar dose-response curve activity is observed for F(ab′)2, Fab, and whole IgG on HIV-infected PBMC (Fig. 3⇓). It is noteworthy that the neutralizing concentration of F(ab′)2 or Fab is comparable on PBMC and on MDM. The lower neutralizing activity observed for F(ab′)2 or Fab, compared with whole IgG, on HIV-infected MDM strongly suggests the participation of the Fc portion of IgG in HIV-1 inhibition on MDM. Different groups have shown the potent role of IgA in HIV-1 neutralization (21, 22, 23). Thus, we have measured on MDM the neutralizing activity of polyclonal IgA, previously described to inhibit HIV-1 replication on PBMC (21). No significant difference in the neutralizing titer of the three polyclonal IgA purified from HIV-infected patients was found whether the neutralization experiments were performed in MDM or PBMC (Table III⇓). Moreover, F(ab′)2 of IgA no. 8 exhibit similar neutralizing titer as the whole corresponding IgA. Thus, contrary to IgG samples, we could not detect an increased inhibitory effect of IgA when MDM are used as HIV-target cells. These results show that the increased inhibitory activity observed for these IgG is related to Fcγ part of polyclonal IgG that could play a role in the clearance of the virus, but not to the F(ab′)2 or Fab-bound or the IgA-bound virus particles.

FIGURE 2.
FIGURE 2.

Neutralizing activities of polyclonal IgG, Fab, and F(ab′)2 purified from HIV-infected patients on HIV-1BaL infection of MDM and PBMC. Twenty four hours after the infection, virus neutralization was determined by measurement of one round infection using immunofluorescent staining of p24 Ag by flow cytometry. Percentage of infected PBMC or MDM were determined in the presence of increasing concentrations of purified IgG or corresponding Fab or F(ab′)2 fractions from either sample nos. 44 (A) or 8 (B). Values are the mean of three independent wells from one representative experiment.

FIGURE 3.
FIGURE 3.

Competition experiments between IgG or Fc fragments from non-HIV-infected donors and polyclonal or monoclonal neutralizing IgG. Fc fragments or IgG from nonseronegative donors were added to MDM (A, B, and C) or PBMC (D) infected with HIVBaL-IgG immune complexes. The percentages of infected cells were measured after intracellular staining of p24 Ag by flow cytometry. Increasing concentrations of purified IgG sample nos. 11 (A) or 8 (B and D) or monoclonal 2F5 Ab (C) were used in the presence of IgG or Fc fragments from uninfected individuals. (•, untreated HIVBaL-infected cells; ▵, 0.1 mg/ml and □, 0.05 mg/ml purified IgG from uninfected donors; ○: 0.1 mg/ml Fc fragments from uninfected donors). Values are the mean of three independent wells from one representative experiment.

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Table III.

Neutralizing activity of human polyclonal IgG and IgA from sera of infected individuals and their corresponding F(ab′)2 and Fab when MDM or PBMC are used as HIV-target cellsa

Participation of the FcγR in the inhibition of HIV-1 in MDM

In an effort to determine the implication of the FcγRs in the clearance of IgG-HIV immune complexes, we performed Ab competition experiments between IgG from HIV-1-infected individuals and IgG or Fcγ fragments of polyclonal IgG purified from sera of a HIV-1-negative donor. Incubation of human normal IgG or their Fcγ fragments with MDM allows to considerably diminish the inhibitory activities of polyclonal IgG from HIV individuals (Fig. 3⇑, A and B). Similarly, Ab competition of Fcγ fragments of IgG from non-HIV-infected donors with monoclonal 2F5 Ab markedly decreased the inhibitory activity of this mAb in a concentration-dependent manner, as shown in Fig. 3⇑C. Similar results were obtained when FcγRs on MDM were saturated with human IgG from a HIV-negative donor, 10 min before the addition of immune complexes or when competition experiments were performed using multiple rounds of infection (data not shown). In contrast, incubation of PBMC with purified IgG from non-HIV-infected individuals did not affect the neutralizing activity of polyclonal IgG sample nos. 8 (Fig. 3⇑D) and 44 (data not shown). The decrease of the inhibitory activity observed in the presence of IgG or their Fc fragments strongly suggests that FcγRs participate for a major part to this inhibitory activity recorded. To evaluate which type of FcγR is implicated in this mechanism, Ab competition experiments with different purified monoclonal IgG directed against specific human FcγRI (CD64), FcγRII (CD32), or FcγRIII (CD16) were added to virus and Ab before incubation on MDM. As recorded in Fig. 4⇓, purified human anti-CD64 mAb decrease the HIV inhibitory activity of polyclonal IgG sample nos. 8 or 11 (Fig. 4⇓, A and B) and mAb 2F5 (Fig. 4⇓C), whereas both purified anti-human CD16 or CD32 mAbs have no effect on the inhibitory activity of these polyclonal or monoclonal IgG tested. These data demonstrated the specific participation of the human FcγRI or CD64 in the inhibition of HIV-1 replication on MDM.

FIGURE 4.
FIGURE 4.

Competition experiments between anti-human monoclonal IgG against FcγR and polyclonal or monoclonal neutralizing IgG. Purified mouse anti-human monoclonal IgG against the different FcγR (CD16, CD32, or CD64) were added concomitantly to MDM infected with HIVBaL-IgG immune complexes. The percentages of infected cells were measured after intracellular staining of p24 Ag by flow cytometry. Increasing concentrations of purified IgG sample nos. 11 (A) or 8 (B) or monoclonal 2F5 Ab (C) were used in the presence of purified anti-human monoclonal IgG1κ directed against FcγR. (•, untreated HIVBaL-infected cells; ▵, 6.25 μg/ml purified IgG anti-CD16; □, 6.25 μg/ml purified IgG anti-CD32; ⋄, 6.25 μg/ml purified IgG anti-CD64). Values are the mean of three independent wells from one representative experiment.

No correlation between viral inhibition and early chemokine production

MDM are mainly infected through the chemokine receptor CCR5, highly expressed on these cells (3, 24). Several studies have shown that the down-regulation of CCR5 expression or ligation by CC chemokines in MDM is associated to a reduction of R5 virus entry and replication (25). Chemokines such as MIP-1α, RANTES, and MIP-1β are natural ligands of the CCR5 receptor, and are well identified as inhibitors of R5 HIV strains (26). To exclude that these chemokines can contribute to virus inhibition, we have measured the production of MIP-1α, β, and RANTES in the supernatants early, 2, and 6 h, after infection in parallel to the Ab competition experiments (Table IV⇓). RANTES, MIP-1α, and -1β were detected as early as 2 h after infection and an ∼3-fold increase of production of these chemokines was measured 6 h after infection (Table IV⇓). In our culture conditions, the amount of chemokines induced was similar when infected cells were treated with monoclonal 2F5 Ab or polyclonal IgG sample no. 8 in the presence or the absence of the Fc part of IgG from HIV-negative donors. Thus, chemokine production detected early after infection is not correlated with HIV inhibition observed at 24 h (Table III⇑).

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Table IV.

β-Chemokine secretion in culture supernatants of HIV-1BaL-infected monocyte-derived macrophages at different times after infectiona

Discussion

These experiments were designed to analyze the mechanisms of inhibition of HIV replication by human monoclonal and polyclonal IgG in MDM 24 h postinfection. For this purpose, we set up a single cycle infection assay using either MDM or PBMC as HIV-target cells. We have found the neutralization of HIV-1 primary isolates by monoclonal IgG and polyclonal IgG samples purified from HIV-1-infected individuals to be considerably higher when MDM instead of PBMC were used. Moreover, purified IgG samples, which did not exhibit neutralizing activity in HIV-infected PBMC, exhibited potent inhibitory activity on MDM. Zhuge et al. (15) and Ruppach et al. (16) previously reported results showing higher neutralization of SIV or HIV when MDM were used as target cells instead of PBMC in multiple round neutralization assay. Ruppach et al. (16) analyzed autologous neutralization of virus isolated early after infection by their corresponding sera and the experimental procedure for neutralization using either PBMC or MDM was not strictly identical. As we found a comparable increase neutralization of macrophage infection using polyclonal IgG from individuals infected for a long period, we demonstrate that this augmentation of neutralizing activity is not restricted to early autologous IgG. Moreover, we used a single cycle neutralization assay with identical experimental conditions for both PBMC and MDM. By considering a single round of infection, an eventual increase in neutralization due to lower virus production in human primary macrophages is minimized. Indeed, we effectively observed a higher neutralizing titer of IgG samples in multiple rounds of HIV replication assay compared with single cycle neutralization assay when MDM were used as HIV-target cells.

The higher inhibitory activity is not only due to the implication of a subpopulation of Abs, present in the polyclonal IgG sample, directed to epitopes specifically involved in inhibition of HIV replication in MDM as mAbs IgG1b12, 4E10, and 2F5 also more efficiently neutralized HIV primary isolates in these cells. These mAbs have been described to exhibit broad cross-neutralizing activities against a wide range of primary isolates of HIV-1 in vitro in assays involving PBMC as target cells (27, 28, 29, 30, 31, 32). Stamatatos et al. (33) have already described a similar 10-fold increase in the inhibition of MDM infection for mAb IgG1b12. This latter group proposed that this higher neutralizing titer measured might result from lower expression in numbers of CD4 target sites on MDM. Indeed, MDM express fewer CD4 molecules than PBMC (34) and thus, less IgG1b12 Abs might be necessary to block the interaction of virions with CD4 receptor present in these cells (33). However, we found that blocking virus interaction with CD4 by soluble CD4 was as efficient whether PBMC or MDM were used (data not shown), suggesting that the increased neutralizing activity on MDM is not simply the consequence of the lower expression of CD4 on these human primary cells. We observed a higher increase in neutralizing activity (>300-fold) for both mAbs 2F5 and 4E10 than for IgG1b12; this may suggest that the conserved epitope on gp41 is more relevant than the CD4 binding site epitope for neutralization of MDM infection or that the activity can be modulated by differences in their structures. As the epitope on gp41 is not very accessible on the native viral particle (35), we can further emphasize that binding of Abs to viral particles are not correlated to neutralization of macrophage infection. Such discordance between binding and neutralization was previously reported for other mAbs by Stamatatos et al. (33). The polyclonal IgG samples purified from HIV-infected patients used in this study were all able to efficiently capture the two primary HIV-1 isolates used for the determination of the neutralizing titer.4 However, the efficiency of the purified IgG samples to capture virus was not correlated to the neutralizing titers obtained when either PBMC4 (Refs.21 and 30) or MDM (data not shown) were used as target cells.

As IgA have also been shown to exhibit HIV-neutralizing activity (21, 22, 23), we have evaluated the capacity of some IgA purified from sera of HIV-infected patients to neutralize primary isolates when MDM or PBMC are used as target cells. Contrary to IgG, we could not observe an increased neutralization of HIV on MDM for polyclonal IgA, indicating that the increase of neutralizing titer observed on MDM is specific to IgG.

Contrary to previous reports (15, 16), when MDM are used as target cells, the neutralizing activity of the Fab or F(ab′)2 of polyclonal IgG was reduced to the inhibitory activity detected when PBMC are the HIV-target cells in the neutralization assay.

The two previous reports analyzing neutralization of macrophage infection have found that Fab have similar inhibitory activity than their corresponding whole IgG (15, 16). Such discrepancies between these previous results and our findings could not be attributed to degradation, dilution, or loss of material resulting from enzymatic treatment of our Fab or F(ab′)2 preparations as these fragments conserved their neutralizing potency when PBMC were used as HIV-target cells. The discrepancy could neither be related to the type of assay used, as we have also detected a great difference in the inhibitory activity between whole IgG and Fab or F(ab′)2 in the multiple round neutralization assay, similar to the assay used by Zhuge et al. (15) or Ruppach et al. (16) (data not shown). The difference may be related to the polyclonal samples used. Ruppach et al. (16) have evaluated the neutralizing capacity of sera and one purified IgG sample from recently infected individuals (few months after seroconversion); Zhuge et al. (15) used sera and two purified IgG samples obtained from macaques, 6 mo after SIV inoculation while our polyclonal IgG samples were obtained from asymptomatic individuals collected after a mean duration of 7 years of infection (15). It is noteworthy that Ruppach et al. (16) have adjusted the amounts of IgG and Fab to the amount of IgG corresponding to 10% patient serum estimated by SDS-PAGE. In our neutralizing experimental conditions, our Fab or F(ab′)2 are also capable of completely inhibiting HIV replication in MDM when used at such high concentrations (corresponding to 10% IgG in the serum). However, our polyclonal IgG have >100-fold higher HIV-inhibitory activity than Fab or F(ab′)2, as ∼2 μg/ml purified IgG is sufficient to reduce by 90% the number of infected MDM cells. Nevertheless, this remark does not apply for the results published by Zhuge et al. (15) that have found 1 μg/ml Fab concentration having similar inhibitory activity to whole IgG of sera from SIV-infected macaques (15).

Competition experiments performed between Fc fragments of IgG from non-HIV-infected donors and polyclonal IgG samples from HIV-infected individuals further validate the participation of the Fc domains of IgG in the strong inhibition observed when MDM are used as HIV-target cells. Moreover, we demonstrated that this inhibition of HIV-1 on MDM by polyclonal and monoclonal IgG principally involved the participation of the FcγRI (or CD64). FcγRI is largely expressed (>90%) at the cell surface of macrophages and has a high affinity for IgG, whereas FcγRII and FcγRIII are expressed to a lesser extent (∼10% for FcγRIII on MDM) and have a lower affinity for IgG. These FcγRs are implicated in virus degradation by phagocytosis (3, 36) or in lysis of infected macrophages by Ab-dependent cellular cytotoxicity (ADCC) (37, 38). Both mechanisms have been largely described to account for virus destruction in these cells. Macrophages and other phagocytic cells (neutrophils, monocytes, and dendritic cells) are in charge of rapid degradation of virus, which once opsonized by IgG may cross-link with FcγRs on the cell surface of an effector cell and undergo phagocytosis. For ADCC, Ab attached via the FcγRs at the surface of MDM recognize an infected cell, which express viral Ag at their membrane surface, and mediate lysis of this latter cell. Moreover, the complement-dependent cytotoxicity activated through binding of C1q to the Fc domain of IgG or IgM, complexed with Ags, also account for the lysis of infected cells (38, 39).

As our purified IgG do not contain complement, we can exclude complement-dependent cytotoxicity as a possible mechanism of inhibition. The fact that we were unable to detect an increased number of cells undergoing apoptosis or necrosis in the presence of inhibitory concentrations of Abs after a single cycle of HIV replication (data not shown) is not in favor of a mechanism of ADCC, but it is difficult to quantify a low level of cytotoxicity given that the percentage of HIV-infected cells is very low. Moreover, addition of IgG, 3 h after the addition of virus to the cells, did not protect MDM from HIV-1 infection (data not shown) further disproving the mechanism of ADCC in our experimental culture conditions. Thus, we propose two mechanisms of HIV-1 inhibition in MDM: the one consisting in neutralization of infectivity by Abs that recognize specific HIV epitopes (a mechanism that is common for PBMC and MDM and occurs at similar concentration range) and the second one based on HIV inhibition via FcγRI involved in phagocytosis of IgG-virus complexes by macrophages (capture of immune complexes by FcγRI, internalization via endosomal vesicles, and further degradation of the virus).

Depending on their subtype, IgG bind FcγR with various efficiencies, thus differences in the Fc region of IgG may influence HIV inhibition by endocytosis. However, for the two mAbs used, 2F5 and IgG1b12, the Fc regions are similar (IgG1κ) suggesting that the difference in HIV-1 inhibition observed with these two mAbs is not due to the IgG subtype.

FcγRs have also been implicated in Ab-dependent enhancement of infection. Trischmann et al. (40) could detect an enhancement of virus production in human primary macrophages when HIV-immune complexes formed with certain Abs bound to the CD16 receptor or FcγRIII. In our study, we did not observe any enhancement of human MDM infection with the polyclonal and monoclonal Abs tested using the cycle round infection assay.

Recently, Perez-Bercoff et al. (41) proposed that FcγR stimulation by human IgG from non-HIV-infected donors could mediate inhibition of HIV-1 replication in macrophages in vitro when coated on a plate. These authors speculated that macrophage activation through FcγR cross-linking, alone or in synergy with other stimuli such as LPS or cytokines, could contribute to natural protection against HIV-1 infection in some exposed uninfected individuals by inhibiting viral transmission. Our findings agree for a pivotal role of FcγRs present at the cell membrane of macrophages, which may contribute to the clearance of the HIV-IgG immune complexes in vitro. In contrast, FcγR cross-linking on human monocytes and MDM has been reported to induce a panel of chemokines and cytokines, notably GM-CSF (42). Chemokines, such as MIP-1α, MIP-1β, and RANTES, have been shown to inhibit HIV-1 replication when added during and after infection (43) and could thus contribute to the suppression of HIV-1 replication detected in our assay. Therefore, we have quantified the levels of these chemokines in the supernatant of infected MDM in parallel with the Ab competition experiments. Amounts of MIP-1α, MIP-1β, and RANTES detected in the supernatant of MDM, 2 and 6 h after the infection, were below the inhibitory concentration required to inhibit HIV replication in these cells and are not correlated with viral inhibition observed after a single round of virus replication. Of note, the chemokine secretion recorded 24 h after infection was of the same order of magnitude as that measured at 6 h (our unpublished results).

Macrophages represent a leukocyte population involved in the first line of defense against many infections, including HIV infection. Through presentation of Ags to T cells and production of cytokines and chemokines, macrophages constitute an important link between the innate and adaptive immune systems. Multiple studies including our data have suggested the potential role of macrophages in HIV inhibition, either by phagocytosis of immune complexes, by mediating ADCC, or even through chemokine production. However, although Abs highly efficient in inhibiting infection of MDM in vitro were found in the sera of almost all infected individuals, HIV spreads and persists in these individuals.

A lack of clearance of HIV-IgG immune complexes may be attributed to impaired FcγR-specific phagocytosis as described in some AIDS patients (44). Indeed, after in vitro infection with HIV-1BaL, phagocytosis of immune complexes mediated by both FcγR and complement receptors is impaired in human-infected MDM (45). These authors have shown that infection of macrophages by HIV did not affect surface expression of either FcγR or the major complement receptor, but in HIV-infected macrophages signal transduction is decreased and clearance of IgG-opsonized targets via FcγR is altered. Thus, human IgG present in most HIV-infected individuals are able to bind HIV particles and form immune complexes but phagocytosis and degradation of these immune complexes is impaired in infected macrophages.

Not only virus-IgG complexes could be inefficient to inhibit HIV replication in vivo, IgG bound to virus may even favor their persistence when trapped by follicular dendritic cells (FDC). Indeed, a recent study has shown that optimal maintenance of HIV infectivity requires both Abs against particle-associated determinants and FDC-FcγR (46). In addition to impaired phagocytosis and virus trapping by FDC, immune escape, infection of CD4+ T lymphocytes, complement-mediated and Ab-dependent enhancement of infection in MDM may contribute to the failure of neutralizing Abs to control HIV infection.

Nevertheless, before infection is established and HIV reach FDC and infect numerous macrophages, one may expect that such Abs, which efficiently inhibit HIV when MDM were used as target cells in vitro, could participate in the reduction of plasma viral load in the early asymptomatic phase.

Passive transfer of HIVIG (polyclonal IgG derived from the plasma of multiple HIV of positive donors) alone in macaques, 24 h prior to i.v. challenge with SHIV89.6PD virus, reduced the decline in CD4+ T cell count and prevented clinical AIDS during the 14 wk of observation (47). In this work, a decline in plasma viral load was observed despite HIVIG not being very efficient in neutralizing PBMC infection. As we do not know whether HIVIG is able to inhibit SHIV89.6P when MDM are used as target cells, we cannot conclude whether this decline could be attributed to clearance of virus by macrophages. In another passive transfer study, Binley et al. (48) have infused Abs collected from SIVmac251-infected macaques to other infected macaques that had negligible Ab response. These Abs were not able to neutralize PBMC infection but should form immune complexes as they bind virus particles. Transfer of such Abs results in 3-fold decrease in plasma RNA load concomitant to a similar drop in cell-associated viral RNA within 2 h after infusion. The inhibition is however only transient. These authors conclude from the kinetics of inhibition that the decrease of virus observed is inconsistent with neutralization of new cycles of infection or the immune complex clearance mechanism and that the best likely explanation is that infused Abs killed SIV-infected cells, via an effector mechanism such as ADCC.

Further studies should be performed to clarify the clinical implications of Abs able to inhibit in vitro HIV replication when macrophages are the target cells; as, for example, the analysis of the neutralization of autologous virus isolates by the corresponding IgG samples in appropriate subjects (i.e.: long-term nonprogressor vs rapid progressor). If such Abs play a role in vivo in protection against infection, they should also be one of the components to be induced by vaccination, and present at the site of infection to allow early and efficient clearance of HIV-1 by macrophages. As such Abs are detected at much higher levels after infection than Abs neutralizing PBMC infection, one may speculate that their induction by vaccination would also be achieved more easily.

Acknowledgments

We are grateful to Nadège Lagarde, Sandrine Haessig, and Sigismond Sliwinski for their technical assistance.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by grants from the Agence Nationale de Recherches sur le SIDA and from the European Union (QLK2-CT-199-01321 “EuroVac”).

  • ↵2 Address correspondence and reprint requests to Dr. Vincent Holl, EA 3770, ULP, Institut de Virologie, 3 rue Kœberlé, 67000 Strasbourg, France. E-mail address: Vincent.Holl{at}hemato-ulp.u-strasbg.fr

  • 3 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; MDM, monocyte-derived macrophage; TCID50, 50% tissue culture infective dose; ADCC, Ab-dependent cellular cytotoxicity; FDC, follicular dendritic cell.

  • ↵4 R. Burrer, S. Haessig-Einius, A. M. Aubertin, and C. Moog. Neutralizing as well as non neutralizing polyclonal IgG from infected patients capture HIV-1 via antibodies directed against the principal immunodominant of gp41. Submitted for publication.

  • Received June 25, 2004.
  • Accepted August 25, 2004.

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