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Antimicrobial Agents and Chemotherapy, October 2005, p. 4296-4304, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4296-4304.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inhibition of Coreceptor-Independent Cell-to-Cell Human Immunodeficiency Virus Type 1 Transmission by a CD4-Immunoglobulin G2 Fusion Protein

Berta Bosch,1 Julià Blanco,1 Eduardo Pauls,1 Imma Clotet-Codina,1 Mercedes Armand-Ugón,1 Boyan Grigorov,2 Delphine Muriaux,2 Bonaventura Clotet,1 Jean-Luc Darlix,2 and José A. Esté1*

Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain,1 LaboRetro, Unité de Virologie Humaine, INSERM #412, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69 364 Lyon, France2

Received 29 March 2005/ Returned for modification 4 June 2005/ Accepted 28 July 2005


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ABSTRACT
 
We have previously shown (J. Blanco et al., J. Biol. Chem. 279:51305-51314, 2004) that the contact between HIV producing cells and primary CD4+ T cells may induce the uptake of human immunodeficiency virus (HIV) particles by target cells in the absence of HIV envelope-mediated membrane fusion or productive HIV replication. HIV uptake by CD4+ T cells was dependent on cellular contacts mediated by the binding of gp120 to CD4 but was independent of the expression of the appropriate HIV coreceptor, CCR5 or CXCR4. Here, we have characterized the effect of agents blocking gp120 binding to CD4 on cell-to-cell HIV transmission. A recombinant CD4-based protein (CD4-immunoglobulin G2 [IgG2]), that is currently being evaluated in clinical trials, completely inhibited the uptake of HIV particles by CD4+ T cells from persistently infected cells expressing R5, X4, or X4/T-20-resistant HIV-1 envelope glycoproteins. Consequently, both the release of viral particles from endocytic vesicles and the infection of reporter U87-CD4 cells were also prevented. The polyanionic anti-HIV agent dextran sulfate failed to prevent the intracellular uptake of virions by CD4+ T cells. Indeed, it increased HIV uptake in a dose-dependent manner, suggesting functional differences between the specific gp120-targeting CD4-IgG2 agent and nonspecific HIV binding inhibitors. Thus, the inhibition of the specific interaction between gp120 and CD4 protein could be an effective strategy to inhibit HIV binding to CD4+ T cells, and the mechanism by which CD4+ T cells lacking the appropriate coreceptor may be converted in HIV carriers.


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INTRODUCTION
 
Human immunodeficiency virus type 1 (HIV-1) cell-to-cell transmission implies the polarization of viral production in the infected cell and the viral receptors and coreceptors in the target cell. This polarization leads to the formation of a functional virological synapse, triggering a rapid and efficient infection of target cells (19). Moreover, during these cellular contacts, large amounts of HIV particles may be transferred from infected cells to CD4+ T cells in the absence of membrane fusion in a manner that is independent of coreceptor expression or any later step of HIV virus cycle but appears to depend on cellular contacts mediated by the binding of surface (SU; gp120) to CD4. HIV particles taken up by CD4+ T cells may reside in large intracellular vesicles and may be released to extracellular compartments and finally transmitted to a third uninfected cell (9).

Productive HIV-1 entry into target cells is a process initiated by the binding of the HIV envelope, SU/transmembrane (TM; gp41), to CD4, triggering conformational changes that enable SU binding to chemokine receptors and, subsequently, TM-mediated membrane fusion. CD4-immunoglobulin G2 (CD4-IgG2; PRO-542) inhibits HIV-1 entry, blocking the interaction between the envelope glycoprotein gp120 and CD4 protein. CD4-IgG2 is a tetravalent recombinant antibody-like fusion protein wherein the heavy- and light-chain variable domains of human IgG2 were replaced with the V1 and V2 domains of human CD4 (1, 31). CD4-IgG2 binds the HIV-1 SU with nanomolar affinity, inhibits syncytium formation, and has a 90% reduction in virus infectivity (90% inhibitory concentration) at 20 µg/ml (1, 18). Phase I and phase II clinical studies show antiviral activity of CD4-IgG2 in HIV-1-infected adults, especially in advanced disease (17, 18).

Taking into account that binding of SU to CD4 is a necessary step to trigger HIV-1 coreceptor-independent cell-to-cell HIV-1 transmission and that CD4-IgG2 inhibits SU-CD4 binding, we have studied the effect of CD4-IgG2 in coreceptor-independent transmission of HIV. Our results demonstrate that CD4-IgG2 inhibits HIV coreceptor-independent transmission in a dose-dependent manner, with a 50% effective dose (EC50) comparable to its anti-HIV activity.


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MATERIALS AND METHODS
 
Cells. Peripheral blood mononuclear cells (PBMC) from healthy donors were purified by Ficoll-Hypaque sedimentation. CD4+ T cells were immediately purified (>95%) from PBMC by negative selection using the CD4+ T cell enrichment kit (StemCell Technologies, Vancouver, Canada). Primary cells were used without previous stimulation. Media were supplemented with 10% heat-inactivated fetal calf serum (Invitrogen, Madrid, Spain), 100 U/ml penicillin, 100 µg/ml streptomycin. HIV-1 chronically infected MOLT-4/CCR5 cells were generated in the laboratory after infection of MOLT-4/CCR5 cells with the following viruses: recombinant viruses carrying the HIV-1 envelope (Env) sequences corresponding to the X4 HIV-1 strain NL4-3 or the R5 HIV-1 strain BaL constructed in an HIVHXB2 backbone as described previously (6); the primary isolate 168.1 (13, 15); and a C34-resistant and T-20-cross-resistant HIV-1 NL4-3 strain (2), NT38, generated by sequential passages of the NL4-3 strain in vitro in MT-4 cells in the presence of increasing concentrations of T-20 (3). After infection peak, persistently infected cultures of MOLT-NL4-3, MOLT-BaL, MOLT-168.1, and MOLT-NT38 cells were grown and characterized for Env expression and virus production (7). Uninfected MOLT-4/CCR5 (MOLT-uninfected) cells were used as negative controls in all experiments.

Cocultures of infected and uninfected cells. Primary cells (purified CD4+ T cells; 2 x 105 cells) were cultured in 96-well plates with effector (uninfected or infected) MOLT-4/CCR5 cells at a ratio of 1:1 in the absence or the presence of the following HIV inhibitors: 10 µg/ml of murine IgG (SantaCruz Biotechnologies) as an isotype control; 5 µg/ml of the CCR5 antagonist TAK779 (14) (National Institutes of Health [NIH] AIDS Reagent Program); 5 µg/ml of the TM inhibitor C-34 (2) (Service of Peptide Synthesis, University of Barcelona); 1 µg/ml of the reverse transcriptase (RT) inhibitor zidovudine (AZT; Sigma, Madrid, Spain); 0.08 to 50 µg/ml of the binding inhibitor dextran sulfate (DS) (Sigma, Madrid, Spain); 10 µg/ml of the CXCR4 antagonist, the bicyclam AMD3100; 0.0005 to 10 µg/ml of the TM inhibitor T-20 (Service of Peptide Synthesis, University of Barcelona); 0.0003 to 20 µg/ml of neutralizing anti-gp120 monoclonal antibody (MAb) IgGb12 (National Institute for Biological Standards and Control AIDS Reagent Project); and 0.0003 to 20 µg/ml of recombinant CD4-based protein (17, 18) (CD4-IgG2, kindly provided by M. Franti, Progenics Pharmaceutical, Tarrytown, NY). Cocultures were incubated at 37°C for 6 h or 24 h to evaluate HIV transfer and fusion or cell death, respectively, as described below.

Antiviral assay. Anti-HIV activities of CD4-IgG2, MAb IgGb12, DS, T-20, AZT, and AMD3100 were measured against NL4-3 HIV-1 virus in MT-4 cells by a tetrazolium-based colorimetric method (MTT method) as described previously (11, 27).

Evaluation of cell death. After 24 h of coculture, cells were extensively washed with phosphate-buffered saline (PBS) and fixed with 1% formaldehyde. Single-cell death was quantified by morphological parameters (forward versus side scatter plots) in a FACScalibur flow cytometer (BD, Madrid, Spain) as described previously (3, 4). Dead cells showed increased side and reduced forward scatter values compared with those of living cells.

Evaluation of HIV transfer. After 6 h of coculture, cells were trypsinized. For trypsin treatment, cells were washed and were treated (10 min at room temperature) with 0.25% trypsin solution (Invitrogen, Madrid, Spain). The action of trypsin was controlled by the disappearance of the Leu3a epitope of CD4+, uninfected, trypsin-treated cells (9, 29). Trypsin action was stopped by addition of fetal calf serum. Cells were then washed and stained with Leu3a anti-CD4 MAb. After surface staining, cells were fixed, permeabilized (Fix & Perm; Caltag, Burlingame, CA), and stained with KC57 anti-HIV capsid p24 antigen (CA p24) MAb (Coulter, Barcelona, Spain) and analyzed in a FACScalibur flow cytometer (BD, Madrid, Spain). Cells were identified by morphological parameters. Quantification of HIV transfer was either assessed by the percentage of CA p24+ cells (using uninfected cells as a control) or by the mean fluorescence intensity.

Fifty-percent effective concentration (EC50) calculation. Effective concentrations that inhibit 50% of HIV cell-to-cell transfer, HIV infection, or single-cell death were calculated by a nonlinear regression using the Enzfitter software in which the background levels are subtracted.

Evaluation of HIV transfer in a 293T cell model. 293T cells (4 x 106) were seeded in 100-mm dishes. After 24 h, cells were transfected with 9 µg of the HIV-1 NL4-3 expression plasmid lacking a functional envelope (pNL4-3.Luc.R-E-; NIH AIDS Reagent Program), 9 µg of the Env gp160 expression plasmid pHenv, and 9 µg of the vesicular stomatitis virus envelope protein expression vector (pVSV-G; Clontech) using a CalPhos transfection system (Clontech). Forty-eight hours posttransfection, intracellular CA p24 antigen levels in 293T cells were assessed by staining with KC57 anti-HIV-CA p24 antigen MAb as described above. Mock-transfected 293T cells or 293T cells transfected with pNL4-3.Luc.R-E-, pNL4-3.Luc.R-E- with pHenv, or pNL4-3.Luc.R-E- with pVSV-G were cocultured with purified CD4+ T cells for 24 h at a ratio of 1:1 in the presence or absence of the following HIV inhibitors: 1 µg/ml of the reverse transcriptase (RT) inhibitor AZT (Sigma, Madrid, Spain); 1 µg/ml of the CXCR4 antagonist, the bicyclam AMD3100; and 20 µg/ml of neutralizing anti-gp120 MAb IgGb12 (National Institute for Biological Standards and Control AIDS Reagent Project). After 24 h, intracellular CA p24 antigen transfer was analyzed in CD4+ T cells identified by forward and side scatter values.

Immunofluorescent staining. After 6 h of coculture, cells were trypsinized and fixed in 3% paraformaldehyde for 20 min. Cells were then washed with PBS and stuck over a coverslip, and the free aldehydes were quenched with 50 mM NH4Cl (diluted in PBS). Cells were permeabilized using 0.2% Triton X-100 for 5 min and blocked in 1% bovine serum albumin (diluted in PBS) for 15 min. Cells were then incubated for 1 h at room temperature with the following primary monoclonal antibodies: rabbit anti-HIV-matrix p17 antigen (MAp17; NIH AIDS Reagent Program), nondirectly conjugated with a fluorocrome, at 1/1,000 dilution, and mouse L120.3 anti-human-CD4-fluorescein isothiocyanate (BD, Madrid, Spain) at 1/10 dilution. Cells were washed three times in 1% bovine serum albumin and incubated for 1 h at room temperature with the secondary antibody donkey anti-rabbit Alexa 546 (2 mg/ml; Molecular Probes, Orlando, FL) at 1/3,000 dilution. Coverslips were washed three times with PBS and mounted with Mowiol (Sigma). Samples were observed on an Axioplan 2 Zeiss CLSM 510 confocal microscope with Argon 488/458 and HeNe 543 lasers and a 63x (1.4 numerical aperture) plan Apochromat oil objective, supplied with LSM 510 3.4 software.

Analysis of HIV transfer in primary cells. PBMC were stimulated for 72 h with 4 µg/ml phytohemagglutinin (PHA) and 6 U/ml interleukin-2 (IL-2) and then infected with the HIV-1BaL strain R5 (100 ng of CA p24 antigen/107 cells). Cells were then cultured at 106 cells/ml in RPMI containing 20% fetal calf serum and 10 U/ml IL-2. In the following days, the expression of CA p24 antigen and CD4 were monitored by flow cytometry as described above. At day 7 after infection, the percentage of CD4–/CA p24+ cells reached a plateau, and CD8+ and CD4+ cells were depleted from the culture using a combination of CD8+ and CD4+ T-cell enrichment kits. Only CD4–/CA p24+ cells were considered to be productively infected because of the complete disappearance of CD4 from the surface of HIV-infected cells. Selected cells were reanalyzed for CA p24 antigen expression and were cocultured for 6 h with unstimulated uninfected fresh 5-chloromethylfluorescein diacetate (CMFDA)-labeled CD4+ T cells (ratio, 1:1) in the presence of IL-2 at 10 U/ml to maintain HIV expression by infected cells. Transfer of CA p24 antigen from infected to uninfected cells labeled with CMFDA was assayed as described above.

Infectivity of captured virions. To analyze infectivity of virions captured by CD4+ T cells, we purified them after coculture with MOLT-BaL cells. Briefly, 10 x 106 CD4+ T cells were cultured with 10 x 106 MOLT-BaL cells in 15-ml tubes in a final volume of 1 ml in the absence or presence of TAK779 (5 µg/ml), MAb IgGb12 (10 µg/ml), DS (50 µg/ml), and CD4-IgG2 (4 µg/ml). After 6 h of coculture, cells were recovered and CD4+ T cells were purified again by negative selection using the CD4+ T-cell enrichment kit (StemCell Technologies, Vancouver, Canada). Purified cells were assayed for the percentage of contaminating MOLT cells by both morphological parameters and CA p24 staining and were treated with trypsin for 10 min in order to remove extracellular attached viruses, MAb IgGb12, and CD4-IgG2. After extensive washes in PBS, cells were cultured in RPMI supplemented with 10% fetal calf serum in the absence or the presence of 1 µg/ml AZT to prevent productive infection. The content of CA p24 antigen was evaluated at 12, 24, and 48 h in the supernatant (Innogenetics ELISA kit; Barcelona, Spain) and in the purified cells by flow cytometry as indicated above. Supernatants were recovered at 12 h of culture, and their infective titers were evaluated in U87.CD4+, CCR5+ cells. Syncytium formation was evaluated at day 6 postinfection by visualizing cultures in a Nikon Eclipse TE200 fluorescence microscope after staining with Hoechst 33324 (1 µM). Wells showing syncytia with more than four nuclei were scored as positive to calculate infectious titers.


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RESULTS
 
CD4-SU binding inhibition blocked the uptake of HIV-1 particles by CD4+ T cells. Short cocultures (6 h) of unstimulated CD4+ T cells with chronically infected cell line MOLT-NL4-3 or MOLT-BaL elicited an uptake of HIV-1 particles by CD4+ T cells as seen by confocal microscopy or flow cytometry (Fig. 1A and B). In the absence of HIV-1, CD4 was found to distribute all along the plasma membrane of CD4+ T cells (Fig. 1B, upper panel). In contrast, after 6 h of coculture with MOLT-BaL cells, CD4 localization drastically changed at the surface of CD4+ T cells. In fact, CD4 was localized at one pole of the CD4+ T-cell surface together with HIV-1 Gag, most probably illustrating the recruitment of CD4 molecules at the cell-cell contact interface (Fig. 1B, lower panel). As observed for longer cocultures (24 h) (9), HIV antigens accumulated in trypsin-resistant compartments in CD4+ T cells. The transfer of CA p24 BaL antigen to CD4+ T cells was not blocked by C-34, TAK779, or AZT (data not shown). When MOLT-NL4-3 cells were used, C-34 and the CXCR4 coreceptor antagonist AMD3100 increased the amount of transferred NL4-3 antigens from 39% ± 6% of HIV transfer without drug up to 57% ± 2% or 62% ± 5% in the presence of C-34 at 5 µg/ml or AMD3100 at 10 µg/ml, respectively (data not shown).



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  		FIG. 1. CD4+ T cells internalized HIV antigens into trypsin-resistant compartments by a CD4 receptor- and HIV envelope-dependent process. (A) CD4+ T cells were cocultured with MOLT-uninfected (solid peak), MOLT-NL4-3 (empty peak), or MOLT-BaL (empty peak) cells. After 6 h of coculture, cells were stained with or without prior trypsin treatment and analyzed by flow cytometry. Cells were identified by morphological parameters. Values of one representative experiment are shown. (B) Six-hour cocultures between CD4+ T cells and MOLT-uninfected (upper panel) or MOLT-BaL (lower panel) cells were treated with trypsin and stained for CD4 (green) and MAp17 (red). Colocalization of both markers is shown in the merge picture (yellow). One CD4+ T cell representative of each coculture is shown. (C) 293T cells mock transfected (Mock) or 293T cells transfected with pNL4-3.Luc.R-E- (Luc), pNL4-3.Luc.R-E- and pHenv (pHenv), or pNL4-3.Luc.R-E- and pVSV-G (VSV) were cocultured with purified CD4+ T cells in the presence or absence of the following drugs or antibodies: 1 µg/ml of the reverse transcriptase (RT) inhibitor AZT; 1 µg/ml of the CXCR4 antagonist, the bicyclam AMD3100; and 20 µg/ml of neutralizing anti-gp120 MAb IgGb12. Intracellular CA p24 antigen was measured by flow cytometry. The percentage of CA p24+ CD4+ T cells was analyzed. One of two independent experiments done in duplicate is shown.

In order to analyze the role of the SU/TM envelope in cell-to-cell HIV transfer, 293T cells loaded with viral particles were obtained upon DNA transfection with an HIV-1 NL4-3 expression plasmid lacking a functional envelope. 293T cells were also cotransfected with plasmids expressing Env gp160 HIV (pHenv) or that of vesicular stomatitis virus (pVSV). As expected, HIV envelope expressing 293T cells conferred the capacity of transferring HIV particles to resting purified CD4+ T cells (Fig. 1C). Virus transfer was dependent on SU-CD4 interaction, as it could only be inhibited with the MAb IgGb12. Conversely, 293T cells expressing VSV-G protein were not able to transfer CA p24 antigen to CD4+ T cells. 293T cells expressing NL4-3 (Luc) without envelope protein did not show HIV transfer.

In order to analyze the effect of CD4-IgG2 on cell-to-cell HIV transmission, we performed cocultures of MOLT-uninfected, MOLT-BaL, MOLT-168.1, or MOLT4-NL4-3 cells with unstimulated CD4+ T cells. After 6 h of coculture, intracellular CA p24 antigen staining revealed high levels of HIV in CD4+ T cells (40% ± 7%, 87% ± 4%, and 14% ± 2% for NL4-3, BaL, and 168.1, respectively). CD4-IgG2 inhibited the transfer of HIV NL4-3 or BaL antigen to unstimulated CD4+ T cells in a dose-dependent manner (Fig. 2A) with an EC50 of 0.09 ± 0.03 µg/ml or 0.05 ± 0.01 µg/ml, respectively. CD4-IgG2 could also inhibit the transfer of HIV-1 antigens from MOLT-168.1 cells, infected with the R5 using primary isolate 168.1, with similar potency to the inhibition of HIV-1 NL4-3 or BaL (EC50 of 0.07 ± 0.02 µg/ml). The anti-gp120 MAb IgGb12 was also able to block virus transfer to CD4+ T cells (EC50, 0.28 ± 0.05 µg/ml or 0.33 ± 0.05 µg/ml for HIV-1 NL4-3 or BaL, respectively) (Fig. 2B). CD4-IgG2 and MAb IgGb12 inhibited HIV-1 NL4-3 infection in MT-4 cells with calculated EC50s of 0.05 ± 0.05 and 0.2 ± 0.1 µg/ml, similar to the inhibition of virus transfer (Table 1).



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  		FIG. 2. Inhibition of CD4-SU binding blocked coreceptor-independent HIV transfer. CD4+ T cells were cultured for 6 h with MOLT-uninfected (solid circles), MOLT-NL4-3 (empty circles), or MOLT-BaL (empty squares) cells in the absence or the presence of CD4-IgG2 (A) or MAb IgGb12 (B). Data are the means ± standard deviations of two independent experiments.


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  		TABLE 1. Inhibition of HIV infection and transfer

Cocultures of CD4+ T lymphocytes with MOLT-NL4-3 cells or MOLT-NT38 cells, chronically infected cells that express X4/T-20-resistant envelope on their surface (3), showed 33% ± 2% and 42% ± 10% of single CD4+ T-cell death, respectively. T-20 inhibited single-cell death induced by chronically infected MOLT-NL4-3, with a calculated EC50 of 0.36 ± 0.21 µg/ml, but was completely ineffective at blocking cell death in cocultures with chronically infected MOLT-NT38 cells (Fig. 3A, left), confirming the phenotype of the NT38 envelope. Conversely, CD4-IgG2 inhibited single CD4+ T-cell death induced by cells that were chronically infected with NL4-3 or NT38 with a calculated EC50 of 0.05 ± 0.02 µg/ml or 1.03 ± 0.95 µg/ml, respectively (Fig. 3A, right). Inhibition of cell-to-cell NL4-3 or BaL antigen transmission and single-cell death shown by CD4-IgG2 led us to investigate if CD4-IgG2 could also inhibit HIV antigen transfer from MOLT-NT38 cells to CD4+ T lymphocytes. Cocultures of MOLT-NT38 cells with CD4+ T cells were performed in the absence or the presence of CD4-IgG2 in a range of 0.0003 to 20 µg/ml. CD4-IgG2 inhibited cell-to-cell transmission of NT38 virus particles with an EC50 of 2.2 ± 0.8 µg/ml (Fig. 3B). Taken together, these results suggest that SU-CD4 binding is the necessary step of the HIV life cycle to permit cell-to-cell HIV transmission and that CD4-IgG2 may block coreceptor-independent HIV-1 transmission, including the transfer from infected cells expressing T-20-resistant envelope on their surface.



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  		FIG. 3. CD4-IgG2 inhibited single-cell death and CA p24 antigen transfer from T-20-resistant HIV chronically infected cells. (A) CD4+ T cells were cocultured for 24 h with MOLT-uninfected (solid circles), MOLT-NL4-3 (empty circles), or MOLT-NT38 (empty triangles) cells. T-20 (left) and CD4-IgG2 (right) activities were assayed at different concentrations. Single CD4+ T-cell death was quantified by morphological parameters; dead cells showed reduced forward and increased side scatter values compared to those of living cells. (B) CA p24 antigen transfer was analyzed in 6-h cocultures of CD4+ T cells with MOLT-NL4-3 (empty circles) or MOLT-NT38 (empty triangles) cells. Cocultures were treated with CD4-IgG2 at different drug concentrations. Data are the means ± standard deviations of two independent experiments.

Dextran sulfate did not block HIV-1 transmission. Dextran sulfate (DS) is a polyanion that inhibits HIV binding to target cells (8, 12, 25). To test whether DS was able to block cell-to-cell transfer, we cocultured MOLT-uninfected, MOLT-NL4-3, or MOLT-BaL cells with unstimulated CD4+ T lymphocytes in the absence or the presence of DS. DS did not block HIV NL4-3 or BaL antigen transfer to CD4+ T cells at concentrations of up to 50 µg/ml. In fact, DS increased NL4-3 antigen transfer in a dose-dependent manner, from 34.7% ± 0.1% of HIV transfer without DS up to 71% ± 5% in the presence of DS at 50 µg/ml (Fig. 4), in spite of complete inhibition of syncytium formation between MOLT-NL4-3 and CD4+ T cells (data not shown) and anti-HIV-1 NL4-3 activity with a calculated EC50 of 0.067 ± 0.001 µg/ml in MT-4 cells. Taken together, these results suggest functional differences between specific, i.e., CD4-IgG2, and nonspecific (polyanionic) virus binding inhibitors in the context of cell-to-cell HIV transmission.



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  		FIG. 4. Dextran sulfate did not inhibit coreceptor-independent HIV transmission. After 6 h of coculture of CD4+ T cells with MOLT-uninfected (solid circles), MOLT-NL4-3 (empty circles), or MOLT-BaL (empty squares) cells, CA p24 antigen staining was performed. Data are the means ± standard deviations of two independent experiments.

CD4-IgG2 inhibited HIV-1 CA p24 transfer from productively infected to unstimulated CD4+ T lymphocytes. To evaluate the potential of CD4-IgG2 to inhibit coreceptor-independent HIV transfer of viruses propagated in primary cells, we purified HIV-producing cells from PHA-, IL-2-stimulated PBMC that were infected with HIV-1BaL. Seven days postinfection, 3% of PBMC were considered productively infected, CD4–, and CA p24+ (Fig. 5A left). After CD4–, CD8–, CA p24+ enrichment by depleting CD4+ and CD8+ cells, final preparations of effector cells had 59% infected cells (Fig. 5A, right). Effector cells were then cocultured with unstimulated CD4+ T lymphocytes labeled with CMFDA, a green cell tracker. After 6 h of coculture (Fig. 5B), 16% ± 6% of target cells were transferred with HIV particles from enriched infected effector cells. This transfer was not inhibited by C-34 or AZT, but MAb IgGb12 or the recombinant fusion protein CD4-IgG2 completely inhibited HIV transfer at 20 µg/ml. Surprisingly, DS at 50 µg/ml inhibited 44% ± 7% of HIV uptake by target cells. These results suggest that coreceptor-independent HIV transfer occurred between productively infected and unstimulated CD4+ T lymphocytes and was completely blocked by CD4-IgG2.



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  		FIG. 5. CD4-IgG2 blocked HIV transfer from HIV-infected primary cells to unstimulated CD4+ T cells. PHA-, IL-2-activated PBMC were infected with HIV-1BaL. In order to evaluate HIV transfer, 7 days postinfection the cell culture was enriched for CD4–/p24+ cells, and purified HIV-1BaL-infected cells were cocultured with unstimulated CD4+ T cells. (A) Dot plots showing an infected culture before (left) and after (right) depletion of CD4+ and CD8+ T cells. Values shown indicate the percentage of cells in each quadrant. (B) Percentage of CA p24+ CMFDA-labeled CD4+ target T lymphocytes after 6 h of coculture with purified HIV-1BaL-infected T lymphocytes as effector cells shown in panel A. DS was used at 50 µg/ml, C-34 at 5 µg/ml, AZT at 1 µg/ml, MAb IgGb12 at 20 µg/ml, and CD4-IgG2 at 20 µg/ml. Data are the means ± standard deviations of two experiments.

CD4-IgG2 inhibited the release of infectious HIV particles. HIV particles that are taken up by CD4+ T lymphocytes can be released into supernatants as infectious viruses (9). Thus, inhibition of virus transfer by CD4-IgG2 should prevent the subsequent release of infectious particles. Purified CD4+ T cells were cocultured with uninfected MOLT cells or MOLT-BaL cells in the presence or absence of TAK779, MAb IgGb12, DS, and CD4-IgG2. CD4-IgG2 was used at 4 µg/ml, the lowest concentration that completely blocked HIV transfer (Fig. 2A). After purification, CD4+ T cells were contaminated by less than 18% of MOLT cells (data not shown). Once purified, CD4+ T cells were trypsinized to remove extracellular virions, cultured in the presence of AZT to avoid viral replication, and analyzed for intracellular and extracellular CA p24 content. CD4+ T cells released CA p24 (Fig. 6A, left) to the supernatant concomitantly with a reduction of intracellular CA p24 (Fig. 6A, right), and as expected, TAK779 did not inhibit this process. CD4-IgG2 inhibited the uptake of HIV particles and, subsequently, blocked the release of CA p24 antigen into the supernatant. Conversely, DS could not block HIV cell-to-cell transfer and the release of CA p24 antigen that was similar to the untreated control or TAK779-treated cocultures. The experiment shown in Fig. 6, contaminated by 6% MOLT cells, was representative of another experiment with higher contamination (18%). To analyze the infectivity of released HIV particles, 12-h supernatants from transferred and subsequently purified CD4+ T cells were recovered and assayed for infectivity in U87.CD4+, CCR5+ cells. Supernatants from CD4-IgG2-treated cocultures showed lower infectivity (31% ± 1% respective to the untreated control without drug) than untreated or TAK779-treated cocultures (Fig. 6B), whereas supernatants from DS-treated cocultures showed higher infectivity (173% ± 61% respective to the untreated control without drug) than the untreated coculture. MAb IgGb12 was used as a control for CD4-SU binding inhibition. Taken together, these data suggest that CD4-IgG2, in addition to inhibiting HIV infection, would be able to block coreceptor-independent HIV transfer and, as a consequence, the release of HIV infectious particles and transmission to a third permissive cell. The release of HIV particles reached a plateau at roughly 15 h of culture, suggesting that not all internalized virus was able to re-escape from the CD4+ carrier cells.



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  		FIG. 6. CD4-IgG2 inhibited the release of infectious HIV particles from CD4+ T cells. Purified CD4+ T cells were cultured for 6 h with MOLT-uninfected or MOLT-BaL cells. After coculture, cells were purified by negative selection, trypsinized, and cultured for 48 h without stimulation. (A) Time course of HIV release (left) and CA p24 content of purified CD4+ T cells (right). Prior to purification, CD4+ T cells had been cultured with MOLT-uninfected (solid squares) or MOLT-BaL cells in the absence (No drug, empty squares) or the presence of TAK779 at 5 µg/ml (empty triangles), neutralizing MAb IgGb12 at 10 µg/ml (solid triangles), CD4-IgG2 at 4 µg/ml (solid circle), or DS at 50 µg/ml (empty circles). Data shown correspond to one representative experiment of two performed. (B) Supernatants from purified CD4+ T cells cocultured in these conditions were recovered 12 h after purification and were used to infect U87.CD4+, CCR5+ cells. The panel shows the infectious titers of these preparations. Data are the means ± standard deviations of two independent experiments. MFI, mean fluorescence intensity.


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DISCUSSION
 
HIV fusion and entry is a multistep process that requires the expression of the appropriate receptors and coreceptors in the target cell. Multiple factors may affect receptor and coreceptor expression and, in turn, alter HIV entry, infection, and disease progression (21-23). Agents that block virus-receptor interactions block HIV infection and replication (14, 24) but may not necessarily block all events associated with the interaction of HIV-infected cells with uninfected CD4+ T cells (10). Coreceptor-independent cell-to-cell transmission is a process by which HIV may use CD4+ T cells lacking the appropriate coreceptor as an itinerant virus reservoir and appears not to be prevented by coreceptor antagonists or TM-dependent fusion inhibitors. In CCR5-using strains, HIV uptake was coreceptor independent up to 85 to 95%, since only 5 to 15% of primary CD4+ T cells express the CCR5 coreceptor. Conversely, in CXCR4-using strains, cell-to-cell HIV transmission was mainly dependent on coreceptor expression and on TM-envelope-mediated fusion. Nevertheless, when coreceptor-dependent fusion events are blocked by CXCR4 antagonists or fusion inhibitors, the main route used by HIV transmission is independent of CXCR4 expression.

CD4-SU binding has been described as the necessary interaction that triggers coreceptor-independent HIV transfer (9). The CD4-SU binding inhibitor CD4-IgG2 is a fusion protein that incorporates four copies of the CD4 virus-binding domains (1, 31). Phase I and phase II clinical trials revealed its antiviral activity, especially in advanced HIV-1 disease (17, 18). As shown in Fig. 1, coculture of CD4+ T cells with chronically infected cells (MOLT-NL4-3 and MOLT-BaL) triggered a polarization of the CD4 receptor and an uptake of CA p24 into trypsin-resistant intracellular compartments, suggesting a coreceptor-independent but CD4-dependent HIV transfer, since only a few percent of CD4+ T lymphocytes are positive for CCR5 receptor and antigen transfer could not be blocked by HIV coreceptor antagonists. Moreover, the presence of the appropriate coreceptor (i.e., CXCR4 for X4 strains) led to an apparent increase of HIV transfer in the presence of fusion inhibitors or coreceptor antagonists. This observation suggests that when fusion events, such as syncytium formation or single-cell death (which are excluded from the analysis), are inhibited, the route used by HIV could be coreceptor-independent transfer. Thus, drug-treated samples appear to have increased cell-to-cell transfer compared to the untreated control.

In the 293T cell model, cells expressing the VSV-G envelope were not able to transfer viral particles to CD4+ T cells. However, VSV-G-expressing cells were able to induce syncytium formation between effector (VSV-G+) and CD4+ T target cells (data not shown). Thus, these data confirm the absolute requirement of the CD4-SU interaction as a necessary step that triggers coreceptor-independent HIV transfer to mononuclear T cells.

HIV transfer was completely blocked by CD4-IgG2 when both wild-type (X4 or R5 phenotype) (Fig. 2A) or X4/T-20-resistant envelope (Fig. 3B) was expressed on the surface of chronically infected cells. CD4-IgG2 inhibited, with comparable EC50s, coreceptor-independent HIV transfer in both R5-using viruses, BaL and 168.1, suggesting that CD4-IgG2 was able to block cell-to-cell virus transmission from different R5 strains or an HIV-1 clinical isolate. The inhibition shown when the X4/T20-resistant envelope was expressed would confirm that the mechanism of HIV transfer and CD4-IgG2 inhibition would be independent of fusion events and occurred against a virus strain that has been made resistant to a clinically relevant anti-HIV agent. In addition, the ability of HIV to use CD4+ T lymphocytes lacking the appropriate coreceptor as a virus reservoir could lead infectious viral particles to infect a third permissive cell. As a result of the inhibition of HIV transfer by CD4-IgG2, the release of infectious particles was blocked (Fig. 6A), and the subsequent infection of permissive cells by transferred and released HIV particles was also prevented (Fig. 6B). CD4-IgG2 could also inhibit HIV-1BaL transfer from infected to uninfected CD4+ T lymphocytes (Fig. 5), showing that coreceptor-independent HIV transfer may occur between primary cells and that CD4-IgG2 would be able to inhibit this process. Conversely, the binding inhibitor DS did not block coreceptor-independent cell-to-cell transmission in cocultures with MOLT-NL4-3 and MOLT-BaL cells (Fig. 4). DS binds SU glycoprotein (5, 16, 26) through the interaction of negatively charged sulfate groups on DS to positively charged regions in SU, including the hypervariable V3 loop (5, 12, 26). It has been suggested that DS may bind to SU and prevent HIV infection without blocking the interaction between CD4 and SU (5, 12). CD4 binding to SU induces conformational changes that expose the chemokine receptor binding site in SU (20). Moreover, several reports described the V3 loop as the principal determinant of chemokine receptor specificity (20, 28, 30). Our results suggest that DS could bind the V3 loop inhibiting coreceptor-dependent events like syncytium formation, virus-cell fusion, and single-cell death, which are more pronounced with the X4 strain, without interfering with the binding of CD4 and SU. The only route of virus transmission would be that of cell-to-cell, fusion-independent transfer. Thus, an increased cell-to-cell transfer of virus could be expected in the presence of DS compared to the untreated control in which all other fusion-dependent events may occur (Fig. 4). An alternative hypothesis is that HIV attachment to target cells involves envelope-dependent but nonspecific contacts that lead to CD4 binding and fusion. These initial virus-cell contacts can be inhibited by DS. In the cell-to-cell contact model of coreceptor-independent virus transmission, electrostatic interactions that are needed for virus-to-cell contacts may not be required or, alternatively, may be compensated for by interactions mediated by adhesion molecules. DS would fail to block SU binding to CD4 and virus transmission. Nevertheless, DS induced a partial block of HIV transfer from infected to uninfected primary CD4+ T lymphocytes at a relatively high concentration (50 µg/ml) (Fig. 5B), suggesting that when both effector and target cells are primary lymphocytes, electrostatic interactions needed for viral SU and cellular CD4 contact could be required. Conversely, CD4-IgG2, a binding inhibitor which specifically blocks the interaction between the envelope SU and CD4 protein, inhibited cell-to-cell transfer from chronically infected MOLT cells (Fig. 2A) or acutely infected CD4+ T lymphocytes (Fig. 5B) to primary target cells in addition to all other fusion-dependent events. Thus, the interaction between SU and CD4 protein appears to be the only event required to trigger coreceptor-independent HIV transmission.

It remains to be elucidated if cell-to-cell, coreceptor-independent virus transmission has any relevance in the pathogenesis of HIV infection. CD4-IgG2, currently studied in clinical trials, could then be able to prevent the transport of infectious virus particles to other body compartments where cells permissive to HIV infection may reside.


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ACKNOWLEDGMENTS
 
We thank M. Franti from Progenics Pharmaceutical, Tarrytown, N.Y., for providing CD4-IgG2, as well as the National Institutes of Health (AIDS Research and Reference Reagent Program) and the National Institute for Biological Standards and Control (AIDS Reagent Program) for reagents.

This work was supported in part by the Spanish Ministerio de Educación y Ciencia, project BFI-2003-00405, the Fondo de Investigación Sanitaria (FIS), project 02/0879, the FIS Red Temática Cooperativa de Investigacion en SIDA (RIS), the European TRIoH Consortium (LSHB-CT-203-503480), and Fundació Marató de TV3 project 020930. J.B. is a researcher from Fundació de Recerca Hospital Germans Trias i Pujol. I.C. holds an FI scholarship from Generalitat de Catalunya.


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FOOTNOTES
 
* Corresponding author. Mailing address: Fundació irsiCaixa, Laboratori de Retrovirologia, Hospital Universitari Germans Trias i Pujol, Ctra. Del Canyet s/n, 08916 Badalona, Spain. Phone: 34-934656374. Fax: 34-934653968. E-mail: jaeste{at}irsicaixa.es. Back


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Antimicrobial Agents and Chemotherapy, October 2005, p. 4296-4304, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4296-4304.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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