Human immunodeficiency virus type 1 (HIV-1) infects its host cells by the initial specific interaction of the HIV-1 envelope glycoprotein Env (gp120/gp41) with the CD4 molecule found primarily on T helper lymphocytes and macrophages [1Düzgünes N, Ed. Mechanisms and Specificity of HIV Entry into Host Cells Advances in Experimental Medicine and Biology. New York: Plenum Press 1991; 300.,2Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease Annu Rev Immunol 1999; 17: 657-700.]. Entry of HIV-1 into target cells requires formation of a complex between the Env glycoprotein gp120, the primary receptor CD4, and a co-receptor. The chemokine receptors CXCR4 (X4) and CCR5 (R5) have been identified as the principal co-receptors for X4 (T cell tropic) and R5 (macrophage tropic) viruses, respectively. Binding of gp120 to the N-terminal domain of CD4 induces a pH-independent conformational change in both molecules, resulting in the interaction of the fusion domain at the N terminus of the transmembrane gp41 with the target cell membrane. gp41 then folds back on itself and forms a six-helix bundle, bringing the viral and cellular membranes into close proximity, and also forcing the fusion of the two membranes [3Melikyan GB. Common principles and intermediates of viral protein-mediated fusion: the HIV-1 paradigm Retrovirology 2008; 5: 111.].

Broadly cross-reactive, neutralizing human monoclonal antibodies can inhibit HIV-1 infection in vitro; however administration of these antibodies to HIV-1-infected patients has resulted in only modest antiviral activity [4Chen W, Dimitrov DS. Human monoclonal antibodies and engineered antibody domains as HIV-1 entry inhibitors Curr Opin HIV AIDS 2009; 4: 112-7.]. There may be a number of reasons for this observation, including access of the antibodies to sites of active virus infection. It is also possible that, while these antibodies inhibit virus-cell fusion, they do not inhibit cell-cell fusion, and hence enable the spread of the virus via syncytium formation.

The human monoclonal antibody 2F5 neutralizes a variety of HIV-1 laboratory strains and clinical isolates, and interacts with the gp41 ectodomain, close to the transmembrane region [5Muster T, Steindl F, Purtscher M, et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 J Virol 199; 67: 6642-7.] (Table 1). The inhibitory activity of the antibody was evaluated by pre-incubating it with the virus, adding the mixture to AA-2 cells, and measuring syncytium formation 5 days later. The IC₅₀ values were 3.7, 0.7 and 0.8 µg/ml for the IIIB, MN and SF2 isolates, respectively [6Purtscher M, Trkola A, Gruber G, et al. A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 AIDS Res Human Retroviruses 1994; 10: 1651-8.]. By contrast, the 50%-effective concentration of 2F5 for inhibiting the fusion of chronically infected H9 cells with AA-2 cells was 21.0, 19.3 and 17.7 µg/ml, respectively, indicating that much higher concentrations of antibody were necessary to inhibit cell-cell fusion in this system. The 4E10 antibody inhibited the infection of AA-2 cells by IIIB, MN and RF isolates, measured by the presence or absence of syncytium formation 5 days after inoculation, at IC₅₀s of 1.0, 0.3 and 12.5 µg/ml, respectively [7Stiegler G, Kunert R, Purtscher M, et al. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 AIDS Res Human Retroviruses 2001; 17: 1757-65.].

Table 1

The Molecular Targets and IC₅₀ Values of the Antibodies Evaluated in this Study, and the HIV-1 Strains they Inhibit

The recombinant monoclonal antibody, IgG1 b12, is directed to the CD4-binding site of gp120 from the IIIB strain of HIV-1 [8Burton DR, Pyati J, Koduri R, et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody Science 1994; 266: 1024-7.]. It had 50% neutralization titers of 3 ng/ml against the MN strain, and 7 ng/ml against IIIB, as determined by the inhibition of plaque formation. The monoclonal antibody, 2G12, binds to a conformational carbohydrate-based epitope on gp120 [9Buchacher A, Predl R, Strutzenberger K, et al. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization AIDS Res Hum Retroviruses 1994; 10: 359-69.], and neutralizes HIV-1 strains IIIB, RF, and MN at ID₅₀s of 0.02, 0.13 and 1.56 µg/ml, respectively [10Trkola A, Purtscher M, Muster T, et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1 J Virol 1996; 70: 1100-08.]. It also neutralizes a broad range of primary isolates in a PBMC-based assay at ID₅₀ concentrations in the range of <0.01–0.15 µg/ml, but it is ineffective against other isolates. In a syncytium inhibition assay involving AA-2 cells, 2G12 antibody had EC₅₀s of 4.6 and 0.5 µg/ml for IIIB and RF, respectively, but it did not inhibit syncytium formation directed by the MN isolate (> 60 µg/ml), consistent with its inability to achieve 90% neutralization with this virus with respect to inhibition of virus production in AA-2 cells. The antibody IgG1 m14 originated from a human Fab phage display library based on inhibition of gp120 binding to soluble CD4 [11Zhang MY, Xiao X, Sidorov IA. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody J Virol 2004; 78: 9233-42.]. Fab m14 neutralized the HxB2 isolate with an IC₅₀ of 1 µg/ml.

The fusion activity of Env can be studied conveniently by the fusion of HIV-1-infected cells with uninfected CD4+ cells [12Lifson JD. Fusion of human immunodeficiency virus-infected cells with uninfected cells Methods Enzymol 1993; 221: 3-12.]. Alternatively, cells expressing the Env protein in the absence of infection can be utilized for this purpose. We developed a new HIV fusion assay, using Clone69TRevEnv cells that express Env cloned from the IIIB (HXB) strain when tetracycline is removed from the medium [13Yu H, Rabson AB, Kaul M, Ron Y, Dougherty JP. Inducible human immunodeficiency virus type 1 packaging cell lines J Virol 1996; 70: 4530-7.]. SupT1 cells that express CD4 at high levels [14Smith SD, Shatsky M, Cohen PS, Warnke R, Link MP, Glader BE. Monoclonal antibody and enzymatic profiles of human malignant T lymphoid cells and derived cell lines Cancer Res 1984; 44: 5657-60.,15Konopka K, Düzgünes N. Expression of CD4 controls the susceptibility of THP-1 cells to infection by CCR5- and CXCR4-dependent HIV Type 1 isolates AIDS Res Hum Retrovirus 2002; 18: 123-31.] were used as target cells. The use of Clone69TRevEnv cells cultured in tetracycline also provides a convenient control in which Env is not expressed. For the fluorescence assay, Clone69TRevEnv cells were labeled with a green fluorescent dye that is trapped in the cytoplasm following permeation through the plasma membrane. SupT1 cells were labeled with a red fluorescent dye that is trapped in the same manner. Env-mediated fusion between these fluorescently labeled cells is observed by the formation of orange syncytia.

Several fluorescence methods to study HIV-induced cell-cell fusion have been described previously. These include the transfer of the membrane probe DiI or the cytoplasmic probe (BCECF) from labeled cells to unlabeled cells [23Golding H, Dimitrov DS, Blumenthal R. LFA-1 adhesion molecules are not involved in the early stages of HIV-1 env-mediated cell membrane fusion AIDS Res Hum Retroviruses 1992; 8: 1593-8.,24Broder CC, Dimitrov DS, Blumenthal R, Berger EA. Envelope glycoprotein-mediated membrane membrane fusion in animal cells expressing human CD4 can be overcome by a human cell component(s) Virology 1993; 193: 483-91.]. Frey et al. [25Frey S, Marsh M, Günther S, et al. Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 J Virol 1995; 69: 1462-72.] described a method where a fluorescent fatty acid probe was incorporated metabolically into SupT1 cells, and the dilution of the probe into unlabeled cells was monitored. The principle of the assay used in this study is similar to that described by Muñoz-Barroso et al. [26Muñoz-Barroso I, Durell S, Sakaguchi K, Appella E. Blumenthal R: Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41 J Cell Biol 1998; 140: 315-23.], who used B cell-derived TF228.1.6 cells that express constitutively Env, and SupT1 cells. They labeled the former cells with the blue dye, CMAC, which interacts with free sulfhydryl groups inside cells, and the latter cells with both calcein and the membrane probe DiI. We used Calcein AM to label the cytoplasm of adherent Clone69TRevEnv cells, and the dye CellTrace™ Calcein red-orange to label the suspension SupT1 cells. An advantage of the Clone69TRevEnv cells is that they can be induced to express Env (Env⁺), enabling us to use identical cells that do not express Env (Env^–) as controls. The use of an adherent-suspension cell pair to monitor fusion is also advantageous, because the shape of the fusion product resembles that of adherent cells, in that it is spread out. This may be advantageous in a cell imaging-based screening system. Although there are many drugs that inhibit the HIV enzymes, reverse transcriptase and protease, there are only two approved drugs that inhibit virus entry into cells, Enfuvirtide and Maraviroc. The fluorescence assay described here may be useful in screening potential inhibitors of HIV-induced membrane fusion in high-throughput cell imaging assays.

The plant-derived, carbohydrate-binding proteins, HHA and GNA, and the peptide, T-20 (Enfuvirtide), markedly inhibited fusion in this assay system. CBP are thought to act by binding to the glycans on the HIV-1 gp120 envelope, and by cross-linking several glycans during virus-cell interaction [21Balzarini J. Inhibition of HIV entry by carbohydrate-binding proteins Antiviral Res 2006; 71: 237-47.]. They may freeze the conformation of gp120, consequently preventing further interaction with the HIV-1 co-receptor(s) on the cell surface. Viruses resistant to inhibition by 2G12 antibody maintain their full sensitivity to various mannose-specific lectins [27Huskens D, Van Laethem K, Vermeire K, Balzarini J, Schols D. Resistance of HIV-1 to the broadly HIV-1-neutralizing, anti-carbohydrate antibody 2G12 Virology 2007; 360: 294-304.]. Moreover, a 2G12-resistant NL4.3 strain generated in vitro by antibody pressure was even more sensitive to mannose-specific lectins. Since T-20 inhibits the formation of the six-helix bundle of gp41 [28Wexler-Cohen Y, Johnson BT, Puri A, Blumenthal R, Shai Y. Structurally altered peptides reveal an important role for N-terminal heptad repeat binding and stability in the inhibitory action of HIV-1 peptide DP178 J Biol Chem 2006; 281: 9005-10.], our results indicate that cell-cell fusion in our assay system is mediated by the gp41 protein component of Env.

Surprisingly, monoclonal antibodies that bind either of the two components of Env, gp120 and gp41, and that reportedly inhibit infection by a large number of HIV-1 isolates, had little or no inhibitory effect on cell-cell fusion. We showed previously that a monoclonal antibody to the gp120-CD4 complex (Mab F-91-55) that bound the D1/D2 domains of CD4 inhibited syncytium formation between chronically HIV-1-infected H9 cells and either A3.01 or Sup-T1 cells, even at a concentration of 0.1 µg/ml [29Konopka K, Pretzer E, Celada F, Düzgünes N. A monoclonal antibody to the gp120-CD4 complex has differential effects of on HIV-induced syncytium formation and viral infectivity J Gen Virol 1995; 76: 669-79.]. However, the antibody had a very limited effect on HIV-1 infection of A3.01 or Sup-T1 cells. This observation is in contrast to our current observations with the anti-gp120 and anti-gp41 antibodies that inhibit HIV-1 infection, but are not very effective in inhibiting syncytium formation. Nevertheless, Mab F-91-55 inhibited both the infectivity of HIV-1 in H9 cells, and syncytium formation between these cells and chronically infected H9 cells. We proposed earlier that cellular systems used for syncytium assays differ in their susceptibility to antibodies, and that the interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion [29Konopka K, Pretzer E, Celada F, Düzgünes N. A monoclonal antibody to the gp120-CD4 complex has differential effects of on HIV-induced syncytium formation and viral infectivity J Gen Virol 1995; 76: 669-79.]. This difference may also explain our current results. The molecular organization and surface density of gp120/gp41 may be different in Env-expressing cells and HIV-1 virions. Purtscher et al. [6Purtscher M, Trkola A, Gruber G, et al. A broadly neutralizing human monoclonal antibody against gp41 of human immunodeficiency virus type 1 AIDS Res Human Retroviruses 1994; 10: 1651-8.] have explained their observations that significantly higher concentrations of antibody are necessary to inhibit fusion between infected cells and target cells by suggesting that higher levels of gp120/gp41 are expressed on the surface of infected cells. Choudhoury et al. [30Choudhry V, Zhang MY, Harris I, et al. Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentrations Biochem Biophys Res Commun 2006; 348: 1107-5.] have found that the IC₅₀ of 2F5 and 4E10 antibodies decreases by up to two orders of magnitude in cell lines with low cell surface expression of CCR5. Thus, the relatively high level of CXCR4 in the SupT1 cells used in our assay [15Konopka K, Düzgünes N. Expression of CD4 controls the susceptibility of THP-1 cells to infection by CCR5- and CXCR4-dependent HIV Type 1 isolates AIDS Res Hum Retrovirus 2002; 18: 123-31.] may have also contributed to the limited ability of the tested antibodies to inhibit cell-cell fusion.

CBPs are smaller in size and bind in a much less specific manner to HIV gp120/gp41 than the mAbs. Thus, a higher number of CBPs can bind simultaneously to cell-surface Env. If the conformation of viral Env is different from that of cell membrane Env, the efficiency of mAb binding to the latter may be compromised, whereas the affinity of the CBP binding to the gp120 glycans would be expected to be much less affected by conformational differences at the protein level. Therefore, the CBPs used in our study are very effective in blocking the fusion capacity even in the presence of a high density of Env, whereas the anti-gp120 and anti-gp41 mAbs are rather poor inhibitors.

The limited inhibitory effect of the antibodies used in our experiments against Env-induced cell-cell fusion raises the possibility that such antibodies may not be able to inhibit efficiently the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. Two broadly neutralizing antibodies (PG9 and PG16) that recognize an epitope expressed on trimeric Env, and comprising conserved variable loops of gp120, have been identified recently [31Walker LM, Phogat SK, Chan-Hui PY, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target Science 2009; 326: 285-9.]. Antibodies (VRC01 and VRC02) to the CD4 receptor-binding site that neutralize over 90% of circulating HIV-1 strains have also been identified [32Wu X, Yang ZY, Li Y, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1 Science 2010; 329: 856-61.]. It has been suggested that vaccine-induced antibodies of similar specificity may provide protection against many HIV-1 strains currently circulating. It will be of interest to examine the potential inhibitory effects of PG9, PG16, VRC01 and VRC02 antibodies on syncytium formation in our experimental system.

The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. The inability of HIV neutralizing antibodies to inhibit effectively HIV-Env-mediated syncytium formation fusion raises concerns about the ability of such neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. CBPs appear to interact more efficiently with cell membrane Env than antibodies against gp120 and gp41, and are highly inhibitory to cell-cell fusion.