Immunology Letters, 30 (1991) 219- 228

Elsevier IMLET 01692

Inhibition of human immunodeficiency virus infection in monocytes by monoclonal antibodies against leukocyte adhesion molecules D. C h e s t e r K a l t e r l, 2, H o w a r d E. G e n d e l m a n 1, 2 and M o n t e S. M e l t z e r 1 tH1V Immunopathogenesis Program, Department of Cellular Immunology, Walter Reed Army Institute of Research, Washington, DC, U.S.A. and 2Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville, MD, U.S.A.

(Received21 June 1991; accepted24 June 1991)

1. Summary

distinct from cell-cell adhesion.

CD4 is the surface receptor for HIV envelope. Some evidence exists, however, that other cell surface receptors may be involved in viral entry subsequent to the initial binding of gpl20 to CD4. Antibodies to leukocyte integrin LFA-1, a major component of intercellular adhesive interactions, have been shown to inhibit HIV-induced syncytia formation. Using a stringent system for in vitro HIV infection of human leukocytes, we examine the ability of some monoclonal antibodies (mAb) against various adhesion-related molecules to block or partially inhibit productive viral replication. HIV-1 infection of target monocytes or T cells by cell-free virus was blocked completely or partially by some mAb that prevent cell-cell interactions (CD4, HLA-DR, LFA-1, LFA-3), but not by others (ICAM-1, MAC-l, gp150.95, CD2, CD3, CD14). The capacity for mAb to block HIV infection appears to be epitope-specific, and does not relate to the ability to block homotypic adhesion. HIV transmission from infected cells was more difficult to block than was infection by cell-free virus. Adhesion molecules may be involved in facilitating early stages of HIV infection, following g p l 2 0 / C D 4 binding but prior to viral integration, in a manner

2. Introduction

Key words: HIV; Monocyte;Leukocyteadhesion molecule; In-

hibition of infection Correspondence to: D. Chester Kaher, WRAIR and the H. M. Jackson Foundation, 9620 Medical Center Drive, Suite 200, Rockville, MD 20850, U.S.A.

HIV infection of T cells and monocytes typically results in obvious virus-associated cytopathic effects with cell lysis in at least a subpopulation of cells in culture. With T cells and T cell lines, HIVinfected and uninfected cells coalesce into balloonlike syncytia, while HIV-infected monocytes form characteristic multinucleated giant cells in culture [1,2]. Similar multinucleated giant ceils are evident within the central nervous system of infected patients [3]. Such cytopathic effects are implicated in cell-to-cell spread of virus and the ultimate depletion of CD4 ÷ T cells associated with the acquired immune deficiency syndrome [1, 4]. The high affinity interaction between HIV gpl20 on virus-infected cells and CD4 on the plasma membrane of uninfected cells provides a molecular basis for cell syncytia formation in T cells and monocytes [1, 5]. Indeed, mAb against CD4 completely prevent cell fusion between infected and uninfected T cells and cell-to-cell spread o f virus [5, 6]. Recent reports document participation of the LFA adhesion receptor family in HIV-induced syncytia [4, 7, 8]. These leukocyte integrins (LFA1, CR3, and p150,95) are heterodimers of a unique o~subunit (CD1 la, CD1 Ib, and CD1 lc) and a common /32 subunit (CD18). Hildreth and Orentas showed inhibition of syncytia between 8E5 cells (a constitutively infected T cell line that produces defective noninfectious HIV particles) and uninfected

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T cells by mAb against CD18 and CDI la [7]. Studies by Valentin et al. showed inhibition of HIVinduced syncytia by mAb against CD18 with infectious HIV (HTLVm~) in both T cell and myeloid cell lines [8]. In the continuous presence of anti-CDl8, the number of syncytia and the frequency of infected cells at 3 - 4 weeks in the U937 myeloid cell line infected with HIV were both suppressed. These effects were freely reversible after removal of antibody 1 week after infection. The most convincing evidence for participation of leukocyte integrins in HIV-induced syncytia derives from studies with PBMC of patients with leukocyte adhesion deficiency, a genetic disorder in which leukocytes lack all three members of the LFA family because of mutations in the common 132 subunit. HIV infection of PBMC from such patients induces no cell fusion or syncytia formation even though levels of virus replication in these cells are not very different from those of control infected cells [4]. The preceding observations show that while HIV-associated cell fusion and syncytia formation require participation of both CD4 and LFA-1, HIV infection from the fluid-phase and cell-to-cell spread of virus requires only CD4. Although CD4 is the specific and primary receptor for HIV, other cell surface determinants may also be involved in viral binding and entry. Such determinants can participate as viral attachment sites during HIV infection and function either as an alternative receptor for virus or as an accessory molecule that facilitates or suppresses CD4-mediated infection. In a search for cell surface determinants that affect HIV attachment and entry, we surveyed more than 20 different mAb against intercellular adhesion receptors and ligands. Most mAb had little or no effect on the replication of HIV in T cells or monocytes. Several mAb directed against different adhesion molecules in CD4/MHC class II, LFA-I/ICAM-1, and CD2/ LFA-3 interactions inhibited the replication of HIV. Such inhibition was epitope-specific and bore no direct correlation to inhibition of PMA-induced homotypic aggregation of PBMC. The surface determinants involved jn the initiation of infection of susceptible cells by, fluid-phase HIV are distinct from those involved in HIV-induced syncytium formation.

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3. Materials and Methods

3.1. Isolation and culture o f monocytes and PBL

Monocytes were recovered from PBMC of HIV and hepatitis B-seronegative donors after leukapheresis and purified by countercurrent centrifugal elutriation. Cell suspensions were >98% monocytes by cell morphology criteria on Wrightstained cytosmears, by granular peroxidase, and by nonspecific esterase. Monocytes were cultured as adherent cells in DMEM (Sigma Chemical Co., St. Louis, MO) with 10°70 heat-inactivated A + human serum, 50/~g/ml gentamicin, and 1000 U/ml highly purified (< 0.01 ng/ml endotoxin) recombinant human MCSF (lot FAP-809, Cetus Corp., Emeryville, CA) [9]. PBL free of monocytes and NK cells were isolated as above and cultured in RPMI1640 medium (Gibco, Grand Island, NY) with 5 ttg/ml PHA (Sigma), 10°70 purified human IL-2 (Advanced Biotechnologies, Inc., Columbia, MD), gentamicin, and 15070 heat-inactivated FCS (Sterile Systems, Inc., Logan, UT). 3.2. Antibodies and other reagents

Murine mAb were against: CD2 (Leu 5b), CD3 (Leu 4, SK7), CD4 (Leu-3a), CD1 lb (Mac-l, CR3, Leu-15), CD15 (Leu M1, MMA), HLA-DR (L243) (Becton Dickinson, Mountainview, CA); C D l l a (LFA-1 s-chain, SPV-L7) (Accurate Chemical and Scientific Corp., Westbury, NY); CD1 la (25.3.1), C D l l b (Bearl), CDllc (BU15), CD14 (RM052), CD18 (BL5, to an epitope distinct from the active binding site of the leukocyte integrin common/32chain) and CD54 (ICAM-1, 84H10) (AMAC Inc., Westbrook, ME); CDI la (TS 1/22), CD18 (TS1/ 18), CD54 (RR 1/1), and CD58 (TS 2/9) (gifts from Dr. Timothy A. Springer, Boston, MA and Dr. Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT); CD18 (H-52), CD18 (PLM-2, to an epitope distinct from the active binding site of the leukocyte integrin common ~32-chain), CD18 (MHM.23), CD18 (60.3) (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA), and CD1 la (MHM.24) (gifts from Dr. James E. K. Hildreth, Baltimore, MD) and C D l l a (NKI-L16, to an epitope involved in activation of LFA-1 binding, [10]) (gift from Dr. Carl Figdor, Amsterdam).

All mAbs containing preservatives were dialyzed twice against PBS and filter-sterilized (0.22/zm) prior to use.

3.3. Virus infection of target ceils Adherent MCSF-treated monocytes were cultured in 48-well plates 7 - 10 days prior to infection. PHA/IL-2-treated PBL were cultured in 75cm 2 flasks for 4 days,.then added to 48-well plates for infection or were infected in the flask. Cells were treated with mAb at 4 ° C for 15min, then exposed at a multiplicity of infection (MOI) of 0.01 infectious virus/target cell to any of several HIV strains (monocyte tropic strains, ADA and 24; T cell tropic strain, HTLVII m [2, 9]). All viral stock was tested and free of mycoplasma contamination (Gen-probe If, Gen-probe Inc., San Diego, CA). After a 2-h viral adsorption interval at 4 °C, cultures were washed twice, and re-fed with fresh medium with antibody. For some experiments, infected PBL blasts were added to uninfected monocyte monolayers. Culture medium was half-exchanged every 3 days with medium and no antibody. Cultures were examined for cytopathic effects for a period of 6 weeks. Levels of p24 antigen and reverse transcriptase (RT) activity in culture fluids were determined as described [2]. 3.4. Qualitative homotypic aggregation assay PBL free of monocytes and NK cells were suspended at 2 × 106 cells/ml RPMI-1640 with 5 mM Hepes buffer (Sigma) in flat-bottomed 96-well microtiter plates with and without 50ng/ml PMA (Sigma) and 20~g/ml mAb. Cell aggregates were scored at 3 h by light microscopy: 0, no inhibition of PMA-induced aggregation (up to 100%0 of cells in aggregates); 1 + , < 5 0 % of cells in aggregates; 2 + , < 10% of cells in aggregates; and 3 + , no cell aggregates [11]. 4. Results

MCSF-treated monocytes were treated with mAb for 15 min at 4 °C, and then exposed to HIV at a multiplicity of infection of 0.01 infectious virus/cell. After a 2-h viral adsorption, cultures were washed twice and re-fed with fresh medium

with mAb. Culture fluids were half-exchanged every 3 days for 6 weeks with medium without mAb. Levels of virus-associated RT activity in culture fluids were assayed at intervals. MAb against CD4 inhibited HIV replication in monocytes. Similarly, cells treated with mAb against HLA-DR, CD1 la, and CD58 also inhibited replication of HIV in a dose dependent fashion, though not so effectively as did anti-CD4 (Figs. ! - 3). Levels of RT activity in culture fluids of cells treated with equal concentrations of mAb against CD54, the ligand for CD1 la, or CDI lb, a leukocyte integrin that shares a common 32-chain with CD1 la, were equal to or greater than that of cells cultured in medium alone. MAb against CD4 inhibited HIV replication in monocytes at a concentration range of 1 - 10/zg/ ml, while the other mAb were effective in the range of 10-100/zg/ml. Correspondingly, typical HIVassociated cytopathic effects in monocyte (cell 400

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Inhibition of human immunodeficiency virus infection in monocytes by monoclonal antibodies against leukocyte adhesion molecules.

CD4 is the surface receptor for HIV envelope. Some evidence exists, however, that other cell surface receptors may be involved in viral entry subseque...
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