Chemically Modified Bovine β-Lactoglobulin Blocks Uptake of HIV-1 by Colon- and Cervix-Derived Epithelial Cell Lines (1996)

To the Editor: We recently showed that bovine β-lactoglobulin, a protein present in milk and whey, modified by droxyphthalic anhydride (3HP-β-LG) inhibits infection by human (HIV-1 and HIV-2) and simian (SIV) immunodeficiency viruses (1). The inexpensive and widely available source (whey) for production of this inhibitor suggests its potential for large-scale prevention of sexual (mucosal) HIV transmission, the most frequent (86%) route of adult HIV transmission worldwide (2). The mechanism by which 3HP-β-LG inhibits HIV infection has been ascribed to blockade of the binding site on the CD4 receptor molecule for the HIV surface glycoprotein (1). There is evidence suggesting that Langerhans and dendritic cells, which express CD4 molecules, are the primary target for HIV and SIV infection, especially in the case of heterosexual viral transmission (3-6). However, it was proposed that CD4- epithelial cells in rectal and genital tract mucosae may also contribute to HIV-1 transmission (7-9). Therefore, it was of interest to determine whether 3HP-β-LG blocks HIV-1 transmission through CD4- epithelial cells. Since it is controversial that HIV-1 can efficiently replicate in CD4- epithelial cells, we used an assay, modified from a method described by Fantini et al. (7), to determine the inhibitory activity of 3HP-β-LG on the uptake of HIV-1 by epithelial cell lines. Briefly, 4 ml of HT-29 (ATCC HTB38, derived from human colonic adenocarcinoma; ATCC, Rockville, MD, U.S.A.) or ME-180 (ATCC HTB 33, derived from human cervical carcinoma) cells in McCoy's 5A containing 10% fetal bovine serum (2.5 × 105 cells/ml) were placed into wells of six-well plates. After incubation at 37°C for 24 h, the supernatants were removed. Two milliliters of 3HP-β-LG at different concentrations in the culture medium and 2 ml of HIV-1 IIIB at a 0.02 multiplicity of infection (MOI) were added to the wells. After incubation at 37°C for 16 h, cells were washed four times with medium without serum. Then, 4 ml of phosphate-buffered saline (PBS) containing trypsin (0.25 mg/ml) were added. After 2 min at 37°C, the trypsin-containing solution was removed. Cells were washed twice with PBS, lysed with 5% Triton X-100, and assayed for p24 antigen by enzyme-linked immunosorbent assay (ELISA) (Coulter Immunology, Hialeah, FL, U.S.A.) as described (10). As shown in Fig. 1A, there was a marked uptake of HIV-1 IIIB by these two epithelial cells lines (≈0.0003 pg of p24 per cell) and this uptake was blocked by 3HP-β-LG in a dose-dependent manner. In addition, we tested whether 3HP-β-LG blocked the binding of epithelial cells to magnetic beads coated with an HIV-1 V3 loop peptide. Briefly, 5 mg of streptavidin magnetic beads (PerSeptive BioResearch Products, Cambridge, MA, U.S.A.) in 1 ml PBS containing 1 mg/ml bovine serum albumin (PBS-BSA) were incubated with 1 ml (60 μg) biotinylated HIV-1 BH10 V3 loop peptide (sequence 303-338) (11) at 25°C overnight. Beads were washed 10 times with PBS-BSA and stored at 4°C until use. The binding of ME-180 cells to these magnetic beads was measured in the presence or absence of 3HP-β-LG (100 μg/ml), as previously described (1). The results showed that 97% of ME-180 cells bound to the magnetic beads and the binding was significantly blocked by 3HP-β-LG (Fig. 1B), indicating that the binding of gp120 V3 loop on the HIV-1 particles to a cell surface is the first step of uptake of HIV-1 by CD4- epithelial cells and that inhibition of HIV-1 gp120 V3 loop binding to the cells by 3HP-β-LG results in the block of uptake of HIV-1 by CD4- epithelial cells. The exact mechanism(s) whereby 3HP-β-LG blocks the uptake of HIV-1 by ME-180 and HT-29 cells is unclear. It was reported that HT-29 cells expressed galactosyl ceramide, serving as an alternative receptor for HIV-1 (7), but others demonstrated that galactosyl ceramide did not apparently act as an HIV coreceptor (12). In addition, there was no detectable galactosyl ceramide on ME-180 cells (own unpublished data). ME-180 cells could take up HIV-1 IIIB as efficiently as HT-29 cells, and 3HP-β-LG blocked equally the uptake of HIV-1 IIIB by both cell lines, indicating that ME-180 and HT-29 cells may share a common pathway for the uptake of HIV-1. The results presented here suggest that 3HP-β-LG has potential for prevention of sexual transmission of HIV-1 through epithelial cells. Acknowledgment: We thank Dr. Asim K. Debnath for advice. HIV-1 IIIB was provided by the NIH AIDS Research and Reference Reagent Program and contributed by Dr. R. Gallo. This study was supported by NIH grant CA-43315. Shibo Jiang; Kang Lin; Nathan Strick; Yun-Yao Li; A. Robert Neurath Laboratory of Biochemical Virology; The Lindsley F. Kimball Research Institute of; The New York Blood Center; New York, New York, U.S.A.FIG. 1. A:: Inhibitory effect of 3HP-β-LG on HIV-1 uptake by HT-209 and ME-180 cells, respectively. B: Binding of ME-180 cells to magnetic beads with immobilized V3 loop peptide from HIV-1 gp120 BH10 and inhibition of binding by 3HP-β-LG.