A Human Milk Factor Susceptible to Cathepsin D Inhibitors Enhances Human Immunodeficiency Virus Type 1 Infectivity and Allows Virus Entry into a Mammary Epithelial Cell Line

Abstract

The cell source of prolonged production of human immunodeficiency virus type 1 (HIV-1) in breast milk (8, 11, 21-23, 26, 27, 29) is not yet clear, since lymphocyte counts in milk decline rapidly after delivery (15). The risk of mother-to-child transmission through breast-feeding has been reemphasized recently, when discontinuation of antiretroviral therapy was followed by an increase in HIV-1 RNA load (10, 13). Therapy withdrawal in a mother after childbirth might also increase viral load in milk, although a short course of oral zidovudine during the peripartum period provided an important protection of the child despite breast-feeding (7).We have previously shown (12) that HIV-1 variants incubated in vaginal wash samples showed increased infectivity for lymphocyte cultures and acquired the ability to grow in a CD4-negative epithelial cell line obtained from a cervical epidermoid carcinoma. This effect was attributed to cathepsin D, which we purified from vaginal secretions. This protease might react with the viral gp120 and modify affinities for coreceptors.In the present work, we found that human breast milk also enhanced the infectivity of some HIV-1 variants. Two experiments showed that the enhancing factor was susceptible to cathepsin D inhibitors.Enhancing effect of milk is destroyed by inhibitors of cathepsin D. To demonstrate the effect of pepstatin A, an experiment was run in two steps. Milk sample 2 or buffer was first preincubated for 10 min at 37°C with 20 g of pepstatin A per ml or with buffer, and these mixtures were further incubated with HIV-1 variants before inoculation into primary lymphocyte cultures. Milk preincubated with pepstatin A lost its enhancing effect on HIV-1 IIIb, but enhancement was not completely lost when the same milk sample was assayed on fresh syncytium-inducing isolates (SI), 492 isolates, or non-syncytium-inducing isolates (NSI), 75 isolates (Fig. 1). Chymotrypsin and trypsin inhibitors did not modify the enhancing effect (not shown). A similar experimental protocol was applied to study the effect of a polyclonal anti-cathepsin D antibody on enhancing properties of milk, vaginal wash, or cathepsin D (Table 1).Enhancement of SI 121 and NSI 114 isolates was decreased by treatment with the anti-cathepsin D antibody, although it was not completely abolished in some of the mixtures. Table 2 summarizes data obtained in various experiments and shows that the enhancement effect of milk sample 2 on the three isolates simultaneously tested was greater than that of milk sample 1, although this difference did not appear in the assay of the laboratory strain, HIV-1 IIIb. A survey of the whole table does not disclose a general trend distinguishing a difference between SI and NSI isolates regarding their susceptibility to milk enhancement. This table also shows the effect of various concentrations of milk sample 2. It was striking that the final dilution of 1:2 showed a lower enhancing effect than did the dilution of 1:10. At higher concentrations, there may be a balance with HIV-1 inhi...

Lewis X component in human milk binds DC-SIGN and inhibits HIV-1 transfer to CD4+ T lymphocytes

“…The 2-fold dilution of human milk demonstrated a significant degree of viral inhibition (>90%; P < 0.05) in comparison with the PBS control ( Figure 1A), which corresponds with previous reports (44)(45)(46)(47)(48)(49). We also identify an enhancement to infection at the 1:10 dilution similar to findings from a previous study (38). Preincubation of the same human milk sample (1:2) with HIV-1 before addition to CD4 + T lymphocytes ( Figure 1B) showed no inhibitory effect in comparison to the PBS control, indicating that the inhibition shown in Figure 1A is not due to a direct virucidal effect of the milk and that the effect is likely conferred on the CD4 + lymphocyte.…”

“…Macrophages in human milk are found at varying concentrations during lactation (34) and are preferentially infected with viruses that utilize the CCR5 coreceptor for viral entry (R5) and that are typically associated with HIV-1 transmission (35,36). Mammary epithelial cells are present in human milk, and these cells are predominantly infected with viruses utilizing the CXCR4 coreceptor (X4), virus strains that seldom undergo transmission (37,38). CD4 + T lymphocytes are also present in human milk, and these cells can be infected with both R5 and X4 strains, depending on the cellular phenotype and cell activation status (39,40).…”

Cell-Type-Dependent Effect of Transforming Growth Factor β, a Major Cytokine in Breast Milk, on Human Immunodeficiency Virus Type 1 Infection of Mammary Epithelial MCF-7 Cells or Macrophages

“…In addition to macrophages and lymphocytes, mammary epithelial cells are thought to be the sources of HIV-1 in breast milk. Interestingly, the susceptibility of those cells to HIV-1 strains differs: macrophages support replication of R5-tropic HIV-1 much better than that of X4-tropic HIV-1, lymphocytes support replication of R5-and X4-tropic HIV-1 equally well, and mammary epithelial cells support replication of X4-tropic rather than R5-tropic HIV-1 (18,28,30). It is, however, unclear which cell type plays a major role in mother-to-child transmission.…”

“…Macrophages are susceptible to R5-tropic HIV-1 but much less to X4-tropic virus (1, 3) which utilizes CXCR4 as an entry coreceptor. Although the major source of HIV-1 in mature (Ͼ2 weeks after delivery) milk remains unknown, mammary epithelial cells, the predominant cell type in mature milk (8), have been shown to be susceptible to certain (mostly X4-tropic) HIV-1 strains (18,28,30).…”

Heroin-Induces Differential Protein Expression by Normal Human Astrocytes (NHA)

“…Further studies have also shown that vaginal secretions contain cathepsin D and exposure of lymphocyte cultures to these secretions increased HIV-1 replication in these cells [45] . Furthermore, human breast milk also enhanced HIV-1 infection of lymphocyte cultures which was inhibited by anti-cathepsin D antibodies or pepstatin A, an inhibitor of cathespin D suggesting that cathepsin D may interact with gp120 to modify the affinity of HIV-1 co-receptors thereby enhancing HIV-1 infectivity [45,46] . The current study demonstrates for the first time that heroin enhances the expression of the preprotein form of cathepsin D in NHA.…”