Deactivation of human immunodeficiency virus type 1 in medium by copper oxide-containing filters

Abstract

Human immunodeficiency virus type 1 (HIV-1) can be transmitted through breast-feeding and through contaminated blood donations. Copper has potent biocidal properties and has been found to inactivate HIV-1 infectivity. The objective of this study was to determine the capacity of copper-based filters to inactivate HIV-1 in culture media. Medium spiked with high titers of HIV-1 was exposed to copper oxide powder or copper oxide-impregnated fibers or passed through copper-based filters, and the infectious viral titers before and after treatment were determined. Cell-free and cell-associated HIV-1 infectivity was inhibited when exposed to copper oxide in a dose-dependent manner, without cytotoxicity at the active antiviral copper concentrations. Similar dose-dependent inhibition occurred when HIV-1 was exposed to copper-impregnated fibers. Filtration of HIV-1 through filters containing the copper powder or copper-impregnated fibers resulted in viral deactivation of all 12 wild-type or drug-resistant laboratory or clinical, macrophage-tropic and T-cell-tropic, clade A, B, or C, HIV-1 isolates tested. Viral inactivation was not strain specific. Thus, a novel means to inactivate HIV-1 in medium has been developed. This inexpensive methodology may significantly reduce HIV-1 transmission from "mother to child" and/or through blood donations if proven to be effective in breast milk or plasma and safe for use. The successful application of this technology may impact HIV-1 transmission, especially in developing countries where HIV-1 is rampant.

FIG. 1.

Inhibition of cell-free HIV-1 infectivity by cupric and cuprous oxide powder mix. cMAGI cells were exposed to HIV-1_BaL in the presence of 0 to 1% of copper powder mix. After 2 h of incubation, the cells were washed thoroughly. After 3 days of culture, viral growth (•) was assessed by counting the number of HIV-1-infected cMAGI cells. The results shown are the means ± standard deviations (error bars) of triplicate samples. In parallel, cMAGI cells were exposed for 2 h to 0 to 1% copper powder mix in the absence of HIV-1, and the cytotoxicity was determined (○).

FIG. 2.

Inhibition of cell-associated HIV-1 infectivity by cupric and cuprous oxide powder mix. H9+ cells, which were exposed for 2 h to different concentrations of copper powder mix, were cocultured with attached cMAGI target cells for 3 h, before being thoroughly removed. The cMAGI cells were then cultured for 3 days, and the number of cells infected with HIV-1 was then determined (▪). In addition, the supernatants containing HIV virions that budded out from the H9+ cells during the period when these cells were exposed to the copper were added to uninfected cMAGI cells. After 3 days of incubation, the number of infected cMAGI cells was determined (▾). The viabilities of the H9+ cells exposed to the various copper concentrations are also shown (•). Cell viability is expressed as a percentage of a control using untreated cells. The data shown are the average of duplicate samples. The differences between the duplicate samples were not more than 5%.

FIG. 3.

Cytotoxicity and inhibition of HIV-1 infectivity by filters containing copper powder mix. (a) Sketch of copper oxide-containing filter. (b) The cytotoxicity when applied on MT2 cells of 1:20, 1:50, or 1:100 final dilution of medium that eluted from filters containing 50 mg copper powder mix and no carbon layer or a 100 mg carbon layer is presented. The cytotoxicity after overnight incubation of the cells in a moist 5% CO₂ incubator at 37°C was determined by the trypan blue exclusion assay. (C) The cytotoxicity of 1:2, 1: 5, and 1:10 final dilutions of medium that eluted from filters containing 50 mg copper powder mix and 100, 200, 300, 400, or 500 mg carbon, is presented. cMAGI cells were then exposed to these HIV-1-spiked and filtered samples. After 3 days of culture, the number of HIV-1-infected cMAGI cells (stained blue) was determined (see Table 1). The data shown are averages of duplicate samples. The differences between the duplicate samples were not more than 5%.

FIG. 4.

Copper oxide-impregnated polypropylene fibers. (a) Scanning electron microscopy (JEOL JMS 5410 LV scanning electron microscope, Japan) of a copper oxide-impregnated polypropylene fibers. Bar, 50 μm. (b) X-ray photoelectron spectrum analysis (Link IV, ISIS, Oxford Instruments, England) of the surfaces of the copper-impregnated fibers used to fill filters for deactivation of HIV-1.

FIG. 5.

Inactivation of HIV-1 by filters containing copper oxide-impregnated polypropylene fibers. (a) One hundred twenty-five milliliters of medium containing HIV-1_IIIB was passed through filters containing 10 (circles), 20 (squares), or 30 (triangles) grams of copper-impregnated fibers. The first milliliter (black symbols) and the 100 ml (open symbols) that eluted from each filter were collected and subjected to sequential fourfold dilutions before they were added to MT2 target cells. For each viral dilution, four replicate wells were used. The eluate from the control filter (black diamonds) served as a positive control. (b) One hundred twenty-five milliliters of medium containing HIV-1 clinical isolate clade B, resistant to AZT and PI, were passed through filters containing 30 g of copper-impregnated fibers. The first milliliter (black triangles) and the 100 ml (white triangles) that eluted from each filter were collected and subjected to sequential fourfold dilutions before they were added to MT2 target cells. For each viral dilution, six replicate wells were used. The eluate from the control filter (black diamonds) served as a positive control. (c) One hundred twenty-five milliliters of medium containing HIV-1 clinical isolate clade C, resistant to AZT, was passed through filters containing 30 g of copper-impregnated fibers. The first milliliter (black triangles) and the 100 ml (white triangle) that eluted from each filter were collected and subjected to sequential fourfold dilutions before they were added to MT2 target cells. For each viral dilution, four replicate wells were used. The eluate from the control filter (black diamonds) served as a positive control. With all three isolates tested, viral infectivity was determined after 5 days of culture at 37°C by examining syncytium formation. Each well in which even one syncytium was seen was considered a positive well, i.e., infected with HIV-1. No cytotoxicity was observed in all examined wells. (d) Ten milliliters of medium containing HIV-1 clinical isolate clade B, resistant to AZT and PI, were passed through filters containing 10 g of copper-impregnated fibers (○) or control fibers without copper (•). The eluate was fourfold sequentially diluted and then added to PHA-activated PBMC in 96-well plates (triplicate wells). After overnight incubation at 37°C, the cells were thoroughly washed with RPMI 1640 medium and then cultured for additional 8 days at 37°C. MT2 cells were then cocultured with the PBMC (at a MT2/PBMC ratio of 5:1). After an additional 5 days of culture, the presence of syncytia was determined. Each well in which even one syncytium was seen was considered a positive well, i.e., infected with HIV-1. The experiments shown are representative of two similar experiments performed for each virus.