Jenabian, Mohammad-Ali DVM, PhD*; Auvert, Bertran MD, PhD†‡§; Saïdi, Héla PhD*; Lissouba, Pascale MSc§; Matta, Mathieu BSc*; Bélec, Laurent MD, PhD*
From the *Université Paris Descartes (Paris V), and Laboratoire de Virologie, Hôpital Européen Georges Pompidou, Paris, France; †Université de Versailles-St Quentin-en-Yvelines, Versailles, France; ‡Hôpital Ambroise Paré, Boulogne, France; and §INSERM U1018, Hôpital Paul Brousse, Villejuif, France.
Received for publication March 16, 2010; accepted April 20, 2010.
Grants supports from the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales and Institut National de la Santé et de la Recherche Médicale, France.
We have no financial conflict of interest.
Correspondence to: Laurent Bélec, MD, PhD, Université Paris Descartes (Paris V), Laboratoire de Virologie, Hôpital Européen Georges Pompidou, 15-20 rue Leblanc, 75 908 Paris Cedex 15, France (e-mail: email@example.com).
Soap and water solutions have been shown to be highly efficient at destroying both nonenveloped and enveloped viruses, such as the HIV.1,2 A previous in vitro study demonstrated the virucidal efficacy of a solution of common soap and water against CXCR4-specific HIV-1SF33 strain, reporting a 30-fold viral inactivation and a marked reduction in viable peripheral blood mononuclear cells (PBMCs) in a mixture of cervicovaginal lavage and seminal fluids and suggesting that soap and water may be used to inactivate HIV and HIV-infected cells in genital secretions.3
The aim of the present study was to confirm and extend under different experimental conditions these latter observations on HIV-1 inactivation and cytotoxicity by soap and water solutions carried out with common soap bars. Indeed, effective virus inactivation may depend on many factors, such as viral concentration, viral phenotype, the degree of virucidal activity of antiviral molecules, and the duration of incubation.4 In particular, both CCR5-tropic and CXCR4-tropic HIV-1 are present in genital secretions from HIV-1-infected individuals,5 and CCR5-tropic HIV-1 has been demonstrated to be largely selected during heterosexual HIV-1 acquisition via transmucosal genital crossing,5 emphasizing the need to evaluate soap-associated inactivation on both CCR5-tropic and CXCR4-tropic HIV-1 strains. Finally, in the present study, we evaluated whether soap and water could inactivate both cell-free virus particles (virucidal potency) and HIV-1-infected cells (cytotoxic potency), using both CCR5-tropic and CXCR4-tropic HIV-1 strains, purified lymphocytes, which are virus target cells thought to be highly involved during mucosal HIV passage6 and 2 common soap bars commercially available in South Africa.
MATERIALS AND METHODS
Primary CXCR4-tropic HIV-1NDK was donated by F. Barré-Sinoussi (Institut Pasteur, Paris, France). CCR5-tropic HIV-1BaL was provided by the National Institute of Health (NIH, MD). The viral stocks were amplified in monocyte-derived macrophages of healthy donors and quantified by p24 antigen capture enzyme-linked immunosorbent assay measurement (DuPont de Nemours, Les Ulis, France).
RPMI 1640 (with L-glutamine) was provided by Cambrex (Biosciences, Verviers, Belgium). Penicillin/streptomycin was provided by Invitrogen (Cergy-Pontoise, France). Medium of separation for lymphocytes and fetal calf serum were obtained from PAA Laboratories GmbH (Les Mureaux, France) and Eurobio (Les Ulis, France). rhIL-2 was obtained from Eurobio and phytohemagglutinin-P from Sigma-Aldrich (St. Louis, MO).
Two commercial soaps widely available in South Africa, Sunlight (sodium palmate, sodium palm kernelate, palm fatty acid, tetrasodium EDTA, and trace chemicals; Unilever) and Protex Herbal (sodium palmate, sodium palm kernelate, PEG-12, sodium chloride, and trace of various chemicals; Colgate-Palmolive) were used for experimentation. Solutions of soap and distilled water were prepared at final concentrations of 1/200 and 1/400 g/mL corresponding to soap dilutions reported during usual hand washing.3
Purification of Peripheral Blood Lymphocytes
PBMCs were isolated from buffy coats of healthy adult donors by Ficoll density gradient centrifugation on Medium for Separation of Lymphocytes (Eurobio, Les Ulis, France) as previously described.7 PBMCs were resuspended in RPMI 1640 medium supplemented with glutamine, penicillin (100 IU/mL) and streptomycin (100 μg/mL). Cells were seeded into 24-well plates (Costar, Cambridge, MA) at concentration of 1 × 106 adherent cells per milliliter and incubated at +37°C for 45 minutes. Nonadherent cells were removed with 4 washes and used to prepare peripheral blood lymphocytes (PBLs) by culture for 48 hours in fresh medium supplemented with phytohemagglutinin-P at 2.5 μg/mL concentration and interleukin-2 (rhIL-2) at 1 μg/mL concentration. PBLs were then washed and cultured in growth medium containing rhIL-2 (1 μg/mL) for 48 hours.7
Virucidal Assay for Free HIV-1 Particles
The capacity of soap and water solutions to disrupt cell-free HIV-1 particles and inactivate their infectiousness was evaluated by virucidal assay. HIV-1BaL and HIV-1NDK (1 ng of p24 antigen) were coated on poly-L-lysine precoated 96-well plates (Greiner Bio-One, Germany) at +4°C overnight. After washing, plates were incubated with solutions of each soap diluted in sterile distilled water at final concentrations of 1/200 and 1/400 g/mL, for 1 and 2 minutes at +37°C. In positive and negative control wells, 1% Triton X-100 and culture medium were added, respectively. After 4 washes, 3 × 105 stimulated PBLs were added in each well. After 6 days of incubation, viral production was assessed by p24 antigen capture enzyme-linked immunosorbent assay.8,9
Cytotoxicty Assays Using HIV-Infected Lymphocytes
HIV-1BaL-infected PBLs were seeded onto 96-well plates at a density of 2 × 105 cells per well and incubated for 1 minute at 37°C with each soap solution. In positive and negative control wells, 1% Triton X-100 and culture medium were added, respectively. After the end of incubation, plates were centrifuged during 5 minutes, and the cytotoxicity of increasing soap concentrations on PBLs were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Sigma-Aldrich, St-Louis, MO) as previously described.7 The absorbance of each well was measured in a microtitre reader at 490 nm. To translate the OD490 values into the number of live cells in each well, the OD490 values were compared with those of standard OD490 versus cell number curves generated for the PBLs.8 The survival index was calculated using the formula: Survival index = live cell number (test)/live cell number (control).
To evaluate the soap-associated cytotoxicity occurring during rapid soap washing (around 30 seconds), 4 × 105 HIV-1BaL-infected PBLs were collected in 100 μL of complemented RPMI1640 cell culture medium in 15-mL tubes. Once the soap and water solutions were added, the tubes were incubated at +37 °C during 30 seconds. The soap cytotoxic effect was then stopped by adding 15 mL of water to the tubes, thus decreasing the soap concentration. The cells were then centrifuged, and the MTT test was carried out.
Virucidal Effect of Soap and Water Solutions on Cell-Free HIV-1 Particles
As clearly depicted in Figure 1, both Sunlight and Protex Herbal soaps diluted in water at 1/200 and 1/400 g/mL concentrations were able to disrupt nearly completely the replication of CCR5-tropic and CXCR4-tropic HIV-1 particles. Similar observations were obtained for exposures of 2 minutes.
Cytotoxicity of Soap and Water Solutions on HIV-1-Infected Lymphocytes
The 1/200 and 1/400 g/mL solutions of Sunlight or Protex Herbal soaps were largely cytotoxic against the HIV-1BaL-infected PBLs after 1 minute of incubation, decreasing cell viability by more 95%. When the HIV-1BaL-infected PBLs were treated by solutions of Sunlight and Protex Herbal soaps diluted at 1/200 and 1/400 g/mL during only 30 seconds, followed by quick neutralization of soap activity by adding 15 mL of water, a dramatic decrease of cell viability was observed, reaching over 90% (Fig. 2). The viability of cells treated with a solution of 1% Triton X-100, used as positive control for toxicity was zero (data not shown).
We demonstrated herein the potent virucidal effect of soap and water solutions against both CCR5-tropic and CXCR4-tropic cell-free HIV-1 strains and their cytotoxicity against HIV-1-infected lymphocytes. These observations were obtained with short incubation durations ranging from 30 seconds to 2 minutes, suggesting a rapid action of soap and water solutions on viral infectivity. Furthermore, Sunlight and Protex Herbal soaps diluted in distilled water at concentrations of 1/200 and 1/400 g/mL and incubated during 1 minute with stimulated PBLs (3 × 105 per well), then washed, and finally incubated with HIV-1BaL and HIV-1NDK (1 ng of p24 antigen) under standard culture conditions, inhibited completely HIV-1 production in vitro (not shown). These observations confirm the previously found marked virucidal effect of soap and water solutions against HIV-1 in genital fluids.3 In addition, the virucidal effect of soap and water solutions was demonstrated against CCR5-tropic HIV-1 strain, which is primarily selected during HIV genital crossing,5 and against both cell-free viral particles and cell-associated virus, which are both likely involved in HIV sexual transmission.10 Potent cytotoxicity was observed against HIV-1-infected lymphocytes, which represents important targets for HIV-1 within mucosae.6 Finally, these results were obtained by using 2 common soap bars commercially available in South Africa, confirming those previously reported by using a North American soap.3 Taken together, these findings confirm that solutions of soap of various origins diluted in water at concentrations used at durations corresponding to standard cleansing should be effective in inactivating cell-free HIV and HIV-infected cells, including macrophage-tropic HIV-1 strain and suggest that they could act as topical virucidal agents against HIV to prevent its mucosal acquisition through simple hygiene practice.
AIDS vaccines are proving difficult to develop, and it is likely to be many years before effective vaccines, suitable for diverse geographic regions, will be available on a large scale. Thus, new methods of prevention are urgently needed to prevent heterosexual HIV transmission. Our findings suggest that soap and water could be an effective topical microbicide against HIV, especially among men, and a likely candidate for testing on human subjects via a randomized controlled trial.
1. Kampf G, Kramer A. Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs. Clin Microbiol Rev. 2004;17:863-893.
2. Sickbert-Bennett EE, Weber DJ, Gergen-Teague MF, et al. Comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses. Am J Infect Control. 2005;33:67-77.
3. Li JZ, Mack EC, Levy JA. Virucidal efficacy of soap and water against human immunodeficiency virus in genital secretions. Antimicrob Agents Chemother. 2003;47:3321-3322.
4. Aranda-Anzaldo A, Viza D, Busnel RG. Chemical inactivation of human immunodeficiency virus in vitro. J Virol Methods. 1992;37:71-81.
5. Margolis L, Shattock R. Selective transmission of CCR5-utilizing HIV-1: the ‘gatekeeper’ problem resolved? Nat Rev Microbiol. 2006;4:312-317.
6. Hladik F, Sakchalathorn P, Ballweber L, et al. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity. 2007;26:257-270.
7. Saïdi H, Nasreddine N, Jenabian MA, et al. Differential in vitro inhibitory activity against HIV-1 of alpha-(1-3)- and alpha-(1-6)-D-mannose specific plant lectins: implication for microbicide development. J Transl Med. 2007;5:28.
8. Saïdi H, Eslahpazir J, Carbonneil C, et al. Differential modulation of human lactoferrin activity against both R5 and X4-HIV-1 adsorption on epithelial cells and dendritic cells by natural antibodies. J Immunol. 2006;177:5540-5549.
9. Saïdi H, Jenabian MA, Gombert B, et al. Pre-clinical development as microbicide of zinc tetra-ascorbo-camphorate, a novel terpenoid derivative: potent in vitro inhibitory activity against both R5- and X4-tropic HIV-1 strains without significant in vivo mucosal toxicity. AIDS Res Ther. 2008;5:10.
10. Zhu T, Wang N, Carr A, et al. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol. 1996;70:3098-3107.
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