Infection of Macaques after Vaginal Exposure to Cell-Associated Simian Immunodeficiency Virus

MAJOR ARTICLE Infection of Macaques after Vaginal Exposure to Cell-Associated Simian Immunodeficiency Virus Bettina Sallé,1,2,3 Patricia Brochard,1,2 Olivier Bourry,1,2 Abdelkrim Mannioui,1,2 Thibault Andrieu,1,2 Sophie Prevot,4 Nathalie Dejucq-Rainsford,5 Nathalie Dereuddre-Bosquet,1,2 and Roger Le Grand1,2 Commissariat à L’Energie Atomique (CEA), Division of Immuno-Virology, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses, 2Unité Mixte de Recherche (UMR) E01, Université Paris-Sud 11, Orsay, 3Centre de Primatologie, Centre International de Recherches Médicales de Franceville, Franceville, Gabon, 4Service d’Anatomie et de Cytologie Pathologiques, Hôpital Antoine Béclère, AP-HP, Clamart, 5Institut National de la Santé et de la Recherche Médicale (INSERM) U625-GERHM, Rennes, France. (See the editorial commentary by Anderson, on pages 333–336.) Background. The contribution of infected semen cells to sexual transmission of human immunodeficiency virus (HIV) is still debated. We addressed this issue in the model of experimental infection of macaques with simian immunodeficiency virus (SIV). Methods. Frozen stocks of cells obtained from the spleen of macaques at the peak of SIVmac251 viremia were prepared. After being thawed and washed, cells were deposited at different concentrations in the vaginas of adult macaques treated with medroxyprogesterone acetate (Depo-Provera). To unravel mechanisms of infection, stock cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) were inoculated intravaginally. Followup testing of samples from the mucosa and different lymphoid tissues obtained 21 and 45 h later was performed by flow cytometry, immunohistochemical analysis, and in situ hybridization. Results. Systemic and persistent infection was achieved after vaginal exposure of macaques to SIV-infected cells. The dose needed to infect 50% of females was 6.69 ⫻ 10 5  2.08 ⫻ 10 5 viral DNA copies. At days 1 and 2 after exposure to cell-associated SIV labeled with CFSE, SIV-positive cells were detected in proximal and distal lymphoid tissues. Conclusions. Infection with SIV after exposure of vaginal and cervical mucosa to cell-associated virus represents a new mechanism of sexual transmission of HIV and SIV that may have significant impacts in the development of preventive approaches like microbicides. The development of strategies for the control of the human immunodeficiency virus (HIV) epidemic requires an improved understanding of the mechanism underlying sexual transmission of the virus [1]. Studies of AIDS performed in vitro with reconstituted mucosal barriers or in nonhuman primate models highlight the diversity of mechanisms by which HIV and simian im- Received 21 July 2009; accepted 22 February 2010; electronically published 22 June 2010. Potential conflicts of interest: none reported. Financial support: EMPRO European program (grant LSH-2002-2.3.0-2), the EUROPRISE European Network of Excellence (grant LSHP-CT-2006-037611), the Dormeur Investment Service Ltd, the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS, Paris, France), the “Coopération Française” (Paris, France) and the Commissariat à l’Energie Atomique (CEA, France). Reprints or correspondence: Dr Roger Le Grand, Service d’Immuno-virologie, Institut des maladies émergentes et des thérapies innovantes, CEA, 18, route du Panorama, 92265, Fontenay-aux-Roses, Cedex, France (). The Journal of Infectious Diseases 2010; 202(3):337–344  2010 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2010/20203-0002$15.00 DOI: 10.1086/653619 munodeficiency virus (SIV) virions may cross the cervico-vaginal or rectal mucosa and disseminate into the host to establish persistent infection [2]. Many studies have emphasized the role played by infectious viral particles in contaminating fluids. However, genital fluids, especially semen, may also contain significant amounts of infected cells that may contribute to transmission of HIV infection. Semen of untreated patients contains high levels of cell-free HIV particles and significant amounts of HIV-infected cells [3]. HIV DNA is present in the leukocyte cell fractions of semen in 4%–65% of HIV-infected patients, at levels of !10–80,000 viral DNA copies/mL [3–5], which may represent high numbers of infected cells assuming that infected cells contains few [1–3] copies of integrated DNA. In addition, leukocytes in semen may also actively replicate the virus because intracellular HIV RNA (27,000–70,000 copies/ mL) could be detected in significant amounts [4]. Provirus load may significantly increase during inflammation of the genital tract [5] in individuals with ureCell-Associated SIV Transmission in Macaques • JID 2010:202 (1 August) • 337 1 MATERIALS AND METHODS Ethics statement. The studies we described used nonhuman primate models of HIV and AIDS in accordance with European Union guidelines for animal care (European Union Directive 86/609/EEC), and protocols were approved by the Ethical Committee “Ile-De-France SUD,” as requested by our Institution (CEA). Animals and cell-associated virus stocks. Fourteen adult female cynomolgus macaques (Macaca fascicularis), each weighing between 4 and 5 kg and imported from Mauritius, were maintained and handled in accordance with European guidelines for animal care (Directive 86/609/EEC). Animals were anesthetized with 10 mg/kg of ketamin (Imalgene). Cells were isolated, using Ficoll, from the spleen of 2 adult female cynomolgus macaques (K833 and X801) at day 12 after infection with SIVmac251 strain (provided by A.-M. Aubertin, Strasbourg, France). Spleen cell stocks were then frozen in 10% dimethyl sulfoxide (DMSO). Stocks K833 and X801 contained 107 spleen cells per ampoule. Cynomolgus macaque females were treated with 30 mg/kg of medroxyprogesterone acetate (Depo-Provera) and then inoculated 30 days later by the vaginal route. Stock cells were thawed 1 h before challenge and washed twice before being suspended in 1 mL Roswell Park Memorial Institute 1640 medium (RPMI 1640) for administration to macaques. Before inoculation, the lower female reproductive tract was inspected for signs of preexisting inflammation. A lubricated nasogastric tube was used (Centravet) for the inoculation of cells in the vaginal vault. Animals were then kept in prone position for 5 min. Twelve animals were followed up over 6 months after inoculation with infected cells; 2 macaques were killed at days 1 and 2 after inoculation. Carboxyfluorescein succinimidyl ester labeling of stock cells. Spleen cells from stock K833 were labeled with 5 mmol/L of carboxyfluorescein diacetate succinimidyl ester (CFSE), using 338 • JID 2010:202 (1 August) • Sallé et al the CellTrace TM CFSE Proliferation Kit (C34554 Molecular probes; Invitrogen) as described elsewhere [3]. Plasma viral load. Viral RNA was prepared from 200 mL of cell-free plasma or supernata (...truncated)