4th International AIDS Society Conference on HIV Pathogenesis and Treatment Sydney, Australia - July 22 - 25, 2007

FLUORESCENT DETECTION OF INDIVIDUAL HIV VIRIONS WITHIN GENITAL TISSUES

IAS Conf HIV Pathog Treat 2007 Jul 22-25;4th: Abstract No. MOPDA05

McCoombe S.G., Hope T.J.
Northwestern University, Cell and Molecular Biology, Chicago, United States

OBJECTIVES: Sexual transmission accounts for over 80% of all HIV infections. How HIV breaches the genital epithelia of men and women remains poorly defined. Previous attempts to observe HIV interactions in tissue explant and primate models have been hindered by high background. To study single virion interactions with genital tissues, we developed a new technology utilising photoactivatable GFP (PA-GFP) labeled HIV and fluorescent deconvolution microscopy. Using this system, tissue autofluorescence is defined, the sample is activated and newly detected signal is labeled HIV.

METHODS: Modifying existing labeling techniques, HIV virions were produced containing PA-GFP labeled Vpr. Photoactivation of PA-GFP is achieved by exposure to 400-430nm light. Before photoactivation, PA-GFP fluorescence is minimal. After photoactivation, PA-GFP fluoresces like regular GFP and is distinguishable from background. To study initial HIV interactions with the genitalia, we incubated human foreskin and cervical explants for 4-24 hours with PA-GFP labeled HIV. These experiments were replicated in the rhesus macaque non-human primate model. Following inoculation, genital samples were frozen, sectioned and stained, enabling observation of tissue structure, HIV target cells and PA-GFP labeled virions.

RESULTS: Within 4 hours, HIV penetrates the superficial squamous epithelia of the ectocervix. Penetrating virions are observed between differentiated squamous epithelial cells. We also observe HIV penetration of the columnar endocervical epithelium with virions observed in the sub-basal milieu. Penetrating virions regularly co-localise with both Langerhans cells and surface CD4 T cells. In cervical explants, mucus harbors numerous virions, but when present reduces the incidence of epithelial penetration. Complimentary results were obtained using the rhesus macaque model.

CONCLUSIONS: Understanding the initial events of HIV sexual transmission is essential for the development of new preventative strategies. Observing early HIV interactions in genital tissues both in vitro and in vivo has provided important insight into HIV transmission across epithelial barriers, and highlights the importance of local target cells.

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2007-07-22
MOPDA05
Models for Mucosal Immunity and Transmission

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