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Published February 15, 2017 | Supplemental Material
Journal Article Open

Longitudinal imaging of HIV-1 spread in humanized mice with parallel 3D immunofluorescence and electron tomography


Dissemination of HIV-1 throughout lymphoid tissues leads to systemic virus spread following infection. We combined tissue clearing, 3D-immunofluorescence, and electron tomography (ET) to longitudinally assess early HIV-1 spread in lymphoid tissues in humanized mice. Immunofluorescence revealed peak infection density in gut at 10-12 days post-infection when blood viral loads were low. Human CD4+ T-cells and HIV-1-infected cells localized predominantly to crypts and the lower third of intestinal villi. Free virions and infected cells were not readily detectable by ET at 5-days post-infection, whereas HIV-1-infected cells surrounded by pools of free virions were present in ~10% of intestinal crypts by 10-12 days. ET of spleen revealed thousands of virions released by individual cells and discreet cytoplasmic densities near sites of prolific virus production. These studies highlight the importance of multiscale imaging of HIV-1-infected tissues and are adaptable to other animal models and human patient samples.

Additional Information

© 2017, Kieffer et al. This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited. Received November 15, 2016; Accepted February 13, 2017; Published February 15, 2017. We thank Andres Collazo and the Caltech Biological Imaging Center for use of the Zeiss LSM-710 and 880 confocal microscopes and help with image capture and analysis, Carol Garland and Alasdair McDowall for help maintaining electron microscopes, Viviana Gradinaru and Ben Deverman for advice about tissue clearing methodologies, Wesley Sundquist for a rabbit polyclonal anti-HIV-1 p24 antibody, and Michel Nussenzweig for the pNL4-3envYU2 HIV-1. This work was supported by the National Institutes of Health (2 P50 GM082545-06; WI Sundquist, PI), Ragon Institute and Rosalind W. Alcott post-doctoral fellowships (CK), gifts from the Gordon and Betty Moore Foundation and the Agouron Institute to support electron microscopy at Caltech, and funds provided by The Regents of the University of California, Research Grants Program Office, California HIV/AIDS Research Program, Grant Number ID15-CT-017. The opinions, findings, and conclusions herein are those of the author and not necessarily represent those The Regents of the University of California, or any of its programs. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Competing Interests: The authors declare that they have no competing interests.

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