Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Kateena Addae Konadu; Ming Bo Huang; William Roth; Wendy Armstrong; Michael Powell; Francois Villinger; Vincent Bond

doi:10.3791/53495

Method Article

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

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DOI:

10.3791/53495

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January 5th, 2016

Kateena Addae Konadu¹ , Ming Bo Huang¹ , William Roth¹ , Wendy Armstrong² , Michael Powell¹ , Francois Villinger³^,⁴ , Vincent Bond¹

¹Department of Microbiology, Biochemistry, Immunology, Morehouse School of Medicine, ²Department of Medicine, Emory University School of Medicine, ³Department of Pathology and Laboratory Medicine, Emory University School of Medicine, ⁴Yerkes National Primate Research Center

Summary

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Abstract

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.

Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.

To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Introduction

The HIV-1 epidemic continues to have a significant impact throughout the world. As of 2013, approximately 35 million people worldwide were living with HIV, and 2.1 million of these were newly infected individuals¹. Prevention strategies and increased access to antiretroviral therapy have been helpful in reducing the overall acquisition of HIV. However, individual populations are still experiencing rises in the acquisition of HIV¹. Thus, there is a need for continued efforts to address this epidemic.

One of the strongest predictors of HIV disease progression is chronic immune activation (CIA)^2-10. Defined by ....