|
5th International AIDS Society Conference on HIV Pathogenesis and TreatmentCape Town - July 19 - 22, 2009 |
IDENTIFICATION OF ARCHIVAL VIRUS FROM LATER-STAGE SAMPLES USING ULTRA-DEEP SEQUENCING
IAS Conf HIV Pathog Treat 2009 Jul 19-22;5th: Abstract No. MOPDA101
D.M. Dudley
1, M.L. Budde2, S.L. O'Connor1, B.N. Bimber2, R.W. Wiseman3, D.H. O'Connor1,3
1University of Wisconsin-Madison, Pathology and Laboratory Medicine, Madison, United States, 2University of Wisconsin-Madison, Cellular and Molecular Biology, Madison, United States, 3University of Wisconsin-Madison, Wisconsin National Primate Research Center, Madison, United States
BACKGROUND: Early immune responses that target HIV remain elusive but important to guide vaccine development. Understanding what immune responses present a selection pressure on the virus robust enough to cause the first escape mutations will provide insight into what responses may be most effective in a vaccine. However, acute stage HIV samples are difficult to obtain. We are developing a method to sequence known archival SIV from rhesus macaque samples collected at later-stage disease. We intend to apply this method to human clinical samples to identify early archival HIV.
METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from Mamu-A*01+ macaques infected with SIVmac239 for more than two years. Genomic DNA containing provirus was isolated from bulk PBMCs, CD4+ T-cells, or CD4 + resting T-cells. The Tat SL8 epitope previously found to escape by 3 weeks post-infection in Mamu-A*01+ macaques was PCR amplified from all samples, purified, and subjected to either ultra-deep sequencing using a Genome Sequencer FLX or cloning and sequencing by the Sanger method.
RESULTS: We were unable to identify archival virus containing a wildtype (pre-escape) Tat SL8 epitope in up to 96 clones from any PBMC subset from multiple animals using Sanger sequencing. As expected, plasma contained only mutant epitope. On the other hand, wildtype epitopes were found at detectable levels using ultra-deep sequencing from a 4-year post-infection time point from bulk PBMC and CD4+ resting T-cells.
CONCLUSIONS: We have developed a method utilizing ultra-deep sequencing of subsets of PBMCs to identify early archival virus. We are currently using this method with clinical human samples to study early CTL escape in HIV. Identification of HIV escape mutants from a presumably strong early CTL response may inform which CTL responses to elicit in an effective HIV vaccine. This method will also hopefully provide viral sequence information when acute-stage samples are unavailable.
Download PDF of this abstract.
2009-07-22
MOPDA101
Poster Discussion: MOPDA1 - HLA Pathogen Interactions
Copyright © 2009 - International AIDS Society (IAS). All information and content relating to the abstracts from the 5th International AIDS Society Conference on HIV Pathogenesis and Treatment, such as text, graphics, logos, button icons, images, audio clips, and software is protected by copyright. Permission is hereby granted for the non-commercial use or reproduction of the information on this web site, provided that the use of such information is accompanied by an acknowledgement that IAS is the source of the information and the name of the author of the article.
AEGiS is a 501(c)3, not-for-profit, tax-exempt, educational corporation. AEGiS is made possible through unrestricted funding from the Elton John AIDS Foundation, National Library of Medicine, AIDS Walk of Orange County, and donations from users like you. Always watch for outdated information. This article first appeared in 2009. This material is designed to support, not replace, the relationship that exists between you and your doctor.
AEGiS presents published material, reprinted with permission and neither endorses nor opposes any material. All information contained on this website, including information relating to health conditions, products, and treatments, is for informational purposes only. It is often presented in summary or aggregate form. It is not meant to be a substitute for the advice provided by your own physician or other medical professionals. Always discuss treatment options with a doctor who specializes in treating HIV.
Copyright ©1980, 2009. AEGiS. All materials appearing on AEGiS are protected by copyright as a collective work or compilation under U.S. copyright and other laws and are the property of AEGiS, or the party credited as the provider of the content. Permission is hereby granted for the non-commercial use or reproduction of the information herein, provided that the use of such information is accompanied by an acknowledgement that IAS is the source of the information and the name of the author of the article.