A 454 Sequencing Study Reveals New Insights in Drug Resistance and Tropism Associated with the New HIV Integrase and Entry Inhibitor Therapy Classes
Research study results may have potential implications for therapy selection in HIV patients in the future
The presentations at CROI outline research carried out by scientists from the University of Rome "Tor Vergata" and Virco BVBA, in the latest in a series of joint studies performed at Virco's laboratory to analyze and interpret data generated by sequencing HIV-infected research samples with the Genome Sequencer FLX System. The studies explore resistance to a new class of drug called integrase inhibitors, as well as an in-depth evaluation of viral tropism. The ultra-deep method enabled by 454 Sequencing systems offers dramatically improved sensitivity in detecting low-frequency viral variants, also known as viral quasispecies, compared to traditional population based sequencing.
The first study analyzed long-term antiretroviral users receiving a combination therapy of an integrase-inhibitor, raltegravir, and optimized backbone therapy. The investigators analyzed samples at multiple time-points using both traditional Sanger genotyping, in vitro phenotyping and the ultra-deep sequencing method. The researchers found, at levels far below normal detectability, an abundance of viral quasispecies in patients whose treatment failed, in contrast to a far lower number of quasispecies found among patients whose treatment was succesful. They hypothesized that the co-presence of many different strains may favor the development of drug resistance and consequently drug failure.
Furthermore, the researchers suggest that the correlation between quasispecies identified at baseline and treatment failure may be occurring via pathways which are much more complex than simple drug-driven selection of the primary mutations currently identified. "The ability of the technology of the 454 Sequencing System to identify mutants at very low levels provides new insights on how HIV may develop resistance to new drugs such as Integrase inhibitors," said Professor Perno from Tor Vergata University. His colleague Dr. Ceccherini-Silberstein added, "This technology will compliment existing techniques and can provide a significant added value to HIV research."
In the second study, the researchers analyzed isolates from HIV-infected patients to characterize viral tropism by both traditional phenotyping and genotyping, as well as deep sequencing analysis. HIV tropism refers to the preference of a patient's particular HIV virus to select one of two co-receptors for entry into the patient's CD4 immune cell, where the virus reproduces. Determining the co-receptor that a HIV strain uses, either CCR5, CXCR4 or a combination of both, is a critical component of monitoring and treating HIV. Dual-tropic virus types have the ability to use either or both co-receptors for cell entry. In the research study, ten randomly-selected dual-tropic isolates were analyzed by V3 deep sequencing analysis with the 454 Sequencing System, and tested for research purposes in human immune cells for sensitivity to therapies, including the CCR5 antagonist maraviroc and the CXCR4 antagonist AMD3100.
From these research samples, they found that dual tropic viruses are mostly characterized as quasispecies with potential to use both R5 and CXCR4 receptors as opposed to quasispecies with mixtures of R5 and X4 using viruses, a new insight into how the HIV virus adapts in choosing a co-receptor to enter human target cells CD4 cells. "This analysis helps develop better understanding of the complex mechanisms of HIV co-receptor tropism," said Dr. Lieven Stuyver, Vice President R&D at VIRCO BVBA. "We believe that the use of current technologies such as phenotyping, combined with the newer tools such as ultra-deep sequencing with the Genome Sequencer FLX System, linked to clinical outcome data may allow us to unravel these complex viral mechanisms and ultimately could lead to better HIV treatment in future."
"These studies continue to demonstrate the utility of the 454 Sequencing System for research on viral diseases and their potential treatment," explains Christopher McLeod, President and CEO of 454 Life Sciences. "The development of novel therapies, such as the new classes of HIV drugs, often requires companion tools for proper selection of suitable treatment regimens for patient candidates. We believe that that the sensitivity, accuracy, and speed of the 454 Sequencing System will transform the way HIV is monitored and treated in the near future."
454 Life Sciences, a center of excellence of Roche Applied Science, develops and commercializes the innovative 454 Sequencing System for ultra-high-throughput DNA sequencing. Specific applications include de novo sequencing and re-sequencing of genomes, metagenomics, RNA analysis, and targeted sequencing of DNA regions of interest. The hallmarks of the 454 Sequencing System are its simple, unbiased sample preparation and long, highly accurate sequence reads, including paired-end reads. The technology of the 454 Sequencing System has enabled hundreds of peer-reviewed studies in diverse research fields, such as cancer and infectious disease research, drug discovery, marine biology, anthropology, paleontology and many more.
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Headquartered in Basel, Switzerland, Roche is one of the world's leading research-focused healthcare groups in the fields of pharmaceuticals and diagnostics. As the world's biggest biotech company and an innovator of products and services for the early detection, prevention, diagnosis and treatment of diseases, the Group contributes on a broad range of fronts to improving people's health and quality of life. Roche is the world leader in in-vitro diagnostics and drugs for cancer and transplantation, and is a market leader in virology. It is also active in other major therapeutic areas such as autoimmune diseases, inflammatory and metabolic disorders and diseases of the central nervous system. In 2008 sales by the Pharmaceuticals Division totalled 36.0 billion Swiss francs, and the Diagnostics Division posted sales of 9.7 billion francs. Roche has R&D agreements and strategic alliances with numerous partners, including majority ownership interests in Genentech and Chugai, and invested nearly 9 billion Swiss francs in R&D in 2008. Worldwide, the Group employs about 80,000 people. Additional information is available on the Internet at www.roche.com.
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