HIV Tropism - Genotypic Testing Methods As A Screen For Co-Receptor Antagonist Therapy
The identification of HIV tropism has led to the introduction of a new class of HIV drugs – co-receptor antagonists. Dr. Richard Harrigan, Director of the Research Laboratories at the British Columbia Centre for Excellence in HIV/AIDS, discusses the nature of HIV tropism and how tropism screening provides a basis for this new therapeutic option.
Understanding HIV Tropism
Viral tropism is the ability of viruses to enter and infect specific host cells and is based on the ability of viruses to bind to receptors on those cells. The HIV-1 virus uses the CD4 cell receptor, together with a co-receptor, to enter a cell. One form of the HIV virus (“R5” virus) uses a co-receptor called CCR5 to enter cells, while another kind (“X4” virus) uses a co-receptor CXCR4 for entry. There are also dual/mixed-tropic viruses that utilize both co-receptors.1
HIV tropism tends to reflect the stage of a patient’s infection. As Dr. Harrigan explains, “It seems that early in infection most people harbor the R5 virus, but over time the proportion of people who harbor detectable amounts of the X4 virus increases. By the time CD4s have declined with HIV disease progression, the proportion of people with X4 virus goes up to about 50% or more.”
The Basis For A New Therapy
A new class of drugs, CCR5 co-receptor antagonists, have been developed, which work by binding to the CCR5 receptor and preventing HIV from entering the cell. Maraviroc is currently the only FDA approved agent in this class.2 Identifying a patient’s viral tropism is critical to determine whether this drug will be effective. “If you’ve got X4 virus, the CCR5 antagonists don't work,” notes Dr. Harrigan, “so we need to screen whether you’ve got detectable R5 virus only. If that’s the case you would expect the maraviroc to work.”
The DHSS guidelines for the use of antiretroviral agents recommend that co-receptor tropism assays are used whenever the use of a CCR5 inhibitor is being considered.3
Screening For HIV Tropism
The original test used to identify tropism was a phenotypic test. A version of this test, the Trofile assay, was the method used to screen for the CCR5 antagonist clinical trials. “In this method,” explains Dr. Harrigan, “a piece of the virus is amplified by polymerase chain reaction (PCR), put into a different backbone and grown up on one of two cell lines, either one that expresses CCR5 or one that expresses CXCR4. A light signal then indicates which cell type is being infected.” A more advanced version of this test was subsequently developed, which is called enhanced sensitive tropism assay or ESTA.
In discussing this assay Dr. Harrigan notes, “It’s a fine assay, though it does have some practical drawbacks as a clinical test. From our viewpoint the limitations were that it’s rather expensive to perform and it takes several weeks to get the results back. Additionally, the test is also only available in the U.S.”
An Alternative Method For Screening - Genotypic Testing
To address the shortcomings of the phenotypic test researchers investigated other approaches for tropism testing. “The one that made the most sense is called genotypic testing,” he says, “This is based on knowledge of associations between variations in the virus sequence and what they expect the phenotype to be. Advantages of this method are that it’s faster, it’s cheaper and we can exploit new developments in DNA sequencing without having to invent everything ourselves. The genotypic testing approach is quite analogous to how a lot of HIV resistance tests are performed, so the methodology tends to be available everywhere.”
Accuracy of the genotypic test is consistent with that of the phenotypic assay. “When we conducted retrospective analyses of the clinical trials of maraviroc it was shown that the ability of the genotypic test to predict clinical response to that drug is as good as the original Trofile assay4, using standard sequencing methods” observes Dr. Harrigan.
Use of genotypic testing has become widespread outside the United States. European guidelines recently issued for tropism testing indicate the use of either genotypic or phenotypic testing, with the expectation that greater access to the genotypic test, combined with its lower cost and faster turnaround time, will support its adoption in European clinical practice.5
Interpretation Of Genotypic Testing Results
A genotypic test generates a string of DNA bases, which are most commonly interpreted using a methodology known as “genotopheno”, an algorithm, which indicates the likelihood of a patient’s tropism based on their DNA sequence. “Essentially it takes this string of 105 bases,” explains Dr. Harrigan “and converts it to a number. This reflects the likelihood that the sample is actually an X4 virus - the smaller the number the more likely that it’s X4.” A result of “X4 not detected” is consistent with eligibility for treatment with a CCR5 antagonist, while alternative therapy should be considered if the test result is “X4 detected.”
When To Screen
A patient is screened for HIV tropism when being considered for a CCR5 antagonist drug. “Because your virus can evolve over time, the ideal time to screen is just before going on the drug,” says Dr. Harrigan. “The intent is that after going onto the drug the HIV levels measured for tropism should essentially disappear – your HIV should become undetectable and you should never, in an ideal world, see that virus again.”
The Role Of Deep Sequencing
Genotypic testing also provides a basis for DNA sequencing approaches used to monitor HIV therapy, both in tropism and in HIV drug resistance. Dr Harrigan points out that these deep sequencing methods allow you to look at an individual’s virus population in a completely new way: “Instead of just getting the overall picture of the most common virus variant, or the most common virus variants, you get a detailed breakdown of every single virus variant within that population. It’s equivalent to having an election and instead of just seeing the result, you see every single vote in that election. It increases the sensitivity, enabling you to detect minority species at a lower level.”
This approach may help direct therapy more effectively. “There is a concern that sequencing methods used for resistance and for tropism might miss clinically significant minor variants that exist within the HIV population,” says Dr. Harrigan. “The deep sequencing methodology allows us to identify those people, who have somewhere between 2% of an X4 virus population and 25% or 50% of that virus population. So in a minority of people, the deep sequencing approach offers enhanced sensitivity.”
Dr. Harrigan expects that in the coming years there will be an increasing use of deep sequencing methods for monitoring all the different HIV genes in a single test, while simultaneously being able to provide some human genetic predictors of virological response.
Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Berger EA et al. Annu Rev Imunol. 1999;17:657-700.
Approval of Maraviroc, CCR-5 co-receptor antagonist for treatment of HIV. U.S. Food and Drug Administration. Aug 6, 2007.
Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. Department of Health and Human Services. October 14, 2011; 1–167. Available at
http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. Accessed 12/19/11
Genotypic tropism testing: evidence-based or leap of faith? P. Richard Harriganand Anna Maria Gerettib AIDS 2011, 25:257–264
European guidelines on the clinical management of HIV-1 tropism testing. L P R Vandekerckhove et al. The Lancet Published Online March 22, 2011 DOI:10.1016/S1473- 3099(10)70319-4