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One of the tricky parts of HIV drug therapy is determining how well the drugs have worked. The HIV DNA (provirus) in resting cells is usually too defective to replicate itself the way intact provirus can. But most current tests cannot tell the difference between the two. However, researchers from Johns Hopkins University School of Medicine in Baltimore have developed an accurate and scalable assay to easily count the cells in the HIV reservoir.
A stable, latent reservoir for HIV-1 in resting CD4+ T cells is “the principle barrier to a cure,” the researchers say. Quantitative outgrowth assays and assays for cells that produce viral RNA after T-cell activation may underestimate the reservoir size because 1 round of activation does not induce all proviruses. Many studies, the researchers say, rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of those assays is unclear since the vast majority of proviruses are defective.
In their study, supported by the National Institute of Allergy and Infectious Diseases, the researchers analyzed DNA sequences from > 400 HIV proviruses from 28 people with HIV. They mapped 2 types of flaws: deletions and lethal mutations. They then developed strategically placed “genetic probes” that could distinguish between deleted or highly mutated proviruses and intact ones. Finally, they developed a nanotechnology-based method to analyze 1 provirus at a time to determine how many in a sample are intact.
The researchers say their findings show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. Their hope is that their method will speed HIV research by allowing scientists to easily quantify the number of proviruses in an individual, which must be eliminated to achieve a cure.
One of the tricky parts of HIV drug therapy is determining how well the drugs have worked. The HIV DNA (provirus) in resting cells is usually too defective to replicate itself the way intact provirus can. But most current tests cannot tell the difference between the two. However, researchers from Johns Hopkins University School of Medicine in Baltimore have developed an accurate and scalable assay to easily count the cells in the HIV reservoir.
A stable, latent reservoir for HIV-1 in resting CD4+ T cells is “the principle barrier to a cure,” the researchers say. Quantitative outgrowth assays and assays for cells that produce viral RNA after T-cell activation may underestimate the reservoir size because 1 round of activation does not induce all proviruses. Many studies, the researchers say, rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of those assays is unclear since the vast majority of proviruses are defective.
In their study, supported by the National Institute of Allergy and Infectious Diseases, the researchers analyzed DNA sequences from > 400 HIV proviruses from 28 people with HIV. They mapped 2 types of flaws: deletions and lethal mutations. They then developed strategically placed “genetic probes” that could distinguish between deleted or highly mutated proviruses and intact ones. Finally, they developed a nanotechnology-based method to analyze 1 provirus at a time to determine how many in a sample are intact.
The researchers say their findings show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. Their hope is that their method will speed HIV research by allowing scientists to easily quantify the number of proviruses in an individual, which must be eliminated to achieve a cure.
One of the tricky parts of HIV drug therapy is determining how well the drugs have worked. The HIV DNA (provirus) in resting cells is usually too defective to replicate itself the way intact provirus can. But most current tests cannot tell the difference between the two. However, researchers from Johns Hopkins University School of Medicine in Baltimore have developed an accurate and scalable assay to easily count the cells in the HIV reservoir.
A stable, latent reservoir for HIV-1 in resting CD4+ T cells is “the principle barrier to a cure,” the researchers say. Quantitative outgrowth assays and assays for cells that produce viral RNA after T-cell activation may underestimate the reservoir size because 1 round of activation does not induce all proviruses. Many studies, the researchers say, rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of those assays is unclear since the vast majority of proviruses are defective.
In their study, supported by the National Institute of Allergy and Infectious Diseases, the researchers analyzed DNA sequences from > 400 HIV proviruses from 28 people with HIV. They mapped 2 types of flaws: deletions and lethal mutations. They then developed strategically placed “genetic probes” that could distinguish between deleted or highly mutated proviruses and intact ones. Finally, they developed a nanotechnology-based method to analyze 1 provirus at a time to determine how many in a sample are intact.
The researchers say their findings show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. Their hope is that their method will speed HIV research by allowing scientists to easily quantify the number of proviruses in an individual, which must be eliminated to achieve a cure.