Monday, 10 July 2017

HIV Reservoirs: Does The Virus Hide in the Brain?

Treatment advances over the last several decades have dramatically extended the lifespan of patients with human immunodeficiency virus (HIV). However, a continuing problem in HIV treatment is the ability of the virus to remain dormant in a small number of cells throughout the body, including some areas of the brain. Researchers at the University of Montreal in Canada recently discovered a way to locate and destroy hidden HIV viral particles, a finding that could eventually lead to a complete cure for the disease.

HIV currently affects an estimated 36.7 million people worldwide, and infects 2.1 million new individuals each year. HIV infection targets a group of immune cells called CD4+ T cells. These cells are responsible for recognizing and attacking harmful bacteria and viruses that enter the body. When a large proportion of these cells are destroyed by HIV, the body can no longer defend against infections and other diseases. This stage is called acquired immunodeficiency syndrome, or AIDS, and frequently results in premature death due to opportunistic infections and cancer.

The present treatments for HIV include reverse transcriptase inhibitors and protease inhibitors, among several others. These drugs work by targeting the enzymes that convert viral RNA into DNA and produce mature viral particles. These drugs are highly effective and have drastically increased the life expectancy of patients. However, a complete cure for HIV has remained elusive because of the ability of the virus to remain in so-called reservoirs in the body. If a patient in remission forgoes their medication, the virus can become re-activated.

Researchers from several universities, led by Daniel Kauffman of the University of Montreal, recently discovered a way to identify and potentially destroy HIV reservoirs. Their findings are published in the journal Cell Host and Microbe.

First, the researchers sought a new way to identify HIV-infected CD4+ T cells using a method called flow-FISH, which combines two techniques for identifying proteins and nucleotides in cells. Flow cytometry uses a machine to sort and identify cell subpopulations based on surface protein markers, but it is not sensitive or specific enough to identify HIV-infected cells. Fluorescence in situ hybridization, or FISH, uses fluorescent probes directed against specific nucleotide sequences to identify DNA or RNA in a cell with local and temporal specificity. Flow-FISH was used to detect the mRNA of two specific HIV genes in CD4+ T cells, gag and pol.

Using this method, the researchers were able to detect virus-containing cells at concentrations as low as 0.5–1 per million CD4+ T cells. This assay was combined with polychromatic flow cytometry, which analyzes multiple cell surface markers simultaneously. Together, these techniques allowed the researchers to quantify and identify the characteristics of HIV-infected cells in patient blood samples. HIV-infected CD4+ T-cells showed an increase in markers of “immune exhaustion” (PD-1, CTLA-4, and TIGIT), which occurs when T cells have been chronically activated and are no longer responsive. They also were able to estimate the size of the viral reservoir, which was on average 3.56 cells per million CD4+ T cells, but a wide variation was observed among patients on anti-viral therapy.

In the next part of the study, two latency-reversal agents (LRA) were tested on cells from HIV-infected patients. LRAs are used in a “shock and kill” technique that activates dormant viral particles in the body, allowing them to be eradicated by antiretroviral agents. Bryostatin modulates protein kinase C, an enzyme involved in cell signaling pathways, and is currently being tested for use as an anti-cancer, anti-HIV, and memory-enhancing agent. Ingenol is a plant-derived drug with several proposed mechanism of action that include protein kinase C activation, which leads to the activation of immune cells.

The researchers determined that bryostatin and ingenol differed in their ability to activate two subpopulations of CD4+ T cells. Ingenol activated central memory cells, which persist for several years in patients, whereas bryostatin was not as effective against this T-cell subpopulation. This finding suggested that ingenol may be a more effective treatment for latent HIV. While both drugs were capable of killing the HIV virus in vitro, their effectiveness in the body remains unclear. The researchers suggest that their viral detection method will be useful for assessing the effectiveness of LRAs in clinical trials.

This method of reactivating latent HIV provides hope for one day eradicating the virus, but it is not without the potential for side effects. Researchers at Johns Hopkins University recently tested the effect of viral reactivation by LRAs in monkeys with simian immunodeficiency virus (SIV), the correlate of HIV in monkeys. They found that the drug caused a harmful form of brain inflammation. The researchers suspect that this side effects was caused by the existence of HIV reservoirs in the brain.

SIV-infected monkeys were treated with a cocktail of antiretroviral agents (tenofovir, darunavir, ritonavir, and the integrase inhibitor L-870812). Following 400 days on this regimen, viral suppression was confirmed by measuring SIV in plasma. Then two of the monkeys were treated for ten days with the latency-reversing agents ingenol-B and vorinostat. Vorinostat is a histone deacetylase inhibitor that works by changing the expression of genes involved in cell differentiation. The animals continued on the antiretroviral drug cocktail during the treatment with LRAs.

One of the treated monkeys remained healthy under this treatment regimen. However, the second monkey developed symptoms of brain inflammation, including lethargy and lack of appetite. Active SIV and markers of inflammation were detected in the cerebrospinal fluid (CSF), the fluid that surrounds the brain and spinal cord. After the animal was euthanized due to the illness, they removed the brain for examination. They were careful to first remove the monkey’s blood to ensure that the brain tissue wasn’t contaminated by infected blood cells. They found that SIV was present in a part of the brain called the occipital cortex.

The discovery that viral reactivation can cause brain inflammation, possibly by reactivating brain reservoirs of the virus, is a potential drawback for using LRAs to treat latent HIV in humans. Reassuringly, this side effect occurred in only one of the two animals, so these findings are still preliminary. Further, while there are innate differences between SIV and HIV, clinical trials of LRA in humans with HIV must be carried out with caution. Monitoring the CSF for evidence of viral reactivation in the brain should accompany LRA treatment to prevent such side effects.

References

Lugli E., Troiano L, Cossarizza A. (2009) Investigating T cells by polychromatic flow cytometry. Methods Mol Biol. 514:47-63. doi: 10.1007/978-1-60327-527-9_5.

Wherry, J.E. and Kurachi M. (2015) Molecular and cellular insights into T cell exhaustion. Nature Reviews Immunology 15:486–499. doi: 10.1038/nri3862

Lorenzo-Redondo R., Fryer H.R., Bedford T., Kim E.Y., Archer J., Kosakovsky Pond S.L., et al. (2016) Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature 530:51–56. doi: 10.1038/nature16933

Gama L., Abreu C.M., Shirk E.N., Price S.L., Li M., Laird G.M. et al. (2017) Reactivation of simian immunodeficiency virus reservoirs in the brain of virally suppressed macaques. AIDS. Jan 2;31(1):5-14. DOI: 10.1097/QAD.0000000000001267

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