Researchers identify lower GI tract tissues as source of initial rebound of HIV-like virus after stopping ART

Antiretroviral treatment (ART) can suppress HIV to the point it is undetectable in the blood. However, when ART is stopped, even after decades of treatment, the virus reignites and the infection progresses.

This persistent “rebound competent viral reservoir” is where dormant, virus-infected cells lay in wait. Pinpointing where in the body the virus begins its comeback has long been a challenge for researchers.

In a new study using a monkey model of human HIV infection, scientists in the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research and collaborators used an innovative method to track in unprecedented detail the anatomic origin of this viral rebound.

They used the technique to show that after antiretroviral treatment is stopped, the virus rebound originates in the lower gastrointestinal tract and associated lymph nodes. These results provide scientists with a target for potential treatments to prevent or control HIV spread.

The study, reported in the journal Nature Microbiology, used simian immunodeficiency virus (SIV) infection of rhesus macaques, a well-established experimental model that mimics key aspects of HIV infection in humans.

Barcode model helps locate site of virus rebound

The study employed a barcoded virus model developed by AIDS and Cancer Virus Program Senior Principal Scientist and study first author Brandon Keele.

Twenty-four rhesus macaques were infected with a virus stock containing thousands of SIV variants that were identical, except for distinct, unique genetic barcodes to allow detailed tracking of each variant by viral sequence analysis, including identification of anatomic sites where different variants were present. After infection, the animals began ART that was continued for more than a year.

In the final phase of the study, some of the animals were humanely euthanized in accordance with approved standards while still receiving antiretroviral treatment. The scientists analyzed these tissue samples for levels of SIV DNA and SIV RNA and sequencing of barcodes, providing a baseline level of viral expression when ART was on board and suppressing viral replication.

The remaining animals were humanely euthanized at five days or seven days after stopping ART, times calculated to capture the earliest stages of viral rebound.

Taina Immonen, head of the AIDS and Cancer Virus Program Computational Virology Core, analyzed and interpreted the data from more than 2,200 tissue samples taken from 60 different major and minor sites known to harbor residual virus while on ART. SIV DNA was found in tissues throughout the body.

Her analysis of tissues sampled after discontinuation of ART identified rare barcodes in tissue sites designated “outliers,” with levels of SIV RNA for these individual barcodes greater than the level expected for the corresponding level of individual barcode SIV DNA while on ART.

These outlier sites showed a higher ratio of SIV RNA to SIV DNA, indicating the virus was replicating and spreading. In a compelling illustration of the power of the barcoded virus approach, some of these rare tissue outlier barcodes were also shown to be present in the earliest detectable rebound virus in the blood, implicating them as the likely source of viral rebound.

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Data points to GI tract as source of virus rebound

Tissues from the lower GI tract and associated lymph nodes were found to be the predominant tissues harboring “outlier” barcodes and therefore the major presumptive origin sites of the rebound competent viral reservoir. Immonen, a computational scientist, said she did not anticipate this finding.

“This was a massive team effort that created a truly one-of-a-kind data set,” Immonen said. “The barcoding let us find needles in a haystack, and what was striking was how clearly the GI signal came through despite all that biological complexity.”

Jeff Lifson, director of the AIDS and Cancer Virus Program, said he, too did not expect to see such clearcut evidence identifying tissues in the GI tract as the earliest tissue origin for viral rebound. It was an important discovery that sets the stage for refining treatment approaches targeting the rebound competent viral reservoir.

“Use of the barcoded virus approach enabled us to characterize the biology of the rebound competent viral reservoir and the critical initial stages of post-treatment viral rebound with unprecedented sophistication and detail and is the type of work that is only possible in a nonhuman primate model,” Lifson said.

Some of the tissues collected in this study will be used to characterize the viral, immunologic and tissue microenvironmental factors that lead some tissue sites to be the source of the viral comeback after ART is stopped, while others do not.

This should help guide future therapeutic approaches intended to stop the reignition and spread of the virus in the absence of ongoing ART.

Study collaborators include researchers from the Oregon National Primate Research Center, Oregon Health and Sciences University; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School; and Ragon Institute of MGH, MIT and Harvard.