Evaluating the Intactness of Persistent Viral Genomes in Simian Immunodeficiency Virus-Infected Rhesus Macaques after Initiating Antiretroviral Therapy within One Year of Infection

Abstract

The major obstacle to more-definitive treatment for HIV infection is the early establishment of virus that persists despite long-term combination antiretroviral therapy (cART) and can cause recrudescent viremia if cART is interrupted. Previous studies of HIV DNA that persists despite cART indicated that only a small fraction of persistent viral sequences was intact. Experimental simian immunodeficiency virus (SIV) infections of nonhuman primates (NHPs) are essential models for testing interventions designed to reduce the viral reservoir. We studied the viral genomic integrity of virus that persists during cART under conditions typical of many NHP reservoir studies, specifically with cART started within 1 year postinfection and continued for at least 9 months. The fraction of persistent DNA in SIV-infected NHPs starting cART during acute or chronic infection was assessed with a multiamplicon, real-time PCR assay designed to analyze locations that are regularly spaced across the viral genome to maximize coverage (collectively referred to as “tile assay”) combined with near-full-length (nFL) single-genome sequencing. The tile assay is used to rapidly screen for major deletions, with nFL sequence analysis used to identify additional potentially inactivating mutations. Peripheral blood mononuclear cells (PBMC) from animals started on cART within 1 month of infection, sampled at least 9 months after cART initiation, contained at least 80% intact genomes, whereas those from animals started on cART 1 year postinfection and treated for 1 year contained intact genomes only 47% of the time. The most common defect identified was large deletions, with the remaining defects caused by APOBEC-mediated mutations, frameshift mutations, and inactivating point mutations. Overall, this approach can be used to assess the intactness of persistent viral DNA in NHPs. IMPORTANCE Molecularly defining the viral reservoir that persists despite antiretroviral therapy and that can lead to rebound viremia if antiviral therapy is removed is critical for testing interventions aimed at reducing this reservoir. In HIV infection in humans with delayed treatment initiation and extended treatment duration, persistent viral DNA has been shown to be dominated by nonfunctional genomes. Using multiple real-time PCR assays across the genome combined with near-full-genome sequencing, we defined SIV genetic integrity after 9 to 18 months of combination antiretroviral therapy in rhesus macaques starting therapy within 1 year of infection. In the animals starting therapy within a month of infection, the vast majority of persistent DNA was intact and presumptively functional. Starting therapy within 1 year increased the nonintact fraction of persistent viral DNA. The approach described here allows rapid screening of viral intactness and is a valuable tool for assessing the efficacy of novel reservoir-reducing interventions.

The Paradox of HIV Blood–Brain Barrier Penetrance and Antiretroviral Drug Delivery Deficiencies

“…In studies using simian immunodeficiency virus (SIV) in rhesus macaques, SIV-infected CD4 + T cells, macrophages, and dendritic cell markers were found in brain and bone marrow tissues. Additionally, SIV RNA was observed to be actively dividing, thus supporting the theory of clonal expansion of latent viral reservoirs in brain compartments [ 24 , 25 ]. Mice infected with a chimeric form of HIV, termed EcoHIV, have been shown to express the viral genome that was illuminated by the increased expression of C3, IL-1β, IL-6, CCL2, and STAT-1, which are factors that influence inflammatory responses to HIV in the brain [ 26 ].…”

“…It was found that induction of HIV expression is possible ex vivo , suggesting latency was pre-established in these isolated CD4 + T cells in vivo [ 30 ]. Real-time PCR assays and genomic sequencing have also confirmed the presence of viral reservoirs within CD4 + T cells that can cause rebound viremia if ART drugs are terminated [ 25 , 28 , 31 , 32 ]. More recently, the novel development of an intact proviral DNA assay has enabled selective detection of the levels of proviral DNA within CD4 + T cells, thus discerning from any present defective proviruses [ 33 ].…”

Predictors of SIV recrudescence following antiretroviral treatment interruption

“…For example, if SIV DNA were 100% replication competent at day 4, and 1% replication competent at day 27, this could explain a 100-fold change in reactivation rates. To investigate this, we sequenced near full length SIV DNA from PBMCs taken just prior to ATI from animals treated on day 10 (n = 4) and day 27 (n = 5)(we note that this data has also been included as part of another study; Long et al, 2019) (Figure 5). We found that 50/60 sequences from the day 10 treated and 86/101 sequences from day 27 treated animals were intact and presumptively replication competent (mean of 84% intact, range of 75–100% in individual animals).…”

Therapeutic vaccination of SIV-infected, ART-treated infant rhesus macaques using Ad48/MVA in combination with TLR-7 stimulation

“…While quantitative virus outgrowth assays could not be performed here due to limited blood volumes, the newly described Intact Proviral DNA Assay (IPDA) [ 35 , 36 ] could prove useful for pediatric studies. Interestingly, though, work from the Siliciano and Keele groups suggests that in nonhuman primates total cell-associated SIV DNA may more closely approximate the intact reservoir than in humans [ 35 , 37 ]. A recent study by Garcia-Broncano et al .…”