Genotypic Resistance Testing of HIV-1 DNA in Peripheral Blood Mononuclear Cells

Abstract

HIV-1 DNA exists in nonintegrated linear and circular episomal forms and as integrated proviruses. In patients with plasma viremia, most peripheral blood mononuclear cell (PBMC) HIV-1 DNA consists of recently produced nonintegrated virus DNA while in patients with prolonged virological suppression (VS) on antiretroviral therapy (ART), most PBMC HIV-1 DNA consists of proviral DNA produced months to years earlier. Drug-resistance mutations (DRMs) in PBMCs are more likely to coexist with ancestral wild-type virus populations than they are in plasma, explaining why next-generation sequencing is particularly useful for the detection of PBMC-associated DRMs. In patients with ongoing high levels of active virus replication, the DRMs detected in PBMCs and in plasma are usually highly concordant. However, in patients with lower levels of virus replication, it may take several months for plasma virus DRMs to reach detectable levels in PBMCs. This time lag explains why, in patients with VS, PBMC genotypic resistance testing (GRT) is less sensitive than historical plasma virus GRT, if previous episodes of virological failure and emergent DRMs were either not prolonged or not associated with high levels of plasma viremia. Despite the increasing use of PBMC GRT in patients with VS, few studies have examined the predictive value of DRMs on the response to a simplified ART regimen. In this review, we summarize what is known about PBMC HIV-1 DNA dynamics, particularly in patients with suppressed plasma viremia, the methods used for PBMC HIV-1 GRT, and the scenarios in which PBMC GRT has been used clinically.

Keywords: DNA sequencing; HIV-1; adaptive mutations; antiviral therapy; drug resistance evolution; peripheral blood mononuclear cells; provirus.

FIG 1

Sensitivity of peripheral blood mononuclear cell (PMBC) genotypic resistance testing (GRT) in patients with plasma virus suppression while receiving antiretroviral therapy (ART). In the first scenario (A), in which previous episodes of virological failure (VF) were prolonged and associated with high plasma HIV-1 RNA levels, the likelihood of detecting drug-resistance mutations (DRMs) in proviral DNA will be high. In the second scenario (B), in which previous episodes of VF were short and associated with low plasma HIV-1 RNA levels, the likelihood of detecting DRMs in proviral DNA will be low. Rx1, Rx2, and Rx3 followed by an arrow indicate periods of antiviral therapy. Proviral DNA containing DRMs are indicated by red asterisks in the boxes at the right side of the figure.

FIG 2

Sensitivity of PMBC GRT in patients with the recent development of low-level viremia while receiving ART. Rx followed by an arrow indicates the period of antiviral therapy. The dashed line indicates a threshold of between 200 and 500 copies/mL below which plasma virus GRT is often not successful. The likelihood of detecting DRMs in proviral DNA is likely to be low. Detecting DRMs in unintegrated linear and episomal viral DNA may be possible because these are more likely to reflect recently circulating viruses. Proviral and unintegrated viral DNA containing DRMs are indicated by red asterisks. In the boxed figure on the right, unintegrated linear viral DNA is shown outside the nucleus. Episomal DNA is not shown; its dynamics are likely to be closer to unintegrated linear DNA than proviral DNA.

FIG 3

Sensitivity of plasma virus and PMBC GRT in patients who discontinue ART following VF and emergent HIV drug resistance (HIVDR). Rx followed by an arrow indicates the period of antiviral therapy. Most DRMs will no longer be detectable by plasma virus GRT within 2 to 3 months following ART discontinuation because plasma viruses containing DRMs are often rapidly outcompeted by ancestral wild-type viruses established in viral reservoirs prior to ART initiation. Although recently emergent DRMs may have begun to seed the proviral DNA reservoir, their levels in this compartment will be low unless VF has been prolonged. In addition, the presence of unintegrated linear and episomal viral DNA, which are in equilibrium with circulating plasma virus, may also reduce the proportion of viral DNA molecules containing DRMs. In the boxed figure on the right, proviral DNA containing DRMs are indicated by red asterisks. Unintegrated linear viral DNA is shown outside the nucleus. Episomal DNA is not shown; its dynamics are likely to be closer to unintegrated linear DNA than proviral DNA.