Development of Methods for Coordinate Measurement of Total Cell-Associated and Integrated Human Immunodeficiency Virus Type 1 (HIV-1) DNA Forms in Routine Clinical Samples: Levels Are Not Associated with Clinical Parameters, but Low Levels of Integrated HIV-1 DNA May Be Prognostic for Continued Successful Therapy

Abstract

We have adapted our established Alu PCR assay for proviral DNA and PCR for total cellular DNA to a real-time PCR format and applied these to human immunodeficiency virus (HIV)-positive specimens collected for routine determination of the plasma viral load (pVL). In a cohort of five patients, measurements of integrated viral load (iVL) and cell-associated viral load (cVL) in CD4؉ cells isolated by a single positive selection step were not indicative of HIV DNA levels in the circulation, and further analysis was performed on peripheral blood mononuclear cells (PBMC). In a cohort of 46 samples total cVL was quantitated in most samples, but iVL could be quantitated in only 47.8%, since in 26% iVL was undetectable and in 21.7% the results were invalid due to high levels of unintegrated HIV DNA. There was no correlation of cVL or iVL with pVL, CD4 count, or duration of successful antiretroviral treatment. Out of 26 patients with undetectable pVL, 4 patients failed therapy within the subsequent 12 months and had higher than average iVL, but this was not the case for cVL. Among nine patients with long-term undetectable pVL, no consistent decline in cVL or iVL was seen with time, and changes in cVL and iVL within a patient could be concordant or discordant. These results show that cVL and iVL can be coordinately measured in PBMC from clinical samples but do not correlate with pVL, CD4 counts, or length of suppressive antiretroviral therapy. Interestingly, a high iVL (but not a high cVL) in patients with undetectable pVL was associated with subsequent treatment failure.

A more precise HIV integration assay designed to detect small differences finds lower levels of integrated DNA in HAART treated patients

“…Furthermore, results obtained with the assay suggest that the level of integrated DNA is lower in patients on effective HAART than in patients not on HAART. Although this result may seem obvious, two prior reports failed to detect this difference (Carr et al, 2007; Ibanez et al, 1999). Our ability to detect this difference suggests our assay may be more precise than prior assays.…”

“…Second, the level of integration in HIV-infected individuals is low and detection is a further challenge since only a fraction of integration events are detectable because of the widely distributed integration sites (Agosto et al, 2007). While many studies have reported the level of total viral DNA in cells from HIV-infected individuals (Brenchley et al, 2004; Burgard et al, 2000; Debiaggi et al, 2000; Douek et al, 2002; Gibellini et al, 2004; Jubault et al, 1998; Kabamba-Mukadi et al, 2005; Kostrikis et al, 2002; Lillo et al, 2004; Ngo-Giang-Huong et al, 2001; Rouzioux et al, 2005; Viard et al, 2004; Vitone et al, 2005), only a few have reported the level of integrated DNA (Carr et al, 2007; Chun et al, 1997a; Chun et al, 1995; Chun et al, 1997b; Ibanez et al, 1999; Izopet et al, 2002; Koelsch et al, 2008; Ostrowski et al, 1999). …”

“…However, none of the kinetic PCR assays that have been developed are sensitive enough to measure integration in patient samples. For example, a recent study proposed a kinetic PCR integration assay for clinical use, but the assay did not detect integration in ~50% of patient PBMCs (Carr et al, 2007). Thus, integration data was only obtained for half the patients.…”

Prognostic value of peripheral blood mononuclear cell-associated HIV-1 DNA for virological outcome in asymptomatic HIV-1 chronic infection

“…Given that there is not a standardized and accurate quantitative method for intracellular HIV-1 DNA quantification, the comparison of the results obtained from the different studies [21][22][23][24][25][26][27][28][29][30][31][32][33][38][39][40][41][42][43][44][45][46][47][48] is without doubt a difficult task.…”

An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

“…In vivo, this inefficiency of integration results in an accumulation of unintegrated HIV-1 DNA (uDNA) in blood CD4 ϩ T cells and monocytes (7,8,11,12), as well as in lymphoid tissue (8-10), brain (13,14), and untreated patients with high or low viral loads (15,16). During untreated chronic infection, the majority of infected cells have been reported to contain only unintegrated HIV-1 DNA (7).…”