Twenty years after the discovery of HIV-2, the second human immunodeficiency virus , the reasons for the observed differences in its epidemiology and pathogenicity relative to HIV-1 are still unclear. Relative to HIV-1, HIV-2 viraemia is less frequently detectable and plasma viral load is lower in patients with similar clinical and immunological status [2–4]. Previous quantitative studies of HIV-2 DNA, including our own, indicated that DNA load in HIV-2-infected patients was not significantly different from DNA load in HIV-1-infected patients [5–8]. However, the lack of specific tools allowing truly comparable PCR quantification of the two viral genomes restricted the conclusions that could be drawn from these differences. We therefore designed a ‘co-plasmid standard’ containing both HIV-1 and HIV-2 genomic sequences. We then used this HIV-1+HIV-2 co-plasmid to calibrate real-time PCR-based HIV-1 and HIV-2 genomic amplification methods, allowing us to compare in identical conditions the amounts of DNA circulating in antiretroviral-naive patients at various stages of immunodeficiency.
Patients and methods
Samples were obtained, with written consent, from HIV-1- and HIV-2-infected subjects never exposed to antiretroviral drugs and living in France. Peripheral blood mononuclear cells (PBMC) were collected on Ficoll Lymphocyte Separation Medium (Eurobio, Courtaboeuf, France) .
Quantification of DNA HIV by real-time PCR
Preparation of the HIV-1+HIV-2 co-plasmid
We synthesized a co-plasmid for use as a standard and calibrator for PCR quantification. It contains the regions of the long terminal repeats (LTR) that are amplified during PCR quantification of total HIV-1 and HIV-2 DNA. We first synthesized the HIV-1 and HIV-2 LTR fragments with their usual primers and then extended these fragments with overlapping primers matching the PCR products (Table 1). For this purpose we used Herculase HotStart DNA Polymerase (Stratagene, Amsterdam, the Netherlands), as follows: the 50 μl reaction mixtures consisted of 5 μl buffer, 0.5 μl Taq, 0.25 μM each primer, 0.8 μM an equimolar mix of dNTP, and 2 μl PCR product. Amplification was carried out as follows: 95°C for 2 min (1 cycle), 95°C for 10 s then 55°C for 45 s and 68°C for 1 min (30 cycles), with a final extension step at 68°C for 7 min, on a GeneAmp 9700 (Applied Biosystems, Courtaboeuf, France). These extended fragments were polymerized with the same programme as above, in a primer-free PCR mix using the Expand Long Template kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions, with the two extended fragments in equal quantities. The overlapping sequences permit self-priming. The long fragment was then amplified by PCR with the same programme and the same mix containing the most external primers at 0.6 μM. The PCR product was cloned with the TOPO TA cloning pCR 2.1 kit (Invitrogen, Cergy Pontoise, France), and produced in competent Escherichia coli. The HIV-1+HIV-2 co-plasmid products were sequenced with a CEQ 8000 automaton (Beckman Coulter, Roissy, France) and quantified by measuring OD at 260 nm. For each quantification run, the co-plasmid was serially diluted 10-fold in human DNA, in duplicate, from 50 000 to 50 copies/μg DNA.
Nucleic acid extraction and real-time PCR
Cellular DNA was extracted in a Magna Pure automaton (Roche Diagnostics) as previously reported . Primers and probes were selected in the LTR–gag region (Table 1) [11,12]. Amplification was performed with the Platinum Quantitative Supermix-UDG kit (Invitrogen) and an ABI PRISM 7900HT device (Applied Biosystems, Courtaboeuf France). Reagent conditions were as follows: master mix 25 μl; primer 1 (10 μM) 1 μl; primer 2 (10 μM) 1 μl; Taqman probe (10 μM) 1 μl; 1 μg of DNA extract; final volume 50 μl. DNA denaturation and Taq activation at 95°C for 10min (one cycle) was followed by amplification at 95°C for 15 s then at 57°C for 1 min (50 cycles). The results are expressed as log10 HIV DNA copies/μg total DNA extract.
Determination of sensitivity, specificity and reproducibility
Specificity was determined by testing 20 extracts of DNA obtained from HIV-seronegative blood donors and a panel of DNA obtained from positive PBMC cultures: 10 HIV-1 subtypes (A, B, C, D, F, G, H, J, CRF01 and CRF02) and 5 HIV-2 group A (n = 3) and group B strains (n = 2). Between-run reproducibility was determined by calculating, for 13 different runs, the mean and standard deviation for the crossing threshold (CT) of two points of the range and the coefficient of variation of the point with a theoretical value of 2.7 log10 copies/μg. The sensitivity of the PCR assays was determined by 10-fold analysis of plasmid dilutions of 5, 10 and 20 copies/μg of human DNA in the same run.
Plasma viral RNA load assay
HIV-1 RNA plasma load was determined with the Cobas Amplicor HIV-1 Monitor v1.5 method (Roche Diagnostics) and HIV-2 RNA plasma load was determined with our real-time RT–PCR method as described elsewhere [13,14].
The total amount of HIV DNA in PBMC was reported as the median and interquartile range (IQR). Total viral DNA load in HIV-1- and HIV-2-infected patients was compared with the Wilcoxon test (α risk ≤5%). Each group of patients was divided into three balanced subgroups defined by the CD4 cell counts, with cut points of 300 (untreated HIV-2-infected patients with counts < 200/μl are extremely rare in France) and 500/μl.
Samples under the detection limit were given an arbitrary value of 10 copies/μg for statistical analysis (SAS software, version 8.2, SAS Institute Inc., Cary, North Carolina, USA).
Highly sensitive and reproducible DNA quantification
No HIV-negative samples gave positive results. HIV-1 DNA PCR amplified all the samples (HIV-1 subtypes A, B, C, D, F, G, H, J, CRF01, CRF02) and HIV-2 PCR correctly amplified all the HIV-2 group A and B samples. As expected, given the high degree of divergence between the HIV-1 and HIV-2 genomes, the primers did not cross-hybridize.
PCR quantification of total HIV-1 and HIV-2 DNA was strictly comparable and had excellent reproducibility thanks to the use of the common standard. The CT in 13 different runs of the 50 000 copies/μg standard point showed excellent reproducibility, with CT values of 25.68 (SD, 0.50) cycles for HIV-1 and 24.16 (SD, 0.37) for HIV-2. For a point with a theoretical value of 2.7 log10 copies/μg, we obtained inter-assay coefficients of variation of 2.2% for HIV-1 and 3.3% for HIV-2. The detection limit was 10 genome copies/μg for HIV-1 and 20 genome copies/μg for HIV-2.
Quantitative differences between HIV-1 and HIV-2 DNA in patients' PBMC
Thirty-seven of the 40 samples from HIV-1-infected patients and 31 of the 42 samples from HIV-2-infected patients were positive and quantifiable for total viral DNA in PBMC. The amounts of total HIV DNA in these 82 never-treated patients were compared at various stages of infection. Median values in each of the three CD4 cell count categories (> 500, 300–500, < 300 cells/μl) were lower in subjects infected by HIV-2 than in subjects infected by HIV-1 (Fig. 1). Below a CD4 cell count of 300/μl, the difference between the median amount of HIV-1 DNA (n = 14; median, 2.9; IQR, 2.2–3.2) and HIV-2 DNA (n = 9; median, 2.7; IQR, 2.2–3.3) was not significant (P = 0.9256). In contrast, the amount of HIV-1 DNA was significantly higher (P = 0.0119) than the amount of HIV-2 DNA in the subgroups with 300–500 CD4 cells/μl (HIV-1: n = 12; median, 2.7; IQR, 2.3–2.8; HIV-2: n = 11; median, 2.0; IQR, 1.0–2.4). Above 500 CD4 cells/μl, the difference was highly significant (P = 0.0019) between HIV-1 (n = 14; median, 2.5; IQR, 2.1–2.7) and HIV-2 (n = 22; median, 1.6; IQR, 1.0–2.0).
Correlation between HIV-2 DNA load, viral RNA load and the CD4 cell count
HIV-2 DNA viral load correlated negatively with the CD4 cell count (r, −0.557; n = 42; P = 0.0001; Spearman rank correlation) and the correlation was stronger than in the group of HIV-1-infected patients (r, −0.378; n = 40; P = 0.0161). The HIV-2 RNA viral loads were under the detection limit (100 copies/ml) in 30 patients, explaining the moderate correlation found between HIV-2 RNA and DNA (r, 0.488; n = 42; P = 0.0011). Only one HIV-1-infected patient had RNA viral load under the detection limit, and a stronger correlation was found between RNA and DNA in this group (r, 0.638; n = 40; P < 0.0001).
Viral DNA load was significantly lower in HIV-2-infected patients with CD4 cell counts > 300/μl than in their HIV-1-infected counterparts. Values were also lower in HIV-2-infected patients with CD4 cell counts < 300/μl, but the difference with HIV-1-infected patients was no longer significant. It is noteworthy that we had no samples from HIV-2-infected patients with counts < 50/μl, such patients being rare in France.
Previous studies showed no major difference in the amount of total DNA between HIV-1- and HIV-2-infected patients, whatever the CD4 cell count [5,6,15]. However, the PCR assays used in these studies targeted different genomic regions in the two viruses, and there was no common standard. In addition, the viral load differences observed here, although statistically significant, were relatively small. HIV DNA values have a narrower range and less variability than RNA values. DNA loads > 3.5 log10/μg DNA extract are rare, even in HIV-1-infected patients , and the differences observed in our study between HIV-1 and HIV-2 DNA loads can be considered relatively large. The use of our HIV-1+HIV-2 co-plasmid containing a single copy as a similar calibrator for the two viruses explains the excellent reproducibility of our results, which are compatible with the natural history of human HIV infection.
Most quantitative studies of HIV-2 proviral load have shown a negative correlation with the CD4 cell count [5,7,8]. Conversely, as previously reported, HIV-2 genomic diversity has little or no impact on DNA amplification, and HIV-2 groups A and B are associated with the same immunological and clinical differences relative to HIV-1 . We also confirmed the strong negative correlation between HIV-2 proviral DNA and the CD4 cell count (r, −0.557; n = 42; P < 0.001), which was of the same strength as that found by Berry et al. (r, −0.54; n = 63; P < 0.001) . We also found that DNA and RNA values correlated with the CD4 cell count in HIV-1-infected patients (r, 0.638 and −0.378, respectively), as reported by Rouzioux et al.. Our study population was similar to that of other authors, and the significant differences we observed are only due to the improvement in technical conditions.
HIV-1-infected ‘slow progressors’ have lower viral DNA loads than other patients , and the lower proviral DNA load that we found in HIV-2-infected patients relative to HIV-1-infected patients is compatible with the slower clinical progression of HIV-2 infection. HIV-1 DNA load is predictive of progression to AIDS, independently of HIV RNA load and the CD4 T cell count . In HIV-2-infected-patients, viral RNA is often undetectable while DNA is generally quantifiable; however, the small range of DNA values, in addition to PCR variability, limit the use of this marker in clinical practice. Finally, the moderate correlations observed between RNA viral load, DNA viral load and the CD4 cell count in both HIV-1- and HIV-2-infected patients confirm the complex relationship between these markers in HIV infection .
Clotilde Bertin, Fanny Lemerchain, Eléonore Wach, Sébastien Delannoy and Arnaud Fleury performed the technical part of this work. Blood was kindly provided by the Normandy bloodbank (ETS).
Sponsorship: This work was supported by ANRS.
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