Figure 1.
Linear correlation between Ln (Ct of Alu-gag) and Ln (HIV DNA copies per well) at low and high DNA concentrations.
The integration standard was diluted in PBMC DNA from HIV-negative donors at 2 (A) or 40 (B) µg/ml as indicated for each standard curve in order to obtain samples containing known numbers of integrated HIV DNA copies. 25 µl of the standard were then assayed per well after adding 25 µl of the PCR master mixture for a total of 50 µl in each reaction. The final concentration of HIV DNA in each well is therefore, 1 and 20 µg/ml for the low and high DNA concentrations, respectively. Each point represents the average Ln(Ct) for 42 replicates.
Figure 2.
Elite suppressors have lower levels of integration than other HIV+ patients on and off HAART.
HIV DNA Integration levels from ten ES patients were compared to the levels found in 21 patients on and 8 patients off HAART. Individual levels of total and integrated HIV DNA have been previously published for 10 of the patients on and 6 of the patients off HAART [21], [22]. The level of integration in ES patients was significantly lower than patients off and on HAART. The level in the on HAART group was also statistically lower than off HAART (p<0.001, statistical test described in methods). The line represents the median value.
Table 1.
Elite suppressor characteristics.
Figure 3.
Total HIV DNA is not significantly different in patients on HAART and ES patients.
Total HIV DNA was measured by RU5 quantitative PCR in PBMC from 2 non-controllers off and 4 on HAART and elite suppressor patients. Serial two-fold dilutions (from 1e6 to 2.5e5 cells per well) were measured in triplicates for each sample. This was repeated on a separate extraction for all ES. In all cases inhibitors were demonstrated to be absent by dose response in 3 serial two-fold dilutions. Data from 6 off and 17 on HAART patients previously measured was also included for analysis [21], [22]. Patients off HAART have higher levels of total HIV DNA than other patient populations. The level of total HIV DNA was not significantly different between patients on HAART and ES, while both were significantly lower than patients off HAART (p = 0.002 for both). The line represents the median value.
Figure 4.
ES patients have a large excess of unintegrated HIV DNA.
Total and integrated HIV DNA was compared in ES patients (A). The ratio was then calculated by dividing the number of total HIV DNA copies per cell by the number of integrated HIV DNA copies per cell for each patient sample, and these were compared to patients on and off HAART. The excess of unintegrated HIV DNA was significantly higher in the ES patient population than either patients on or off HAART (B). However, the levels were not different between patients on and off HAART (p = 0.172). The line represents the median value.
Figure 5.
ES patients harbor higher levels of 2-LTR circles than HIV+ patients on HAART.
DNA samples from ten ES patients were analyzed for 2-LTR circular HIV DNA. 2-LTR circular HIV DNA levels were compared to those in samples from HIV+ patients on and off-HAART. Only ES and on HAART could be compared due to small samples size. The level of 2-LTR circles was higher in ES as was the frequency with which they were detected (p = 0.007, chi-square test). The line represents the median value.
Figure 6.
ES CD4+ T cells are not resistant to in vitro infection.
CD4+ T cells from 10 ES and 6 uninfected donors were inoculated with NL4-3 (A). Reverse transcripts and integrated HIV DNA were measured at 2 days post-infection. From these values, integration efficiency was calculated (i.e. integrated copies per reverse transcript). ES were compared to normal donors with a Wilcoxon rank-sum test. Neither reverse transcription, integration nor integration efficiency measurements were statistically different between the two populations. Six ES were also inoculated with 3 serial ten-fold dilutions of the same virus (B). The bars represent the averages of multiple experiments (n is indicated in figures) and the error bars represent the standard deviations.
Table 2.
Percentage of integrated HIV DNA per reverse transcript.