Limits...
Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

Graf EH, Mexas AM, Yu JJ, Shaheen F, Liszewski MK, Di Mascio M, Migueles SA, Connors M, O'Doherty U - PLoS Pathog. (2011)

Bottom Line: To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC.We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells.Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

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Related in: MedlinePlus

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.
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ppat-1001300-g001: 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.

Mentions: Viral burden in HIV infected patients can be measured as viral particles containing RNA, cell associated viral RNA, and total and integrated viral DNA. Viral burden quantification in ES has been limited to measurements of viral RNA in plasma, cell-associated viral RNA or total DNA (often described as “proviral DNA”) in PBMC, and estimates of Infectious Units per Million cells (IUPM) [14]–[17]. To our knowledge, there have been no previous attempts to specifically measure integrated DNA in ES patients. Integrated viral DNA is believed to be of great importance in the establishment of a latent reservoir that is resistant to HAART and measuring integration may serve as a surrogate measure of the viral reservoir in the absence of ongoing replication [18]. The establishment of this latent reservoir is thought to occur early in the course of infection [19], but the contribution of integrated HIV DNA to viral persistence in ES patients remains unknown. Here, we apply a unique, sensitive and precise method to measure integrated HIV DNA in PBMC samples obtained from ES patients. In order to accurately measure very low levels of HIV integration in this cohort, we increased the sensitivity of our previously described repetitive sampling Alu-gag PCR integration assay [20] by increasing the number of genomes assayed per well. We find low but measurable levels of integrated DNA in 10 out of 10 ES patients with a wide distribution of levels of integration between patients. The level of integration in ES patients was significantly lower than that in equally suppressed patients on HAART. This is intriguing, given that we and others [17] find the level of total HIV DNA in ES patients is similar to that in HAART treated patients, albeit with small data sets for comparison. To determine if the unintegrated HIV DNA in the ES samples was due to the accumulation of 2-LTR circular DNA, we measured 2-LTR circular HIV DNA in these (and other) samples. We found 2-LTR circles were present at detectable levels more often in samples from ES patients, compared to samples from patients on HAART and off HAART. Furthermore, the levels of 2-LTR circular HIV DNA were higher in ES than in on-HAART patients. As shown through in vitro inoculations, the excess of 2-LTR circles was not due to an innate restriction at the level of integration in the ES cells since integration occurred with similar efficiency in CD4+ T cells from ES and normal donors after in vitro inoculation with HIV.


Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

Graf EH, Mexas AM, Yu JJ, Shaheen F, Liszewski MK, Di Mascio M, Migueles SA, Connors M, O'Doherty U - PLoS Pathog. (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044690&req=5

ppat-1001300-g001: 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.
Mentions: Viral burden in HIV infected patients can be measured as viral particles containing RNA, cell associated viral RNA, and total and integrated viral DNA. Viral burden quantification in ES has been limited to measurements of viral RNA in plasma, cell-associated viral RNA or total DNA (often described as “proviral DNA”) in PBMC, and estimates of Infectious Units per Million cells (IUPM) [14]–[17]. To our knowledge, there have been no previous attempts to specifically measure integrated DNA in ES patients. Integrated viral DNA is believed to be of great importance in the establishment of a latent reservoir that is resistant to HAART and measuring integration may serve as a surrogate measure of the viral reservoir in the absence of ongoing replication [18]. The establishment of this latent reservoir is thought to occur early in the course of infection [19], but the contribution of integrated HIV DNA to viral persistence in ES patients remains unknown. Here, we apply a unique, sensitive and precise method to measure integrated HIV DNA in PBMC samples obtained from ES patients. In order to accurately measure very low levels of HIV integration in this cohort, we increased the sensitivity of our previously described repetitive sampling Alu-gag PCR integration assay [20] by increasing the number of genomes assayed per well. We find low but measurable levels of integrated DNA in 10 out of 10 ES patients with a wide distribution of levels of integration between patients. The level of integration in ES patients was significantly lower than that in equally suppressed patients on HAART. This is intriguing, given that we and others [17] find the level of total HIV DNA in ES patients is similar to that in HAART treated patients, albeit with small data sets for comparison. To determine if the unintegrated HIV DNA in the ES samples was due to the accumulation of 2-LTR circular DNA, we measured 2-LTR circular HIV DNA in these (and other) samples. We found 2-LTR circles were present at detectable levels more often in samples from ES patients, compared to samples from patients on HAART and off HAART. Furthermore, the levels of 2-LTR circular HIV DNA were higher in ES than in on-HAART patients. As shown through in vitro inoculations, the excess of 2-LTR circles was not due to an innate restriction at the level of integration in the ES cells since integration occurred with similar efficiency in CD4+ T cells from ES and normal donors after in vitro inoculation with HIV.

Bottom Line: To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC.We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells.Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

Show MeSH
Related in: MedlinePlus